ROLE OF PLASMA MEMBRANE IN STIMULUS-SECRETION COUPLING

这项研究计划的长期目标是

The long-range objective of this research proposal is to determine the role of the plasma membrane of a glucose- responsive insulinoma in stimulus-secretion coupling by analyzing the mechanisms by which it controls calcium fluxes. Specifically, we wish to accomplish the following: 1) to characterize voltage-dependent calcium channels and study their influence on the intracellular calcium ion concentration and insulin release; 2) to study glucose transport by the beta-cell plasma membrane; 3) to study the effect of glucose and other insulin secretagogues on Ca2+-ATPase activity and transmembrane calcium fluxes; 4) to investigate the effect of plasma membrane antibodies from insulin-dependent diabetics on calcium channels, interacellular calcium ion concentration, insulin release, glucose transport and Ca2+-ATPase activity. To accomplish these goals, intact beta-cells or a plasma membrane preparation will be used. In intact beta-cells, insulin release and changes in cytosol calcium concentrations in response to secretagogues will be studied in the presence and absence of the calcium channel agonist Bay K 8644 and the antagonist nitrendipine in a perifusion system. Simultaneously insulin release and changes in intracellular calcium will be studied in cell suspensions under identical conditions. In addition, acute and chronic effects of secretagogues on the binding of (3H)- nitrendipine will be studied in intact insulinoma cells. Calcium channels will also be characterized in a plasma membrane preparation. The plasma membrane preparation or membrane vesicles will also be used to study the effects of glucose and other hexoses on the "calcium-pump, Ca2+-Mg2+-ATPase. Since the plasma membrane has been the target of antibodies found in patients with insulin dependent diabetes, we will use intact cells or the plasma membranes preparation from this tumor to study the effect of an immunoglobulin fraction from diabetics on all proposed experiments. These studies will give important insight on the function of the plasma membrane as a regulator of insulin release and on its involvement in the pathogenesis of diabetes.