calcimycin and anandamide

2-arachidonoylglycerol (2-AG) is an endogenous ligand for the cannabinoid receptors with a variety of potent biological activities. In this study, we first examined the effects of potassium-induced depolarization on the level of 2-AG in rat brain synaptosomes. We found that a significant amount of 2-AG was generated in the synaptosomes following depolarization. Notably, depolarization did not affect the levels of other molecular species of monoacylglycerols. Furthermore, the level of anandamide was very low and did not change markedly following depolarization. It thus appeared that the depolarization-induced accelerated generation is a unique feature of 2-AG. We obtained evidence that phospholipase C is involved in the generation of 2-AG in depolarized synaptosomes: U73122, a phospholipase C inhibitor, markedly reduced the depolarization-induced generation of 2-AG, and the level of diacylglycerol was rapidly elevated following depolarization. A significant amount of 2-AG was released from synaptosomes upon depolarization. Interestingly, treatment of the synaptosomes with SR141716A, a CB1 receptor antagonist, augmented the release of glutamate from depolarized synaptosomes. These results strongly suggest that the endogenous ligand for the cannabinoid receptors, i.e. 2-AG, generated through increased phospholipid metabolism upon depolarization, plays an important role in attenuating glutamate release from the synaptic terminals by acting on the CB1 receptor.

Topics: Animals; Arachidonic Acids; Brain; Calcimycin; Calcium Channel Blockers; Camphanes; Diglycerides; Endocannabinoids; Estrenes; Fatty Acids; Glycerides; Male; Membrane Potentials; Neuromuscular Depolarizing Agents; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Pyrrolidinones; Rats; Rats, Wistar; Receptors, Cannabinoid; Rimonabant; Synaptosomes

We examined the effects of the endocannabinoide-anandamide (AEA), the synthetic cannabinoid, WIN55,212-2, and the active phorbol ester, 4-beta-phorbol 12-myristate 13-acetate (4-beta-PMA), on the release of [(3)H]d-Aspartate ([(3)H]d-ASP) from rat hippocampal synaptosomes. Release was evoked with three different stimuli: (1) KCl-induced membrane depolarization, which activates voltage-dependent Ca(2+) channels and causes limited neurotransmitter exocytosis, presumably from ready-releasable vesicles docked in the active zone; (2) exposure to the Ca(2+) ionophore-A23187, which causes more extensive transmitter release, presumably from intracellular reserve vesicles; and (3) K(+) channel blockade by 4-aminopyridine (4-AP), which generates repetitive depolarization that stimulates release from both ready-releasable and reserve vesicles. AEA produced concentration-dependent inhibition of [(3)H]d-ASP release stimulated with 15 mM KCl (E(max)=47.4+/-2.8; EC(50)=0.8 microM) but potentiated the release induced by 4-AP (1mM) (+22.0+/-1.3% at 1 microM) and by A23187 (1 microM) (+98.0+/-5.9% at 1 microM). AEA's enhancement of the [(3)H]d-ASP release induced by the Ca(2+) ionophore was mimicked by 4-beta-PMA, which is known to activate protein kinase C (PKC), and the increases produced by both compounds were completely reversed by synaptosome treatment with staurosporine (1 microM), a potent PKC blocker. In contrast, WIN55,212-2 inhibited the release of [(3)H]d-ASP evoked by KCl (E(max)=47.1+/-2.8; EC(50)=0.9 microM) and that produced by 4-AP (-26.0+/-1.5% at 1 microM) and had no significant effect of the release induced by Ca(2+) ionophore treatment. AEA thus appears to exert a dual effect on hippocampal glutamatergic nerve terminals. It inhibits release from ready-releasable vesicles and potentiates the release observed during high-frequency stimulation, which also involves the reserve vesicles. The latter effect is mediated by PKC. These findings reveal novel effects of AEA on glutamatergic nerve terminals and demonstrate that the effects of endogenous and synthetic cannabinoids are not always identical.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Benzoxazines; Calcimycin; Calcium Channel Blockers; Capsaicin; Endocannabinoids; Glutamates; Hippocampus; Male; Morpholines; Naphthalenes; Polyunsaturated Alkamides; Presynaptic Terminals; Rats; Rats, Wistar; Synaptosomes; Tetradecanoylphorbol Acetate

