The aim of this proposal is to pursue studies on the regulatory function of peptides present in neurons of the myenteric and submucosal plexuses of the intestine. We have elected to study isolated intestinal muscle cells as the postjunctional targets of myenteric neuropeptides, and an endocrine cell (CCK) and putative paracrine cell (somatostatin) as the targets of submucosal neuropeptides. Several muscle cell preparations are currently being used to characterize peptide receptors pharmacologically with selective antagonists and immunochemically by radioligand binding. The preparations include suspensions of muscle cells isolated separately from the longitudinal and circular muscle layers, single perfused muscle cells, and cultures of human intestinal muscle cells. Receptors for the following myenteric neuropeptides will be examined: opioid peptide derivatives of proenkephalin and prodynorphin; mammalian tachykinins (SP, SK and NK); mammalian bombesins (GRP1-27, GRP18-27 and neuromedin B); CCK-8 and CCK-33; VIP and PHI/PHM. Receptor enrichment with protective ligands and site-directed alkylating agents will be used as an adjunct in characterization of multiple receptor subtypes. Interaction between co-localized neuropeptides will be examined with respect to enhancement or desensitization of specific receptors. Signal transduction mechanisms for contractile and relaxant neuropeptides will be determined in intact and permeabilized muscle cells. In addition to measurement of contraction and relaxation, the following types of measurements have been validated for use in muscle cells: cytosolic free Ca2+ with quin2 and fura2, net 45Ca2+ flux, membrane potential with voltage-sensitive fluorescent dyes, cyclic AMP and GMP and the phosphorylated fraction of myosin light chain. The role of inositol trisphosphate in Ca2+ mobilization, protein kinase C in sustained contraction, and cyclic nucleotides in relaxation will be evaluated. The regulation of CCK and somatostatin secretion by submucosal neuropeptides will be examined in three versatile in vitro preparations: vascularly perfused intestinal segments, superfused mucosal slices, and intestinal mucosal membranes mounted in an Ussing chamber. In earlier studies on the stomach, we have shown these preparations to be suitable for characterization of the regulatory influence of cholinergic and peptidergic neurons on endocrine and paracrine secretion.
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