The overall goal of this project is to identify and characterize abnormalities in the electrophysiological function of beta-cells associated with diabetes or induced by exposure to high concentrations of glucose. We will test the hypothesis that these conditions are associated with specific defects in the ion channels which regulate depolarization and repolarization of the beta-cell membrane or of Ca2+ homeostatic processes within the beta-cell. The techniques of fura-2 fluorimetry and digital image analysis will be applied in combination with whole cell and perforated patch recordings and single channel measurements to study the patterns of Ca2+ signalling within individual insulin secreting cells and cell groups. Experiments will be conducted in dispersed islet beta-cells isolated from normal animals or from animals with beta-cell dysfunction induced by infusion of glucose or with spontaneous diabetes resulting from a reduction in beta-cell mass (the GK rat) or autoimmune beta-cell destruction (the diabetes prone BB/Wor rat). The various components of the normal beta-cell and beta-cell derived lines which are concerned in the regulation of Ca2+ signalling will be studied including Ca2+ channels, K+ channels and various types of intracellular Ca2+ homeostatic mechanisms such as Ca2+ stores, pumps and exchange mechanisms. We shall attempt to determine how these factors contribute to the production of Ca2+ signals in beta-cells in response to various regulators including glucose, amino acids, sulfonuylureas and neurotransmitters. An insulin secreting beta- cell line, the betaTC3 cell line which retains many of the secretory properties of normal beta-cells has been demonstrated to increase its secretory response to a secretory stimulus after incubation in low glucose compared with high glucose. The electrophysiologic correlates of this increase in beta-cell function will be studied. Finally the electrophysiologic properties of novel ion channel genes expressed in the beta-cell will be characterized using heterologous expression systems. Using antisense oligonucleotides and antibodies the effects of these genes on cellular electrophysiology and Ca2+ signalling will be determined. It is anticipated that these studies will provide insights into the role of the ion channels in the regulation of insulin secretion in normal beta-cell physiology and in the secretory dysfunction of early IDDM.
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