TRANSIENT AND PROLONGED INCREASE IN ENDOTHELIAL PERMEABILITY INDUCED BY HISTAMINE AND THROMBIN - ROLE OF PROTEIN-KINASES, CALCIUM, AND RHOA

In the present study, we differentiated between short- and long-term e ffects of vasoactive compounds on human endothelial permeability in an in vitro model. Histamine induced a rapid and transient (<3 minutes) decrease in barrier function, as evidenced by a decreased transendothe lial electrical resistance and an increased passage of Na-22 ions. Thi s increase in permeability was inhibited completely by chelation of in tracellular calcium ions by BAPTA-AM and inhibition of calmodulin acti vity and myosin light chain (MLC) phosphorylation. The presence of ser um factors prolonged the barrier dysfunction induced by histamine. Thr ombin by itself induced a prolonged barrier dysfunction (>30 minutes) as evidenced by an increased passage of peroxidase and 40 kDa dextran. It was dependent only partially on calcium ions and calmodulin. The p rotein tyrosine kinase inhibitors genistein and herbimycin A, but not the inactive analogue daidzein, inhibited to a large extent the increa se in permeability induced by thrombin. Genistein and BAPTA-AM inhibit ed the thrombin-induced permeability in an additive way, causing toget her an almost complete prevention of the thrombin-induced increase in permeability. Inhibition of protein tyrosine kinase was accompanied by a decrease in MLC phosphorylation and a reduction in the extent of F- actin fiber and focal attachment formation. Inhibition of RhoA by C3 t ransferase toxin reduced both the thrombin-induced barrier dysfunction and MLC phosphorylation. Genistein and C3 transferase toxin did not e levate the cellular cAMP levels. No evidence was found for a significa nt role of protein kinase C in the thrombin-induced increase in permea bility or in the accompanying MLC phosphorylation. These data indicate that in endothelial cell monolayers that respond to histamine in a ph ysiological way, thrombin induces a prolonged increase in permeability by ''calcium sensitization,'' which involves protein tyrosine phospho rylation and RhoA activation.