The overall aim is to understand how the cytosol free Ca++ concentration is controlled in the pancreatic B-cell, and how it, in turn, controls the rate at which insulin is secreted. Cytosol Ca++ will be measured using Quin 2 fluorescence after incorporation of the acetoxymethylester of Quin 2 into B-cells. We plan to investigate the relationship between the cytosol Ca++ concentration and 1. the stimulation of insulin release, 2. the biphasic pattern of insulin release in response to glucose, 3. the possible Ca++ independence of secretion, 4. the postulated action of cyclic AMP to sensitize the exocytotic machinery to Ca++, 5. inhibition of insulin release by epinephrine and somatostatin, 6. the fluxes of Ca++ across the plasma membrane, and 7. the actions of TMB-8 (8-N.N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride) and dantrolene, two agents which block Ca++ release by intracelullar stores, but which can potentiate insulin release. From these and other data we have developed a """"""""set point"""""""" hypothesis of the control of insulin release which we propose to test. Further studies will investigate the way in which mitochondria and endoplasmic reticulum, regulate the cytosol Ca++ concentration. One series will employ Ca++ sensitive electrodes to monitor the concentration of Ca++ in the medium in the presence of mitochondria and/or endoplasmic reticulum when subjected to 1. different loads of Ca++ to assess their ability to take up and regulate the ambient Ca++ concentration, 2. intermediates in metabolism thought to be involved in the mobilization or sequestration of Ca++ and 3. pharmacological agents such as TMB-8 and dantrolone which are thought to block Ca++ efflux, but not influx, in an intracellular store. A second series of studies will employe 45CA++ as a tracer to measure Ca++ fluxes into, and out of, mitochondria and endoplasmic reticulum. These latter studies, which isolate the separate transport systems involved, will be concerned with control mechanisms and the actions of intermediates and drugs. The studies should enhance our knowledge of the way that cytosol Ca++ affects the rate of insulin release, and the manner in which intracellular organelles help in the regulation of the concentration of Ca++. The information gained should be useful to our understanding of endocrine pancreatic function both in health, and in disease states such as diabetes.
