Master Document: April 18

[0026] Fatty acid amide hydrolase (FAAH) is an enzyme that hydrolyzes the fatty acid amide (FAA) family of endogenous signaling lipids. General classes of FAAs include the N- acylethanolamines (NAEs) and fatty acid primary amides (FAPAs). Examples of NAEs include anandamide (AEA), palmitoylethanolamide (PEA) and oleoylethanolamide (OEA). An example of
FAPAs includes 9-Z-octadecenamide or oleamide. (McKinney MK, Cravatt BF. 2005. Annu Rev Biochem 74:411-32)].
[0027] Diseases, disorders, syndromes and/or conditions, that would benefit from inhibition of FAAH enzymatic activity include, for example, Alzheimer's Disease, schizophrenia, depression, alcoholism, addiction, suicide, Parkinson's disease, Huntington's disease, stroke, emesis, miscarriage, embryo implantation, endotoxic shock, liver cirrhosis, atherosclerosis, cancer, traumatic head injury, glaucoma, and bone cement implantation syndrome.
Other diseases, disorders, syndromes and/or conditions that would benefit from inhibition of FAAH activity, include, for example, multiple sclerosis, retinitis, amyotrophic lateral sclerosis, immunodeficiency virus-induced encephalitis, attention-deficit hyperactivity disorder, pain, nociceptive pain, neuropathic pain, inflammatory pain, non-inflammatory pain, painful hemorrhagic cystitis, obesity, hyperlipidemia, metabolic disorders, feeding and fasting, alteration of appetite, stress, memory, aging, hypertension, septic shock, cardiogenic shock, intestinal inflammation and motility, irritable bowel syndrome, colitis, diarrhea, ileitis, ischemia, cerebral ischemia, hepatic ischemia, myocardial infarction, cerebral excitotoxicity, seizures, febrile seizures, neurotoxicity, neuropathies, sleep, induction of sleep, prolongation of sleep, insomnia, and inflammatory diseases. [0028] Carbamic acid esters have shown promise as selective FAAH inhibitors (Kathuria et al., Nat. Med. 2003, 9:76-81). In particular, a series of alkylcarbamic acid aryl esters, such as, for example, the compound of Formula (I) disclosed herein, have been shown to be potent and selective inhibitors of FAAH activity. Mor et al. (J. Med. Chem. 2004, 47:4998-5008; incorporated by reference) and Piomelli et al. (International Patent Publication No. WO 2004/033422; incorporated by reference) disclosed representative alkylcarbamic acid aryl esters of Formula (I) that are potent inhibitors of FAAH activity, but do not significantly interact with selected serine hydrolases or with cannabinoid receptors. Among the disclosed alkylcarbamic acid esters of Formula (I), the compound cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-yl ester (also known as 5'- carbamoylbiphenyl-3-yl cyclohexyl carbamate, UCM597 and URB597; referenced herein as compound KDS-4103) was identified as a potent and selective inhibitor of FAAH activity.
First, chronic unpredictable stress, used to model anhedonia, a core depression symptom, is associated with decreased endocannabinoid 2-arachidonoylglycerol in the rat hippocampus (Hill et al., 2005). Second, in humans, upregulation of PFC CB1R in suicidal depressives may indicate an adaptive response to decreased endocannabinoids (Hungund et al., 2004). Third, randomized trials of the CB1R antagonist rimonabant for obesity management increased adverse effects of depression and anxiety (Bronander and Bloch, 2007).
Treatment with Delta(9)-THC did not produce significant changes in oxidative stress markers or in mRNA levels of CB1 and CB2 receptors in the liver of mice, but attenuated the increase in the selenium-dependent GPx activity (Delta(9)-THC: 8%; VCtrl: 23% increase) and the GSH/oxidized GSH ratio (Delta(9)-THC: 61%; VCtrl: 96% increase), caused by treatment with the vehicle. Delta(9)-THC administration did not show any harmful effects on lipid peroxidation, protein carboxylation or DNA oxidation in the healthy liver of mice but attenuated unexpected effects produced by the vehicle containing ethanol/cremophor EL.
Whereas the CA1 region appears to process features of space, the DG processes orientation in space. Both phenomena are strongly associated with the expression of LTD in these hippocampal subregions. Taken together with our findings that persistent expression of LTP occurs in the DG and CA1 regions following exposure to novel empty space, one could speculate that LTP is generated as an instantaneous response to exposure to empty space. Superimposed on this phenomenon comes the processing of novel spatial orientation through LTD in the DG and the processing of novel spatial features through LTD in the CA1 region. The integration of these individual aspects of space results in the basis for a complete spatial map of a novel environment. This implies that feature-rich spatial memory is acquired by bidirectional synaptic plasticity and that in the subregions different types of functional spatial information trigger LTD.
Each of the 3 cannabis-associated cases of cerebellar infarction was confirmed by biopsy (1 case) or necropsy (2 cases) and showed inflammatory cellular reactions characteristic of a 1- to 3-day-old infarct (case 1) or a 3- to 7-day-old infarct (cases 2 and 3).3 In all 3 cases, the central nervous system infarction was acute and localized to cerebellum.
We previously presented a case of biopsy-proven cerebellar infarction involving multiple branch cerebellar arterial territories with a benign cerebral arteriogram in a 15-year-old male that was temporally related to heavy marijuana use, confirmed by toxicologic study. This patient presented with a 3-day history of headache, nausea, and unsteadiness of gait after a binge of marijuana smoking.
Brainstem compromise caused by cerebellar and cerebral edema led to death in the 2 fatal cases.
From the literature, it is clear that marijuana use can cause systemic hypotension, impair peripheral vasomotor reflexes, and may alter central nervous system blood flow and cerebral vascular autoregulation. Marijuana use has been associated with stroke in adults, but acute central nervous system infarction related to marijuana use is not well described in children. Although the mechanism of neurologic injury and its localization to the posterior circulation in these cases remains uncertain, our observation of acute cerebellar infarction in 3 adolescents shortly after marijuana use suggests that this drug may contribute to cerebellar vascular injury, possibly by causing vasospasm, especially in the inexperienced or episodic user, resulting in cerebellar ischemia.
Hemopressin is a 9 amino acid peptide derived from alpha-hemoglobin, the main constituent of red blood cells.
Hemopressin is a short, nine amino acid peptide (H-Pro-Val-Asn-Phe-Lys-Leu-Leu-Ser-His-OH) isolated from rat brain that behaves as an inverse agonist at the cannabinoid receptor CB1, and is shown here to inhibit agonist-induced receptor internalization in a heterologous cell model. Since this peptide occurs naturally in the rodent brain, we determined its effect on appetite, an established central target of cannabinoid signaling. Hemopressin dose-dependently decreases night-time food intake in normal male rats and mice, as well as in obese ob/ob male mice, when administered centrally or systemically, without causing any obvious adverse side effects. The normal, behavioral satiety sequence is maintained in male mice fasted overnight, though refeeding is attenuated. The anorectic effect is absent in CB1 receptor null mutant male mice, and hemopressin can block CB1 agonist-induced hyperphagia in male rats, providing strong evidence for antagonism of the CB1 receptor in vivo. We speculate that hemopressin may act as an endogenous functional antagonist at CB1 receptors and modulate the activity of appetite pathways in the brain.
On the other hand, the 11- and 12-residue forms of hemopressin are likely to represent the endogenous forms, and further studies in the field should use these peptides if the goal is to characterize the functions of naturally-occurring peptides.
Che FY, Zhang X, Berezniuk I, Callaway M, Lim J, Fricker LD (2007) Optimization of neuropeptide extraction from the mouse hypothalamus. J Proteome Res 6: 4667-4676.
Fricker LD (2010) Analysis of mouse brain peptides using mass spectrometry-based peptidomics: implications for novel functions ranging from non-classical neuropeptides to microproteins. Mol Biosyst.
Gelman JS, Sironi J, Castro LM, Ferro ES, Fricker LD (2010) Hemopressins and other hemoglobin-derived peptides in mouse brain: Comparison between brain, blood, and heart peptidome and regulation in Cpefat/fat mice. J Neurochem in press.
Gomes I, Grushko JS, Golebiewska U, Hoogendoorn S, Gupta A, Heimann AS, Ferro ES, Scarlata S, Fricker LD, Devi LA (2009) Novel endogenous peptide agonists of cannabinoid receptors. FASEB J 23: 3020-3029.
Heimann AS, Gomes I, Dale CS, Pagano RL, Gupta A, de Souza LL, Luchessi AD, Castro LM, Giorgi R, Rioli V, Ferro ES, Devi LA (2007) Hemopressin is an inverse agonist of CB1 cannabinoid receptors. Proc Natl Acad Sci U S A 104: 20588-20593.
Rioli V, Gozzo FC, Heimann AS, Linardi A, Krieger JE, Shida CS, Almeida PC, Hyslop S, Eberlin MN, Ferro ES (2003) Novel natural peptide substrates for endopeptidase 24.15, neurolysin, and angiotensin-converting enzyme. J Biol Chem 278: 8547-8555.
Among all food additive-containing foods, the highest contributors were: soft drinks to benzoates intake, nuts and canned juices to sulphites intake, bread and biscuits to BHA intake and chewing gum to BHT intake.
We examined the food additive, butylated hydroxyanisole (BHA), for its capacity to modulate the cytotoxic effects of Δ9-tetrahydrocannabinol (THC). THC was not cytotoxic when added to cultures of A549 lung tumor cells at concentrations<5 μg/ml, but induced cell necrosis at higher levels with an LC50=16–18 μg/ml. BHA alone, at concentrations of 10–200 μM, produced limited cell toxicity but significantly enhanced the necrotic death resulting from concurrent exposure to THC. In the presence of BHA at 200 μM, the LC50 for THC decreased to 10–12 μg/ml. Similar results were obtained with smoke extracts prepared from marijuana cigarettes, but not with extracts from tobacco or placebo marijuana cigarettes (containing no THC). Two different mechanisms for this synergistic cytotoxicity were investigated. Experiments were repeated in the presence of either diphenyleneiodonium or dicumarol as inhibitors of the redox cycling pathway. Neither of these compounds protected cells from the effects of combined THC and BHA, but rather enhanced necrotic cell death. Measurements of cellular ATP revealed that both THC and BHA reduced ATP levels in A549 cells, consistent with toxic effects on mitochondrial electron transport. The combination was synergistic in this respect, reducing ATP levels to <15% of control. Exposure to marijuana smoke in conjunction with BHA, a common food additive, may promote deleterious health effects in the lung.
The effects of butylated hydroxyanisole (BHA), a commonly used food antioxidant, on oxygen consumption, ATPase activity, and the redox state of some electron carriers of rat liver mitochondria have been studied. It was observed that BHA slightly stimulated state 4 respiration but strongly inhibited ADP- and uncoupler-stimulated respiration on NAD+- and FAD-linked substrates. ATPase activity and vectorial H+ ejection were affected only slightly by BHA, suggesting that BHA predominantly inhibits mitochondrial electron flow. Experiments to determine its site of action showed that BHA did not noticeably affect electron flow through cytochrome oxidase; in contrast, NADH:duroquinone reductase activity and electron flow through ubiquinone-cytochrome b-cytochromec complex were inhibited strongly because the oxidation of duroquinol was affected markedly. The BHA block of electron transport was bypassed by both N,N,N′,N′-tettamethyl-p-phenyleaediamine and 2,6-dichlorophenolindophenol. Also, the presence of BHA changed the redox state of cytochrome b and C1 to a more oxidized level. These observations suggest that electron transport is inhibited by BHA at the NADH-ubiquinone and at the ubiquinone-cytochrome b levels. From Hill plots, it is clear that more than one binding site is involved in complete inhibition; in addition, available evidence suggests that there may be two sites at the substrate side of ubiquinone and another two sites at the oxygen side of ubiquinone. Consequently, mitochondrial ATP synthesis would be interrupted. This event could be related to the toxicity of BHA.
Exposure to marijuana smoke in conjunction with BHA, a common food additive, may promote deleterious health effects in the lung." BHA & BHT are human-made fat preservatives, and are found in many packaged foods including: plastics in boxed cereal, Jello, Slim Jims, and more
BHA is added to butter, lard, meats, cereals, baked
goods, sweets, beer, vegetable oils, potato chips, snack foods, nuts and
nut products, dehydrated potatoes, and flavoring agents. It is used in
sausage, poultry and meat products, dry mixes for beverages and desserts,
glazed fruits, chewing gum, active dry yeast, defoaming agents for beet
sugar and yeast, and emulsion stabilizers for shortenings.
BHA stabilizes the petroleum wax
coatings of food packaging (Sax and Lewis 1987). BHA is considered a
GRAS (generally recognized as safe) compound by the FDA when the
content of the antioxidant is not greater than 0.02% w/w of the total fat
or oil content of the food.
Fast food service personnel who normally cook and serve fried and oily
foods have the potential for high exposure to BHA.
Potential occupational exposure exists for workers in certain
industries, including food producers, animal feed producers, livestock
producers, cosmetic manufacturers, some petroleum workers, and
rubber producers and those who handle the end products such as tires.
BHA is used as a preservative and antioxidant in pharmaceutical
preparations and cosmetic formulations containing fats and oils (Osol
1980). Cosmetic-grade BHA reportedly contains a minimum of 90%
3-BHA and approximately 8% 2-BHA (IARC 1986). In a 1981 FDA
survey, BHA was reported to be used in 3,217 to 21,279 cosmetic
formulations; the majority (88%) of the reported concentrations was
≤0.1% (IARC 1986). One product, a lipstick, was reported to contain
>10% BHA. In this survey, lipstick formulations (1,256 products)
represented the highest use of BHA, with eye shadows being the next
highest (410 products). A widely used antioxidant mixture for
cosmetics contained 20% BHA, 6% propyl gallate, 4% citric acid, and
70% propylene glycol (Kirk-Othmer 1979a, IARC 1986).
Potential for consumer exposure to BHA by ingestion and dermal
contact is widespread. In 1975, the estimated average daily intake of
BHA in the diet was 4.3 mg (IARC 1986). It is a widely used food
additive in products containing vegetable oils or animal fats. It retains
its antioxidant properties even at high temperatures. The general
population may be exposed to BHA in butter, lard, meats, cereals,
baked goods, sweets, beer, vegetable oils, potato chips, snack foods,
nuts, dehydrated potatoes, flavoring agents, sausage, poultry and meat
products, dry mixes for beverages and desserts, glazed fruits, chewing
gum, active dry yeast, defoaming agents for beet sugar and yeast, and
emulsion stabilizers for shortenings. The estimated U.S. consumption
of BHA in food increased to 660,000 lb/yr during 1970 to 1982, up
from 374 lb/yr in 1960 (Anonymous 1984, IARC 1986). Reported
annual consumption for BHA in the mid 1970s was 990,000 lb
compared with 3.1 million lb for butylated hydroxytoluene (BHT)
(Kirk-Othmer 1978). Industrial use of BHA has largely been replaced
by tert-butylhydroquinone (TBHQ) (Kirk-Othmer 1980).
Theoretical and experimental.
studies have revealed that at microwave frequencies, geometrical
resonances occur in the mammalian brain that are manifested in non-uniform
distributions of absorbed energy which, for example, may result in maximum
absorption at the anterior hypothalamus, the thermoregulatory center of the
brain. Evidence derived from neurophysiological studies suggests that a
0 temperature rise of 0.01 C could result in a 3% alteration in the firing rate
of thermosensitive neurons of the preoptic nucleus of the hypothalamus.
Consequently, low-level microwave heating of such neural elements could conceivably
induce thermocompensatory responses which would be detected as an
alteration in the physiological status of the experimental subject. Such
effects would be dependent upon microwave-specific thermal changes. The
validity of this concept is dependent upon the creation of non-uniform temperature
distributions within the brain due to non-uniform energy absorption.
Since the brain is a vascularized organ, conductive cooling by blood circulation
would act to reduce non-uniform temperature distributions.
Experimental studies have been conducted at various frequencies ranging
from extra low frequencies (ELF) to microwave electromagnetic field effects.
Tinney exposed isolated poikilothermic turtle hearts to 960 MHz
continuous wave (CW) fields. In opposition to the anticipated tachycardia
which results from generalized heating, microwave exposure in the range of
absorbed powers of 2-10 mW/g resulted in significant bradycardia. The authors
hypothesize that microwave radiation causes neurotransmitter release by excitation
of the nerve remnants in the heart. Atropine, a blocker of the
parasympathetic system, and propranol hydrochloride, a sympathetic system
blocking agent, resulted in a suppression of the microwave effect when added
to the isolated hearts. By administering the drugs separately, evidence was
obtained that microwave radiation affects neurotransmitter release; the mechanism
for the effect is, however, unknown.
In vivo studies of the effects of low intensity microwaves on neurotransmitter
release have also been reported. Syngajevskaja found that 0.5 mW/cm2 decimeter wave exposure produced a significant decrease in acetylcholinesterase (Ache) activity in rabbit brains. An increase in acetylcholine and a decrease in Ache in the blood of irradiated animals has also been reported.
Repeated exposure of rats to 3 GHz microwaves at intensities
of from 10 to 40 mW/cm2 led to an initial increase followed by a
decrease in Ache blood activity.
Similar results were reported by
Baranski following irradiations of rabbits for two and one half to five
months, whereas an eight month exposure at an intensity of 1 mW/cm2 was
without effect. Three hour exposures of guinea pigs to 3 GHz CW radiation
at intensities of 3.5 and 25 mW/cm2 had no effect on Ache brain activity,
wnereas pulsed wave (PW) exposures caused a significant decrease in activity.
Repeated exposures resulted in increases in Ache activity, the effect being
more pronounced following pulsed field exposures. Ache activity in
rabbits repeatedly exposed to CW and PW microwaves for four months was found
to decrease in the case of CW exposure and increase as a result of PW irradiation.
The midbrain regions were found to be most affected. Microwaveinduced
alterations in lipid and nucleo-protein metabolism in the brains of
experimental animals have also been reported, the alterations again being
more pronounced following pulsed field exposure.
Merritt compared the effects of 1.6 GHz microwave and nonradiation
heating on neurotransmitter concentrations in the rat brain. Rats exposed for ten minutes to 80 mW/cm experienced a 4 C rise in rectal temperature
as did thermal controls exposed to elevated environmental temperatures.
Hypothalamic norepinephrine decreased in irradiated and hyperthermal control
animals compared to normothermal controls. Serotonin concentrations were
decreased in the hippocampal regions of irradiated rats but not in hyperthermal
controls. Similar results were obtained for the dopamine concentrations
in the corpus striatum and hypothalamus. Thermographic analyses of
the absorbed microwave power distributions indicated that the neurotransmitter
alterations were the result of microwave-induced hyperthermia.
Histological effects of low-intensity microwave exposure have been reported
by Albert and DeSantis. Chinese hamsters were irradiated with 1.7- 2
GHz CW microwaves at power densities of 10 and 25 mW/cm. Exposure for
thirty minutes to two hours induced cytopathology in hypothalamic and subthalamic
neurons.
