Ebata S, Muto S, Okada K, Nemoto J, Amemiya M, Saito T, Asano Y
Department of Nephrology, Jichi Medical School, Minamikawachi Tochigi, Japan.
Kidney Int. 1999 Oct;56(4):1400-12. doi: 10.1046/j.1523-1755.1999.00674.x.
In vascular smooth muscle cells (VSMCs), Na+/H+ exchange (NHE) plays an important role in intracellular pH (pHi) 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.
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.
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 down-regulation (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 disruptors of microtubule (colchicine) and of filamentous-actin (cytochalasin B). Long-term exposure of ALDO caused a threefold increase in NHE (NHE-1) mRNA levels.
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.
在血管平滑肌细胞（VSMC）中，钠氢交换体（NHE）在细胞内pH（pHi）调节中起重要作用。最近，有研究提示醛固酮（ALDO）对VSMC的NHE活性存在非基因组效应。然而，ALDO对NHE的非基因组和基因组效应以及这些效应的细胞内信号传导机制在VSMC中尚未完全明确。
采用pH敏感染料2'7'-双（羧乙基）-5（6）-羧基荧光素通过单细胞pHi测量法，检测短期（3小时）和长期（24小时）暴露于ALDO对大鼠胸主动脉培养VSMC中NHE活性的影响。NHE活性根据酸负荷后Na+依赖的pHi恢复初始速率计算得出。
VSMC短期和长期暴露于ALDO（10⁻⁶ M）后，NHE活性显著增加。基因转录抑制剂（放线菌素D）和蛋白质合成抑制剂（环己酰亚胺）对ALDO的短期效应无影响，但抑制了ALDO的长期效应。盐皮质激素受体（MR）拮抗剂（螺内酯）和糖皮质激素受体（GR）拮抗剂（RU38486）对ALDO的短期效应无影响，但抑制了ALDO的长期效应。两种蛋白激酶C（PKC）抑制剂（星形孢菌素A和钙泊三醇）以及PKC下调（预先暴露于佛波酯12-肉豆蔻酸酯13-乙酸酯24小时）均抑制了ALDO的短期和长期效应。VSMC暴露于佛波酯3小时可模拟ALDO对NHE活性的短期效应。ALDO显著增加VSMC中的PKC活性。ALDO的短期效应被微管破坏剂（秋水仙碱）和丝状肌动蛋白破坏剂（细胞松弛素B）抑制。长期暴露于ALDO导致NHE（NHE-1）mRNA水平增加三倍。
ALDO对NHE活性的短期效应不是通过MR或GR介导，其发生独立于基因转录和蛋白质合成，且通过涉及细胞骨架结构元件的机制发生。ALDO对NHE活性的长期效应通过MR和GR发生，且需要基因转录和蛋白质合成。ALDO的短期和长期效应均通过PKC激活介导。因此，ALDO通过非基因组和基因组机制在VSMC中激活NHE。
