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RationaleAdolescent exposure to ∆9-tetrahydrocannabinol (THC), the psychotropic constituent of cannabis, might affect brain development, and in rodent models leads to long-term behavioral and physiological alterations. Yet, the basic pharmacology of this drug in adolescent rodents, especially when ingested via ecologically relevant routes like aerosol inhalation, commonly referred to as "vaping," is still poorly characterized. Moreover, sex differences exist in THC metabolism, kinetics, and behavioral effects, but these have not been rigorously examined after vapor dosing in adolescents.ObjectivesWe investigated the pharmacokinetics and pharmacodynamics of aerosolized THC (30 min inhalation exposure, 25 or 100 mg/ml) in adolescent Wistar rats of both sexes.MethodsLiquid chromatography/mass spectrometry analysis of THC and its major metabolites was conducted on blood plasma and brain tissue at 5, 30, 60, and 120 min following a 30-min aerosol dosing session. Effects on activity in a novel environment for 120 min after aerosol, and temperature, were measured in separate rats.ResultsWe found sex-dependent differences in the pharmacokinetics of THC and its active (11-OH-THC) and inactive (11-COOH-THC) metabolites in the blood and brain, along with dose- and sex-dependent effects on anxiety-like and exploratory behaviors; namely, greater 11-OH-THC levels accompanied by greater behavioral effects in females at the low dose but similar hypothermic effects in both sexes at the high dose.ConclusionsThese results provide a benchmark for dosing adolescent rats with aerosolized (or "vaped") THC, which could facilitate adoption by other labs of this potentially human-relevant THC exposure model to understand cannabis effects on the developing brain.

RationaleAdolescence is characterized by endocannabinoid (ECB)-dependent refinement of neural circuits underlying emotion, learning, and motivation. As a result, adolescent cannabinoid receptor stimulation (ACRS) with phytocannabinoids or synthetic agonists like "Spice" cause robust and persistent changes in both behavior and circuit architecture in rodents, including in reward-related regions like medial prefrontal cortex and nucleus accumbens (NAc).Objectives and methodsHere, we examine persistent effects of ACRS with the cannabinoid receptor 1/2 specific agonist WIN55-212,2 (WIN; 1.2 mg/kg/day, postnatal day (PD) 30-43), on natural reward-seeking behaviors and ECB system function in adult male Long Evans rats (PD 60+).ResultsWIN ACRS increased palatable food intake, and altered attribution of incentive salience to food cues in a sign-/goal-tracking paradigm. ACRS also blunted hunger-induced sucrose intake, and resulted in increased anandamide and oleoylethanolamide levels in NAc after acute food restriction not seen in controls. ACRS did not affect food neophobia or locomotor response to a novel environment, but did increase preference for exploring a novel environment.ConclusionsThese results demonstrate that ACRS causes long-term increases in natural reward-seeking behaviors and ECB system function that persist into adulthood, potentially increasing liability to excessive natural reward seeking later in life.

The endogenous lipid amides, palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), exert marked antinociceptive and anti-inflammatory effects in animal models by engaging nuclear peroxisome proliferator-activated receptor-α. PEA and OEA are produced by macrophages and other host-defense cells and are deactivated by the cysteine amidase, N-acylethanolamine acid amidase (NAAA), which is highly expressed in macrophages and B-lymphocytes. In the present study, we examined whether a) NAAA might be involved in the inflammatory reaction triggered by injection of complete Freund's adjuvant (CFA) into the rat paw and b) administration of 4-cyclohexylbutyl-N-[(S)-2-oxoazetidin-3-yl]-carbamate (ARN726), a novel systemically active NAAA inhibitor, attenuates such reaction. Injection of CFA into the paw produced local edema and heat hyperalgesia, which were accompanied by decreased PEA and OEA content (assessed by liquid chromatography/mass spectrometry) and increased NAAA levels (assessed by Western blot and ex vivo enzyme activity measurements) in paw tissue. Administration of undec-10-ynyl-N-[(3S)-2-oxoazetidin-3-yl] carbamate (ARN14686), a NAAA-preferring activity-based probe, revealed that NAAA was catalytically active in CFA-treated paws. Administration of ARN726 reduced NAAA activity and restored PEA and OEA levels in inflamed tissues, and significantly decreased CFA-induced inflammatory symptoms, including pus production and myeloperoxidase activity. The results confirm the usefulness of ARN726 as a probe to investigate the functions of NAAA in health and disease and suggest that this enzyme may provide a new molecular target for the treatment of arthritis.

BackgroundSystemic nitroglycerin (NTG) activates brain nuclei involved in nociceptive transmission as well as in neuroendocrine and autonomic functions in rats. These changes are considered relevant for migraine because NTG consistently provokes spontaneous-like migraine attacks in migraineurs. Several studies have suggested a relationship between the endocannabinoid levels and pain mediation in migraine. URB937, a peripheral inhibitor of fatty acid amide hydrolase (FAAH)-the enzyme that degrades anandamide, produces analgesia in animal models of pain, but there is no information on its effects in migraine.AimWe evaluated whether URB937 alters nociceptive responses in the animal model of migraine based on NTG administration in male rats, using the tail flick test and the plantar and orofacial formalin tests, under baseline conditions and after NTG administration. Furthermore, we investigated whether URB937 affects NTG-induced c-Fos expression in the brain.ResultsDuring the tail flick test, URB937 showed an antinociceptive effect in baseline conditions and it blocked NTG-induced hyperalgesia. URB937 also proved effective in counteracting NTG-induced hyperalgesia during both the plantar and orofacial formalin tests. Mapping of brain nuclei activated by NTG indicates that URB937 significantly reduces c-Fos expression in the nucleus trigeminalis caudalis and the locus coeruleus.ConclusionsThe data suggest that URB937 is capable of changing, probably via indirect mechanisms, the functional status of central structures that are important for pain transmission in an animal model of migraine.

Background and purposeTo understand how anandamide transport inhibition impacts the regulation of nausea and vomiting and the receptor level mechanism of action involved. In light of recent characterization of an anandamide transporter, fatty acid amide hydrolase-1-like anandamide transporter, to provide behavioural support for anandamide cellular reuptake as a facilitated transport process.Experimental approachThe systemic administration of the anandamide transport inhibitor ARN272 ([(4-(5-(4-hydroxy-phenyl)-3,4-diaza-bicyclo[4.4.0]deca-1(6),2,4,7,9-pentaen-2-ylamino)-phenyl)-phenylamino-methanone]) was used to evaluate the prevention of LiCl-induced nausea-induced behaviour (conditioned gaping) in rats, and LiCl-induced emesis in shrews (Suncus murinus). The mechanism of how prolonging anandamide availability acts to regulate nausea in rats was explored by the antagonism of cannabinoid 1 (CB1) receptors with the systemic co-administration of SR141716.Key resultsThe systemic administration of ARN272 produced a dose-dependent suppression of nausea-induced conditioned gaping in rats, and produced a dose-dependent reduction of vomiting in shrews. The systemic co-administration of SR141716 with ARN272 (at 3.0 mg·kg(-1)) in rats produced a complete reversal of ARN272-suppressed gaping at 1.0 mg·kg(-1). SR141716 alone did not differ from the vehicle solution.Conclusions and implicationsThese results suggest that anandamide transport inhibition by the compound ARN272 tonically activates CB1 receptors and as such produces a type of indirect agonism to regulate toxin-induced nausea and vomiting. The results also provide behavioural evidence in support of a facilitated transport mechanism used in the cellular reuptake of anandamide.

