NEM inhibits transcytosis, endocytosis, and capillary permeability: implication of caveolae fusion in endothelia.
Various vesicular carriers transport select molecular cargo between intracellular compartments utilizing a budding mechanism with docking and fusion of individual vesicles with their target membranes. This fusion requires key intracellular component(s) that are sensitive to alkylation with N-ethylmaleimide (NEM). In endothelium, caveolae may mediate endocytosis and transcytosis of select macromolecules. If caveolae utilize a mechanism similar to other vesicular carriers, then their transport should also be sensitive to NEM. The following tracers were chosen based on their pathway specificity: 1) albumin-gold complexes (A-Au) that bind gp30 and gp18 for endocytosis by caveolae, 2) native albumin that binds albondin and is transported by caveolae, 3) ferritin as a fluid-phase probe transcytosed by caveolae, and 4) inulin as a paracellular probe. In culture, NEM significantly inhibited A-Au uptake and delivery to endosomes but not endothelial cell surface binding. In rat lung, NEM reduced capillary permeability to albumin and the tissue uptake of ferritin and A-Au, but not inulin, indicating inhibition of caveolae-mediated but not paracellular transport. Neutral but not charged alkylating agents inhibited A-Au uptake, consistent with their relative abilities to cross membranes and modify intracellular factors. Like other vesicular pathways, endothelial caveolae transport their select ligands utilizing a NEM-sensitive mechanism, apparently requiring vesicle-membrane fusion.
Department of Pathology, Harvard Medical School, Beth Israel Hospital, Boston, Massachusetts 02215.
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Schnitzer
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Allard
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D000818:Animals; D001011:Aorta; D001692:Biological Transport; D002196:Capillaries / Q000187:drug effects / Q000502:physiology; D002199:Capillary Permeability / Q000187:drug effects*; D002417:Cattle; D002478:Cells, Cultured; D004705:Endocytosis / Q000187:drug effects*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000502:physiology*; D005033:Ethylmaleimide / Q000494:pharmacology*; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D008168:Lung / Q000502:physiology*; D008833:Microcirculation; D010663:Phenylmercury Compounds / Q000494:pharmacology; D011652:Pulmonary Circulation; D051381:Rats; D017207:Rats, Sprague-Dawley; D012710:Serum Albumin, Bovine
D006902:Hydroxymercuribenzoates; D010663:Phenylmercury Compounds; C030601:4-hydroxymercuribenzoate; C002910:4-hydroxymercuribenzenesulfonate; D012710:Serum Albumin, Bovine; D005033:Ethylmaleimide