Modulated low intensity electromagnetic fields have been reported to
alter calcium binding in cerebral tissue. Bawin and Adey25 exposed isolated
chick and cat cerebral tissues for twenty minutes to 0.05, 0.1, 0.56 and
1.0 V/cm fields at frequencies of 1, 6, 16, 32, or 75 Hz. A biphasic respouse
of Ca2 + in brain tissue was detected with a maximum suppression of
Ca2+ efflux occurring at 6 and 16 Hz and for field strengths of 0.10 and
0.56 V/cm. As in the case of alteration in heart rate reported by Tinney,
et aZ 16 , these results suggest the existence of a biphasic field strength
dependence since increasing the field strength to magnitudes greater than
0.56 V/cm reduced the treatment effect. Direct cortical stimulation of the
intact cat brain with 200 Hz, 10 msec pulses at intensities of 20-50 mV/cm,
on the other hand, resulted in a 20% increase in ca2+ efflux26 • Radiofrequency
fields with a carrier wave frequency of 147 MHz, amplitude modulated
at 6, 9, 11, and 16 Hz produced a statistically significant increase27
in ca2+ efflux from isolated chick forebrain at intensities of 1 to 2 mW/cm2•
A possible mechanism for the effect of low intensity radiofrequency radiation
on ca2+ efflux is suggested by the authors. They state17 that the
electrochemical equilibrium that exists in cerebral tissues between ions,
polyanionic macromolecules, and glycoproteins of the cell surface may be
disrupted by small variations of either the surrounding ionic concentrations
or local electrical gradients. Modulations of the radiofrequency field, reflected
as slow modulations of the extracellular electric field could thus
affect the Ca 2+ binding to the neuronal membrane. The fact that no effect
on Ca 2+ fluxes was produced by unmodulated fields and that a pulse repetition
rate dependent maximum rate of efflux was detected at rates of 11 and
2+ 16 Hz indicates that Ca movement was critically related to specific slow
components of the radiation field. They draw attention to the fact that this
frequency effect is consistent with results of other studies using various
electrophysiological endpoints and that it supports the finding that specific
modulation effects are involved in the reported induction of EEG changes in
the cat.
Bawin and Adev in discussing the inverse relationship of ca2+ binding
effects in neural tissue in response to modulated VHF and ELF fields indicate
that the results suggest field interaction with a common substrate which
is dependent on the interaction energy provided by the field. The basis for
the amplitude selectivity or biphasic amplitud~ response of ca2+ binding is
suggested as being related to the mode of Ca 2+ binding to neuronal biomolecules
which may consist of strong or weak bonding depending upon sitespecific
molecular binding sites2'. The authors state that no completely satisfactory explanation can be given ~or the biphasJc frequency response;
\they note that subjective time estimates in monkeys'are speeded up in the
presence of a 7 Hz, 0.10 V/cm sinusoidal field but in order to induce this
effect at 45 to 75 Hz larger field strengths are required. Bawin and Adey'
also state that EEG patterns in the cat are reinforced by VHF fields amr
plitude modulated at the dominant EEG frequencies but not by CW fields or
fields modulated at other frequencies. It is suggested that the frequency
specificity of the responses is an indication of a direct field effect on
the CNS, rather than a more generalized peripheral transduction.
It is indicated that the current density induced by a 10 mW/cm2
microwave field is 1000 times greater than the polarizing current densities required to produce an alteration in the firing patterns of ganglia.
Servantie, et aZ 32 studied the synchronization of cortical neurons in the rat by pulsed 3 GHz radiation at an average intensity of 5 mW/cm and a pulse repetition rate of 500-600 Hz. Experimental animals were exposed in free space conditions for ten days after which occipital EEG's were recorded.
The exposure resulted in the induction of an electrocortical frequency synchronous
with the microwave pulse modulation frequency. The induced electrocortical
frequency, which is significantly higher than those generally recorded,
persisted for a period of hours following the termination of exposure.
Thomas, et aZ 36 as previously
noted, found that exposure of rats to low intensity 5 mW/cm2
pulse modulated microwaves altered the animal's ability to judge the passage
of time, a result previously reported by Justesen 3 ~ in the same species.
Thomas and coworkers~ 0 have recently reported that PW 2.45 GHz microwaves at
5, 10, or 15 mW/cm 2 caused a dose-dependent increase in the frequency of premature
switching in a fixed consecutive-number schedule in rats following
thirty minute exposures, suggesting an alteration in time perception. Using
a markedly lower radiation frequency, Gavalas-Medici and Day~ 1 found exposure
of rhesus monkeys to 7 or 75 Hz fields at field strengths as low as 0.10 V/cm
caused a reduction in interresponse times in agreement with the results of an
earlier study employing a 0.07 V/cm, 7Hz field~ 2 • At a field strength of
0.56 V/cm interresponse times were significantly shorter for both 7 and 75 H?
fields and the differences were almost three times greater than at a field
strength of 0.10 V/cm. Another indication of an alteration in the "biological
clock" was reported by Bliss and Heppner~ 3 who used a different temporally dependent
endpoint, a different species, and a different type of exposure. They
investigated the effects of exposure to a zero magnetic field on the entrainment
of the circadian activity rhythm of the House Sparrow, Passer domesticus.
Periodogram analysis showed that the activity period lengths of control birds
were longer than the experimental birds. Midpoints of activity time were significantly
later for controls than for experimentals.
Studies of the interaction of microwave radiation and various drugs that
affect the mammalian CNS have also yielded evidence of the sensitivity of
neural elements to low field intensities. Goldstein and Sisko~~ and Cleary
and Wangemann~ 5 detected alterations in the response of rabbits to anesthesia
as a result of low intensity CW microwaves at 9.3 and 2.45 GHz respectively.
Baranski and Edelwejn~ 6 investigated microwave-drug interactions in
rabbits given CNS stimulants or depressants. Chloropromazine induced rapid
synchronization of previously desynchronized EEG patterns which led the
authors to conclude that there was an antagonistic interaction of microwaves
withthis drug which most likely occurred in the activating part of thereticular
formation and the cerebral cortex~ 6 • Servantie, et al~ 7 also found
that the susceptibility of mice to the effects of certain drugs was significantly
altered by exposure to pulse modulated 3 GHz microwaves at an average
intensity of 5 mW/cm2.
Altered drug tolerance in humans occupationally exposed to microwaves
has also been reported by Edelwejn and Baranski~ 5 • Intravenous injection of
cardiazole (Metrazol), a drug which acts on the human CNS, did not induce any
effects in normal adult males at a dosage level of 7 mg/kg body weight. However,
the drug produced alterations in EEG patterns (theta waves, theta discharges
and spike discharges) in microwave workers with over three years of
exposure; convulsions occurred forcing the abandonment of the study as being
hazardous to the patients. A marked decrease incardiazole tolerance has also
been demonstrated in microwave exposed rabbits.
Evidence has also been reported that occupational microwave exposure
leads to alterations in human CNS function. The effects, many of which are of
a subjective nature such as headaches and fatigue, reportedly occur during
the first three months of employment and then recur after another three to
four months. After one year of occupational exposure, the symptoms appear to
remit for various lengths of time, suggested as being due to an adaptive .
response, but objective symptoms of various neurovegetative disturbances recur
after approKimately five years of exposure 50
• The chronic overexposure syndrome
in humans is characterized by the occurrence of headaches, irritability,
sleep disturbances, weakness, decrease in libido, chest pains, and generalized
psycho-physiological depression.
The adaptive response of rats to microwave exposure following irradiation
with 2.86 - 2.88 GHz CW microwaves at intensities between 10 to 120
mW/cm2 was illustrated by the experiments of Mikolajczyk.
The survival time of hypophysectomized rats exposed to 120 mW/cm2 fields was increased
relative to unoperated animals.
Alterations in carbohydrate metabolism, a possible indirect neuroendocrine effect of microwave exposure, were induced by exposure of rabbits to 3 GHz CW fields at 5 mW/cm2.
Low intensity microwave exposures,
which would involve low level gradual heating, would be expected to
result in a generalized suppression of neuroendocrine response as reflected
in metabolic suppression.
Although data on the human response to low-level
microwave exposure is very limited, the reported decrease in thyroid function
in chronically exposed workers is consistent with this hypothesis.
A rather consistent finding has been that
microwave exposure causes transient lymphocytosis and leukocytosis. Erythrocytes
appear to exhibit a somewhat lower sensitivity to exposure effects,
with some suggestion of an exposure dependent decrease in erythrocyte
production.
Alterations in nuclear structure and in the mitotic activity
of erythroblasts, bone marrow cells, and lymphatic cells in both lymph nodes
and spleen in guinea pigs and rats following prolonged exposures to 3 GHz microwaves at an intensity of 3.5 mW/cm have been reported by Baranski.
Low intensity (3 mW/cm2) exposures at 2.95 GHz for periods of one to three
months altered the circadian rhythms in hematopoietic cell mitosis in rabbits.
Repeated daily exposures of mice and rabbits to 0.5 and 5 mW/cm
intensities respectively, to 2.95 GHz microwaves for periods of up to six
months, resulted in an increase in lymphoblasts in lymph nodes and in lymphoblastoid
transformations during the first two months of exposure and for one
month post-exposure, an effect which Czerski68 indicates to be uniquely related
to microwave exposure.
The mobilization of bone marrow granulocyte reserve pools
in response to six weeks of exposure at 3 mW/cm2 , as well as a reduction in
lysozyme activity in the blood, has been reported by Szmigielski69 • Microwave
exposure has also been reported to increase the number of colony forming units
(CFU) in the bone marrow and spleen of mice following a five minute exposure
to 2.45 GHz CW microwaves at an intensity of 100 mW/cm2, evidence of an induced
transition of hematopoietic stem cells from a resting to an active state.
The obvious problem encountered in the establishment of causal relationships
for low intensity microwave-induced alterations in biological systems is
the lack of physical interaction mechanisms to explain such phenomena. In
spite of the fact that a number of hypothetical mechanisms, such as fieldinduced
alterations in macromolecular hydrogen bonding, proton tunneling and
disruption of bound water, have been suggested, no theoretical or experimental
basis has been established for the occurrence of such mechanisms in either in
vivo or in vitro biological systems. It may be concluded that since the energy
of a microwave photon is far smaller than the activation energies estimated
for such effects, quantized frequency-specific microwave absorption would not
appear to be a feasible mode of interaction.
This conclusion, however, must be qualified due to the fact that the activation energies are by and large based on estimates derived from in vitro molecular studies rather than in
vivo systems in which high potential energy states are known to exist that
could markedly alter activation energies for the above mentioned phenomena.
Relatively weak interactions or secondary forces are responsible for
the molecular conformation as well as for the stabilization of macromolecular
complexes such as biomembranes. In view of the fact that many biomolecular
functions are dependent upon precise steric interactions, the integrity of
the noncovalent secondary bonding is essential for biological function. Such
secondary forces, including hydrophobic interactions, hydrogen bonds, and
London-van der Waals interactions, correspond generally to significantly
lower energies than covalent bonding. Although the actual energies associated
with secondary bonding in in vivo systems are not known, the potentially
low magnitude of such forces in cooperatively bonded in vivo macromolecular
assemblages suggest that such systems could be subject to perturbation by low
intensity microwave or radiofrequency fields.
The most well-characterized interaction mechanism of alternating electromagnetic
fields with biomolecules is field-induced rotation, Polar molecules,
such as proteins or water, exhibit dielectric relaxation w~th frequency dispersions
that are dependent upon molecular parameters such as size, shape,
molecular weight, and dipole moment. Characteristic frequencies, the moleculespecific
frequencies of maximum energy exchange with impressed fields, are in
the region of 1 to 10 MHz for proteins in aqueous solution, and in the 20 GHz
region for free water. A particular dispersion, referred to as the 8 dispersion,
which has been detected in the 0.1 to 1 GHz regions, is attributed to
either field-induced rotation of protein polar sidechains or the rotation of
bound water. Although dielectric relaxation of proteins and other biomolecules
has been extensively investigated, whether or not this type of interaction results
in functional alterations in living systems has not been determined. On
the basis of data derived from in vitro dielectric dispersion studies of proteins,
it appears that microwave or radiofrequency intensities of 10 mW/cm 2
or greater would be required for dielectric relaxation in living systems, although
there is much uncertainty in this estimate 72 • It is thus difficult, in
view of the pa~city of the available information, to determine the role of
dielectric relaxation in low intensity microwave or radiofrequency effects on
biological systems.
Rotation of water is characteristic of dielectric dispersion of small
polar molecules and even the polar side chain of larger molecules such as proteins.
Thus, Pennock and Schwan 3 determined that dispersion in hemoglobin
solution between 50 and 100 MHz was due to rotation of polar side chains. The
dispersion in the regions between 1 MHz and 100 MHz is further complicated by
the finding5 that there exists an additional dispersion due to the induced dipole
moment of the protein backbone which has a dielectric constant greater
than 20. Other large macromolecular constituents of the cell may also be expected
to have such induced dipole moments. Finally, the helix-coil transition
in proteins also has a relaxation in this region sometimes called the
"chemical" relation6 • Such dispersions due to conformational transitions may
contribute little to the overall dielectric constant even at their maximum
dispersion; however, conformational transition induced by electromagnetic radiation
may have functional effects, although the displacement from the already
rapid transitions due to thermal agitation is quite small.
At lower frequencies, larger cellular structures become responsible for
dispersion. In the 8 region, Schwan7 has attributed the dispersion to the
change in polarization which occurs at the boundary of phases of different dielectric
constant, the so-called Maxwell-Wagner effect. In the cell, the most
likely boundary is the cell membrane where a lipid-water transition occurs.
Such an interpretation was further verified using the dielectric dispersion of
yeast at 100 KHz. Upon treatment with cetyl trimethyl ammonium bromide, a
detergent which disrupts the cell membrane, the 100 KHz dispersion disappeared.
Most tissues and cells exhibit a strong dispersion in the a region at
100 Hz and below, with the apparent dielectric constants increasing to very
large values, with even DNA having a dielectric constant of 240-340 at 100 Hz 9 •
At least part of the dispersion at these low frequencies can be ascribed to
movement of ionic countercharges in the fixed charge latticework of large
assemblies of macromolecules such as bacterial cell walls; the same dispersion
can be seen in ionic exchange resins.
While the bulk dielectric constants of living matter can be fairly well
understood by the foregoing discussion, other more highly specific absorptions
of electromagnetic radiation may be important but not be observable against
the background of strong absorption by the more non-specific mechanisms.
It was pointed out by Lowdin several years ago that the DNA double
helix formed by complementary hydrogen-bonded base pairs could have the position
of the proton shifted from one nucleic acid to its complementary neighbor
by a tunneling process. Subsequent replication of the sequence would then
yield a complete alteration of the base pair from A-T to G-C or vice versa.
In the same paper11 , the author suggested that such a tunneling process might
be induced by electromagnetic radiation of the proper frequency. No experimental
evidence of such assisted tunneling in DNA has yet been reported. However,
it is interesting to note that recent experiments indicate that at
low temperatures (< 25°K) carbon monoxide can tunnel to form a ligand in heme
proteins. The possibility of tunneling of large atomic masses such as CO and
assistance by electromagnetic radiation at lower frequencies bears further
investigation.
The highly oriented assemblies of macromolecules which make up functioning
units of the cell such as ribosomes or mitochondria depend on correct
positioning of neighboring molecules for sequential reactions. The induction
of rotation of one or more molecules within such an assembly might disrupt
function transiently or permanently if the rotation is to a new stable
position. No experimental evidence is available concerning such rotation,
but measurements of the functioning of the assembly would probably be much
more sensitive to such an effect than measurements of dielectric constant.
Another possible interaction of electromagnetic fields in living cells
might occur by bringing into forced oscillations "gating" molecules which
are postulated to control ionic conductivity in excitable membranes.
Armstrong and Bezanilla 1 ~ have attributed the frequency dependent capacitance
of squid axons to these gating molecules 15 • Polarization currents
dependent on membrane potential in muscle membrane are also thought to be
due to gating molecules with large permanent dipole moments 16
While single peak-to-peak amplitude of external a-c fields would need to be large to
cause the gate molecules to undergo a transition, much smaller amplitudes
could cause transitions over a period of time if the gating molecules were
brought into gradually increasing forced oscillations.
Whatever the mechanisms of absorption of electromagnetic radiation,
the energy absorbed must be dissipated and thermalized. The rate and pathways
of thermalization of absorbed energy in the living system have been
little explored. Recent studies 17using laser enhanced chemical reactions
in the gas phase, have indicated that equipartition of electronic energy
into vibrational modes is not immediate but may take as long as 10-5sec.
Such slow distribution of energy allows time for enhanced reactivity of one
particular vibrational mode 18• If such unexpected delays in reaching thermal
equilibrium occur in rotational and translational excitations, enhanced
reaction rates might be observed in living systems excited by electromagnetic
radiation of comparatively low frequencies.
Barriers to thermalization might well be cellular structures with low
thermal conductivity such as the cell membrane. As a consequence of electromagnetic
radiation absorption within a relatively thermally isolated volume,
energy dissipation would be slowed enough so that temperature rise would
occur in that volume. At least in one model membrane system, detergent
vesicles, temperature rise was seen to occur and persist after visible light excitation of a dye within the vesicles.Highly localized temperature
elevations due to thermal barriers might then cause susceptible proteins to denature even if the temperature elevation is not more than 8-10 C. Some
denaturations of proteins can occur over very narrow temperature ranges.
In addition, temperature dependent phase transitions in lipid membranes
can be induced over the entire physiological range. In several membranebound
enzyme systems the transition of the lipid phase is accompanied by
functional changes in certain membrane-bound enzyme activity.
Because of the thickness of the cell membranes (-100 A), even a small
absolute difference in temperature between two sides of the membrane results
in a large thermal gr~dient across the cell membrane. Such thermal gradients
can be coupled to flow of solutes and solvents known as the LudwigSoret
effect. The phenomenological equation can be written in terms of nonequilibrium
thermodynamics 23 • In cell membranes, the thermal diffusion coefficient
is undetermined but with large thermal gradients both ionic and
osmotic gradients could be established.
These and related studies further imply that:
(a) the refinement of theoretical biophysical models beyond the heuristic
state now extant, and (b) their test and refinement via millimeter wave and
far infrared spectroscopy of biological materials may serve to elucidate
fundamental aspects of biophysics, including enzyme selectivity and catalysis,
coding of cellular differentiation, and aspects of neurological
function.
As Frohlich24 suggests, the raison d'etre, in an evolutionary sense,
for the extraordinarily high intrinsic electric fields in biological membranes
(- 10 5 V/cm) may be the establishment of the resultant unusual dielectric
properties of in situ biomolecular systems which, via collective behavior,
permit biological systems to perform tasks (such as information acquisition
and discrimination in the ubiquitous presence of inherent thermal noise) and
whose sophistication may border on the limits set by quantum-mechanical principles.
In this connection, the extent to which quasi-resonant interactions
between coherent electromagnetic fields and biological systems may be operative
in the millimeter wave and far infrared region is of crucial importance
to the question of the existence of bioeffects.
Qualitatively, while theprediction, on the basis of the Debye model, of a constant (asymptotic)
attenuation distance for H20 in the millimeter wave region and the low frequency
portion of the far infrared region is correct, it is expected that
there may exist a window (relatively large attenuation distance) between the
broad rotational relaxation spectrum in the millimeter wave and far infrared
region and the discrete, resonant (intramolecular-vibrational) transitions in
the infrared region. An additional complication arises from the existence of
translational and librational quasi-lattice modes in liquid H20 in the far
infrared region 10 • Inasmuch as the relaxation absorption spectrum of free
H20 serves as an efficient attenuator of millimeter wave and low-frequency
far infrared radiation (the attenuation distance, [a(w)]- 1 ),is of the order
of 10-2 em in the 10- 1 em wavelength range, and since, in contrast, this constant
attenuation due to the relaxation regime cannot persist in the highfrequency
limit, an accurate characterization of this contribution to a(w)
over the entire range in which (quasi-resonant) coherent oscillations may be
expected to occur, is of considerable interest. Fig. (3) represents a(w)
for the relaxation spectrum of free H20 as computed from the collisionalperturbation model.
Structural H20 is a salient entity in the stabilization of conformational
structures of biopolymers in general , and appears to be a crucialcomponent in the formation of metastable states associated, in particular,
with coherent electromagnetic oscillations. As a component in the free-H20-structural-
H20 mixture, the latter contributes a broad relaxation regime
absorption, with a maximum value for x"(w) at a frequency inte~diate to the
corresponding critical frequencies for free liquid H20 and ice.
