A pyruvate‒proton symport and an H+-ATPase regulate the intracellular pH of Trypanosoma brucei at different stages of its life cycle

Abstract

Regulation of intracellular pH (pHi) and H+ efflux were investigated in Trypanosoma brucei bloodstream and procyclic trypomastigotes using the fluorescent dyes 2ʹ,7ʹ-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) acetoxymethyl ester and free BCECF respectively. pHi in bloodstream and procyclic trypomastigotes was 7.47±0.06 and 7.53±0.07 respectively. Differences in the mechanisms for the regulation of pHi were noted between bloodstream and procyclic forms. Procyclic trypomastigotes maintained their pHi at neutral over a wide range of external pH values from 6 to 8, and in the absence of K+ or Na+. The H+-ATPase inhibitors N,Nʹ-dicyclohexylcarbodi-imide (DCCD), diethylstilboestrol and N-ethylmaleimide substantially decreased the steady-state pHi and inhibited its recovery from acidification. The rate of H+ efflux in these forms was determined to be 62±6.5 nmol/min per mg of protein, and was substantially decreased by H+-ATPase inhibitors. The data support the presence of an H+-ATPase as the major regulator of pHi in procyclic trypomastigotes. In contrast, bloodstream trypomastigotes were unable to maintain a neutral pH under acidic conditions, and their steady-state pHi and recovery from acidification were unaffected by H+-ATPase inhibitors, except for DCCD (100 μM). Their steady-state pHi was markedly decreased in glucose-free buffer or by ≥ 10 mM pyruvate, whereas procyclic trypomastigotes were unaffected by similar treatments. The rate of H+ efflux in bloodstream trypomastigotes was 534±38 nmol/min per mg of protein, and was decreased in the absence of glucose and by the addition of pyruvate or DCCD. Pyruvate efflux in these forms was calculated to be 499±34 nmol/min per mg of protein, and was significantly inhibited by DCCD, 4,4ʹ-di-isothiocyanatodihydrostilbene-2,2ʹ-disulphonic acid and α-cyanohydroxycinnamic acid. The pyruvate analogues β-hydroxypyruvate, 3-bromopyruvate, 3-oxoglutarate, oxaloacetate, 3-oxoisovalerate and 3-oxoisohexanoate significantly decreased pHi, as well as proton and pyruvate efflux, whereas lactate had only a small effect, and no effect was observed with citrate or fumarate. The inhibition by pyruvate analogues of pyruvate efflux, proton efflux and acidification of pHi supports the hypothesis that pyruvate efflux is accompanied by proton efflux and that this is the major pHi control mechanism in bloodstream forms. Inhibition by H+-ATPase inhibitors of residual H+ efflux in the absence of glucose or in the presence of high extracellular pyruvate indicates a minor role for H+-ATPase(s) in control of pHi in bloodstream forms.