Background and purposeTo understand how anandamide transport inhibition impacts the regulation of nausea and vomiting and the receptor level mechanism of action involved. In light of recent characterization of an anandamide transporter, fatty acid amide hydrolase-1-like anandamide transporter, to provide behavioural support for anandamide cellular reuptake as a facilitated transport process.Experimental approachThe systemic administration of the anandamide transport inhibitor ARN272 ([(4-(5-(4-hydroxy-phenyl)-3,4-diaza-bicyclo[4.4.0]deca-1(6),2,4,7,9-pentaen-2-ylamino)-phenyl)-phenylamino-methanone]) was used to evaluate the prevention of LiCl-induced nausea-induced behaviour (conditioned gaping) in rats, and LiCl-induced emesis in shrews (Suncus murinus). The mechanism of how prolonging anandamide availability acts to regulate nausea in rats was explored by the antagonism of cannabinoid 1 (CB1) receptors with the systemic co-administration of SR141716.Key resultsThe systemic administration of ARN272 produced a dose-dependent suppression of nausea-induced conditioned gaping in rats, and produced a dose-dependent reduction of vomiting in shrews. The systemic co-administration of SR141716 with ARN272 (at 3.0 mg·kg(-1)) in rats produced a complete reversal of ARN272-suppressed gaping at 1.0 mg·kg(-1). SR141716 alone did not differ from the vehicle solution.Conclusions and implicationsThese results suggest that anandamide transport inhibition by the compound ARN272 tonically activates CB1 receptors and as such produces a type of indirect agonism to regulate toxin-induced nausea and vomiting. The results also provide behavioural evidence in support of a facilitated transport mechanism used in the cellular reuptake of anandamide.

Background and purposeURB937 is a peripherally restricted inhibitor of the anandamide-deactivating enzyme fatty-acid amide hydrolase (FAAH). Despite its limited access to the CNS, URB937 produces marked antinociceptive effects in rodents. URB937 is actively extruded from the CNS by the ATP-binding cassette (ABC) membrane transporter, Abcg2. Tissue Abcg2 levels are markedly different between males and females, and this transporter is known to limit the access of xenobiotics to the fetoplacental unit in gestating female rodents. In the present study, we investigated the tissue distribution and antinociceptive properties of URB937 in female mice and rats.Experimental approachWe studied the systemic disposition of URB937 in female mice and the antinociceptive effects of this compound in models of visceral (acetic acid-induced writhing) and inflammatory nociception (carrageenan-induced hyperalgesia) in female mice and rats. Furthermore, we evaluated the interaction of URB937 with the blood-placenta barrier in gestating mice and rats.Key resultsAbcg2 restricted the access of URB937 to the CNS of female mice and rats. Nevertheless, URB937 produced a high degree of antinociception in female mice and rats in models of visceral and inflammatory pain. Moreover, the compound displayed a restricted access to placental and fetal tissues in pregnant mice and rats.Conclusions and implicationsPeripheral FAAH blockade with URB937 reduces nociception in female mice and rats, as previously shown for males of the same species. In female mice and rats, Abcg2 limits the access of URB937, not only to the CNS, but also to the fetoplacental unit. LINKED ARTICLES This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.167.issue-8.

Social deprivation in early life disrupts emotionality and attentional processes in humans. Rearing rats in isolation reproduces some of these abnormalities, which are attenuated by daily handling. However, the neurochemical mechanisms underlying these responses remain poorly understood. We hypothesized that post-weaning social isolation alters the endocannabinoid system, a neuromodulatory system that controls emotional responding. We characterized behavioral consequences of social isolation and evaluated whether handling would reverse social isolation-induced alterations in behavioral reactivity to context and the endocannabinoid system. At weaning, pups were single or group housed and concomitantly handled or not handled daily until adulthood. Rats were tested in emotionality- and attentional-sensitive behavioral assays (open field, elevated plus maze, startle and prepulse inhibition). Cannabinoid receptor densities and endocannabinoid levels were quantified in a separate group of rats. Social isolation negatively altered behavioral responding. Socially-isolated rats that were handled showed less deficits in the open field, elevated plus maze, and prepulse inhibition tests. Social isolation produced site-specific alterations (supraoptic nucleus, ventrolateral thalamus, rostral striatum) in cannabinoid receptor densities compared to group rearing. Handling altered the endocannabinoid system in neural circuitry controlling emotional expression. Handling altered endocannabinoid content (prefrontal and piriform cortices, nucleus accumbens) and cannabinoid receptor densities (lateral globus pallidus, cingulate and piriform cortices, hippocampus) in a region-specific manner. Some effects of social isolation on the endocannabinoid system were moderated by handling. Isolates were unresponsive to handling-induced increases in cannabinoid receptor densities (caudal striatum, anterior thalamus), but were sensitive to handling-induced changes in endocannabinoid content (piriform, prefrontal cortices), compared to group-reared rats. Our findings suggest alterations in the endocannabinoid system may contribute to the abnormal isolate phenotype. Handling modifies the endocannabinoid system and behavioral reactivity to context, but surmounts only some effects of social isolation. These data implicate a pivotal role for the endocannabinoid system in stress adaptation and emotionality-related disturbances.

Background and purposeMonoacylglycerol lipase (MGL) is a presynaptic serine hydrolase that inactivates the endocannabinoid neurotransmitter, 2-arachidonoyl-sn-glycerol. Recent studies suggest that cysteine residues proximal to the enzyme active site are important for MGL function. In the present study, we characterize the role of cysteines in MGL function and identify a series of cysteine-reactive agents that inhibit MGL activity with nanomolar potencies by interacting with cysteine residue 208.Experimental approachA series of cysteine traps were screened for the ability to inhibit MGL in vitro. Rapid dilution assays were performed to determine reversibility of inhibition. Molecular modelling and site-directed mutagenesis were utilized to identify cysteine residues targeted by the inhibitors.Key resultsThe screening revealed that 2-octyl-4-isothiazolin-3-one (octhilinone) inhibited purified rat recombinant MGL (IC(50)= 88 +/- 12 nM) through a partially reversible mechanism. Initial structure-activity relationship studies showed that substitution of the n-octyl group of octhilinone with a more lipophilic oleoyl group increased inhibitor potency (IC(50)= 43 +/- 8 nM), while substitution with a methyl group produced the opposite effect (IC(50)= 239 +/- 68 nM). The inhibitory potency of octhilinone was selectively decreased by mutating cysteine 208 in MGL to glycine (IC(50); wild-type, 151 +/- 17 nM; C208G, 722 +/- 74 nM), but not by mutation of other cysteine residues (C32, C55, C201, C208 and C242).Conclusions and implicationsThe results indicated that cysteine 208 plays an important role in MGL function and identified a novel class of isothiazolinone-based MGL inhibitors with nanomolar potency in vitro.

Neuropathic pain consequent to peripheral nerve injury has been associated with local inflammation. Following noxious stimulation afferent fibres release substance P (SP) and calcitonin-gene related peptide (CGRP), which are closely related to oedema formation and plasma leakage. The effect of the anandamide transport blocker AM404 has been studied on plasma extravasation after chronic constriction injury (CCI) which consists in a unilateral loose ligation of the rat sciatic nerve (Bennett and Xie, 1988). AM404 (1-3-10 mg kg(-1)) reduced plasma extravasation in the legated paw, measured as mug of Evans Blue per gram of fresh tissue. A strong effect on vascular permeability was also produced by the synthetic cannabinoid agonist WIN 55,212-2 (0.1-0.3-1 mg kg(-1)). Using specific antagonists or enzyme inhibitors, we demonstrate that cannabinoids act at several levels: data on the 3rd day suggest a strong involvement of substance P (SP) and calcitonin gene-related peptide (CGRP) in the control of vascular tone, whereas at the 7th and 14th days the major role seems to be played by prostaglandins (PGs) and nitric oxide (NO). Capsaicin injection in ligated paws of AM404- or WIN 55,212-2-treated rats resulted in an increase of Evans Blue extravasation, suggesting the involvement of the cannabinergic system in the protective effect of C fibres of ligated paws. Taken together, these data demonstrate the efficacy of cannabinoids in controlling pain behaviour through the modulation of several pain mediators and markers of vascular reactivity, such as SP, CGRP, PGs and NO.

