The role of a H+-ATPase in the regulation of cytoplasmic pH in Trypanosoma cruzi epimastigotes

Abstract

Cytoplasmic pH (pH_i) regulation was studied in Trypanosoma cruzi epimastigotes using fluorescent probes. Steady-state pH_i was maintained even in the absence of extracellular Na⁺ or K⁺, but was significantly decreased in the absence of Cl^-. Acid-loaded epimastigotes regained normal pH_i by a process that was ATP-dependent and sensitive to N-ethylmaleimide, dicyclohexylcarbodi-imide and diethylstilboestrol, suggesting involvement of a H⁺-pumping ATPase. Recovery from an acid load was independent of extracellular Na⁺ or K⁺ and insensitive to omeprazole, vanadate and low concentrations of bafilomycin A₁ . Using the fluorescent probe bisoxonol to measure the membrane potential of intact cells, acid loading of epimastigotes was shown to result in a dicyclohexylcarbodi-imide-sensitive hyperpolarization, which suggests electrogenic pumping of protons across the plasma membrane. Addition of glucose, but not of 6-deoxyglucose, produced a transient cellular acidification of possible metabolic origin, and increased the rate of recovery from an acid load. Taken together, these results are consistent with an important role of a H⁺-ATPase in the regulation of pH_i homoeostasis in T. cruzi.