Evidence for the role of alveolar epithelial gp60 in active transalveolar albumin transport in the rat lung

1. Transcytosis of albumin, involving the 60 kDa albumin-binding glycoprote in, gp60, was studied in cultured type II alveolar epithelial cells obtaine d from rat lungs. 2. Type II cells internalized the interfacial fluorescent dye RH 414, which marks for plasmalemma vesicles. Fluorescent forms of albumin and anti-gp60 antibody colocalized in the same plasmalemma vesicles. 3. Antibody (100 mug ml(-1)) cross-linking of gp60 for brief periods (15 mi n) markedly stimulated vesicular uptake of fluorescently tagged albumin. Th e caveolar disrupting agent, filipin (10 nM), abolished the stimulated inte rnalization of albumin. 4. The vast majority of plasmalemmal vesicles carrying albumin also immunos tained fur caveolin-1; however, lysosomes did not stain for caveolin-1. Fil ipin depleted the epithelial cells of the caveolin-1-positive, albumin-tran sporting plasmalemma vesicles. 5. Prolonged (> 1 h) stimulation of type II cells with cross-linking anti-g p60 antibody produced loss of cell-surface gp60 and abolished endocytic alb umin uptake. 6. Transalveolar transport of albumin was also studied in the isogravimetri c rat lung preparation perfused at 37 degreesC. I-125-labelled albumin was instilled into distal airspaces of lungs, and the resulting I-125-labelled albumin efflux into the vascular perfusate was determined. 7. Unlabelled albumin (studied over a range of 0-10 g (100 instilled ml)(-1 )) inhibited 40 % of the transport of labelled albumin ((5.7 +/- 0.4) x 10( 5) counts(instilled ml)(-1)) with an IC50 value of 0.34 g (100 ml) 8. Filipin blocked the displacement-sensitive component of I-125-labelled a lbumin transport, but had no effect on the transport of the paracellular tr acer (3)[H]mannitol. 9. Displacement-sensitive I-125-labelled albumin transport. had a significa ntly greater Q(10) (27-37 degreesC) than the non-displaceable component. 10. Cross-linking of gp60 by antibody instillation stimulated only the disp lacement-sensitive I-125-labelled albumin transalveolar transport in intact , rat lungs. 11. To estimate the transport capacity of the displacement-sensitive system , the percentage of instilled I-125-labelled albumin counts remaining in lu ng tissue was compared in lungs treated with instillates containing either 0.05 g (100 ml)(-1) unlabelled albumin or 5 g (100 ml)(-1) unlabelled album in. Approximately 25 % of instilled I-125-labelled albumin was cleared from the lung preparations per hour by the displacement-sensitive transport pat hway. This component was blocked by filipin.