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 maintain a hyperpolarized plasma membrane which is primarily set by a dicyclohexylcarbodiimide (DCCD)-sensitive electrogenic H(+)-pump. We studied here the possible contribution of the membrane potential (Vm) and the transmembrane proton gradient (delta pH) to the mediated uptake of amino acids and their intracellular accumulation. Proline transport and 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 Vm and considerably less by changes in delta pH. The most pronounced effects were obtained by treatment with either the H(+)-uncoupler carbonylcyanide chlorophenylhydrazone (CCCP), the cation ionophore gramicidin or by 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 4,4'-diisothiocyanodihydrostilbene-2,2'-disulfonic acid (H2DIDS). No significant effects were found with cells exposed to K+ in the presence of nigericin, to Na+ in the presence of monensin or to other cations substituting for Na+. These results suggest that neither extracellular Na+ or K+, per 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 intracellular alkalinization. The present observations indicate that uphill nutrient uptake by Leishmania promastigotes is largely determined by Vm. The relatively high intracellular pools of amino acids might be of physiological relevance to osmoregulation by parasites.
Vieira
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LL
Department of Biological Chemistry, Institute of Life Sciences Hebrew University, Jerusalem, Israel.
Cabantchik
Z I
ZI
D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002462:Cell Membrane / Q000502:physiology; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D018320:Leishmania major / Q000502:physiology*; D008564:Membrane Potentials; D011392:Proline / Q000378:metabolism*; D017494:Proton Pumps / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors; D014316:Tritium; D014475:Uncoupling Agents / Q000494:pharmacology
D000596:Amino Acids; D004791:Enzyme Inhibitors; D017494:Proton Pumps; D014475:Uncoupling Agents; D014316:Tritium; D004024:Dicyclohexylcarbodiimide; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D011392:Proline; D006180:Proton-Translocating ATPases