Cannabinoid signaling in rat cerebellar granule cells: G-protein activation, inhibition of glutamate release and endogenous cannabinoids.
Previous studies have indicated that cannabinoids inhibit presynaptic neurotransmitter release in brain through CB1 receptors. To examine this issue in a primary neuronal culture system, rat cerebellar granule cells (CGCs) were prepared. [35S]GTPgammaS binding assays in saponin-permeabilized CGCs showed that G-protein activation by the CB1 agonist, WIN55212-2, and adenosine A1 agonist, phenylisopropyladenosine, was maximal during the second week in culture. Delta9-tetrahydrocannabinol stimulated [35S]GTPgammaS binding to a lesser degree than WIN55212-2, and the antagonists SR141716A and AM281 acted as inverse agonists in intact CGCs, but not in CGC membrane preparations. Ten micromolar WIN55212-2 and Delta9-tetrahydrocannabinol decreased depolarization-evoked efflux of [3H]-D-aspartate from CGCs by 32% and 13%, respectively. SR141716A and AM281 increased [3H]-D-aspartate release by 28%. The fatty acid amide hydrolase (FAAH) inhibitor phenylmethylsulfonyl fluoride (PMSF) and the anandamide uptake inhibitor AM404 inhibited transmitter release, implying that the antagonist effects were mediated by blockade of endocannabinoid activity. Levels of endocannabinoids (both anandamide and 2-arachidonyl glycerol [2-AG]) in extracts of the cells and cell incubation buffer were increased by PMSF pre-treatment. Depolarization with KCl significantly decreased the amount of anandamide and 2-AG in PMSF-treated CGCs. These results suggest that endogenous cannabinoids inhibit neurotransmitter release in CGCs, which may also release endocannabioids upon neural stimulation.
Breivogel
Christopher S
CS
Department of Pharmaceutical Sciences, Campbell University School of Pharmacy, P.O. Box 1090, Buies Creek, NC 27506, USA. breivogel@mailcenter.campbell.edu
Walker
J Michael
JM
Huang
Susan M
SM
Roy
Mary Beth
MB
Childers
Steven R
SR
D000818:Animals; D001095:Arachidonic Acids / Q000494:pharmacology; D001224:Aspartic Acid / Q000378:metabolism; D002186:Cannabinoids / Q000494:pharmacology*; D002478:Cells, Cultured; D002531:Cerebellum / Q000187:drug effects / Q000502:physiology*; D063388:Endocannabinoids; D005260:Female; D019204:GTP-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D053284:Polyunsaturated Alkamides; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
D001095:Arachidonic Acids; D002186:Cannabinoids; D063388:Endocannabinoids; D053284:Polyunsaturated Alkamides; D001224:Aspartic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D018698:Glutamic Acid; D019204:GTP-Binding Proteins; C078814:anandamide