Impaired intestinal NHE3 activity in the PDK1 hypomorphic mouse.
In vitro experiments have demonstrated the stimulating effect of serum- and glucocorticoid-inducible kinase (SGK)1 on the activity of the Na+/H+ exchanger (NHE3). SGK1 requires activation by phosphoinositide-dependent kinase (PDK)1, which may thus similarly play a role in the regulation of NHE3-dependent epithelial electrolyte transport. The present study was performed to explore the role of PDK1 in the regulation of NHE3 activity. Because mice completely lacking functional PDK1 are not viable, hypomorphic mice expressing approximately 20% of PDK1 (pdk1(hm)) were compared with their wild-type littermates (pdk1(wt)). NHE3 activity in the intestine and PDK1-overexpressing HEK-293 cells was estimated by utilizing 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein fluorescence for the determination of intracellular pH. NHE activity was reflected by the Na+-dependent pH recovery from an ammonium prepulse (DeltapH(NHE)). The pH changes after an ammonium pulse allowed the calculation of cellular buffer capacity, which was not significantly different between pdk1(hm) and pdk1(wt) mice. DeltapH(NHE) was in pdk1(hm) mice, only 30 +/- 6% of the value obtained in pdk1(wt) mice. Conversely, DeltapH(NHE) was 32 +/- 7% larger in PDK1-overexpressing HEK-293 cells than in HEK-293 cells expressing the empty vector. The difference between pdk1(hm) and pdk1(wt) mice and between PDK1-overexpressing and empty vector-transfected HEK cells, respectively, was completely abolished in the presence of the NHE3 inhibitor S3226 (10 microM). In conclusion, defective PDK1 expression leads to significant impairment of NHE3 activity in the intestine, pointing to a role of PDK1-dependent signaling in the regulation of NHE-mediated electrolyte transport.
Sandu
Ciprian
C
Department of Physiology I, University of Tübingen, Germany.
Artunc
Ferruh
F
Palmada
Monica
M
Rexhepaj
Rexhep
R
Grahammer
Florian
F
Hussain
Azeemudeen
A
Yun
Chris
C
Alessi
Dario R
DR
Lang
Florian
F
D064413:3-Phosphoinositide-Dependent Protein Kinases; D000465:Algorithms; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D015194:Diffusion Chambers, Culture; D004232:Diuretics / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005243:Feces / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000235:genetics; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007422:Intestines / Q000033:anatomy & histology / Q000201:enzymology*; D008689:Methacrylates / Q000494:pharmacology; D051379:Mice; D018345:Mice, Knockout; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014162:Transfection
C118956:3-(2-(3-guanidino-2-methyl-3-oxo-propenyl)-5-methylphenyl)-N-isopropylidene-2-methyl-acrylamide dihydrochloride; D004232:Diuretics; D004791:Enzyme Inhibitors; D006146:Guanidines; D008689:Methacrylates; D000644:Quaternary Ammonium Compounds; C000613385:SLC9A3 protein, human; C000613386:Slc9a3 protein, mouse; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D049452:Green Fluorescent Proteins; D000584:Amiloride; D012964:Sodium; D064413:3-Phosphoinositide-Dependent Protein Kinases; C578562:PDPK1 protein, human; C578564:Pdpk1 protein, mouse; D017346:Protein Serine-Threonine Kinases