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

Na/K泵是维持机体代谢的主要代谢引擎， 跨膜离子梯度对兴奋性至关重要， 心脏的收缩。我们最近的数据表明心室细胞 两种功能不同类型的Na/K泵，称为h型和 I型，因为它们对强心苷二氢- 哇巴因（DHO）。I型电流受生理学调节， 细胞外K+（[K]o）和细胞外H+（[H]o或pHo）的变化 而type=h不是。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] i） Na+（[Na]o），尽管phi/i和[Na]o效应在 这两种类型的泵。自主输入还调节泵电流。高城、 等，1992年，表明β受体的刺激引起泵电流， 根据细胞内Ca +2（[Ca]i）增加或减少。的 β刺激的作用是phi/i依赖性的， 乙酰胆碱（ACh），并专门针对I型泵。在 与心室细胞中的这些观察相反，Cohen等，1987， 发现浦肯野细胞中的Na/K泵的DHO阻断和[K] o激活 电池仅与H型泵紧密对应。这些有趣 结果表明，组织特异性，微调调节钠/钾泵 哺乳动物心脏的电流。病理状态如缺血， 而在[A]和[B]之间，则会有明显的差异。 心室和浦肯野钠/钾泵不同，这些差异 会导致传导异常此外，糖苷疗法 在浦肯野细胞中比心室细胞早得多， 治疗剂量对动作电位有不同的影响 [i][i][i]本提案的重点是了解 负责调节这两种类型的Na/K的机制 心脏里的泵 全细胞膜片钳技术将用于急性分离的 豚鼠心脏的心室肌细胞h型电流为 测量为被5 μ M DHO阻断，而总的（h型+l型） 电流被1 μ M DHO阻断。自主神经输入对 这两种类型的泵将进行研究，目的是了解 级联反应中的每一步都是由受体的激活启动的， 最终导致Na/K泵电流的变化。Gao等人，1994 a，显示了 对β刺激的反应是通过磷酸化步骤， 激酶A（PKA），但这种级联不是Ca+2依赖性的。因此 β诱导的泵响应的[Ca]i调节机制是 特别的兴趣。此外，两种类型泵的不同之处在于， 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有差异。

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.