A plasma membrane P-type H(+)-ATPase regulates intracellular pH in Leishmania mexicana amazonensis.
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.
Marchesini
Norma
N
Laboratory of Molecular Parasitology, Department of Pathobiology, College of Verterinary Medicine, University of Illinois at Urbana Champaign, 2001 South, Lincoln Avenue, Urbana, IL 61802, USA.
Docampo
Roberto
R
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001639:Bicarbonates / Q000494:pharmacology; D015153:Blotting, Western; D002021:Buffers; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002713:Chlorine / Q000494:pharmacology; D004594:Electrophysiology; D004795:Enzyme Stability / Q000187:drug effects; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D007894:Leishmania mexicana / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D009994:Osmolar Concentration; D011188:Potassium / Q000494:pharmacology; D006180:Proton-Translocating ATPases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology
D001639:Bicarbonates; D002021:Buffers; D002713:Chlorine; C018564:dihydro-DIDS; D012964:Sodium; D006180:Proton-Translocating ATPases; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D011188:Potassium