Adenosine induces dephosphorylation of myosin II regulatory light chain in cultured bovine corneal endothelial cells
Adenosine induces dephosphorylation of myosin II regulatory light chain in cultured bovine corneal endothelial cells
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DOI：
10.1016/j.exer.2004.06.027
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发表时间：
2004-10-01
影响因子：
3.4
通讯作者：
Van Driessche, W
中科院分区：
文献类型：
--
作者：
Srinivas, SP;Satpathy, M;Van Driessche, W
目的：肌球蛋白 II 调节轻链 (MLC) 的去磷酸化通过松弛肌动蛋白细胞骨架来促进细胞单层屏障的完整性。本研究探讨了腺苷（ADO）对培养的牛角膜内皮细胞（BCEC）MLC磷酸化的影响。方法：通过尿素-甘油凝胶电泳和免疫印迹评估MLC磷酸化。 cAMP 响应元件结合蛋白 (CREB) 的磷酸化证实了 A(2b) 受体（P1 嘌呤能受体亚型）激动剂引起的 cAMP 升高，并通过蛋白质印迹法测定。通过蛋白质印迹法评估 MAP 激酶的激活（即激活的 ERK1 和 ERK2），以检查它们对 MLC 磷酸化的影响。测量在多孔过滤器上生长的细胞的跨上皮电阻 (TER)，以评估改变的屏障完整性。结果：暴露于已知的 A(2b) 受体激动剂 ADO（200 微米；30 分钟）和 N-乙基（甲酰胺）腺苷（NECA；50 微米；30 分钟），诱导 CREB 磷酸化，类似于直接激活剂毛喉素（FSK，下午 20 点；30 分钟）腺苷酸环化酶。暴露于 ADO、NECA 和 FSK 分别导致 MLC 去磷酸化 51%、40% 和 47%。 ADO 诱导的去磷酸化呈剂量依赖性，1 mum ADO 时去磷酸化高达 31%。 CGS-21680 是一种选择性 A(2a) 激动剂，既不诱导 MLC 去磷酸化，也不诱导 CREB 磷酸化。 ADO 磷酸化 MAP 激酶可通过暴露于 MAP 激酶特异性抑制剂 U0126 (10 muM) 来预防。与 ADO 类似，NECA 和 FSK 也诱导 ERK1 和 ERK2 激活。暴露于 U0126 会在基础条件下抑制 MLC 磷酸化 17%。同时暴露于 U0126 可以增强 ADO 诱导的 MLC 去磷酸化（去磷酸化增加 25%）。暴露于ADO导致TER从17欧姆cm(2)增加至22欧姆cm(2)。结论：(1)响应ADO和NECA的CREB磷酸化表明cAMP-PKA轴的激活，表明BCEC中A2b受体的表达。 (2) ERK1和ERK2被cAMP和A2b受体激活，促进MLC磷酸化。然而，cAMP 升高的最终结果是 MLC 去磷酸化，大概是因为涉及 MLCK 和/或 ROCK 失活的竞争途径占主导地位（Rho 相关卷曲线圈蛋白激酶或 Rho 激酶）。 (3) 与 MLC 去磷酸化一致，暴露于 ADO 会增加 TER，这表明屏障完整性增加。 (C) 2004 Elsevier Ltd. 保留所有权利。
Purpose: Dephosphorylation of the myosin II regulatory light chain (MLC) promotes barrier integrity of cellular monolayers through relaxation of the actin cytoskeleton. This study has investigated the influence of adenosine (ADO) on MLC phosphorylation in cultured bovine corneal endothelial cells (BCEC).Methods: MLC phosphorylation was assessed by urea-glycerol gel electrophoresis and immunoblotting. Elevation of cAMP in response to agonists of A(2b) receptors (subtype of P1 purinergic receptors) was confirmed by phosphorylation of the cAMP response element binding protein (CREB), which was determined by Western blotting. Activation of MAP kinases (i.e. activated ERK1 and ERK2) was assessed by Western blotting to examine their influence on MLC phosphorylation. Transepithelial electrical resistance (TER) of cells grown on porous filters was measured to assess the altered barrier integrity.Results: Exposure to ADO (200 mum; 30 min) and N-ethyl (carboxamido) adenosine (NECA; 50 mum; 30 min), known agonists of A(2b) receptors, induced phosphorylation of CREB similar to forskolin (FSK, 20 pm; 30 min), a direct activator of adenylate cyclase. Exposure to ADO, NECA, and FSK led to dephosphorylation of MLC by 51, 40, and 47%, respectively. ADO-induced dephosphorylation was dose-dependent with as much as 31% dephosphorylation at 1 mum ADO. CGS-21680, a selective A(2a) agonist, neither induced MLC dephosphorylation nor CREB phosphorylation. ADO phosphorylated MAP kinases which could be prevented by exposure to the MAP kinase-specific inhibitor, U0126 (10 muM). NECA and FSK also induced ERK1 and ERK2 activation similar to ADO. Exposure to U0126 inhibited MLC phosphorylation under basal conditions by 17%. ADO-induced MLC dephosphorylation was enhanced by a simultaneous exposure to U0126 (25% increase in dephosphorylation). Exposure to ADO caused an increase in TER from 17 to 22 ohms cm(2).Conclusions: (1) CREB phosphorylation in response to ADO and NECA, which indicates activation of the cAMP-PKA axis, suggests expression of A2b receptors in BCEC. (2) ERK1 and ERK2, activated by cAMP and A2b receptors, promote MLC phosphorylation. However, the net result of cAMP elevation is MLC dephosphorylation, presumably because the competing pathways involving inactivation of MLCK and/or ROCK are dominant (Rho-associated coiled coil-containing protein kinase or Rho kinase). (3) Consistent with MLC dephosphorylation, exposure to ADO increases TER, which suggests increased barrier integrity. (C) 2004 Elsevier Ltd. All rights reserved.