Master Document: Edited

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.
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.
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.
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.
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).
CB1 Cannabinoid Receptor and Sphingomyelin Hydrolysis 957
As shown in Fig. 4B, THC induced 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).
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.
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.
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.
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.
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
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.
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.
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).
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.
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)].
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.
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.
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.
Mice fed diets containing 3% or 6% coffee for 5 days had increased levels of mRNA for NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferase class Alpha 1 (GSTA1) of between 4- and 20-fold in the liver and small intestine. Mice fed 6% coffee also had increased amounts of mRNA for UDP-glucuronosyl transferase 1A6 (UGT1A6) and the glutamate cysteine ligase catalytic (GCLC) subunit of between 3- and 10-fold in the small intestine. Up-regulation of these mRNAs was significantly greater in mice possessing Nrf2 (NF-E2 p45 subunit-related factor 2) than those lacking the transcription factor. Basal levels of mRNAs for NQO1, GSTA1, UGT1A6 and GCLC were lower in tissues from nrf2(-/-) mice than from nrf2(+/+) mice, but modest induction occurred in the mutant animals. Treatment of mouse embryonic fibroblasts (MEFs) from nrf2(+/+) mice with either coffee or the coffee-specific diterpenes cafestol and kahweol (C+K) increased NQO1 mRNA up to 9-fold. MEFs from nrf2(-/-) mice expressed less NQO1 mRNA than did wild-type MEFs, but NQO1 was induced modestly by coffee or C+K in the mutant fibroblasts. Transfection of MEFs with nqo1-luciferase reporter constructs showed that induction by C+K was mediated primarily by Nrf2 and required the presence of an antioxidant response element in the 5'-upstream region of the gene. Luciferase reporter activity did not increase following treatment of MEFs with 100 mumol/l furan, suggesting that this ring structure within C+K is insufficient for gene induction. Priming of nrf2(+/+) MEFs, but not nrf2(-/-) MEFs, with C+K conferred 2-fold resistance towards acrolein.
Epidemiological and laboratory studies have highlighted the potent chemopreventive effectiveness of both dietary selenium and cruciferous vegetables, particularly broccoli. Sulforaphane (SFN), an isothiocyanate, was identified as the major metabolite of broccoli responsible for its anti-cancer properties. An important mechanism for SFN chemoprevention is through the enhancement of glutathione (GSH), the most abundant antioxidant in animals and an important target in chemoprevention. Enhancement of GSH biosynthetic enzymes including the rate-limiting glutamate cysteine ligase (GCL), as well as other Phase II detoxification enzymes results from SFN-mediated induction of the nuclear factor-erythroid 2-related factor 2 (Nrf2)/antioxidant response elements (ARE) signaling pathway. While isothiocyanate compounds such as SFN are among the most potent Nrf2 inducers known, we hypothesized that substitution of sulfur with selenium in the isothiocyanate functional group of SFN would result in an isoselenocyanate compound (SFN-isoSe) with enhanced Nrf2 induction capability. Here we report that SFN-isoSe activated an ARE-luciferase reporter in HepG2 cells more potently than SFN. It was also found that SFN-isoSe induced GCL and GSH in MEF cells in an Nrf2-dependent manner. Finally, we provide evidence that SFN-isoSe was more effective in killing HepG2 cancer cells, yet was less toxic to non-cancer MEF cells, than SFN. These data support our hypothesis, and suggest that SFN-isoSe and potentially other isoselenocyanates may be highly effective chemoprotective agents in vivo due to their ability to induce Nrf2 with low toxicity in normal cells and high efficiency at killing cancer cells.
Oxidative stress and loss of cellular Ca2+ homeostasis are closely linked and are common denominators in the pathophysiology of many neurodegenerative diseases and acute disorders of the nervous system. Mitochondria are major targets of oxidative stress and abnormal intracellular Ca2+, as both can cause bioenergetic failure through synergistic activation of the mitochondrial inner membrane permeability transition pore. Opening of this molecularly ill-defined pore causes both collapse of the membrane potential, which drives oxidative phosphorylation, and release of small metabolites, including pyridine nucleotides and glutathione, which are necessary for energy metabolism and defense against oxidative stress. Expression of genes coding for many antioxidant defense proteins is regulated by the Nrf2 transcriptional activating factor. Translocation of this protein from the cytosol to the nucleus is stimulated by oxidative stress and by specific agents that either react with cysteine sulfhydryl groups present on the protein KEAP1, that normally binds and restricts Nrf2 translocation, or that stimulate serine phosphorylation of Nrf2. Recent evidence indicates that mitochondria are a target of the cytoprotective gene expression induced by Nrf2 and that this pathway can increase resistance to redox-regulated opening of the permeability transition pore. Pharmacologic stimulation of the Nrf2 system and its protection against mitochondrial bioenergetic dysfunction may therefore constitute a powerful mechanism for both pre-conditioning against neurodegeneration and for post-conditioning against neural cell death associated with acute neurologic injury.
