Cannabinoid Receptor Agonist Anandamide Research Articles

Anandamide in the anterior hypothalamus diminishes defensive responses elicited in mice threatened by Epicrates cenchria constrictor serpents

The purpose of this study was to investigate whether the panicolytic‑like effect of different doses of anandamide microinjected into the anterior hypothalamus (AH) follows the same pattern of a bell‑shaped dose‑response curve observed with the same dose treatment in dorsomedial and ventromedial hypothalamus. We investigated this assumption by administering the cannabinoid and vanilloid receptor agonist anandamide into the anterior hypothalamus of mice and exposing them to the real threatening situation by using our experimental model based on confrontations between rodents and wild snakes. Our findings showed a gradual decay of response, with a significant attenuation of the panic attack‑like responses with anandamide at the highest dose but no effect was found after anandamide at the lowest or intermediate doses. An immunohistochemical procedure showed a lower degree of TRPV1 receptor and moderate to higher degree of Cb1 receptors in anterior hypothalamus. In conclusion, the pattern of dose‑response curve of anandamide microinjected in the AH does not seem to be the same classical pattern compared with other hypothalamic nuclei.

Comparing the effects of endogenous and synthetic cannabinoid receptor agonists on survival of gastric cancer cells

AimsAnti-neoplastic activity induced by cannabinoids has been extensively documented for a number of cancer cell types; however, this topic has been explored in gastric cancer cells only in a limited number of approaches. Thus, the need of integrative and comparative studies still persists. Materials and methodsIn this study we tested and compared the effects of three different cannabinoid receptor agonists-anandamide (AEA), (R)-(+)-methanandamide (Meth-AEA) and CP 55,940 (CP)- on gastric cancer cell morphology, viability and death events in order to provide new insights to the use of these agents for therapeutic purposes. Key findingsThe three agents tested exhibited similar concentration-dependent effects in the induction of changes in cell morphology and cell loss, as well as in the decrease of cell viability and DNA laddering in the human gastric adenocarcinoma cell line (AGS). Differences among the cannabinoids tested were mostly observed in the density of cells found in early and late apoptosis and necrosis, favoring AEA and CP as the more effective inducers of apoptotic mechanisms, and Meth-AEA as a more effective inducer of necrosis through transient and rapid apoptosis. SignificanceThrough a comparative approach, our results support and confirm the therapeutic potential that cannabinoid receptor agonists exert in gastric cancer cells and open possibilities to use cannabinoids as part of a new gastric cancer therapy.

The effects of endocannabinoid receptor agonist anandamide and antagonist rimonabant on opioid analgesia and tolerance in rats.

The role of the cannabinoid (CB) system in the tolerance to analgesic effect of opioid remains obscure. The aim of the present study was to evaluate the effects of the endocannabinoid nonselective receptor agonist anandamide (AEA) and CB1 receptor antagonist rimonabant (SR141716) on morphine analgesia and tolerance in rats. Male Wistar albino rats weighing 215-230 g were used in these experiments. To constitute morphine analgesic tolerance, a 3-day cumulative dosing regimen was used. The analgesic effects of AEA (10 mg/kg), SR141716 (10 mg/kg), and morphine (5 mg/kg) were considered at 30-min intervals by tail flick (TF) and hot plate (HP) analgesia tests. The analgesic effects of the drugs were measured as TF and HP latencies in all groups for each rat and converted to %MPE. The data were analysed by analysis of variance followed by Tukey test. The findings suggested that AEA in combination with morphine produced a significant increase in expression of analgesic tolerance to morphine. Conversely, cannabinoid receptor antagonist SR141716 attenuated morphine analgesic tolerance. In addition, administration of AEA with morphine increased morphine analgesia. In conclusion, we observed that the cannabinoid receptor agonist anandamide and CB1 receptor antagonist SR141716 plays a significant role in the opioid analgesia and tolerance.

