The luminal plasmalemma of continuous and fenestrated microvascular endothelia (obtained from bovines and rats) will be mapped at the macromolecular level by selecting promising antigens (glycoproteins and proteoglycans) from cultured cells for the generation of monoclonal or polyclonal antibodies. The latter will be used to localize the cognate antigens to specific differentiated plasmalemmal microdomains, e.g., plasmalemmal vesicles, transendothelial channels, apertured fenestrae and intercellular junctions. Immunocytochemical tests will be carried out in the intact animal (rat) to find out whether the antigen distribution is the same in situ as in culture. Since we have evidence suggesting that albumin is transported across the endothelium by receptor-mediated transcytosis, we'll attempt to identify and characterize this receptor, and to extend the inquiry to the transcytosis of other plasma proteins. Antibodies to the putative receptor will be tested to find out whether they block ligand binding and transport. Experiments will be carried out on the mesenteric microvasculature of the frog in an attempt to localize the sites of exit (small pores) for albumin and other smaller proteins (e.g., peroxidases). Albumin (or peroxidases) conjugated to photoactivatable groups will be perfused in individual capillaries, crosslinked to surrounding structures (by light exposure) seconds after the beginning of perfusion, and localized close to its exit sites by immunocytochemistry, using a tagged antibody (or a peroxidatic reaction). The mesenteric microvasculature will also be used to investigate the ability of pericytes to control local blood flow. Work on the contraction-associated proteins of pericytes will be expanded to new antigens, especially vascular smooth muscle-specific Alpha-actin, to find out how close these cells come to regular vascular smooth muscle. TEM and SEM work on endothelial surface fine structure will be continued and extended to antigen localization via tagged (or untagged) antibodies.
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