Cruciferous vegetables contain glucosinolates that, after conversion to isothiocyanates (ITC), are capable of inducing cytoprotective genes. We examined whether broccoli seeds can elicit a chemoprotective response in mouse organs and rodent cell lines and investigated whether this response requires nuclear factor-erythroid 2 p45-related factor 2 (Nrf2). The seeds studied contained glucosinolate at 40 mmol/kg, of which 59% comprised glucoiberin, 19% sinigrin, 8% glucoraphanin, and 7% progoitrin. Dietary administration of broccoli seeds to nrf2(+/+) and nrf2(-/-) mice produced a approximately 1.5-fold increase in NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferase (GST) activities in stomach, small intestine, and liver of wild-type mice but not in mutant mice; increased transferase activity was associated with elevated levels of GSTA1/2, GSTA3, and GSTM1/2 subunits. These seeds also increased significantly the level of glutamate cysteine ligase catalytic (GCLC) subunit in the stomach and the small intestine of nrf2(+/+) mice but not nrf2(-/-) mice. An aqueous broccoli seed extract was prepared for treatment of cultured cells that contained ITC at approximately 600 mumol/L, composed of 61% 3-methylsulfinylpropyl ITC, 30% sulforaphane, 4% allyl ITC, and 4% 3-butenyl ITC. This extract induced GSTA1/2, GSTA3, NQO1, and GCLC between 3-fold and 10-fold in mouse Hepa-1c1c7 and rat liver RL-34 cells. The broccoli seed extract affected increases in GSTA3, GSTM1, and NQO1 proteins in nrf2(+/+) mouse embryonic fibroblasts but not in nrf2(-/-) mouse embryonic fibroblasts. These experiments show that broccoli seeds are effective at inducing antioxidant and detoxication proteins, both in vivo and ex vivo, in an Nrf2-dependent manner.
We demonstrate that Keap1/Nrf2 and NF-kappaB respond differently to electrophiles that bind proteins covalently and the redox perturbation associated with glutathione depletion, and that crosstalk may enable NF-kappaB to partly influence Nrf2 expression during cellular stress. We have investigated two transcription factors known to be sensitive to electrophilic stress and redox perturbation, Nrf2 and NF-kappaB, in mouse liver cells. Cellular stress was induced by the probes: N-acetyl-p-benzoquinineimine (NAPQI), the reactive metabolite of acetaminophen; dinitrochlorobenzene (DNCB), a model electrophile; and buthionine (S,R)-sulfoximine (BSO), an inhibitor of glutamate-cysteine ligase. NAPQI, DNCB and BSO can all cause glutathione (GSH) depletion; however only NAPQI and DNCB can covalently bind proteins.
