The cannabinoids have a long history as drugs of abuse as the active psychoactive ingredients in marijuana. Nevertheless, the molecular mechanisms of action of these drugs are still not clear. A crucial breakthrough in this field occurred with the discovery that delta9- tetrahydrocannabinol (THC) and its analogs bind to brain membrane receptors which are coupled to G-proteins to inhibit adenylyl cyclase. While these cannabinoid receptors may not be responsible for all CNS actions of cannabinoids, they explain many of the specific neuronal actions of these compounds. Moreover, since many other neurotransmitters and neuromodulators bind to G-protein-coupled receptors, these results suggest that cannabinoids may not simple be exogenous drugs of abuse but also be members of a novel class of neurotransmitters. The current study will explore components of the putative endogenous cannabinoid system in the brain by utilizing both traditional cannabinoid ligands as well as a novel class of cannabinoid ligands known as the aminoalkylindoles. These receptors can be studied by radioreceptor binding to brain membranes, by GTP-dependent inhibition of adenylyl cyclase in cerebellar membranes and cerebellar granule cells, by stimulation of low k/m GTPase in cerebellar membranes, and by inhibition of electrically-induced contractions of mouse vas deferens. This project will focus on cannabinoid actions in cultured cerebellar granule cells, which represent a novel, non-transformed cell culture system for the study of cannabinoid receptors. In the cerebellar granule cells, the effects of cannabinoids on glutamate release will be explored, in order to demonstrate that these compounds act like other inhibitory agonists in cerebellum to inhibit glutamate release. Specific antibody and antisense experiments will be utilized to identify the specific G-proteins coupled to cannabinoid receptors in different areas of the brain. A major component of this project will continue to characterize endogenous cannabinoid ligands from brain extracts. The previous grant period isolated one such compound from bovine brain. Although its structure is not yet known, many of its chromatographic properties suggest that it is structurally different from anandamide, an ethanolamine derivative of arachidonic acid recently identified as a potential endogenous cannabinoid. These studies will determine the structure and localization of this novel cannabinoid liqand, and compare these properties to those of other compounds isolated by other laboratories.
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