Objective: To examine how cell-substrate adhesion is regulated during barrier changes produced by exposure to inflammatory mediators. Methods: Lung microvascular endothelial monolayers were treated with test agents ± blockers, and barrier was measured by transendothelial resistance; cell-substrate adhesion was assessed by surface area conservation after trypsin treatment of monolayers. Protein phosphorylation and distribution were assayed by immunoblotting and fluorescent microscopy, respectively. Results: H[SUB2]O[SUB2], histamine, bradykinin, and thrombin, decreased endothelial barrier function, and enhanced adhesion to the substratum. H[SUB2]O[SUB2] enhanced cell adhesion to the substrate in a concentration (0-1 mM)- and time (0-60 minutes)-dependent fashion. This effect of H[SUB2]O[SUB2] reversed within 120 minutes of removal of H[SUB2]O[SUB2] and was blocked by the mean arterial pressure (MAP) kinase inhibitor, PD98059 and by chelating cytoplasmic Ca[SUP2+] but not PKC or PKG inhibition. H[SUB2]O[SUB2] also stimulated tyrosine phosphorylation of several proteins and increased the association of the focal adhesive proteins paxillin, talin, and vin-culin with the cytoskeleton and may promote localization of these proteins to junctions. Conclusions: Our data indicate that inflammatory mediators reduce cell-cell contact, contributing to reduced solute barrier and simultaneously enhanced substrate binding, which may be reciprocal events in barrier regulation in vitro and in vivo. [ABSTRACT FROM AUTHOR]