| To investigate whether small G protein RhoA's downstream effector Rho kinase mediates burn induced endothelial hyperpermeability, a series of experiments were done in tissue and in cell level by using microsurgical technique, fluorescence ratio technique, laser confocal scanning microscopy and cellular biology method.PartIIn the tissue level research, rats were subjected to scalding local ventral skin and venules were isolated from scalded skin and cannulated with micropipette. The venular permeability was measured with a fluorescence ratio technique and expressed with the permeability coefficient to albumin (Pa). The venular F-actin filaments were detected by staining with rhodamine phalloidin and observed under laser confocal scanning microscope. A specific Rho kinase inhibitor Y-27632 was added into vessel bathing solution and incubated with vessels to evaluate the role of Rho kinase in regulating of vascular barrier function. The main results are as follow:1. Scalding increased Pa value of skin venule about threefold compared to normal skin venules (P<0.01).2. Y-27632 did not influence Pa value in normal venule. However, it significantly attenuated the hyperpermeability responses to scalding in a dose and time dependent fashion. After 30 |Amol/L of Y-27632 appilication, the Pa value reduced gradually within 10-15 min. 50 min later Pa was remarkably lower then basal value (P<0.05). Pa value was gradually recovered by washing and changing to solution without Y-27632. At hight dose (60 umol/L), Y-27632 attenuated the burn-induced hyper-permeability responsemore effectively tnan 30 uamo1/L Y-27632 application. The Pa decreased significantly at 20 min and sustained for 90 min, which did not reverse by changing to solution without Y-27632.3. In normal rat dermal venule, F-actms appeared to be peripheral fibers at outer area of cell and near the cell-cell junctions, formed dense fiber bundle, which was called peripheral actin rim (PAR). The PARs were arranged regularly and almost continuously along cell junctions. The width is about 6 to 15 um, and its length is 10 to 40 um Occasional breaks in the PAR were observed where accompanied by little of FITC-albumin leakage.4. Burn injury induced the depolymerization of PAR disappeared regular net construction. Only disrupted F-actin knots remained and a large amount of FITC-albumin leaked out5. After treatment of Y-27632 (60 umo1/L, 37), arrangement of actin filament were dramatically recovered in burn injuried venule. PARs were clear with occasional disruption and only little of FITC-albumin leaked out, which was similar to that in control group.PartIn the cellular level research, primary cultured rat dermal microvascular endothelial cells (DMECs) and human umbilical vein endothelial cells (ECV304 cell line) were exposed to serum isolated from burned or sham burn rats, or lysophosphatidic acid (LPA 13umol/L), a RhoA agonist. Cells were or were not pretreated or post-treated with Y-27632 (30 umol/L), a specific inhibitor of Rho kinase. ECs were then prepared for routine scanning electron microscopy observation, or stained with rhodamine-phalloidin and Oregon Green-DNase I for F-actin and G-actin visualization. The actin content was measured by using flow cytometry. Permeability to FITC-albumin was evaluated using EC monolayers. The main results are as follow:1. Stimulation with 15% burn serum for 6 h changed the ultrastructure on cellular surface of DMECs and ECV304 cells with appearance of ripple marks instead of microvillus. The small protuberances at cellular lateral wereshorten and the gaps were seen between adjacent cells. Pretreatment or post-treatment of Y-27632 reversed the changes of ultrastructure on the cellular surface. There was little ripple marks while the microvillus were seen at the same time and the small protuberances at the intercellular contacts link with each other tightly between adjacent endothelial cells.2. In normal endothelial cell, F-actin mainly distributed in the cellular peripheral site and formed peripheral fi...
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