Northern blotting has shown that mouse small intestine contains relatively large amounts of the nuclear factor-E2 p45-related factor (Nrf) 2 transcription factor but relatively little Nrf1. Regulation of intestinal antioxidant and detoxication enzymes by Nrf2 has been assessed using a mouse line bearing a targeted disruption of the gene encoding this factor. Both Nrf2-/- and Nrf2+/+ mice were fed a control diet or one supplemented with either synthetic cancer chemopreventive agents [butylated hydroxyanisole (BHA), ethoxyquin (EQ), or oltipraz] or phytochemicals [indole-3-carbinol, cafestol and kahweol palmitate, sulforaphane, coumarin (CMRN), or alpha-angelicalactone]. The constitutive level of NAD(P)H:quinone oxidoreductase (NQO) and glutathione S-transferase (GST) enzyme activities in cytosols from small intestine was typically found to be between 30% and 70% lower in samples prepared from Nrf2 mutant mice fed a control diet than in equivalent samples from Nrf2+/+ mice. Most of the chemopreventive agents included in this study induced NQO and GST enzyme activities in the small intestine of Nrf2+/+ mice. Increases of between 2.7- and 6.2-fold were observed in wild-type animals fed diets supplemented with BHA or EQ; increases of about 2-fold were observed with a mixture of cafestol and kahweol palmitate, CMRN, or alpha-angelicalactone; and increases of 1.5-fold were measured with sulforaphane. Immunoblotting confirmed that in the small intestine, the constitutive level of NQO1 is lower in the Nrf2-/- mouse, and it also showed that induction of the oxidoreductase was substantially diminished in the mutant mouse. Immunoblotting class-alpha and class-mu GST showed that constitutive expression of most transferase subunits is also reduced in the small intestine of Nrf2 mutant mice. Significantly, induction of class-alpha and class-mu GST by EQ, BHA, or CMRN is apparent in the gene knockout animal. No consistent change in the constitutive levels of the catalytic heavy subunit of gamma-glutamylcysteinyl synthetase (GCS(h)) was observed in the small intestine of Nrf2-/- mice. However, although the expression of GCS(h) was found to be increased dramatically in the small intestine of Nrf2+/+ mice by dietary BHA or EQ, this induction was essentially abolished in the knockout mice. It is apparent that Nrf2 influences both constitutive and inducible expression of intestinal antioxidant and detoxication proteins in a gene-specific fashion. Immunohistochemistry revealed that induction of NQO1, class-alpha GST, and GCS(h) occurs primarily in epithelial cells of the small intestine. This suggests that the variation in inducibility of NQO1, Gsta1/2, and GCS(h) in the mutant mouse is not attributable to the expression of the enzymes in distinct cell types but rather to differences in the dependency of these genes on Nrf2 for induction.
tert-Butyl-4-hydroxyanisole (BHA) and its demethylated analog, tert-butyl-hydroquinone (TBHQ), are antioxidants used in food. Both BHA and TBHQ have been shown to promote kidney and bladder carcinogenesis in the rat. We have previously demonstrated that glutathione (GSH) conjugates of a variety of hydroquinones are nephrotoxic and proposed that GSH conjugation serves to target these compounds to the kidney. In the present study, we examined the metabolism of TBHQ, focusing on the formation of potentially nephrotoxic sulfur-containing metabolites. 2-tert-Butyl-5-glutathion-S-ylhydroquinone, 2-tert-butyl-6-glutathion-S-ylhydroquinone, and 2-tert-butyl-3,6-bisglutathion-S-ylhydroquinone were identified as biliary metabolites of TBHQ (1.0 mmol/kg, ip) in male F344 rats, accounting for 2.2% of the dose. Liquid chromatography/mass spectroscopic analysis of urine also revealed the presence of additional sulfur-containing metabolites, tentatively identified as 2,5-dihydroxy-3-tert-butyl-thiophenol, 2,5-dihydroxy-4-tert-butylthiophenol, and their S-methyl derivatives. No mercapturic acids of TBHQ were found in the urine. The major biliary and urinary metabolites were TBHQ-glucuronide and TBHQ-sulfate, with a trace of TBHQ excreted unchanged. The results indicate that TBHQ undergoes oxidation and GSH conjugation in vivo in the male F344 rat. These conjugates are excreted into bile and undergo further metabolism prior to excretion in urine. Formation of the S-containing metabolites of TBHQ may occur in amounts sufficient to play a role in the toxicity of TBHQ to kidney and bladder.
