Biomedical subjects

A C Howlett

Publications and source records attributed to A C Howlett.

At least 37 records · Page 2Linked to original sources

PubMed1998

Derivation of a pharmacophore model for anandamide using constrained conformational searching and comparative molecular field analysis.

Constrained molecular dynamics simulations on anandamide, together with a systematic distance comparison search, have revealed a specific low-energy conformer whose spatial disposition of the pharmacophoric elements closely matches that of HHC. This conformer enables near superposition of the following: (1) the oxygen of the carboxyamide and the phenolic hydroxyl group of HHC, (2) the hydroxyl group of the ethanol and the cyclohexyl hydroxyl group of HHC, (3) the alkyl tail and the lipophilic side chain of HHC, and (4) the polyolefin loop and the tricyclic ring structure of HHC. The close matching of common pharmacophoric elements of anandamide with HHC offers persuasive evidence of the biological relevance of this conformer. The proposed pharmacophore model was capable of discriminating between structurally related compounds exhibiting different pharmacological potency for the CB1 cannabinoid receptor, i.e., anandamide and N-(2-hydroxyethyl)prostaglandinamide. Furthermore, a 3D-QSAR model was derived using CoMFA for a training set of 29 classical and nonclassical analogues which rationalized the binding affinity in terms of steric and electrostatic properties and, more importantly, which predicted the potency of anandamide in excellent agreement with experimental data. The ABC tricyclic HU-210/HU-211 and ACD tricyclic CP55,243/CP55,244 enantiomeric pairs were employed as test compounds to validate the present CoMFA model. For each enantiomeric pair, the CoMFA-predicted log Ki values correctly identified that enantiomer exhibiting the higher affinity for the receptor.

Arachidonic Acids↗

PubMed1998

The CB1 cannabinoid receptor in the brain.

The CB1 cannabinoid receptor in brain is a G-protein-coupled receptor that exists as a protein possessing seven transmembrane helices that span the membrane. The intracellular surface is able to interact with f1p4oteins of the Gi/o family to regulate effector proteins, including adenylate cyclase, Ca2+ channels, and K+ channels, and to stimulate the mitogen-activated protein kinase pathway. The CB1 cannabinoid receptor recognizes three classes of agonist ligands: cannabinoid, eicosanoid, and aminoalkylindole. These agonist subtypes may interact with the CB1 cannabinoid receptor by some common points of association, yet may have subtle differences in the way that they interact with the receptor protein. This may be evident in the allosteric regulation by monovalent cations and individual agonists. The juxtamembrane region of the C-terminal is able to activate G-proteins. It is proposed that conformational changes in the receptor induced by agonist ligands may alter the conformation or exposure of the juxtamembrane C-terminal region extending from helix VII.

Allosteric Regulation↗

PubMed1998

CB1 cannabinoid receptor: cellular regulation and distribution in N18TG2 neuroblastoma cells.

In order to characterize cellular regulation of CB1 cannabinoid receptors, synthesis and turnover studies were performed. Metabolic labeling of N18TG2 cells with 35S-labeled amino acids was followed by immunoprecipitation from cell lysates using an affinity-purified antibody generated to the N-terminal 14-amino-acid segment of the CB1 receptor. During a 2 h labeling period, CB1 receptors were rapidly and constitutively synthesized (rate: 0.86%/min). The majority of newly synthesized CB1 cannabinoid receptors (70%) was degraded rapidly by a first-order process (t1/2=4.8 h). The remaining nascent receptors, which were degraded slowly (t1/2>24 h), may represent the pool of potentially functional receptors. Trypsin treatment of intact confluent cells, designed to cleave the extracellular antibody recognition site, did not alter the recovery of metabolically labeled immunoprecipitated CB1 receptors. This suggests that a large percentage of newly synthesized receptors was inaccessible to the protease and is probably intracellular. Immunocytochemistry revealed CB1 cannabinoid immunoreactivity both intracellularly and on the cell surface. Subcellular membrane fractions exhibited receptor binding activity on plasma membranes and nuclear-associated membranes. Only low-affinity binding was seen in the chromatin fraction. An hypothesis has been developed to explain these results and form the basis for future studies.

Animals↗

PubMed1998

Differential receptor-G-protein coupling evoked by dissimilar cannabinoid receptor agonists.

