Stimulation of Na+ transport by AVP is independent of PKA phosphorylation of the Na-K-ATPase in collecting duct principal cells.
Arginine-vasopressin (AVP) stimulates Na(+) transport and Na-K-ATPase activity via cAMP-dependent PKA activation in the renal cortical collecting duct (CCD). We investigated the role of the Na-K-ATPase in the AVP-induced stimulation of transepithelial Na(+) transport using the mpkCCD(c14) cell model of mammalian collecting duct principal cells. AVP (10(-9) M) stimulated both the amiloride-sensitive transepithelial Na(+) transport measured in intact cells and the maximal Na pump current measured by the ouabain-sensitive short-circuit current in apically permeabilized cells. These effects were associated with increased Na-K-ATPase cell surface expression, measured by Western blotting after streptavidin precipitation of biotinylated cell surface proteins. The effects of AVP on Na pump current and Na-K-ATPase cell surface expression were dependent on PKA activity but independent of increased apical Na(+) entry. Time course experiments revealed that in response to AVP, the cell surface expression of both endogenous Na-K-ATPase and hybrid Na pumps containing a c-myc-tagged wild-type human alpha(1)-subunit increased transiently. Na-K-ATPase cell surface expression was maximal after 30 min and then declined toward baseline after 60 min. Immunoprecipitation experiments showed that PKA activation did not alter total phosphorylation levels of the endogenous Na-K-ATPase alpha-subunit. In addition, mutation of the PKA phosphorylation site (S943A or S943D) did not alter the time course of increased cell surface expression of c-myc-tagged Na-K-ATPase in response to AVP or to dibutyryl-cAMP. Therefore, stimulation of Na-K-ATPase cell surface expression by AVP is dependent on PKA but does not rely on alpha(1)-subunit phosphorylation on serine 943 in the collecting duct principal cells.
Mordasini
David
D
Service de Néphrologie, Fondation pour Recherches Médicales, CH-1211 Geneva 4, Switzerland.
Bustamante
Mauro
M
Rousselot
Martine
M
Martin
Pierre-Yves
PY
Hasler
Udo
U
Féraille
Eric
E
D000818:Animals; D015153:Blotting, Western; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D005786:Gene Expression Regulation; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000502:physiology*; D051379:Mice; D009481:Neurophysins / Q000096:biosynthesis / Q000502:physiology*; D010766:Phosphorylation; D011498:Protein Precursors / Q000096:biosynthesis / Q000502:physiology*; D012964:Sodium / Q000493:pharmacokinetics*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D014667:Vasopressins / Q000096:biosynthesis / Q000502:physiology*
C438657:AVP protein, human; D009481:Neurophysins; D011498:Protein Precursors; D014667:Vasopressins; D012964:Sodium; D017868:Cyclic AMP-Dependent Protein Kinases; D000254:Sodium-Potassium-Exchanging ATPase