The overall objectives of this proposal are to uncover putative differences in the intracellular signal transduction pathways resulting from stimulation of different phosphoinositide turnover-coupled neurotransmitter receptors in the same nerve cell, and to understand the functional significance of such differences. Although all PI-turnover-coupled receptors stimulate phospholipase C, much evidence indicates that protein kinase C is not always activated, and that the kinetics of this and of subsequent intracellular reactions are not identical in all systems. Such distinctive intracellular pathways may be important in understanding mechanisms of transmembrane signaling, neuronal communication, and of the acquisition of long-term memory. The immediate aims are to compare several intracellular biochemical events resulting from stimulating muscarinic histamine and bradykinin receptors in SK-N-SH human neuroblastoma cells, a cell line that expresses all three of these receptors. Thus, cells will be stimulated with various combinations of each of these agonists, and the following resulting biochemical changes will be studied: a) the dose-response relationship and the additivity of the resulting PI hydrolysis; b) the pertussis toxin, neomycin, and calcium sensitivities of the resulting PI hydrolysis; c) the detailed kinetics of accumulation of each measurable phosphoinositide; d) the resulting activation of protein kinase C isozymes; e) the phosphorylation of membrane proteins resulting from activation of each of these receptors; f) the kinetics and spatial distribution of calcium mobilization with fura2-loaded cells. In addition, the electrophysiological and functional consequences of stimulating each of these receptors will be studied. Other receptor-mediated intracellular changes will also be studied, including cAMP, cGMP, and arachidonic acid levels. In order to determine whether differences can be detected in the signal transduction pathways elicited from stimulating two different muscarinic receptor subtypes that coupled preferentially to PI turnover (m1 and m3 subtypes), these studies will be extended to include A9L cells transfected with the m1 and the m3 muscarinic receptor subtypes.
