GLUCOSE-TRANSPORT IN AMASTIGOTES AND PROMASTIGOTES OF LEISHMANIA-MEXICANA MEXICANA

Promastigotes and amastigotes of Leishmania mexicana mexicana transpor ted 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 pro mastigote and a K-m of 29 +/- 8 mu M and V-max of 0.13 nmol min(-1) (m g protein)(-1) for the amastigote stage. Amastigotes incorporated 2-DO G 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 s tereospecific carrier-mediated process which was competitively inhibit ed by D-glucose and D-mannose but not L-glucose. Transport was depende nt upon temperature, with a Q10 in promastigotes of 1.83 and an optimu m rate at 35 degrees C (+/-4 degrees C) with an activation energy of 5 0.12 kJ mol(-1). Stationary phase promastigotes accumulated 2-DOG at a pproximately twice the rate of mid-log phase promastigotes. Cytochalas in B, forskolin and phloretin were all found to inhibit human erythroc yte 2-DOG uptake but only cytochalasin B was found significantly to in hibit promastigote 2-DOG uptake. Interestingly, leishmanial 2-DOG upta ke was inhibited by a series of membrane potential antagonists includi ng the ionophore monensin, the H(+)ATPase inhibitor N,N'-dicyclohexylc arbodiimide (DCCD) and uncoupling agent carbonylcyanide-4-(triflourome thoxy) phenylhydrazone (FCCP), as well as, the tricyclic drugs chlomip ramine and imipramine, but was insensitive to the Na+/K(+)ATPase inhib itor ouabain and the antitrypanosomal drugs Pentostam and Suramin. We therefore conclude that there are significant structural and mechanist ic differences between the D-glucose uptake systems of Leishmania and the mammalian host to merit the inclusion of glucose transporters as p utative targets for rational drug design.