Multiple affinity states are revealed by agonist competition for radioantagonist [3H]SR141716A binding to rat brain CB1 cannabinoid receptors. Desacetyllevonantradol (DALN), a tricyclic cannabinoid, and WIN55212-2, an aminoalkylindole, both bound in two discrete affinity states (30% high affinity), but the ratios of the IC50 revealed distinct differences. Other affinity-state differences between the agonists were: Na+ reduced the affinity for the membrane-bound receptor by 10-fold for DALN but minimally for WIN55212-2; a nonhydrolysable GTP analogue decreased the fraction of high-affinity WIN55212-2 binding but not that of DALN unless Na+ was also present. Detergent solubilisation increased the fraction of high-affinity binding for both agonists but eliminated any effect of Na+ on the agonist affinities. In detergent solution, the GTP analogue reduced the WIN55212-2 high-affinity fraction but not that of DALN, even though the IC50 values increased for both DALN and WIN55212-2. The differential modulation of CB1 receptor-G-protein coupling by Na+ and guanine nucleotides is dependent upon the cannabimimetic agonist bound.

Animals↗

PubMed1998

Structural requirements for arachidonylethanolamide interaction with CB1 and CB2 cannabinoid receptors: pharmacology of the carbonyl and ethanolamide groups.

Analogs of arachidonylethanolamide (anandamide) were prepared to investigate the structural requirements for ligand binding to and activation of the CB1 and CB2 cannabinoid receptors. The importance of the presence and the placement of the carbonyl was examined with analogs lacking the carbonyl or with the carbonyl amide order reversed. The presence and location of the carbonyl is essential for high-affinity binding to both cannabinoid receptor subtypes, and for determination of signal transduction via G-proteins. Methyl groups were substituted on the 1'- and 2'-positions of arachidonylethanolamide and the significance of chirality was examined. Stereochemical differences in the ethanolamide group influence the affinity for both cannabinoid receptor subtypes and the signal transduction capabilities of the methanandamide derivatives.

Adenylyl Cyclases↗

PubMed1998

Characterization of CB1 cannabinoid receptors using receptor peptide fragments and site-directed antibodies.

The mechanism by which CB1 cannabinoid receptors are coupled to the Gi/Go class of G proteins was studied. A peptide representing the juxtamembrane carboxyl terminus robustly stimulated guanosine-5'-O-(3-thio)triphosphate binding. Peptides simulating subdomains of the third intracellular loop (IL3) activated minimally when present alone but produced additive effects when present in combination. Peptides representing the amino-side IL3 and the juxtamembrane carboxyl terminus autonomously inhibited adenylate cyclase, and this response was not significantly augmented or inhibited by peptides representing other intracellular domains. Site-directed antipeptide antibodies developed against the domains of the amino terminus, first extracellular loop, amino-side IL3, and juxtamembrane carboxyl terminus of CB1 receptors failed to influence binding of [3H]CP-55940. However, IgG raised against the amino-side IL3 diminished the agonist-dependent inhibition of adenylate cyclase. These experiments suggest that the juxtamembrane carboxyl terminus is critical for G protein activation by CB1 cannabinoid receptors and that the amino-side IL3 also may interact with Gi proteins leading to inhibition of adenylate cyclase.

Adenylyl Cyclase Inhibitors↗

PubMed1998

Effect of environmental estrogens on IL-1beta promoter activity in a macrophage cell line.

Environmental estrogens or estrogen disrupters have recently received a great deal of attention because of their potential health impact on reproductive tissues. Few, if any, studies have been made on the impact of these compounds on the immune system. We sought to determine the activities of various environmental estrogens on the modulation of the interleukin-1beta (IL-1beta) gene in a model monocytic cell line, hER + IL-1beta-CAT+. This cell line stably transfected with the human estrogen receptor, and an IL-1beta promoter construct fused to the CAT reporter gene allows us to monitor the effect of estrogenic compounds on IL-1beta promoter activity. 17beta-estradiol (E2) markedly enhanced lipopolysaccharide- (LPS) induced IL-1beta promoter-driven CAT activity in a dose-dependent manner. The mycotoxins alpha-zearalenol and zearalenone both exhibited full agonist activity, but at lower potencies, with EC50 values of 1.8 and 54 nM, respectively, compared with E2 at 0.5 nM. In addition, genistein was a very low-potency agonist, having an EC50 of 1.5 microM. Similar to the E2 response, the slope factors for alpha-zearalenol, zearalenone, and genistein were close to 3.0, suggesting positive cooperativity in the estrogenic response. The activity of the mycotoxins appeared to be mediated through the estrogen receptor, since both the antiestrogens H1285 and ICI 182,780 effectively inhibited their agonist activity in a dose-dependent manner. Representative environmental estrogenic compounds both from plant and industrial sources were also tested. Unlike the mycoestrogens, none of the compounds, with the exception of genistein, synergized with LPS to enhance IL-1beta promoter activity. When tested for antiestrogenic activity, the industrial compound 4-octylphenol was able to antagonize the response to E2; however, the response was three orders of magnitude less potent than H 1285. Naringenin, a plant flavonoid, showed little or no ability to antagonize the response to E2. Overall, the results show that some environmental estrogens that display agonist activity in reproductive tissue also have an effect on IL-1 gene expression in hemopoietic-derived tissue.

