钠钾ATP酶、其同工型以及对哇巴因在离体衰竭人心脏中的变力反应的表征

Characterisation of Na/K-ATPase, its isoforms, and the inotropic response to ouabain in isolated failing human hearts.

作者信息

Shamraj O I, Grupp I L, Grupp G, Melvin D, Gradoux N, Kremers W, Lingrel J B, De Pover A

机构信息

Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, College of Medicine, Ohio.

出版信息

Cardiovasc Res. 1993 Dec;27(12):2229-37. doi: 10.1093/cvr/27.12.2229.

DOI:10.1093/cvr/27.12.2229

PMID:8313433

Abstract

OBJECTIVE

The aim was to determine whether failing human hearts have increased sensitivity to the inotropic and toxic effects of ouabain, and to examine alterations in Na/K-ATPase that might explain the observed higher ouabain sensitivity.

METHODS

For contractility studies, a total of 57 trabeculae were isolated from two non-failing (death from head injury) and 10 terminally failing, explanted human hearts. After the experiment, each trabecula was inspected under the light microscope for morphological alterations consistent with heart failure. Samples for biochemical and molecular studies were obtained from five non-failing and 13 failing hearts. Total Na/K-ATPase was measured in desoxycholate treated homogenates and expressed per unit of tissue wet or dry weight, DNA, protein, or myosin. Interference from residual bound digoxin due to previous therapy was excluded. The expression of the three alpha isoforms was studied at both the mRNA level using northern blots and the protein level by analysis of dissociation kinetics of the [3H]ouabain-enzyme complex.

RESULTS

Trabeculae showing morphological alterations and decreased contractility were sensitive to lower concentrations of ouabain (3-100 nM) than control trabeculae (100-1000 nM); the inotropic EC50 and the minimum toxic concentration were both reduced. [3H]Ouabain binding was significantly lower (p << 0.001) in failing than in non-failing hearts, at 293(SD 74) v 507(48) pmol.g-1 wet weight. No significant change was observed in maximum ATPase turnover rate, or in sensitivities to Na+, K+, vanadate, and dihydro-ouabain. All three alpha isoforms were expressed at the mRNA level in both normal and failing hearts.

CONCLUSIONS

This study shows conclusively, for the first time, that failing human hearts are more sensitive to ouabain. This may be at least partly due to a mean reduction of 42% (95% confidence interval, 26 to 56%) in the concentration of Na/K-ATPase (decrease in Na,K pump reserve), but not to an alteration in its catalytic properties or in its isoform composition.

摘要

目的

确定衰竭的人类心脏对哇巴因的变力作用和毒性作用的敏感性是否增加，并研究钠钾 -ATP 酶的改变，以解释观察到的更高的哇巴因敏感性。

方法

为进行收缩性研究，从两颗非衰竭心脏（因头部受伤死亡）和 10 颗终末期衰竭的离体人类心脏中总共分离出 57 条小梁。实验结束后，在光学显微镜下检查每条小梁是否有与心力衰竭一致的形态学改变。用于生化和分子研究的样本取自 5 颗非衰竭心脏和 13 颗衰竭心脏。在脱氧胆酸盐处理的匀浆中测量总钠钾 -ATP 酶，并以每单位组织湿重或干重、DNA、蛋白质或肌球蛋白表示。排除了先前治疗导致的残留结合地高辛的干扰。使用 Northern 印迹在 mRNA 水平以及通过分析 [³H]哇巴因 - 酶复合物的解离动力学在蛋白质水平研究三种α同工型的表达。

结果

显示形态学改变和收缩性降低的小梁对哇巴因的浓度（3 - 100 nM）比对照小梁（100 - 1000 nM）更敏感；变力作用的半数有效浓度（EC50）和最小毒性浓度均降低。在衰竭心脏中，[³H]哇巴因结合显著低于非衰竭心脏（p << 0.001），分别为 293（标准差 74）与 507（48）pmol·g⁻¹湿重。最大 ATP 酶周转率或对 Na⁺、K⁺、钒酸盐和双氢哇巴因的敏感性未观察到显著变化。三种α同工型在正常和衰竭心脏的 mRNA 水平均有表达。