Endocytosis of hyaluronan in rat Kupffer cells.
The binding, uptake and degradation of hyaluronan (HA) labelled with 3H in its acetyl group were studied in cultured rat Kupffer cells (KC). At 4 degrees C the binding increased with increasing concentrations of HA in the culture medium up to at least 1 microgram/ml, when saturation occurred. Binding could be prevented efficiently by the addition of an excess of unlabelled HA, and to a lesser extent by chondroitin sulphate and oligosaccharide fragments of HA, consisting of four sugars or more. The labelled HA bound to the cells could be removed by incubating the cells with Streptomyces hyaluronidase, or trypsin, indicating that the HA-binding sites are located on the cell surface. At 37 degrees C HA was internalized in a concentration-dependent manner, and degradation products appeared in the supernatant after 1-5 h, depending on the concentration applied. At 50 ng of free HA/ml, each KC accumulated 60 ag of the polysaccharide/min in the first 1 h, and degraded a total amount of 10 fg of HA during an 8 h period. Addition of the negatively charged polysaccharide dextran sulphate reduced binding, and to an even greater extent internalization, of HA in KC, while no effect was observed with dextran. Depletion of intracellular potassium caused a marked reduction in the rate of endocytosis of cell-membrane-associated HA into KC, without affecting binding. Addition of KCl to the culture medium returned endocytosis of [3H]HA to normal levels. There was no effect on binding and a partial effect on internalization by depletion of bivalent cations or in the presence of EDTA. The degradation of [3H]HA by KC cultures was abolished in the presence of weak bases, NH4Cl and chloroquine, supporting the idea that HA is endocytosed into lysosomes prior to degradation. The fluid-phase marker [14C]sucrose was internalized in the cells at much lower rate than was HA. Rates of binding, internalization and degradation of HA in KC point therefore to a specific endocytosis followed by an intracellular degradation to low-M(r) compounds. It was estimated that, under physiological conditions, KC only clear a minor proportion of circulating HA.
Alston-Smith
J
J
Institute of Medical and Physiological Chemistry, University of Uppsala, Sweden.
Pertoft
H
H
Laurent
T C
TC
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent; D002414:Cations, Monovalent; D002478:Cells, Cultured; D004705:Endocytosis; D006820:Hyaluronic Acid / Q000378:metabolism*; D007728:Kupffer Cells / Q000187:drug effects / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008297:Male; D010477:Perfusion; D010873:Pinocytosis; D011188:Potassium / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013395:Sucrose / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology
D002413:Cations, Divalent; D002414:Cations, Monovalent; D013395:Sucrose; D006820:Hyaluronic Acid; D014357:Trypsin; D008274:Magnesium; D011188:Potassium; D002118:Calcium