Animals↗

PubMed1997

Failure of cannabinoid compounds to stimulate estrogen receptors.

delta 9-Tetrahydrocannabinol (THC), the primary active compound in Cannabis sativa (marihuana), and other cannabinoid receptor agonists exert potent effects on luteinizing hormone and prolactin release in animal models and humans. Compounds possessing the tricyclic cannabinoid structure, including delta 9-THC and cannabidiol, have been reported to interact with rodent uterine estrogen receptors in ligand binding assays. The present study tested the hypothesis that cannabinoid compounds produce a direct activation of estrogen receptors. We investigated whether cannabinoid compounds exhibit estrogen-induced mitogenesis in MCF-7 breast cancer cells. Under conditions in which 10 pM estradiol promoted MCF-7 cell proliferation, no response was observed with biologically relevant concentrations (< = 10 microM) of delta 9-THC or its tricyclic analog desacetyllevonantradol. No response was observed with cannabidiol, a bicyclic cannabinoid compound that exhibits no cannabimimetic behavioral effects but has been reported to bind to the estrogen receptor in vitro. delta 9-THC also failed to antagonize the response to estradiol under conditions in which the antiestrogen LY156758 (keoxifene; raloxifene) was effective. The phytoestrogen formononetin behaved as an estrogen at high concentrations, and this response was antagonized by LY156758. We also investigated the ability of cannabinoid compounds to stimulate transcription of an EREtkCAT reporter gene transiently transfected into MCF-7 cells. Neither delta 9-THC, desacetyllevonantradol, nor cannabidiol stimulated transcriptional activity. We conclude that psychoactive or inactive compounds of the cannabinoid structural class fail to behave as agonists in appropriate assays of estrogen receptor responses in vitro.

Breast Neoplasms↗

PubMed1997

Nitric oxide, an endogenous regulator of Dictyostelium discoideum differentiation.

We have previously demonstrated that nitric oxide (NO)-generating compounds inhibit D. discoideum differentiation by preventing the initiation of cAMP pulses (Tao, Y., Howlett, A. and Klein, C. (1996) Cell. Signal. 8, 37-43). In the present study, we demonstrate that cells produce NO at a relatively constant rate during the initial phase of their developmental cycle. The addition of oxyhemoglobin, an NO scavenger, stimulates cell aggregation, suggesting that NO has a negative effect on the development of aggregation competence. Starvation of cells in the presence of glucose, which has been shown to prevent the initiation of cAMP pulses (Darmon, M. and Klein, C. (1978) Dev. Biol. 63, 377-389), results in an increased production of NO. The inhibition of cell aggregation by glucose treatment can be reversed by oxyhemoglobin. These findings indicate that NO is a signaling molecule for D. discoideum cells and that physiological or environmental conditions that enhance external NO levels will delay the initiation of cAMP pulses, which are essential for cell differentiation.

Animals↗

PubMed1996

(Aminoalkyl)indole isothiocyanates as potential electrophilic affinity ligands for the brain cannabinoid receptor.