Effects of the dietary antioxidants alpha-tocopherol (alpha-Toc), t-butylhydroquinone (TBHQ), propyl gallate (PG) and butylated hydroxytoluene (BHT) were examined using a multi-organ carcinogenesis model. Groups of 20 F344 male rats were treated with a single intragastric administration of 100 mg/kg body weight N-methyl-N'-nitro-N-nitrosoguanidine, a single intragastric administration of 750 mg/kg body weight N-ethyl-N-hydroxyethylnitrosamine, two subcutaneous injections of 0.5 mg/kg body weight N-methylbenzyl-nitrosamine and four subcutaneous injections of 40 mg/kg body weight 1,2-dimethylhydrazine. At the same time the rats were given 0.1% N-dibutylnitrosamine for 4 weeks and then 0.1% 2,2'-dihydroxy-di-n-propylnitrosamine for 2 weeks in the drinking water, for a total carcinogen exposure period of 6 weeks. Starting 3 days thereafter the rats received 1% alpha-Toc, 1% TBHQ, 1% PG or 0.7% BHT in the diet, or basal diet alone. Further groups of 10-15 animals each were treated with antioxidant alone or basal diet alone as controls. Surviving animals were killed at the end of week 36. Histopathological examination showed that alpha-Toc increased the incidence of glandular stomach atypical foci but reduced the incidence and multiplicity of kidney atypical tubules. TBHQ significantly elevated the incidences of esophageal papillary or nodular (PN) hyperplasias and papillomas, as well as forestomach papillomas, but significantly decreased the multiplicity of colon adenocarcinomas. PG was only effective in reducing the multiplicity of kidney atypical tubules. BHT enhanced the development of thyroid hyperplasias, but strongly reduced the incidence and multiplicity of colon adenocarcinomas. This compound was also associated with lowered incidence and multiplicity of renal cell tumors. None of the agents studied was unequivocal in exerting either positive or negative influence.
To determine whether the disruption of thyroid hormone and retinoid homeostasis that occurs after exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can be mediated by the arylhydrocarbon receptor (AhR), pregnant AhR-heterozygous (AhR+/-) mice were administered a single oral dose of 10 microg kg(-1) TCDD at gestation day 12.5. Serum and liver were collected on postnatal day 21 from vehicle-treated control or TCDD-treated AhR+/- and AhR-null (AhR-/-) mouse pups. Whereas TCDD exposure resulted in a marked reduction of total thyroxin (TT4) and free T4 (FT4) levels in the serum of AhR+/- mice, TCDD had no effects on AhR-/- mice. Gene expression of UDP-glucuronosyltransferase (UGT)1A6, cytochrome P450 (CYP)1A1, and CYP1A2 in the liver was induced markedly by TCDD in AhR+/- but not AhR-/- mice. Induction of CYP1A1 in response to TCDD was confirmed by immunohistochemical evidence in that CYP1A1 protein was conspicuously localized in the cytoplasm of hepatocytes in the centrilobular region. Levels of retinyl palmitate were greatly reduced in the liver of TCDD-exposed AhR+/- mice, but not in vehicle-treated AhR+/- mice. No effects of TCDD on retinoid levels in the liver were found in AhR-/- mice. We conclude that disruption of thyroid hormone and retinoid homeostasis is mediated entirely via AhR. Induction of UGT1A6 is thought to be responsible at least partly for reduced serum thyroid hormone levels in TCDD-exposed mice.
The aryl hydrocarbon receptor (AHR) plays a central role in 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) hepatotoxicity, regulation of xenobiotic metabolism, and hepatovascular development. Each of these processes appears to be dependent on binding of the AHR to dioxin- responsive elements (DREs) within the genome. The Cyp1a1 and Cyp1a2 loci represent linked genes thought to play important roles in AHR biology. In the mouse, 8 DREs are located in the 14-kb intergenic region between the Cyp1a1 and Cyp1a2 genes. Seven of these DREs, collectively known as the DRE cluster (DREC), are located 1.4 kb upstream of the Cyp1a1 transcriptional start site and 12.6 kb upstream of the Cyp1a2 start site. To investigate the role of the DREC in each aspect of AHR biology, we generated a DREC-deficient mouse model through homologous recombination. Using this mouse model, we demonstrate that the DREC controls the adaptive up-regulation of both Cyp1a1 and Cyp1a2 genes in vivo. Using selected aspects of acute hepatic injury as endpoints, we also demonstrate that DREC null mice are more sensitive to dioxin-induced hepatotoxicity than WT mice. The results of parallel toxicologic studies using individual Cyp1a1 and Cyp1a2 null mice support the observation that up-regulation of these P450s is not the cause of many aspects of dioxin hepatotoxicity. Finally, we observed normal closure of the ductus venosus (DV) in DREC null mice. Given the 100% penetrance of patent DV in Ahr null mice, these results indicate that Cyp1a1 and Cyp1a2 do not play a dominant role in AHR-mediated vascular development.
We report the first genome-wide association study of habitual caffeine intake. We included 47,341 individuals of European descent based on five population-based studies within the United States. In a meta-analysis adjusted for age, sex, smoking, and eigenvectors of population variation, two loci achieved genome-wide significance: 7p21 (P = 2.4×10(-19)), near AHR, and 15q24 (P = 5.2×10(-14)), between CYP1A1 and CYP1A2. Both the AHR and CYP1A2 genes are biologically plausible candidates as CYP1A2 metabolizes caffeine and AHR regulates CYP1A2.
