Affinity and distribution of surface and intracellular hyaluronic acid receptors in isolated rat liver endothelial cells

¹²⁵I-Hyaluronic acid (HA) uniquely modified only at the reducing end (Raja, R. H., LeBoeuf, R. D., Stone, G. W., and Weigel, P. H. (1984) Anal. Biochem. 139, 168-177) binds specifically to rat liver endothelial cells in suspension or in culture. About 67-85% of the HA binding sites in isolated cells in suspension and 50% in cultured cells were intracellular, since they were exposed after permeabilizing cells with digitonin. Specific ¹²⁵I-HA binding at 4°C varied from 60 to 80% for intact cells and from 70 to 90% for permeabilized cells. Freshly isolated permeabilized cells bound about 500,000 HA molecules/cell at saturation. Within 5 h of culture, however, total HA binding decreased to 250,000 molecules/cell and then remained constant for at least 36 h. Surface HA receptor activity was essentially the same on cultured cells or cells in suspension (~ 10⁵/cell). Cultured cells had 1.8 x 10⁵ fewer intracellular receptors/cell. The affinities of surface and intracellular receptors of cells in culture and in suspension were essentially the same. The average K(d), determined by equilibrium binding studies, was 5.8 ± 2.8 x 10^-8 M (n = 12). Dissociation of bound ¹²⁵I-HA from permeable cultured cells was rapid (t( 1/2 ) = 30.9 min; k(off) = 3.7 x 10^-4 s^-1). A variety of carbohydrates had essentially identical effects on ¹²⁵I-HA binding to surface or total cellular receptors in cells in culture or in suspension. Chondroitin sulfate and heparin competed almost as effectively as unlabeled HA for ¹²⁵I-HA binding at 4°C. Other saccharides including polygalacturonic acid, dextran, glucuronic acid, and N-acetylglucosamine competed poorly or not at all. We conclude that (i) the ¹²⁵I-HA binding sites within liver endothelial cells are HA receptors, identical in affinity and specificity to those on the cell surface; (ii) the distribution of cellular HA receptors is similar to other receptor systems with about 50-80% being intracellular; (iii) the liver endothelial cell HA receptor recognizes several glycosaminoglycans; and (iv) the liver endothelial receptor is different in function and characteristics than the fibroblast HA receptor.