Cai Haiping, Wu Liang, Qu Weikai, Malhotra Deepak, Xie Zijian, Shapiro Joseph I, Liu Jiang
Department of Medicine, University of Toledo College of Medicine, 3120 Glendale Ave., Toledo, OH 43614-5089, USA.
Am J Physiol Cell Physiol. 2008 Feb;294(2):C555-63. doi: 10.1152/ajpcell.00475.2007. Epub 2007 Dec 12.
The long-term effects of ouabain on transepithelial Na(+) transport involve transcriptional downregulation of apical Na(+)/H(+) exchanger isoform 3 (NHE3). The aim of this study was to determine whether ouabain could acutely regulate NHE3 via a posttranscriptional mechanism in LLC-PK1 cells. We observed that the basolateral, but not apical, application of ouabain for 1 h significantly reduced transepithelial Na(+) transport. This effect was not due to changes in the integrity of tight junctions or increases in the intracellular Na(+) concentration. Ouabain regulated the trafficking of NHE3 and subsequently inhibited its activity, a process independent of intracellular Na(+) concentration. Ouabain-induced NHE3 trafficking was abolished by either cholesterol depletion or Src inhibition. Moreover, ouabain increased the intracellular Ca(2+) concentration. Pretreatment of cells with the intracellular Ca(2+) chelator BAPTA-AM blocked ouabain-induced trafficking of NHE3. Also, blockade of Na(+)-K(+)-ATPase endocytosis by a phosphatidylinositol 3-kinase inhibitor was equally effective in attenuating ouabain-induced NHE3 trafficking. These data indicate that ouabain acutely stimulates NHE3 trafficking by activating the basolateral Na(+)-K(+)-ATPase signaling complex. Taken together with our previous observations, we propose that ouabain can simultaneously regulate basolateral Na(+)-K(+)-ATPase and apical NHE3, leading to inhibition of transepithelial Na(+) transport. This mechanism may be relevant to proximal tubular Na(+) handling during conditions associated with increases in circulating endogenous cardiotonic steroids.
哇巴因对上皮细胞钠（Na⁺）转运的长期影响涉及顶端Na⁺/H⁺交换体3（NHE3）的转录下调。本研究的目的是确定哇巴因是否能通过转录后机制在LLC-PK1细胞中急性调节NHE3。我们观察到，在基底外侧而非顶端施加哇巴因1小时可显著降低上皮细胞钠（Na⁺）转运。这种效应并非由于紧密连接完整性的改变或细胞内Na⁺浓度的增加。哇巴因调节NHE3的转运并随后抑制其活性，这一过程独立于细胞内Na⁺浓度。胆固醇耗竭或Src抑制均可消除哇巴因诱导的NHE3转运。此外，哇巴因可增加细胞内Ca²⁺浓度。用细胞内Ca²⁺螯合剂BAPTA-AM预处理细胞可阻断哇巴因诱导的NHE3转运。同样，磷脂酰肌醇3激酶抑制剂对Na⁺-K⁺-ATP酶内吞作用的阻断在减弱哇巴因诱导的NHE3转运方面同样有效。这些数据表明，哇巴因通过激活基底外侧Na⁺-K⁺-ATP酶信号复合物急性刺激NHE3转运。结合我们之前的观察结果，我们提出哇巴因可同时调节基底外侧Na⁺-K⁺-ATP酶和顶端NHE3，从而导致上皮细胞钠（Na⁺）转运受到抑制。这一机制可能与循环内源性强心甾体增加时近端小管对Na⁺的处理有关。
