PGE, RECEPTOR AND POST-RECEPTOR ACTIONS IN THE ISLET

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The overall goal of this proposal is to determine whether PGE2 has receptors on pancreatic islet beta cells through which PGE2 exercises important regulatory functions over insulin secretion that are mediated by the stimulatory (Ns) are inhibitory (Ni) subunits of beta cell adenylate cyclase. A cloned beta cell line (HIT cells) will be used because of the unique advantages it provides for this area of investigation. The specific aims of this proposal are (1) to fully characterize the effects of PGE2 on insulin secretion and c-AMP generation by HIT cells; (2) to fully characterize PGE2 synthesis and catabolism in HIT cells; (3) to fully characterize one PGE2 receptor in HIT cells, to examine regulation of this receptor by PGE2, and to ascertain whether the receptor is on the plasma membrane; (4) to determine whether variable effects of PGE2 on insulin secretion can be explained by variable PGE2 effects on c-AMP generation and to ascertain whether down-regulation of the PGE2 receptor by PGE2 is associated with homologous or heterologous changes in beta cell adenylate cyclase activation; (5) to determine whether effects of PGE2 on insulin secretion can be related specifically to the activity of Ns and/or Ni subunits of adenylate cyclase. Insulin secretion from HIT cells will be assessed during perifusion so that both first and second phase insulin secretion in response to a variety of secretagogues can be assessed. PGE2 synthesis and catabolism will be examined through the use of radio- chromatograms and HPLC. Studies of PGE2 receptors will be performed through the use of (3H)PGE2 binding experiments. Post-receptor consequences will be assessed by measurements of c-AMP and adenylate cyclase activity in response to a variety of stimulators so that it can be ascertained whether homologous or heterologous desensitization is associated with PGE2 receptor down-regulation. Extensive studies specifically designed to assess the effects of choleratoxin and pertussis toxin on Ns and Ni will be performed. These experiments will involve (32P)NAD- ribosylation of HIT cells, SDS gel analysis and reconstitution of cell lines deficient in these subunits. At the conclusion of these studies we plan to have established whether the complex actions of PGE2 on pancreatic beta cell function are intimately involved with these subunits of adenylate cyclase and anticipate that these observations will at least partially clarify the controversies that exist in this research area.