Characterization of an intracellular hyaluronic acid binding site in isolated rat hepatocytes

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Abstract
125I-HA, prepared by chemical modification at the reducing sugar, specificially binds to rat hepatocytes in suspension or culture. Intact hepatocytes have relatively few surface 125I-HA binding sites and show low specific binding. However, permeabilizaion 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 125-I-HA binding sites. Scatchard analysis of equilibrium 125I-HA binding to permeabilized hepatocytes in suspensions at 4.degree. C indicates a Kd = 1.8 .times. 10-7 M and 1.3 .times. 106 molecules of HA (Mr .apprx. 30,000) bound per cell at saturation. Hepatocytes in primary cultures for 24 h show the same affinity but the total number of HA molecules bound per cell at saturation decrease to .apprx.6.2 .times. 105. Increasing the ionic strength above physiologic concentrations decreases 125I-HA binding to permeable cells, whereas decreasing the ionic strength causes a .apprx.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.degree. C increased >10-fold at pH 5.0 as compared to pH 7. The kinetics of 125I-HA binding to intact hepatocytes at 37.degree. C was rapid and similar to the kinetics of 125I-HA binding at 4.degree. C (t1/2 .apprx. 5 min). Very little 125I-HA was internalized after 4 h at 37.degree. C (460 molecules cell-1 h-1). This rate is extremely slow (.apprx.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.