In the extreme of highly efficient interconversion,
the strongly selective intervention of external electromagnetic
radiation, in the region below 10 GHz, into the properties of structural
H20, would be unlikely. However, the actual efficiency of this intercoversion,
as a function of T, and, in particular, any resulting biologically important
selectivity, is as yet an unresolved question.
A number of studies indicate that cannabinoids
impair memory acquisition in mammals by suppressing
excitation in the hippocampus, one consequence
of which is that hippocampal neural activity remains
below levels required for long-term potentiation
(LTP; Terranova et al., 1995; Stella et al., 1997;
Hampson and Deadwyler, 2000; Sullivan; 2000). That
is, activation of CB1 receptors impairs LTP via inhibition
of presynaptic calcium channels, thereby reducing
excitatory neurotransmitter release below levels
that are required to remove the magnesium block from
postsynaptic NMDA receptors (Misner and Sullivan,
1999; Sullivan, 1999; Hoffman and Lupica, 2000).
Prior work has shown that cannabinoid exposure of zebra finches during sensorimotor stages of vocal development alters song patterns produced in adulthood. We are currently working to identify physiological substrates for this altered song learning. FoxP2 is a transcription factor associated with altered vocal development in both zebra finches and humans. This protein shows a distinct pattern of expression within Area X of striatum that coincides with peak expression of CB1 cannabinoid receptors during sensorimotor learning. Coincident expression in a brain region essential for song learning led us to test for a potential signaling interaction. We have found that cannabinoid agonists acutely increase expression of FoxP2 throughout striatum. When administered during sensorimotor song learning, cannabinoids increase basal levels of striatal FoxP2 expression in adulthood. Thus, song-altering cannabinoid treatments are associated with persistent increases in basal expression of FoxP2 in zebra finch striatum.
Vocal learning and production
in zebra finches is associated with marked physiological
changes within distinct regions of telencephalon (e.g.
lMAN, Area X, auditory Field L2, RA) and thalamus
(DLM, ovoidalis) known to be critical for song perception,
production and learning. Each of these regions distinctly
and densely expresses CB1 receptors [3]. Normal
development in song regions is associated with gross
anatomical changes in region volume, neuron number
and density, both increases and decreases in axonal
interconnections between song regions, and changes in
synaptic densities. Cannabinoid-altered vocal development
implies that exogenous agonist exposure must
somehow alter some or all of these processes responsible
for critical periods of song learning. We are currently
working to identify which processes are modified by
developmental cannabinoid exposure and the mechanism
(s) responsible.
Indeed, Grant~ 9 hasdirected attention to the potential role of structural H20 as principal absorber
(via its relaxation spectrum) in the low-frequency portion of the relaxation
spectrum of free H20 below - 10 GHz. In addition, in view of the
role that structural H20 plays in the establishment and stabilization of the
metastable state, selective interactions involving structural H20 in the millimeter
wave and far infrared region would appear to be possible.
Considerable controversy exists as yet concerning the equilibria and
dynamics of H20 molecules in general cellular systems, particularly, with respect
to the proportion of free H2 and structural H20. In part, this arises
from the ambiguities inherent in the analyses of relaxation dynamics of
rotationally- and irrotationally-bound and (rotationally and translationally)
free H20 from proton magnetic resonance spectroscopy • e attenuation
function for structural H20 has been characterized~'~ 2 to --30 GHz,
but no experimental data is available in the millimeter wave and far infrared
region. As indicated, structural H20 is considered to be an indispensible
building block in the system associated with the long-range collective interactions
and the coherent electromagnetic oscillations.
Conversely, if such controlled (beneficial) biomedical applications
were possible, it is requisite to inquire into possible bioeffects and health
implications of technological electromagnetic radiation in the coherent regime
frequency region. In point of fact, several technological applications must
be considered in this context: (a) multichannel millimeter wave communications
systems [waveguide and open transmission systems], (b) millimeter wave
high-resolution radar, and (c) microwave power transmission, along terrestrial
and proposed satellite-based transmission routes throuph the earth's
I
atmosphere 58 ' 59 • In connection with these existing and proposed man-made
electromagnetic radiation fields, one may ask the general question concerning
the coupling of electromagnetic radiation with the entire biophysical
model envisaged by Frohlich24 ' 29 , encompassing the coherent regime oscillations
associated with the metastable state and the coupled ELF oscillations
arising from the oscillatory biochemical reactions. Fig. (5) provides a
schematic view of components of these systems. On the basis of current evidence,
and the (as yet incomplete) understanding of underlying biophysical
mechanisms, one may speculate along the following lines concerning external
electromagnetic field interactions with this system. Three possible leverage
points for perturbation may be identified: (a) the membrane intrinsic electric
field, which is responsible for setting up the metastable state(s), (b)
the coherent regime frequency branch in the - 10-10+3 GHz region, which is
associated with the long range molecular interactions, leading to the coupled
biochemical reactions, and (c) the resulting ELF frequency branch in the
-10-100 Hz region.Thermal fluctuations cause time-variant electromagnetic
fields, with a vanishing time average, and may superimpose on the average
membrane intrinsic field, and cause occasional sporadic nerve impulses~ 9 • In
a system close to the triggering threshold, at a given instant in time, an
external field may, in principle, add to the fields caused by thermal fluctuations
and lead to sporadic impulses 60 • However, strong intervention in the
average membrane field, and hence significant perturbations of its time average
properties by external fields is not predicted except for very high (static)
field strengths, close to the dielectric breakdown of bulk tissue. Intervention
in the ELF branch <~ 10-100 Hz) of the cooperative system by external
fields is probably weak, while coupling with coherent electromagnetic radiation
in the coherent regime branch <~ 10-1000 GHz) may occur. This (highly
tentative) analysis appears to be in harmony with the type of (unperturbed)
environmental terrestrial electromagnetic fields 5 that exist, if one may
assume that evolutionary development would emerge in a fashion (for a pervasive
biophysical system) so as to minimize coupling with the unperturbed environment
and optimize the operation of the biophysical system and associated
biological function. While atmospheric electric fields in the ELF region are
prevalent and may reach substantial field strengths, the millimeter wave
region of the terrestrial background electromagnetic field is a quiet zone,
contributed to only by the (extremely low field strength) low frequency tail
of terrestrial blackbody radiation, which peaks in the infrared region, and
the (extremely low field strength) contribution of the cosmic millimeter wave
blackbody radiation arising from the (drastically red-shifted) electromagnetic
pulse attendant to the initial phase of the formation of the universe.
in DNA there are some water molecules
which are hydrogen-bonded to the biopolymer itself; the water molecule is in
the framework of the biopolymer. That is certainly different from water
molecules which are hydrogen-bonded to the cells. The efficiency of the interconversion
of rotational relaxation of the two types of water must be far
different.
We are referring to an assembly of biopolymers within the membrane
intrinsic field which produce oscillations which are coherent electromagnetically.
The sources of the intrinsic oscillations are vibrations. The whole
theory has a formal resemblance to the Einstein condensation of a gas.
Frohlich suggests that the frequencies may be as low as 10 gigahertz and may
go up into the hundred or thousand gigahertz region.
Since 1975, evidence has accumulated that the binding and release of
calcium in brain tissue involves processes radically different from simple
equilibrium states 1
• Calcium ions are essential in the release of transmitter
substances from presynaptic nerve terminals and in transductive
coupling of immunological, endocrine and neurophysiogical stimuli at the
membrane surface, as well as in ensuing transmembrane coupling. This new
evidence suggests that for cerebral tissue, at least, it may be necessary
to substantially revise generally accepted views on the structural and functional
basis of excitation, particularly in regard to the role of weak electrical
gradients in the immediate environment of brain cells.
The response of cerebral tissue to certain low level oscillating environmental
electromagnetic fields is anomalous in that a sharply modified
release of calcium has been found only in a narrow range of frequencies
between 6 and 20 Hz. Responses also occur with radiofrequency fields amplitude
modulated in the same narrow range of low frequencies, but are qualitatively
different. The second quite unexpected finding is that these sensitivities
occur only within a range of field strength or a power window. The
appearance of sharp upper and lower field intensity thresholds distinguishes
these findings from what is expected of chemical reactions in simple equilibrium
systems.
The data strongly suggest that the binding and release of calcium occurs
cooperatively as the result of long-range interactions between anionic charge
sites on the binding substrate. A striking aspect of these studies has been
the consistent occurrence of major shifts in calcium efflux (in excess of 10
per cent) with fields that produce very small gradients in the extracellular
space surrounding cell membranes. The extracellular fields are about 10- 7V /cm
and 10- 3V /cm for the low frequency and modulated radio frequency experiments
respectively. They are thus far below transmembrane gradients of 10 3V /cm
associated with a typical synaptic depolarization. Moreover, the gradients
induced in the extracellular space along the membrane surface would be about
76
10 3 times larger than any transmembrane components of the same field, because
-1
the low specific resistance of extracellular fluid, about 4 ohm-em , shunts
a current around the membrane, which has a transmembrane resistance of about
2 5,000 ohms/em •
Thus, on biophysical grounds alone, large shifts in calcium efflux induced
by the imposed fields would be expected to occur primarily in the extracellular
fluid and at cell membrane surfaces. This a priori consideration
fits well with the huge differences in calcium concentration between the
extracellular space and the general cytoplasm of the cell. Typically, calcium
concentration in cerebral extracellular space is 2.4 mM; within the
cytoplasm, it is around 10- 8M . Organelles, such as mitochondria, which have
much higher concentrations 2 , may not participate in the dynamic exchanges
associated with membrane excitation.
FREQUENCY AND POWER WINDOWS IN SENSITIVITY
OF CEREBRAL TISSUE TO OSCILLATING EM FIELDS
Extremely low frequency (ELF) sinusoidal fields from 1 to 75 Hz have
been tested for effects on calcium efflux from freshly isolated neonate chick
cerebral hemisphere and cat cerebral cortex~. Electric gradients in air
ranged from 0. 05 to 1. 0 V/ em. Tissue gradients could not be measured, but were
estimated to be of the order of 0.1 ~V/cm. Both frequency and amplitude
sensitivities were observed. A decrease in calcium efflux of 12 to 15 per cent
occurred at frequencies of 6 and 16 Hz. For chick tissue, the field threshold
in air for this response was around 0.1 V/cm, and for cat cortex around
0.6 V/cm. At intensities above or below these levels, trends towards a decreased
efflux that were not statistically significant were observed (Fig. 1).
Studies with very high frequency (VHF), 147 MHz, radio fields, amplitude
modulated in the same low frequency band (0.5 to 35Hz), also produced shifts
in calcium efflux from chick cerebral tissue 5
• At modulation frequencies from
6 to 20 Hz, there was a significant increase in calcium efflux, reaching a
maximum of more than 15 per cent, and following a smooth "tuning curve" over
this range.
It should be noted
that the increased efflux observed with these 147 MHz fields mirrors the curve
for decreased efflux produced by ELF fields. This point is discussed below,
although no adequate biophysical or structural model can be offered at this time. Fields were maintained constant at 0.8 mW/cm in these studies. Tissue
gradients were not measured, but would be expected to be about 10 mV/cm, a
value consistent with actual measurements at 450 MHz described below.
The cell membrane has long been described in terms of a lipid bilayer.
With the progression from light to electron microscopy, the membrane became
identified with the plasma membrane in standard electron micrographs. It is
now known8 that the plasma membrane is part of a much broader bounding
structure, the "greater membrane". In this model, there are macromolecular
lamellae on .the outer and inner surfaces of the bilayer. In part, at least,
these covering macromolecules are protrusions from intramembranous particles
(IMP) that lie within the bilayer. This complex organization and its possible
significance in transductive coupling of weak electrical and chemical
stimuli has been reviewed elsewhere.
COHERENT STATES OF FIXED CHARGES ALONG THE MEMBRANE SURFACE
Singer and Nicolson9 have extended the greater membrane concept in the
"fluid mosaic" model, emphasizing the lateral mobility of certain IMPs within
the lipid layers. These protruding terminals of protein molecules are composed
of sugar molecules. They are acidic and have numerous negative charge
sites. The surface formed by these negatively charged binding sites constitute
a polyanionic sheet. From studies of biopolymer sheets, including
poly-1-glutamic acid, Schwarz 10 ' 11 concluded that these charges may behave
coherently, with adjacent elements having the same energy levels. A coherent
domain of anionic fixed surface charge sites at the membrane surface requires
energy to raise them above the ground state. In some membrane surface phenomena,
energy is supplied by metabolic processes, as in the immunological
"patching" reaction at the surface of lymphocytes. There is as yet no precise
knowledge about sources of energy that might contribute to coherent
surface charge states.
Implicit in the concept of a coherent domain is the expectation that for
the duration of the coherent state, this patch of membrane surface would be
thermally "quiet", that is, isolated from the randomization of energy levels
which is characteristic of the non-coherent condition. Such a system could
be restored to the ground state by an extremely weak trigger, at the level of
thermal noise or below. The action of a weak trigger at one point in a coherent
system resulting in an effect at a remote site elicits a far greater
release of energy than the trigger itself.There is an avalanche or domino
effect, best expressed in the term "quantum amplification".
COOPERATIVE PROCESSES BETWEEN SURFACE FIXED CHARGES
Cooperative processes occur, or are suspected to occur, in a variety of
biological systems. For example, a single photon of visible light with an
energy of 0.6 eV produces a perception of light. Its absorption by the disc
within a retinal receptor causes the release of about 1400 calcium ions,
which migrate outward to the receptor membrane.The triggering energy of
the single quantum is several orders of magnitude less than that required to
release these calcium ions from their binding sites.
For brain tissue, evidence from our electrom·agnetic field experiments
suggests that binding of calcium ions is highly sensitive to weak imposed
fields. A fully successful model must specify the location of the calcium
ions susceptible to the electromagnetic fields, and the competitive binding
of other cations (including the monovalent ions sodium, potassium and hydrogen)
to the same or adjacent sites on a suitable macromolecular, polyanionic substrate.
This first step is impeded because the binding energies for these
ions remain conjectural. For calcium, at least, binding may occur directly
to carboxyl groups, whereas a-sulfate groups offer a looser electrostatic
attraction through an atmosphere of water molecules.
In dielectric measurements over the spectrum from 10 MHz to 50 GHz water molecules close to macromolecular surfaces exhibit dielectric properties different from those of
molecules remotely located. These measurements show that it is no longer
possible to regard water as behaving uniformly throughout the extracellular
and intracellular compartments.
Electrophysiological evidence also supports the concept of interaction
between calcium and cell surface macromolecules in functional modulation of
the intercellular space. An apparent reduction in extracellular calcium concentration
by as much as 20 per cent occurs during cerebellar stimulation,
but measuring techniques would not distinguish between actual removal of
calcium from the extracellular space and an alteration in its state of binding
to surface polyanions. The latter explanation would appear more probable
with respect to the major part of the extracellular calcium. There is a
sharp increase in the electrical impedance of cerebral tissue associated with
increased extracellular calcium levels 15 • In these measurments, the major
part of the impedance measuring current travels in extracellular channels
The reciprocal contours of calcium efflux curves from cerebral tissue in
ELF and VHF/UHF field exposures suggest that calcium ions may be bound to
surface macromolecules in at least two different ways. As discussed elsewhere,
a plausible but so far untested explanation may lie in the concept of
strong attachment to single molecular strands and weaker bonding between
strands. These different modes of bonding might be amenable to manipulation
by the widely differing energies in weak ELF and much stronger radiofrequency
field interactions.
DIELECTRIC CHARACTERISTICS OF THE COUNTER ION LAYER
Micron diameter resin particles with porous surfaces have effective dielectric
constants as high as 10 6 at frequencies below 1KHz. The f indings
may be relevant to interactions of the numerous finer branches of cerebral
dendrites with surrounding electric fields. This model considers the case of
porous particles with a uniform volume distribution of fixed charges. The
boundary region is characterized by a very large, radially-directed static
field with a corresponding radial variation in the distribution of mobile
ions. The external electric field causes a polarization of the ionic atmosphere,
an effect that can be expressed by an additional "apparent" dielectric
constant of the particle, exceeding the actual dielectric constant by several
orders of magnitude at low frequencies. The magnitude of the low frequency
dielectric constant is proportional to the size of the particle and the square
root of the fixed charge concentration in the porous material.
As discussed elsewhere, this phenomenon at the surface of small particles
or tubes with diameters in the range of most branches of cerebral neuropil
suggests that ionic movements in oscillating electric fields close to such a
membrane surface may be severely attenuated except at very low frequencies.
This attenuation would apply to the transfer of thermoelectric noise. With
a specific resistance for brain tissue of 300 oh~cm and an effect~e frequency
bandwidth from 0 to 100 Hz, the equivalent noise voltage would be of
the order of 10-8 V/cm. This tentative model is of interest in view of observed
sensitivities of 10-8 V/cm in marine vertebrates and the behavioral
and neurochemical effects seen in birds and man and other primates attributable
to oscillating gradients less than an order of magnitude larger.
Preliminary models of membrane excitation based on quantum mechanical
aspects of cooperative charge interactions have been formulated by
Frohlich18- 20 • Frohlich has modeled long range coherence and energy storage
based on dipole interactions and the recurrence of certain bonds, such as
H-bonds, in macromolecules. High sensitivity of biological systems to weak
electromagnetic fields would then relate to a mechanism through which they
store energy, and in so doing, overcome thermal noise. Extremely high frequency
oscillations at 10 11 to 101 2 Hz would occur simultaneously with limitcycle
behavior at very low frequencies. Grodsky has considered the
membrane lipid bilayer as a sheet of dipoles under an electric strain attributable
both to the dipoles' mutual interactions and to the local electric
field generated by cations in the polyanionic glycoproteins of the outer
membrane. This system will respond to a surrounding electric field with a
change in long range order, a phase transition occurring over a narrow range
of fixed temperatures below a critical point (The Neel temperature). The
system will oscillate with most energy in a narrow low frequency band.
1. The effects of weak electromagnetic fields have been tested on the efflux
of calcium from cerebral tissue of chick and cat. The data strongly suggest
that the binding and release of calcium occurs cooperatively as the
result of long-range interactions between anionic charge sites on the
binding substrate.
2. Extremely low frequency (ELF) fields at frequencies of 6 and 12 Hz and
gradients in air of 0.1 to 0.5 V/cm decreased calcium efflux by 12 to 15
per cent. Higher and lower frequencies were without significant effect.
For chick tissue, the field threshold in air was 0.1 V/cm and for the cat
around o".6 V/m. At intensities above and below these levels, effects became
statistically insignificant.
3. With 147 MHz amplitude modulated fields, calcium afflux from chick cerebral
tissue increased for modulation frequencies from 6 to 20 Hz, with a
maximum of more than 15 per cent. No significant changes occurred at
hi£her or lower modulation frequencies, nor with an unmodulated carrier
wave.
4. With 450 MHz fields amplitude modulated at 16 Hz, increased calcium efflux
from chick cerebral tissue occurred at field intensities between 0.1 and
2 1.0 mW/cm • No increase was noted above or below these levels.
5. This series of amplitude and frequency windows is discussed in relation to
possible modes of cooperative organization of cell membrane surface glycoproteins
in the binding and release of calcium.
You indicate that these extremely long waves were in some way involved
in the transfer of information from the brain. It is well known that the
information that cane tPansmitted on a aarrieP signat is a function of the
fPequenay of the aarPieP signat. A aarPier signat in the t6 HePtz Pange aan
at most tPansmit onty a few bits peP seaond.
We examined cardiovascular responses to intravenous administration of anandamide and the synthetic cannabinoid, (WIN55212-2), in conscious male Wistar rats made acutely hypertensive by infusion of angiotensin II (AII) and arginine vasopressin (AVP). Rats were chronically instrumented for measurement of arterial blood pressure and vascular conductances in the renal, mesenteric and hindquarters beds.