The ability of CB(1) receptors to regulate the release of glutamate in the striatum, together with the finding that, in experimental models of Huntington disease (HD), both endocannabinoid levels and CB(1) receptor densities are reduced, has prompted the investigation on the neuroprotective role of the cannabinoids in HD. Quinolinic acid (QA) is an excitotoxin that, when injected in the rat striatum reproduces many features of HD and that acts by stimulating glutamate outflow. The aim of the present study was to test the ability of the cannabinoid receptor agonist WIN 55,212-2 to prevent the effects induced by QA in the rat striatum. In microdialysis experiments, probe perfusion with WIN 55,212-2 significantly and dose-dependently prevented the increase in extracellular glutamate induced by QA. In electrophysiological recordings in corticostriatal slices, the application of WIN 55,212-2 prevented QA-induced reduction of the field potential amplitude. Both effects of WIN 55,212-2 were prevented by the CB(1) receptor antagonist AM 251. In in vivo experiments, intrastriatal WIN 55,212-2 significantly attenuated the striatal damage induced by QA, although no significant effects were observed on a behavioural ground. These data demonstrate that the stimulation of CB(1) receptors might lead to neuroprotective effects against excitotoxic striatal toxicity.

Brief depolarization of postsynaptic neurons in hippocampus and cerebellum results in a transient depression of GABAergic inhibitory input, called "depolarization-induced suppression of inhibition" (DSI). We studied whether a similar phenomenon occurs in the rat neocortical neurons. Using patch-clamp technique in neocortical cell cultures, we examined the effects of a 5-second depolarization of postsynaptic neurons on evoked GABAergic inhibitory post-synaptic currents (IPSCs). We found that the depolarization evoked a suppression of IPSC amplitude in 6 out of 26 neuronal pairs tested. The suppression of IPSC amplitude lasted for approximately 70 seconds and was accompanied by changes of paired-pulse ratio and IPSC coefficient of variation (CV), which is suggestive of a presynaptic mechanism. These results are in agreement with previous observations in hippocampal cell cultures and suggest that neocortical neurons express DSI.

The endocannabinoid system may serve important functions in the central and peripheral regulation of pain. In the present study, we investigated the effects of the endocannabinoid transport inhibitor AM404 [N-(4-hydroxyphenyl)-eicosa-5,8,11,14-tetraenamide] on rodent models of acute and persistent nociception (intraplantar formalin injection in the mouse), neuropathic pain (sciatic nerve ligation in the rat), and inflammatory pain (complete Freund's adjuvant injection in the rat). In the formalin model, administration of AM404 (1-10 mg/kg i.p.) elicited dose-dependent antinociceptive effects, which were prevented by the CB(1) cannabinoid receptor antagonist rimonabant (SR141716A; 1 mg/kg i.p.) but not by the CB2 antagonist SR144528 (1 mg/kg i.p.) or the vanilloid antagonist capsazepine (30 mg/kg i.p.). Comparable effects were observed with UCM707 [N-(3-furylmethyl)-eicosa-5,8,11,14-tetraenamide], another anandamide transport inhibitor. In both the chronic constriction injury and complete Freund's adjuvant model, daily treatment with AM404 (1-10 mg/kg s.c.) for 14 days produced a dose-dependent reduction in nocifensive responses to thermal and mechanical stimuli, which was prevented by a single administration of rimonabant (1 mg/kg i.p.) and was accompanied by decreased expression of cyclooxygenase-2 and inducible nitric-oxide synthase in the sciatic nerve. The results provide new evidence for a role of the endocannabinoid system in pain modulation and point to anandamide transport as a potential target for analgesic drug development.

Although anecdotal reports suggest that cannabis may be used to alleviate symptoms of depression, the psychotropic effects and abuse liability of this drug prevent its therapeutic application. The active constituent of cannabis, delta9-tetrahydrocannabinol, acts by binding to brain CB1 cannabinoid receptors, but an alternative approach might be to develop agents that amplify the actions of endogenous cannabinoids by blocking their deactivation. Here, we show that URB597, a selective inhibitor of the enzyme fatty-acid amide hydrolase, which catalyzes the intracellular hydrolysis of the endocannabinoid anandamide, exerts potent antidepressant-like effects in the mouse tail-suspension test and the rat forced-swim test. Moreover, URB597 increases firing activity of serotonergic neurons in the dorsal raphe nucleus and noradrenergic neurons in the nucleus locus ceruleus. These actions are prevented by the CB1 antagonist rimonabant, are accompanied by increased brain anandamide levels, and are maintained upon repeated URB597 administration. Unlike direct CB1 agonists, URB597 does not exert rewarding effects in the conditioned place preference test or produce generalization to the discriminative effects of delta9-tetrahydrocannabinol in rats. The findings support a role for anandamide in mood regulation and point to fatty-acid amide hydrolase as a previously uncharacterized target for antidepressant drugs.

Brief depolarization of postsynaptic neurons in hippocampus and cerebellum results in a transient depression of GABAergic inhibitory input, called "depolarization-induced suppression of inhibition" (DSI). We studied whether a similar phenomenon occurs in the rat neocortical neurons. Using patch-clamp technique in neocortical cell cultures we examined the effects of a 5-second depolarization of postsynaptic neurons on evoked GABAergic inhibitory post-synaptic currents (IPSCs). We found that the depolarization evoked a suppression of IPSC amplitude in 6 out of 26 neuronal pairs tested. The suppression of IPSC amplitude lasted for approximately 70 seconds and was accompanied by changes of paired-pulse ratio and IPSC coefficient of variation (CV), which is suggestive of a presynaptic mechanism. These results are in agreement with previous observations in hippocampal cell cultures and suggest that neocortical neurons express DSI.

Fatty acid amide hydrolase (FAAH) and monoglyceride lipase (MGL) catalyse the hydrolysis of the endocannabinoids anandamide and 2-arachidonoyl glycerol. We investigated their ultrastructural distribution in brain areas where the localization and effects of cannabinoid receptor activation are known. In the hippocampus, FAAH was present in somata and dendrites of principal cells, but not in interneurons. It was located mostly on the membrane surface of intracellular organelles known to store Ca(2+) (e.g. mitochondria, smooth endoplasmic reticulum), less frequently on the somatic or dendritic plasma membrane. MGL immunoreactivity was found in axon terminals of granule cells, CA3 pyramidal cells and some interneurons. In the cerebellum, Purkinje cells and their dendrites are intensively immunoreactive for FAAH, together with a sparse axon plexus at the border of the Purkinje cell/granule cell layers. Immunostaining for MGL was complementary, the axons in the molecular layer were intensively labelled leaving the Purkinje cell dendrites blank. FAAH distribution in the amygdala was similar to that of the CB(1) cannabinoid receptor: evident signal in neuronal somata and proximal dendrites in the basolateral nucleus, and hardly any labelling in the central nucleus. MGL staining was restricted to axons in the neuropil, with similar relative signal intensities seen for FAAH in different nuclei. Thus, FAAH is primarily a postsynaptic enzyme, whereas MGL is presynaptic. FAAH is associated with membranes of cytoplasmic organelles. The differential compartmentalization of the two enzymes suggests that anandamide and 2-AG signalling may subserve functional roles that are spatially segregated at least at the stage of metabolism.

