Activation of proteinase-activated receptor 1 stimulates epithelial chloride secretion through a unique MAP kinase- and cyclo-oxygenase-dependent pathway
Activation of proteinase-activated receptor 1 stimulates epithelial chloride secretion through a unique MAP kinase- and cyclo-oxygenase-dependent pathway
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DOI：
10.1096/fj.02-0039com
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发表时间：
2002-10-01
期刊：
影响因子：
4.8
通讯作者：
MacNaughton, WK
中科院分区：
文献类型：
--
作者：
Buresi, MC;Buret, AG;MacNaughton, WK
蛋白酶激活受体1 （PAR-1）被凝血酶激活，诱导肠上皮细胞分泌氯化物。为了进一步阐明PAR -1刺激分泌的机制，我们在改良的Ussing室中研究了SCBN肠上皮细胞的单层。在存在或不存在各种信号转导和环加氧酶（COX）途径抑制剂的情况下，在基础侧应用凝血酶和PAR-1激活肽，ala - parfluoro - ph - arg -cyclohexyl- ala - citruline - tyr （cte - nh2）后，测定短路电流响应。通过免疫沉淀和靶蛋白的Western blot分析来监测激酶活性的增加。EGF受体酪氨酸激酶、src -激酶、MEK1/2抑制剂以及胞质磷脂酶（cPL） A(2)、COX-1和COX-2抑制剂可显著减弱PAR 1诱导的氯化物分泌反应。通过磷酸化丝氨酸残基的Western blot显示，在MEK抑制剂U0126处理的细胞中，par -1诱导的cPLA活化(2)被阻断，这表明MEK- erk1 /2 MAP激酶途径介导了par -1诱导的cPLA2磷酸化。我们的数据显示，PAR 1诱导的SCBN细胞中氯化物分泌涉及Src、EGF受体反式激活、MAPK通路激活、cPLA2磷酸化、COX活性，但不包括PGF（2 α）或PGE(2)。这些发现可能对肠道炎症性疾病具有临床意义，其中分泌功能障碍明显和凝血酶水平升高。
Proteinase-activated receptor 1 (PAR-1) is activated by thrombin and induces chloride secretion by intestinal epithelial cells. To elucidate further the mechanisms whereby PAR -1 stimulates secretion, monolayers of SCBN intestinal epithelial cells were studied in modified Ussing chambers. Short circuit current responses were determined after basolateral application of thrombin and the PAR-1 -activating peptide, Ala-parafluoro-Phe-Arg-cyclohexyl-Ala-Citrulline-Tyr (Cit-NH2) in the presence or absence of a variety of signal transduction and cyclo-oxygenase (COX) pathway inhibitors. Increased kinase activity was monitored by immunoprecipitation and Western blot analysis of target phosphoproteins. The PAR 1-induced chloride secretory response was significantly attenuated by inhibitors of the EGF receptor tyrosine kinase, Src-kinase, MEK1/2, as well as by inhibitors of cytosolic phospholipase (cPL) A(2), COX-1 and COX-2. PAR-1-induced activation of cPLA(2), as shown by Western blot of phosphoserine residues, was blocked in cells treated with the MEK inhibitor U0126, indicating that the MEK-ERK1/2 MAP kinase pathway mediated PAR-1-induced cPLA2 phosphorylation. Our data show that PAR 1-induced chloride secretion in SCBN cells involves Src, EGF receptor trans-activation, activation of a MAPK pathway, phosphorylation of cPLA2, COX activity, but not PGF(2alpha) or PGE(2). These findings may be of clinical importance in inflammatory diseases of the intestine where secretory dysfunction is evident and thrombin levels are elevated.