PH REGULATION IN VENTRICULAR CELLS

描述(改编自申请者的摘要)：长期目标 这项研究的目的是更好地了解细胞内的机制 成人心室肌的pH(Phi)调节。在这个项目中， 申请者将专注于心脏PHI控制系统的两个调节器， 血管活性多肽(血管紧张素II、内皮素1)和慢性(4-5 天)酸碱失衡(代谢性酸中毒和碱中毒)。这些 摘要多肽是一种强效正性肌力调节剂，作用范围广泛。 对心脏的影响，目前被认为会加剧 缺血/再灌流时的心肌损伤。它们对心脏的影响可能 在一定程度上是通过phi的变化来调节的。类似地， 心脏PHI控制系统上的长期酸中毒和碱中毒可能 是对慢性心肌缺血的一种重要的适应性反应。 申请者将测试这两个调制器都改变 Na-HCO3与Na/H交换的动力学性质及其与pH的关系 共转运和Na-非依赖的Cl-HCO3交换。实验也是 旨在识别介导Phi的肌膜受体 多肽的作用。申请者还将调查 CaI、钙调蛋白、钙/钙调蛋白依赖的细胞内信号转导作用 蛋白激酶II和蛋白激酶C在介导PHI效应中的作用 ATII和ET1。实验将在37oC的脑室上进行 从正常成年兔、豚鼠、 和人类(只有兔子将被用于慢性酸碱研究)。 细胞内H+和Na+的活性将用 荧光指示剂(SNARF-1、BCECF)和SBFI。在一些 实验中，将对PHI和NaI进行测量 用吸液吸管对细胞进行透析。快速解决方案切换者将 用于快速(小于3毫秒)的外部涂抹 多肽。

DESCRIPTION (adapted from the applicant's abstract): The long term goal of this research is to better understand the mechanisms of intracellular pH (pHi) regulation in adult ventricular muscle. In this project the applicant will focus on two modulators of cardiac pHi control systems, vasoactive peptides (angiotensin II, endothelin 1) and chronic (4-5 days) acid-base disturbances (metabolic acidosis and alkalosis). These peptides are potent positive inotropic agents which exert a wide range of effects on the heart and are currently thought to exacerbate myocardial damage during ischemia/reperfusion. Their cardiac effects may be mediated, in part, by changes in pHi. Similarly, the effect of prolonged acidosis and alkalosis on cardiac pHi control systems may represent an important adaptive response to chronic myocardial ischemia. The applicants will test the hypothesis that both modulators alter the kinetic properties and pHi dependence of Na/H exchange, Na-HCO3 cotransport, and Na- independent Cl-HCO3 exchange. Experiments are also designed to identify the sarcolemmal receptor which mediates the pHi action of the peptides. The applicant will also investigate the intracellular signaling role of Cai, calmodulin, Ca/calmodulin-dependent protein kinase II, and protein kinase C in mediating the pHi effects of ATII and ET1. The experiments will be performed at 37oC on ventricular myocytes, enzymatically isolated from normal adult rabbits, guinea pigs, and humans (only rabbits will be used in the chronic acid-base studies). Intracellular activities of H+ and Na+ will be measured with the fluorescent indicators (SNARF-1, BCECF) and SBFI, respectively. In some experiments, measurement of pHi and Nai will be made during internal dialysis of cells with a suction pipette. A rapid solution switcher will be used for rapid (less than 3 msec) external application of the peptides.