The endogenous cannabinoid anandamide is removed from the synaptic space by a high-affinity transport system present in neurons and astrocytes, which is inhibited by N-(4-hydroxyphenyl)-arachidonamide (AM404). After internalization, anandamide is hydrolyzed by fatty-acid amide hydrolase (FAAH), an intracellular membrane-bound enzyme that also cleaves AM404. Based on kinetic evidence, it has recently been suggested that anandamide internalization may be mediated by passive diffusion driven by FAAH activity. To test this possibility, in the present study, we have investigated anandamide internalization in wild-type and FAAH-deficient (FAAH(-/-)) mice. Cortical neurons from either mouse strain internalized [(3)H]anandamide through a similar mechanism, i.e., via a rapid temperature-sensitive and saturable process, which was blocked by AM404. Moreover, systemic administration of AM404 to either wild-type or FAAH(-/-) mice enhanced the hypothermic effects of exogenous anandamide, a response that was prevented by the CB(1) cannabinoid antagonist rimonabant (SR141716A). The results indicate that anandamide internalization in mouse brain neurons is independent of FAAH activity. In further support of this conclusion, the compound N-(5Z, 8Z, 11Z, 14Z eicosatetraenyl)-4-hydroxybenzamide (AM1172) blocked [(3)H]anandamide internalization in rodent cortical neurons and human astrocytoma cells without acting as a FAAH substrate or inhibitor. AM1172 may serve as a prototype for novel anandamide transport inhibitors with increased metabolic stability.

The endogenous cannabinoids (endocannabinoids) are lipid molecules that may mediate retrograde signaling at central synapses and other forms of short-range neuronal communication. The monoglyceride 2-arachidonoylglycerol (2-AG) meets several criteria of an endocannabinoid substance: (i) it activates cannabinoid receptors; (ii) it is produced by neurons in an activity-dependent manner; and (iii) it is rapidly eliminated. 2-AG inactivation is only partially understood, but it may occur by transport into cells and enzymatic hydrolysis. Here we tested the hypothesis that monoglyceride lipase (MGL), a serine hydrolase that converts monoglycerides to fatty acid and glycerol, participates in 2-AG inactivation. We cloned MGL by homology from a rat brain cDNA library. Its cDNA sequence encoded for a 303-aa protein with a calculated molecular weight of 33,367 daltons. Northern blot and in situ hybridization analyses revealed that MGL mRNA is heterogeneously expressed in the rat brain, with highest levels in regions where CB(1) cannabinoid receptors are also present (hippocampus, cortex, anterior thalamus, and cerebellum). Immunohistochemical studies in the hippocampus showed that MGL distribution has striking laminar specificity, suggesting a presynaptic localization of the enzyme. Adenovirus-mediated transfer of MGL cDNA into rat cortical neurons increased MGL expression and attenuated N-methyl-D-aspartate/carbachol-induced 2-AG accumulation in these cells. No such effect was observed on the accumulation of anandamide, another endocannabinoid lipid. The results suggest that hydrolysis by means of MGL is a primary mechanism for 2-AG inactivation in intact neurons.

Oleylethanolamide (OEA) is a natural analogue of the endogenous cannabinoid anandamide. Like anandamide, OEA is produced in cells in a stimulus-dependent manner and is rapidly eliminated by enzymatic hydrolysis, suggesting a function in cellular signalling. However, OEA does not activate cannabinoid receptors and its biological functions are still unknown. Here we show that, in rats, food deprivation markedly reduces OEA biosynthesis in the small intestine. Administration of OEA causes a potent and persistent decrease in food intake and gain in body mass. This anorexic effect is behaviourally selective and is associated with the discrete activation of brain regions (the paraventricular hypothalamic nucleus and the nucleus of the solitary tract) involved in the control of satiety. OEA does not affect food intake when injected into the brain ventricles, and its anorexic actions are prevented when peripheral sensory fibres are removed by treatment with capsaicin. These results indicate that OEA is a lipid mediator involved in the peripheral regulation of feeding.

We investigated the transduction mechanisms mediating formation of the endogenous cannabinoid (endocannabinoid) lipids, anandamide (arachidonylethanolamide) and 2-arachidonylglycerol, in primary cultures of rat cortical neurons. Unstimulated neurons contained 0.3 +/- 0.1 pmol of anandamide and 16.5 +/- 3.3 pmol of 2-arachidonylglycerol per mg of protein, as determined by gas chromatography/mass spectrometry. Ca(2+) entry into the neurons via activated glutamate N-methyl-D-aspartate (NMDA) receptors increased 2-arachidonylglycerol levels approximately three times, but had no effect on anandamide levels. By contrast, anandamide formation was stimulated five times by simultaneous activation of NMDA and acetylcholine receptors. Alone, acetylcholine receptor activation had no effect on anandamide or 2-arachidonylglycerol levels. The formation of fatty acid ethanolamides that do not activate cannabinoid receptors, including palmitylethanolamide and oleylethanolamide, was stimulated by coactivation of NMDA and acetylcholine receptors. Pharmacological experiments suggest that the cholinergic contribution to anandamide formation was mediated by alpha7 nicotinic receptors (antagonized by methyllycaconitine), whereas the contribution to palmitylethanolamide and oleylethanolamide formation was mediated by muscarinic receptors (antagonized by atropine). These findings indicate that cortical neurons produce anandamide and 2-arachidonylglycerol in a receptor-dependent manner, and that brain neurons may generate different endocannabinoid lipids depending on their complement of neurotransmitter receptors.

Smoking marijuana or administration of its main active constituent, delta9-tetrahydrocannabinol (delta9-THC), may exert potent dilating effects on human airways. But the physiological significance of this observation and its potential therapeutic value are obscured by the fact that some asthmatic patients respond to these compounds with a paradoxical bronchospasm. The mechanisms underlying these contrasting responses remain unresolved. Here we show that the endogenous cannabinoid anandamide exerts dual effects on bronchial responsiveness in rodents: it strongly inhibits bronchospasm and cough evoked by the chemical irritant, capsaicin, but causes bronchospasm when the constricting tone exerted by the vagus nerve is removed. Both effects are mediated through peripheral CB1 cannabinoid receptors found on axon terminals of airway nerves. Biochemical analyses indicate that anandamide is synthesized in lung tissue on calcium-ion stimulation, suggesting that locally generated anandamide participates in the intrinsic control of airway responsiveness. In support of this conclusion, the CB1 antagonist SR141716A enhances capsaicin-evoked bronchospasm and cough. Our results may account for the contrasting bronchial actions of cannabis-like drugs in humans, and provide a framework for the development of more selective cannabinoid-based agents for the treatment of respiratory pathologies.

The biological actions of the endogenous cannabinoid anandamide are terminated by carrier-mediated transport into neurons and astrocytes, followed by enzymatic hydrolysis. Anandamide transport is inhibited by the compound N-(4-hydroxyphenyl)arachidonylamide (AM404). AM404 potentiates several responses elicited by administration of exogenous anandamide, suggesting that it may also protect endogenous anandamide from inactivation. To test this hypothesis, we studied the effects of AM404 on the plasma levels of anandamide using high-performance liquid chromatography/mass spectrometry (HPLC/MS). Systemic administration of AM404 (10 mg kg(-1) intraperitoneal, i.p. ) caused a gradual increase of anandamide in rat plasma, which was significantly different from untreated controls at 60 and 120 min after drug injection. In plasma, both AM404 and anandamide were associated with a plasma protein, which we identified as albumin by non-denaturing polyacrylamide gel electrophoresis. AM404 (10 mg kg(-1), i.p.) caused a time-dependent decrease of motor activity, which was reversed by the cannabinoid CB(1) receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide.hydrochloride (SR141716A, 0.5 mg kg(-1), i.p). These results are consistent with the hypothesis that AM404 inhibits anandamide inactivation in vivo.

