Modulation of neuronal muscarinic acetylcholine receptor sensitivity by phosphoinositide hydrolysis will be investigated in mouse neuroblastoma cells (clone N1E-115). Receptor-mediated increase in phosphoinositide hydrolysis results in the formation of inositol trisphosphate and diacylglycerol. The former has been implicated in releasing Ca2+ from intracellular stores, while the latter has been suggested to be the endogenous activator of protein kinase C. Activation of muscarinic receptors in mouse neuroblastoma cells results in Ca2+ release and cyclic GMP formation; with the latter response showing an absolute dependence on Ca2+. Furthermore, muscarinic agonists elicit a rapid Ca2+- independent increase in phosphoinositide hydrolysis in these cells. The present proposal aims at investigating the relationship between muscarinic receptor-mediated formation of inositol trisphosphate and the induction of Ca2+ release and cyclic GMP synthesis using several experimental designs. In addition, the role of inositol trisphosphate in desensitization of muscarinic receptor-mediated increase in Ca2+ release and cyclic GMP formation will be tested. Finally, the effects of diacylglycerol on receptor sensitivity will be examined. Thus, the present proposal aims mainly at elucidating the possible role of phosphoinositide hydrolysis in fine-tuning the sensitivity of muscarinic receptors, and also at demonstrating the existence of a possible """"""""cross-talk"""""""" between the various muscarinic receptor second messengers. The plausibility of this hypothesis is supported by several pieces of evidence which are presented in the proposal, both from the literature and from preliminary data. Investigation of such mechanisms which underlie the modulation of muscarinic receptor sensitivity in nervous tissue is of utmost value, due to the important role of muscarinic receptors in brain function, in addition to their implication in serious side effects of psychotropic drugs and in diseases of the central nervous system.
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