Phenylephrine and sustained acidosis activate the neonatal rat cardiomyocyte Na+/H+ exchanger through phosphorylation of amino acids Ser770 and Ser771.
The mammalian Na(+)/H(+) exchanger isoform 1 (NHE1) is a ubiquitously expressed membrane protein that regulates intracellular pH in the myocardium. NHE1 is also important in mediating myocardial hypertrophy, and the blockage of NHE1 activity prevents hypertrophy and reduces ischemia-reperfusion injury in animal models. We recently demonstrated that extracellular-regulated kinase (ERK)-mediated activation of NHE1 occurs during ischemia-reperfusion of the myocardium. To understand the regulation of NHE1 in the myocardium by phosphorylation, we expressed a series of adenoviruses that express wild-type and mutant cDNA for NHE1. All exogenous cDNA for NHE1 had additional mutations [Leu(163)Phe/Gly(174)Ser], which increases NHE1 resistance to EMD-87580 (a specific blocker of NHE1) 100-fold, and allowed the measurement of exogenous NHE1 while inhibiting endogenous NHE1. By examining the effects of a series of mutations of the NHE1 cytosolic region, we determined that the amino acids Ser(770) and Ser(771) were essential for the acute activation of NHE1 activity in rat cardiomyocytes. The specific mutation of either residue prevented the rapid activation of exchanger activity by a sustained intracellular acidosis through ERK-dependent pathways. The same amino acids were critical to phenylephrine-mediated, ERK-dependent activation of NHE1 activity and increased the phosphorylation in intact rat cardiomyocytes. The results demonstrate that both sustained intracellular acidosis and phenylephrine rapidly activate the NHE1 protein in intact cardiac cells through ERK-dependent pathways that act on a common pathway mediated by amino acids Ser(770) and Ser(771) of the cytosolic tail of the protein.
Coccaro
Ersilia
E
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
Karki
Pratap
P
Cojocaru
Cicerone
C
Fliegel
Larry
L
D000138:Acidosis / Q000378:metabolism*; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000831:Animals, Newborn; D002316:Cardiotonic Agents / Q000494:pharmacology*; D002478:Cells, Cultured; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000502:physiology; D016296:Mutagenesis / Q000502:physiology; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology*; D010656:Phenylephrine / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D051381:Rats; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics* / Q000378:metabolism*
D002316:Cardiotonic Agents; C452850:Slc9a1 protein, rat; D000074082:Sodium-Hydrogen Exchanger 1; D017923:Sodium-Hydrogen Exchangers; D010656:Phenylephrine; D012694:Serine; D038744:Ribosomal Protein S6 Kinases, 90-kDa; D048049:Extracellular Signal-Regulated MAP Kinases