Qiao Jing, Huang Fei, Lum Hazel
Department of Pharmacology, Rush Presbyterian St. Luke's Medical Center, Chicago, Illinois 60612, USA.
Am J Physiol Lung Cell Mol Physiol. 2003 Jun;284(6):L972-80. doi: 10.1152/ajplung.00429.2002. Epub 2003 Feb 14.
Much evidence indicates that cAMP-dependent protein kinase (PKA) prevents increased endothelial permeability induced by inflammatory mediators. We investigated the hypothesis that PKA inhibits Rho GTPases, which are regulator proteins believed to mediate endothelial barrier dysfunction. Stimulation of human microvascular endothelial cells (HMEC) with thrombin (10 nM) increased activated RhoA (RhoA-GTP) within 1 min, which remained elevated approximately fourfold over control for 15 min. The activation was accompanied by RhoA translocation to the cell membrane. However, thrombin did not activate Cdc42 or Rac1 within similar time points, indicating selectivity of activation responses by Rho GTPases. Pretreatment of HMEC with 10 micro M forskolin plus 1 micro M IBMX (FI) to elevate intracellular cAMP levels inhibited both thrombin-induced RhoA activation and translocation responses. FI additionally inhibited thrombin-mediated dissociation of RhoA from guanine nucleotide dissociation inhibitor (GDI) and enhanced in vivo incorporation of (32)P by GDI. HMEC pretreated in parallel with FI showed >50% reduction in time for the thrombin-mediated resistance drop to return to near baseline and inhibition of approximately 23% of the extent of resistance drop. Infection of HMEC with replication-deficient adenovirus containing the protein kinase A inhibitor gene (PKA inhibitor) blocked both the FI-mediated protective effects on RhoA activation and resistance changes. In conclusion, the results provide evidence that PKA inhibited RhoA activation in endothelial cells, supporting a signaling mechanism of protection against vascular endothelial barrier dysfunction.
大量证据表明，环磷酸腺苷（cAMP）依赖性蛋白激酶（PKA）可防止炎症介质诱导的内皮通透性增加。我们研究了一个假说，即PKA抑制Rho GTP酶，这些调节蛋白被认为介导内皮屏障功能障碍。用凝血酶（10 nM）刺激人微血管内皮细胞（HMEC），1分钟内活化的RhoA（RhoA-GTP）增加，在15分钟内其水平比对照升高约四倍。这种活化伴随着RhoA转位至细胞膜。然而，凝血酶在相似时间点未激活Cdc42或Rac1，表明Rho GTP酶激活反应具有选择性。用10 μM福斯高林加1 μM异丁基甲基黄嘌呤（FI）预处理HMEC以提高细胞内cAMP水平，可抑制凝血酶诱导的RhoA活化和转位反应。FI还抑制凝血酶介导的RhoA与鸟嘌呤核苷酸解离抑制剂（GDI）的解离，并增强GDI在体内对（32）P的掺入。与FI平行预处理的HMEC显示，凝血酶介导的阻力下降恢复至接近基线的时间减少>50%，阻力下降幅度抑制约23%。用含有蛋白激酶A抑制剂基因的复制缺陷型腺病毒（PKA抑制剂）感染HMEC，可阻断FI介导的对RhoA活化和阻力变化的保护作用。总之，结果提供了证据表明PKA在内皮细胞中抑制RhoA活化，支持了一种防止血管内皮屏障功能障碍的信号传导机制。