We characterized the pharmacological properties of the anandamide transport inhibitor N-(4-hydroxyphenyl)-arachidonamide (AM404) in rats and investigated the effects of this drug on behavioral responses associated with activation of dopamine D(2) family receptors. Rat brain slices accumulated [(3)H]anandamide via a high-affinity transport mechanism that was blocked by AM404. When administered alone in vivo, AM404 caused a mild and slow-developing hypokinesia that was significant 60 min after intracerebroventricular injection of the drug and was reversed by the CB1 cannabinoid receptor antagonist SR141716A. AM404 produced no significant catalepsy or analgesia, two typical effects of direct-acting cannabinoid agonists. However, AM404 prevented the stereotypic yawning produced by systemic administration of a low dose of apomorphine, an effect that was dose-dependent and blocked by SR141716A. Furthermore, AM404 reduced the stimulation of motor behaviors elicited by the selective D(2) family receptor agonist quinpirole. Finally, AM404 reduced hyperactivity in juvenile spontaneously hypertensive rats, a putative model of attention deficit hyperactivity disorder. The results support a primary role of the endocannabinoid system in the regulation of psychomotor activity and point to anandamide transport as a potential target for neuropsychiatric medicines.

We developed a high-performance liquid chromatography/mass spectrometry (HPLC/MS) method for the identification and quantification of anandamide, an endogenous cannabinoid substance, and other fatty acid ethanolamides (AEs) in biological samples. Using a mobile-phase system of methanol/water and gradient elution, we achieved satisfactory resolution of all major AEs, including anandamide, palmitylethanolamide (PEA), and oleylethanolamide (OEA). Electrospray-generated quasi-molecular species were used as diagnostic ions and detected by selected ion monitoring (SIM). Synthetic deuterium-labeled AEs were used as internal standards, and quantification was carried out by isotope dilution. A linear correlation (r2 = 0.99) was observed in the calibration curves for standard AEs over the range 0-0.5 nmol. Detection limits between 0.1 and 0.3 pmol per sample and quantification limits between 0.5 and 1.2 pmol per sample were obtained. The method was applied to the quantification of anandamide, PEA, and OEA in plasma prepared from rat blood collected either by cardiac puncture or by decapitation. After cardiac puncture, AE levels were in the low-nanomolar range: anandamide, 3.1 +/- 0.6 pmol/ml; PEA, 9.4 +/- 1.6 pmol/ml; OEA, 9.2 +/- 1.8 pmol/ml (mean +/- SE, n = 9). By contrast, after decapitation AEs were dramatically elevated (anandamide, 144 +/- 13 pmol/ml; PEA, 255 +/- 55 pmol/ml; OEA, 175 +/- 48 pmol/ml). Thus, disruptive procedures of blood collection may result in gross overestimates in the concentrations of circulating AEs.

CB1, a cannabinoid receptor enriched in neuronal tissue, was found in high concentration in retinas of rhesus monkey, mouse, rat, chick, goldfish, and tiger salamander by using a subtype-specific polyclonal antibody. Immunolabeling was detected in the two synaptic layers of the retina, the inner and outer plexiform layers, of all six species examined. In the outer plexiform layer, CB1 was located in and/or on cone pedicles and rod spherules. Labeling was detected in some amacrine cells of all species and in the ganglion cells and ganglion cell axons of all species except fish. In addition, sparse labeling was found in the inner and/or outer segments of the photoreceptors of monkey, mouse, rat, and chick. Using GC/MS to detect possible endogenous cannabinoids, we found 3 nmol of 2-arachidonylglycerol per g of tissue, but no anandamide was detectable. Cannabinoid receptor agonists induced a dramatic reduction in the amplitude of voltage-gated L-type calcium channel currents in identified retinal bipolar cells. The presence and distribution of the CB1 receptor, the large amounts of 2-arachidonylglycerol found, and the effects of cannabinoids on calcium channel activity in bipolar cells suggest a substantive role for an endogenous cannabinoid signaling system in retinal physiology, and perhaps vision in general.

We measured endogenous cannabinoid release in dorsal striatum of freely moving rats by microdialysis and gas chromatography/mass spectrometry. Neural activity stimulated the release of anandamide, but not of other endogenous cannabinoids such as 2-arachidonylglycerol. Moreover, anandamide release was increased eightfold over baseline after local administration of the D2-like (D2, D3, D4) dopamine receptor agonist quinpirole, a response that was prevented by the D2-like receptor antagonist raclopride. Administration of the D1-like (D1, D5) receptor agonist SKF38393 had no such effect. These results suggest that functional interactions between endocannabinoid and dopaminergic systems may contribute to striatal signaling. In agreement with this hypothesis, pretreatment with the cannabinoid antagonist SR141716A enhanced the stimulation of motor behavior elicited by systemic administration of quinpirole. The endocannabinoid system therefore may act as an inhibitory feedback mechanism countering dopamine-induced facilitation of motor activity.

The structural similarities between the anandamide transport inhibitor N-(4-hydroxyphenyl)-arachidonylamide (AM404) and the synthetic vanilloid agonist olvanil [(N-vanillyl)-9-oleamide], prompted us to investigate the possibility that olvanil may interfere with anandamide transport. The intracellular accumulation of [3H]anandamide by human astrocytoma cells was prevented by olvanil with a Ki value of 14.1+/-7.1 microM. By contrast, capsaicin [(8-methyl-N-vanillyl)-6-noneamide], a plant-derived vanilloid agonist, and capsazepine (N-[2-(4-chlorophenyl)ethyl]-1,3,4,5-tetrahydro-7,8-dihydroxy-2 H-2-benzazepine-2-carbothioamide), a vanilloid antagonist, had no such effect (Ki > 100 microM). These results indicate that, although less potent than AM404 (Ki 2.1+/-0.2 microM), olvanil may reduce anandamide clearance at concentrations similar to those needed for vanilloid receptor activation.

The potent analgesic effects of cannabis-like drugs and the presence of CB1-type cannabinoid receptors in pain-processing areas of the brain and spinal cord indicate that endogenous cannabinoids such as anandamide may contribute to the control of pain transmission within the central nervous system (CNS). Here we show that anandamide attenuates the pain behaviour produced by chemical damage to cutaneous tissue by interacting with CB1-like cannabinoid receptors located outside the CNS. Palmitylethanolamide (PEA), which is released together with anandamide from a common phospholipid precursor, exerts a similar effect by activating peripheral CB2-like receptors. When administered together, the two compounds act synergistically, reducing pain responses 100-fold more potently than does each compound alone. Gas-chromatography/mass-spectrometry measurements indicate that the levels of anandamide and PEA in the skin are enough to cause a tonic activation of local cannabinoid receptors. In agreement with this possibility, the CB1 antagonist SR141716A and the CB2 antagonist SR144528 prolong and enhance the pain behaviour produced by tissue damage. These results indicate that peripheral CB1-like and CB2-like receptors participate in the intrinsic control of pain initiation and that locally generated anandamide and PEA may mediate this effect.

Anandamide and allied fatty acylethanolamides (AEs) may act as signalling molecules in brain and peripheral tissues. In the present study, we describe an electron-impact gas chromatography/mass spectrometry (GC/MS) method based on isotope dilution, which may be used for the identification and quantification of anandamide and other AEs in biological matrices. The calibration curves for standard AEs were linear over the range 0-1000 pmol (r2 = 0.99) with a coefficient of variation of 4% at 2.5 pmol. Detection and quantification limits were in the high fmol to low pmol range for all AEs. Using this method we measured nanomolar concentrations of three endogenous AEs in deproteinated rat blood plasma (anandamide: 5.2 pmol/ml; palmitylethanolamide: 16.7 pmol/ml; oleylethanolamide: 8.1 pmol/ml). These results are consistent with a regulatory role of anandamide and other AEs in peripheral tissues.