A series of (aminoalkyl)indole compounds, naphthalene analogs of pravadoline (1), has been shown to exhibit cannabinoid agonist activities such as antinociception in animals, inhibition of adenylate cyclase in brain membranes, and binding to the cannabinoid receptor. These pravadoline analogs were selected for the preparation of potential electrophilic affinity ligands based on the synthesis of isothiocyanate derivatives. One isothiocyanatonaphthalene derivative (8) displaced [3H]CP-55940 binding to a rat brain P2 membrane preparation with an IC50 of 690 nM, which was 10-fold less potent than the parent molecule (IC50 = 73 nM). Isothiocyanate substitution at various positions on the naphthalene moiety of the desmethyl analog 10 gave compounds that displaced [3H]CP-55940 with IC50 values between 400 and 1000 nM, compared with 46 nM for the parent compound 10. However, 6-isothiocyanato substitution on the indole ring of the desmethyl analog provided isothiocyanate 12 that displaced [3H]CP-55940 binding with an IC50 and 160 nM. After pretreatment of brain membranes with this high-affinity isothiocyanato ligand followed by washing out the ligand, the membranes were depleted of 90% of the cannabinoid receptor binding capacity. Loss of receptor binding capacity was half-maximal at 300 nM of the derivative under standard assay conditions. As a control, pretreatment with the parent compound at concentrations that were 20 times the Kd failed to alter subsequent binding activity. This study demonstrates that an isothiocyanato (aminoalkyl)-indole (12) can behave as an affinity ligand which binds irreversibly to the cannabinoid receptor in brain and which precludes subsequent binding of the cannabinoid ligand [3H]CP-55940.

Adenylyl Cyclases↗

PubMed1996

Cerebrodiene, arachidonyl-ethanolamide, and hybrid structures: potential for interaction with brain cannabinoid receptors.

Cerebrodiene (cis-9, 10-octadecenoamide) was recently isolated from cerebral fluid of sleep-deprived cats and shown to induce sleep in rats. Because this lipid amide is related to arachidonylethanolamide (AEA), and because AEA binds to the cannabinoid receptor with high affinity, we investigated the binding affinity of cerebrodiene and some analogs to the cannabinoid receptor. In addition, we tested the ability of these compounds to act as cannabinoid receptor agonists by determining GTP gamma S binding. Each of the analogs competed for [3H] CP55940 binding, but with relatively low affinity (Ki = 26-44 microM). These analogs were not able to stimulate binding of GTP gamma S at concentrations of 100 microM or 1 mM. We conclude that the sleep-inducing actions of cerebrodiene are not mediated via activation of the cannabinoid receptor.

Animals↗

PubMed1995

Rat brain cannabinoid receptors are N-linked glycosylated proteins.

To study the N-linked glycosylation properties of the CB1 receptor, rat brain membranes were treated with exo- and endoglycosidases. For visualizing CB1 receptors, an antipeptide antibody was raised against the N-terminal 14 amino acids and used to specifically detect the protein by Western blotting. We found that the apparent molecular weight of mature CB1 receptors was 64 kDa. Treatment of membranes with endoglycosidase F shifted the 64 kDa band to the 59 kDa and 53 kDa bands. The latter is consistent with the calculated molecular weight of deglycosylated CB1 receptors. Treatment of membranes with endoglycosidase H and alpha-mannosidase partially shifted the 64 kDa band to 53 kDa band, indicating a portion of the oligosaccharides was of the high mannose type. These data confirmed that the CB1 receptors in brain are N-linked glycoproteins with heterogeneous carbohydrate composition. Among three potential N-linked glycosylation sites on the N-terminus of the CB1 receptor, only two sites are actually glycosylated.

Animals↗

PubMed1995

Pharmacology of cannabinoid receptors.

Two subtypes of cannabinoid receptors, CB1 and CB2, have been described to date, although future investigations may elucidate other receptors. The actions of cannabimimetic agents via CB1 receptors in brain are mediated by GI/O to inhibit adenylate cyclase and Ca2+ channels. Little is known about signal transduction mechanisms utilized by CB2 receptors. Three classes of agonist ligands regulate cannabinoid receptors: cannabinoid, aminoalkyl-indole, and eicosanoid derivatives. Cannabinoid receptors produce analgesia and modify cognition, memory, locomotor activity, and endocrine functions in mammals.

Adenylyl Cyclase Inhibitors↗

PubMed1995

Structure-activity relationships defining the ACD-tricyclic cannabinoids: cannabinoid receptor binding and analgesic activity.