PP-122 RED CELL DISTRIBUTION WIDTH HAS A PREDICTABLE VALUE FOR DIFFERENTIATION OF PROVOKED AND UNPROVOKED VENOUS THROMBOEMBOLISM

Corporal smooth muscle relaxation is mediated mainly but not completely by nitric oxide. Endocannabinoids modulate the various neurotransmitter systems.In the present study, a possible role of endocannabinoids on non-nitrergic nonadrenergic noncholinergic (NANC)-mediated relaxations was investigated.In precontracted tissues, control electrical field stimulation (EFS)-induced NANC relaxation responses were obtained using varying frequencies of stimulation in the presence of L-arginine methyl ester (L-NAME), guanethidine, and atropine. To investigate the effects of cannabinoids on EFS-evoked non-nitrergic NANC relaxation responses, a similar experimental procedure was applied in the presence of cannabinoid receptor antagonists AM251 or AM630; vanilloid receptor antagonist capsazepine; or cannabinoid receptor agonists anandamide, arachidonyl-2-chloroethylamide (ACEA), or JHW015.Effects of cannabinoid receptor antagonists and agonists on EFS-evoked non-nitrergic NANC relaxation responses.L-NAME abolished EFS-induced relaxation responses at lower frequencies (2–4 Hz) and inhibited the relaxation responses at higher frequencies (8–32 Hz). AM251 and AM630 either together or separately inhibited, whereas anandamide, ACEA, and JHW015 enhanced non-nitrergic NANC relaxation responses. Anandamide did not alter EFS-induced non-nitrergic NANC relaxations in the presence of AM251 and AM630. Capsazepine enhanced non-nitrergic NANC relaxation responses.These results suggest that non-nitrergic NANC relaxations may be mediated partially by cannabinoid-like neuronal factors acting at both cannabinoid CB1 and cannabinoid CB2 receptors. Vural IM, Ozturk GS, and Sarioglu Y. Functional characterization of nonadrenergic noncholinergic neurotransmitter release via endocannabinoids: An in vitro study in rabbit corpus cavernosum. J Sex Med 2009;6:717–729.

Role of Pre‐Junctional CB1, But not CB2, TRPV1 or GPR55 Receptors in Anandamide‐Induced Inhibition of the Vasodepressor Sensory CGRPergic Outflow in Pithed Rats

Stimulation of the perivascular sensory outflow in pithed rats produces vasodepressor responses mediated by CGRP release. Interestingly, endocannabinoids such as anandamide (which interacts with CB1 , CB2 , TRPV1 and GPR55 receptors) can regulate the activity of perivascular sensory nerves in dural blood vessels by modulating CGRP release. Yet, as no publication has reported whether this mechanism is operative in the healthy systemic vasculature, this study has specifically analysed the receptors mediating the potential inhibitory effects of the cannabinoid (CB) receptor agonists anandamide (non-selective), JWH-015 (CB2 ) and lysophosphatidylinositol (GPR55) on the rat vasodepressor sensory CGRPergic outflow (an index of systemic vasodilatation). Healthy pithed rats were pre-treated with consecutive i.v. continuous infusions of hexamethonium, methoxamine and the above agonists. Electrical spinal (T9 -T12 ) stimulation of the vasodepressor sensory CGRPergic outflow or i.v. injections of α-CGRP produced frequency-dependent or dose-dependent vasodepressor responses. The infusions of anandamide in a dose-dependent manner inhibited the vasodepressor responses by electrical stimulation (remaining unaffected by JWH-015 or lysophosphatidylinositol), but not those by α-CGRP. After i.v. administration of antagonists, the inhibition by 3.1 μg/kg min anandamide was: (i) potently blocked by 31-100 μg/kg NIDA41020 (CB1 ), (ii) unaffected by 180 μg/kg AM630 (CB2 ), 31 μg/kg cannabidiol (GPR55) or 31-100 μg/kg capsazepine (TRPV1) and (iii) slightly blocked by 310 μg/kg AM630. The above doses of antagonists were enough to block their respective receptors. These results suggest that anandamide-induced inhibition of the vasodepressor sensory CGRPergic outflow is mainly mediated by pre-junctional activation of CB1 receptors, with no pharmacological evidence for the role of CB2 , TRPV1 or GPR55 receptors.

