Uptake Mechanism of Fractionated Heparin by Isolated Rat Kupffer Cells: Involvement of Scavenger Receptors

Abstract

The uptake of fractionated [³H]heparin was examined to elucidate the uptake mechanism in isolated rat Kupffer cells. The equilibrium binding of high molecular weight fractionated [³H]heparin (HMWFH; 23000 Da.) to Kupffer cells was concentration-dependent with the dissociation constant of 5.7 nM and the maximum binding capacity of 1.5 pmol/ 10⁶ cells. Several ligands of scavenger receptors inhibited the binding of HMWFH to Kupffer cells competitively and also the internalization of HMWFH, suggesting the involvement of scavenger receptors in the uptake of HMWFH. HMWFH was also suggested to be internalized according to first order kinetics with the apparent internalization rate constant of 0.010 min^-1. Lowering temperature from 37° to 4°C reduced the fraction internalized from 33% to 6% without affecting the total association, while the fraction internalized at 25° was comparable with that at 37°C. Metabolic inhibitors (2, 4-dinitrophenol and rotenone), an inhibitor of receptor-mediated and absorptive endocytosis of polypeptides (phenylarsine oxide) and phagocytosis inhibitors (cytocalasine B and colchicine) did not inhibit the internalization of HMWFH. These results suggested the scavenger receptor-mediated uptake of HMWFH is ATP-independent and different from the receptor-mediated and absorptive endocytosis of polypeptides and phagocytosis, although, as for temperature dependency, it showed the typical characteristics of receptor-mediated endocytosis. Low molecular weight fractionated [³H]heparin (LMWFH; 10000 Da.) was also suggested to be taken up by scavenger receptors. The binding affinity to scavenger receptors and apparent internalization rate constant were reduced with the decrease in molecular weight, suggesting that the molecular weight of fractionated heparin is one of the major factors determining the uptake by scavenger receptors.