EFFECTS OF GLUCOCORTICOIDS ON NA+/H+ EXCHANGE AND GROWTH IN CULTURED VASCULAR SMOOTH-MUSCLE CELLS
EFFECTS OF GLUCOCORTICOIDS ON NA+/H+ EXCHANGE AND GROWTH IN CULTURED VASCULAR SMOOTH-MUSCLE CELLS
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DOI：
10.1002/jcp.1041370302
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发表时间：
1988-12-01
影响因子：
5.6
通讯作者：
ALEXANDER, RW
中科院分区：
文献类型：
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作者：
BERK, BC;VALLEGA, G;ALEXANDER, RW
我们研究了氢化可的松对培养的大鼠主动脉血管平滑肌细胞（VSMC）生长和Na+/H+交换的影响。通过3 H-胸苷掺入和汇合时的细胞数测定，氢化可的松（2 μ M）处理生长停滞的VSMC显著降低了VSMC对10%小牛血清的反应生长。这种效应与改变的细胞表型的出现有关，其特征是大而扁平的VSMC，不形成典型的“小丘”。氢化可的松处理的细胞中的Na+/H+交换也发生了改变，这是通过二甲氨酰苯胺敏感的22 Na+流入酸负载细胞或通过使用荧光染料BCECF的细胞内pH（pHi）变化来测定的。静息pHi为7.25 ±。0.04和7.15 .+-。0.05对照组和氢化可的松处理组的细胞凋亡率分别为0.1 < P < 0.05。在不存在外部Na+的情况下细胞内酸化后，在氢化可的松处理的细胞中，相对于对照，加入Na+后的pHi恢复增加了89%。 这是由于Na+/H+交换剂的Vmax从17.5 ± 1增加。2.4至25.9 .+-。2.0 nmol Na+/mg蛋白质×min（P < 0.01），而Km无明显变化。用放线菌素D（1 μ g/ml）或放线菌酮（10 μ M）处理VSMC完全抑制氢化可的松介导的Na+/H+交换增加，表明RNA和蛋白质合成都需要。由于氢化可的松改变了Na+/H+交换的Vmax，与激动剂如血清或血管紧张素II分别改变细胞内H+或细胞外Na+的Km相反，我们研究了氢化可的松对这些激动剂激活Na+/H+交换的影响。在维持在生理pHi（7.2）的细胞中，血管紧张素II刺激的碱化的初始速率（2分钟）增加了66 ± 0.5。29%，与对照细胞相比，氢化可的松处理。氢化可的松没有引起血管紧张素II刺激的磷脂酶C活性的变化，通过测量细胞内Ca 2+或甘油二酯形成的变化。然而，血管紧张素II和血清刺激只有小的增加Na+/H+交换酸负荷（pHi = 6.8）氢化可的松处理的细胞。这些结果表明，氢化可的松介导的VSMC Na+/H+交换增加与Na+/H+交换激活调节改变的非增殖表型相关。我们建议，氢化可的松介导的生长抑制可能是一个有用的模型，用于研究Na+/H+交换在细胞生长反应中的作用。
We have examined the effects of hydrocortisone on growth and Na+/H+ exchange in cultured rat aortic vascular smooth muscle cells (VSMC). Hydrocortisone (2 .mu.M) treatment of growth-arrested VSMC significantly decreased VSMC growth in response to 10% calf serum assayed by 3H-thymidine incorporation and cell number at confluence. This effect was associated with the appearance of an altered cell phenotype characterized by large, flat VSMC that did not form typical "hillocks." Na+/H+ exchange was also altered in hydrocortisone-treated cells assayed by dimethylamiloride-sensitive 22Na+ influx into acid-loaded cells or by intracellular pH (pHi) change using the fluorescent dye BCECF. Resting pHi was 7.25 .+-. 0.04 and 7.15 .+-. 0.05 in control and hydrocortisone-treated cells, respectively (0.1 < P < 0.05). Following intracellular acidification in the absence of external Na+, pHi recovery upon addition of Na+ was increased 89% in hydrocortisone-treated cells relative to control. This was due to an increase in the Vmax for the Na+/H+ exchanger from 17.5 .+-. 2.4 to 25.9 .+-. 2.0 nmol Na+/mg protein .times. min (P < 0.01) without a significant change in Km. Treatment of VSMC with actinomycin D (1 .mu.g/ml) or cycloheximide (10 .mu.M) completely inhibited the hydrocortisone-mediated increase in Na+/H+ exchange, indicating a requirement for both RNA and protein synthesis. Because hydrocortisone altered the Vmax for Na+/H+ exchange, in contrast to agonists such as serum or angiotensin II which alter the Km for intracellular H+ or extracellular Na+, respectively, we studied the effect of hydrocortisone on activation of Na+/H+ exchange by these agonists. In cell maintained at physiological pHi (7.2), the initial rate (2 min) of angiotensin II-stimulated alkalinization was increased 66 .+-. 29% in hydrocortisone-treated compared with control cells. Hydrocortisone caused no change in angiotensin II-stimulated phospholipase C activity assayed by measurement of changes in intracellular Ca2+ or diacylglycerol formation. However, angiotensin II and serum stimulated only small increases in Na+/H+ exchange in acid-loaded (pHi = 6.8) hydrocortisone-treated cells. These findings suggest that hydrocortisone-mediated increases in VSMC Na+/H+ exchange occur in association with a nonproliferating phenotype that has altered regulation of Na+/H+ exchange activation. We propose that hydrocortisone-mediated growth inhibition may be a useful model for studying the role of Na+/H+ exchange in cell growth responsiveness.