Histamine-induced myosin light chain phosphorylation breaks down the barrier integrity of cultured corneal epithelial cells
Histamine-induced myosin light chain phosphorylation breaks down the barrier integrity of cultured corneal epithelial cells
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DOI：
10.1007/s11095-007-9309-1
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发表时间：
2007-10-01
影响因子：
3.7
通讯作者：
Srinivas, Sangly P.
中科院分区：
文献类型：
--
作者：
Guo, Ying;Ramachandran, Charanya;Srinivas, Sangly P.
目的.研究组胺对角膜上皮细胞肌球蛋白轻链（MLC）磷酸化的影响及其对角膜上皮细胞屏障完整性的影响。实验在牛角膜上皮细胞（BCEC）中进行。RT-PCR和Western blotting检测H1受体和MLC激酶（MLCK）的表达。通过尿素-甘油凝胶电泳和蛋白质印迹法评估MLC的磷酸化。屏障完整性通过辣根过氧化物酶（HRP; 44 kDa）穿过多孔过滤器上生长的单层的渗透性来确定。H1受体和MLCK在BCEC中均有表达。暴露于组胺诱导显着的MLC磷酸化伴随着HRP渗透性的增加。此外，在静息细胞中发现的皮质肌动蛋白的组织被破坏。与组胺相反，ATP（一种P2 Y受体激动剂）诱导MLC的去磷酸化。预先暴露于ATP可降低组胺对HRP通透性和皮层肌动蛋白破坏的影响。MLC磷酸化是肌动蛋白细胞骨架收缩性增加的生化先决条件，导致组胺诱导的角膜上皮细胞屏障完整性的破坏。这是由于肌动蛋白收缩性增加产生的向心力减弱了紧密连接处的束缚力。
Purpose. To investigate changes in the phosphorylation of myosin light chain (MLC) in response to histamine and its effect on the barrier integrity of corneal epithelial cells.Materials and Methods. Experiments were performed in bovine corneal epithelial cells (BCEC). RT-PCR and Western blotting were employed to characterize expression of H1 receptors and MLC kinase (MLCK). Phosphorylation of MLC was assessed by urea-glycerol gel electrophoresis and Western blotting. Barrier integrity was determined as permeability to horseradish peroxidase (HRP; 44 kDa) across monolayers grown on porous filters.Results. Expression of both H1 receptors and MLCK was found in BCEC. Exposure to histamine induced significant MLC phosphorylation concomitant with an increase in HRP permeability. In addition, organization of the cortical actin found in resting cells was disrupted. In contrast to histamine, ATP (a P2Y receptor agonist) induced dephosphorylation of MLC. Pre-exposure to ATP reduced the effect of histamine on HRP permeability and disruption of cortical actin.Conclusion. MLC phosphorylation, a biochemical pre-requisite for increased contractility of the actin cytoskeleton, led to histamine-induced breakdown of the barrier integrity in the corneal epithelial cells. This is attributed to weakening of the tethering forces at the tight junctions by the centripetal forces produced by increased actin contractility.