CB1 and CB2 Cannabinoid Receptor Agonists Induce Peripheral Antinociception by Activation of the Endogenous Noradrenergic System

Cannabinoid agonists induce norepinephrine release in central, spinal, and peripheral sites. Previous studies suggest an interaction between the cannabinoid and adrenergic systems on antinociception. In this study, we sought to verify whether the CB1 and CB2 cannabinoid receptor agonists anandamide and N-palmitoyl-ethanolamine (PEA), respectively, are able to induce peripheral antinociception via an adrenergic mechanism. All drugs were administered locally into the right hindpaw of male Wistar rats. The rat paw pressure test was used, with hyperalgesia induced by intraplantar injection of prostaglandin E2 (2 μg). Anandamide, 12.5 ng/paw, 25 ng/paw, and 50 ng/paw elicited a local peripheral antinociceptive effect that was antagonized by CB1 cannabinoid receptor antagonist AM251, 20 µg/paw, 40 µg/paw, and 80 µg/paw, but not by CB2 cannabinoid receptor antagonist AM630, 100 µg/paw. PEA, 5 µg/paw, 10 µg/paw, and 20 µg/paw, elicited a local peripheral antinociceptive effect that was antagonized by AM630, 25 µg/paw, 50 µg/paw, and 100 µg/paw, but not by AM251, 80 µg/paw. Antinociception induced by anandamide or PEA was antagonized by the nonselective α2 adrenoceptor antagonist yohimbine, 05 µg/paw, 10 µg/paw, and 20 µg/paw, and by the selective α2C adrenoceptor antagonist rauwolscine, 10 µg/paw, 15 µg/paw, and 20 µg/paw, but not by the selective antagonists for α2A, α2B, and α2D adrenoceptor subtypes, 20 μg/paw. The antinociceptive effect of the cannabinoids was also antagonized by the nonselective α1 adrenoceptor antagonist prazosin, 0.5 µg/paw, 1 µg/paw, and 2 µg/paw, and by the nonselective β adrenoceptor antagonist propranolol, 150 ng/paw, 300 ng/paw, and 600 ng/paw. Guanethidine, which depletes peripheral sympathomimetic amines (30 mg/kg/animal, once a day for 3 days), restored approximately 70% the anandamide-induced and PEA-induced peripheral antinociception. Furthermore, acute injection of the norepinephrine reuptake inhibitor reboxetine, 30 µg/paw, intensified the antinociceptive effects of low-dose anandamide, 12.5 ng/paw, and PEA, 5 µg/paw. This study provides evidence that anandamide and PEA induce peripheral antinociception activating CB1 and CB2 cannabinoid receptors, respectively, stimulating an endogenous norepinephrine release that activates peripheral adrenoceptors inducing antinociception.

Rapid glucocorticoid-induced activation of TRP and CB1 receptors causes biphasic modulation of glutamate release in gastric-related hypothalamic preautonomic neurons

Glucocorticoids rapidly regulate synaptic input to neuroendocrine cells in the hypothalamic paraventricular nucleus (PVN) by inducing the retrograde release of endogenous messengers. Here we investigated the rapid effects of dexamethasone (DEX) on excitatory synaptic input to feeding-related, preautonomic PVN neurons using whole-cell patch-clamp recordings. In ∼50% of identified gastric-related preautonomic PVN neurons, DEX elicited a biphasic synaptic response characterized by an initial rapid and transient increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs), followed by a decrease in mEPSC frequency within 9 min; remaining cells displayed only a decrease in mEPSC frequency. The late-phase decrease in mEPSC frequency was mimicked by the cannabinoid receptor agonists anandamide (AEA) and WIN 55,212-2, and it was blocked by the CB1 receptor antagonist AM251. The biphasic DEX effect was mimicked by AEA. The early increase in mEPSCs was mimicked by activation of transient receptor potential vanilloid type 1 (TRPV1) receptors with capsaicin and by activation of TRPV4 receptors with 4-α-PDD. The increase was reduced, but not blocked, by selective TRPV1 antagonists and in TRPV1 knockout mice; it was blocked completely by the broad-spectrum TRPV antagonist ruthenium red and by combined application of selective TRPV1 and TRPV4 antagonists. The DEX effects were prevented entirely by intracellular infusion of the G-protein inhibitor, GDPβS. Thus, DEX biphasically modulates synaptic glutamate onto a subset of gastric-related PVN neurons, which is likely mediated by induction of a retrograde messenger. The effect includes a TRPV1/4 receptor-mediated transient increase and subsequent CB1 receptor-mediated suppression of glutamate release. Multiphasic modulation of glutamate input to PVN neurons represents a previously unappreciated complexity of control of autonomic output by glucocorticoids and endogenous cannabinoids.

