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

Abstract

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 Ca2+ concentration. Pretreatment of cells with the intracellular Ca2+ 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.

Involvement of Reactive Oxygen Species in a Feed-forward Mechanism of Na/K-ATPase-mediated Signaling Transduction

“…Although disruption of the Na/K-ATPase⅐c-Src signaling (as in PY-17 and C2-9 cells) attenuated ouabain-stimulated protein carbonylation, studies with SYF/SYF ϩ c-Src cells and Src kinase inhibitor PP2 are consistent with our previous observations that ouabain stimulates a c-Src-dependent regulation of RPT Na/K-ATPase and NHE3 (13,21). Interestingly, GO-stimulated protein carbonylation was also attenuated in SYF cells, suggesting that the Na/K-ATPase⅐c-Src signaling complex is capable of functioning as a receptor complex for extracellular H 2 O 2 .…”

“…In LLC-PK1 cells, a low concentration of ouabain (up to 100 nM, about 1/10th of IC 50 ) does not inhibit 86 Rb ϩ uptake nor alter intracellular Na ϩ concentration when administered for up to 15 min (13, 47), but it does inhibit 86 Rb ϩ and Na ϩ uptake when administered for longer periods as we reported previously (13,47,48), due to ouabain-induced Na/K-ATPase endocytosis via Na/KATPase signaling (11,13,21,47,48). In LLC-PK1 cells, ouabain (1 h)-induced inhibition of transepithelial 22 Na ϩ flux is mostly dependent on the coordinated regulation of Na/K-ATPase and NHE3 through Na/K-ATPase signaling (13,21,47). Ouabain induced redistribution of Na/K-ATPase and NHE3 in LLC-PK1 cells, with a resultant reduction in cell surface levels of both transporters to depress apical Na ϩ entry through NHE3 (and other Na ϩ -coupled Na ϩ transporters) and basolateral Na ϩ extrusion through Na/K-ATPase.…”

“…We have demonstrated that activation of this Na/K-ATPase signaling function inhibits RPT sodium reabsorption to correct sodium retention-related volume expansion and blood pressure increase. We conclude that, rather than contributing to development and maintenance of hypertension, properly regulated RPT Na/K-ATPase signaling has a protective effect under physiological settings (12,13,21,22). However, a fundamental unanswered question is the underlying mechanism to "turn on/off" the RPT Na/K-ATPase signaling.…”

“…Isolation of Early Endosome (EE) Fractions-The EE fractions (EEA-1-positive) were isolated by sucrose floatation centrifugation and identified previously as described (13,21). The enrichment of EE fractions was assessed by the EE marker EEA-1.…”

“…Active transepithelial 22 Na ϩ flux (from apical to basolateral compartment) was determined by counting radioactivity in the basolateral aspect 1 h after 22 Na ϩ addition to the apical compartment as described previously (13). Each experiment was performed in triplicate.…”