Inflammatory signaling plays a key role in tumor progression, and the pleiotropic cytokine interleukin-6 (IL-6) is an important mediator of protumorigenic properties. Activation of the aryl hydrocarbon receptor (AHR) with exogenous ligands coupled with inflammatory signals can lead to synergistic induction of IL6 expression in tumor cells. Whether there are endogenous AHR ligands that can mediate IL6 production remains to be established. The indoleamine-2,3-dioxygenase pathway is a tryptophan oxidation pathway that is involved in controlling immune tolerance, which also aids in tumor escape. We screened the metabolites of this pathway for their ability to activate the AHR; results revealed that kynurenic acid (KA) is an efficient agonist for the human AHR. Structure-activity studies further indicate that the carboxylic acid group is required for significant agonist activity. KA is capable of inducing CYP1A1 messenger RNA levels in HepG2 cells and inducing CYP1A-mediated metabolism in primary human hepatocytes. In a human dioxin response element-driven stable reporter cell line, the EC(25) was observed to be 104nM, while in a mouse stable reporter cell line, the EC(25) was 10muM. AHR ligand competition binding assays revealed that KA is a ligand for the AHR. Treatment of MCF-7 cells with interleukin-1beta and a physiologically relevant concentration of KA (e.g., 100nM) leads to induction of IL6 expression that is largely dependent on AHR expression. Our findings have established that KA is a potent AHR endogenous ligand that can induce IL6 production and xenobiotic metabolism in cells at physiologically relevant concentrations.
Maltol is used extensively as a flavor-enhancing agent, food preservative, antioxidant, and also in cosmetic and pharmaceutical formulations. However, a number of studies have shown that maltol may induce carcinogenicity and toxicity but the mechanisms involved remain unknown. Therefore, we examined the ability of maltol to induce the cytochrome P450 1a1 (Cyp1a1), an enzyme known to play an important role in the chemical activation of xenobiotics to carcinogenic derivatives. Our results showed that treatment of Hepa 1c1c7 cells with maltol significantly induced Cyp1a1 at mRNA, protein, and activity levels in a concentration-dependent manner. The RNA synthesis inhibitor, actinomycin D, completely blocked the Cyp1a1 mRNA induction by maltol, indicating a requirement of de novo RNA synthesis through transcriptional activation. In addition, maltol induced aryl hydrocarbon receptor (AhR)-dependent luciferase reporter gene expression in stably transfected H1L1.1c2 cells, suggesting an AhR-dependent mechanism. This is the first demonstration that the food flavoring agent, maltol, can directly induce Cyp1a1 gene expression in an AhR-dependent manner and represents a novel mechanism by which maltol promotes carcinogenicity and toxicity.
BACKGROUND AND PURPOSE: There is a strong correlation between cytochrome P450 (P450)-dependent arachidonic acid metabolism and the pathogenesis of cardiac hypertrophy. Several aryl hydrocarbon receptor (AhR) ligands were found to alter P450-dependent arachidonic acid metabolism. Here, we have investigated the effect of 3-methylcholanthrene (3-MC) and benzo(a)pyrene (BaP), two AhR ligands, on the development of cardiac hypertrophy.
EXPERIMENTAL APPROACH: Male Sprague Dawley rats were injected (i.p.) daily with either 3-MC (10 mg kg(-1)) or BaP (20 mg kg(-1)) for 7 days. Then hearts were removed, and the heart to body weight ratio and the gene expression of the hypertrophic markers and P450 genes were determined. Levels of arachidonic acid metabolites were determined by liquid chromatography-electron spray ionization-mass spectrometry.
KEY RESULTS: Both 3-MC and BaP increased the heart to body weight ratio as well as the hypertrophic markers, atrial natriuretic peptide and brain natriuretic peptide. 3-MC and BaP treatment increased the gene expression of CYP1A1, CYP1B1, CYP2E1, CYP4F4, CYP4F5 and soluble epoxide hydrolase. Both 3-MC and BaP treatments increased the dihydroxyeicosatrienoic acids (DHETs) : epoxyeicosatrienoic acids (EETs) ratio and the 20-hydroxyeicosatetraenoic acid (20-HETE) : total EETs ratio. Treatment with benzo(e)pyrene, an isomer of BaP that is a poor ligand for the AhR, did not induce cardiac hypertrophy in rats, confirming the role of AhR in the development of cardiac hypertrophy. Treatment with the omega-hydroxylase inhibitor, HET0016, significantly reversed BaP-induced cardiac hypertrophy.
