We examined the contribution of actin-myosin contraction to the modulation of human umbilical vein endothelial cell focal adhesion caused by histamine and thrombin. Focal adhesion was measured as the electrical resistance across a cultured monolayer grown on a microelectrode. Actin-myosin contraction was measured as isometric tension of cultured monolayers grown on a collagen gel. Histamine immediately decreased electrical resistance but returned to basal levels within 3-5 min. Histamine did not increase isometric tension. Thrombin also immediately decreased electrical resistance, but, however, resistance did not return to basal levels for 40-60 min. Thrombin also increased isometric tension. ML-7, an inhibitor of myosin light chain kinase, prevented increases in myosin light chain phosphorylation and increases in tension development in cells exposed to thrombin. ML-7 did not prevent a decline in electrical resistance in cells exposed to thrombin. Instead, ML-7 restored the electrical resistance to basal levels in a shorter period of time (20 min) than cells exposed to thrombin alone. Also, histamine subsequently increased electrical resistance to above basal levels, and thrombin initiated an increase in resistance during the time of peak tension development. Hence, histamine and thrombin modulate endothelial cell focal adhesion through centripetal and centrifugal forces. (

Histamine and thrombin increase myosin light-chain kinase-mediated phosphorylation of myosin light chain (MLC) in human umbilical vein endothelial cells (HUVEC). The increase in MLC phosphorylation caused by thrombin persists longer (330 min) than the increase caused by histamine (<5 min), although both increase cell calcium similarly. We hypothesized that some of the longer duration of the increase in MLC phosphorylation caused by thrombin was because of inhibition of myosin dephosphorylation by thrombin. Calyculin A, an inhibitor of type 1 and 2A protein phosphatases, caused a time-dependent increase in MLC phosphorylation in unstimulated HUVEC. As thrombin-stimulated phosphorylation approached its peak at 15 min, calyculin A caused progressively less of an increase in MLC phosphorylation in thrombin-stimulated HUVEC, and no increase at the peak of thrombin stimulation. In HUVEC in which cell calcium was maintained at 600 nM, thrombin increased MLC phosphorylation above the level caused by increased calcium alone at a time coinciding with the peak of thrombin stimulation. However, when phosphatase activity was already inhibited with calyculin A, thrombin did not further increase MLC phosphorylation in cells in which calcium was maintained at 600 nM calcium. Thrombin increases MLC phosphorylation in HUVEC not only by increasing cell calcium but also by inhibiting calyculin A-sensitive dephosphorylation of MLC.

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Pseudomonas aeruginosa pneumonia causes a vasculitis of small pulmonary arteries. While the fully developed lesion demonstrates vessel wall necrosis, the early lesion is remarkable for preservation of viable endothelium despite vessel wall invasion by bacteria. Pyocyanin, an exoproduct of P. aeruginosa, markedly inhibited prostacyclin production by pulmonary artery endothelial cells without causing cell lysis. Pyocyanin might alter vascular homeostasis in the absence of cytolysis. Pseudomonas aeruginosa pneumonia causes a distinctive vascular lesion which begins with Pseudomonas penetration of the wall of small pulmonary arteries and evolves into necrosis and hyalinization of the endothelial and muscular layers (4). When combined, two exoproducts of P. aeruginosa, pyocyanin and ferripyochelin, form cytolytic amounts of hydroxyl radical in the presence of endothelial cells, suggesting one possible mechanism for the necrosis (1, 1a). However, early P. aeruginosa pneumonia vascular lesions, as well as early systemic icthyma lesions, are remarkable for a paucity of histologically evident endothelial injury despite considerable bacterial invasion of the vessel wall (4, 12). Hence, it was of interest to identify noncytolytic effects of P. aeruginosa exoproducts on cells of the vessel wall. Release of prostaglandin I 2 (PGI 2) or PGE 2 is an important antithrombotic, antiinflammatory, and vasoregulatory activity of endothelium that is sensitive to redox potential (3, 5). Pyocyanin is a phenazine dye that is an effective electron acceptor that might interfere with redox-sensitive endothelial prostanoid production (1, 1a, 9). Porcine pulmonary artery endothelial (PPAE) cells were cultured in 24-well plates (Costar, Cambridge, Mass.) until they were 2 days postconfluency, as previously described (1, 8). At the time of the experiment, the medium was aspirated and the cells were washed three times with phosphate-buffered saline, and then 250 l of Hanks balanced salt solution was added to each well. Pyocyanin or vehicle (H 2 O) was then added at various concentrations for 30 min. When the cells were to be stimulated with the calcium ionophore A23187 (1 M in dimethyl sulfoxide) or arachidonic acid (in ethanol), either agonist (or vehicle) was added after the initial 30-min incubation, and the cells were incubated an additional 30 min in the presence of both pyocyanin and agonist. Crystalline pyocyanin was prepared by Charles Cox as previously reported (2). PGI 2 was measured in the supernatant fluid by radioimmunoassay according to the manufacturer's directions (Advanced Magnetics Inc., Cambridge, Mass.). Briefly, the medium was acidified to pH 3.0 with HCl, extracted with 2 ml of ethyl acetate, and dried under nitrogen. The sample was then redis