Glucose-induced changes in activity and phosphorylation of the Na +/H + exchanger, NHE-1, in vascular myocytes from Wistar-Kyoto and spontaneously hypertensive rats

Abstract

Increased Na +/H + exchanger (NHE) activity has been demonstrated in cells from patients with hypertension and diabetic nephropathy. Vascular myocytes from the spontaneously hypertensive rat (SHR) also exhibit increased NHE activity as compared with cells from the normotensive Wistar Kyoto rat (WKY). The interaction of increased glucose concentrations with NHE activity is unclear. The effect of glucose on NHE activity, NHE-1 (isoform 1) protein expression, and phosphorylation of cultured vascular myocytes from these rat strains was thus investigated. NHE activity was determined fluorometrically with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). A rabbit NHE-1—specific polyclonal antibody was used (1) to measure NHE-1 abundance in Western blots of cell extracts and (2) for immunoprecipitating 32P-labeled NHE-1. Cells from SHR exhibited increased NHE activity and NHE-1 phosphorylation as compared with cells from WKY, with similar NHE-1 protein content per cell. Incubation in 25 mmol · L −1 glucose for 24 hours led to increased NHE activity only in WKY cultures, with no change in NHE-1 protein but a concomitantly reduced NHE-1 phosphorylation. Changes in NHE activity in WKY cells were reversed by inhibition of protein kinase C. Incubation of SHR cells with 25 mmol · L −1 glucose did not enhance the increased NHE activity or NHE-1 phosphorylation present in these cells. Thus, high glucose levels have disparate effects on NHE activity and NHE-1 phosphorylation in cells from different rat strains. The glucose-induced increase in NHE-1 turnover number in WKY cells is not mediated by an increase in its direct phosphorylation, but is dependent on protein kinase C.