Studies On Effects Of CGMP-dependent Protein Kinase On Vascular Endothelial Cell Permeability

Vascular hyperpermeability is one of the important cause'in hypovole-mia and organs disfunction after various kinds of shock,and its treatment is still one of the problems in the medical field. For example,the operative methods of preventing hyperpermeability and plasm bleeding is still unreliable after severity burn,and traditional treatment just passively transfuses according to the burn clysis formula instead of inhibiting permeability by injecting drugs. The principal reason is that pathogenesis of vascular endothelial cell permeabilty has not been entirely revealed at the level of molecular biology.In the recent 20 years,evidences have proved that endothelial cell barrier function and vascular permeability depend on the endothelial morphologic changes and the formation of intercellular gap. It is thought that change of endothelial contractility is the last congenerous pathway of different signal transduction in permeability changes. Endothelial cell form and contractility are regulated by cytoskeleton proteins,such as actin and myosin. A number of evidences have proved that one is the pathway composed of calcium,nitric oxide synthase,cGMP and cGMP-dependent protein kinase,and another is the pathway of protein kinase C in the vascular hyperpermeability signal transduction. The pathway of protein kinase has cross talk with that of cGMP-dependent protein kinase by partly regulating nitric oxide synthase activity. At present,there are many studies about detailed effects of PKC on intracellular signal trunsduction of microvascular hyperpermeability,but the importance of PKG is justly realized,and the effects of cGMP on vascular permeability are still argued. In this research,we study relations between exogenous cGMP and vascular permeabilty transformation,and the role of PKG in the intracellular signal transduction pathway of microvascular hyperpermeability after burn shock and septicshock,in order to reveal their molecular biology pathogenesis,and instructthe treatments of them fundamentally.In the research,confluent endothelial cells were disintegrated and centrifugated to obtain cell lysates after being treated with exogenous cGMP(also cGMP-dependent protein kinase activator)8-Br-cGMP or 10% burn serum(including burn toxin) or lipopolysaccharide respectively. The cGMP-dependent protein kinase activity of lysates was measured with radioactive isotope label method in a reaction system of phosphorylation of specific substrate H2b by cGMP-dependent protein kinase,and the shape and the distribution of intracellular filamentous actin were detected by specific fluorescence staining. For the control study,the cGMP-dependent protein kinase specific inhibitor KT5823 were used to pretreat the endothelial cells before the administration of 8-Br-cGMP or burn serum or lipopolysaccharide. The results showed that cytoplasmic filamentous actin obviously increased with the polar distribution,and there was obvious stress fibre with time-dependent increase of intracellular PKG activity after administration of 8-Br-cGMP or burn serum or lipopolysaccharide. Howere,pretreatment with KT5823 inhibited these changes.Our results indicate that cGMP induces endothelial hyperpermeability with stress changes of intracellular cytoskeleton,and that vascular hyperpermeability after burn shock has positive correlation with increase of cGMP-dependent protein kinase activity,and that lipopolysaccharide of septic shock results in endothelial cell hyperpermeability with disfunction of endothelial cell barriers through activating cGMP-dependent protein kinase which influences endothelial cell cytoskeleton system. This suggests that antibody or inhibition of cGMP-dependent protein kinase may prevent vascular hyperpermeability of severity burn shock and septic shock,and fundamentally take on a new look in treatment of shocks instead of traditional and passive clysis.