Endogenous cannabinoid receptor agonist anandamide induces peripheral antinociception by activation of ATP-sensitive K + channels

Aims The effects of several potassium (K +) channel blockers were studied to determine which K + channels are involved in peripheral antinociception induced by the cannabinoid receptor agonist, anandamide. Main methods Hyperalgesia was induced by subcutaneous injection of 250 μg carrageenan into the plantar surface of the hind paw of rats. The extent of hyperalgesia was measured using a paw pressure test 3 h following carrageenan injection. The weight in grams (g) that elicited a nociceptive response, paw flexion, during the paw pressure test was used as the nociceptive response threshold. Key findings Doses of 50, 75, and 100 ng of anandamide elicited a dose-dependent antinociceptive effect. Following a 100 ng dose of anandamide no antinociception was observed in the paw that was contralateral to the anandamide injection site, which shows that anandamide has a peripheral site of action. Pretreatment with 20, 40 and 80 μg AM251, a CB 1 receptor antagonist, caused a dose-dependent decrease in anandamide-induced antinociception, suggesting that the CB 1 receptor is directly involved in anandamide effect. Treatment with 40, 80 and 160 μg glibenclamide, an ATP-sensitive K + channel blocker, caused a dose-dependent reversal of anandamide-induced peripheral antinociception. Treatment with other K + channel antagonists, tetraethylammonium (30 μg), paxilline (10 μg) and dequalinium (50 μg), had no effect on the induction of peripheral antinociception by anandamide. Significance This study provides evidence that the peripheral antinociceptive effect of the cannabinoid receptor agonist, anandamide, is primarily caused by activation of ATP-sensitive K + channels and does not involve other potassium channels.

Opioid receptor and NO/cGMP pathway as a mechanism of peripheral antinociceptive action of the cannabinoid receptor agonist anandamide

Aims In this study, we investigated whether the opioid system and the nitric oxide pathway were involved in the peripheral antinociception induced by a cannabinoid receptor agonist anandamide. Main methods Hyperalgesia was induced by a subcutaneous injection of carrageenan (250 μg) into the plantar surface of the rat's hindpaw and measured by the paw pressure test 3 h after injection. The weight in grams (g) required to elicit a nociceptive response, paw flexion, was determined as the nociceptive threshold. Key findings Anandamide elicited a dose-dependent (50, 75, and 100 ng per paw) antinociceptive effect. The highest dose of anandamide did not produce antihyperalgesia in the contralateral paw, indicating a peripheral site of action. The CB 1 receptor antagonist AM251 (20, 40, 80 and 160 μg per paw) antagonized peripheral antihyperalgesia induced by anandamide (100 ng), in a dose-dependent manner, suggesting CB 1 receptor activation. Anandamide-induced peripheral antihyperalgesia was reverted by blockers of the l-arginine/NO/cGMP pathway N G-nitro- l-arginine (NOARG; 24, 36 and 48 μg per paw) and 1H-[1,2,4] Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 25, 50 and 100 μg per paw), in a dose-dependent manner. Furthermore, opioid receptor antagonist naloxone (12.5, 25 and 50 μg per paw) antagonized the peripheral antihyperalgesia induced by anandamide. Significance This study provides evidence that the peripheral antinociceptive effect of the cannabinoid receptor agonist anandamide may result from l-arginine/NO/cGMP pathway activation and that the opioid system is also involved.

