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

Na/K泵是维持身体健康的主要代谢引擎 跨膜离子梯度对兴奋性和 心脏的收缩。我们最近的数据表明，心室细胞 两种功能不同的Na/K泵，称为h型和 L对心脏糖苷二氢化合物的高亲和力和低亲和力 哇巴因(DHO)。L电流受生理因素的调制 胞外-K([K]o)和胞外-H([H]o或pho)的变化 而类型=h则不是。两者都受到生理变化的调节 在胞内Na([Na]i)、膜电压(Phi/I)和胞外- Na([Na]o)，尽管Phi/I和[Na]o的影响在 这两种类型的泵。自主输入也调节泵的电流。高， 等人，1992年，发现刺激β受体导致泵电流 增加或减少取决于细胞内钙离子([Ca]i)。这个 β-刺激的作用依赖于phi/i，被 乙酰胆碱(ACh)，并专门针对L泵。在……里面 与在心室细胞中观察到的这些相反，Cohen等人，1987， 在Purkinje发现Na/K泵的DHO阻断和[K]O激活 细胞仅与h型泵紧密对应。这些耐人寻味 结果表明，Na/K泵具有组织特异性，调节精细 在哺乳动物的心脏里。像缺血这样的病理状态， 导致[K]O和[H]O的增加，将明显影响 脑室和浦肯野Na/K泵的不同以及这些差异 会在传导异常中起作用。此外，糖苷类药物 对浦肯野细胞的毒性远早于心室细胞，而且 治疗剂量对动作电位有不同的影响。 以及[Na]i。这项建议的重点是理解 调节这两种类型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 取自豚鼠心脏的心室肌细胞。H类电流是 测量为被5微米DHO阻断而总(h型-L) 电流被1微米DHO阻断。自主神经输入对小鼠脑功能的影响 将对这两种类型的泵进行调查，以了解 级联中的每个步骤都是由一个受体的激活和 最终导致Na/K泵电流的变化。Gao等人，1994a，展示了 对β刺激的反应是通过磷酸化的步骤。 蛋白激酶A(PKA)，但这种级联反应不依赖于钙离子。因此， [Ca]i调节β诱导的泵反应的机制是 特别感兴趣。此外，两种泵的不同之处在于 将研究对[K]o、Pho和Phi/I的答复，并与之相关 他们对自主输入的反应不同。

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.