REGULATION OF CALCIUM CURRENT BY CGMP IN HEART CELLS

CGMP 对心脏细胞钙电流的调节

• 批准号: 2839053
• 负责人: Rajiv Kumar
• 金额: $ 10.49万
• 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 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2839053
• 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(改编自申请者摘要)：本报告的具体目标 项目是了解cGMP在心脏调节中的作用。 L型钙电流(Ica，pA/pF)，特别是新生(Nb)兔 心。不同组织中cGMP对ICa调控的各种研究 物种表现出不一致，cGMP的作用尚不清楚 有争议的。在心脏细胞中，关于特定亚型的知识 CGMP依赖的蛋白激酶(PKG)及其底物非常有限。 最近，库马尔博士表明，在NB心脏细胞中，基本的Ica是 显著增加cGMP水平并抑制cGMP水平降低 CGMP水平(由鸟苷酸环化酶抑制剂亚甲基蓝或 LY-83583)。亚甲蓝的作用可被8BrcGMP阻断。基本图像 在成人(AD)心肌细胞中不受这些药物的影响。依赖cAMP 蛋白激酶(PKA)抑制剂阻断cAMP的刺激作用 非8CPT-cGMP对Nb心肌细胞Ica的影响。这一根本区别 Nb和AD心脏细胞之间的关系，导致Kumar博士研究了Ica的调节 NB型心肌细胞中cGMP的表达。他将专门测试cGMP的假说 是NB细胞Ica的重要调节因子。生理上的相关性 CGMP对ICa的刺激作用将通过增加或降低cGMP来评估 级别。CGMP和cAMP在ICa调节中的相互作用将是 在存在专用于激酶的调节剂的情况下进行检查， 磷酸二酯酶和磷酸酶。他将检验这一假设，即 CGMP和cAMP在神经细胞Ica调节中的作用未见报道 对抗性。他将调查表情上的差异和 AD和NB细胞中PKG的水平及其磷酸化底物的鉴定 PKA和PKG。库马尔博士将测试不同的假说 实验方法：1)记录全细胞Ica并改变 灌流吸管内溶于离体兔脑室 细胞，2)记录单个通道的活动及其由各种不同的 干预，3)测量PKG活性及其调节，亚细胞 不同亚型脑白质中PKG的分布、特异性mRNA及其亚型 分子生物学技术，如Western blotting、Northern blotting和 分子克隆，以及4)磷酸化分析以特异地识别 PKG和PKA的生理底物。了解这些 依赖cGMP对ICa调节的发育差异 机制可以提供对离子通道所涉及的机制的深入了解 监管，并可能有助于更好地理解治疗 婴幼儿心功能不全的治疗方法。