The overall goal of the proposed research is to elucidate the mechanisms involved in the cross-talk that occurs between the major signaling pathways controlling secretion in mouse parotid glands. The hypotheses to be tested in this proposal are that muscarinic receptors link to several signaling pathways, independently of PLC, via different receptor subtypes and GTP-binding proteins to regulate cAMP metabolism; and that regulation of cAMP metabolism involves an interrelationship between calcium, cGMP and cyclic ADP-ribose (cADPR); and protein kinase C.
Specific aims are: 1) Determination of the role of phospholipases D and A2 in muscarinic regulation of cAMP metabolism. - Phospholipase pathways, muscarinic receptor subtypes, and GTP-binding proteins linked to augmentation and inhibition of accumulation will be identified in intact acini and/or cellular fractions by: enzymatic assays using radiolabeled substrates and/or immunoblot analysis , muscarinic receptor antagonists, and heterotrimeric and monomeric recombinant GTP-binding proteins and/or specific antisera. Modulation of agonist-induced phospholipase activation by phosphorylation will be examined in isolated cellular fractions and permeabilized acini by using radiolabeled [32P]ATP and [32P] orthophosphate, respectively. 2) Determination of the role of cGMP and cADPR in muscarinic regulation of cAMP. - The mechanism(s) involved in muscarinic regulation of cAMP accumulation will be assessed by determining the involvement of nitric oxide synthase (NOS); and by correlating the effects of cGMP and cADPR on calcium influx and release from IP3-sensitive and insensitive intracellular stores with the time course of cAMP accumulation. Modulation of cADPR action will be determined by examining the effects of cGMP on intracellular calcium release; and by phosphorylation of ADP-ribosyl cyclase and the ryanodine receptor. ) Identification and regulation of the enzymes involved in cAMP synthesis and degradation. - Adenylate cyclase(s) will be identified by anti sense ribroprobes and, and by use of transgenic mice deficient in the different types of cyclases. Phosphodiesterases (PDEs) will be identified by measuring the time course and decay of cAMP accumulation in the absence and presence of PDE inhibitors; and by immunoblot analysis using specific PDE antisera. Modulation of PDE activity by phosphorylation will be determined in cell-free extracts and intact acini using radiolabled [32P], followed by immunoprecipitation, immunoblot analysis and enzymatic analysis of the immunoprecipitate. The studies proposed will provide a framework for examining cellular biochemical defects in diseases related to salivary dysfunction.