CONCLUSIONS AND IMPLICATIONS: 3-MC and BaP induce cardiac hypertrophy by increasing the ratio of DHETs : EETs and/or the ratio of 20-HETE : total EETs, through increasing soluble epoxide hydrolase activity.
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).
In addition, the aryl hydrocarbon receptor (AHR) antagonist, resveratrol, inhibited the increase in Cyp1a1 activity by tBHQ.
In contrast to the beneficial effects of tert-butylhydroquinone (tBHQ) as a food antioxidant, a number of studies have shown that chronic exposure to tBHQ may induce carcinogenicity. Therefore, we examined the ability of tBHQ to induce the cytochrome P450 1a1 (Cyp1a1), an enzyme known to play an important role in the chemical activation of xenobiotics to carcinogenic derivatives. A significant concentration-dependent increase in Cyp1a1 mRNA, protein, and activity occurred after treatment of murine hepatoma Hepa 1c1c7 cells with tBHQ. The increase in mRNA was apparent 3 h after treatment. The RNA polymerase inhibitor, actinomycin D, completely blocked the Cyp1a1 induction by tBHQ, indicating a requirement of de novo RNA synthesis through transcriptional activation. The protein synthesis inhibitor cycloheximide superinduced the tBHQ-mediated induction of Cyp1a1 mRNA and completely prevented the increase in Cyp1a1 activity, indicating that the induction of enzyme activity by tBHQ is dependent on de novo protein synthesis. In addition, the aryl hydrocarbon receptor (AHR) antagonist, resveratrol, inhibited the increase in Cyp1a1 activity by tBHQ. Gel electrophoretic mobility shift assays showed that tBHQ causes activation or transformation of the AHR in nuclear extracts, indicating that AHR-dependent mechanisms contributed to the Cyp1a1 induction. Similar to murine Hepa 1c1c7 cells, tBHQ caused a concentration-dependent increase in CYP1A1 at the mRNA and activity levels in human HepG2 cells. This is the first demonstration that the phenolic antioxidant, tBHQ, can directly induce Cyp1a1 gene expression in an AHR-dependent manner and may represent a novel mechanism by which tBHQ promotes carcinogenicity.
We recently demonstrated that heavy metals, Hg2+, Pb2+, and Cu2+ induced Cyp1a1 gene expression, yet the mechanisms involved remain unknown. To explore the molecular mechanisms involved in the modulation of Cyp1a1 by heavy metals, Hepa 1c1c7 cells were treated with the metals in the presence and absence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent Cyp1a1 inducer. Time-dependent effect study showed that all metals significantly induced the basal Cyp1a1 mRNA. This was apparent 3 h after treatment, and levels remained elevated for at least 24 h. At the inducible level, Hg2+ and Pb2+ further increased, while Cu2+ decreased, the TCDD-mediated induction of Cyp1a1 mRNA. The RNA synthesis inhibitor, actinomycin D, completely blocked the Cyp1a1 induction by heavy metals. The protein synthesis inhibitor, cycloheximide, and 26S proteasome inhibitor, carbobenzoxy-L-leucyl-L-leucyl-leucinal (MG-132), superinduced the metal-mediated induction of Cyp1a1 mRNA. In addition, all three metals induced aryl hydrocarbon receptor/xenobiotic-responsive element (AhR/XRE) binding, suggesting an AhR-dependent mechanism. Cyp1a1 mRNA and protein decay experiments showed that the three metals did not significantly affect the half-life of mRNA; however, they significantly decreased the degradation rate of its protein, implying a posttranslational regulation of the Cyp1a1 by the heavy metals. A significant decrease in TCDD-mediated induction of Cyp1a1 activity associated with an increase in HO-1 mRNA and a decrease in cellular heme content was observed after all metals treatment. This suggests that heme degradation plays a role in reducing Cyp1a1 activity. This is the first demonstration that heavy metals can directly induce Cyp1a1 gene expression in an AhR-dependent manner through transcriptional and posttranslational mechanisms.
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.
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.
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.
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.

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