PREVENTION OF INCREASED VASCULAR PERMEABILITY

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Beta-adrenergic agonists such as isoproterenol and terbutaline have been shown to attenuate the edema that various agents can produce in systemic and pulmonary vascular beds. We hypothesize that beta-agonists can prevent and possibly reverse the development of edema by a permeability-decreasing effect on vascular endothelial cells as well as hemodynamic effects and that this effect is unique to beta-agonists as opposed to other vasodilator substances. Furthermore, we hypothesize that this permeability-decreasing effect is mediated via cyclic AMP which alters the intracellular microfilaments and/or microtubules in such a way as to change the shape of the cell. This proposal will utilize in vitro and in vivo experimentation to demonstrate whether beta-agonists have a permeability-decreasing effect and to elucidate the mechanism for this activation the in vitro studies will utilize cultured endothelial cells from arteries. These studies will determine if beta-agonists can prevent and reverse the increased endothelial permeability produced by various permeability-increasing mediators such as thrombin, endotoxin, and histamine. The measurement for endothelial permeability will be the clearance of 125I-albumin across the cultured endothelial cell monolayers seeded on Nucleopore (0.8 um) filters. Radioligand binding studies will be done to demonstrate the presence of beta-receptors on the cultured endothelial cells. The in vitro studies will also determine if the permeability-increasing agents alter cell shape and the number and organization of cellular microfilaments and microtubules and if beta-agonists can prevent these alterations. Cell shape and microfilament changes will be assessed by time-lapse video, phase contrast and immunofluorescent microscopy. The proposed studies will focus on the pulmonary endothelium. With the information gained by the in vitro experimentation, in vivo studies, utilizing the chronic sheep lung lymph fistula preparation, will be done to differentiate between the effects of beta-agonists on vascular permeability and hemodynamics in the awake animal. These in vivo sheep studies will also focus on the pulmonary vasculature. In parallel with these objectives is the long-term objective of determining the transcellular signal pathways that may alter the shape of endothelial and epithelial cells and influence their function as semipermeable membranes.