Anandamide, an endogenous ligand for central cannabinoid receptors, is released from neurons on depolarization and rapidly inactivated. Anandamide inactivation is not completely understood, but it may occur by transport into cells or by enzymatic hydrolysis. The compound N-(4-hydroxyphenyl)arachidonylamide (AM404) was shown to inhibit high-affinity anandamide accumulation in rat neurons and astrocytes in vitro, an indication that this accumulation resulted from carrier-mediated transport. Although AM404 did not activate cannabinoid receptors or inhibit anandamide hydrolysis, it enhanced receptor-mediated anandamide responses in vitro and in vivo. The data indicate that carrier-mediated transport may be essential for termination of the biological effects of anandamide, and may represent a potential drug target.

Cannabinoid receptors are molecular targets for marijuana and hashish, the widespread drugs of abuse. These receptors are expressed in areas of the central nervous system that contribute in important ways to the control of memory, cognition, movement and pain perception. Indeed, such functions can be strongly influenced by cannabinoid drugs, with consequences that include euphoria, analgesia, sedation and memory impairment. Although the pharmacology of cannabinoid drugs is now beginning to be understood, we still lack essential information on the endogenous signalling system(s) by which cannabinoid receptors are normally engaged. An endogenous ligand for cannabinoid receptors, anandamide, has been described. Here we report that sn-2 arachidonylglycerol (2-AG), a cannabinoid ligand isolated from intestinal tissue, is present in brain in amounts 170 times greater than anandamide. 2-AG is produced in hippocampal slices by stimulation of the Schaffer collaterals, an excitatory fibre tract that projects from CA3 to CA1 neurons. Formation of 2-AG is calcium dependent and is mediated by the enzymes phospholipase C and diacylglycerol lipase. 2-AG activates neuronal cannabinoid receptors as a full agonist, and prevents the induction of long-term potentiation at CA3-CA1 synapses. Our results indicate that 2-AG is a second endogenous cannabinoid ligand in the central nervous system.

It has been suggested that anandamide (N-arachidonoylethanolamine), an endogenous cannabinoid substance, may be produced through Ca2+-stimulated hydrolysis of the phosphatidylethanolamine (PE) derivative N-arachidonoyl PE. The presence of N-arachidonoyl PE in adult brain tissue and the enzyme pathways that underlie its biosynthesis are, however, still undetermined. We report here that rat brain tissue contains both anandamide (11 +/- 7 pmol/gm wet tissue) and N-arachidonoyl PE (22 +/- 16 pmol/gm), as assessed by gas chromatography/mass spectrometry. We describe a N-acyltransferase activity in brain that catalyzes the biosynthesis of N-arachidonoyl PE by transferring an arachidonate group from the sn-1 carbon of phospholipids to the amino group of PE. We also show that sn-1 arachidonoyl phospholipids are present in brain, where they constitute approximately 0.5% of total phospholipids. N-acyltransferase activity is Ca2+ dependent and is enriched in brain and testis. Within the brain, N-acyltransferase activity is highest in brainstem; intermediate in cortex, striatum, hippocampus, medulla, and cerebellum; and lowest in thalamus, hypothalamus, and olfactory bulb. Pharmacological inhibition of N-acyltransferase activity in primary cultures of cortical neurons prevents Ca2+-stimulated N-arachidonoyl PE biosynthesis. Our results demonstrate, therefore, that rat brain tissue contains the complement of enzymatic activity and lipid substrates necessary for the biosynthesis of the anandamide precursor N-arachidonoyl PE. They also suggest that biosynthesis of N-arachidonoyl PE and formation of anandamide are tightly coupled processes, which may concomitantly be stimulated by elevations in intracellular Ca2+ occurring during neural activity.

Anandamide, an endogenous canabinoid substance, is hydrolyzed by an amidohydrolase activity present in rat brain and liver. We report that the bromoenol lactone, (E)-6-(bromomethylene) tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (BTNP), is a potent inhibitor of this enzyme activity. BTNP prevented anandamide hydrolysis in rat brain microsomes with an IC50 of 0.8 +/- 0.3 microM. Kinetic and dialysis experiments indicated that this effect was non-competitive and irreversible. After chromatographic fractionation of the enzyme activity, BTNP was still effective, suggesting that it interacts directly with the enzyme. Anandamide hydrolysis was 12-fold greater in rat cortical neurons (1.94 +/- 0.1 pmol/min/mg protein) than in cortical astrocytes (0.16 +/- 0.01 pmol/min/mg protein) and, in either cell type, it was inhibited by BTNP (IC50 = 0.1 microM in neurons). These results suggest that BTNP may provide a useful lead for the development of novel inhibitors of anandamide hydrolysis.

It has been suggested that anandamide (N-arachidonoylethanolamine), an endogenous cannabinoid substance, may be produced through Ca2+-stimulated hydrolysis of the phosphatidylethanolamine (PE) derivative N-arachidonoyl PE. The presence of N-arachidonoyl PE in adult brain tissue and the enzyme pathways that underlie its biosynthesis are, however, still undetermined. We report here that rat brain tissue contains both anandamide (11 +/- 7 pmol/gm wet tissue) and N-arachidonoyl PE (22 +/- 16 pmol/gm), as assessed by gas chromatography/mass spectrometry. We describe a N-acyltransferase activity in brain that catalyzes the biosynthesis of N-arachidonoyl PE by transferring an arachidonate group from the sn-1 carbon of phospholipids to the amino group of PE. We also show that sn-1 arachidonoyl phospholipids are present in brain, where they constitute approximately 0.5% of total phospholipids. N-acyltransferase activity is Ca2+ dependent and is enriched in brain and testis. Within the brain, N-acyltransferase activity is highest in brainstem; intermediate in cortex, striatum, hippocampus, medulla, and cerebellum; and lowest in thalamus, hypothalamus, and olfactory bulb. Pharmacological inhibition of N-acyltransferase activity in primary cultures of cortical neurons prevents Ca2+-stimulated N-arachidonoyl PE biosynthesis. Our results demonstrate, therefore, that rat brain tissue contains the complement of enzymatic activity and lipid substrates necessary for the biosynthesis of the anandamide precursor N-arachidonoyl PE. They also suggest that biosynthesis of N-arachidonoyl PE and formation of anandamide are tightly coupled processes, which may concomitantly be stimulated by elevations in intracellular Ca2+ occurring during neural activity.

We studied the localization of N-acyl phosphatidylethanolamine (NAPE), a putative cannabinoid precursor, in primary cultures of striatal and cortical neurons from the rat brain. We probed intact neurons with various exogenous phospholipases, including S. chromofuscus phospholipase D (PLD). S. chromofuscus PLD does not penetrate into neurons (as demonstrated by a lack of internalization of 125I-labeled PLD), and does not cause gross damage to the neuronal membrane (as demonstrated by a lack of effect of PLD on [3H]gamma-aminobutyric acid release). When neurons, labeled to isotopic equilibrium with [3H]ethanolamine, were incubated for 10 min with S. chromofuscus PLD, approximately 50% of neuronal NAPE was hydrolysed. This hydrolysis was accompanied by the release of a family of N-acyl ethanolamines (NAE) (assessed by high performance liquid chromatography), which included the cannabinoid receptor agonist, anandamide. Exogenous phospholipase A2 (PLA2) (Apis mellifera) and PLC (B. cereus) mobilized [3H]arachidonate and [3H]diacylglycerol, respectively, but had no effect on NAE formation under these conditions. These experiments indicate that approximately 50% of neuronal NAPE is localized in a compartment that is easily accessible to extracellular PLD, possibly the plasmalemma, where it would also be easily hydrolyzed upon stimulation to produce NAE.

