PGE, RECEPTOR AND POST-RECEPTOR ACTIONS IN THE ISLET

这项提案的总体目标是确定PGE2是否 胰岛β细胞上有受体，PGE2通过 对胰岛素分泌起着重要的调节作用 由刺激性(NS)介导的是抑制性(Ni) β细胞腺苷环化酶亚单位。一种克隆的β细胞系 (点击单元格)将被使用，因为它具有独特的优势 规定了这一领域的调查。这样做的具体目的是 建议如下：(1)充分描述前列腺素E_2对 HIT细胞的胰岛素分泌和c-AMP的产生；(2)充分 鉴定HIT细胞中PGE2的合成和分解代谢；(3) 充分鉴定HIT细胞中的一种PGE2受体，以检测 通过PGE2对该受体的调节，并确定是否 受体在质膜上；(4)确定 前列腺素E_2对胰岛素分泌的不同影响可以用以下方式解释 可变前列腺素E_2对c-AMP生成的影响 前列腺素E_2是否下调前列腺素E_2受体 与β细胞的同源或异源变化相关 腺苷环化酶激活；(5)确定 前列腺素E_2对胰岛素分泌的影响可能与 腺苷环化酶NS和/或Ni亚基的活性。胰岛素 HIT细胞的分泌将在灌流过程中进行评估，以便 第一时相和第二时相胰岛素分泌对 分泌促进剂的种类可以被评估。前列腺素E_2的合成和 分解代谢将通过使用无线电进行检查- 色谱图和高效液相色谱图。对PGE2受体的研究将是 通过使用(~3H)PGE2结合实验进行。 受体后的后果将通过以下测量进行评估 C-AMP和腺苷环化酶活性对多种药物的响应 刺激器，以便可以确定它是同源的还是 异源脱敏与PGE2受体相关 下调监管。广泛的研究专门设计用于评估 霍乱毒素和百日咳毒素对NS和Ni意志的影响 被执行。这些实验将涉及(32P)NAD- HIT细胞的核糖化、SDS凝胶分析及重组 缺乏这些亚单位的细胞系。在这些结束时 我们计划进行的研究已经确定了复杂的行动 前列腺素E_2对胰岛β细胞功能的影响 腺苷环化酶的这些亚单位，并预计这些 观察将至少部分澄清以下争议 存在于这一研究领域。

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.