Functional Characterization of Nonadrenergic Noncholinergic Neurotransmitter Release via Endocannabinoids: An in Vitro Study in Rabbit Corpus Cavernosum

Corporal smooth muscle relaxation is mediated mainly but not completely by nitric oxide. Endocannabinoids modulate the various neurotransmitter systems. In the present study, a possible role of endocannabinoids on non-nitrergic nonadrenergic noncholinergic (NANC)-mediated relaxations was investigated. In precontracted tissues, control electrical field stimulation (EFS)-induced NANC relaxation responses were obtained using varying frequencies of stimulation in the presence of L-arginine methyl ester (L-NAME), guanethidine, and atropine. To investigate the effects of cannabinoids on EFS-evoked non-nitrergic NANC relaxation responses, a similar experimental procedure was applied in the presence of cannabinoid receptor antagonists AM251 or AM630; vanilloid receptor antagonist capsazepine; or cannabinoid receptor agonists anandamide, arachidonyl-2-chloroethylamide (ACEA), or JHW015. Effects of cannabinoid receptor antagonists and agonists on EFS-evoked non-nitrergic NANC relaxation responses. L-NAME abolished EFS-induced relaxation responses at lower frequencies (2-4 Hz) and inhibited the relaxation responses at higher frequencies (8-32 Hz). AM251 and AM630 either together or separately inhibited, whereas anandamide, ACEA, and JHW015 enhanced non-nitrergic NANC relaxation responses. Anandamide did not alter EFS-induced non-nitrergic NANC relaxations in the presence of AM251 and AM630. Capsazepine enhanced non-nitrergic NANC relaxation responses. These results suggest that non-nitrergic NANC relaxations may be mediated partially by cannabinoid-like neuronal factors acting at both cannabinoid CB(1) and cannabinoid CB(2) receptors.

CB1Receptors Mediate the Analgesic Effects of Cannabinoids on Colorectal Distension-Induced Visceral Pain in Rodents

Activation of cannabinoid receptors (CB(1), CB(2) and GPR(55)) produces analgesic effects in several experimental pain models, including visceral pain arising from the gastrointestinal tract. We assessed the role of CB(1), CB(2), and GPR(55) receptors and the endogenous cannabinoid system on basal pain responses and acute mechanical hyperalgesia during colorectal distension (CRD) in rodents. The effects of cannabinoid receptor agonists and antagonists on pain-related responses to CRD were assessed in rats and in wild-type and CB(1) receptor knock-out mice. The dual CB(1/2) agonist, WIN55,212-2, and the peripherally acting CB(1)-selective agonist, SAB-378, inhibited pain-related responses to repetitive noxious CRD (80 mmHg) in a dose-related manner in rats. The analgesic effects of WIN55,212-2 and SAB-378 were blocked by the selective CB(1) antagonist SR141716, but were not affected by the selective CB(2) antagonist SR144528. SR141716, per se, increased the responses to repetitive noxious CRD, indicative of hyperalgesia, and induced pain-related responses during non-noxious CRD (20 mmHg), indicative of allodynia. The cannabinoid receptor agonists anandamide, virodhamine and O-1602 had no effect. At analgesic doses, WIN55,212-2 did not affect colonic compliance. In accordance to the rat data, WIN55,212-2 produced analgesia, whereas SR141716 induced hyperalgesia, during noxious CRD (55 mmHg) in wild-type but not in CB(1)-knock-out mice. These data indicate that peripheral CB(1) receptors mediate the analgesic effects of cannabinoids on visceral pain from the gastrointestinal tract. The allodynic and hyperalgesic responses induced by SR141716 suggest the existence of an endogenous cannabinoid tone and the activation of CB(1) receptors during noxious CRD.

Activation of CB1 cannabinoid receptors in the dorsolateral periaqueductal gray reduces the expression of contextual fear conditioning in rats

