INTERACTIONS BETWEEN CA & CAMP REGULATION IN MYOCYTES

CA 之间的互动

• 批准号: 2029291
• 负责人: RICHARD D GREEN
• 金额: $ 21.95万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-04-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2029291
• 关键词: adenylate cyclase; calcium channel; calcium flux; cyclic AMP; enzyme feedback; heart cell; intermolecular interaction; isozymes; muscle cells; phosphorylation; protein kinase A; protein kinase C; tissue /cell culture

This is a proposal to examine the role of Ca in regulating adenylyl cyclase(AC) activity in chick cardiac myocytes. Evidence is presented that the myocytes express type V nd VI AC isoforms, both of which are inhibited by Ca. The PI has found that lowering external Ca stimulates cAMP production in response to either ISO or forskolin. The applicant proposes to identify the AC isoforms at the protein level using isoform- specific antibodies, and to alter expression levels of types V and VI isozymes in embryonic chick heart myocytes using adenovirus-mediated expression techniques (sense or antisense), an to examine the effects of these alterations on the Ca-AC interaction. AC activities will be measured in myocytes expressing known levels of AC V and VI, and the effects of Ca will be measured in intact cells (variations in extracellular Ca+/- nifedipine), in permeabilized cells and in isolated membranes. The second specific aim will focus on the long term effects of exposure to Ca channel blockers on myocyte AC levels, using a CRE- luciferase reporter construct, to assess cellular cAMP levels. The third specific aim will investigate potential phosphorylation of th AC isoforms by PKA and PKC, and associated effects on activity levels and the"cross- talk" between Ca and cAMP production.

这是一项研究 Ca 在调节腺苷酸中的作用的提案 鸡心肌细胞环化酶（AC）活性。 证据已出 肌细胞表达 V 型和 VI 型 AC 亚型，这两种亚型都是 被Ca抑制。 PI 发现降低外部 Ca 会刺激 cAMP 的产生响应 ISO 或毛喉素。 申请人 提议使用异构体在蛋白质水平上鉴定 AC 异构体 特异性抗体，并改变 V 型和 VI 型的表达水平 使用腺病毒介导的胚胎鸡心肌细胞中的同工酶 表达技术（有义或反义），以检查效果 这些改变对 Ca-AC 相互作用的影响。 交流活动将 在表达已知水平的 AC V 和 VI 的肌细胞中进行测量，并且 Ca 的影响将在完整细胞中测量（变化 细胞外 Ca+/- 硝苯地平），在透化细胞和分离的细胞中 膜。 第二个具体目标将侧重于长期影响 使用 CRE- 暴露于 Ca 通道阻滞剂对肌细胞 AC 水平的影响 荧光素酶报告基因构建体，用于评估细胞 cAMP 水平。 第三个 具体目标是研究 AC 亚型的潜在磷酸化 PKA 和 PKC 的作用，以及对活动水平和“交叉”的相关影响 Ca 和 cAMP 生产之间的对话。