The research program outlined in this application is designed to extend our prior observations on the pathogenesis of inflammatory edema. It is built around the observations that inflammatory edema, whether caused by histamine, bradykinin or activated neutrophils, is invariably associated with the retraction of adjacent endothelial cells. In the past we have identified neutrophil derived mediators that activate signal transduction pathways that cause retraction of endothelial cells. Histamine and bradykinin activate these same pathways and also cause cell retraction. We have recently found that these pathways lead to phosphorylation of the light chain of myosin (MLC), and phosphorylation of the light chain is necessary for cell retraction in response to histamine or when cell attachment to substrate is interrupted by chelating extracellular calcium. In the proposed experiments we will determine if histamine, bradykinin, and thrombin increase centripetal tension in endothelial cells, and if they do, if the increase in tension is dependent on phosphorylation of MLC. We will also determine if histamine, bradykinin or thrombin cause endothelial cells to release from substrate and cell-cell attachments, and if they do, if this is occurs in the absence of an increase in centripetal tension. We have new preliminary data that are consistent with a paradigm in which agonists such as histamine and bradykinin release endothelial cells from attachments without increasing centripetal tension. In our recent experiments we have also observed that the magnitude and duration of MLC phosphorylation in response to histamine and bradykinin are very limited. These same experiments identify a potentially important role for phosphatases in the control of light chain phosphorylation. We will extend these observations to more precisely determine the role of phosphatases in controlling the extent of MLC phosphorylation in endothelium. We expect these investigations to increase our understanding of how inflammatory edema occurs; and to direct our future investigations into understanding how cell attachments can be regulated and whether some edemagenic agonists may directly alter the activity of endothelial phosphatases.
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