CHARACTERIZATION OF AN INTRACELLULAR HYALURONIC-ACID BINDING-SITE IN ISOLATED RAT HEPATOCYTES

125I-HA, prepared by chemical modification at the reducing sugar, specifically binds to rat hepatocytes in suspension or culture. Intact hepatocytes have relatively few surface 125I-HA binding sites and show low specific binding. However, permeabilization of hepatocytes with the nonionic detergent digitonin results in increased specific 125I-HA binding (45-65%) and a very large increase in the number of specific 125I-HA binding sites. Scatchard analysis of equilibrium 125I-HA binding to permeabilized hepatocytes in suspension at 4 °C indicates a Kd = 1.8 × 10−7 M and 1.3 × 106 molecules of HA (Mr ~ 30000) bound per cell at saturation. Hepatocytes in primary culture for 24 h show the same affinity but the total number of HA molecules bound per cell at saturation decreases to ~6.2 × 105. Increasing the ionic strength above physiologic concentrations decreases 125I-HA binding to permeable cells, whereas decreasing the ionic strength causes a ~ 4-fold increase. The divalent cation chelator EGTA does not prevent binding nor does it release 125I-HA bound in the presence of 2 mM CaCl2, although higher divalent cation concentrations stimulate 125I-HA binding. Ten millimolar CaCl2 or MnCl2 increases HA binding 3-6-fold compared to EGTA-treated cells. Ten millimolar MgCl2, SrCl2, or BaCl2 increased HA binding by 2-fold. The specific binding of 125I-HA to digitonin-treated hepatocytes at 4 °C increased > 10-fold at pH 5.0 as compared to pH 7. The kinetics of 125I-HA binding to intact hepatocytes at 37 °C was rapid and similar to the kinetics of 125I-HA binding at 4 °C (t1/2 ~ 5 min). Very little 125I-HA was internalized after 4 h at 37 °C (460 molecules cell−1 h−1). This rate is extremely slow (~1–3%) compared to the rate of receptor-mediated internalization of other ligands and indicates that HA uptake occurs by a noncoated pit pathway, probably reflecting general membrane pinocytosis. There is no evidence for recycling of the surface HA binding sites or use of the large intracellular reservoir for endocytosis. © 1990, American Chemical Society. All rights reserved.