DIGITALIS: MECHANISM AND REVERSAL STUDIES

所提出的研究旨在进一步了解基本原理

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.