Anandamide dose-dependently decreased the mean arterial blood pressure of rats made hypertensive by AII-AVP infusion, but not normotensive rats. Interestingly, acute hypertension also revealed a hypotensive response to WIN55212-2, which caused hypertension in normotensive animals. The enhanced depressor effects of the cannabinoids in acute hypertension were associated with increased vasodilatation in hindquarters, renal and mesenteric vascular beds. Treatment with URB597, which inhibits anandamide degradation by fatty acid amide hydrolase, potentiated the depressor and mesenteric vasodilator responses to anandamide. Furthermore, haemodynamic responses to WIN55212-2, but not to anandamide, were attenuated by the CB1 receptor antagonist, AM251.
The sphingomyelin cycle plays and important role in the regulation of cell physiology in the central nervous system. Ceramide generated by challenge of astroglial cells to cannabinoids may serve as a second messenger in the control of several functions [e.g., metabolic regulation and induction of apoptosis. The notion that the CB1 cannabinoid receptor, like the 55- kDa TNF receptor, may control the activity of the sphingomyelin cycle and of mitogen-and stress-activated protein kinases points to a general role of cannabinoids as modulators of glial cell fate. By showing that the CB1 cannabinoid receptor may be coupled to FAN independently of Gi/o proteins, this report opens a new conceptual view on the mechanism of cannabinoid action and contributes to the novel idea that G protein-coupled receptors may signal via ceramide as well as by mechanisms alternative to the classical heterotrimeric-G protein paradigm.
The DCLHK sequence is conserved in rat, human, mouse and cat CB1 receptors, located in the C-terminal portion of the CB1 receptor in a domain of the protein potentially involved in the interaction with cytoplasmic effectors.
The cannabinoid 1 (CB1) receptor antagonist SR141716A reversed the actions of URB-597 and OMDM-2, but not PMSF, without affecting reward thresholds by itself.
Results indicate that ACEA (2.5 mg/kg, i.p.) co-administered with PMSF (30 mg/kg, i.p.), significantly enhanced the anticonvulsant activity of phenobarbital.
THC induced a transient breakdown of sphingomyelin in cells expressing dominant-negative factor associated with neutral sphingomyelinase activation.
As expected, THC-induced sphingomyelin breakdown occurred in concert with an elevation of intracellular ceramide levels.
THC increases mRNA encoding the immediate early genes zif-268, c-fos and c-jun and FosB protein in the cingulate area of the rat prefrontal cortex.
THC-induced working memory deficits in a delayed alternation T-maze task are associated with altered dopamine and noradrenaline turnover in the rat prefrontal cortex.
Rats must shift attentional bias (or ‘set’) between different abstract features of stimuli, a process termed ‘attentional set shifting’. In contrast, in order to perform reversal-learning discriminations, rats must update contingencies between stimuli and reward presentation when these are reversed. Acute administration of D9-THC impairs performance on reversal learning stages of the task, whilst attentional set shifting ability is unaffected.
Initial evidence that antagonism of CB1 receptors may improve certain memory processes was obtained using an olfactory recognition task. As mature rodents normally spend more time investigating unfamiliar than familiar conspecific animals, the olfactory recognition task measures social short-term working memory capacity. In this task, administration of SR141716A alone improves olfactory recognition memory in both aged rats and mice. In addition, SR141716A improves working memory performance on the 8-arm radial maze when long delay periods are included. These studies have therefore, suggested that SR141716A may exert nootropic effects when administered alone, and, by extension, indicate that the endocannabinoid system may negatively influence some mnemonic processes.
Analysis of these results has led to the suggestion that the effects of SR141716A administration on working memory are dependent on temporal components of the task, and that CB1 blockade may prolong the duration of memory rather than facilitating learning.
SR141716A did not block URB597-mediated attenuation of KainicAcid-evoked burst firing.
CB1 receptors are involved in the control of excitatory glutamatergic neurotransmission. Blockade
of AEA metabolism could therefore confer inhibitory effects via suppression of excitatory synapses under hyperexcitable conditions.
AEA has been shown to directly modulate a number of seven-transmembrane and nuclear receptors, including GPR55 and peroxisome proliferator-activated receptors (reviewed in Alexander and Kendall, 2007), and voltage and ligand-gated ion channels, in some cases at intracellular sites, including T-type calcium channels, voltage-gated, and background potassium channels, a7-nicotinic acetylcholine receptors, TRPV1 receptors, and 5-HT3 serotonin receptors.
This work found that administration of a CB1 antagonist during febrile seizures blocked seizure induced potentiation of DSI, eliminated seizure-induced CB1 receptor upregulation, and prevented the development of long-term limbic hyperexcitability.
Furthermore, this study indicates that by inhibiting the degradation of AEA and other endocannabinoids, specific FAAH inhibitors such as URB597 may provide us with new hope for effectively lowering IOP by enhancing aqueous humor outflow.
But the FAAH inhibitors also produced large elevations in the levels of two anandamide analogs, palmitoylethanolamide and oleoylethanolamide, whose biological effects are independent of CB1 receptors.
We have developed a new class of agents that prevents anandamide
inactivation by targeting the intracellular enzymatic activity
of FAAH. URB597, the most potent member of this class,
inhibited FAAH activity with an IC50 value of 4 nM in brain membranes
and 0.5 nM in intact neurons, and an ID50 value of 0.15
mg kg–1 after systemic administration in the rat. This compound
had much greater selectivity for FAAH than other cannabinoid related
targets, including cannabinoid receptors (selectivity
index, >25,000) and MGL, an enzyme involved in the deactivation
of the endogenous cannabinoid ester 2-AG (selectivity
index, >7,500). Such target discrimination was matched by a lack
of overt cannabimimetic effects in vivo. Thus, at doses that almost
abolished FAAH activity and substantially raised brain anandamide
levels, URB597 and its analog URB532 did not evoke
catalepsy, reduce body temperature or stimulate feeding, three
key symptoms of cannabinoid intoxication in the rodent11.
Nevertheless, the compounds did elicit marked anxiolytic-like
responses, which paralleled their ability to inactivate FAAH and
were prevented by the CB1 receptor antagonist rimonabant. We
interpret these findings to indicate that URB597 and URB532 may
selectively modulate anxiety-like behaviors by enhancing the
tonic actions of anandamide on a subset of CB1 receptors which
may normally be engaged in controlling emotions. Forebrain sites
that might be implicated in such actions include the basolateral
amygdala, the anterior cingulate cortex and the prefrontal cortex,
all key elements of an “emotion circuit”33 that contains high densities
of CB1 receptors21,22. CB1 receptors in these structures are localized
to the axon terminals of a subpopulation of GABAergic
interneurons, which also express the peptide cholecystokinin23,34
(CCK). The anxiogenic properties of CCK35 and the ability of CB1
agonists to inhibit K+-evoked CCK release from hippocampal
slices36 indicate that interactions between this peptide and anandamide
may participate in the control of anxiety.
In addition to their anxiolytic-like actions, URB597 and
URB532 exerted modest anti-nociception in a model of acute
pain, which also was sensitive to CB1 receptor blockade. These
findings are similar to those reported for Faah–/– mice9 and support
the proposed roles of anandamide in the intrinsic modulation
of pain37. However, as emotional states may strongly
influence pain sensation, it is possible that anxiolysis might
have contributed to the mild anti-nociceptive effects of the
FAAH inhibitors.
URB597 and URB532 increased brain anandamide levels without
modifying those of the second endogenous cannabinoid,
2-AG. It is therefore likely that the pharmacological actions of
these compounds, which are sensitive to the CB1 antagonist rimonabant,
are primarily due to anandamide accumulation. But
the FAAH inhibitors also produced large elevations in the levels
of two anandamide analogs, palmitoylethanolamide and
oleoylethanolamide, whose biological effects are independent of
CB1 receptors7,8. Thus, we cannot exclude the possibility that additional
properties of URB597 and URB532, mediated by these
fatty ethanolamides, remain to be discovered.
We have recently reported
that THC induces sphingomyelin breakdown and intracellular
ceramide accumulation in primary astrocytes in a timeand
dose-dependent manner (Sa´nchez et al., 1998b; Bla´zquez
et al., 1999). Because rat primary astrocytes express the CB1
receptor mRNA (Bouaboula et al., 1995a) and protein
(Sa´nchez et al., 1998a), experiments were conducted to test
whether THC-evoked sphingomyelin breakdown was dependent
on this receptor. As shown in Fig. 1, the effect of 1 mM
THC was prevented by 1 mM SR141716, a selective CB1
receptor antagonist, but not by 1 mM SR144528, a selective
CB2 receptor antagonist. The synthetic cannabinoid agonist
HU-210 at 50 nM also stimulated sphingomyelin hydrolysis.
The endogenous cannabinoid ligand anandamide induced
sphingomyelin breakdown at 50 but not at 25 mM. Because
primary astrocytes have a high capacity to take up and
degrade anandamide (Di Marzo et al., 1994), we tested the
effect of PMSF, an inhibitor of anandamide hydrolysis, on
anandamide action. When coincubated with 100 mM PMSF,
25 mM anandamide was able to evoke maximal sphingomyelin
breakdown (Fig. 1). Likewise, methanandamide, a stable
synthetic analog of anandamide, induced maximal sphingomyelin
hydrolysis at 25 mM, indicating that anandamide
action is blunted by cellular degradation. The synthetic CB2
selective agonist JWH-133 at 50 nM did not significantly
affect sphingomyelin levels (Fig. 1). Interestingly, although it
is well established that cannabinoid receptors are coupled to
Gi/o proteins, 50 ng/ml pertussis toxin was unable to prevent
THC-induced sphingomyelin breakdown (Fig. 1), whereas
under identical experimental conditions, pertussis toxin fully
blocked THC-induced activation of extracellular signal-regulated
kinase (Bouaboula et al., 1995b) and protein kinase B
(Go´mez del Pulgar et al., 2000) (results not shown).
To test whether THC-induced sphingomyelin hydrolysis
also occurs in other cells expressing the CB1 receptor, primary
neurons, U373 MG astrocytoma cells, and CHO cells
transfected with the CB1 receptor cDNA were examined.
However, THC was unable to induce a significant breakdown
of sphingomyelin in these cells (Fig. 2).
The Adaptor Protein FAN Couples the CB1 Cannabinoid
Receptor of Astrocytes to Sphingomyelin Breakdown.
Extensive studies on the 55-kDa TNF receptor have
finely defined the domains of the receptor that allow coupling
to sphingomyelinase activation (Adam-Klages et al., 1998;
Kolesnick and Kro¨nke, 1998). A protein designated as FAN
(for factor associated with neutral sphingomyelinase activation)
has been shown by Kro¨nke and coworkers (Adam-
Klages et al., 1996; Kreder et al., 1999) to couple the NSD (for
neutral sphingomyelinase activation domain) of the 55-kDa
TNF receptor to neutral sphingomyelinase activation. FAN
contains five WD repeats in its C-terminal portion that serve
as functional motifs to facilitate defined protein-protein interactions.
Interestingly, b subunits of heterotrimeric G proteins
are also WD-repeat proteins (Adam-Klages et al., 1998).
We therefore examined whether the CB1 receptor may be
coupled to FAN in primary astrocytes and in U373 MG astrocytoma
cells, the latter selected as an example of cells in
which THC is unable to induce sphingomyelin hydrolysis.
For this purpose, the CB1 receptor was precipitated with a
specific antibody and FAN was subsequently detected by
immunoblotting. As shown in Fig. 3, FAN from primary
astrocytes was precipitated by the anti-CB1 receptor antibody.
Furthermore, pretreatment of astrocytes with THC
favored the binding of FAN to the CB1 receptor, whereas
SR141716 prevented this association. By contrast, the effect
of THC was not evident in U373 MG astrocytoma cells, despite
the presence of FAN in these cells.
To test whether the FAN-CB1 receptor interaction is functionally
relevant, we used ECV304 cells stably transfected
with either full-length FAN or a truncated, dominant-negative
form of FAN that does not bind to the NSD domain of the
55-kDa TNF receptor and therefore blocks TNF-induced
sphingomyelin breakdown and ceramide generation (Adam-
Klages et al., 1996; Se´gui et al., 1999). In line with a previous
report (Liu et al., 2000), ECV304 cells were shown to express
the CB1 receptor (Fig. 4A). As shown in Fig. 4B, THC induced
CB1 Cannabinoid Receptor and Sphingomyelin Hydrolysis 957
a transient breakdown of sphingomyelin in cells expressing
wild-type FAN but not in cells expressing dominant-negative
FAN. The involvement of the CB1 receptor in THC-induced
sphingomyelin hydrolysis was proved by the antagonistic
effect of SR141716. As expected, THC-induced sphingomyelin breakdown occurred in concert with an elevation of intracellular ceramide levels (Fig. 4C). HU-210 also induced
sphingomyelin hydrolysis in ECV304 cells expressing wildtype
FAN, and this effect was prevented by SR141716 (results
not shown).
The notion that FAN is coupled to the CB1 receptor is
strengthened by the homology between the NSD of the 55-
kDa TNF receptor and a region of the CB1 cannabinoid
receptor. NSD is a short, nine-amino-acid motif that includes
the sequence DSAHK (Adam-Klages et al., 1996), and the
CB1 receptor contains the highly homologous sequence
DCLHK at amino acid positions 431 to 435 (Matsuda et al.,
1990). The DCLHK sequence 1) is conserved in rat, human, mouse and cat CB1 receptors is located in the C-terminal portion of the CB1 receptor (i.e., in a domain of the protein potentially involved in the interaction with cytoplasmic effectors); and 3) is not
present in the CB2 receptor, which, in HL-60 cells at least, is
not coupled to sphingomyelin hydrolysis (results not shown).
It is difficult to evaluate whether the aforementioned sequence
homology between the NSD of the 55-kDa TNF receptor
and the NSD-like portion of the CB1 receptor is actually
significant. In this respect, it is worth noting that CD40,
a member of the TNF receptor superfamily, has been recently
shown to evoke sphingomyelin hydrolysis via FAN (Se´gui et
al., 1999). The sequence QETLH in the cytoplasmic domain of
CD40, the one that fits better with a portion of the NSD of the
55-kDa TNF receptor (EDSAH), shares a lower similarity
with the latter than the CB1 receptor. Nevertheless, as we
are aware, because the CB1 receptor is not coupled to FAN in
U373 MG astrocytoma cells, which express FAN, additional,
still-unknown factors may be necessary for the CB1 receptor
to activate sphingomyelin hydrolysis.
The sphingomyelin cycle plays and important role in the
regulation of cell physiology in the central nervous system
(Kolesnick and Kro¨nke, 1998). Ceramide generated by challenge
of astroglial cells to cannabinoids may serve as a second
messenger in the control of several functions [e.g., metabolic
regulation (Sa´nchez et al., 1998b; Bla´zquez et al.,
1999) and induction of apoptosis (Galve-Roperh et al., 2000)].
The notion that the CB1 cannabinoid receptor, like the 55-
kDa TNF receptor, may control the activity of the sphingomyelin
cycle (the present report) and of mitogen- (Bouaboula
et al., 1995b) and stress-activated protein kinases (Rueda et
al., 2000) points to a general role of cannabinoids as modulators
of glial cell fate. By showing that the CB1 cannabinoid
receptor may be coupled to FAN independently of Gi/o proteins,
this report opens a new conceptual view on the mechanism
of cannabinoid action and contributes to the novel idea
that G protein-coupled receptors may signal via ceramide
(Limatola et al., 1999) as well as by mechanisms alternative
to the classical heterotrimeric-G protein paradigm (Hall et
al., 1999).
The physical description of a completely free electron differs substantially
from the description of a bound electron. Because the mass of the electron is so
small, it reflects a behavior that is near the boundary between being described
as a particle and being described as a wave. Traveling far from any nucleus, the
electron behaves more like a particle. When it enters the region of a charged
nucleus that has an unoccupied electron state, the wave nature predominates
in response to the vibrating field of the nucleus, and the electron settles into
a stable standing wave pattern. If the nucleus were not vibrating, there would
be nothing to stop the electron from penetrating as a particle to the center
and interacting directly with the nucleus. Thus, these random vibrations of
the nucleons caused by the neutrinos and their tendency to behave like targets
that are very unfeasible for electrons to impact are what allow for stable atomic
structures.
The above description of the neutrino as the coordinator and sustainer of
atomic behavior encourages an alternate and more fundamental explanation of
black hole behavior. We believe that the absence of a fully developed omnidirectional
neutrino distribution will prevent some stable electron states from
persisting in super dense stars. As soon as the total path-integrated collision
cross section along any essentially radial path in such a star is large enough to
shield the central core from a large fraction of the incident neutrinos (and their
interaction with nucleons as described above), that core coulombically collapses
to higher density [15
Current information indicates that our universe is expanding at the rate of 50−100km/sec/megaparsec
With any external neutrino source, the ultimate fate of our universe has the same expansion or collapse possibilities as are provided in a Newtonian theory. However, a neutrino-induced gravity theory with outside neutrino sources, as contrasted with a Newtonian theory, would favor an expansion because the formation of black holes shields significant quantities of mass from neutrinos and thus prevents that mass, once “hidden”, from having any gravitational influence on other mass bodies.
The estimated average mass density of the universe is 10^-29 grams/cm^3, and this can be alternately
expressed as an average relativistic energy density of 3 x 10^-9 erg/cm^3.
The average neutrino energy density required to account for induced gravity is 10^27 erg/cm^3, and most of this neutrino energy continuously escapes from the universe at speeds in excess of other known material and without interacting with any mass particles.
Approximating the diameter of a nucleon as 10^-13cm, then an atomic nucleus
with atomic weight of 35 has a diameter of 3 x 10^-13cm. Average neutrino
spacing is 1.5 x 10^-11cm and they are moving at speed c and traveling in
random directions. A 2.5MeV neutrino has a mass of 1/400 AMU. Electrons
have a mass of 1/1862 AMU, and the inner electron states have a diameter
of 10^-8cm. This large void between the nucleus and the average radius of the
inner most electron states can be explained by the inability to establish a stable
electron standing wave pattern at distances closer to the nucleus because of the
large (and multipole) electromagnetic fluctuations (some being destabilizing to
a standing wave) in the near field region of a charged nucleus that is subjected
to such small and random displacements hundreds or thousands of times each
second.
The inner-most electron states behave as standing waves (closed complete
surfaces with non-zero thickness) whose fundamental frequency is determined
by the dipolar electromagnetic disturbances produced in the nucleons by the
shorter collision events. These are the most tightly bound electrons and are
more sensitive (than are more removed electrons) to this high frequency electromagnetic
mode. An electron further from that nucleus behaving as a looselycoupled
standing wave (also a closed surface) establishes the pattern whose
fundamental frequency is matched to one of the lower frequency (associated
with longer collision events) dipolar electromagnetic disturbances. This more
remote electron standing wave pattern is more loosely coupled (lower coupling
constant) to the motion of the nucleons, and the lower frequency electromagnetic
disturbances do not exert much of an influence on the above-mentioned
inner electron states. Since the higher frequency disturbances for the outer electron
are too high and too weak to completely upset the fundamental frequency
behavior of that electron, these higher frequencies act mainly to superimpose
higher multipole perturbations on that standing wave. These electromagnetic
disturbances do not of themselves determine the energies of the electron states
that respond to them; they merely are able to transfer energy when necessary to
either establish or maintain those states. The average positions of these standing
waves relative to the nucleus establish their principle quantum numbers, and
the perturbations to the standing waves provide energy differentiation between
the states belonging to the same principle quantum number. Thus, quantization
of the electron states results from the quantizing of the electron standing wave
patterns (by integral wavelength relationships characteristic of standing waves)
caused indirectly by neutrino bombardment, and the nucleon electromagnetic
frequencies sustain certain of those candidate electron standing wave patterns
but do not of themselves impose quantization. Other quantization effects that
are similarly facilitated by neutrino bombardment are discussed [13, 14].
