Na + -H + Exchanger Isoform 1 Phosphorylation in Normal Wistar-Kyoto andSpontaneously Hypertensive Rats

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Abstract
Increased activity of the cellular Na+-H+ exchanger (NHE) has been documented in various cell types in essential hypertension and in vascular myocytes of the spontaneously hypertensive rat (SHR). The mechanism underlying this abnormality is unclear. Because the NHE can be activated by phosphorylation, we examined phosphorylation of the Na+-H+ exchanger isoform 1 (NHE-1) as one possible mechanism for its increased turnover number in cultured vascular myocytes of the SHR. A polyclonal rabbit antibody against a fusion protein consisting of β-galactosidase and the C-terminus of NHE-1 was used to immunoprecipitate 32P-labeled NHE-1 from cell extracts of SHR and Wistar-Kyoto (WKY) rat vascular myocytes in the absence and presence of 10% fetal calf serum. Immunoprecipitates were separated by SDS-PAGE, and 32P-labeled NHE-1 was quantified from autoradiographs. Similar amounts of NHE-1 protein were detected on Western blots of the cultured vascular myocytes from SHR and WKY rats. In quiescent cells, NHE-1 was significantly more phosphorylated in SHR myocytes than in WKY myocytes (2.17±0.06-fold enhancement [mean±SEM]; P<.001, n=8). The addition of fetal calf serum to quiescent cells had no significant effect on the phosphorylation of NHE-1 in SHR myocytes. However, NHE-1 phosphorylation fell transiently in serum-treated WKY myocytes, with recovery to control levels after 20 minutes. Measurement of NHE activity using fluorometry confirmed elevated activity in the quiescent SHR myocytes compared with WKY myocytes. Fetal calf serum led to further enhancement of NHE activity in both cell types. These findings suggest that the increased NHE activity in quiescent SHR myocytes may be correlated with enhanced NHE-1 phosphorylation and that serum stimulates NHE activity in both cell types without a further increase in total NHE-1 phosphorylation, indicating a role for non–phosphorylation-dependent regulatory mechanisms.