NA-H+ EXCHANGER ISOFORM-1 PHOSPHORYLATION IN NORMAL WISTAR-KYOTO AND SPONTANEOUSLY HYPERTENSIVE RATS()

Increased activity of the cellular Na+-H+ exchanger (NHE) has been doc umented in various cell types in essential hypertension and in vascula r myocytes of the spontaneously hypertensive rat (SHR). The mechanism underlying this abnormality is unclear. Because the NHE can be activat ed by phosphorylation, we examined phosphorylation of the Na+-H+ excha nger isoform 1 (NHE-1) as one possible mechanism for its increased tur nover number in cultured vascular myocytes of the SHR. A polyclonal ra bbit antibody against a fusion protein consisting of beta-galactosidas e and the C-terminus of NHE-1 was used to immunoprecipitate P-32-label ed NHE-1 from cell extracts of SHR and Wistar-Kyoto (WKY) rat vascular myocytes in the absence and presence of 10% fetal calf serum. Immunop recipitates were separated by SDS-PAGE, and P-32-labeled NHE-1 was qua ntified from autoradiographs. Similar amounts of NHE-1 protein were de tected on Western blots of the cultured vascular myocytes from SHR and WKY rats. In quiescent cells, NHE-1 was significantly more phosphoryl ated in SHR myocytes than in WKY myocytes (2.17+/-0.06-fold enhancemen t [mean+/-SEM]; P<.001, n=8). The addition of fetal calf serum to quie scent cells had no significant effect on the phosphorylation of NHE-1 in SHR myocytes. However, NHE-1 phosphorylation fell transiently in se rum-treated WKY myocytes, with recovery to control levels after 20 min utes. Measurement of NHE activity using fluorometry confirmed elevated activity in the quiescent SHR myocytes compared with WKY myocytes. Fe tal calf serum led to further enhancement of NHE activity in both cell types. These findings suggest that the increased NHE activity in quie scent SHR myocytes may be correlated with enhanced NHE-1 phosphorylati on and that serum stimulates NHE activity in both cell types without a further increase in total NHE-1 phosphorylation, indicating a role fo r non-phosphorylation-dependent regulatory mechanisms.