Muto S, Ebata S, Okada K, Saito T, Asano Y
Departments of Nephrology and Endocrinology and Metabolism, Jichi Medical School, Tochigi, Japan.
Kidney Int. 2000 Jun;57(6):2319-33. doi: 10.1046/j.1523-1755.2000.00092.x.
In vascular smooth muscle cells (VSMCs), Na+/H+ exchange (NHE) plays an important role in intracellular pH (pHi) regulation. The genomic effect of glucocorticoid (GC) on NHE activity has been suggested in VSMCs. However, the nongenomic and genomic effects of GC on NHE activity and the underlying intracellular signaling mechanisms have not yet been demonstrated in VSMCs. Also, it is not known whether there are specific surface-binding sites of GC to the plasma membrane of VSMCs.
The effects of short (3 h)- and long (24 h)-term exposure to corticosterone (CORTI) on NHE activity were studied in cultured rat aortic VSMCs by using pHi measurement with the pH-sensitive fluorescent dye 2'7'-bis(carboxyethyl)-5(6)-carboxyfluorescein. The NHE activity was calculated from the initial rate of Na+-dependent pHi recovery after the acid load.
Short-term exposure of VSMCs to CORTI (10-6 mol/L) increased NHE activity, whereas long-term exposure to CORTI decreased it. The inhibitors of gene transcription (actinomycin D) and of protein synthesis (cycloheximide) did not affect the short-term effect of CORTI on NHE activity, but inhibited the long-term effect of CORTI on NHE activity. The cytosolic GC receptor (GR) antagonist (RU38486) inhibited both the short- and long-term effects of CORTI on NHE activity, but the cytosolic mineralocorticoid receptor antagonist (spironolactone) did not influence either the short- or long-term CORTI effects. Two protein kinase C (PKC) inhibitors (staurosporine A and calphostin C) and PKC down-regulation [24-h pre-exposure to phorbol 12-myristate 13-acetate (PMA)] inhibited both short- and long-term CORTI effects. Exposure to PMA for three hours mimicked the short-term CORTI effect. The short-term CORTI effect was inhibited by the disruptor of microtubule (colchicine), but not by the disruptor of filamentous-actin (cytochalasin B). The long-term exposure to CORTI decreased NHE (NHE-1) mRNA levels to 0.65 times the control level, whereas the short-term exposure to CORTI caused no effect. Scatchard analysis of [3H]CORTI surface binding to VSMCs showed a single class of CORTI binding sites with a Bmax of 876.2 fmol per mg of cell protein and a Kd of 12.2 nmol/L. RU38486 also inhibited [3H]CORTI surface binding to VSMCs.
In VSMCs, NHE activity is stimulated by short-term exposure to CORTI, but is inhibited by long-term exposure to CORTI. The short-term stimulatory effect of CORTI on NHE activity is independent of gene transcription and protein synthesis, is mediated through the CORTI surface receptor, and occurs through a microtubule-dependent process. The long-term inhibitory effect of CORTI on NHE activity requires gene transcription and protein synthesis and occurs only through the cytosolic GR. The short- and long-term effects of CORTI on NHE activity occur via PKC activation. Therefore, CORTI differentially modulates NHE activity in VSMCs by nongenomic and genomic mechanisms.
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。糖皮质激素（GC）对VSMC中NHE活性的基因组效应已被提出。然而，GC对NHE活性的非基因组和基因组效应及其潜在的细胞内信号传导机制尚未在VSMC中得到证实。此外，尚不清楚GC是否存在与VSMC质膜的特异性表面结合位点。
通过使用对pH敏感的荧光染料2'7'-双（羧乙基）-5（6）-羧基荧光素测量pHi，研究了在培养的大鼠主动脉VSMC中短期（3小时）和长期（24小时）暴露于皮质酮（CORTI）对NHE活性的影响。NHE活性根据酸负荷后Na +依赖的pHi恢复的初始速率计算。
VSMC短期暴露于CORTI（10 -6 mol/L）可增加NHE活性，而长期暴露于CORTI则降低NHE活性。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）不影响CORTI对NHE活性的短期作用，但抑制CORTI对NHE活性的长期作用。胞质糖皮质激素受体（GR）拮抗剂（RU38486）抑制CORTI对NHE活性的短期和长期作用，但胞质盐皮质激素受体拮抗剂（螺内酯）对CORTI的短期或长期作用均无影响。两种蛋白激酶C（PKC）抑制剂（星形孢菌素A和钙泊三醇C）和PKC下调[24小时预先暴露于佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）]均抑制CORTI的短期和长期作用。暴露于PMA三小时模拟了CORTI的短期作用。CORTI的短期作用被微管破坏剂（秋水仙碱）抑制，但未被丝状肌动蛋白破坏剂（细胞松弛素B）抑制。长期暴露于CORTI使NHE（NHE-1）mRNA水平降至对照水平的0.65倍，而短期暴露于CORTI则无影响。对[3H]CORTI与VSMC表面结合的Scatchard分析显示存在一类CORTI结合位点，Bmax为每毫克细胞蛋白876.2 fmol，Kd为12.2 nmol/L。RU38486也抑制[3H]CORTI与VSMC的表面结合。
在VSMC中，短期暴露于CORTI可刺激NHE活性，但长期暴露于CORTI则抑制NHE活性。CORTI对NHE活性的短期刺激作用独立于基因转录和蛋白质合成，通过CORTI表面受体介导，并通过微管依赖的过程发生。CORTI对NHE活性的长期抑制作用需要基因转录和蛋白质合成，并且仅通过胞质GR发生。CORTI对NHE活性的短期和长期作用均通过PKC激活发生。因此，CORTI通过非基因组和基因组机制差异性地调节VSMC中的NHE活性。
