DIGITALIS--MECHANISM AND REVERSAL STUDIES

提出的研究试图进一步了解基本的 参与控制心脏功能的机制在细胞和 分子水平，以及荷尔蒙如何调节心脏功能， 神经递质，特别是由一组被称为心脏的药物 苷类(洋地黄)。使用自发跳动的单层培养 心肌细胞，我们将检验心脏糖苷的假设 肌膜上NAK-ATPase的结合和抑制 形成正性变力反应所必需的步骤。 单向和净Na+、K+和Ca++通量将在 变力反应期间的间歇。区分NAK泵 抑制和其他假定的变力机制，我们将改变 低[K+]O条件下培养心肌细胞NAK-ATPase的酶位点数 媒体和测试变力和毒性反应的心脏糖苷类药物。 我们将利用我们的能力在化学物质中培养心脏细胞 明确的、无血清的介质，证实并扩展了我们的发现 细胞被快速通道激活并对毒鼠强高度反应 激动剂。在无血清或常规培养基中生长的细胞将被比较 Na~+、K~+、Ca~(++)流量和含量；NAK-ATPase 特性；β-肾上腺素能和M受体特性；以及 膜成分包括胆固醇含量。特别感兴趣的 将研究电压依赖的快钠和慢钙通道 通过放射性配基结合和膜片钳分析。其他研究使用 培养的心脏细胞将检验甲状腺激素直接 诱导NAK-ATPase位点的数量增加，并且 甲亢状态下对洋地黄的抵抗现象是由于 增强了NaK泵的能力。高场傅里叶变换核磁共振研究 我将扩展我们以前的工作，用 被限制在细胞外空间并起到 各向同性超精细位移试剂。使用完整的灌流的心脏，我们将 测定跨肌膜Na+变化的幅度和时间进程 心脏糖苷类药物和其他干预措施的分布 用~(23)Na核磁共振仪。信号采集与心脏周期的门控将是 用来检验钠离子瞬变过程中Na+i增加的假设 动作电位是正性肌力的基础或贡献。 洋地黄在亚毒性水平的作用。其他核磁共振实验将 使用稀土移位试剂检测心脏中的K+I和K+O，以及 31P-核磁共振将被用来描绘E1-P和E2-P磷酸酶形式 NAK-ATPase和SR-Ca-ATPase。

The studies proposed seek to gain further understanding of fundamental mechanisms involved in the control of cardiac function at the cellular and molecular level, and how cardiac function is modulated by hormones, neurotransmitters, and particularly by the group of drugs known as cardiac glycosides (digitalis). Using spontaneously beating monolayer cultures of cardiac myocytes, we will test the hypothesis that cardiac glycoside binding to and inhibition of NaK-ATPase in the sarcolemmal membrane are steps requisite to the development of a positive inotropic response. Unidirectional and net Na+, K+ and Ca++ fluxes will be measured at intervals during the inotropic response. To distinguish between NaK pump inhibition and other postulated mechanisms of inotropy, we will alter the number of NaK-ATPase sites in cultured heart cells by growth in low [K+]o media and test inotropic and toxic responsiveness to cardiac glycosides. We will exploit our ability to grow cultured heart cells in chemically defined, serum free media, confirming and extending our finding that these cells are fast channel activated and highly responsive to muscarinic agonists. Cells grown in serum-free or conventional media will be compared with respect to Na+, K+ and Ca++ fluxes and contents; NaK-ATPase properties; beta-adrenergic and muscarinic receptor properties; and membrane composition including cholesterol content. Of particular interest will be studies of voltage-dependent fast sodium and slow calcium channels by radioligand binding and patch clamp analyses. Additional studies using cultured heart cells will test the hypotheses that thyroid hormone directly induces an increase in the number of NaK-ATPase sites, and that the phenomenon of resistance to digitalis in the hyperthyroid state is due to enhanced NaK pump capacity. Studies using high-field Fourier transform NMR will extend our previous work on the delineation of Na+i and Na+o using lanthanide chelates that are confined to the extracellular space and act as isotropic hyperfine shift reagents. Using intact perfused hearts, we will determine the magnitude and time course of changes in transsarcolemmal Na+ distribution in response to cardiac glycosides and other interventions using 23Na NMR. Gating of signal acquisition to the cardiac cycle will be used to test the hypothesis that augmentation of the Na+i transient during the action potential underlies or contributes to the positive inotropic effects of digitalis at subtoxic levels. Other NMR experiments will examine K+i and K+o in the heart using lanthanide shift reagents, and 31P-NMR will be used to delineate E1-P and E2-P phosphoenzyme forms of NaK-ATPase and SR Ca-ATPase.