Stimulation of apical NHE3 endocytosis
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By using LLC-PK1 cells (a porcine renal proximal tubule cell line), we have demonstrated that the long-term effects of ouabain and other cardiotonic steroids on transepithelial Na+ transport involved transcriptional down-regulation of NHE3 inMoreBy using LLC-PK1 cells (a porcine renal proximal tubule cell line), we have demonstrated that the long-term effects of ouabain and other cardiotonic steroids on transepithelial Na+ transport involved transcriptional down-regulation of NHE3 in previous studies. In this study, we investigated the acute effect of ouabain on NHE3. We observed that administration of ouabain in basolateral aspect of LLC-PK1 cells, but not in apical aspect, significantly reduced transepithelial Na+ transport. This ouabain effect was not due to changes in the barrier function of tight junction or increases in intracellular Na+ concentration, but resulted from ouabain-stimulated the endocytosis of NHE3 and subsequent inhibition of apical NHE3 activity. Moreover, ouabain-induced NHE3 endocytosis was abolished by either cholesterol depletion or Src inhibitor PP2. Likewise, blocking of Na/K-ATPase endocytosis by PI3K inhibitor was equally effective in inhibiting ouabain-induced NHE3 endocytosis. In addition, ouabain increased intracellular Ca2+ and pre-treatment of cells with an intracellular Ca2+ chelator (BAPTA-AM) completely blocked ouabain-induced endocytosis of NHE3. Taken together with these observations, it indicates that ouabain can stimulate apical NHE3 endocytosis by activating the basolateral Na/K-ATPase signaling complex. This pathway may be relevant to proximal tubular sodium handling during conditions associated with increases in circulating endogenous cardiotonic steroids.