Pharmacodynamic tolerance, which is characterized by adaptive changes within affected systems, has been widely described for many structurally diverse agents among which drugs of abuse have been the most extensively studies. Cannabinoids, including the primary psychoactive component of marihuana, delta9-THC, are agents which have been established to readily produce this phenomenon. Despite the fact that tolerance has been described ina variety of experimental systems, the mechanisms responsible for these adaptive changes are not well understood. The overall goal of this 4 year research plan is to utilize=e an immunologic experimental model system to investigate cellular elements responsible for signal transduction through the cannabinoid receptor to identify their role in mediating cannabinoid-induced tolerance by testing the following Hypothesis: Tolerance to immune suppression by delta9-THC is mediated through a decrease in signaling through the cannabinoid receptor present on lymphocytes. This diminution in receptor associated signal transduction is mediated either through desensitization and/or down regulation of cannabinoid receptors following extended receptor agonist stimulation. This hypothesis is based on the following critical observations made from work conducted in our laboratory: (1) Mouse splenocytes possess a functional G-protein coupled cannabinoid receptor which is responsible for mediating the immunosuppressive effects associated with exposure to cannabimimetic agents; (2) Characterization thus far of this receptor on spleen cells strongly suggests that it is very similar if not identical to that present in neuronal tissue; (3) Immune suppression by cannabinoids is at least in part mediated through inhibition of adenylate cyclase activity in mouse spleen cells; (4) Tolerance of splenocytes to immune inhibition by delta9-THC is readily induced by chronic delta9-THC treatment of mice or through the incubation of spleen cells in vitro in the presence of delta9- THC; and (5) Immune tolerance to delta9-THC is paralleled by a loss in the ability of cannabinoids to inhibit adenylate cyclase in splenocytes. Based on these observation, our hypothesis as it relates to cannabinoid-induced tolerance will be tested using the following specific aims; (1) Compare changes in receptor function in tolerized and nontolerized splenocytes, specifically, (a) cannabinoid receptors number (Kd); (b) affinity of receptor-ligand binding (Bmax); and (c0 cannabinoid receptor mRNA; (2) Investigate the mechanism responsible for the loss of adenylate cyclase inhibition by delta9-THC following chronic delta9-THC exposure in murine lymphocytes; (30 Identify differences in Gi protein function between tolerized and nontolerized lymphocytes; and (40 Identify differences in protein kinase A activity between tolerized and nontolerized lymphocytes in the presence and absence of cannabinoids. One of the greatest strengths of this proposal is the model system which; (1) capitalizes on the use of isolated cells in primary culture, rather than cell-lines; (2) possess a functional receptor which is coupled to an identified second messenger system; and (3) can be manipulated both in vivo and in vitro in order to correlate biochemical changes to functional changes as they relate to the mechanisms of tolerance.