Understanding the mechanisms involved in the biogenesis of N-arachidonoylethanolamine (anandamide) and N-palmitoylethanolamine is important in view of the possible role of these lipids as endogenous cannabinoid substances. Anandamide (which activates cannabinoid CB1 receptors) and N-palmitoylethanolamine (which activates a CB2-like receptor subtype in mast cells) may both derive from cleavage of precursor phospholipid, N-acylphosphatidylethanolamine (NAPE), catalyzed by Ca(2+)-activated D-type phosphodiesterase activity. We report here that the de novo biosynthesis of NAPE is enhanced in a Ca(2+)-dependent manner when rat cortical neurons are stimulated with the Ca(2+)-ionophore ionomycin or with membrane-depolarizing agents such as veratridine and kainate. This reaction is likely to be mediated by a neuronal N-acyltransferase activity, which catalyzes the transfer of an acyl group from phosphatidylcholine to the ethanolamine moiety of phosphatidylethanolamine. In addition, we show that Ca2+-dependent NAPE biosynthesis is potentiated by agents that increase cAMP levels, including forskolin and vasoactive intestinal peptide. Our results thus indicate that NAPE levels in cortical neurons are controlled by Ca2+ ions and cAMP. Such regulatory effect may participate in maintaining a supply of cannabimimetic N-acylethanolamines during synaptic activity, and prime target neurons for release of these bioactive lipids.

5-Lipoxygenase-activating protein (FLAP) is an 18-kDa integral membrane protein required, in peripheral cells, for the activation of 5-lipoxygenase (5-LO) and for the resulting synthesis of leukotrienes from arachidonic acid. In the brain, the leukotrienes have been implicated in several pathophysiological events and in the electrophysiological effect of somatostatin, yet the cellular origin and role of these messenger molecules are still poorly understood. In the present study, we used reverse transcriptase-polymerase chain reaction, in situ hybridization, and immunohistochemistry to demonstrate that 5-LO and FLAP are expressed in various regions of the rat brain, including hippocampus, cerebellum, primary olfactory cortex, superficial neocortex, thalamus, hypothalamus, and brainstem. Highest levels of expression were observed in cerebellum and hippocampus. In the latter we demonstrate the colocalization of 5-LO and FLAP in CA1 pyramidal neurons. Moreover, electrophysiological experiments show that selective inhibition of FLAP with the compound MK-886 (0.25-1 microM) prevents the somatostatin-induced augmentation of the hippocampal K+ M-current. Our results provide necessary evidence for the presence and signaling role of 5-LO and FLAP in central neurons and strongly support their proposed participation in somatostatin-receptor transmembrane signaling.

Recent reports have suggested that N-arachidonoylethanolamine (anandamide) acts as an endogenous ligand for cannabinoid receptors in mammalian brain. Here we describe methods for the extraction, purification and analysis of anandamide and related N-fatty acyl-ethanolamines (NAEs). Liquid-phase extraction, silica gel G column chromatography and thin-layer chromatography (TLC) were employed for sample fractionation. Three analytical high-performance liquid chromatography (HPLC) methods for purification of NAEs were developed. Finally, analyses of NAEs by gas chromatography/mass spectrometry (GC/MS) are described. The applications of these analytical methods to the identification of anandamide and related NAEs in cell cultures as well as of artifacts in biosynthetic studies are described.

Anandamide, an endogenous arachidonic acid derivative that is released from neurons and activates cannabinoid receptors, may act as a transcellular cannabimimetic messenger in the central nervous system. The biological actions of anandamide and the identity of its target cells are, however, still poorly documented. Here we show that anandamide is a potent inhibitor of gap-junction conductance and dye permeability in striatal astrocytes. This inhibitory effect is specific for anandamide as compared to co-released congeners or structural analogues, is sensitive to pertussis toxin and to protein-alkylating agents, and is neither mimicked by cannabinoid-receptor agonists nor prevented by a cannabinoid-receptor antagonist. Glutamate released from neurons evokes calcium waves in astrocytes that propagate via gap junctions, and may, in turn, activate neurons distant from their initiation sites in astrocytes. We find that anandamide blocks the propagation of astrocyte calcium waves generated by either mechanical stimulation or local glutamate application. Thus, by regulating gap-junction permeability, anandamide may control intercellular communication in astrocytes and therefore neuron-glial interactions.

An amidohydrolase activity present in rat brain microsomes catalyzes the hydrolysis of N-arachidonoyl-[3H]ethanolamine ([3H]anandamide), an endogenous cannabimimetic substance, forming [3H]ethanolamine and arachidonic acid. Amidohydrolase activity is maximal at pH 6 and 8, is independent of divalent cations, has an apparent Km for [3H]anandamide of 12.7 +/- 1.8 microM, and has a Vmax of 5630 +/- 200 pmol/min/mg of protein. Phenylmethylsulfonyl fluoride, a serine protease inhibitor, and p-bromophenacyl bromide, a histidine-alkylating reagent, inhibit the activity, whereas N-ethylmaleimide and various nonselective peptidase inhibitors (EDTA, o-phenanthroline, bacitracin) have no effect. Brain amidohydrolase activity exhibits high substrate specificity for [3H]anandamide; N-gamma-linolenoyl-, N-homo-gamma-linolenoyl-, and N-11,14-eicosadienoyl- are hydrolyzed at markedly slower rates. Moreover, N-11-eicosaenoyl- and N-palmitoyl-[3H]ethanolamine are not hydrolyzed. [3H]Anandamide hydrolysis is inhibited competitively by nonradioactive anandamide and by other N-acylethanolamines with the following rank order of potency: anandamide > N-linoleoyl- = N-cis-linolenoyl- = N-gamma-linolenoyl- = N- homo-gamma-linolenoyl- > N-11,14-eicosadienoyl- > N-oleoyl- > N- docosahexaenoyl- > N-docosatetraenoyl > N-linoelaidoyl- > N-eicosaenoyl- > N- palmitoyl > or = N-elaidoyl- = N-eicosanoyl-ethanolamine = no effect. Amidohydrolase activity is high in liver and brain and low in heart, kidney, intestine, stomach, lung, spleen, and skeletal muscle. Within the central nervous system, highest activity is found in globus pallidus and hippocampus, two regions rich in cannabinoid receptors, and lowest activity is found in brainstem and medulla, where cannabinoid receptors are sparse. The results, showing that brain amidohydrolase activity is selective for anandamide and enriched in areas of the central nervous system with high density of cannabinoid receptors, suggest that this activity may participate in the inactivation of anandamide at its sites of action.

Anandamide (N-arachidonoyl-ethanolamine) was recently identified as a brain arachidonate derivative that binds to and activates cannabinoid receptors, yet the mechanisms underlying formation, release and inactivation of this putative messenger molecule are still unclear. Here we report that anandamide is produced in and released from cultured brain neurons in a calcium ion-dependent manner when the neurons are stimulated with membrane-depolarizing agents. Anandamide formation occurs through phosphodiesterase-mediated cleavage of a novel phospholipid precursor, N-arachidonoyl-phosphatidylethanolamine. A similar mechanism also governs the formation of a family of anandamide congeners, whose possible roles in neuronal signalling remain unknown. Our results and those of others indicate therefore that multiple biochemical pathways may participate in anandamide formation in brain tissue. The life span of extracellular anandamide is limited by a rapid and selective process of cellular uptake, which is accompanied by hydrolytic degradation to ethanolamine and arachidonate. Our results thus strongly support the proposed role of anandamide as an endogenous neuronal messenger.

