Stimulation of Na⁺transport by AVP is independent of PKA phosphorylation of the Na-K-ATPase in collecting duct principal cells

Abstract

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 mpkCCDc14 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 α1-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 α-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 α1-subunit phosphorylation on serine 943 in the collecting duct principal cells.

Vasotocin and isotocin regulate aquaporin 1 function in the sea bream

“…These findings reinforce the importance of the AVT/AVP system in AQP-mediated water balance in vertebrates (Mahlmann et al, 1994;Mordasini et al, 2005;Schafer et al, 1990;Warne, 2001;Warne et al, 2002). mRNA expression of Aqp1 paralogs, i.e.…”

Tonicity-Responsive Enhancer Binding Protein Is an Essential Regulator of Aquaporin-2 Expression in Renal Collecting Duct Principal Cells

“…*P Ͻ 0.05. electrogenic Na ϩ transport. Moreover, this highly differentiated state is maintained in transfected cells (24,34). In this respect, we believe that the changes that were observed in cultured mpkCCD cl4 cells that were transiently transfected with TonEBP siRNA or DN-TonEBP more accurately reflect direct consequences of altered TonEBP activity than changes that were observed in cells that displayed a reprogrammed phenotype as a result of decreased TonEBP activity.…”

“…Transfection was performed by electroporating cells as described previously (24) in the presence of 1.2 nmol of small interfering RNA (siRNA); 8 pmol of plasmid that contained either eGFP or human dominant-negative TonEBP (DN-TonEBP) (2); or 8 pmol of plasmid that contained luciferase constructs. Sense primers for Stealth siRNA (Invitrogen, San Diego, CA) were the following: 5Ј-GGUGUUGCAGGUAUUUGUGGGCAAU-3Ј, 5Ј-GGAUUCUAUCAGGCCUGUAGAGUAA-3Ј, and 5Ј-CCUAGUUCU-CAAGAUCAGCAAGUAA-3Ј.…”

“…Electroporation of mpkCCD cl4 cells yields high transfection efficiencies. Cultured mpkCCD cl4 cells were electroporated in the presence of 8 pmol of plasmid that contained eGFP reporter gene as described previously (24) and grown on a plastic support. Forty-eight hours after transfection, the transfection efficiency was estimated by comparing cells that were visualized under bright field (A) with the eGFP fluorescence signal emitted by the same cells (B).…”

Aldosterone Activates NF-κB in the Collecting Duct

“…55 Briefly, 4 ϫ 10 6 cells, along with 400 l of culture medium supplemented with 10% serum and 1.25% DMSO and 2 nmol siRNA or 8 pmol of plasmid constructs, were transferred to electroporation cuvettes (Gene Pulser cuvette 0.4 cm; Bio-Rad, Hercules, CA), electroporated (300 mV, 960 pF, pulse duration 15 to 17 ns in mpkCCD cl4 cells and 22 to 24 ns in mCCD cl1 cells) using a Bio-Rad Gene Pulser. Cells were seeded on permeable filters at a density of 500,000 cells per well, allowed to recover for 24 h in culture medium containing 10% serum, and then maintained in serum and hormone-free medium for another 24 h for siRNA or 48 h for plasmids.…”