Conditioned fear to context causes freezing and cardiovascular changes in rodents and has been used to measure anxiety. It also activates the dorsolateral column of the periaqueductal gray (dlPAG). Microinjections of cannabinoid agonists into the dlPAG produced anxiolytic-like effects in the elevated plus maze, but the effects of these treatments on fear conditioning remains unknown. The objective of this study was to verify if intra-dlPAG injection of the CB1 cannabinoid receptor agonist anandamide (AEA) or the anandamide transport inhibitor AM404 would attenuate behavioral (freezing) and cardiovascular (increase of arterial pressure and heart rate) responses of rats submitted to a contextual fear-conditioning paradigm. Male Wistar rats with cannulae aimed at the dlPAG were re-exposed to a chamber where they had received footshocks 48 h before. Fifteen minutes before the test, the animals received a first intra-dlPAG injection of vehicle or AM251, a CB1 receptor antagonist (100 pmol/200 nl), followed 5 min later by vehicle, AEA (5 pmol/200 nl) or AM404 (50 pmol/200 nl). Freezing and cardiovascular responses were recorded for 10 min. Freezing and cardiovascular responses were reduced by administration of either AEA or AM404 into the dlPAG before re-exposition to the aversively conditioned context. These effects were abolished when the animals were locally pretreated with AM251. The latter drug, even at a higher dose (300 pmol), was ineffective when administered alone into the dlPAG. The results suggest that facilitation of endocannabinoid-mediated neurotransmission in the dlPAG, through activation of local CB1 receptors, attenuates the expression of contextual fear responses.

Synergistic antinociception by the cannabinoid receptor agonist anandamide and the PPAR-α receptor agonist GW7647

The analgesic properties of cannabinoid receptor agonists are well characterized. However, numerous side effects limit the therapeutic potential of these agents. Here we report a synergistic antinociceptive interaction between the endogenous cannabinoid receptor agonist anandamide and the synthetic peroxisome proliferator-activated receptor-α (PPAR-α) agonist 2-(4-(2-(1-Cyclohexanebutyl)-3-cyclohexylureido)ethyl)phenylthio)-2-methylpropionic acid (GW7647) in a model of acute chemical-induced pain. Moreover, we show that anandamide synergistically interacts with the large-conductance potassium channel (K Ca1.1, BK) activator isopimaric acid. These findings reveal a synergistic interaction between the endocannabinoid and PPAR-α systems that might be exploited clinically and identify a new pharmacological effect of the BK channel activator isopimaric acid.

Anandamide Inhibits Endothelin-1 Production by Human Cultured Endothelial Cells: A New Vascular Action of This Endocannabinoid

The endogenous cannabinoid receptor agonist anandamide (AEA) exerts vascular effects such as vasodilatation and hypotension. In this study, we determined the effect of AEA on endothelin-1 production by cultured human umbilical vein endothelial cells. Anandamide (≧5 µmol/l) significantly decreased endothelin-1 production in a dose-dependent manner, a response not affected by the specific CB₁ receptor antagonist/inverse agonist SR-141716A. Adenosine, via activation of adenosine receptors (also targets for SR-141716A), was not involved in these effects. Conversely, AEA increased nitric oxide (NO) production, an effect inhibited by SR-141716A, indicating the involvement of CB₁ receptors. Therefore, we hypothesize that AEA effects on endothelial cells may lead to vasodilatation through independent concerted mechanisms, involving a non-CB₁ receptor-dependent inhibition of endothelin-1 production and a CB₁-mediated increase of NO.

Role of cannabinoid receptor agonists in mechanisms of suppression of central pain syndrome

We studied the effect of cannabinoid receptor agonists anandamide and WIN 55,212-2 on the central pain syndrome induced by intraspinal injection of penicillin sodium salt in rats. Cannabinoids suppressed allodynia and spontaneous attacks in rats with the central pain syndrome. The analgesic effect was most pronounced after intrathecal injection of cannabinoid receptor agonist in a dose of 100 microg in 10 microl. After systemic treatment the analgesic effect was produced by only WIN 55,212-2 in a dose of 1 mg/kg. WIN 55,212-2 was superior to anandamide by the duration and intensity of the effect on allodynia and spontaneous attacks.

