Regulation of apical NHE3 trafficking by ouabain-induced activation of the basolateral Na+-K+-ATPase receptor complex.

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.