Anandamide (N-arachidonoylethanolamine, AEA), an endogenous cannabinoid receptor agonist, causes potent vasodilation in the cerebral circulation through an endothelial-dependent or -independent mechanism. We have investigated the processing of [3H]AEA in cultured mouse cerebral microvascular endothelial cells (MEC) in order to better understand its mechanism of action in the cerebral vasculature. These cells took up anandamide very quickly, reaching a maximum value in 5 min and remaining at that level for at least 8 h. Analysis of the cell lipids demonstrated that, in addition to free anandamide, radioactivity was incorporated into phosphatidylcholine (PC), phosphatidylinositol (PI), and phosphatidylethanolamine (PE) in a time-dependent manner. Analysis of the hydrolyzed cell lipids indicated that anandamide was converted to arachidonic acid, a process that was inhibited by the selective fatty acid amide hydrolase inhibitor oleyl trifluoromethyl ketone (OTMK). Phospholipase A2 (PLA2) hydrolysis of the PC, PI, and PE fractions indicated that the arachidonic acid formed from anandamide was esterified predominately into sn-2 position of the endothelial phospholipids. Furthermore, anandamide and arachidonic acid were released when the cells were incubated with A23187. These results suggest that the biological activity of anandamide might be regulated by its rapid uptake and calcium-dependent release in endothelial cells, and conversion of anandamide to arachidonic acid might serve as an inactivation process in the cerebral microcirculation.

Topics: Amidohydrolases; Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cannabinoid Receptor Agonists; Cannabinoid Receptor Modulators; Capillaries; Cerebellum; Endocannabinoids; Endothelium, Vascular; Ionophores; Ketones; Mice; Phospholipases A; Phospholipases A2; Phospholipids; Polyunsaturated Alkamides; Receptors, Cannabinoid

We investigated whether 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, is involved in acetylcholine- and calcium ionophore A23187-induced relaxations in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME) and indomethacin, which is considered to be mediated by endothelium-derived hyperpolarizing factor (EDHF). In rabbit mesenteric arterial rings pre-constricted with noradrenaline, 2-arachidonoylglycerol caused concentration-dependent relaxation. The 2-arachidonoylglycerol-induced relaxations were not affected by endothelium removal. N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-caroxamide (SR141716A) and 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morholinyl-1H-pyrazole-3-carboxamide (AM281), cannabinoid CB(1) receptor antagonists, significantly attenuated 2-arachidonoylglycerol-induced relaxation and the acetylcholine-induced relaxation only slightly, but not the calcium ionophore A23187-induced relaxation. On the other hand, charybdotoxin plus apamin, K(+) channel blockers, significantly attenuated acetylcholine and calcium ionohore A23187-induced relaxations but not 2-arachidonoylglycerol-induced relaxations. These results suggest that 2-arachidonoylglycerol can cause relaxations via cannabinoid CB(1) receptors, but is not involved in EDHF-mediated relaxations.

Topics: Animals; Arachidonic Acids; Biological Factors; Calcimycin; Calcium Channel Blockers; Endocannabinoids; Glycerides; Ionophores; Male; Neurotransmitter Agents; Piperidines; Polyunsaturated Alkamides; Pyrazoles; Rabbits; Receptors, Cannabinoid; Receptors, Drug; Rimonabant; Vasodilation

Polyunsaturated N-acylethanolamines (NAEs), including anandamide (20:4n-6 NAE), elicit a variety of biological effects through cannabinoid receptors, whereas saturated and monounsaturated NAEs are inactive. Arachidonic acid mobilization induced by treatment of intact mouse peritoneal macrophages with Ca2+ ionophore A23187 had no effect on the production of NAE or its precursor N-acylphosphatidylethanolamine (N-acyl PE). Addition of exogenous ethanolamine resulted in enhanced NAE synthesis by its N-acylation with endogenous fatty acids, but this pathway was not selective for arachidonic acid. Incorporation of (18)O from H2 (18)O-containing media into the amide carbonyls of both NAE and N-acyl PE demonstrated a rapid, constitutive turnover of both lipids.

Topics: Acylation; Animals; Arachidonic Acids; Calcimycin; Carbon Radioisotopes; Endocannabinoids; Ethanolamine; Ethanolamines; Ionophores; Macrophages, Peritoneal; Male; Mice; Mice, Inbred ICR; Polyunsaturated Alkamides

Anandamide (arachidonylethanolamide), 5,8,11,14-eicosatetraenamide, (N-2-hydroxyethyl), was tested for bronchodilator and anti-inflammatory activities. Conscious guinea pigs were given cumulative i.v. doses of anandamide (1.0, 3.0, and 10.0 mg/kg) to assess its effect on dynamic compliance (Cdyn), total pulmonary resistance (RL), tidal volume (VT) and breathing frequency (f). Other guinea pigs were exposed to an aerosol of A23187 (6S-[6alpha(2S*,3S*),8beta(R*),9beta,11alpha]-5- (methylamino)-2-[[3,9,11-trimethyl-8-[1-methyl-2-oxo-2-(1H-pyrrol-2-yl)e thyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-4-benzoxazole carboxylic acid) until Cdyn decreased by 50% (approximately 5 min) and at 20 min, cumulative i.v. doses of anandamide (1.0, 3.0, and 10.0 mg/kg) were administered and reversal of Cdyn examined. After the final dose of anandamide, the animals were killed and excised lung gas volumes (ELGV), i.e., pulmonary gas trapping, measured. Other animals were treated i.v. with anandamide (10.0 mg/kg), exposed to an aerosol of A23187 until labored breathing began, and then killed 1 h later. Anandamide did not significantly affect Cdyn, RL, VT and f. ELGV values of anandamide-treated guinea pigs were not different from those of vehicle-treated animals. Anandamide failed to reverse A23187-induced decreases in Cdyn and to reduce A23187-associated ELGV increases. Also, it did not prevent the prolonged airway obstruction caused by A23187. Histological evaluation revealed that anandamide significantly reduced A23187-related airway epithelial injury and pulmonary leukocytosis. However, it did not prevent A23187-induced peribronchiolar granulocytic accumulation. Our results suggest that in vivo anandamide has minimal direct airway smooth muscle-related actions, however it may possess modest anti-inflammatory properties.

