The goal of this research is to continue studies of receptor-signal transduction subserving the actions of neurotransmitters in the colon. Our focus in this renewal is to broaden and integrate our efforts from studies of the actions of individual agents (particularly acetylcholine) to the study of other important agonists, as well as ACh, acting in concert. Our interest is focused on questions developed as a result of the overall efforts of the program. Thus, we will focus on questions developed as a result of the overall efforts of the program. Thus, we will focus on the actions of those compounds, known to be released from colonic nerves, that regulate motility on a moment to moment basis. We will examine proximal colon circular and longitudinal muscles and the cells prepared from them. Cells will be employed as both freshly isolated cells and cells grown in culture. We will continue studies in our canine model as well as human surgical specimens and now add normal primate (rhesus and cynomologus) tissues. We will determine the origin and distribution of receptors subserving both contraction and relaxation of colonic smooth muscle and their biochemical signaling as follows: i] Do receptors for excitatory neurokinins (SubP and NKA) and receptors for the inhibitory transmitter vasoactive intestinal peptide (VIP) exist on interstitial cells of Cajal (ICC) and/or on smooth muscle cells in the proximal colon? ii] What is the nature and distribution of receptors for nitric oxide (guanylate cyclase) in the ICC and smooth muscle cells of the colon? iii] Do ICC possess a distinct compliment of muscarinic receptors? iv] Are there purinergic (nucleoside or nucleotide) receptors in the colonic cells and what is their nature and distribution between ICC and smooth muscle cells? v] What biochemical signal- transduction events (phospholipase activation, cyclic nucleotide production, protein kinase activation) follow the actions of these neurotransmitters in ICC and smooth muscle cells in the colon and how do these pathways interact? vi] What signals accrue following stimulation of M4 receptors recently discovered in longitudinal muscle? vii] What are the functional consequences of adenylate and guanylate cyclase-coupled receptor stimulation and phosphodiesterase inhibitor-mediated cyclase inhibition in colonic muscle contraction? Are the inhibitory actions of the cyclic nucleotides additive or synergistic? In addition to these primary specific aims we will collaborate with projects 1 and 5 in order to learn; a) what role(s) do cAMP and cGMP play in the agonist-mediated regulation of intracellular Ca2+? b) What is the role of receptor mediated IP3 and IP4 generation in the release of Ca2+ in ICC and smooth muscle cells? Our experiments will improve our understanding of the regulation of colonic function and because we can employ human and primate tissues, we have an opportunity to probe the application of our theories to humans.
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