Protein kinase C-alpha mediates endothelial barrier dysfunction induced byTNF-alpha

We tested the hypothesis that protein kinase C-alpha (PKC-alpha) mediates t umor necrosis factor-alpha (TNF-alpha) induced alterations in permeability of pulmonary microvessel endothelial monolayers (PEM). The permeability of PEM was assessed by the clearance rate of Evans blue-labeled albumin. PEM l ysates were analyzed for PKC-alpha mRNA (Northern cDNA blot), protein (West ern immunoblot), and activity (translocation and phosphorylation of myristo ylated arginine-rich C kinase substrate). Incubation of PEM with TNF-alpha (1,000 U/ml) for 4 h resulted in increases in 1) PKC-alpha protein, 2) cyto skeletal-associated PKC-alpha, 3) PKC-alpha activity, and 4) permeability t o albumin. The TNF-alpha-induced increase in PKC-alpha protein, PKC-alpha a ctivity, and permeability was prevented by a 4-h pretreatment with PKC-alph a antisense oligonucleotide but not by the scrambled nonsense oligonucleoti de. The TNF-alpha-induced increase in permeability to albumin was prevented by myristoylated protein kinase C inhibitor (an inhibitor of PKC-alpha/bet a, 100 mu M) and calphostin (an inhibitor of the classic and novel PKC isot ypes, 200 nM). The treatment with calphostin from 0.5 to 3.0 h after TNF-al pha still prevented barrier dysfunction induced by 4 h of TNF-alpha treatme nt. The data indicate that prolonged activation of PKC-alpha, maintained by a translation-dependent pool of PKC-alpha protein, mediates TNF-alpha-indu ced increases in endothelial permeability in PEM.