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.
Cai
Haiping
H
Department of Medicine, University of Toledo College of Medicine, 3120 Glendale Ave., Toledo, OH 43614-5089, USA.
Wu
Liang
L
Qu
Weikai
W
Malhotra
Deepak
D
Xie
Zijian
Z
Shapiro
Joseph I
JI
Liu
Jiang
J
D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D002614:Chelating Agents; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D007424:Intracellular Fluid / Q000737:chemistry / Q000378:metabolism; D017136:Ion Transport / Q000187:drug effects / Q000502:physiology; D007687:Kidney Tubules, Proximal / Q000201:enzymology* / Q000648:ultrastructure; D010042:Ouabain / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D011505:Protein-Tyrosine Kinases / Q000187:drug effects / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000187:drug effects / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000187:drug effects / Q000378:metabolism*; D034421:Sus scrofa; D019061:src-Family Kinases
D000081247:CSK Tyrosine-Protein Kinase; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D010042:Ouabain; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011505:Protein-Tyrosine Kinases; D012964:Sodium; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D000254:Sodium-Potassium-Exchanging ATPase; D019061:src-Family Kinases