Because the electromagnetic disturbances that drive the electron states are
classical and derive from mechanical motion, the statistical relationships between
expectation values of conjugate variables that appear in the Heisenberg
Uncertainty Principle apply as well to these electron states, and their numerical
products are expected to remain considerably higher than the value of h/(2_)
(resulting normally in lack of spontaneous photon emission). As applied to these
electron states, the uncertainty relationship is used to describe the fundamental
delocalization of the electron mass (and associated charge) that is characteristic
of a standing wave (probability amplitude) description even in the absence of
external stimuli such as interrogating radiation.
The physical description of a completely free electron differs substantially
from the description of a bound electron. Because the mass of the electron is so
small, it reflects a behavior that is near the boundary between being described
as a particle and being described as a wave. Traveling far from any nucleus, the
electron behaves more like a particle. When it enters the region of a charged
nucleus that has an unoccupied electron state, the wave nature predominates
in response to the vibrating field of the nucleus, and the electron settles into
a stable standing wave pattern. If the nucleus were not vibrating, there would
be nothing to stop the electron from penetrating as a particle to the center
and interacting directly with the nucleus. Thus, these random vibrations of
the nucleons caused by the neutrinos and their tendency to behave like targets
that are very unfeasible for electrons to impact are what allow for stable atomic
structures.
The above description of the neutrino as the coordinator and sustainer of
atomic behavior encourages an alternate and more fundamental explanation of
black hole behavior. We believe that the absence of a fully developed omnidirectional
neutrino distribution will prevent some stable electron states from
persisting in super dense stars. As soon as the total path-integrated collision
cross section along any essentially radial path in such a star is large enough to
shield the central core from a large fraction of the incident neutrinos (and their
interaction with nucleons as described above), that core coulombically collapses
to higher density [15]. This onset condition is specified by
Atoms in the outer region similarly coulombically collapse, and the region of
collapse continues to rapidly spread outward, releasing large amounts of energy.
In the black hole extreme, there are no sustainable electron states, and the
only radiation emission that can emanate from such a black hole comes from
impinging external atoms just before they encounter the surface or from purely
nuclear phenomena especially transient ones.
Induced Gravity Model Based on External Impinging Neutrinos : Calculation of G in Terms of Collision Phenomena and Inferences to Inertial Mass and Atomic Quantization
Herein, we present a particle-based mechanism and mathematical formulation of gravity, focusing on the neutrino as the gravity-inducing particle. The mechanism is based on the primacy of momentum conservation and postulates an omni-directional distribution throughout the universe of fast small particles of finite mass that have a low probability of colliding with nucleons. The measured acceleration between two neighboring mass bodies results from an alteration of this distribution caused by nucleons of each body interacting with some of those particles. Based on findings establishing that the neutrino has mass, we evaluate the various neutrinos as external particle candidates. We show that for mass quantities up to several times that of the sun the form of the time rate of momentum transfer to each body is proportional to the product of the two body masses because of the probability nature of any collision process, and inversely proportional to the square of the distance between them because of the mathematical properties of an altered particle flux. A derived expression involving the neutrino momentum flux, the neutrino-nucleon collision cross section, and the nucleon mass replaces the constant G from the classical gravitational model. The neutrino momentum flux that is required to account for gravity is so large as to cause us herein to re-evaluate conventional notions in kinematics and the cause of inertial properties and to examine neutrino-nucleon collisions as a possible source of electromagnetic standing waves essential to establish electron shell states. This reasoning indicates that in a much more massive body that is accreting mass, a coulombic collapse to a black hole will ensue when external neutrinos lose the ability to penetrate in sufficient numbers to the central region.
Our work, and our interpretation of the Shnoll work
[1, 2, 3], and many other works by Shnoll, correlates very
well with the positron annihilation work of Vikin [19] showing
that the production of positronium from Na-22 undergoes
a maximum near the time of the New Moon, and a minimum
near the time of the Full Moon. At the time of the New Moon,
the Earth laboratory (whether measurements are of gravitational
interactions or of radioactive decay phenomena) faces
in the general direction of the line of the Moon and the Sun
for a short period of the day, and then rotates such that the laboratory
faces free and open space and distant stars during the
duration of the day, so that a large complement of neutrinos
falls uninterrupted onto the measuring device; also neutrinos
that are emitted by the Sun may be scattered by the Moon to
aff ect the data. During the Full Moon, however, the Earth laboratory
is always between the Moon and the Sun, and hence
the overall collision physics is considerably different.
Shnoll sums the interpretation of the work that he describes
within [1] by stating “Taken together, all these facts
can mean that we deal with narrowly directed wave fluxes”,
which he refers to as beams that are more narrow than the
aperture of the collimators of the apparatus (0.9 mm). Our
model and theory of gravity [11] is based on a flux of particles,
and the “narrow beam” is interpreted due to very lowangle
elastic scattering of external particles by the nucleons of
the celestial bodies [11, 12], particularly the Moon (near body
in [12]) and Sun (far body), such that some particles never
reach the detecting apparatus such as pendula, gravimeter, or
radioactive source-detector system.
Fundamental to Shnoll’s work is his assertion that these
periodic characteristic histograms relate to a wide variety of
phenomena ranging from bio-chemical phenomena, to the
noise in a gravitational antenna, to alpha decay. This is in
agreement with my own work and that of others, and I have
found that anomalies in gravity, radioactive decay of Po-210
(and Co-60), and changes in plant growth, orientation, and
physiology, as well as embryonic centriole-centriole separation
phenomena, and even DNA and its sheathing H2O, are
affected by the Earth-Moon-Sun relationship [10, 11, 12, 14,
19, 20, 17]. It has been shown by Gershteyn et al. [21] that
the value of G varies at least 0.054% with the orientation of
the torsion pendula masses with the stars, and that G is periodic
over the sidereal year [21]—this periodicity arguing for
a strong link between the Shnoll radioactive decay data and
gravity. Furthermore the Shnoll work [1] cites the possibility
of a space-time anisotropy in a preferential direction, and
refers to the drift of the solar system toward the constellation
Hercules. Our theoretical work in collision-induced gravity
shows that G is a function of collision cross-section of the
neutrino-nucleon interaction [11], and experimental work indicates
that G is a function also of at least temperature, phase,
and shape [22, 10]. Our very recent experimental work determined
that G=6.692_10��11 cubic meters per kg sec2 [15]
which compares very favorably with the slightly earlier work
of Fixler et al [22] using precision a interferrometric method
in conjunction with cold Cs atoms and a known Pb mass,
yielding G=6.693_10��11 cubic meters per kg sec2 — these
values being considerably larger than the normally utilized
value of 6.67_10��11. These data are in accord with an increasing
trend in G that could possibly be related to other
trends such as that cited by Shnoll [1].
Shnoll reports [1] that the subject histograms have a fine
structure that shows what he refers to as “macroscopic fluctuations”.
We have reported gravitational fluctuations [10]
that appear at random, and are associated with time intervals
of _0.13 sec, indicating another correlation between gravity
data and radioactive decay data. The gravitational fluctuations
that we detected were observed in the form of two Newton
cradle pendula dwelling near each other for prolonged periods
of time, but occurring in an unpredictable manner. We
tentatively correlated these events with signals arriving from
supernovae events that had occurred somewhere in the vastness
of the universe. We also had detected on 27 August 2001
a peak in the radioactive decay of our Po-210 source, far in
excess of two-sigma Poisson statistics, and later correlated
with the arrival of radiation from supernovae explosion SN
2001 dz in UGC 47, emitting energy in all neutrinos of the
order of 1046 joules.
All of the above points to the ubiquity of a model of nature
based on elementary impinging momentum-transferring
external particles that can be interrupted by mass particles,
rather than nature being based on the conventional four axiomatic
forces and their respective field theory. Furthermore,
in an external particle based model for gravity, there is no
need to invoke a purely mathematical “fabric” to space-time
curvilinearity according to geodesics or warping, nor is it necessary
to invoke Riemanian space, nor Minkowski space, but
instead space-time is considered to be of a fractal geometry,
and the trajectory of mass particles and photons through space
is curved because of collisions with neutrinos (WIMPS).
Although the collision cross-section of the neutrino with the photon is extremely low, the flux density of the neutrino in our region of the universe is extremely high, and we postulate that
the bending of light is due to that interaction. It seems that astrophysics is now poised to affirm modifications to Einstein’s theory of General Relativity, and this is not unexpected in that many recent findings have indicated that gravity is quantized. Understanding the nature and details of
this quantization is one of the very major challenges and objectives in physics of this new century.
See also [27] for corroboration by private communication
of periodic behavior of radioactive decay data during New
Moon
Oxidation of catechols to semiquinones and quinones is a mechanism of tumor initiation, not only for endogenous estrogens, but also for synthetic estrogens such as DES and BPA, a human carcinogen, because quinone reacts with DNA [15]
Recently, alkylphenols such as p-octylphenol (p-OP), p-nonylphenol (p-NP), and bisphenol A (2,2-bis(4-hydroxyphenyl)propane, BPA) have been recognized as endocrine-disrupting chemicals to interfere with hormones of animals as well as phthalic esters and styrene.
Among alkylphenols, BPA is widely used as a material for polycarbonate resins and epoxy resins,
and it may contaminate food from the inner coating of the can and migrate from polycarbonate tableware and plastic packaging.
Chemical and biological degradation of BPA have been reported by several researchers. Horikoshi et al. reported that nonylphenol polyethoxylate was photodegraded in the presence of titanium dioxide. Nonylphenol ethoxylate was oxidized by ozone and hydrogen peroxide.
On the other hand, there are many reports for biologically degrading and polymerizing alkylphenols such as BPA using microorganisms and enzyme
Ronen and Abeliovich observed microbial degradation of BPA and tribromophenol by sediments. Lobos et al. reported that Gram-negative aerobic bacteria could degrade BPA and its analogues. Furusawa et al. also found microorganisms which could degrade p-NP.
cytochrome c oxidase subunit II, GRB2-associated binding protein 2, adaptor-related protein complex 2, and guanine nucleotide exchange factor p532
A Novel Rodent Model of Autism: Intraventricular Infusions of Propionic Acid Increase Locomotor Activity and Induce Neuroinflammation and Oxidative Stress in Discrete Regions of Adult Rat Brain http://www.autismcanada.org/pdfs/ajbb.pdf
Propionic acid is commonly used as a food preservative in wheat and dairy food products and may be involved in the recent postulated increase in the incidence of autistic spectrum disorder cases in North America (approximately 1 in 166). A number of effects have been shown following PPA treatment, including catecholamine and proenkephalin gene induction[46], cytoskeletal phosphorylation[47], histone modulation[48], second messenger metabolism[49], impaired mitochondrial respiratory transport chain function[50,51], modulation of gap junctions[52] and immune system activation[53]. There is also evidence of variable PPA metabolism in a number of metabolic disorders such as propionic and methylmalonic acidemia[54], disorders of biotin[55] and B12 metabolism[56], as well as following valproate[57] and ethanol exposure[58]. These disorders are associated with developmental delay, seizure and episodes of paroxysmal metabolic dysfunction resulting in increased oxidative stress and are to some degree reminiscent of autism[59,60]. We have hypothesized that PPA may be a candidate environmental factor putatively involved in the diverse behavioral, neuropathological and gastrointestinal aspects of ASD[61]. In our initial studies we found that repeated microinfusions of PPA into the lateral cerebral ventricles of adult rats produced behavioral, electrophysiological and neuropathological effects consistent with ASD[61]. PPA treated animals exhibited increased locomotor activity and repetitive behaviors such as dystonic limb movements, retropulsion and axial hyperextension. Simultaneous electrophysiological examination of cortical, hippocampal and striatal EEG evidenced electrographic changes reminiscent of human complex partial seizure and movement disorder. These effects were apparent within minutes of microinfusion. Furthermore, repeated exposures resulted in increased behavioral and electrographic effects suggestive of a kindling process. Collectively these findings indicated that PPA may have permanent effects on brain function. Neuropathological analysis of hippocampus and external capsule white matter tissue revealed PPA related increases in reactive astrocytes and activated microglia in the absence of gross neuronal loss and apoptotic effects. Additional analyses of whole brain homogenates produced evidence of increased oxidative stress and impaired glutathione metabolism in PPA treated animals. These findings are consistent with the neuropathological changes found in ASD autopsy brains[8] as well as biochemical studies in ASD patients[27,31]. The present study extended the examination of the effects of intraventricular PPA infusion on behavior, neuropathology and oxidative stress levels in the adult rodent ASD model. In particular, we characterized PPA induced increases in locomotor activity with additional automated measures of horizontal and vertical activity. We also examined the neuropathological responses following the twice-daily treatment schedule, utilizing additional indices of microglial activation and neurotoxicity. Lastly, we examined pro- and antioxidative stress pathways in brain regions which have been previously implicated in ASD pathophysiology.
Propionic acid is also used as an inert ingredient in pesticide formulations.
Propionic acid (PPA) is a dietary and gut bacterial short chain fatty acid which can produce brain and behavioral changes reminiscent of ASD following intraventricular infusion in rats. Impairments in GSH and catalase levels may render CNS cells more susceptible to oxidative stress from a variety of toxic insults.
The cannabinoid 1 (CB1) receptor antagonist SR141716A reversed the actions of URB-597 and OMDM-2, but not PMSF, without affecting reward thresholds by itself.
THC increases mRNA encoding the immediate early genes zif-268, c-fos and c-jun and FosB protein in the cingulate area of the rat prefrontal cortex.
THC-induced working memory deficits in a delayed alternation T-maze task are associated with altered dopamine and noradrenaline turnover in the rat prefrontal cortex.
Rats must shift attentional bias (or ‘set’) between different abstract features of stimuli, a process
termed ‘attentional set shifting’. In contrast, in order to perform reversal-learning discriminations, rats must update contingencies between stimuli and reward presentation when these are reversed. Acute administration of D9-THC impairs performance on reversal learning stages of the task, whilst attentional set shifting ability is unaffected.
Initial evidence that antagonism of CB1 receptors may improve certain memory processes was obtained using an olfactory recognition task. As mature rodents normally spend more time investigating unfamiliar than familiar conspecific animals, the olfactory recognition task measures social short-term working memory capacity. In this task, administration of SR141716A alone improves olfactory recognition memory in both aged rats and mice. In addition, SR141716A improves working memory performance on the 8-arm radial maze when long delay periods are included. These studies have therefore, suggested that SR141716A may exert nootropic effects when administered alone, and, by extension, indicate that the endocannabinoid system may negatively influence some mnemonic processes.
Analysis of these results has led to the suggestion that the effects of SR141716A administration on working memory are dependent on temporal components of the task, and that CB1 blockade may prolong the duration of memory rather than facilitating learning.
SR141716A did not block URB597-mediated attenuation of KA-evoked burst firing.
CB1 receptors are involved in the control of excitatory glutamatergic neurotransmission. Blockade
of AEA metabolism could therefore confer inhibitory effects via suppression of excitatory synapses under hyperexcitable conditions.
AEA has been shown to directly modulate a number of seven-transmembrane and nuclear receptors, including GPR55 and peroxisome proliferator-activated receptors (reviewed in Alexander and Kendall, 2007), and voltage and ligand-gated ion channels, in some cases at intracellular sites, including T-type calcium channels, voltage-gated, and background potassium channels, a7-nicotinic acetylcholine receptors, TRPV1 receptors, and 5-HT3 serotonin receptors.
This work found that administration of a CB1 antagonist during febrile seizures blocked seizure induced potentiation of DSI, eliminated seizure-induced CB1 receptor upregulation, and prevented the development of long-term limbic hyperexcitability.
Furthermore, this study indicates that by inhibiting the degradation of AEA and other endocannabinoids, specific FAAH inhibitors such as URB597 may provide us with new hope for effectively lowering IOP by enhancing aqueous humor outflow.
We have developed a new class of agents that prevents anandamide
inactivation by targeting the intracellular enzymatic activity
of FAAH. URB597, the most potent member of this class,
inhibited FAAH activity with an IC50 value of 4 nM in brain membranes
and 0.5 nM in intact neurons, and an ID50 value of 0.15
mg kg–1 after systemic administration in the rat. This compound
had much greater selectivity for FAAH than other cannabinoid related
targets, including cannabinoid receptors (selectivity
index, >25,000) and MGL, an enzyme involved in the deactivation
of the endogenous cannabinoid ester 2-AG (selectivity
index, >7,500). Such target discrimination was matched by a lack
of overt cannabimimetic effects in vivo. Thus, at doses that almost
abolished FAAH activity and substantially raised brain anandamide
levels, URB597 and its analog URB532 did not evoke
catalepsy, reduce body temperature or stimulate feeding, three
key symptoms of cannabinoid intoxication in the rodent11.
Nevertheless, the compounds did elicit marked anxiolytic-like
responses, which paralleled their ability to inactivate FAAH and
were prevented by the CB1 receptor antagonist rimonabant. We
interpret these findings to indicate that URB597 and URB532 may
selectively modulate anxiety-like behaviors by enhancing the
tonic actions of anandamide on a subset of CB1 receptors which
may normally be engaged in controlling emotions. Forebrain sites
that might be implicated in such actions include the basolateral
amygdala, the anterior cingulate cortex and the prefrontal cortex,
all key elements of an “emotion circuit”33 that contains high densities
of CB1 receptors21,22. CB1 receptors in these structures are localized
to the axon terminals of a subpopulation of GABAergic
interneurons, which also express the peptide cholecystokinin23,34
(CCK). The anxiogenic properties of CCK35 and the ability of CB1
agonists to inhibit K+-evoked CCK release from hippocampal
slices36 indicate that interactions between this peptide and anandamide
may participate in the control of anxiety.
In addition to their anxiolytic-like actions, URB597 and
URB532 exerted modest anti-nociception in a model of acute
pain, which also was sensitive to CB1 receptor blockade. These
findings are similar to those reported for Faah–/– mice9 and support
the proposed roles of anandamide in the intrinsic modulation
of pain37. However, as emotional states may strongly
influence pain sensation, it is possible that anxiolysis might
have contributed to the mild anti-nociceptive effects of the
FAAH inhibitors.
URB597 and URB532 increased brain anandamide levels without
modifying those of the second endogenous cannabinoid,
2-AG. It is therefore likely that the pharmacological actions of
these compounds, which are sensitive to the CB1 antagonist rimonabant,
are primarily due to anandamide accumulation. But
the FAAH inhibitors also produced large elevations in the levels
of two anandamide analogs, palmitoylethanolamide and
oleoylethanolamide, whose biological effects are independent of
CB1 receptors7,8. Thus, we cannot exclude the possibility that additional
properties of URB597 and URB532, mediated by these
fatty ethanolamides, remain to be discovered.
Other members of the acylethanolamide lipid family are also produced by neurons and act through
G-protein-coupled receptors: homo-linolenylethanolamide (HEA) and docosatetraenylethanolamide (DEA) act through CB1 receptors
80% of the population have IQs ranging between 80 and 120 with 10% lying below 80, and 10% falling above 120.
Key to understanding this relationship is
the unification between leptonic neutrinos and gluons.
This manifests at lower energy values of particle couplings
and is observed in decay patterns of the highquark
meson complexes, such as the top- and bottomquarks,
but these are resonant energies of the up-quark
and the down-quark.
The proton-neutron interconversion acts to cause a mixing of wave functions and the exchange of a mesonic mediator. This is known as Yukawa coupling, and it is the Yukawa meson that carries the antiquark which couples to an up-quark of the proton. These couplings necessarily relate to
the Heisenberg zero-point energy (ZPE) metric background.
The net result is that the strong gluonic coupling can be
assumed by the weak antineutrino coupling in terms of
a neutral weak-interaction current. The current arises
from the triquark complex of a nucleon, and thus can
re-circulate; therefore the original nucleon (such as the
neutron in the +n inelastic interaction) can be rematerialized.