Topics: Aerosols; Airway Obstruction; Airway Resistance; Animals; Anti-Inflammatory Agents; Arachidonic Acids; Bronchodilator Agents; Calcimycin; Endocannabinoids; Guinea Pigs; Injections, Intravenous; Lung; Lung Compliance; Male; Polyunsaturated Alkamides; Pulmonary Ventilation; Receptors, Cannabinoid; Receptors, Drug; Tidal Volume

The hypothesis that the capability of agents to mobilize arachidonic acid (AA) could predict increased anandamide (ANA) synthesis in a macrophage cell line has been examined. Lipopolysaccharide (LPS), platelet-activating factor (PAF) and cannabinoids such as Delta9-tetrahydrocannabinol (THC) and anandamide were all found to be agonists for the release of AA and led to increased ANA synthesis in RAW264.7 mouse macrophage cells. Nitric oxide, in contrast, stimulated AA release without raising ANA levels. ANA stimulation of its own synthesis indicates the existence of a positive feedback mechanism. The possible involvement of the CB2 receptor in THC-mediated AA release and ANA synthesis is addressed using the antagonist SR144528. ANA synthesis is also increased by the combination of calcium ionophore and indomethacin, suggesting that ANA is metabolized by a cyclooxygenase in this system. The data imply that ANA could play a role in the response of the immune system to cannabinoids and bacterial endotoxins and that AA mobilization is a predictor for increased ANA synthesis.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cell Line; Dronabinol; Endocannabinoids; Immune System; Indomethacin; Ionophores; Lipopolysaccharides; Macrophages; Mice; Platelet Activating Factor; Polyunsaturated Alkamides; Receptors, Cannabinoid; Receptors, Drug

Glutamate-induced formation of N-acylethanolamine (NAE) and N-acylphosphatidylethanolamine (NAPE) was studied in primary cultures of mouse neocortical neurons prelabeled with [14C] ethanolamine. The formation of these two lipids was dependent on the maturity of the cell culture; i.e., no glutamate-induced formation was seen in 2-day-old cultures, whereas glutamate induced a pronounced formation in 6-day-old cultures. The calcium ionophore A23187 (2 microM) stimulated, within 2 h, formation of NAPE in 2-day-old cultures (fourfold) as well as in 6-day-old cultures (eightfold). Glutamate exerted its effect via NMDA receptors as seen by the inhibitory action of the NMDA-selective receptor antagonists D-(-)-2-amino-5-phosphonovalerate and N-(1-(2-thienyl)cyclohexyl)piperidine and the lack of effect of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate-receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In 6-day-old cultures, exposure to NMDA (100 microM for 24 h) induced a linear increase in the formation of NAPE and NAE as well as a 40-50% neuronal death, as measured by a decrease in cellular formazan formation [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay]. The increase in NAPE and NAE could be detected earlier than the neuronal death. Neither cyclic AMP, cyclic GMP, nitric oxide, protein kinase C, nor peroxidation appears to be involved in the formation of NAPE and NAE, as assessed by the use of different pharmacological agents. Exposure to 5 mM NaN3 for 8 h resulted in a >80% decrease in the cellular MTT staining and a pronounced linear increase in the formation of NAE and NAPE (reaching 25-30% of total labeling). [14C]Anandamide was also formed in [14C]arachidonic acid-labeled neurons exposed to NaN3. No NAPE formation was detected in A23187-stimulated mouse astrocytes, rat Leydig cells and cardiomyocytes, and several other cells. These results suggest that the glutamate-induced formation of NAPE and NAE was mediated by the NMDA receptor and the formation of these lipids may be associated with neuronal death.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cell Death; Cells, Cultured; Cerebral Cortex; Embryo, Mammalian; Endocannabinoids; Ethanolamine; Ethanolamines; Glutamic Acid; Ionophores; Mice; Neurons; Phencyclidine; Phosphatidylethanolamines; Polyunsaturated Alkamides; Receptors, N-Methyl-D-Aspartate; Time Factors