Secretin regulates paracellular permeability in canine gastric monolayers by a Src kinase-dependent pathway.
Secretin regulates paracellular permeability in canine gastric monolayers by a Src kinase-dependent pathway.
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促胰液素通过 Src 激酶依赖性途径调节犬胃单层细胞的细胞旁通透性。
DOI：
10.1152/ajpgi.00429.2001
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发表时间：
2002
期刊：
影响因子：
--
通讯作者：
Soll,AndrewH
中科院分区：
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作者：
Chen,MonicaC;Solomon,TravisE;PerezSalazar,Eduardo;Kui,Robert;Rozengurt,Enrique;Soll,AndrewH
以往的研究发现，表皮生长因子(EGF)可降低胃粘膜细胞旁通透性，但其他生理调节因子以及介导这些反应的分子机制尚不清楚。我们研究了促胰液素和Src在调节细胞旁通透性中的作用，因为促胰液素分别调节胃主细胞功能和Src介导涉及细胞骨架-膜界面的事件。犬胃上皮细胞在Transwell滤片上短期培养形成融合单层。在含有或不含有特定激酶抑制剂的促胰液素存在的情况下，监测耐药性。用位点特异性磷酸酪氨酸抗体检测酪氨酸416位点的Src的酪氨酸磷酸化。在浓度为1-100 nM的基础侧，而不是顶端，促胰液素可剂量依赖性地增加耐药性；这种反应迅速且持续时间长达数小时。PP2(10μM)是一种选择性的Src酪氨酸激酶抑制剂，但不是非活性的异构体PP3，它可以消除促胰液素增加的耐药性，但仅适度减弱顶端表皮生长因子的作用。特异性的EGF受体酪氨酸激酶抑制剂AG-1478(100 NM)可减弱对EGF的耐药性增加，但不能抑制分泌素。促胰液素，而不是EGF，以剂量依赖的方式诱导Tyr416处的Src酪氨酸磷酸化，最大反应在1min观察到。PP2，而不是PP3，显著地抑制了这种酪氨酸磷酸化。促胰液素通过Src介导的途径增加胃粘膜的细胞旁抵抗，而EGF的作用不依赖于Src。SRC似乎介导了这种Gs偶联受体在原代上皮细胞中的生理作用。
Previous studies found that epidermal growth factor (EGF) decreased paracellular permeability in gastric mucosa, but the other physiological regulators and the molecular mechanisms mediating these responses remain undefined. We investigated the role of secretin and Src in regulating paracellular permeability because secretin regulates gastric chief cell function and Src mediates events involving the cytoskeletal-membrane interface, respectively. Confluent monolayers were formed from canine gastric epithelial cells in short-term culture on Transwell filter inserts. Resistance was monitored in the presence of secretin with or without specific kinase inhibitors. Tyrosine phosphorylation of Src at Tyr416was measured with a site-specific phosphotyrosine antibody. Basolateral, but not apical, secretin at concentrations from 1 to 100 nM dose dependently increased resistance; this response was rapid and sustained over hours. PP2 (10 μM), a selective Src tyrosine kinase inhibitor, but not the inactive isomer PP3, abolished the increase in resistance by secretin but only modestly attenuated apical EGF effects. AG-1478 (100 nM), a specific EGF receptor tyrosine kinase inhibitor, attenuated the resistance increase to EGF but not secretin. Secretin, but not EGF, induced tyrosine phosphorylation of Src at Tyr416in a dose-dependent fashion, with the maximal response observed at 1 min. PP2, but not PP3, dramatically inhibited this tyrosine phosphorylation. Secretin increases paracellular resistance in gastric mucosa through a Src-mediated pathway, while the effect of EGF is Src independent. Src appears to mediate the physiological effects of this Gs-coupled receptor in primary epithelial cells.