Glucose transport in amastigotes and promastigotes of Leishmania mexicana mexicana

Promastigotes and amastigotes of Leishmania mexicana mexicana transported 2-deoxy-D-glucose (2-DOG) by a saturable process with a K-m of 24 +/- 3 mu M and V-max of 2.21 nmol min(-1) (mg protein)(-1) for the promastigote and a K-m of 29 +/- 8 mu M and V-max of 0.13 nmol min(-1) (mg protein)(-1) for the amastigote stage. Amastigotes incorporated 2-DOG maximally at pH 5.0, while for promastigotes the optimum was at pH 7.0. Mid-log phase promastigotes were found to accumulate 2-DOG via a stereospecific carrier-mediated process which was competitively inhibited by D-glucose and D-mannose but not L-glucose. Transport was dependent upon temperature, with a Q10 in promastigotes of 1.83 and an optimum rate at 35 degrees C (+/-4 degrees C) with an activation energy of 50.12 kJ mol(-1). Stationary phase promastigotes accumulated 2-DOG at approximately twice the rate of mid-log phase promastigotes. Cytochalasin B, forskolin and phloretin were all found to inhibit human erythrocyte 2-DOG uptake but only cytochalasin B was found significantly to inhibit promastigote 2-DOG uptake. Interestingly, leishmanial 2-DOG uptake was inhibited by a series of membrane potential antagonists including the ionophore monensin, the H(+)ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD) and uncoupling agent carbonylcyanide-4-(triflouromethoxy) phenylhydrazone (FCCP), as well as, the tricyclic drugs chlomipramine and imipramine, but was insensitive to the Na+/K(+)ATPase inhibitor ouabain and the antitrypanosomal drugs Pentostam and Suramin. We therefore conclude that there are significant structural and mechanistic differences between the D-glucose uptake systems of Leishmania and the mammalian host to merit the inclusion of glucose transporters as putative targets for rational drug design.