A plasma membrane P-type H +-ATPase regulates intracellular pH in Leishmania mexicana amazonensis

• Published in: Molecular and biochemical parasitology Vol. 119; no. 2; pp. 225 - 236
• Main Authors: Marchesini, Norma, Docampo, Roberto
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.02.2002
• Subjects: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid - analogs & derivatives; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid - pharmacology; Animals; Bicarbonates - pharmacology; Blotting, Western; Buffers; Cell Membrane - chemistry; Cell Membrane - metabolism; Chlorine - pharmacology; Electrophysiology; Enzyme Stability - drug effects; Hydrogen-Ion Concentration - drug effects; Intracellular Fluid - drug effects; Intracellular Fluid - metabolism; Kinetics; Leishmania amazonensis; Leishmania mexicana - cytology; Leishmania mexicana - drug effects; Leishmania mexicana - enzymology; Leishmania mexicana - metabolism; Membrane potential; Membrane Potentials - drug effects; Osmolar Concentration; Potassium; Potassium - pharmacology; Proton-Translocating ATPases - analysis; Proton-Translocating ATPases - antagonists & inhibitors; Proton-Translocating ATPases - immunology; Proton-Translocating ATPases - metabolism; Sodium - pharmacology; MES, 2-[ N-morpholino]ethanesulfonic acid; NEM, N-ethylmaleimide; DES, diethylstilbestrol; H 2DIDS, 4,4′-di-isothiocyanatodihydrostilbene-2,2′-disulphonic acid; pH; BCECF, 2′,7′-bis-(carboxyethyl)-5(and-6)-carboxyfluorescein; DCCD, dicyclohexylcarbo-diimide; Membrane potential; Potassium; Leishmania amazonensis
• ISSN: 0166-6851; 1872-9428
• DOI: 10.1016/S0166-6851(01)00419-4

A recent report (Mukherjee et al., J. Biol. Chem. 276 (2001) 5563) has proposed that the plasma membrane Mg +-ATPase of promastigotes of Leishmania donovani, that is involved in its intracellular pH regulation, is an electroneutral H +/K + antiporter rather than an electrogenic H + pump. Since this proposition has important implications for the use of the pump as a target for chemotherapy, we investigated its nature in the mammalian stage (amastigote) of L. mexicana amazonensis and compared it with that present in promastigotes. Intracellular pH and H + efflux were measured using the acetotoxymethyl ester and the free form of 2′,7′-bis-(carboxyethyl)-5(and-6)-carboxyfluorescein, respectively. Intracellular pH in amastigotes (at an external pH of 5.5) and promastigotes (at an external pH of 7.4) was 6.36±0.02 and 6.83±0.07, respectively. Differences in the mechanisms for regulation of intracellular pH were noted between amastigote and promastigote forms. Amastigotes maintained their intracellular pH neutral over a wide range of external pHs in the absence of K + or Na +. The H +-ATPase inhibitors N, N′-dicyclohexylcarbodi-imide, diethylstilbestrol and N-ethylmaleimide, substantially decreased their steady-state intracellular pH, inhibited proton efflux, and their recovery from acidification. The data support the presence of an H +-ATPase as the major regulator of intracellular pH in amastigotes. In contrast, promastigotes were unable to maintain a neutral pH under acidic conditions and although their steady-state intracellular pH and recovery from acidification were affected by H +-ATPase inhibitors, bicarbonate was able to overcome intracellular acidification. Bicarbonate was also able to raise the steady-state intracellular pH from 6.80±0.03 to 7.25±0.09 and induce membrane hyperpolarization. No evidence was found of the possible involvement of a K +/H +-ATPase in intracellular pH regulation in both developmental stages of L. m. amazonensis.