In this study, we tested the role of Src-dependent tyrosine phosphorylation to modulate endothelial contraction and monolayer barrier function with the use of the myosin phosphatase inhibitor calyculin A (CalA) to directly elevate MLCP with the Src family tyrosine kinase inhibitor herbimycin A (HA) in bovine pulmonary artery endothelial cells (EC). CalA stimulated an increase in MLCP, Src kinase activity, an increase in the tyrosine phosphorylation of paxillin and focal adhesion (FA) kinase (p125 FAK ), and monolayer hyperpermeability. Microscopic examination of CalAtreated EC revealed a contractile morphology characterized by peripheral contractile bands of actomyosin filaments and stress fibers linked to phosphotyrosine-containing FAs. These CalA-dependent events were HA sensitive. HA alone stimulated an improvement in monolayer barrier formation by reducing the levels of MLCP and phosphotyrosine-containing proteins and the number of large paracellular holes. These data show that Src kinase plays an important role in regulating monolayer hyperpermeability through adjustments in tyrosine phosphorylation, MLCP, and EC contraction. nonmuscle myosin; myosin light chain phosphorylation; paxillin; immunofluorescent digital imaging; size-selective solute permeability THE VASCULAR ENDOTHELIUM provides the permeability barrier that controls the passage of fluid and solutes into the perivascular space. Inflammation of this barrier initiates a hyperpermeability state characterized by the formation of large paracellular holes between adjacent endothelial cells (EC). These events occur most frequently in the systemic postcapillary venule and pulmonary arteriole circulations. The formation of paracellular gaps is the result of reorganization of the endothelial cell-cell junctional morphology that permits the enhanced leakage of plasma proteins and fluid into the tissues, thus causing edema and organ dys-

These findings indicate that the rat glomerular filtration barrier restricts the transport of polysaccharide macromolecules as a function of their size and configuration but not negative charge.

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