Vanderheyden N, Benaim G, Docampo R
Department of Veterinary Pathobiology, University of Illinois at Urbana-Champaign, IL 61801, USA.
Biochem J. 1996 Aug 15;318 ( Pt 1)(Pt 1):103-9. doi: 10.1042/bj3180103.
Cytoplasmic pH (pHi) regulation was studied in Trypanosoma cruzi epimastigotes using fluorescent probes. Steady-state pHi 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 pHi by a process that was ATP-dependent and sensitive to N-ethylmaleimide, dicyclohexyl-carbodi-imide and diethylstiboestrol, 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 pHi homoeostasis in T. cruzi.
利用荧光探针研究了克氏锥虫前鞭毛体中的细胞质pH（pHi）调节。即使在没有细胞外Na⁺或K⁺的情况下，稳态pHi也能维持，但在没有Cl⁻的情况下会显著降低。酸负载的前鞭毛体通过一个依赖ATP且对N - 乙基马来酰亚胺、二环己基碳二亚胺和己烯雌酚敏感的过程恢复正常pHi，这表明有一个H⁺泵ATP酶参与其中。从酸负载中恢复与细胞外Na⁺或K⁺无关，且对奥美拉唑、钒酸盐和低浓度的巴弗洛霉素A1不敏感。使用荧光探针双苯甲酰羟肟酸测量完整细胞的膜电位，结果显示前鞭毛体的酸负载会导致二环己基碳二亚胺敏感的超极化，这表明质子通过质膜进行电致泵出。添加葡萄糖而非6 - 脱氧葡萄糖会产生可能源于代谢的短暂细胞酸化，并提高从酸负载中恢复的速率。综上所述，这些结果与H⁺ - ATP酶在克氏锥虫pHi稳态调节中起重要作用相一致。
