DIGITALIS: MECHANISM AND REVERSAL STUDIES

所提出的研究旨在进一步了解 参与控制心脏功能的细胞和 分子水平，以及心脏功能如何被激素调节， 神经递质，特别是被称为心脏的药物组 糖苷（洋地黄）。 使用自发搏动的单层培养物， 心肌细胞，我们将测试的假设， 与肌膜NaK-ATP酶的结合和抑制作用， 发展正性肌力反应所必需的步骤。 单向和净Na+、K+和Ca++通量将在 正性肌力反应期间的间隔。 NaK泵的区别 抑制和其他假定的变力机制，我们将改变 低[K+]浓度培养心肌细胞NaK-ATP酶位点数 对强心苷的中、试验变力性和毒性反应。 我们将利用我们的能力培养培养心脏细胞在化学 定义，无血清培养基，确认和扩展我们的发现，这些 细胞是快通道激活的，并且对毒蕈碱具有高度响应性 激动剂 将比较在无血清或常规培养基中生长的细胞 Na ~+、K ~+、Ca ~（++）通量和含量 β-肾上腺素能和毒蕈碱受体特性;和 膜组成包括胆固醇含量。 特别感兴趣的 将研究电压依赖性快钠通道和慢钙通道 通过放射性配体结合和膜片钳分析。 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-ATP酶位点数量的增加， 甲状腺机能亢进状态下对洋地黄的抵抗现象是由于 提高NaK泵的能力。 高场傅里叶变换核磁共振研究 将扩展我们以前关于Na+i和Na+o的划界工作， 镧系元素螯合物被限制在细胞外空间， 各向同性超精细位移试剂。 使用完整的灌注心脏，我们将 确定跨肌膜Na+变化的幅度和时间过程 对强心苷和其他干预措施作出反应的分布 使用23 Na NMR。 将信号采集门控到心动周期， 用于检验Na+i瞬变在 动作电位是正性肌力的基础或促成正性肌力 洋地黄在亚毒性水平的作用。 其他NMR实验将 使用镧系元素位移试剂检查心脏中的K+1和K+0，以及 31 P-NMR将用于描绘E1-P和E2-P磷酸酶形式的 Na K-ATP酶和SR Ca-ATP酶。

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.