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

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 A(1). 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.