Amastigotes (tissue forms) of Leishmania donovani isolated from infected hamster spleens carried out several physiological activities (respiration, catabolism of energy substrates, and incorporation of precursors into macromolecules) optimally at pH 4.0 to 5.5. All metabolic activities that were examined decreased sharply above the optimal pH. Promastigotes (culture forms), on the other hand, carried out the same metabolic activities optimally at or near neutral pH. This adaptation to an acid environment may account in part for the unusual ability of amastigotes to survive and multiply within the acidic environment of the phagolysosomes in vivo.

Intracellular pH and pH gradients of Leishmania donovani amastigotes and promastigotes were determined over a broad range of extracellular pH values. Intracellular pH was determined by 31p NMR and by equilibrium distribution studies with 5,5-dimethyloxazolidine-2,4-dione or methylamine. Promastigotes maintain intracellular pH values close to neutral between extracellular pH values of 5.0 and 7.4. Amastigote intracellular pH is maintained close to neutral at external pH values as low as 4.0. Both life stages maintain a positive pH gradient to an extracellular pH of 7.4, which is important for active transport of substrates. Treatment with ionophores, such as nigericin and carbonyl cyanide mchlorophenylhydrazone and the ATPase inhibitor dicyclohexylcarbodiimide, reduced pH gradients in both stages. Maintenance of intracellular pH in the physiologic range is especially relevant for the survival ofthe amastigote in its acidic in vivo environment. where 59 is a factor for the conversion of ApH to mV and is equal to (2.3 RT)/F; R, T, and F are the gas constant, absolute temperature, and the Faraday constant, respectively (4). Knowledge of the pHi enables the calculation of ApH and ultimately the magnitude of the driving force for active transport. Such knowledge also provides insight into the homeostatic mechanisms of these parasites, which encounter substantial changes of pH in their extracellular environments.Here we report pH, values for Leishmania donovani over a broad range of extracellular pH values (pHe). Promastigote pHi was determined by two methods: the equilibrium distribution of the weak acid 5,5-dimethyl-oxazolidine-2,4-dione (DMO) and the chemical shift of intracellular inorganic phosphate as determined by 31P NMR spectrometry. The high density of cells required for NMR prohibited the use of this technique for amastigote pHi determination; therefore, amastigote pH1 values were determined by only the DMO method. MATERIALS AND METHODSMaintenance and Preparation of Amastigotes. Amastigotes of L. donovani Sudan strain IS were maintained by serial passage in female Syrian golden hamsters. The organism was passaged by i.p. injection of spleen homogenates containing 1 x 107 parasites. Hamsters were sacrificed 8 weeks postinfection, and parasites were isolated from liver and spleens by a described method (5).Maintenance and Preparation of Promastigotes. Promastigotes of L. donovani IS were maintained at 260C in medium 199 (GIBCO) supplemented with 15% fetal bovine serum. Cells were harvested during stationary phase growth by centrifugation at 1100 x g for 10 min and washed twice in a basal salts solution (6).Determination of pH, by DMO or Methylamine. The pHi of L. donovani promastigotes and amastigotes was determined from the distribution of the weak acid DMO or the weak base methylamine as described by Rottenberg (7), a method based on the principle that the nondissociated acid is fully permeable to the membrane but is impermeable in its ionic form. Therefore, as long as the pHi is more alkaline relati...

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The benzimidazole compound omeprazole, used widely for the treatment of peptic ulcer disease, inhibits the growth of Leishmania donovani, the causative agent of visceral leishmaniasis. Promastigotes cultured at acidic pH and amastigotes within infected macrophages are reduced 90% or more with 150 M omeprazole. Antiparasitic action of the drug is due to its inhibition of the P-type K ؉ ,H ؉ -ATPase on the surface membrane. This enzyme is important for pH homeostasis and the maintenance of proton motive force across the membrane in Leishmania. The drug is effective only at acidic pH, a condition that mimics the in vivo environment within the phagolysosomal vesicles where the amastigote form of the parasite resides. Omeprazole deserves consideration as an alternative to currently available chemotherapeutics, which have severe toxic side effects.