RESUMEN
We established a method to study vascular permeability in vitro on perfused endothelial cell monolayer cultured on micropore filter membrane. It can be used to determine filtration coefficient (Kf) and osmotic reflection coefficient (?) to proteins. Hanks balanced salt solution (HBSS) or 5 g/L albumin in HBSS was used to perfuse confluent endothelial monolayer. Control Kf values were 10.1 ?0.75 and 3.6?0.75?l . min-1 . cm-2 . kPa-1 (n = 3, x?sx) respectively for HBSS and albumin HBSS perfusion, suggesting that albumin may decrease endothelial monolayer permeability to water and small molecules. After exposure of endothelial monolayer to 10-8 mol/L platelet-activating factor (PAF) for 30 min, Kf values increased to 193.1% and 133.3% respectively for HBSS and albumin HBSS perfusion. Protein clearance rate (?l. min-1 . cm-2:) and osmotic reflection coefficient of control endothelial monolayer were 8.0?3.22 and 0. 37?0.09 respectively. In PAF treated endothelial monolayer,they were 12.2 ? 2.95ul min-1 cm 2 and 0.18?0.06, revealing increased permeability to albumin. Computer-assisted image processing demonstrated that PAF treatment decreased cell area while increased cell form factor and intercellular space. The results sug-gest that endothelial cells retracted, rounded and it may be an important mechanism in PAF-induced increased vascular permeability.
RESUMEN
The effects of hydrogen peroxide (H2O2) on endothelial-polymorphonuclear cells (EC-PMN) adhesion and their mechanisms wsre studied in cultured bovine pulmonary artery endothelial monolayers in vitro. H2O2 at various concentrations (10-1, 10-2, 10-3mol/L respectively) stimulated EC dependent PMN adhesion, of which l02mol/L H2O2 was the most potent one, increasing adhesion to 2.3 times that of the control. Pretreatment of PMNs with SRI 63-441, a platelet-activating factor (PAF) receptor antagonist, had no effect on H2O2 induced EC-PMN adhesion. Pretreatment of ECs with SRI 63-441 before H2O2 exposure significantly decreased PMN adherence to ECs. Pretreatment of ECs with phospholipase A2 inhibitor p-bromophenacyl-bromide or cahnodulin antagonist chlorpromazine and aildum ion chelate EGTA obviously decreased H2O2 induced increment of EC-PMN adhesion. These results suggest that H2O2 may activate ECs, causing the inflow of extracellular calcium or the release of calcium from intracellular deposits. Increased intracellular Ca2+ may bind with calmodulin to activate phospholipase A2 thus initiating PAF synthesis and promoting EC-PMN adhesion.
RESUMEN
Intravenous administration of E. coli endotoxin (2mg/kg) caused a persistent increase in platelet activating factor (PAF) contents in arterial blood and bronchoalveolar fluid (BALF). Meanwhile phospholipase A2(PLA2) activity in serum and BALF was elevated and mean arterial blood pressure declined. The total cell number, expecially polymorphonuclear leukocytes and lymphocytes, and protein concentration in BALF were markedly increased 6 h after endotoxin injection. Lung index and extravascular lung water content of endotoxin-treated animals were significantly highter than those of controls. Pretreatment with PAF receptor antagonist SRI 63-441 blocked the increase in PLA, activity and attenuated endotoxin-induced hypotension and acute lung injury. The results suggest that PAF mediates endotoxin-induced lung injury, and leukocyte activation by PAF and the subsequent release of oxygen metabolites and lysoenzymes are important intermediate mechanisms leading to high permeability pulmonary edema.
RESUMEN
A method for isolation and primary culture of guinea pig alveolar type Ⅱ epithelial cells is reported. The injurious effects of phospholipase A2 (PLA2) on the cultured cells were studied. Trypsinizing solution was instilled into the bronchoalveolar space. The lungs were digested, sectioned, vortexed and filtered. The yield of original cell mixture was 172.3?106 cells/guinea pig, of which 33.6% were type Ⅱcells. At 48 h of culture following short term selective attachment 7.9?106 cells were obtained, of which 66.1% were type Ⅱ ceils. The purity could further be raised to 76.2% (63.9% -92%) if pretreated with ethyienediarnine tetraacetic acid (EDTA) before trypsinization to remove attached cells. The characteristics of cultured cells were observed under phase contrast microscopy, light microscopy and electron microscopy. After exposure of the cells to 300U/ml PLA2 for 4h at 37℃, detachment and lactic dehydrogenase (LDH) release of the cultured cells increased significantly. It is suggested that PLA, may damage and promote detachment of cultured type Ⅱ cells and it may play an important part in the development of some diseases.
RESUMEN
We investigated whether platelet-activating factor (PAF) mediates endotoxin-induced changes of lipoperoxidation, lysosomal enzyme release and increased extravascular lung water content in dogs using a specific PAF receptor antagonist, SRI 63-441. Endotoxin infusion caused an increase in serum malonyldialdehyde (MDA) from baseline (100%) to 130.04?14.00% and 169.16?32.49% and ?-glucuronidase (?-g) activity to 191.05?86.71% and 242.54?49.09% at 3 and 6h, respectively. The bronchoalveolar lavage fluid (BALF) ?-g activity, lung MDA and extravascular lung water also significantly increased (P