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

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 泵是维持生命的主要代谢引擎 跨膜离子梯度对于兴奋性和 心脏收缩。我们最近的数据表明心室细胞 两种功能不同类型的 Na/K 泵，称为 h 型和 l 型因其对强心苷二氢的高亲和力和低亲和力而被称为 L 型。 哇巴因（DHO）。 l型电流受生理调节 细胞外 K+ ([K]o) 和细胞外 H+（[H]o 或 pHo）的变化 而 type=h 则不然。两者都受到生理变化的调节 细胞内 Na+ ([Na]i)、膜电压 (phi/i) 和细胞外 - Na+ ([Na]o)，尽管 phi/i 和 [Na]o 的影响在 两种类型的泵。自主输入还可以调节泵电流。高， 等人，1992 年，表明刺激 β 受体会导致泵电流 增加或减少取决于细胞内 Ca+2 ([Ca]i)。这 β-刺激的效果是 phi/i 依赖性的，通过以下方式逆转 乙酰胆碱 (ACh)，并专门针对 l 型泵。在 与心室细胞中的这些观察相反，Cohen 等人，1987 年， 发现浦肯野 Na/K 泵的 DHO 阻断和 [K]o 激活 细胞仅与 h 型泵密切对应。这些耐人寻味的 结果表明 Na/K 泵的组织特异性、精细调节调节 目前存在于哺乳动物的心脏中。缺血等病理状态 导致 [K]o 和 [H]o 增加，将明显影响 心室和浦肯野 Na/K 泵不同，这些差异 会对传导异常产生作用。此外，糖苷疗法 早在心室细胞之前就对浦肯野细胞产生毒性，并且 治疗剂量会对动作电位产生不同的影响 以及[Na]i。该提案的重点是了解 负责调节这两种类型的 Na/K 的机制 心脏中的泵。 全细胞膜片钳技术将用于急性分离 来自豚鼠心脏的心室肌细胞。 h 型电流为 测量为 5 microM DHO 阻断，而总（h 型 + l 型） 电流被 1 microM DHO 阻断。自主输入的影响 将对这两种类型的泵进行研究，目的是了解 级联中的每个步骤都是由受体的激活启动的 最终导致 Na/K 泵电流的变化。高等人，1994a，表明 对β-刺激的反应是通过磷酸化步骤进行的 激酶 A (PKA)，但该级联不依赖于 Ca+2。因此， [Ca]i 调节 β 诱导泵响应的机制是 特别感兴趣。此外，两种类型的泵的差异 将研究对 [K]o、pHo 和 phi/i 的响应并与 他们对自主输入的反应存在差异。