Somatostatin, a tetradecapeptide initially from sheep hypothalami, has been localized to the gut and shown to have a variety of actions on gastrointestinal physiology. Although numerous recent advances in research technology have permitted structural analysis of this peptide at the genetic level, little is known regarding the mechanisms of its synthesis, secretion, and biological action. We hope to utilize isolated cell systems from canine stomach to approach these mechanisms. Somatostatin biosynthesis experiments will be conducted in D-cells with labeled amino acid incorporation studies using region-specific antisera that recognize somatostatin tetradecapeptide (S14), the amino terminus of somatostatin octacosapeptide (S28), and the carboxyl- termini of S28(1-12) and S28(1-14). A synthetic peptide corresponding to S42 will be utilized as a substrate for the production of S28 and S14 in order to characterize the principal enzymes responsible for regulating somatostatin synthesis. Studies on regulation of D-cells will be extended to an examination of gene expression by quantifying the influence of various agents on steady state somatostatin mRNA concentrations with dot blots and transcriptional rates using nuclear run-off assays. The mechanisms regulating somatostatin release will be examined with both stimulants and inhibitors, as well as somatostatin itself as an autoregulatory agent. Receptors for these substances will be characterized using pharmacological studies, ligand binding experiments, and affinity cross-linking techniques. The intracellular signal transduction mechanisms responsible for the action of various influences on somatostatin secretion will be assessed by measuring intracellular cAMP by radioimmunoassay, cytosolic free CA++ concentrations using fluorescent dyes, and cell membrane phosphoinositide turnover by measuring changes in membrane phosphatidylinositol content, incorporation of 32P into phosphoinositides, and accumulation of 3H-inositol phosphates. The mechanisms of somatostatin action will be examined by dissecting its inhibitory effect on parietal cells. Somatostatin receptors will be characterized and the effect of the peptide on the various signal transduction mechanisms in the parietal cell will be assessed. The influence of somatostatin on carbonic anhydrase gene expression induced by various secretagogues will be examined by mRNA quantitation and transcriptional rate analysis.
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