During K(+)-induced depolarization of isolated rat brain nerve terminals (synaptosomes), 1 mM Ba2+ could substitute for 1 mM Ca2+ in evoking the release of endogenous glutamate. In addition, Ba2+ was found to evoke glutamate release in the absence of K(+)-induced depolarization. Ba2+ (1-10 mM) depolarized synaptosomes, as measured by voltage-sensitive dye fluorescence and [3H]-tetraphenylphosphonium cation distribution. Ba2+ partially inhibited the increase in synaptosomal K+ efflux produced by depolarization, as reflected by the redistribution of radiolabeled 86Rb+. The release evoked by Ba2+ was inhibited by tetrodotoxin (TTX). Using the divalent cation indicator fura-2, cytosolic [Ca2+] increased during stimulation by approximately 200 nM, but cytosolic [Ba2+] increased by more than 1 microM. Taken together, our results indicate that Ba2+ initially depolarizes synaptosomes most likely by blocking a K+ channel, which then activates TTX-sensitive Na+ channels, causing further depolarization, and finally enters synaptosomes through voltage-sensitive Ca2+ channels to evoke neurotransmitter release directly. Though Ba(2+)-evoked glutamate release was comparable in level to that obtained with K(+)-induced depolarization in the presence of Ca2+, the apparent intrasynaptosomal level of Ba2+ required for a given amount of glutamate release was found to be several-fold higher than that required of Ca2+.

A DNA, cloned after screening a rat genomic bank with probes derived from the sequence of a putative dog histamine H2 receptor [Gantz, I., Schäffer, M., Delvalle, J., Logsdon, C., Campbell, V., Uhler, M. & Yamada, T. (1991) Proc. Natl. Acad. Sci. USA 88, 429-433], was used to prepare a probe for Northern blot analysis and to transfect Chinese hamster ovary (CHO) cells. Distribution of the gene transcripts in guinea pig tissues was consistent with that of H2 receptors. Transfected CHO cells expressed a high density of sites binding [125I]iodoaminopotentidine, a selective H2-receptor ligand. These sites were characterized as typical H2 receptors by using a series of competing agents that displayed apparent dissociation constants closely similar to corresponding values at a reference biological system. In transfected cells, histamine stimulated, with high potency and large receptor reserve, the accumulation of cAMP. In addition, in the same cells, histamine potently inhibited the release of arachidonic acid induced either by stimulation of constitutive purinergic receptors or by application of a Ca2+ ionophore. This inhibition was independent of either cAMP or Ca2+ levels. The results suggest that a single H2 receptor may be linked not only to adenylyl cyclase activation but also to reduction of phospholipase A2 activity. Because H1 receptors have been reported to stimulate arachidonic acid release, inhibition of this release, an unexpected signaling pathway for H2 receptors, may account for the opposing physiological responses elicited in many tissues by stimulation of these two receptors subtypes.

Understanding the actions of the neurotransmitter dopamine in the brain is important in view of its roles in neuropsychiatric illnesses. Dopamine D1 receptors, which stimulate both adenylyl cyclase and phospholipase C, and D2 receptors, which inhibit them, can nevertheless act synergistically to produce many electrophysiological and behavioral responses. Because this functional synergism can occur at the level of single neurons, another, as yet unidentified, signalling pathway activated by dopamine has been hypothesized. We report here that in Chinese hamster ovary (CHO) cells transfected with the D2 receptor complementary DNA, D2 agonists potently enhanced arachidonic acid release, provided that such release has been initiated by stimulating constitutive purinergic receptors or by increasing intracellular Ca2+. In CHO cells expressed D1 receptors, D1 agonists exert no such effect. When D1 and D2 receptors are coexpressed, however, activation of both subtypes results in a marked synergistic potentiation of arachidonic acid release. The numerous actions of arachidonic acid and its metabolites in neuronal signal transduction suggest that facilitation of its release may be implicated in dopaminergic responses, such as feedback inhibition mediated by D2 autoreceptors, and may constitute a molecular basis for D1/D2 receptor synergism.

In preparations of synaptic terminals (synaptosomes) isolated from rat brain, the activity of phospholipase A2 (PLA2), a phospholipid hydrolase that serves a central function in signal transduction, was inhibited in a Ca(2+)-dependent manner by incubation with 60 mM K+ or with the Ca(2+)-selective ionophore ionomycin. Reversal by alkaline phosphatase treatment suggested that this inhibitory effect resulted from phosphorylation of a synaptosomal protein substrate. When lysed synaptosomes were incubated with Ca2+/calmodulin (CaM), purified Ca2+/CAM-dependent protein kinase II (Ca2+/CaM-dependent PK II) and ATP, PLA2 activity in lysates was nearly abolished within 10 min. This effect was accompanied by a marked decrease in the Vmax of the enzyme and little or no change in the Km. Furthermore, Ca2+/CaM with ATP but without exogenous Ca2+/CaM-dependent PK II partially inhibited PLA2 activity, and this effect was prevented by treating the lysates with a selective peptide inhibitor of Ca2+/CaM-dependent PK II. In contrast, incubation of intact synaptosomes with 4 beta-phorbol 12-myristate 13-acetate or of lysed synaptosomes with purified protein kinase C had little or no effect on PLA2 activity. The results strongly suggest that the Ca(2+)-dependent inhibition of PLA2 activity observed in intact nerve endings was produced by activation of the multifunctional Ca2+/CaM-dependent PK II. A membrane-permeable adenylyl cyclase activator, forskolin, enhanced PLA2 activity in intact synaptosomes, and cAMP-dependent protein kinase potentiated PLA2 activity in lysed synaptosomes. Furthermore, another broad-spectrum protein kinase present in synaptic terminals, casein kinase II, also potentiated PLA2 activity in lysed synaptosomes. The effects of both protein kinases were associated with a decrease in Km and no change in Vmax. The results suggest that PLA2 activity in synaptic terminals is subject to bidirectional control by distinct signal transduction pathways. Moreover, mutually antagonistic effects of the Ca2+/CaM-dependent PK II and PLA2 pathways provide a possible molecular mechanism for bidirectional modulation of neurotransmitter release.

A modification of Vane's cascade is reported, allowing the superfusion bioassay of prostaglandin-like substances (PLS) in the outflow of isolated and perfused heart of the frog Rana esculenta L. Using both this technique and radioimmunoassay determination, the cyclo-oxygenase pathway in perfused frog heart has been investigated. Arachidonate (AA) (2-20 micrograms) injected into the perfusing fluid, was transformed by the heart into PLS, as shown by the response of the bioassay tissues (rat stomach strip, chick rectum, rat colon). A compound capable of relaxing rabbit mesenteric artery and a rabbit aorta contracting substance were also generated. The release was inhibited by indomethacin (1.0 X 10(-5)M). Radioimmunoassay determination of PGE2, TXB2 and 6-keto-PGF1 alpha in frog heart effluent, before and after AA injection (20 micrograms), gave the following yields (ng/ml of effluent). Basal: PGE2 = 0.45 +/- 0.15; TXB2 = 0.46 +/- 0.13; 6-keto-PGF1 alpha = 2.21 +/- 0.3. Following AA: PGE2 = 1.55 +/- 0.35; 6-keto-PGF1 alpha = 3.4 +/- 0.4; TXB2 = 1.00 +/- 0.06. Our results suggest that prostacyclin is a major product of the cyclo-oxygenase pathway in frog perfused heart. The biological significance of this finding is discussed in relation to both the absence of a coronary circulation in amphibians and to the spongy nature of frog myocardium.

1. A comparative quantitative study on the occurrence of 5-HT in several tissues and organs of the elasmobranch Scyliorhinus stellaris and the teleost Conger conger has been carried out. 2. In Scyliorhinus, the richest source of the amine is the brain, in which 5-HT is twice as concentrated as in the teleost brain. Significant levels have been also detected in the intestine, followed by gills and heart. 5-HT is also concentrated in other epithelial organs, such as the rectal gland and the olfactory organ. 3. In Conger, the gills show the highest content of 5-HT, in which, like in the kidney, serotonin is 2-3 times more concentrated than in the corresponding organs of the elasmobranch. 4. These differences are discussed in relation to the distinct phylogenetic and ecophysiological features of the two animals. In addition, the possible functional significance of 5-HT in the fish heart is taken into consideration.