pH homeostasis in Leishmania donovani amastigotes and promastigotes.

Abstract

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...

Dual Action of Antimonial Drugs on Thiol Redox Metabolism in the Human Pathogen Leishmania donovani

“…4, C and F) (28). Both promastigotes and amastigotes maintain intracellular pH values close to neutral, independent of external pH (30). In both developmental forms of L. donovani, the reduction potential for the trypanothione couple was lower than the glutathione couple, indicating that the two systems are apparently not at equilibrium.…”

“…However, the physiological relevance of this observation is questionable because promastigotes contain higher intracellular concentrations of T[SH] 2 and GSH than amastigotes (Fig. 4), both stages maintain intracellular pH values close to neutral and independent of external pH (30), and both stages can take up Sb III and Sb V (37). A recently identified thiol-dependent reductase (TDR1) that is more highly expressed in amastigotes has been postulated to play a role (8).…”

Na+-dependent pH Regulation by the Amitochondriate Protozoan Parasite Giardia intestinalis

“…The relative concentrations of the charged and uncharged species is pH-dependent and hence if the intraand extracellular DMO concentrations ([DMO] i and [DMO] o , respectively) and the extracellular pH (pH o ) are known, the pH i can be determined (14). This method is more straightforward than the 31 P NMR method and requires lower cell numbers.…”

Drug Resistance in Leishmaniasis

“…However, the physiological relevance of these observations is open to question since the rates of reduction are rather slow. Moreover, promastigotes contain higher intracellular concentrations of trypanothione and glutathione than amastigotes (7,161) and both stages maintain intracellular pH values close to neutral, independent of external pH (69). Thus, it is difficult to account for the selective action of Sb(V) against the amastigote stage by a nonenzymatic mechanism.…”