Role of c-SRC and ERK in acid-induced activation of NHE3.
BACKGROUND
In the renal proximal tubule, chronic acidosis causes increases in apical membrane NHE3 activity, which serve to increase transepithelial H+ secretion and return systemic pH to normal levels. Incubation of cultured renal epithelial cells in acid media activates c-Src.


METHODS
OKP cells were incubated in control (pH 7.4) or acid (7.0) media, and NHE3 activity measured as cytoplasmic pH (pHi) recovery from an acid load using BCECF. c-Src, ERK, and JNK kinase activities were measured by immune complex kinase assays with enolase, MBP, and GST-c-Jun, respectively, as substrates in the in vitro assays. To determine the role of c-Src in acid-induced NHE3 activation, cells were transfected with vector alone or a dominant negative c-Src (c-SrcK295M).


RESULTS
Expression of dominant negative c-srcK295M in OKP cells prevented acid-induced activation of NHE3. Incubation of OKP cells in acid media increased ERK activity and c-fos expression, but did not increase JNK activity. Acidosis in vivo also activated renal cortical c-Src and ERK kinases, whereas incubation of 3T3 cells in acid media activated c-Src but not ERK kinase. Expression of c-srcK295M did not affect ERK or c-fos activation by acid incubation. Inhibition of MEK with PD98059 inhibited activation of NHE3 by acid incubation.


CONCLUSIONS
These studies suggest that acidosis activates c-Src and MEK/ERK/c-fos. While both pathways are necessary for activation of NHE3, they are activated independently.
Tsuganezawa
Hirohiko
H
Department of Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9003, USA.
Sato
Soichiro
S
Yamaji
Yasuyoshi
Y
Preisig
Patricia A
PA
Moe
Orson W
OW
Alpern
Robert J
RJ
D000138:Acidosis / Q000378:metabolism*; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D002478:Cells, Cultured; D004789:Enzyme Activation; D048031:JNK Mitogen-Activated Protein Kinases; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1 / Q000502:physiology*; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D016760:Proto-Oncogene Proteins c-fos / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D019061:src-Family Kinases
D016760:Proto-Oncogene Proteins c-fos; C000613387:Slc9a3 protein, rat; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D000643:Ammonium Chloride; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; D017346:Protein Serine-Threonine Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases