REGULATION OF NA+/K+ PUMP CURRENT IN THE HEART

心脏中 NA /K 泵电流的调节

• 批准号: 2232214
• 负责人: Richard T Mathias
• 金额: $ 24.44万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-05-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2232214
• 关键词: calcium flux; calmodulin; guinea pigs; heart; membrane potentials; northern blottings; phosphorylation; protein kinase C; protein structure function; sodium potassium exchanging ATPase; voltage /patch clamp; calcium flux; calmodulin; guinea pigs; heart; membrane potentials; northern blottings; phosphorylation; protein kinase C; protein structure function; sodium potassium exchanging ATPase; voltage /patch clamp

The Na/K pump is the primary metabolic engine for maintaining transmembrane ion gradients which are essential to excitability and contraction of the heart. Our recent data suggest ventricular cells have two functionally different types of Na/K pumps, referred to as type-h and type-l for their high and low affinity for the cardiac glycoside dihydro- ouabain (DHO). The type-l current is modulated by physiological variations in extracellular-K+ ([K]o) and extracellular-H+ ([H]o or pHo) whereas the type=h is not. Both are modulated by physiological variations in intracellular-Na+ ([Na]i), membrane voltage (phi/i) and extracellular- Na+ ([Na]o), though phi/i and [Na]o effects differ significantly between the two types of pumps. Autonomic input also regulates pump current. Gao, et al, 1992, showed stimulation of beta-receptors causes pump current to either increase or decrease depending on intracellular-Ca+2 ([Ca]i). The effects of beta-stimulation are phi/i-dependent, reversed by acetylcholine (ACh), and specifically target the type-l pumps. In contrast to these observations in ventricular cell, Cohen, et al, 1987, found DHO-blockade and [K]o-activation of the Na/K pumps in Purkinje cells corresponded closely to the type-h pumps only. These intriguing results suggest tissue specific, finely tuned regulation of Na/K pump current in the mammalian heart. Pathological states like ischemia, which result in an increase in [K]o and [H]o, will clearly affect the ventricular and Purkinje Na/K pumps differently and these differences will have a role in conduction abnormalities. Moreover, glycoside therapy will be toxic in Purkinje cells long before ventricular cells, and therapeutic doses will have different effects on the action potentials as well as [Na]i. The focus of this proposal is to understand the mechanisms responsible for regulating each of these two types of Na/K pumps in the heart. The whole cell patch clamp technique will be used on acutely isolated ventricular myocytes from the guinea pig heart. The type-h current is measured as that blocked by 5 microM DHO whereas total (type-h + type-l) current is blocked by 1 microM DHO. The effects of autonomic input on both types of pumps will be investigated with the goal of understanding each step in the cascades initiated by activation of a receptor and culminating in a change in Na/K pump current. Gao, et al, 1994a, show the response to beta-stimulation is through a phosphorylation step by phospho kinase A (PKA), but this cascade is not Ca+2-dependent. Thus, the mechanism of [Ca]i modulation of the beta-induced pump response is of particular interest. Moreover, differences in the two types of pumps' responses to [K]o, pHo and phi/i will be studied and related to differences in their responses to autonomic input.

Na/k泵是维护的主要代谢发动机 跨膜离子梯度对于兴奋性至关重要 心脏的收缩。我们最近的数据表明心室细胞具有 两种功能上不同类型的Na/K泵，称为type-H和 type-l具有对心脏糖苷二氢的高亲和力的高亲和力 Ouabain（Dho）。 L型电流由生理调节 细胞外K+（[K] O）和细胞外H+（[H] O或PHO）的变化 而类型= h则不是。两者都由生理变化调节 在细胞内-NA+（[Na] i）中，膜电压（PHI/I）和细胞外 - na+（[na] o），尽管Phi/I和[Na] O效应之间的效果显着不同 两种类型的泵。自治输入还调节泵电流。高， 等，1992，表明β受体的刺激导致泵电流到 根据细胞内Ca+2（[Ca] i）增加或减少。这 β刺激的效果是phi/i依赖性的，反向 乙酰胆碱（ACH），并特别针对L型泵。在 与心室细胞中的这些观察结果对比，Cohen等，1987， 在Purkinje中找到了dho-blockade和[k] o激活Na/k泵 细胞仅与H型泵相对应。这些有趣 结果表明组织特异性，调节Na/k泵的调节 哺乳动物心中的最新。缺血等病理状态， 导致[k] o和[h] o的增加将显然影响 心室和浦肯奈na/k泵有不同的差异和这些差异 将在传导异常中发挥作用。此外，糖苷疗法 在心室细胞之前很久会在Purkinje细胞中有毒，并且 治疗剂量将对动作电位产生不同的影响 以及[na] i。该提议的重点是了解 负责调节这两种类型的Na/K的机制 泵送心脏。 整个细胞贴片夹技术将用于急性分离 豚鼠心的心室心肌细胞。 type-H电流是 测量为5 microM Dho的封闭，而总计（型h + type-l） 电流被1 microm Dho阻塞。自主输入对 两种类型的泵将以理解的目的进行研究 通过激活受体和 最终在Na/k泵电流的变化中达到最终形式。 Gao等，1994a，显示了 对β刺激的反应是通过磷酸化的磷酸化步骤 激酶A（PKA），但该级联反应不是Ca+2依赖性。因此， [CA] I调制β诱导的泵响应的机理是 特别的兴趣。而且，两种类型的泵的差异 对[k] o，pho和phi/i的响应将被研究并与 它们对自主性输入的反应差异。