METABOLIC AND HORMONAL CONTROL OF CARDIAC CONTRACTION

本项目拟开展的研究涉及：1.）迅速的作用 在腺苷酸环化酶的调节中翻转蛋白质， 儿茶酚胺和毛喉素，和2.）荧光显微 β肾上腺素能受体在心肌中的定位 培养细胞使用视频增强显微镜和数字 图像处理 在过去的一段时间里，我们 发现并描述的现象表明 腺苷酸环化酶组分或调节分子， 快速翻转并参与RNA和蛋白质的合成。 的 第一个推定的蛋白质参与异源激素 难治性并且由腺苷酸环化酶的产物诱导， 环腺苷酸。 第二个快速翻转蛋白质是必要的 对于毛喉素的直接刺激作用， 必需的儿茶酚胺介导的环AMP积累或 毛喉素的增强作用。 在本提案中，我们建议 分离、识别并最终研究这些基因的调控， 使用重组测定法将蛋白质渗透到毛地黄皂苷中 细胞，维持全细胞腺苷酸环化酶的系统 活性还使得能够添加或重构组分 进入天然细胞膜。 此外，我们将继续研究 使用非洲爪蟾表达这些环化酶组分的mRNA 卵母细胞用于翻译从经历细胞中分离的mRNA 这些蛋白质的合成。 激素刺激的环磷酸腺苷 卵母细胞中的积累和电或离子通道活性，或 卵母细胞膜将被用来识别成功的 翻译的推定蛋白质和作为一种测定 分离或富集它们各自的mRNA种类。 的 该项目的第二个方面是继续发展 强荧光和125 I标记的β-肾上腺素能受体 准确和特异性定位β-肾上腺素能受体的拮抗剂 活细胞中的受体。 我们第一个成功的衍生产品，NBD- 125 I-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 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 在激素难治性和受体内化的研究中， 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，即双重和不同的行动， 异丙肾上腺素在心肌松弛和刺激缓慢 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。

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.