An H+-ATPase regulates cytoplasmic pH in Pneumocystis carinii trophozoites

Pneumocystis carinii is an opportunistic fungus which causes interstitial pneumonia in patients with acquired immunodeficiency syndrome (AIDS). Cytoplasmic pH (pH(i)) regulation in short-term-cultured P. carinii trophozoites was studied using the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(-6)-carboxy-fluorescein. With an extracellular pH of 7.4, the mean baseline pH(i) of P. carinii trophozoites was 7.40 +/- 0.10 (n = 8). This steady-state pH(i) was not significantly affected in the absence of extracellular Na+ or K+. Moreover, steady-state pH(i) was maintained in the nominal absence of HCO3- and was not affected by the Cl-/HCO3--exchanger inhibitor 4,4'-di-isothiocyanatodihydrostilbene-2,2'-disulphonic acid (100 mu M), or the Na+/H+-exchanger inhibitor N-ethyl-N-isopropylamiloride (100 mu M). In contrast, the general inhibitors of ATPases, N-ethylmaleimide (1 mM), and dicyclohexylcarbodi-imide (100 mu M), and the inhibitor of yeast H+-ATPase, diethylstilbestrol (12.5-100 mu M), decreased pH(i), while the K+/H+-ATPase inhibitor omeprazole (50-400 mu M), and the vacuolar-type H+-ATPase inhibitor bafilomycin A(1) (1-5 mu M) only produced a dose-dependent acidification of the cells when used at high concentrations. In addition, steady-state pH(i) depended on the availability of cellular ATP, since it was decreased by the ATP synthase inhibitors oligomycin (1 mu g/ml) and sodium azide (1 mM), and by the uncoupler of oxidative phosphorylation carbonyl cyanide p-trifluorophenyl-hydrazone (1 mu M), agents that were able to deplete significantly the intracellular ATP levels. Taken together, these results are consistent with an important role of an H+-ATPase similar to those found in other fungi in the regulation of pH(i) homoeostasis in P. carinii trophozoites.