Uptake Mechanism of Fractionated [³H]Heparin in Rat Parenchymal Hepatocytes in Primary Culture : Effect of Transport Inhibitors on the Uptake

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In order to elucidate the uptake mechanism of fractionated [³]heparin by rat parenchymal hepatocytes, the concentration dependent uptake was kinetically analyzed in primary culture of rat parenchymal hepatocytes, and the effects of established transport inhibitors and heparin analogues on the uptake were also examined. The uptake rate of heparin measured over an extended period of 60 min was saturable, with the maximum uptake velocity (V_max) of 0.36±0.05 pmol/min/mg protein and the Michaelis constant (K_m) of 21.2±5.4 nM. The uptake was inhibited by the addition of 4, 4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS), and inhibitor of the anion transport system, and rose bengal, an organic anion. Heparin analogues (pentosan polysulphate, or heparan sulphate) also inhibited the uptake of fractionated heparin. However, the uptake was not inhibited by the inhibitors of receptor-mediated endocytosis (phenylarsine oxide). These results suggest that fractionated heparin may be taken up by anion transport system, rather than by receptor-mediated endocytosis, though the fractionated [³H]heparin is a compound with the high molecular weight of about 20000 Da. At least the negative charge or sulphate group in the drug structure is supposed to play an important role in the uptake of fractionated heparin by parenchymal hepatocytes.