Isoform-specific stimulation of cardiac Na/K pumps by nanomolar concentrations of glycosides.
Isoform-specific stimulation of cardiac Na/K pumps by nanomolar concentrations of glycosides.
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DOI：
10.1085/jgp.20028501
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发表时间：
2002-04
影响因子：
3.8
通讯作者：
Mathias, Richard T
中科院分区：
文献类型：
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作者：
Gao, Junyuan;Wymore, Randy S;Wang, Yongli;Gaudette, Glenn R;Krukenkamp, Irvin B;Cohen, Ira S;Mathias, Richard T
众所周知，微摩尔至毫摩尔浓度的强心苷抑制Na/K泵活性，然而，一些早期报告表明纳摩尔浓度的这些苷刺激活性。这些早期的报告是基于多细胞制剂的间接测量，因此，是否离子积累/耗尽而不是泵刺激引起的观察存在一些不确定性。在这里，我们利用分离的心肌细胞的全细胞膜片钳技术，直接测量Na/K泵电流（IP）的条件下，最大限度地减少离子积累/耗尽引起所观察到的影响的可能性。在豚鼠心室肌细胞中，纳摩尔浓度的二氢哇巴因（DHO）引起的外向电流似乎是由于IP的刺激所致，原因如下：（1）在0mM [K +] o中不存在DHO，IP也是如此;（2）在0mM [Na +] i中不存在DHO，IP也是如此;（3）在[Na +] i降低时，外向电流与IP的降低成比例降低;（4）与IP一样，它也被细胞内钒酸盐清除。我们以前的工作表明豚鼠心室肌细胞共表达Na/K泵的α 1和α 2亚型。IP的刺激似乎是通过刺激高糖苷亲和力α 2-亚型而不是α 1-亚型，这是因为：（1）特异性增加α 2-亚型活性的调节信号增加了刺激的幅度;（2）特异性改变α 1-亚型活性的调节信号不影响刺激;（3）[K +] o的变化影响α 1-亚型的活性，但不影响α 2-亚型的活性，不影响刺激;（4）一组豚鼠的心肌细胞表达α 1-亚型，但不表达α 2-亚型，这些心肌细胞不显示刺激。在10 nM DHO时，总IP增加35 ± 10%（平均值± SD，n = 18）。如果接受这种增加是由于仅刺激α 2-亚型的假设，则α 2-亚型的活性增加107 ± 30%。在豚鼠心肌细胞中，纳摩尔哇巴因和DHO都能刺激α 2-亚型，但哇巴因的刺激和抑制浓度都比DHO低10倍。在犬心房和心室肌细胞中观察到纳摩尔DHO对IP的刺激，这些细胞表达Na/K泵的α 1-和α 3-亚型，这表明另一种高糖苷亲和力亚型（α 3-亚型）也受到纳摩尔浓度DHO的刺激。人心房和心室肌细胞表达所有三种亚型，但糖苷类的亚型亲和力太相似，无法区分它们的活性。然而，纳摩尔DHO引起的刺激IP，这是非常相似的，在其他物种。因此，在研究的所有物种中，纳摩尔DHO引起IP的刺激，并且高糖苷亲和力α 2-和α 3-亚型的贡献可以与α 1-亚型的贡献分开，只有高糖苷亲和力亚型被刺激。这些观察结果支持早期报道，纳摩尔浓度的糖苷刺激Na/K泵活性，并提出了一种新的机制，异构体特异性调节IP在心脏中的纳摩尔浓度的内源性哇巴因样分子。
It is well-known that micromolar to millimolar concentrations of cardiac glycosides inhibit Na/K pump activity, however, some early reports suggested nanomolar concentrations of these glycosides stimulate activity. These early reports were based on indirect measurements in multicellular preparations, hence, there was some uncertainty whether ion accumulation/depletion rather than pump stimulation caused the observations. Here, we utilize the whole-cell patch-clamp technique on isolated cardiac myocytes to directly measure Na/K pump current (IP) in conditions that minimize the possibility of ion accumulation/depletion causing the observed effects. In guinea pig ventricular myocytes, nanomolar concentrations of dihydro-ouabain (DHO) caused an outward current that appeared to be due to stimulation of IP because of the following: (1) it was absent in 0 mM [K+]o, as was IP; (2) it was absent in 0 mM [Na+]i, as was IP; (3) at reduced [Na+]i, the outward current was reduced in proportion to the reduction in IP; (4) it was eliminated by intracellular vanadate, as was IP. Our previous work suggested guinea pig ventricular myocytes coexpress the α1- and α2-isoforms of the Na/K pumps. The stimulation of IP appears to be through stimulation of the high glycoside affinity α2-isoform and not the α1-isoform because of the following: (1) regulatory signals that specifically increased activity of the α2-isoform increased the amplitude of the stimulation; (2) regulatory signals that specifically altered the activity of the α1-isoform did not affect the stimulation; (3) changes in [K+]o that affected activity of the α1-isoform, but not the α2-isoform, did not affect the stimulation; (4) myocytes from one group of guinea pigs expressed the α1-isoform but not the α2-isoform, and these myocytes did not show the stimulation. At 10 nM DHO, total IP increased by 35 ± 10% (mean ± SD, n = 18). If one accepts the hypothesis that this increase is due to stimulation of just the α2-isoform, then activity of the α2-isoform increased by 107 ± 30%. In the guinea pig myocytes, nanomolar ouabain as well as DHO stimulated the α2-isoform, but both the stimulatory and inhibitory concentrations of ouabain were ∼10-fold lower than those for DHO. Stimulation of IP by nanomolar DHO was observed in canine atrial and ventricular myocytes, which express the α1- and α3-isoforms of the Na/K pumps, suggesting the other high glycoside affinity isoform (the α3-isoform) also was stimulated by nanomolar concentrations of DHO. Human atrial and ventricular myocytes express all three isoforms, but isoform affinity for glycosides is too similar to separate their activity. Nevertheless, nanomolar DHO caused a stimulation of IP that was very similar to that seen in other species. Thus, in all species studied, nanomolar DHO caused stimulation of IP, and where the contributions of the high glycoside affinity α2- and α3-isoforms could be separated from that of the α1-isoform, it was only the high glycoside affinity isoform that was stimulated. These observations support early reports that nanomolar concentrations of glycosides stimulate Na/K pump activity, and suggest a novel mechanism of isoform-specific regulation of IP in heart by nanomolar concentrations of endogenous ouabain-like molecules.