Role of protein kinase C isoforms in rat epididymal microvascular endothelial barrier function
Role of protein kinase C isoforms in rat epididymal microvascular endothelial barrier function
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DOI：
10.1165/rcmb.2002-0085oc
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发表时间：
2003-05-01
影响因子：
6.4
通讯作者：
Rounds, S
中科院分区：
文献类型：
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作者：
Harrington, EO;Brunelle, JL;Rounds, S
内皮屏障功能障碍涉及多种疾病状态。我们研究了蛋白激酶C（PKC）在单层通透性中的作用，使用过表达PKCalpha（PKCalphaEC）、PKC δ（PKC δ EC）或载体（载体对照EC）cDNA的内皮细胞（EC）。凝血酶在所有EC中诱导渗透性变化，并在PKCalphaEC中诱导单层渗透率显著升高。相反，PKCBEC的基础渗透性水平明显减弱，导致凝血酶诱导的渗透性变化减少。PKC抑制剂Go 6976和rottlerin分别逆转了PKCalpha和PKC δ过表达对通透性的影响。免疫印迹分析表明，在凝血酶处理的PKCaEC中，与细胞骨架亚细胞部分相关的β-连环蛋白显着减少，这一作用可通过Go 6976预处理来阻断。PKCdeltaEC包含每个细胞显著更大数量的焦点接触。凝血酶在所有EC中增强RhoA GT3酶活性;在PKCdeltaEC中活性水平高3倍。在所有EC中，Rottlerin显著减弱RhoA GT3活性。RhoA显性负性cDNA的过表达减少了EC中病灶接触的大小和数量，并显著增强了PKCdeltaEC单层通透性的基础速率。这些发现表明，单层通透性的变化是由PKC同工酶差异调节，这表明PKCalpha促进内皮屏障功能障碍和PKC δ增强基础内皮屏障功能。
Endothelial barrier dysfunction is involved in a variety of diseased states. We investigated the role of protein kinase C (PKC) in monolayer permeability using endothelial cells (EC) overexpressing PKCalpha (PKCalphaEC), PKCdelta (PKCdeltaEC) or vector (vector control EC) cDNAs. Thrombin induced permeability changes in all EC, and induced significantly elevated rates of monolayer permeability in PKCalphaEC. Conversely, the basal level of permeability was significantly blunted in PKCBEC, resulting in diminished thrombin-induced changes in permeability. PKC inhibitors, Go6976 and rottlerin, reversed the effects of PKCalpha and PKCdelta overexpression on permeability, respectively. Immunoblot analyses demonstrated significantly less beta-catenin associated with the cytoskeletal subcellular fraction in thrombin-treated PKCaEC, an effect blocked by pretreatment with Go6976. PKCdeltaEC contained significantly greater numbers of focal contacts per cell. Thrombin enhanced RhoA GTPase activity in all EC; with a 3-fold greater level of activity in PKCdeltaEC. Rottlerin significantly blunted RhoA GTPase activity in all EC. Overexpression of RhoA dominant-negative cDNA diminished the size and number of focal contacts in EC, and significantly enhanced the basal rate of PKCdeltaEC monolayer permeability. These findings demonstrate that monolayer permeability changes are differentially regulated by PKC isoenzymes, suggesting that PKCalpha promotes endothelial barrier dysfunction and PKCdelta enhances basal endothelial barrier function.