PGE, RECEPTOR AND POST-RECEPTOR ACTIONS IN THE ISLET

本提案的总体目标是确定PGE 2是否 在胰岛β细胞上有受体， 对胰岛素分泌起重要的调节作用 由刺激（Ns）介导的是抑制（Ni） β细胞腺苷酸环化酶的亚基。 克隆的β细胞系 (HIT细胞）将被使用，因为它的独特优势， 为这方面的调查提供了条件。 具体目标是 建议是（1）充分表征PGE 2对 HIT细胞分泌胰岛素和产生c-AMP;（2）充分 表征HIT细胞中的PGE 2合成和催化;（3） 充分表征HIT细胞中的一种PGE 2受体，以检查 PGE 2对该受体的调节，并确定 受体在质膜上;（4）确定是否 PGE 2对胰岛素分泌的不同影响可以解释为： 不同的PGE 2对c-AMP生成的影响，并确定 PGE 2对PGE 2受体的下调是否 与β细胞中的同源或异源变化相关 腺苷酸环化酶激活;（5）确定是否影响 PGE 2对胰岛素分泌的影响可能与胰岛素分泌相关。 腺苷酸环化酶的Ns和/或Ni亚基的活性。 胰岛素 在灌流期间评估HIT细胞的分泌， 第一和第二相胰岛素分泌都响应于 可以评估多种促分泌素。 PGE 2合成和 将通过无线电检查category- 色谱图和HPLC。 PGE 2受体的研究将是 通过使用（3 H）PGE 2结合实验进行。 将通过测量以下指标评估受体后后果： c-AMP和腺苷酸环化酶活性对各种 刺激物，以便可以确定是否同源或 外源性脱敏与PGE 2受体有关 下调。 专门设计的广泛研究， 霍乱毒素和百日咳毒素对Ns和Ni的影响 被执行。 这些实验将涉及（32 P）NAD- HIT细胞的核糖基化，SDS凝胶分析和重组 缺乏这些亚基的细胞系。 在这些结束时， 我们计划进行的研究， PGE 2对胰腺β细胞功能的影响密切相关 与腺苷酸环化酶的这些亚基，并预计这些亚基， 观察至少会部分澄清争议， 存在于这个研究领域。

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.