The associated long decay times are ideal
for heat carry-off. The significant point to this is that the quark-antiquark coupling is transmuted into a temporary diquark selfstate following a simple exchange of the state-antistate couplings of the neutral pions.
It is found that small decreases in gravity that are caused by planet syzygy, full moon presence, and cosmogonic events of deep space can cause a sufficient change in gravitational interactions
to affect the nucleons of a single cell
or a small nanocluster of cells after initial mitosis such that small but significant changes in normal centriole separation can occur. These slight modifications can cause modifications in bond lengths and bond angles of sheathing water molecules, and changes of the helical DNA structure, and lead to a mutation with adverse consequences.
This study was performed to investigate the geotropism and geotaxy of a life science system in order to correlate with predictions of our own external particle model for gravity [8-10].
Calculations indicate that the change in centriole-centriole separation due to the decreased gravity of a full moon interval (during which decreased gravity effects would be maximized) is of the order of tens of angstroms.
This variation in length scale is shown to alter the probability of reaching the activation energy to form a covalent bond in an emerging DNA helix, and create the possibility of a change in bond angle, bond length, and the elements entering the bond.
We have recently found indication of cosmogonic phenomena possibly affecting weak force interactions, and recommend also that experimental work in this sub-area be continued as related to unstable nuclides in organic systems.
Because of the modified chemical potential the effect on phosphorus bridging atoms is significant
The probability then of a miscopy in replication during mitosis will be enhanced by at least 10 percent, and thus the probability of a disadvantageous mutation leading to a birth defect will likewise increase.
The catenoid is of particular interest here since it is the same shape that Wheeler (1962) uses in his theory to describe how wormholes act as transducers for higher dimensional energy to influx into our 4D space/time reality. As similar idea for the influx of energy from higher dimensions has also been
proposed (Shacklett, 1993) based on Twistor theory (Peat, 1988) which is based on a unique topology composed of a catenoid inside the hole of a toroid
We have shown this thermodynamically (and through catalysis) during liquid-liquid short-range-order transitions in many molten elemental states to include sulfur, selenium, tellurium, lead, bismuth, tin, and mercury. We reported in those studies that short range order structural configurations become stable or persistently metastable.
Nanomolecular Gravitational Interactions Causing Increased Probability of Birth Defects in Humans During Period from Conception to Early Fetus Formation
Induced change in centriole separation and consequent probability for disadvantageous mutation
G. C. Vezzoli
Department of Physics and Mathematics
Benjamin Franklin Institute of Technology
41 Berkeley Street, Boston, MA 02116;
Research affiliate, The Farlow Herbaria
Harvard University, Cambridge, MA 02139
ABSTRACT
It is found that small decreases in gravity that
are caused by planet syzygy, full moon presence,
and cosmogonic events of deep space can cause
a sufficient change in gravitational interactions
to affect the nucleons of a single cell or a small
nanocluster of cells after initial mitosis such that
small but significant changes in normal centriole
separation can occur. These slight modifications
can cause modifications in bond lengths and
bond angles of sheathing water molecules, and
changes of the helical DNA structure, and lead
to a mutation with adverse consequences.
Key words: centrioles, nanoclusters, mutation,
sheathing water, gravity, cosmogonic
1 Introduction
1.1 Cellular Systems
Recent work employing sea urgin eggs and
embryos has shown that the centriole-centrosome
complex is altered during cell division when
exposed to microgravity, however, control cells
cultured in a centrifuge at 1g in space, and those
cultured on ground appear normal [1]. It was
reported that the separation of centrioles were
observed most significantly in 4% of the cells
that were exposed to microgravity [1]. Sea
urchins were employed for this study because of
their constituting an animal model system which
is ideal for the study of molecular, cell, and
developmental biology [2,3]. Experiments have
also been recently conducted in microgravity
using ceratopteris [4] and indicated that only as a
single-celled spore did the organism appear to
respond to gravity. It was also shown therein
that gravity detection takes place only during a
very crucial time which allows the effect to
influence subsequent cell polarity, cell
differentiation, direction of nuclear migration,
and direction of rhizoid growth – this time period
being referred to as the “polarity-determination
window”. The response is observed to be that
the nucleus migrates down to the bottom of the
front face of the spore to position itself for an
asymmetric division. The two consequent
daughter cells are not of equal size, and the
larger becomes the prothallus (upward growing
aerial part) whereas the smaller becomes the
rhizoid; the nuclear migration positions the first
cell division such that the rhizoid grows
downward.
The ceratopteris work relates to the microcurrent
studies conducted by Levengood and
Gedye [5,6] on impatiens and soybean plants in
which gravitationally polartropic electrical
effects are measured by a charge density pulse
capacitive technique. Their work also shows
that a gravitropic relationship is affected
by the position of the Sun.
1.2 Botanical systems
The above effects, though of small magnitude,
can have profound consequences, and fall under
what is becoming to be known under the general
category of “subtle energy effects”. We have
measured [7] a very close correlation of
enhanced growth/height of the extremity of a
parabolic cactus shoot corresponding to the time
interval of 99% to 100% visibility of the full
moon of August 3rd-5th, 2001 ( a period of a
slight decrease in gravity), and we have
measured a slight decrease in height during the
new moon (increased gravity) a fortnight
subsequent (heights measured vertically
with respect to a ground plane) – the effects
being of the order of 1-2 %. This study was
performed to investigate the geotropism and
geotaxy of a life science system in order to
correlate with predictions of our own external
particle model for gravity [8-10].
We have corroborated subtle gravitational effects
in our study of the heavy vine, Aristolochia
macrophylla (known as Ducthman’s Pipe
because of the pipe-like structure of its calyx) by
mapping the position of the tip of a cordate leaf
correlated with the position of the Sun passing
directly over the plane of support-fence of the
vine [7,11]. This work showed a small decrease
in the effect of gravity during the tine interval of
10:00 AM to 2:00 PM (EST) in experiments on
cloudless days in August 2001, the effect
observed consistently to be about 2%..
1.3 Macroscopic biological systems
Modern research has shown that Human
chorionic gonadotropin (HCG), secreted by the
human embryo, begins to circulate in maternal
blood very early in pregnancy, and established
[12] that HCG is concentrated and excreted in
maternal urine. The HCG hormone is actually
secreted by the embryonic chorionic villi cells
almost from the moment of conception, and
should be initially susceptible to gravitational
effects. The very correlation of the average
typical menstrual cycle and gestation period in
humans with the lunar month(s), and the
development of the normal monthly corpus
luteum cyst on the ovary (alerted to function by a
signal from the pituitary gland), suggest from a
physics standpoint some complex gravitational
correlation. Additional recent research, aimed at
decreasing the probability of premature births,
has shown that as an expectant mother
approaches delivery, a series of electrical signals
take place in the womb that cause muscle
contraction [13]. This effect and much of the
above phenomena may be a result of subtle
changes in the overall thermodynamic
environment to which sheathing water molecule
clusters are exposed, and a compliant response of
the structure of the water molecule and cluster to
that physical and/or chemical
perturbation/alteration. This viewpoint is borne
out by recent measurements giving the change in
the bond length of the water molecule as a
function of very small changes in temperature
and pressure [14 ], as well as indications of
changes in the resistivity of water depending
upon angle of the charge flow path vertical
gravity vector [7,12,14].
2 Theory an experiment
We have shown [8] that for an external
momenta-transfer theory of gravity, what was
thought in Newtonian theory to be the universal
gravitational constant, G, is actually a function of
gravity-bearing particle flux density, and
postulated that the external particle which is
responsible for gravitational interactions may be
the electron neutrino. We have reported
experimental support for G being a function by
showing that gravitational interactions depend
upon the temperature, shape, phase, and level of
disorder of a mass [9.10]. Much of our work that
relates to life sciences has focused on the
gravitational properties of water [14a]. The
water that exists inside living cell cytoplasm
changes between phases of disordered liquid and
ordered gelatinous solid, the fluctuation between
the two phases determined by the polymerization
of the actin cytoskeleton [16]. Water in a
microtubule environment will respond to subtle
energy gravitational effects[5,6,14]. In that
water is within and around DNA/protein
molecules the gravitational effects then become
very important relative to diffusion and life
processes. In addition to direct hydrogen bonds
between protein and DNA there are water
bridges that exist in virtually all complexes.
Nano-clustering of water molecules can range
from five or so as sheathing molecules to 57
molecules of water bound to a DNA fragment of
19 base pairs length [16]. Subtle changes in
gravity will effectuate slight changes in pressure,
as well as in the overall pressure tensor, and
cause consequent changes in the bond angle of
water from its normally established value of 104-
degrees 27-minutes to an altered value which
will consequently alter hydrogen bonding with
proteins. This alteration will have a polarizationinduced
dipole-dipole effect on the polymeric
structure and electrical properties of the protein
and of nerve cells and synapse responses.
We have reported [9.10] that at length scales of
less than 0.1 mm, gravitational properties do not
behave according to inverse square relationship,
but appear to conform to an inverse fourth
relationship. This has been predicted by string
physicists [17] who had reported that at less than
0.1 mm proximity new and extraordinary
physical behavior may be observed. ( The value
of 0.1 mm corresponds to the length scale that
gives rise to Einstein’s cosmological constant).
Our experiments also indicated that gravity has
a statistical property unlike the expected
behavior of a time-independent field [9,10].
The apparatus which we utilized to measure very
small effects of gravity is described in ref 9. We
tested this apparatus by measuring a decrease in
gravity at the time of the May 2001 Sun-planet
syzygy of the line-up of the Sun, Earth, Jupiter,
and Saturn, predicted at 2203 (UT) on May
18,’01. A cusp-like decrease in gravity was
observed in our study on the morning of May
15th between the hours of 0100 and 0500, and a
square dip in gravity was detected at 2010 UT on
May 18th lasting 35 seconds. A major anomaly
was also detected in a charge-density-pulse array
[15,5,6] in early morning hours of May 15th.
3 Calculations and Simulations
On the above basis, simulations and calculations
indicate that the change in centriole-centriole
separation due to the decreased gravity of a full
moon interval (during which decreased gravity
effects would be maximized) is of the order of
tens of angstroms. This variation in length scale
is shown to alter the probability of reaching the
activation energy to form a covalent bond in an
emerging DNA helix, and create the possibility
of a change in bond angle, bond length, and the
elements entering the bond. Because of the
modified chemical potential the effect on
phosphorus bridging atoms is significant. The
probability then of a miscopy in replication
during mitosis will be enhanced by at least
10 per cent, and thus the probability of a
disadvantageous mutation leading to a birth
defect will likewise increase.
Through the use of the Benard cell, it is reported
[18] that nonequilibrium magnifies the effects of
gravitation. The cell has a very small thickness
of only a few millimeters and is utilized to show
that at non-equilibrium gravitation will modify
diffusion flow. Detailed calculations using the
reaction diffusion equation show that
gravitation-induced changes in diffusion are
clearly shown near the bifurcation point of an
unperturbed system. The authors conclude that
“very small gravitational fields can lead to
pattern selection.” This result [18] is entirely
in accord with the results of our simulations
on cellular nanoclusters.
The above is expected also to be particularly true
during a chemical or a biochemical reaction
during which bonding is breaking down and then
new chemical bonding is being reconstructed.
Under these transient conditions, the species is
most susceptible to external stimuli. We have
shown this thermodynamically (and through
catalysis) during liquid-liquid short-range-order
transitions in many molten elemental states to
include sulfur, selenium, tellurium, lead,
bismuth, tin, and mercury [19]. We reported in
those studies that short range order structural
configurations become stable or persistently
metastable. It is reasonable to expect that during
the reconstruction of the thermodynamically
favored species, such as in polymer formation,
there would exist a preferentially high
vulnerability to gravitation and magnetic
influences during this transient interval.
4 Inferences and Further Work
We have endeavored to sensitize particularly
medical field researchers, consultants, and
practitioners regarding the consideration of
gravitational (and electromagnetic) interactions
during transient periods of molecular bonding
emphasizing very early fetus development (as
well as during the biochemical processes of
healing and joining). Subtle gravitational and
magnetic changes can be caused by lunar,
planetary, and deep space supernovae effecfts.
Data relating birth defects to syzygy effects
have been compiled by Gedye from the work of
Jones over a period of 30 years (2). It is strongly
recommended from the simulations and
calculations described herein that more
experimental research be conducted on early
embryo formation during variation of overall
thermodynamic conditions. We have recently
found indication of cosmogonic phenomena
possibly affecting weak force interactions,
and recommend also that experimental work in
this sub-area be continued [21] as related to
unstable nuclides in organic systems.
Cannabis sativa has been known, used, and misused by mankind for centuries, and yet only over the last two decades has research stemming from the chemical constituents specific to this plant, the cannabinoids, started to provide fundamental insights into animal physiology and pathology, resulting in the development of new therapeutics. The discovery of the endocannabinoid system, and its targeting with two new pharmaceutical preparations now on the market in several countries, represent the most recent example of how studies on medicinal plants and on the mechanism of their
biological effects can reveal, through a chain of breakthroughs, new systems of endogenous signals and physiological phenomena that can become the source of novel strategies for unmet therapeutic challenges.
Introduction
The cannabinoid CB1 receptor antagonist, rimonabant, has been marketed since August 2006 in several EU countries under the commercial name of Acomplia as a pharmacological aid to exercise and calorie-intake reduction for the treatment of obesity and its metabolic complications, such as dyslipidemia and glucose intolerance [1]. About a year earlier, Sativex, a pharmaceutical preparation based on extracts of Cannabis sativa, and containing the psychotropic cannabinoid Δ9-tetrahydrocannabinol (THC) together with the nonpsychotropic cannabidiol (Figure 1) in an 1:1 ratio, was introduced in Canada for the treatment of neuropathic pain associated with multiple sclerosis [2]. These recent milestones of the exploitation of the plant with perhaps the oldest history of medicinal and recreational use by mankind are the result of four decades of intensive research. This process started with the identification and chemical synthesis of THC, the controlled study of its pharmacological properties, and the identification of its receptors in animal organisms and of their endogenous ligands, the endocannabinoids (Figure 1), and it continued with the progressive understanding of the physiological and pathological roles of this newly discovered signaling system and the appreciation of the potential therapeutic use not only of THC and endocannabinoids, but also of other plant cannabinoids. Despite this impressive sequence of achievements, whose aspects have been the subject of recent “historical” reviews [3], [4] and [5], there is much more to the original understanding of the THC mechanism of action than the discovery of the endocannabinoid system. The purpose of this article is not merely to highlight the past contribution of chemical biology to our current understanding of endocannabinoid regulation and function, but, more importantly, to discuss the several other discoveries that stemmed, and are still likely to originate in the future, from the finding of cannabinoid receptors and their endogenous ligands.
Figure 1. Chemical Structures of the Most-Studied Endocannabinoids, of a Cannabinoid Receptor Antagonist, and of Some Plant Cannabinoids
Chemical structures of (1) the two best-studied endocannabinoids, anandamide and 2-arachidonoyl glycerol [7], [8] and [9]; (2) the CB1 receptor antagonist/inverse agonist rimonabant, now on the market under the trade name Acomplia, as a pharmacological aid for the therapy of obesity and the metabolic syndrome [1]; (3) the two major plant cannabinoids, the psychotropic Δ9-THC and the nonpsychoactive cannabidiol, which are also the major constituents, in a 1:1 ratio, of Sativex, currently marketed in Canada against neuropathic pain in patients with multiple sclerosis [2].
From Plant Chemicals to Chemical Signals: Are Endocannabinoids Just Like Endorphins?
Thanks to the chemical synthesis of enantiomerically pure THC analogs (Figure 1), it became clear that the pharmacological actions of this compound were not merely due to its capability to alter membrane permeability in a nonspecific way, but rather to the interaction with specific binding sites. The same analogs, once chemically modified and radiolabelled, served as a tool for the identification of these “cannabinoid” receptors in the rat brain [6]. The finding of the first cannabinoid receptor, the CB1, out of a series of previously cloned orphan G protein-coupled receptors (GPCRs), soon to be followed by the homology cloning of the second type of cannabinoid receptors, the CB2, marked the beginning of the discovery of the endogenous cannabinoid signaling system and prompted the search for endogenous ligands, much in the same way as the identification of receptors for another plant natural product, morphine, had led to the identification of the endorphins two decades earlier. This search, however, might have lasted for several years had it not been guided by a chemical concept, i.e., that, by homology to the highly lipophilic THC, physiological cannabinoid receptor ligands were to be looked for among endogenous lipids rather than peptides like the endorphins. This idea was also supported by the observation that CB1 receptors exhibit relatively high homology with the GPCRs for another family of lipid signals, the lysophosphatidic acids. The identification of the fatty acid ethanolamide anandamide (N-arachidonoylethanolamide) [7] and of its glycerol ester analog, 2-arachidonoyl-glycerol (2-AG) [8] and [9], as endogenous agonists of CB1 and CB2 receptors (Figure 1), and the subsequent finding of their metabolic pathways and enzymes (see [10] for an updated review; see Figure S1 in the Supplemental Data available with this article online), provided the basis for the study of the physiological and pathological role of this endogenous signaling apparatus.
Beyond the similar history of their discoveries, the commonalities between opioid and cannabinoid receptors have been known even before the discovery of the latter, and they include similar inhibitory actions on nociception, gastrointestinal and cardiovascular function, anxiety and stress, and facilitatory actions on food intake and reward in laboratory animals (see [11], [12] and [13] for recent reviews; Table S1). The coupling, via inhibitory Gi/o proteins, of these two receptors to similar intracellular signaling pathways [14] underlies, to some extent, these similarities, whereas interactions between these pathways [13] probably explain why antagonists of each receptor type sometimes counteract the pharmacological effects induced by the stimulation of the other. However, one should not get the impression that the endocannabinoid system is a mere functional duplication of opioidergic signaling. The emerging scenario, in fact, distinguishes between the general strategies of endocannabinoid and endogenous opioid signaling based on the fact that, unlike endorphins, anandamide and 2-AG are lipophilic compounds produced from membrane phosphoglycerides via Ca2+-sensitive biosynthetic pathways triggered on demand, rather than being prestored in secretory vesicles. Hence, because of their chemical nature, endocannabinoids do not typically function like hormones, but they instead act as local (autocrine or paracrine) mediators. This is clearly the case with endocannabinoid action in the brain, where the elevation of intracellular Ca2+ caused by postsynaptic neuron depolarization or stimulation of postsynaptic neurotransmitter receptors coupled to Ca2+ mobilization from intracellular stores, or both, stimulates enzymes catalyzing the biosynthesis of anandamide and, particularly, 2-AG. These compounds are then released from the neuron to activate presynaptic CB1 receptors that, in most cases via inhibition of voltage-activated calcium channels, reduce the release of both excitatory (e.g., glutamate) and inhibitory (e.g., GABA) neurotransmitters, thereby producing various effects on neuronal synapses [15]. This “retrograde” mode for endocannabinoid action (Figure S1) has been implicated in several physiopathological functions of the brain, including the control of food intake, habit-forming and mnemonic processes, neuronal plasticity, and excitotoxicity. Since the biosynthetic precursors for endocannabinoids seem to be ubiquitous in membranes, it is the reciprocal pattern of expression of endocannabinoid biosynthesising enzymes and cannabinoid receptors that determines the specificity of endocannabinoid action, whereas the localization of the degrading enzymes sets its duration. Indeed, the sn-1-specific diacylglycerol lipase (DAGL)-α catalyzing 2-AG release from diacylglycerols [16] is expressed in postsynaptic dendritic “spines” that make synapses with axon terminals expressing CB1 receptors [17] and [18]. This allows a lipophilic molecule like 2-AG to reach its target in close proximity to its site of biosynthesis. Also, one of the enzymes responsible for 2-AG degradation, the monoacylglycerol lipase (MAGL), is located in presynaptic neurons, thus allowing for the immediate inactivation of the endocannabinoid signal [19]. Elegant experiments established the time and space frames for endocannabinoid retrograde action [20] and [21], which can distinguish between neighboring CB1-expressing glutamate- and GABA-releasing neurons as targets [22] and [23]. The type and time coincidence of Ca2+-mobilizing stimuli play a major role in determining the occurrence of retrograde signaling by endocannabinoids [24] and [25].