Additive Effects of Endogenous Cannabinoid Anandamide and Ethanol on α7-Nicotinic Acetylcholine Receptor-Mediated Responses in Xenopus Oocytes

The interaction between the effects of the endogenous cannabinoid receptor agonist anandamide and ethanol on the function of homomeric alpha(7)-nicotinic acetylcholine (nACh) receptors expressed in Xenopus oocytes were investigated using the two-electrode voltage-clamp technique. Anandamide and ethanol reversibly inhibited currents evoked with 100 microM acetylcholine in a concentration-dependent manner. Coapplication of anandamide and ethanol caused a significantly greater inhibition of alpha(7)-nACh receptor function than anandamide or ethanol alone. The IC(50) value of 238 +/- 34 nM for anandamide inhibition decreased significantly to 104 +/- 23 nM in the presence of 30 mM ethanol. The inhibition of alpha(7)-mediated currents by coapplication of anandamide and ethanol was not altered by phenylmethylsulfonyl fluoride, an inhibitor of anandamide hydrolyzing enzyme, or N-(4-hydroxyphenyl)-arachidonylamide, an anandamide transport inhibitor. Analysis of oocytes by matrix-assisted laser desorption/ionization technique indicated that ethanol treatment did not alter the lipid profile of oocytes, and there is negligible, if any, anandamide present in these cells. Results of studies with chimeric alpha(7)-nACh-5-HT(3) receptors comprised of the amino-terminal domain of the alpha(7)-nACh receptor and the transmembrane and carboxyl-terminal domains of 5-HT(3) receptors suggest that although ethanol inhibition of the alpha(7)-nACh receptor is likely to involve the N-terminal region of the receptor, the site of action for anandamide is located in the transmembrane and carboxyl-terminal domains of the receptors. These data indicate that endocannabinoids and ethanol potentiate each other's inhibitory effects on alpha(7)-nACh receptor function through distinct regions of the receptor.

Inhibition of methamphetamine self-administration in rats by cannabinoid receptor antagonist AM 251.

Cannabinoids are drugs that are frequently abused not only alone, but also in combination with other drugs. The present study investigated possible functional interactions between the psychostimulant methamphetamine and the cannabinoid receptor agonists anandamide, or R-(+)-methanandamide and cannabinoid antagonist AM 251 in the rat model of i.v. drug self-administration. In rats trained to self-administer methamphetamine, the intake was significantly decreased by the cannabinoid antagonist and tended to be dose-dependently increased by pre-treatment with cannabinoid receptor agonists. Possible mechanisms for these drug interactions are discussed and the use of the cannabinoid antagonist for the treatment of drug abuse is considered.

Effect of cannabinoids on neural transmission in rat gastric fundus.

The purpose of this study was to examine the possible role of cannabinoids on the neuromuscular function of rat gastric fundus. In addition to possible direct effects on smooth muscle, the influence of cannabinoids on contractile (cholinergic) and relaxant (non-adrenergic, non-cholinergic (NANC)) neural innervation of the rat gastric fundus was investigated in vitro. Neither anandamide (an endogenous cannabinoid receptor agonist) nor Win 55,212-2 and methanandamide (synthetic cannabinoid receptor agonists) nor AM 630 (a cannabinoid receptor antagonist) showed any effect on smooth muscle activity at baseline or after precontraction with 5-hydroxytryptamine (5-HT; 10(-7) M). Electrical field stimulation (EFS) of the smooth muscle preparation (40 V; 5 Hz) caused cholinergically mediated twitch contractions that were abolished by atropine (10(-6) M) or tetrodotoxin (TTX; 10(-6) M). Anandamide and Win 55,212-2 reduced these twitch contractions in a concentration-dependent manner, an effect that could be reversed by the cannabinoid receptor antagonist AM 630 for anandamide, but not for Win 55,212-2. When NANC relaxant neural responses (presence of atropine (10(-6) M) and guanethidine (10(-6) M)) were induced by EFS, the cannabinoid receptor agonists anandamide and Win 55,212-2 reduced the relaxant response, an effect that could be reversed by the cannabinoid receptor antagonist AM 630 for anandamide, but not for Win 55,212-2. When given alone AM 630 caused an increase in the EFS-induced relaxant response. The presence of CB1 and CB2 cannabinoid receptor mRNA within the rat stomach was demonstrated by reverse transcription polymerase chain reaction (RT-PCR). The results of this study indicate that cannabinoids modulate excitatory cholinergic and inhibitory NANC neurotransmission in the rat gastric fundus. Endogenous cannabinoids may play a physiological role only in NANC inhibitory transmission, as AM 630 did not modify the electrically induced cholinergic contraction. The involved cannabinoid receptors are most likely located on neuronal structures. The present study also provides evidence that more than one receptor type is involved.