Previous studies of the structure-activity relationships (SAR) for binding of a series of AC-bicyclic cannabinoid structures to the cannabinoid receptors in rat brain (believed to comprise the CB1 subtype) demonstrated the importance of the A-ring aryl C-3 side chain and phenolic hydroxyl substituents, and elucidated the importance of a C-ring hydroxyalkyl substituent [Melvin et al. Mol. Pharmacol. 44, 1008-1015 (1993)]. The present investigation examines the SAR surrounding this region (D-ring) of the molecule that is not present in the structure of delta(9)-THC and other classical cannabinoid compounds. Both rigid fused ring benzo and cyclohexyl derivatives (creating the D-ring) retained binding affinity for the cannabinoid receptor. Extension of ketone or hydroxyl substituents from the C2 position of the D-ring resulted in a 3-fold increase in binding affinity over the unsubstituted structure. However, the fused ring structure is not critical for the interaction with the receptor in as much as opening the ring did not decrease the potency. Extension of the D-ring C-2 alcohol by one carbon in length resulted in a pair of structures, for which the greatest affinity for the CB1 receptor occurred for the hydroxymethyl group in the axial conformation [(+/-)-CP-55,244]. Upon resolution, the latter provided a pair of enantiomers: (-)-CP-55,244 was approximately 3-fold more potent than the racemic mixtures, and (+)-CP-55,244 failed to bind to the CB1 receptor with an IC50 below 1 mM. Opening of the D-ring of these structures resulted in a loss of binding affinity. This study demonstrates that the potency could be optimized in (-)-CP-55,244 for both binding to the CB1 receptor and the biological activity of analgesia. In addition, the rigid positioning of the hydroxypropyl moiety of CP-55,940 enforced by the decalin ring structure of CP-55,244 increased the enantioselectivity by greater than 100-fold. These data define the critical stereochemistry for a region of the nonclassical ACD-tricyclic cannabinoid structure that contributes a potential hydrogen bonding component to the ligand-receptor interaction mechanism. Inasmuch as this region of the molecule is not present on classical ABC-tricyclic cannabinoid compounds, these studies elucidate a unique agonist recognition site on the CB1 receptor.

Analgesics↗

PubMed1994

Inhibitors of arachidonoyl ethanolamide hydrolysis.

Arachidonoyl ethanolamide (anandamide) is a naturally occurring brain constituent that binds to a specific brain cannabinoid receptor (CBR1). An amidase activity (anandamide amidase) in membrane fractions of brain and in cultured neuroblastoma cells rapidly degrades anandamide to arachidonic acid (Deutsch, D. G., and Chin, S. (1993) Biochem. Pharmacol. 46, 791-796). In the current study, analogs of anandamide representing three classes of putative transition-state inhibitor (trifluoromethyl ketones, alpha-keto esters, and alpha-keto amides) were synthesized and tested as inhibitors of anandamide hydrolysis in vitro and as ligands for CBR1. The trifluoromethyl ketones and alpha-keto esters showed nearly 100% inhibition of anandamide hydrolysis in vitro at 7.5 microM inhibitor and 27.7 microM anandamide. Arachidonyl trifluoromethyl ketone was the only synthetic compound in the series of fatty acid derivatives able to displace [3H]CP-55940 binding to CBR1 with a Ki of 0.65 microM. It was also the most effective inhibitor in intact neuroblastoma cells, leading to a 12-fold increase of cellular anandamide levels at 12 microM. From the action of these inhibitors on this hydrolytic enzyme, it seems likely that anandamide is cleaved by a mechanism that involves an active-site serine hydroxyl group. These inhibitors may serve as useful tools to elucidate the role anandamide plays in vivo.

Amidohydrolases↗

PubMed1994

Cannabinoid receptor binding and messenger RNA expression in human brain: an in vitro receptor autoradiography and in situ hybridization histochemistry study of normal aged and Alzheimer's brains.

The distribution and density of cannabinoid receptor binding and messenger RNA expression in aged human brain were examined in several forebrain and basal ganglia structures. In vitro binding of [3H]CP-55,940, a synthetic cannabinoid, was examined by autoradiography in fresh frozen brain sections from normal aged humans (n = 3), patients who died with Alzheimer's disease (n = 5) and patients who died with other forms of cortical pathology (n = 5). In the structures examined--hippocampal formation, neocortex, basal ganglia and parts of the brainstem--receptor binding showed a characteristic pattern of high densities in the dentate gyrus molecular layer, globus pallidus and substantia nigra pars reticulata, moderate densities in the hippocampus, neocortex, amygdala and striatum, and low densities in the white matter and brainstem. In situ hybridization histochemistry of human cannabinoid receptor, a ribonucleotide probe for the human cannabinoid receptor messenger RNA, showed a pattern of extremely dense transcript levels in subpopulations of cells in the hippocampus and cortex, moderate levels in hippocampal pyramidal neurons and neurons of the striatum, amygdala and hypothalamus, and no signal over dentate gyrus granule cells and most of the cells of the thalamus and upper brainstem, including the substantia nigra. In Alzheimer's brains, compared to normal brains, [3H]CP-55,940 binding was reduced by 37-45% in all of the subfields of the hippocampal formation and by 49% in the caudate. Lesser reductions (20-24%) occurred in the substantia nigra and globus pallidus, internal segment. Other neocortical and basal ganglia structures were not different from control levels. Levels of messenger RNA expression did not differ between Alzheimer's and control brains, but there were regionally discrete statistically significant losses of the intensely expressing cells in the hippocampus. The reductions in binding did not correlate with or localize to areas showing histopathology, estimated either on the basis of overall tissue quality or silver staining of neuritic plaques and neurofibrillary tangles. Reduced [3H]55,940 binding was associated with increasing age and with other forms of cortical pathology, suggesting that receptor losses are related to the generalized aging and/or disease process and are not selectively associated with the pathology characteristic of Alzheimer's disease, nor with overall decrements in levels of cannabinoid receptor gene expression.