Experiments are being performed to extend this mode of action also to CB2 receptors or to nonneuronal cells, including lymphocytes, macrophages, and microglial cells (for the control of the immune and inflammatory responses) [26] and [27]; endothelial cells (for the control of vascular tone and angiogenesis) [28]; adipocytes and pancreatic β cells (for the control of adipokine and insulin secretion) [29] or other endocrine cells [30]; and to reproductive tissues [31] and [32]. For example, the tight control in time and space of endocannabinoid biosynthesis and degradation within the framework of the chemical communication between the fertilized egg and cells of the oviduct and uterus is crucial for the correct implantation of the embryo [33]. Thus, although, like the endorphins, they exert a homeostatic function, the endocannabinoids, ultimately due to their chemical nature and peculiar biosynthetic pathways (and to the fact that constitutive CB1 receptors are much more widely distributed than originally thought, whereas CB2 receptors appear to be upregulated under several pathological conditions), play a local and yet more general function, seemingly regarding all aspects of animal health and disease (Table S1).
Faces and Facets of Endocannabinoid Metabolic Pathways and Enzymes
The interplay between chemistry and biology played a major role in the identification of the enzymes catalyzing endocannabinoid synthesis and degradation. The fatty acid amide hydrolase (FAAH), which catalyzes the hydrolysis of anandamide (and of other bioactive fatty acid amides, see below) and 2-AG, was isolated after the synthesis of inhibitors [34], which were then used for affinity chromatography purification of the enzyme. This eventually led to the cloning of FAAH [35], now considered to be a promising target for the development of new anxiolytic and analgesic drugs [36], [37] and [38]. Also, the X-ray structure of FAAH was obtained thanks to the fact that a “modified version” of the purified enzyme yields good crystals only when covalently bound to an irreversible active-site inhibitor [39]. As to the cloning of the DAGL-α and -β catalyzing 2-AG formation from diacylglycerols, this was achieved by using a bioinformatic approach, i.e., starting from the gene encoding a DAGL from Penicillium, and then “fishing” for orthologs in the sequenced genomes of increasingly complex animal organisms, ending with the human genome [16].
FAAH is an unusual enzyme since, unlike most serine hydrolases which employ a Ser-His-Asp triad, its catalytic mechanism involves a Ser-Ser-Lys triad [40] (Figure 2). This is consistent with mutagenesis and enzymological studies indicating that Ser241 plays a critical role as both acid and base in the hydrolytic cycle, whereas Lys142 is the activator of Ser241 [41] and [42]. Mutagenesis studies also involve Ser217, a residue conserved in all enzymes with an amidase signature sequence, in the catalytic mechanism of FAAH. This residue may act as a “bridge” that, through a proton shift, facilitates both the nucleophile attack and the exit of the leaving group. The FAAH crystal structure revealed the existence of an acyl chain-binding (ACB) channel for entry of hydrophobic substrates and a cytoplasmatic access (AC) channel for hydrophilic compounds, i.e., water for substrate hydrolysis and hydrophilic breakdown products. This latter channel is also likely to accommodate the polar head of anandamide and other fatty acid amides [40] and [43] (Figure 2). Despite its atypical catalytic mechanism, FAAH is inhibited by most classical serine hydrolase inhibitors, including trifluoromethyl ketones, fluorophosphonates, carbamates, and α-ketoheterocycle compounds (Table S2). The inhibitory mechanism of these compounds is based on the presence in their structures of a strong electrophile that engages the Ser241 nucleophile in either a covalently reversible or irreversible manner. However, the systematic evaluation of the selectivity of these FAAH inhibitors against other Ser hydrolases is necessary to delineate their therapeutic potential. A chemical proteomic approach known as “activity-based protein profiling” (ABPP) has been used to assess the selectivity of several FAAH inhibitors [44]. ABPP methods, by using active site-directed chemical probes, allow for the screening of inhibitors against multiple enzymes in parallel, and consequently for the detection of possible off-target enzymes [45]. More recently, a multidimensional protein-identification technology (MudPIT) was employed to identify the major off-targets for several FAAH inhibitors [46]. Using this method, it was shown that the carbamate compounds known as URB-597 [36] and BMS-1 (but not SA-72 [47]), the carbamoyl tetrazole LY2077855, and the α-keto-heterocycle OL-135 [37] act on off-targets [44], [45] and [46] (Table S2). Also, the recently developed irreversible inhibitors of DAGLs [48] are ideal candidates to be investigated with proteomics-type strategies. These compounds, known as O-3841 and O-3640, are fluorophosphonate derivatives of oleic acid and are at least selective against other proteins of the endocannabinoid system.
Figure 2. Schematic Representation of the Binding Site of Fatty Acid Amide Hydrolase
Schematic representation of the active site of fatty acid amide hydrolase (FAAH), with “hydrophobic” and “polar head” pockets (“ACB” and “AC” channels, respectively) to accommodate anandamide and other fatty acid amides, as deduced from crystallographic studies [39] and structure-activity relationship studies carried out with several analogs of FAAH inhibitors [43]. Note how the “AC” channel can theoretically accommodate bulkier groups than ethanolamine. Arachidonoyl-trifluoromethylketone (TFMK) and its analogs [34] were used for the affinity chromatography purification of FAAH [35], whereas methyl-arachidonoyl-fluoro-phosphonate (MAFP) was used to obtain crystals from a slightly modified and purified form of FAAH [39]. Carbamate inhibitors like URB597 (see also Table S2) were suggested to enter the active site in a way opposite that of the fatty acid derivatives, with the ester rather than the amide bond being cleaved by the catalytic action of Ser241 [43].
In view of the “only when and where needed” character of their action, and of the increasing evidence that endocannabinoids participate in several pathological conditions [10], it can be foreseen that inhibitors of their biosynthesis or catabolism will be used for the pharmacological treatment of disorders ranging from neuropathic pain and psychiatric/neurological disorders to hypertension and obesity, possibly in a more selective way than with cannabinoid receptor agonists (see [49] for a comprehensive review). However, two peculiar aspects of endocannabinoid metabolic pathways should call for caution when it comes to their therapeutic exploitation (Figure 3): (1) the high degree of redundancy with which endocannabinoids are made and/or degraded—this means that, for example, inhibiting just one of the several pathways that have been suggested so far for anandamide biosynthesis from N-arachidonoyl-phosphatidylethanolamine [50], [51] and [52], or only one of the several enzymes (FAAH, MAGLs, COX-2) that have been suggested to catalyze 2-AG hydrolysis [10] and [53], might not be sufficient to manipulate anandamide or 2-AG tissue levels, respectively; and (2) the observation that endocannabinoids, as with other bioactive lipids, are part of a sequence of enzymatic reactions leading to a corresponding sequence of chemical signals with distinct molecular targets and biological outputs. 2-AG, for example, is at the same time the product of a family of fundamental intracellular signals, the diacylglycerols, and the precursor of prostaglandin glycerol esters (via COX-2), suggested to act at noncannabinoid receptors [54], or of arachidonic acid (via MAGL), the progenitor of a plethora of mediators and an intracellular mediator itself. Thus, the blockade of DAGLs might not only reduce endocannabinoid signaling, but also may enhance protein kinase C-mediated signals, whereas inhibition of MAGL might lead to the accumulation of prostaglandin glycerol esters. Although it is likely that cellular and subcellular compartmentalization intervenes to segregate these potentially concurring pathways, and perhaps also to ensure that only one pathway contributes to the formation or inactivation of anandamide and 2-AG acting at cannabinoid receptors in a certain cell or tissue (Figure 3), much work remains to validate the efficacy and selectivity in vivo of inhibitors of endocannabinoid metabolism.
Figure 3. Redundancy, Functional Plasticity, and Target Multiplicity of Endocannabinoid Metabolic Pathways
(A) The three pathways proposed for N-arachidonoylphosphatidyl-ethanolamine conversion into anandamide as an alternative to the one represented in Figure S1 [50], [51] and [52]. Although speculative, it is possible that the multiplicity of the biosynthetic pathways of anandamide reflects to some extent its capability to interact at a submicromolar concentration with several molecular targets [126], including, in addition to cannabinoid receptors, opposing actions on TRPV1 (activation) and TRPM8 (inhibition) [127] channels (see Figure 5).
(B) Biosynthesis and degradation of 2-arachidonoylglycerol seen as a sequence of different signals acting on different targets at different times. Apart from being converted, as discussed in the text, to arachidonic acid and to a COX-2 derivative with specific targets, 2-AG can be transformed into, and produced from, 2-arachidonoyl-lysophosphatidic acid, which activates LPA receptors with relatively high homology with CB1 receptors [128]. Abbreviations: Abh4, α/β-hydrolase 4; CB1, CB2, cannabinoid receptors of type 1 and 2, respectively; COX-2, cycloxygenase-2; FAAH, fatty acid amide hydrolase; GPCR, G protein-coupled receptor; LPA, lysophosphatidic acid; lyso-PLD, lyso-phospholipase D; MAGL, monoacylglycerol lipase; PKC, protein kinase C; sPLA2, secretory phospholipase A2, PLC, phospholipase C; PGE2, prostaglandin E2; PTPN22, protein tyrosine phosphatase N22; TRPM8, transient receptor potential melastatin type 8 channel; TRPV1, transient receptor potential vanilloid type 1 channel.
Recent data have suggested, however, that some widely used pharmaceuticals might owe some of their therapeutic actions to the interaction with endocannabinoid-biosynthesizing or -inactivating proteins. In particular, some nonsteroidal anti-inflammatory drugs (NSAIDs) (Figure 4) possess this property. Indomethacin exerts anti-inflammatory actions that are partly antagonized by a CB2 receptor antagonist [55], whereas ibuprofen enhances the analgesic actions of anandamide in a way sensitive to both CB1 and CB2 antagonists [56], and flurbiprofen inhibits the spinal release of proalgesic peptides in a way that is antagonized by a CB1 antagonist [57]. Rofecoxib, a COX-2 inhibitor, synergizes with anandamide while elevating the tissue levels of anandamide and other analgesic fatty acid ethanolamides [58]. Indeed, one possible way to explain these findings is by suggesting that these NSAIDs also inhibit FAAH, as already shown by Fowler and collaborators for ibuprofen in 1997 [59]. More recently, the same group extended this property to other acidic NSAIDs, and they showed that it becomes more important when the extracellular pH is lower than 7, as during inflammatory conditions [60]. Meanwhile, other indomethacin derivatives, such as indomethacin methyl ester and indomethacin morpholinylamides (Figure 4), were also found to directly activate CB1 and CB2 receptors, respectively [61] and [62]. More intriguingly, a recent study [63] showed that, in rats and mice, acetaminophen (paracetamol) is converted in vivo into p-aminophenol and then N-arachidonoyl-phenolamine (AM404) (Figure 4), a compound capable of inhibiting both FAAH and anandamide cellular reuptake and elevating anandamide tissue levels [64] and [65]. The second reaction is catalyzed by FAAH (an enzyme that, under certain conditions, can facilitate the condensation of fatty acids with amines), thus opening the possibility that it might also occur with other aromatic amines used in the clinic and lead to aromatic amides capable of interacting with other proteins of the endocannabinoid system. Importantly, the analgesia on a hot plate of acetaminophen is blocked by CB1 receptor antagonists, thus confirming that this widely used drug does also act through enhancement of the endocannabinoid system [66].
Figure 4. Chemical Structures of Widely Used Therapeutic Drugs that Have Been Reported to Interact with Proteins of the Endocannabnoid System
The cycloxygenase inhibitor indomethacin, and some of its derivatives, were suggested to directly bind to and activate CB1 and/or CB2 receptors [61] and [62]. Despite the lack of chemical similarity with anandamide, the NSAIDs, rofecoxib, ibuprofen, and flurbiprofen, and the general anesthetic propofol were suggested to act, in part, by inhibiting FAAH-catalyzed anandamide degradation [56], [57], [58], [59], [60] and [67]. The NSAID acetaminophen, instead, was shown to act, in part, through the conversion into AM404 and subsequent indirect activation of cannabinoid receptors [63] and [66]. The antiobesity drug orlistat (tetrahydrolipstatin) was shown to potently inhibit the biosynthesis of 2-arachidonoylglycerol [16] and [48].
NSAIDs are not the only therapeutic drugs capable of interacting with endocannabinoid enzymes. The general anesthetic propofol (Figure 4) also inhibits FAAH, and a part of its sedating properties is due to the subsequent elevation of the brain levels of anandamide, which, via CB1 receptors, can induce sleep [67]. More recently, the antiobesity drug tetrahydrolipstatin (orlistat) (Figure 4) was shown to inhibit DAGL-α and -β and, hence, to reduce 2-AG levels in intact cells, at concentrations lower than those necessary to inhibit other lipases [16] and [48]. Although this compound inhibits obesity mostly by acting at the level of lipid assimilation, it is tempting to speculate that part of its actions are due to inhibition of 2-AG-induced, and CB1-mediated, gastrointestinal actions [68]. In summary, from these data the intriguing possibility emerges that we might already be using in the clinic, without knowing it, substances that act in part by manipulating endocannabinoid levels.
Back to Plants: Are Endocannabinoids Like Chili Peppers?
The chemical structure of anandamide is not so different from that of capsaicin (Figure 5A), the pungent fatty acid amide component of another plant with a long history of medicinal and nutritional use, the hot chili pepper Capsicum annuum. In fact, capsaicin is even more chemically similar to the aforementioned AM404 (Figure 4), which inhibits anandamide degradation [64] and [65]. After the identification by Caterina and colleagues [69] of the transient receptor potential vanilloid-type 1 channel (TRPV1, originally known as vanilloid VR1 receptor) as the molecular target of capsaicin (Figures 5B and 5C), we and others, based on these chemical considerations, began to investigate the possibility that this receptor and proteins of the endocannabinoid system share common ligands [70]. Compounds were synthesized that bind to CB1 receptors, FAAH, or the putative anandamide membrane transporter (Figure S1), on the one hand, and to TRPV1, on the other hand [71] and [72]. More importantly, it was possible to demonstrate that anandamide activates TRPV1 receptors [73] and [74]. Later, other long-chain fatty acid ethanolamides, i.e., the naturally occurring homologs of anandamide (Figure 5A), and AM404 itself, were found to stimulate the activity of TRPV1 receptors [72], [75], [76], [77] and [78], and to share this property with other derivatives of arachidonic acid [79]. Molecular-modeling techniques were used to demonstrate that the preferential conformations of these compounds in solution overlap with those of capsaicin [78] and [79] (Figure 5D). However, none of these endogenous compounds appeared to be nearly as potent as capsaicin at inducing TRPV1-mediated biological responses. Although we now know that the “vanilloid” activity of these compounds is dramatically increased by several regulatory factors [80], this observation, together with the original belief that vanilloid TRPV1 receptors, by being mostly expressed in sensory afferents, are used as receptors only for external painful stimuli (high temperature, low pH, plant toxins), seemed to rule out the existence of true “endovanilloids.”
Figure 5. Vanilloid Receptors and Endovanilloids
(A) Chemical structures of N-acyldopamines and oleoylethanolamide, identified after the discovery of endocannabinoids and of the receptors for capsaicin and resiniferatoxin, the transient receptor potential vanilloid type 1 (TRPV1) channel.
(B) Three-dimensional representation of a TRPV1 monomer with the binding site for capsaicin according to Jordt and Julius [129]. According to this model, aromatic stacking of the vanillyl moiety of capsaicin (as well as anandamide) with Tyr511 in the intracellular T2-T3 linker region stabilizes the binding of the aliphatic chain with a transmembrane site created by the T3 and T4 domains. The observation that the T2-T3 linker domain plays a crucial role in ligand recognition in both TRPV1 receptor and the menthol and “cold”-receptor, TRPM8, led to the identification of anandamide and NADA as TRPM8 antagonists [127].
(C) Model proposed by Gavva and colleagues according to which capsaicin, anandamide, and N-arachidonoyldopamine (NADA) bind to Tyr511 through hydrophobic binding with their aliphatic chain, whereas residues Trp549 and Thr550 are involved in binding with the vanillyl or cathecolamine moieties of capsaicin and NADA, respectively [130].
(D) Overlap of capsaicin and oleoylethanolamide conformations in solution, according to the molecular model proposed by Movahed et al. [78].
Meanwhile, however, increasing and conclusive evidence was obtained for the presence of TRPV1 receptors in the brain [81], [82] and [83], where these proteins are unlikely to be reached by exogenous stimuli, thus strongly suggesting the existence of endovanilloid ligands [84]. Considerations on the structure-activity relationships of synthetic vanilloid TRPV1 agonists, which indicated the necessity of (1) a long, unsaturated alkyl chain, (2) a secondary amide group, and (3) 3-hydroxy-4-methoxy substituents on the aromatic moiety, in order to achieve optimal interaction with the receptors [85] and [86] (Figure 5C), together with the realization that these prerequisites could be met, to some extent, only by putative endogenous amides of dopamine with long, unsaturated fatty acids, led first to the synthesis [87] and then the isolation from the brain of N-arachidonoyl- and N-oleoyl-dopamine [88] and [89]. These compounds (Figure 5A; Table 1) are the most potent and efficacious endovanilloids identified to date and are most abundant in brain regions rich in dopamine, in agreement with their potential role as endogenous TRPV1 activators and with their possible biosynthetic origin from the N-acylation of dopamine. They are accompanied by saturated homologs that, although inactive per se on TRPV1, are capable of enhancing the activity of other endovanilloids [90]. Thus, once again, the meeting of chemical and biological minds led to the finding of endogenous counterparts of a plant natural product. The importance of the discovery of N-acyl-dopamines, however, went beyond that of finding potent endogenous ligands for TRPV1 receptors, since it supported the hypothesis that animal tissues might make, and use as chemical signals, any combination between fatty acids and biogenic amines.
The possible combinations of amino acids or biogenic amines and the most abundant fatty acids found in mammalian tissues yield new potential endogenous mediators: the N-acyl-ethanolamines, N-acyl-dopamines, N-acyl-amino acids, and N-acyl-taurines [97], [98], [99] and [100]. Fatty acid primary amides have also been identified [135] and [136]. The targets and metabolism of only a few of the several hundred possible compounds have been determined to date [103], [104], [105], [106] and [107] and are summarized here. R2 = C12:0, C14:0, C16:0, C18:0, C18:1, C18:2, C18:3, C20:0, C20:3, C20:4, C22:0, C22:4, C22:5, C22:6, C24:0, C24:1 alkyl chains; R3 = residues of amino acids or GABA. COMT, catecholamine-O-methyl-transferase; FAAH, fatty acid amide hydrolase; PPAR, peroxisome-proliferator-activated receptor; TRPV, transient receptor potential vanilloid-type.
Bioactive Fatty Acid Amides: When Animal Cells Play Combinatorial Chemistry
That cells could “combine” different fatty acids and biogenic amines to make several possible permutations of different fatty acid amides had been deduced already from the discovery in rodents of N-acyl-amino acids such as N-arachidonoyl-glycine (NAGly) and N-arachidonoyl-alanine (Table 1), which share with fatty acid ethanolamides like anandamide a potent analgesic and anti-inflammatory activity and the property of being recognized by FAAH as substrates [91], [92] and [93]. This finding raised the possibility that any long-chain fatty acid available in tissues could be amidated with any of the 20 amino acids and their naturally occurring derivatives. This would lead to the origination of hundreds of potential endogenous mediators with functions and molecular targets to be discovered. This possibility seems to be confirmed by the recent identification of N-arachidonoyl-serine, a brain constituent with vasodilatory and anti-inflammatory activity [94], and by increasing evidence for the presence in rodent tissues of 40 other postulated fatty acyl amides [95] and [96].
