The goal of the experiments proposed in this application is to determine the biochemical events that lead to cannabinoid-induced arachidonic acid release. The studies to be carried out will be focused on the signal transduction processes following cannabinoid binding to its receptor. The question of receptor mediation in this process will be addressed by the use of anti sense oligodeoxynucleotides in addition to the more conventional ligand binding approach. Oligos will be designed to reduce, or eliminate completely, the expression of the cannabinoid receptor genes and compared with subsequent changes in arachidonic acid release. The term cannabinoid is meant to include both exogenous substances, such as THC, and endogenous molecules like anandamide. The systems to be used for these investigations will be isolated, intact cells such as mouse peritoneal macrophages, neuroblastoma and Chinese-hamster ovary lines. The experiments are aimed at defining roles for phospholipase D, protein kinase C, MAP kinase and phospholipase A2 in mediating cannabinoid- stimulated release of arachidonic acid and the subsequent production of eicosanoids. Direct measurement of enzyme activities will be made over a range of cannabinoid concentrations and compared with arachidonic acid release under identical conditions. Time course studies will also be done to help elucidate the sequence of events involved. Specific inhibitors of the various enzymes will be used where possible to supplement the data obtained by direct measurement. Eicosanoids, such as prostaglandin E2, have been shown to be important mediators of cannabinoid action in humans as well as in animal models so that a detailed understanding of how their levels are elevated is of importance. The recent discovery of an endogenous cannabinoid, anandamide, that may function as a brain neuroregulator, lends even greater significance to the aims of this proposal because of its structural nature, namely, the ethanolantide derivative of arachidonic acid. The significance of the anandamide discovery to the """"""""prostaglandin hypothesis"""""""" of cannabinoid action is not clear at this time, however, it is unlikely that the eicosanoid nature of anandamide is only coincidental. A better understanding of THC/anandamide biochemical actions could improve our understanding of processes such as memory, motor coordination cognitive ability, time sense and self-perception. In addition, some insight might be gained into the question of the molecular basis for cannabis abuse. The recent reports on the increasing use of marihuana, especially among preadolescents, has again focussed attention on efforts to understand the actions of this drug.
