The broad and long term objectives of the research are to understand, in complete detail, the mechanisms by which inhibitors of insulin secretion e.g. galanin, norepinephrine and somatostatin, exert their effects on the pancreatic beta-cell. The areas to be studied and understood include the interactions between, a. the inhibitors and their receptors, b. the receptors and their activation of G-proteins, and c. the activated G- proteins and their targets in the beta-cell. While development work will be done on beta-cell lines and rodent islets, the major thrust eventually will be the description of these events in human beta-cells. It is intended to determine the G-proteins which interact with the norepinephrine (alpha2-adrenergic) and somatostatin receptors in the RINm5F cell. This will give us a picture of the 6-protein/receptor interactions for three inhibitors of insulin secretion in one cell type (we have done this for galanin and worked out the methods for these analyses). We will measure the efficiency of the three receptors to activate the interacting G-proteins, by determination of the rate of GTPgammaS binding (i.e. the rate of activation of the G-proteins) and the rate of specific receptor-stimulated GTPase activity (i.e. G-protein turnover rate) in conjunction with the determination of receptor numbers in the cell membranes. Subsequently, we will determine which G-proteins mediate the effects of norepinephrine, somatostatin and galanin on their individual intracellular targets (the KATP and L-type Ca2+ channels, on adenylyl cyclase and on the distal inhibitory site). We will apply these techniques, developed on the RINm5F cell and on rat islets, to the study of the human beta-cell. Using human islets, we will explore the mechanisms of action of inhibitors of insulin release. Thus we will determine: a. Whether the inhibitors of insulin release act on the human beta-cell as they do in rodent beta-cells and cloned cell lines. We will examine the effects of the inhibitors on the KATP channel, the L-type Ca2+ channel, adenylyl cyclase activity and the distal inhibitory mechanism. b. Which G-proteins are present in the human islet. c. Which of the G-proteins present interact with the receptors for norepinephrine and somatostatin. Galanin will be studied only if we can demonstrate that it has an inhibitory effect on stimulated release. d. The efficiency of the different receptors in activating the G-proteins, and the turnover rates of the activated alpha-subunits. If the inhibitors act on the same intracellular targets in the human beta- cell as they do in the rodent cell (this is assumed but not known), we will determine: e. Which G-proteins mediate the effects on those targets (KATP and Ca2+ channels, adenylyl cyclase and distal step). The information gained will advance our knowledge of the signal transduction mechanisms which control insulin secretion in the pancreatic beta-cell, and help in our understanding of some types of diabetes.
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