The mammalian Na+/H+ exchanger isoform 1 (NHE1) is a ubiquitously
expressed membrane protein that regulates myocardial intracellular pH. Inhibition of NHE1 prevents hypertrophy and reduces ischemia-reperfusion (I/R) injury in animal models. To understand the regulation of NHE1 in the myocardium by phosphorylation we constructed adenoviruses, which express wild type or mutant cDNA for NHE1. Additionally, wild type and mutant NHE1 had mutations Leu163Phe/Gly174Ser, which increases NHE1 resistance to EMD87580 (NHE1 inhibitor) by 100-fold. This allowed measurement of exogenous NHE1 activity while inhibiting endogenous NHE1 activity. We examined the effects of a series of mutations of phosphorylation sites in the cytosolic domain of NHE1. Sustained intracellular acidosis and phenylephrine caused an ERK-dependent activation of NHE1 activity and phosphorylation levels. We demonstrated that amino acids Ser770 and Ser771 were essential for activation of NHE1 activity in isolated rat cardiomyocytes by sustained intracellular acidosis and phenylephrine. Furthermore, mutation of Ser770 and Ser771 to Ala prevented increased NHE1 phosphorylation by sustained intracellular acidosis and phenylephrine. This was found to occur in an ERK-dependent manner. Taken together, our results demonstrate that both sustained intracellular acidosis and phenylephrine rapidly activate the NHE1 protein in isolated cardiac cells via an ERK-dependent pathway that acts on the common amino acids Ser770 and Ser771 of the C-terminal tail of NHE1.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:AEU.10048/845 |
| Date | 06 1900 |
| Creators | Coccaro, Ersilia |
| Contributors | Fliegel, Larry (Biochemistry), Holmes, Charles (Biochemistry), Brindley, David (Biochemistry), Young, Howard (Biochemistry), Schulz, Richard (Pharmacology), Walsh, Michael (Biochemistry and Molecular Biology) |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 6362256 bytes, application/pdf |
| Relation | Coccaro E, Mraiche F, Malo M, Vandertol-Vanier H, Bullis B, Robertson M, and Fliegel L. (2007). Expression and characterization of the Na+/H+ exchanger in the myocardium. Mol. Cell Biochem. 302(1-2): 145-155., Coccaro E, Pratap Karki, Cicerone Cojocaru and Larry Fliegel. (2009) Phenylephrine and sustained acidosis actiate the neonatal rat cardiomyocyte Na+/H+ exchangers through phosphorylation of amino acids Ser770 and Ser771. Am J Physiol Heart Circ Physiol. 297 (2): H846-858. |
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