Docampo R, Vanderheyden N M, Shaw M M, Durant P J, Bartlett M S, Smith J W, McLaughlin G L
Department of Veterinary Pathobiology, University of Illinois at Urbana-Champaign 61801, USA.
Biochem J. 1996 Jun 1;316 ( Pt 2)(Pt 2):681-4. doi: 10.1042/bj3160681.
Pneumocystis carinii is an opportunistic fungus which causes interstitial pneumonia in patients with acquired immunodeficiency syndrome (AIDS). Cytoplasmic pH (pHi) regulation in short-term-cultured P. carinii trophozoites was studied using the fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(-6)-carboxyfluorescein. With an extracellular pH of 7.4, the mean baseline pHi of P. carinii trophozoites was 7.40 +/- 0.10 (n = 8). This steady-state pHi was not significantly affected in the absence of extracellular Na+ or K+. Moreover, steady-state pHi was maintained in the nominal absence of HCO3- and was not affected by the Cl-/HCO(3-)-exchanger inhibitor 4, 4'-di-isothiocyanato-dihydrostilbene-2, 2'-disulphonic acid (100 microM), or the Na+/H(+)-exchanger inhibitor N-ethyl-N-isopropylamiloride (100 microM). In contrast, the general inhibitors of ATPases, N-ethylmaleimide (1 mM), and dicyclohexylcarbodi-imide (100 microM), and the inhibitor of yeast H(+)-ATPase, diethylstilbestrol (12.5-100 microM), decreased pHi, while the K+/H(+)-ATPase inhibitor omeprazole (50-400 microM), and the vacuolar-type H(+)-ATPase inhibitor bafilomycin A1 (1-5 microM) only produced a dose-dependent acidification of the cells when used at high concentrations. In addition, steady-state pHi depended on the availability of cellular ATP, since it was decreased by the ATP synthase inhibitors oligomycin (1 microgram/ml) and sodium azide (1 mM), and by the uncoupler of oxidative phosphorylation carbonyl cyanide p-trifluorophenylhydrazone (1 microM), 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 pHi homoeostasis in P. carinii trophozoites.
卡氏肺孢子菌是一种机会性真菌，可导致获得性免疫缺陷综合征（AIDS）患者发生间质性肺炎。使用荧光染料2',7'-双（2-羧乙基）-5-（-6）-羧基荧光素研究了短期培养的卡氏肺孢子菌滋养体中的细胞质pH（pHi）调节。细胞外pH为7.4时，卡氏肺孢子菌滋养体的平均基线pHi为7.40±0.10（n = 8）。在没有细胞外Na+或K+的情况下，这种稳态pHi没有受到显著影响。此外，在名义上没有HCO3-的情况下，稳态pHi得以维持，并且不受Cl-/HCO(3-)-交换体抑制剂4,4'-二异硫氰酸二氢芪-2,2'-二磺酸（100 microM）或Na+/H(+)-交换体抑制剂N-乙基-N-异丙基阿米洛利（100 microM）的影响。相反，ATP酶的一般抑制剂N-乙基马来酰亚胺（1 mM）和二环己基碳二亚胺（100 microM）以及酵母H(+)-ATP酶抑制剂己烯雌酚（12.5 - 100 microM）会降低pHi，而K+/H(+)-ATP酶抑制剂奥美拉唑（50 - 400 microM）和液泡型H(+)-ATP酶抑制剂巴弗洛霉素A1（1 - 5 microM）仅在高浓度使用时才会使细胞产生剂量依赖性酸化。此外，稳态pHi取决于细胞ATP的可用性，因为它会被ATP合酶抑制剂寡霉素（1微克/毫升）和叠氮化钠（1 mM）以及氧化磷酸化解偶联剂羰基氰对三氟甲氧基苯腙（1 microM）降低，这些试剂能够显著耗尽细胞内ATP水平。综上所述，这些结果与一种类似于其他真菌中发现的H(+)-ATP酶在卡氏肺孢子菌滋养体pHi稳态调节中起重要作用一致。
