Aldosterone activates Na+/H+ exchange in vascular smooth muscle cells by nongenomic and genomic mechanisms

Background. In vascular smooth muscle cells (VSMCs), Na+/H+ exchange (NHE) plays an important role in intracellular pH (pH(i)) regulation. Recently, nongenomic effect of aldosterone (ALDO) on NHE activity has been suggested in VSMCs. However, the nongenomic and genomic effects of ALDO on NHE and the intracellular signaling mechanisms for these effects have not fully been determined in VSMCs. Methods. The effects of short- (3 hr) and long- (24 hr) term exposure to ALDO on NHE activity were examined in cultured VSMCs from rat thoracic aortae by using single-cell pHi measurement with the pH-sensitive dye 2'7'-bis(carboxyethyl)5(6)-carboxyfluorescein. The NHE activity was calculated from the initial rate of Na+-dependent pHi recovery after acid load. Results. The NHE activity significantly increased after short-and long-term exposure of VSMCs to ALDO (10(-6) M). The inhibitors of gene transcription (actinomycin D) and of protein synthesis (cycloheximide) had no effect on the short-term ALDO effect, but inhibited the long-term ALDO effect. The antagonists of the mineralocorticoid receptor (MR) (spironolactone) and of the glucocorticoid receptor (GR) (RU38486) caused no effect on the short-term ALDO effect, but inhibited the long-term ALDO effect. Two protein kinase C (PKC) inhibitors (staurosporine A and calphostin C) and PKC downregulation (24 hr pre-exposure to phobol 12-myristate 13-acetate, PMA) inhibited both the short- and long-term ALDO effects. Exposure of VSMCs to PMA for 3 hours mimicked the short-term effect of ALDO on NHE activity. ALDO significantly increased PKC activity in VSMCs. The short-term ALDO effect was inhibited by disrupters of microtubule (colchicine) and of filamentous-actin (cytochalasin B). Long-term exposure of ALDO caused a threefold increase in NHE (NHE-1) mRNA levels. Conclusions. The short-term effect of ALDO on NHE activity is not mediated through either MR or GR, occurs independent of gene transcription and protein synthesis, and occurs through a mechanism involving the structural elements of cytoskeleton. The long-term effect of ALDO on NHE activity occurs through both MR and GR and requires gene transcription and protein synthesis. Both short- and long-term effects of ALDO are mediated through PKC activation. Therefore, ALDO activates NHE by nongenomic and genomic mechanisms in VSMCs.