How is chemical-biological research going to cope with this “invasion” of new small-molecule mediators? How to discover and measure their levels in tissues, reveal their metabolic pathways, understand their physiological and pathological roles and, most importantly, identify their molecular targets? Clearly, the employment of the most modern “omic” approaches for gene, protein, and metabolite profiling will be required to find a function and a “parent receptor” to these orphan mediators, as well as to guide their discovery. Some progress in this direction has already been made. For example, based on the assumption that at least some of these compounds would be FAAH substrates, Cravatt and collaborators used an original global metabolite-profiling strategy, consisting of comparing, by the use of liquid chromatography-electrospray-mass spectrometric (LC-ESI-MS) techniques, the lipid components of tissues from wild-type and FAAH null mice, to identify new fatty amides. This led to the identification of N-acyl-taurines (Table 1) as new endogenous amides whose levels are controlled by this enzyme [97] and [98]. A similar approach, using FAAH null mice treated with anandamide, led to the identification of a potential alternative pathway for the inactivation of this compound and other acylethanolamides through the formation of the corresponding O-phosphorylcholine derivatives [99]. Other comparisons between lipid profiles, for example those of distinct brain areas from mice at different phases of their estrous cycle [100], can be used to understand the function of the analytes. Multidimensional (tandem) mass spectrometric techniques applied to other types of lipidomic-like approaches 101 M.R. Wenk, The emerging field of lipidomics, Nat. Rev. Drug Discov. 4 (2005), pp. 594–610. View Record in Scopus | Cited By in Scopus (204)[101] and [102] also seem to be very promising for the discovery of even trace quantities of fatty acid amides belonging to several families, or for the analysis of already discovered compounds [95] and [100]. Thanks to the use of “common fragment” methods, all of the naturally occurring members of the same family of fatty acid amides with different acyl chains but yielding common fragments after MS-MS (Table 1) can be analyzed in the same run (see Figure S2). Furthermore, the use of the optimal chromatographic conditions and of LC-ESI-MS-MS allows for the analysis of several fatty acid amide classes at the same time.
Progress is also being made in the identification of receptors for bioactive fatty acid amides. Some serendipitous but nevertheless important discoveries, i.e., that oleoylethanolamide binds to and activates peroxisome proliferator-activated receptor α [103], or that N-arachidonoyl-serine activates an as-yet-unidentified endothelial cannabinoid receptor [94], were prompted by the pharmacological actions observed for these compounds. N-acyl-taurines, like acylethanolamides and acyldopamines, activate TRPV1 receptors, but they also gate a related plasma membrane cation channel, the TRPV4 receptor [104]. Also, the finding of two GPCRs, GPR119 and GPR18, as high-affinity receptors for oleoylethanolamide and NAGly, respectively [105] and [106], was the result of targeted approaches. The implications of industrial patents claiming that GPR55 might be a high-affinity target for palmitoylethanolamide have also been recently discussed [107]. However, in view of the observation that bioactive fatty acid amides can be promiscuous in their targets, and that the latter, as with other lipid mediators, encompass all types of receptors (i.e., GPCRs, nuclear receptors, ion channels [Figure 3]), it can be foreseen that more systematic approaches, such as the use of biospecific interaction analysis (BIAcore), protein or mRNA arrays, and even of virtual screening methodologies, will have to be used in the future for the “deorphanization” of these novel compounds and, at the same time, of the 150 G protein-coupled and 24 nuclear “orphan” receptors still awaiting assignment of endogenous ligands. These techniques, together with the MS methods mentioned above, will have to be applied to also investigate if other amides of fatty acids, for example with serotonin, histamine, adrenaline, and noradrenaline, as well as with trace amines, occur in mammalian tissues. Some of these compounds (e.g., N-arachidonoyl-serotonin, Figure 6) have already been synthesized and exhibit interesting pharmacological properties (see below).
Figure 6. “Tinkering” with Nature's Building Blocks
Combining the vanillyl moiety typical of several plant natural products with arachidonic acid or ricinoleic acid, and the further modification of the chemical structures, yields compounds with mixed activity at cannabinoid and vanilloid TRPV1 receptors, i.e., arvanil, O-1861, and phenyl-acetyl-ricinoleoyl-cyclopropylamide [71], [108] and [109]. The synthetic N-arachidonoyl-serotonin mimics Nature's capability to make amides between long-chain fatty acids and biogenic amines, and turned out to be a selective inhibitor of fatty acid amide hydrolase (Table S2) and a potent antagonist at TRPV1 channels [38] and [110]. Iodination of the vanillyl moiety of a natural compound like resiniferatoxin (RTX), or of synthetic natural product derivatives, such as the acetylvanillyl ester of resiniferol orthophenylacetate (ROPA) and nor-dihydrocapsaicin, can produce opposing effects on the activity at TRPV1 channels [111], [112], [113] and [114]. The combination of a natural product like olivetol and a long-chain fatty acid yielded CB-25, a potent cannabinoid receptor ligand with “protean” agonist activity in vitro and in vivo [115], [116] and [117]. Isobutyl analogs of anandamide are more potent than the parent compound at cannabinoid receptors, and this is perhaps the reason why the Echinacea components dodeca-2E,4E,8Z,10Z-tetraenoic acid- and dodeca-2E,4E-dienoic acid-isobutylamide are the only plant natural products apart from cannabinoids that activate cannabinoid CB2 receptors quite potently [118], [119], [120] and [121].
Think Like a Tinker: Making Endocannabinoid-Based Drugs with Nature's Bits and Pieces
If nature can combine chemical moieties to make small, multitarget chemical signals via dedicated metabolic pathways, the synthetic chemist can also put together naturally occurring building blocks to make drugs that can interact with multiple receptor types, thus obtaining, in theory, more efficacious and therapeutically useful pharmacological tools. Given the overlap between the potential clinical applications (pain, inflammation, emesis, cancer, etc.) and binding recognition properties of proteins of the endocannabinoid system and TRPV1 channels, one strategy to develop hybrid therapeutic drugs was to put together fatty acids and the vanillyl moiety of capsaicin, each opportunely modified in order to maximize the interaction of the end product with each postulated target. N-arachidonoyl-vanyllamide (arvanil) and its analog, O-1861 (Figure 6), were synthesized with this idea in mind, and they proved to be capable of potently activating TRPV1 receptors while acting as agonists at CB1 receptors and inhibitors of the cellular reuptake of anandamide [71] and [108]. Both compounds turned out to be more potent and efficacious analgesics than pure cannabinoid or vanilloid receptor agonists with comparable affinities for their respective receptors. The derivatization of the hydroxy group of ricinoleic acid, followed by amidation with chemically modified ethylamine “heads,” yielded compounds with antagonist/inverse agonist activity at CB2 receptors and agonist activity at TRPV1 receptors and, hence, potential application against inflammation [109]. The aforementioned N-arachidonoyl-serotonin not only inhibits FAAH, thus elevating endocannabinoid levels and acting as an indirect cannabinoid receptor agonist [110], but it is also a potent TRPV1 antagonist in vitro and in vivo, thus exhibiting equivalent activity against neuropathic pain as other compounds that are more potent at FAAH [38].
However, tinkering with natural products can also lead to unexpected results. For example, 5′-iodination of resiniferatoxin on its vanillyl moiety (Figure 6) transforms the ultrapotent agonist activity at TRPV1 channels of this plant toxin into the capability to antagonize these receptors, whereas 6′-iodination only weakens its agonist activity [111]. By converse, 6′-, rather than 5′-, iodination of the much less hindered nordihydrocapsaicin (the saturated analog of capsaicin) and of other vanillamide TRPV1 ligands is required to transform their agonist activity into potent TRPV1 antagonists [112], without decreasing the capability of some of these compounds to directly inhibit NF-kB activity, thus yielding dual target anti-inflammatory and proapoptotic agents [113]. Even more intriguingly, 5′-iodination of the acetylvanillyl ester of resiniferol orthophenylacetate, a resiniferatoxin analog (Figure 6), yields a compound that, instead of behaving as an antagonist (as in the case of 5′-I-resiniferatoxin) or of losing activity (as in the case of 6′-I-resiniferatoxin), is 5-fold more potent as an agonist than the noniodinated analog [114]. On the “cannabinoid side,” attaching a C-11 alkylamide “head” to olivetol, a plant-derived compound, led to compounds that, despite their limited conformational freedom compared to endocannabinoids, bind with higher affinity to both CB1 and CB2 receptors [115]. In this case, the surprise came from the dramatically different behavior of one these compounds (CB-25, Figure 6) in vitro and in vivo, where it can act as a CB1 partial agonist and CB2 “silent” antagonist or as a CB1 antagonist, respectively [116], thus belonging to the ever growing family of receptor “protean agonists” (i.e., compounds that can exhibit agonist, antagonist, or inverse agonist activities depending on the constitutive “tone” of their receptors [117]).
If man can imitate nature by making hybrid drugs, Nature can also imitate the human body by making endocannabinoid-like natural products that potently bind to cannabinoid receptors. This is the case for the alkylisobutylamides from the roots of Echinacea angustifolia [118], [119] and [120]. These compounds, and in particular dodeca-2E,4E,8Z,10Z-tetraenoic acid- and dodeca-2E,4E-dienoic acid-isobutylamide (Figure 6), bind to CB2 receptors with higher affinity than endocannabinoids, although this property explains only part of their strong immunomodulatory and anti-inflammatory properties [121]. The crosstalk between man and Nature extends even further since C18:1 and C18:2 fatty acid ethanolamides and their corresponding phospholipid precursors are also found in higher plant seeds and leaves [122] and [123], in which a functional homolog of the mammalian FAAH has been cloned [124]. These anandamide congeners are inactive at cannabinoid receptors but play a role in the control of plant growth via unidentified molecular targets [124] and [125]. These chemical, but not necessarily functional, similarities between plant metabolites and mammalian mediators underscore the global importance of certain metabolic pathways in eukaryotes, and they emphasize how cells from phylogenetically distant species can exploit similar building blocks and anabolic/catabolic strategies to respond to different biological demands.
Take Home Messages and Open Questions
From the data reviewed in this article it is clear that biological and pharmacological studies carried out with a chemical mind can reveal new physiological and pathological mechanisms and, eventually, solve clinical problems. The exciting walk from the psychotropic constituent of Cannabis to the understanding of the mechanism of its biological actions led to the discovery of the endocannabinoid system, a signaling apparatus whose function goes well beyond what could have been imagined from pharmacological studies on THC. The route from the endocannabinoids to the Capsicum pungent principle then led to the finding of endovanilloids, endogenous mediators whose role is yet to be fully understood. The discovery of endocannabinoids and endovanilloids paved the way for the finding of several other lipid signals, and it prompted, on the one hand, new strategies for the chemical synthesis of compounds with multiple pharmacological and therapeutic targets, and, on the other hand, the search for chemically similar compounds back in plants. Many open questions to which the meeting of chemistry and biology can provide an answer still remain. Just to name a few, these questions involve the understanding of the mechanisms and exact physiological meaning of the target and metabolic pathway promiscuity of fatty acid ethanolamides; the identification of the elusive protein(s) facilitating endocannabinoid membrane transport; the receptor “deorphanization” of the several tens of bioactive fatty acid amides that have been discovered, and their testing on the several TRP channels that have been identified so far, many of which are, like TRPV1, activated by plant natural products; the determination of the exact function and molecular targets of fatty acid ethanolamides in plants; and the understanding of the mechanism of action in animals of other phytocannabinoids, like the therapeutically promising cannabidiol. Thus, the exciting promenade from plant natural products to animal physiology and back might soon become a true treasure hunt.
Cannabinoids Potentiate Emotional Learning Plasticity in Neurons of the Medial Prefrontal Cortex through Basolateral Amygdala Inputs.
Laviolette SR, Grace AA.
Departments of Neuroscience, Psychiatry, and Psychology, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Abstract
Cannabinoids represent one of the most commonly used hallucinogenic drug classes. In addition, cannabis use is a primary risk factor for schizophrenia in susceptible individuals and can potently modulate the emotional salience of sensory stimuli. We report that systemic activation or blockade of cannabinoid CB1 receptors modulates emotional associative learning and memory formation in a subpopulation of neurons in the mammalian medial prefrontal cortex (mPFC) that receives functional input from the basolateral amygdala (BLA). Using in vivo single-unit recordings in rats, we found that a CB1 receptor agonist potentiated the response of medial prefrontal cortical neurons to olfactory cues paired previously with a footshock, whereas this associative responding was prevented by a CB1 receptor antagonist. In an olfactory fear-conditioning procedure, CB1 agonist microinfusions into the mPFC enabled behavioral responses to olfactory cues paired with normally subthreshold footshock, whereas the antagonist completely blocked emotional learning. These results are the first demonstration that cannabinoid signaling in the mPFC can modulate the magnitude of neuronal emotional learning plasticity and memory formation through functional inputs from the BLA.
Intra-dorsal periaqueductal gray administration of cannabidiol blocks panic-like response by activating 5-HT1A receptors.
Soares Vde P, Campos AC, Bortoli VC, Zangrossi H Jr, Guimarães FS, Zuardi AW.
Neuroscience and Behavioral Sciences, School of Medicine of Ribeirão Preto, Campus USP, Ribeirão Preto, SP, Brazil. vanpaso@cb.ufrn.br
Abstract
Activation of 5-HT1A receptors in the dorsal periaqueductal gray (dPAG) impairs escape behavior, suggesting a panicolytic-like effect. Cannabidiol (CBD), a major non-psychotomimetic compound present in Cannabis sativa, causes anxiolytic-like effects after intra-dPAG microinjections by activating 5-HT1A receptors. In the present work we tested the hypothesis that CBD could also impair escape responses evoked by two proposed animal models of panic: the elevated T-maze (ETM) and electric stimulation of dPAG. In experiment 1 male Wistar rats with a single cannula implanted in the dPAG received a microinjection of CBD or vehicle and, 10 min later, were submitted to the ETM and open field tests. In experiment 2 escape electrical threshold was measured in rats with chemitrodes implanted in the dPAG before and 10 min after CBD microinjection. In experiment 3 similar to experiment 2 except that the animals received a previous intra-dPAG administration of WAY-100635, a 5-HT1A receptor antagonist, before CBD treatment. In the ETM microinjection of CBD into the dPAG impaired inhibitory avoidance acquisition, an anxiolytic-like effect, and inhibited escape response, a panicolytic-like effect. The drug also increased escape electrical threshold, an effect that was prevented by WAY-100635. Together, the results suggest that CBD causes panicolytic effects in the dPAG by activating 5-HT1A receptors.
Cannabinoids promote embryonic and adult hippocampus neurogenesis and produce anxiolytic- and antidepressant-like effects.
Jiang W, Zhang Y, Xiao L, Van Cleemput J, Ji SP, Bai G, Zhang X.
Neuropsychiatry Research Unit, Department of Psychiatry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
Abstract
The hippocampal dentate gyrus in the adult mammalian brain contains neural stem/progenitor cells (NS/PCs) capable of generating new neurons, i.e., neurogenesis. Most drugs of abuse examined to date decrease adult hippocampal neurogenesis, but the effects of cannabis (marijuana or cannabinoids) on hippocampal neurogenesis remain unknown. This study aimed at investigating the potential regulatory capacity of the potent synthetic cannabinoid HU210 on hippocampal neurogenesis and its possible correlation with behavioral change. We show that both embryonic and adult rat hippocampal NS/PCs are immunoreactive for CB1 cannabinoid receptors, indicating that cannabinoids could act on CB1 receptors to regulate neurogenesis. This hypothesis is supported by further findings that HU210 promotes proliferation, but not differentiation, of cultured embryonic hippocampal NS/PCs likely via a sequential activation of CB1 receptors, G(i/o) proteins, and ERK signaling. Chronic, but not acute, HU210 treatment promoted neurogenesis in the hippocampal dentate gyrus of adult rats and exerted anxiolytic- and antidepressant-like effects. X-irradiation of the hippocampus blocked both the neurogenic and behavioral effects of chronic HU210 treatment, suggesting that chronic HU210 treatment produces anxiolytic- and antidepressant-like effects likely via promotion of hippocampal neurogenesis.
Effect of cannabinoids on platelet serotonin uptake.
Velenovská M, Fisar Z.
Department of Psychiatry, 1st Faculty of Medicine, Charles University, Prague, Czech Republic.
Abstract
Serotonin is involved in many of the same processes affected by cannabinoids; therefore, we investigated in vitro and in vivo effects of these drugs on the function of serotonin transporter. The effect of Delta(9)-tetrahydrocannabinol (Delta(9)-THC), endocannabinoid anandamide and synthetic cannabinoid receptor agonist WIN 55,212-2 on platelet serotonin uptake and membrane microviscosity was examined in 19 marijuana smokers and 20 controls. (1) Serotonin uptake was inhibited at higher doses of Delta(9)-THC (IC(50) = 139 micromol/l), anandamide (IC(50) = 201 micromol/l) or WIN 55,212-2 (IC(50) = 17.4 micromol/l); the inhibition was found non-competitive. Delta(9)-THC, anandamide and WIN 55,212-2 produced different effects on the membrane microviscosity. (2) Maximal velocity of platelet serotonin uptake was significantly increased in a group of chronic marijuana smokers suffering impairment of cognitive functions when compared with controls. Opposite effect of marijuana smoking on the serotonin uptake efficiency was observed in males beside females. In summary, this study provides evidence that (1) Activity of serotonin transporter is acutely affected by cannabinoids at relatively high drug concentrations; this effect is indirect and can be partially accounted for the changes in the membrane microviscosity. (2) Increase of maximal velocity of the serotonin uptake could be understood as adaptation change in the serotonergic system induced by chronic cannabis use. A hypothesis was supported that lowered serotonin uptake may reflect a gender-related differences in effects of psychoactive cannabinoids.
Inhibition of monoamine oxidase activity by cannabinoids.
Fisar Z.
Department of Psychiatry, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Ke Karlovu 11, 120 00, Prague 2, Czech Republic. zfisar@lf1.cuni.cz
Abstract
Brain monoamines are involved in many of the same processes affected by neuropsychiatric disorders and psychotropic drugs, including cannabinoids. This study investigated in vitro effects of cannabinoids on the activity of monoamine oxidase (MAO), the enzyme responsible for metabolism of monoamine neurotransmitters and affecting brain development and function. The effects of the phytocannabinoid Delta(9)-tetrahydrocannabinol (THC), the endocannabinoid anandamide (N-arachidonoylethanolamide [AEA]), and the synthetic cannabinoid receptor agonist WIN 55,212-2 (WIN) on the activity of MAO were measured in a crude mitochondrial fraction isolated from pig brain cortex. Monoamine oxidase activity was inhibited by the cannabinoids; however, higher half maximal inhibitory concentrations (IC(50)) of cannabinoids were required compared to the known MAO inhibitor iproniazid. The IC(50) was 24.7 micromol/l for THC, 751 micromol/l for AEA, and 17.9 micromol/l for WIN when serotonin was used as substrate (MAO-A), and 22.6 micromol/l for THC, 1,668 micromol/l for AEA, and 21.2 micromol/l for WIN when phenylethylamine was used as substrate (MAO-B). The inhibition of MAOs by THC was noncompetitive. N-Arachidonoylethanolamide was a competitive inhibitor of MAO-A and a noncompetitive inhibitor of MAO-B. WIN was a noncompetitive inhibitor of MAO-A and an uncompetitive inhibitor of MAO-B. Monoamine oxidase activity is affected by cannabinoids at relatively high drug concentrations, and this effect is inhibitory. Decrease of MAO activity may play a role in some effects of cannabinoids on serotonergic, noradrenergic, and dopaminergic neurotransmission.

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