Aged↗

PubMed1994

Cannabinoid receptor binding and agonist activity of amides and esters of arachidonic acid.

The cannabinoid receptor in brain (CB1) specifically binds delta 9-tetrahydrocannabinol, the predominant central nervous system-active component of marijuana. An eicosanoid found in brain, N-(2-hydroxyethyl)arachidonylamide (anandamide), binds to CB1 with similar affinity. This report considers structure-activity requirements for a series of novel amides and rigid hairpin conformations typified by N-(2-hydroxyethyl)prostaglandin amides, assayed with phenylmethylsulfonyl fluoride inactivation of esterases/amidases. Arachidonyl esters were 30-fold less potent than N-(2-hydroxyethyl)arachidonylamide, showing a rank order of potency of methyl = ethyl > propyl = isopropyl. Within the N-(hydroxyalkyl)arachidonylamide series, a one-carbon increase in chain length increased the potency 2-fold, but continued extension decreased affinity. Substituting the amide for the N-(2-hydroxyethyl)amide function produced a 4-fold loss of affinity. The N-(propyl)-, N-(butyl)-, and N-(benzyl)arachidonylamide derivatives exhibited a 3-fold increase, no change, and a 5-fold decrease, respectively, in affinity, compared with N-(2-hydroxyethyl)arachidonylamide. Both the methoxy ether and the formamide derivatives suffered > 20-fold loss of potency, compared with N-(2-hydroxyethyl)arachidonylamide. N-(2-Aminoethyl)arachidonylamide interacted poorly with CB1. At 100 microM, N-(2-hydroxyethyl)amide analogs of prostaglandin E2, A2, B2, and B1 failed to alter [3H]CP55940 binding to CB1. N-(2-Hydroxyethyl)arachidonylamide inhibited adenylate cyclase with lesser potency but with similar efficacy, compared with desacetyllevonantradol. Extending the length of the hydroxyalkyl moiety by one carbon increased the apparent potency by 1 order of magnitude. The N-(propyl) derivative exhibited a 5-fold greater potency than did the N-(2-hydroxyethyl) analog. It appears that the bulk and length of the moiety appended to arachidonic acid are more important determinants of affinity for CB1 than is hydrogen-bonding capability.

Adenylyl Cyclase Inhibitors↗

PubMed1994

Endogenous cannabinoid receptor binding activity released from rat brain slices by depolarization.

As previously reported by this laboratory, an endogenous factor capable of inhibiting the specific binding of the radiolabeled cannabinoid agonist [3H]CP-55940 to its receptor can be released from nerve terminals in response to an influx of Ca++ induced by an ionophore (Evans et al., 1992). In the present report, we provide evidence that the endogenous ligand for the cannabinoid receptor can be released in response to a depolarizing stimulus (75 mM K+) in the presence of extracellular Ca++. K(+)-evoked release was not observed in the absence of extra-cellular Ca++ and was reduced by the specific calcium channel blockers verapamil and omega-conotoxin. The efflux of cannabinoid receptor binding activity is greatest within 2 min of stimulation with the Ca++ ionophore A23187. Within this period of time, the cannabinoid receptor binding activity was enhanced by the presence of a cocktail of peptidase inhibitors. Examination of the contribution of individual inhibitors for enhancing high K(+)-released material revealed a selectivity for captopril and thiorphan. The specificity of the released factor for the cannabinoid receptor was corroborated by its ability to compete with the aminoalkylindole radioligand [3H]WIN-55212 for binding to this receptor. Fractions from a semi-purified sample of the effluent demonstrated binding to the cannabinoid receptor and behaved as agonists in that these fractions could inhibit adenylate cyclase activity in neuroblastoma membrane preparations.

Animals↗