The objective of this proposal is to characterize the receptors, signalling pathways and cellular mechanisms involved in relaxation of gastric smooth muscle by transmitters of the myenteric plexus. The focus is on peptide receptors coupled to multiple interactive pathways that mediate or modulate relaxation. The experimental approach exploits a technique for selective, site-directed receptor protection that allows each pathway to be examined separately and is based on the discovery of an important link between the myenteric neuropeptide, VIP release; in turn, VIP stimulates (i.e., re-generates) NO in target muscle cells. The link to VIP confers receptor specificity on NO signalling and engages both limbs of the cyclic nucleotide system that sustains relaxation. Accordingly, the Specific Aims of the proposal are: (1) to characterize receptors for NPY, opioid peptides, acetylcholine and 5-HT, which are coupled to IP, generation and IP-dependent Ca release via one receptor subtype, and to increase (5-HT and muscarinic M) or decrease (NPY Y, opioid mu, delta, kappa, and muscarinic M) in cAMP via a distinct receptor subtype; (2) to characterize receptors for VIP and the myenteric homologous peptide, PACAP, in isolated gastric muscle cells which are coupled to stimulation of adenylate cyclase and NO-dependent stimulation of guanylate cyclase; our continuing studies in plasma membranes derived from isolated muscle cells have demonstrated the presence of a membrane- bound, constitutive, G protein-coupled, Ca2+/calmodulin-sensitive NO synthase which is specifically activated by VIP and PACAP; and (3) to identify the loci of regulatory phosphorylation by cAMP- and cGMP- dependent protein kinase (cA-kinase and cG-kinase) which mediate relaxation. In comparative studies using cA-kinase and cG-kinase inhibitors including PKI (6-22) amide, a highly specific inhibitor of cA-kinase, we have identified concentrations of inhibitors that selectively block cA-kinase activity (H-89, Rp-cAMPS) and cG-kinase activity (the indole carbazole KT5823). This development set the stage for identification of events in the signalling cascade that, based on preliminary results, are preferentially regulated by cA-kinase (IP3 generation, IP3-dependent Ca2+ release) and cG-kinase (sarcoplasmic Ca uptake) or equally regulated by both kinases (Ca/calmodulin sensitivity of MLC kinase and MLC phosphorylation). The combined approach embodied the three aims should advance our understanding of the cellular mechanisms mediating smooth muscle relaxation.
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