AMINO-ACID-UPTAKE AND INTRACELLULAR ACCUMULATION IN LEISHMANIA-MAJOR PROMASTIGOTES ARE LARGELY DETERMINED BY AN H-PUMP GENERATED MEMBRANE-POTENTIAL()

Leishmania major promastigotes maintain a relatively high pool of free amino acids (>100 mM) under in vitro growth conditions. They also mai ntain a hyperpolarized plasma membrane which is primarily set by a dic yclohexylcarbodiimide (DCCD)-sensitive electrogenic H+-pump. We studie d here the possible contribution of the membrane potential (V-m) and t he transmembrane proton gradient (Delta pH) to the mediated uptake of amino acids and their intracellular accumulation. Proline transport an d accumulation were assessed by analysis of time-dependent changes in the internal pools of free amino acids and by uptake of radiolabelled proline. Proline uptake was markedly affected by changes in the V-m an d considerably less by changes in Delta pH. The most pronounced effect s were obtained by treatment with either the H+-uncoupler carbonylcyan ide chlorophenylhydrazone (CCCP), the cation ionophore gramicidin or b y omitting Cl- from the medium (by exchange with gluconate or mannitol ). Relatively smaller effects were obtained in the presence of the H+- ATPase inhibitor DCCD or with the anion transport blocker ,4'-diisothi ocyano-dihydrostilbene-2,2'-disulfonic acid (H2DIDS). No significant e ffects were found with cells exposed to K+ in the presence of nigerici n, to Na+ in the presence of monensin or to other cations substituting for Na+. These results suggest that neither extracellular Na+ or K+ p er se, nor even intracellular pH, play a major role in proline uptake and accumulation. A significant stimulation In proline uptake induced by HCO3- could be associated with membrane hyperpolarization or intrac ellular alkalinization. The present observations indicate that uphill nutrient uptake by Leishmania promastigotes is largely determined by V -m. The relatively high intracellular pools of amino acids might be of physiological relevance to osmoregulation by parasites.