ION CHANNEL TARGETS FOR CARDIAC GLYCOSIDE ACTIONS

强心苷是使用最广泛的强心剂之一， 尽管有很高的毒性发生率。他们的作用机制是思想 完全是对Na，K-ATPase的抑制。其他引人入胜的 然而，有证据表明，还有其他的细胞活动，包括直接 对肌膜和肌浆网(SR)的作用 它们的正性肌力和/或毒性作用。这样做的目的是 项目是研究几种强心剂的新的亚细胞作用 类固醇对心脏离子通道和收缩的调节作用。 以下为本次续费申请的具体目的：L)至 研究多种强心药对SRCRC活性的影响 类固醇以及这些药物的不同结构成分是如何 (包括内酯环的尺寸和饱和度、是否存在 碳水化合物部分)可能有助于它们的正性变力作用； 2)测量强心类固醇改变SRCRC活性的效果 在正常和衰竭的人类心脏中，以确定这种行为是否 可能实际上有助于其在体内的治疗或毒性作用；3) 确定细胞内应用不同的强心剂 类固醇引起肌质网钙离子释放增加，这是一种正性肌力作用 影响和毒性的发展；以及4)研究 强心类固醇对动作电位构型和离子电流的影响 为了确定在特定的肌膜上是否有直接作用 离子通道TO对其正性肌力和/或毒性起作用 效果。 将使用两种互补的实验方法。单个SRCRC 活性将通过掺入SRCRC蛋白来测量，该蛋白分离自 猫脑室，制成人工平面脂质双层。此外，钙离子i 瞬变(用INDO-1荧光测量)、离子电流和 将在分离的猫心室肌细胞中测量收缩。这个 实验将检验以下总体假设： 强心苷通过几种途径产生强心作用 机制包括细胞内对SR钙释放的作用和 对跨膜离子电流的直接影响；这些影响是 独立于他们已知的行动，但与之协同工作 抑制肌膜Na，K-ATPase。 这些实验的结果将有助于定义细胞 心脏正性变力和毒性作用的机制 糖苷类药物。定义和区分 不同心脏糖苷类药物通过SRCRC活性对钙离子的调节作用 而肌膜离子通道则有可能发展成 在这些站点有选择地采取行动以保留其 治疗心脏疾病的疗效，但其毒性继发 对心脏功能的作用可能会减少或取消。

Cardiac glycosides are among the most widely used cardiotonic agents, despite a high incidence of toxicity. Their mechanism of action is thought to be exclusively an inhibition of the Na,K-ATPase. Other compelling evidence, however, suggests additional cellular actions, including direct actions on the sarcolemma and sarcoplasmic reticulum (SR), that contribute to their positive inotropic and/or toxic actions. The purpose of this project is to investigate new subcellular actions of several cardiotonic steroids on regulation of ion channels and contraction in heart. The following are the specific aims of this renewal application: l) to investigate how SRCRC activity is affected by a number of cardiotonic steroids and how the different structural components of these agents (including size and saturation of the lactone ring, presence of the carbohydrate moiety) might contribute to their positive inotropic actions; 2) to measure the effects of cardiotonic steroids to alter SRCRC activity in normal and failing human heart in order to determine if such an action might in fact contribute to its therapeutic or toxic actions in viva; 3) to determine if intracellular application of different cardiotonic steroids causes an increase in SR Ca2+ release, a positive inotropic effect and the development of toxicity; and 4) to study the effects of cardiotonic steroids on action potential configuration and ionic currents in order to determine if there is a direct action on specific sarcolemmal ion channels to contributes to their positive inotropic and/or toxic effects. Two complementary experimental approaches will be used. Single SRCRC activity will be measured by incorporation of SRCRC protein, isolated from cat ventricle, into artificial planar lipid bilayers. In addition, Ca2+i transients (measured with indo-1 fluorescence), ionic currents and contraction will be measured in isolated cat ventricular myocytes. The experiments will test the following Overall Hypothesis: Cardiac glycosides produce their cardiotonic actions via several mechanisms that include an intracellular action on SR Ca2+ release and direct effects on transmembrane ionic currents; these effects are independent from, but work in conjunction with, their known action to inhibit the sarcolemmal Na,K-ATPase. The results of these experiments will help to define the cellular mechanisms for the positive inotropic and toxic actions of cardiac glycosides. The significance of defining and distinguishing between the different cardiac glycoside effects on Ca2+i regulation via SRCRC activity and sarcolemmal ion channels is that it may then be possible to develop new agents that act selectively at these sites to retain their effectiveness in treating cardiac disease but whose toxic secondary actions on cardiac function might be curtailed or abolished.