The goals of this work are to understand the mechanisms of action of inhibitors of insulin release. The work will combine physiological, biochemical and immunological techniques and will be performed on cloned beta-cell lines and rodent islets. The studies will focus on the mechanism underlying the distal inhibitory effect by which norepinephrine and other physiologic inhibitor block exocytosis. We have found a remarkable effect of inhibitors on two proteins, syntaxin and SNAP-25, which most likely inhibits exocytosis We have found a remarkable effect of inhibitors on two proteins, syntaxin and SNAP-25, specifically reduced by inhibitor action. Furthermore, a GST-syntaxin fusion protein binds to SNAP-25 in cell lysates prepared from beta-cells under control conditions but not after the cells are treated with norepinephrine. In addition, we found that inhibitors of protein actylation block the inhibitory effect of norepinephrine at the distal site of action. The other known sites of action of inhibitors have not yet been examined. Thus, one hypothesis to be tested, with respect to the distal site, is that inhibitors of insulin secretion acting at their membrane receptors on the beta-cell activate PTX-sensitive Gi and Go proteins, which in turn activate a protein acyl transferase. Actylation of a protein (e.g. SNAP-25 or a protein that associates with SNAP-25) would then interfere with the interaction between syntaxin and SNAP-25 and prevent exocytosis. Therefore, the overall aims are 1) To determine the effects of inhibitors of insulin secretion on the mechanism of exocytosis. 2) To explore the role of protein actylation block the action of inhibitors of insulin secretion at any of their other sites of action, or whether they affect only the distal site. 4) To determine which G-protein subunits are associated with the inhibitory effects. 5) To determine the targets of protein actylation in the distal action of the inhibitors.
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