METABOLIC AND HORMONAL CONTROL OF CARDIAC CONTRACTION

心脏收缩的代谢和激素控制

• 批准号: 3340149
• 负责人: Gary Brooker
• 金额: $ 18.75万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-09-01 至 1992-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3340149
• 关键词: Anura; RNA biosynthesis; adenylate cyclase; beta adrenergic receptor; calcium flux; catecholamines; cell membrane; cyclic AMP; digitonin; drug metabolism; guanosine triphosphate; heart contraction; heart pharmacology; high performance liquid chromatography; hormone receptor; hormone regulation /control mechanism; laboratory rabbit; membrane channels; messenger RNA; myocardium; protein biosynthesis; radioimmunoassay; radiotracer; tissue /cell culture

Studies proposed in this project relate to; 1.) the role of rapidly turning over proteins in the regulation of adenylate cyclase by catecholamines and forskolin, and 2.) fluorescent microscopic localization of beta adrenergic receptors in the myocardium add cultured cells using video intensification microscopy and digital image processing. During the preceding grant period we have identified and characterized phenomena which suggest the presence of adenylate cyclase components or regulatory molecules which are rapidly turning over and involve RNA and protein synthesis. The first putative protein is involved in heterologous hormone refractoriness and is induced by the product of adenylate cyclase, cyclic AMP. The second rapidly turning over protein is necessary for the direct stimulatory actions of forskolin yet is not necessary for catecholamine mediated cyclic AMP accumulation or the potentiative actions of forskolin. In this proposal we propose to isolate, identify and ultimately study the regulation of these proteins using reconstitution assays into digitonin permeabilized cells, a system which maintains whole cell adenylate cyclase activity yet enables the addition or reconstitution of components into native cell membranes. Furthermore we will continue study the expression of mRNA for these cyclase components using Xenopus oocytes for the translation of mRNA isolated from cells undergoing active synthesis of these proteins. Hormone stimulated cyclic AMP accumulation and electrical or ion channel activity in oocytes or oocyte membranes will be used to identify the successful translation of the putative proteins and as an assay for the isolation or enrichment of their respective mRNA species. The second of this project involves the continued development of intensely fluorescent and 125I labeled beta-adrenergic receptor antagonists to accurately and specifically localize beta-adrenergic receptors in living cells. Our first successful derivative, NBD- 125I-iodopindolol maintains high specificity and affinity of binding to the beta receptor and has enabled its localization by high sensitivity video intensification fluorescence microscopy with digital image processing. This approach will be especially useful in studies of hormone refractoriness and receptor internalization in the myocardium and other cells. With this methodology we can evaluate our hypothesis that the dual and distinct actions of isoproterenol in the myocardium to relax and to stimulate the slow inward current is mediated by beta-receptors localized in two distinct regions of the cell.

该项目提出的研究涉及； 1.) 迅速的作用 通过改变腺苷酸环化酶调节中的蛋白质 儿茶酚胺和毛喉素，以及 2.) 荧光显微镜 β 肾上腺素能受体在心肌中的定位 使用视频增强显微镜和数字技术培养细胞 图像处理。 在之前的资助期内，我们有 识别和表征表明存在的现象 腺苷酸环化酶成分或调节分子 快速周转并涉及RNA和蛋白质的合成。 这 第一个推定的蛋白质与异源激素有关 不应性是由腺苷酸环化酶的产物诱导的， 环磷酸腺苷。 第二次快速周转蛋白质是必要的 对于毛喉素的直接刺激作用尚未 儿茶酚胺介导的环 AMP 积累或 毛喉素的潜在作用。 在本提案中，我们建议 分离、识别并最终研究这些因素的调节 使用洋地黄皂苷重构测定蛋白质 细胞，维持全细胞腺苷酸环化酶的系统 活动但允许添加或重构组件 进入天然细胞膜。 此外，我们还将继续研究 使用 Xenopus 表达这些环化酶成分的 mRNA 卵母细胞用于翻译从经历过的细胞分离的mRNA 这些蛋白质的主动合成。 激素刺激环磷酸腺苷 卵母细胞中的积累和电或离子通道活性或 卵母细胞膜将用于鉴定成功的 假定蛋白质的翻译并作为测定 分离或富集其各自的 mRNA 种类。 这 该项目的第二个涉及持续开发 强荧光和 125I 标记的 β-肾上腺素受体 拮抗剂可准确、特异地定位 β-肾上腺素能 活细胞中的受体。 我们第一个成功的衍生品，NBD- 125I-碘吲哚洛尔保持了高特异性和亲和力 与 β 受体结合并通过以下方式实现其定位 高灵敏度视频增强荧光显微镜 数字图像处理。 这种方法将特别有用 激素不应性和受体内化的研究 在心肌和其他细胞中。 通过这种方法我们可以 评估我们的假设，即双重且不同的行为 异丙肾上腺素对心肌有松弛和缓慢刺激作用 内向电流由位于两个区域的β受体介导 细胞的不同区域。