REGULATION OF CALCIUM CURRENT BY CGMP IN HEART CELLS

CGMP 对心脏细胞钙电流的调节

• 批准号: 6476969
• 负责人: Rajiv Kumar
• 金额: $ 11.46万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-01-01 至 2003-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6476969
• 关键词: active sites; age difference; cGMP dependent protein kinase; calcium channel; calcium flux; cyclic AMP; cyclic GMP; enzyme activity; enzyme substrate; heart cell; heart ventricle; isozymes; laboratory rabbit; mature animal; molecular cloning; newborn animals; northern blottings; phosphorylation; protein kinase A; voltage /patch clamp; voltage gated channel; western blottings

DESCRIPTION (Adapted from Applicant's Abstract): The specific goal of this project is to understand the role of cGMP in the regulation of cardiac L-type calcium current (Ica,pA/pF), particularly in newborn (NB) rabbit heart. Various studies on the modulation of Ica by cGMP in different species shows inconsistency and the role of cGMP remains unclear and controversial. In heart cells, knowledge about specific isoforms of cGMP-dependent Protein Kinase (PKG) and its substrates is very limited. Recently, Dr. Kumar showed that, in NB heart cells, basal Ica was significantly increased by increased levels of cGMP and inhibited by lowered cGMP levels (produced by guanylyl cyclase inhibitors Methylene blue or LY-83583). Effects of Methylene blue were blocked by 8BrcGMP. Basal Ica was not affected by these agents in adult (AD) heart cells. cAMP dependent protein kinase (PKA) inhibitor blocked the stimulatory effect of cAMP but not of 8CPT-cGMP on Ica in NB heart cells. This fundamental difference between NB and AD heart cells, led Dr. Kumar to examine regulation of Ica by cGMP in NB heart cells. He will specifically test the hypotheses that cGMP is an important modulator of Ica in NB cells. Physiological relevance of cGMP stimulation of Ica will be assessed by increasing or lowering cGMP levels. Interactions of cGMP and cAMP in the regulation of Ica will be examined in the presence of modulators specific for kinases, phosphodiesterases and phosphatases. He will test the hypothesis that the roles of cGMP and cAMP in the regulation of Ica in NB cells are not antagonistic. He will investigate the differences in the expression and levels of PKG in AD and NB cells and identify substrates phosphorylated by PKA and PKG. Dr. Kumar will test different hypotheses using multiple experimental approaches: 1) recording whole cell Ica and changing the internal solution by perfusible pipette in isolated NB rabbit ventricular cells, 2) recording single channel activity and its modulation by various interventions, 3) measuring PKG activity and its modulation, subcellular distribution, specific mRNA and isoform types of PKG in NB using different molecular biology techniques, e.g. Western blotting, Northern blotting and molecular cloning, and 4) phosphorylation assays to specifically identify the physiological substrates of PKG and PKA. Understanding these developmental differences in the regulation of Ica by cGMP dependent mechanisms may provide insights into the mechanisms involved in ion channel regulation and may contribute to a better understanding of therapeutic approaches for cardiac dysfunction in infants.

描述（改编自申请人的摘要）：本发明的具体目标是： 本项目旨在了解cGMP在心脏调节中的作用， L-型钙电流（伊卡，pA/pF），特别是在新生（NB）兔中 心 在不同的细胞中，cGMP对伊卡的调节的各种研究 物种显示不一致，cGMP的作用仍不清楚， 争议 在心脏细胞中，关于特定亚型的知识， cGMP依赖性蛋白激酶（PKG）及其底物非常有限。 最近，Kumar博士表明，在NB心脏细胞中，基底伊卡是 cGMP水平升高可显著增加，降低cGMP水平可抑制 cGMP水平（由鸟苷酸环化酶抑制剂亚甲蓝或 LY-83583）。 8BrcGMP可阻断亚甲蓝的作用。 基底伊卡 在成人（AD）心脏细胞中不受这些药物的影响。 cAMP依赖 蛋白激酶（PKA）抑制剂阻断cAMP的刺激作用， 而8 CPT-cGMP对NB心肌细胞伊卡内钙离子浓度无明显影响。 这个根本的区别 之间的NB和AD心脏细胞，导致库马尔博士检查调节伊卡， NB心脏细胞中的cGMP。 他将专门检验cGMP 是NB细胞中伊卡的重要调节剂。 生理相关性 将通过增加或降低cGMP来评估cGMP对伊卡的刺激 程度. cGMP和cAMP在伊卡调节中的相互作用将是 在激酶特异性调节剂存在下检测， 磷酸二酯酶和磷酸酶。 他将检验这个假设， cGMP和cAMP在NB细胞中调节伊卡的作用不是 敌对 他将研究表达的差异， AD和NB细胞中PKG的水平，并鉴定由 PKA和PKG。 库马尔博士将测试不同的假设， 实验方法：1）记录全细胞伊卡并改变细胞内的 可灌注移液管内液灌注法在离体NB兔心室中应用 细胞，2）记录单通道活动及其调制的各种 3）测量PKG活性及其调节，亚细胞 用不同方法检测NB中PKG的分布、特异性mRNA和亚型 分子生物学技术，例如Western印迹、北方印迹和 分子克隆，和4）磷酸化测定，以特异性地鉴定 PKG和PKA的生理底物。 了解这些 cGMP依赖性对伊卡钙调节的发育差异 这些机制可以提供对离子通道中所涉及的机制的深入了解。 调节，并可能有助于更好地了解治疗 治疗婴儿心功能不全的方法