ATP-Dependent colchicine transport by human erythrocyte glutathione conjugate transporter

We have recently demonstrated mutually inhibitory ATP-dependent transport o f dinitrophenyl-S-glutathione (DNP-SG) and doxorubicin by DNP-SG ATPase pur ified from human erythrocyte membranes (S. Awasthi et at, 1998a,b). Our pre vious studies indicate a broad substrate specificity for this transport mec hanism, including some P-glycoprotein substrates. Present studies were carr ied out to determine whether colchicine (COL), a classical P-glycoprotein s ubstrate, could be transported by purified human erythrocyte DNP-SG ATPase reconstituted in artificial liposomes. We also investigated whether leukotr iene C4 (LTC4), an endogenous proinflammatory glutathione-conjugate derived from arachidonic acid, would inhibit colchicine transport. Uptake of COL w as compared in proteoliposomes reconstituted with the purified DNP-SG ATPas e as well as control liposomes in the presence or absence of ATP, Increased colchicine uptake was observed upon addition of ATP to proteoliposomes, bu t not control liposomes. Uptake was linear with respect to the amount of ve sicle protein used. Sensitivity to osmolarity was consistent with intravesi cular COL accumulation. The ATP-dependent colchicine uptake was sensitive t o temperature in a manner consistent with a protein-mediated transport proc ess with activation energy of 7.3 kcal/mol. Time-dependent COL uptake by pr oteoliposomes in the presence of ATP was consistent with a single compartme nt model with an apparent rate constant of 0.21 +/- 0.02 min(-1). Kinetic s tudies indicated a saturable behavior with respect to ATP (K-m 2.3 +/- 0.7 mM) and colchicine (K-m 4.3 +/- 0.2 mu M). LTC4 was found to be a competiti ve inhibitor of COL transport (K-is 16.4 mu M). Since DNP-SG ATPase is pres ent in many tissues, it may play an important role in determining colchicin e accumulation in cells. Increased LTC4 would tend to increase cellular COL accumulation. (C) 1999 Academic Press.