MUSCARINIC RECEPTOR & G PROTEINS IN CULTURED HEART CELLS

毒蕈碱受体

• 批准号: 2217992
• 负责人: Jonas Bernard Galper
• 金额: $ 27.72万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-07-01 至 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2217992
• 关键词: G protein; RNase protection assay; acetylcholine; chick embryo; developmental neurobiology; embryo /fetus tissue /cell culture; gene expression; genetic transcription; growth media; heart cell; heart contraction; heart innervation; mixed tissue /cell culture; molecular cloning; muscarinic receptor; myosins; nonmammalian vertebrate embryology; northern blottings; protein isoforms; protein sequence; receptor expression; reporter genes; sympathetic ganglion; transfection; transforming growth factors

The long range objective of these studies is to elucidate the mechanisms of regulation of gene expression in cardiac tissue which determine the level of responsiveness of the heart to autonomic stimulation. We will test the hypothesis that innervation of the embryonic chick heart results in the coordinate regulation of the expression of members of several families of genes including isoforms of muscarinic receptors, G-proteins, and myosin heavy chains. At 3 1/2 - 5 days in ovo, many biochemical and physiologic processes characteristic of the atrium are not present. We have demonstrated that the development of these processes in ovo is associated with a 6-8 fold increase in mRNA coding for alphai2. Co-culture of heart cells 3 1/2 days in ovo with ciliary ganglia, with medium conditioned by growth of heart cells and ciliary ganglia or with TGF-beta appears to induce the development of cholinergic responsiveness and an increase in alphao and alphai. Using this model system, we will test the hypotheses that during co-culture of embryonic chick heart cells and ciliary ganglia, muscarinic stimulation becomes coupled to inhibition of adenylate cyclase activity and to stimulation of an atrial-specific pertussis toxin sensitive diacylglycerol formation, and that these changes take place in parallel with the appearance of mRNAs coding for isoforms of G-proteins and muscarinic receptors either previously not expressed or expressed at low levels. We will test the hypothesis that TGF-beta is the soluble factor in conditioned medium responsible for the development of a muscarinic response and that a unique TGF-beta family member is induced during co-culture. To determine whether expression of G-protein alpha-subunits and/or muscarinic receptor subtypes which appear during co-culture with ciliary ganglia regulates the development of a muscarinic response, heart cells 3 1/2 days in ovo will be co-transfected with genes coding for these receptors and G- proteins and the effect on chronotropic response to muscarinic stimulation and an acetylcholine dependent K+ current determined. Genomic clones of these G-protein alpha-subunits and receptors will be used to determine the transcription start site an 5' upstream response elements. Deletions of the 5' upstream regulatory region ligated to a luciferase reporter gene transfected into heart cells 3 1/2 days in ovo will be used to determine whether responsiveness to condition medium and TGF-beta depend on common response elements. These studies should help our understanding of the role of innervation in the development and regulation of autonomic responsiveness and may aid in our understanding of the genesis of arrhythmias and control of contractile state.

这些研究的远程目标是阐明 调节心脏组织中基因表达的调节 心脏对自主刺激的反应能力。 我们将测试 假设胚胎雏鸡心脏的神经导致 协调几个家庭成员表达的调节 包括毒蕈碱受体，G蛋白和肌球蛋白的同工型在内的基因 重链。 在OVO的3 1/2-5天时，许多生化和生理学 中庭的过程特征不存在。 我们有 证明这些过程在OVO中的发展是相关的 编码Alphai2的mRNA编码时增加了6-8倍。 心脏共同文化 细胞3 1/2天在OVO中使用睫状神经节，中等条件 心脏细胞和睫状神经节或与TGF-β的生长似乎 诱导胆碱能反应的发展和增加 Alphao和Alphai。 使用此模型系统，我们将测试假设 在胚胎小鸡心脏细胞和纤毛神经节的共同培养期间 毒蕈碱刺激耦合到抑制腺苷酸环化酶 活动和刺激心房特异性百日咳毒素敏感 二酰基甘油形成，这些变化并联 随着MRNA编码为G蛋白的同工型的出现和 毒蕈碱受体先前未表达或以低表达 水平。 我们将检验以下假设：TGF-beta是可溶性因素 有条件的培养基负责发展毒蕈碱反应 并且在共培养过程中引起了独特的TGF-β家庭成员。 到 确定G蛋白α-亚基的表达和/或毒蕈碱是否表达 与睫状神经培养期间出现的受体亚型 调节毒蕈碱反应的发展，心脏细胞3 1/2天 在OVO中，将与编码这些受体和G-编码的基因共转染 蛋白质以及对毒蕈碱刺激对年度反应的影响 确定乙酰胆碱依赖性的K+电流。 基因组克隆 这些G蛋白α-亚基和受体将用于确定 转录启动站点是5'上游响应元素。 删除 5'上游调节区域连接到荧光素酶报告基因 在OVO中转染3 1/2天的心脏细胞将用于确定 对条件培养基和TGF-beta的响应是否取决于常见 响应元素。 这些研究应该有助于我们理解角色 自主神经的神经支配 响应能力，可能有助于我们理解 心律不齐和收缩状态的控制。