METABOLIC AND HORMONAL CONTROL OF CARDIAC CONTRACTION

心脏收缩的代谢和激素控制

• 批准号: 3340148
• 负责人: Gary Brooker
• 金额: $ 21.3万
• 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/3340148
• 关键词: Anura; 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.

本项目建议的研究涉及以下几个方面：速成的作用 转运蛋白在腺苷环化酶调节中的作用 儿茶酚胺和佛斯可林，2.)荧光显微镜 β肾上腺素能受体在心肌ADD中的定位 用视频增强显微镜和数字技术培养细胞 图像处理。在之前的授权期内，我们有 确定和描述了表明存在的现象 腺苷环化酶组分或调节分子 快速翻转，并涉及RNA和蛋白质的合成。这个 第一个推测的蛋白质与异源激素有关 难治性，由腺苷环化酶产物诱导， 环状AMP。第二种快速翻转的蛋白质是必要的。 对于Forsklin的直接刺激作用还不是 儿茶酚胺介导的环状AMP蓄积或 福斯可林的增敏作用。在这项提案中，我们建议 分离、鉴定并最终研究这些基因的调控 重构法测定穿透洋地黄素的蛋白质 细胞，一个维持整个细胞腺苷环化酶的系统 活动仍然允许添加或重新构建组件 进入天然细胞膜。此外，我们会继续研究 利用非洲爪哇表达这些环化酶组分的mRNA 卵母细胞用于翻译从细胞中分离出来的mRNA 这些蛋白质的活性合成。激素刺激的环磷酸腺苷 卵母细胞中的蓄积和电或离子通道活性 将卵母细胞膜用于鉴定成功 推测的蛋白质的翻译并作为一种检测 分离或浓缩其各自的信使核糖核酸。这个 该项目的第二个项目涉及继续开发 强荧光和125I标记的β-肾上腺素能受体 准确和特异地定位β-肾上腺素能的拮抗剂 活细胞中的受体。我们第一个成功的衍生品，NBD- ~(125)I-碘吲哚维持较高的特异性和亲和力 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 在激素不敏感和受体内化的研究中 在心肌和其他细胞中。使用这种方法，我们可以 评估我们的假设，即两个不同的行为 异丙肾上腺素对心肌的舒缓和刺激作用 内向电流由位于两个细胞中的β受体介导 细胞的不同区域。