Anandamide and methanandamide induce both vanilloid VR1- and cannabinoid CB1 receptor-mediated changes in heart rate and blood pressure in anaesthetized rats.

In anaesthetized rats activation of vanilloid receptors on sensory vagal nerves elicits rapid bradycardia and hypotension (Bezold-Jarisch reflex). Recent in vitro experiments revealed that the endogenous cannabinoid ligand anandamide acts as an agonist at the vanilloid VRI receptors. The present study was aimed at examining whether vanilloid VR1 receptors are involved in the cardiovascular effects of anandamide in the anaesthetized rat. Intravenous injection of anandamide, its stable analogue methanandamide and the vanilloid receptor agonist capsaicin produced a dose-dependent immediate and short-lasting decrease in heart rate and blood pressure with the following rank order of potencies: capsaicin > methanandamide > anandamide. This bradycardia was dose-dependently diminished by the selective vanilloid receptor antagonist capsazepine (0.3-3 micromol/kg) and the nonselective inhibitor of these receptors, ruthenium red (1-10 micromol/kg). Both antagonists reduced or tended to reduce the hypotension stimulated by the agonists. Following this bradycardia and hypotension (presumably evoked by the Bezold-Jarisch reflex; phase I), capsaicin, anandamide and methanandamide led to a brief vasopressor effect (phase II). Subsequently both anandamides, but not capsaicin, induced a more prolonged decrease in blood pressure (phase III). Capsazepine and ruthenium red (at doses up to 3 tmol/kg and 10 micromol/kg, respectively) failed to affect these changes in blood pressure. The cannabinoid CB1 receptor antagonist SR 141716 at 3 micromol/kg abolished the prolonged decrease in blood pressure (phase III) induced by anandamide and methanandamide, but had no effect on the reflex bradycardia and hypotension (phase I) and on the subsequent vasopressor effect (phase II) evoked by capsaicin, anandamide and methanandamide. In conclusion, the endogenous cannabinoid receptor agonist anandamide and its stable analogue methanandamide induce reflex bradycardia and hypotension (phase I) by activating the vanilloid VRI receptor. Whereas the mechanism underlying the brief vasopressor effect (phase II) is unknown, the prolonged hypotension (phase III) results from stimulation of the cannabinoid CB1 receptor.

Anandamide Excites Central Terminals of Dorsal Root Ganglion Neurons via Vanilloid Receptor-1 Activation

Recently, the cannabinoid (CB) receptor agonist anandamide (AEA) has been shown to excite perivascular terminals of primary sensory neurons via activation of the vanilloid receptor-1 (VR-1). To determine whether AEA stimulates central terminals of these neurons, via VR-1 activation, we studied the release of calcitonin gene-related peptide (CGRP)- and substance P (SP)-like immunoreactivities (LI) from slices of rat dorsal spinal cord. Mobilization of Ca(2+) in rat dorsal root ganglion (DRG) neurons in culture was also studied. AEA (0.1-10 micrometer) increased the outflow of CGRP-LI and SP-LI from slices of the rat dorsal spinal cord in a Ca(2+)-dependent manner and increased [Ca(2+)](i) in capsaicin-sensitive cultured DRG neurons. Both effects of AEA were abolished by capsaicin pretreatment and by the VR-1 antagonist capsazepine but not affected by the CB receptor antagonists AM281 or AM630. Both neuropeptide release and Ca(2+) mobilization induced by electrical field stimulation (EFS) were inhibited by a low concentration of AEA (10 nm). Inhibition by AEA of EFS-induced responses was reversed by AM281 and AM630, but was not affected by capsazepine. Results indicate that stimulation of VR-1 with high concentrations of AEA excites central terminals of capsaicin-sensitive DRG neurons, thus causing neuropeptide release in the dorsal spinal cord. This novel activity opposes the CB receptor-mediated inhibitory action of low concentrations AEA. However, only if large amounts of endogenous AEA could be produced at the level of the dorsal spinal cord, they may not inhibit, but rather activate, nociceptive sensory neurons.