Activation of protein kinase C pathway contributes to hydrogen peroxide-induced increase in endothelial permeability.
BACKGROUND
We examined the effects of hydrogen peroxide (H2O2) on endothelial permeability and the possible role of protein kinase C (PKC) activation in mediating the response.


METHODS
Pulmonary microvessel endothelial cell monolayers were grown to confluency on gelatin- and fibronectin-coated microporous filters. Endothelial permeability was measured by determining the transendothelial clearance rate of [125I]albumin. The monolayers in all cases were challenged for 1 hour with H2O2. In some experiments, the monolayers were preincubated with PKC inhibitors H7 (an isoquinolinylsulphonamide derivative) (0.05 mM) or calphostin C (5 x 10(-6) mM) or with the inactive isoquinolinylsulphonamide analog, HA1004 (0.05 mM), before the H2O2 challenge.


RESULTS
Addition of H2O2 (0 to 0.5 mM) to endothelial monolayers in the absence of PKC inhibitors resulted in a concentration-dependent increases in endothelial permeability and the response occurred without LDH release and morphologic evidence of cytolysis. The increase in permeability was significantly reduced by H7 and calphostin C, but not by HA1007. Immunocytochemical localization of PKC indicated that PKC isotype II was abundant in these cells and that it was distributed uniformly in the cytosol. H2O2 induced translocation of PKC to the cell membrane indicating enzyme activation. H7 and calphostin C prevented the H2O2-induced PKC translocation, whereas HA1004 had no effect. Both PKC inhibitors also prevented cell "rounding" and formation of interendothelial gaps, whereas HA1004 was ineffective.


CONCLUSIONS
The results indicate that PKC activation is an important determinant of the H2O2-induced increase in endothelial permeability.
Siflinger-Birnboim
A
A
Department of Physiology, Albany Medical College of Union University, New York.
Goligorsky
M S
MS
Del Vecchio
P J
PJ
Malik
A B
AB
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D008841:Actin Cytoskeleton / Q000648:ultrastructure; D000818:Animals; D002417:Cattle; D002470:Cell Survival / Q000187:drug effects; D046508:Culture Techniques; D004730:Endothelium, Vascular / Q000502:physiology*; D004789:Enzyme Activation / Q000187:drug effects; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007546:Isoquinolines / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D009281:Naphthalenes; D010539:Permeability; D010879:Piperazines / Q000494:pharmacology; D011083:Polycyclic Compounds / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013449:Sulfonamides
D007546:Isoquinolines; D009281:Naphthalenes; D010879:Piperazines; D011083:Polycyclic Compounds; D013449:Sulfonamides; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; C043204:N-(2-guanidinoethyl)-5-isoquinolinesulfonamide; D006861:Hydrogen Peroxide; D011493:Protein Kinase C; C058819:calphostin C