The mechanisms regulating the intracellular pH (pHi) in both forms of Trypanosoma brucei brucei (cultured cells) were investigated using the fluorescent probe 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). The pHi values measured were 7.22+/-0.03 in the procyclics and 7.40+/-0.05 in the bloodstream form. In the presence of 24mM HCO3-, pHi values were slightly higher in both forms of trypanosomes suggesting a bicarbonate-linked pH regulation. pHi was more stable in procyclics (between 7.15 and 7.30 in the external pH range 6.4-7.6) than in the bloodstream forms. The amiloride analogue tested decreased pHi, suggesting Na+-driven Na+/H+ antiporters. H+-ATPases also seem to be involved in pHi regulation since the inhibitors N-ethylmaleimide (1 mM) and N,N'-dicyclohexylcarbodiimide (80 microM) induced a rapid acidification in both forms of trypanosomes. Addition of pyruvate caused a cytosol acidification in the bloodstream form only confirming the existence of a diffusion-facilitated carrier for pyruvate, with the cotransport of H+. Our results show that, although similar pH regulation mechanisms seem to exist in both forms of trypanosomes, the procyclics can regulate efficiently their pHi and consequently their plasma membrane potential whereas the bloodstream forms cannot always maintain their pHi and are easily depolarized following a small acid load.

The characteristics of the plasma-membrane potential of procyclic and bloodstream forms of Trypanosoma brucei brucei (cultured cells) were investigated using the fluorescent anionic probe bisoxonol. Observation of a stable and representative plasma-membrane potential in the resting state required careful washing, centrifugation and maintenance of the cells at room temperature before measurement. Bloodstream forms were more prone to depolarization during washing at 4 degrees C than procyclic cells. The higher fluorescence observed in the presence of long slender cells than in the presence of procyclic cells shows that the plasma-membrane potential is more negative in the insect form. Healthy dilute cells can sustain their plasma-membrane potential for hours in the presence of external glucose. The presence of a high K+ concentration in the medium did not promote by itself the depolarization of either type of cell. Study of bisoxonol fluorescence as a function of time allowed us to follow the kinetics of the action of metabolic inhibitors in the presence of various ions. o-Vanadate (1 mM) was found to depolarize bloodstream-form cells rapidly but only in a phosphate-free NaCl buffer. Omeprazole and strophanthidin also specifically depolarized bloodstream-form trypanosomes. However, NN'-dicyclohexylcarbodi-imide depolarized both types of cell, but more rapidly for bloodstream-form cells. Bloodstream-form trypanosomes appear to use mainly a vanadate-sensitive Na+ pump to maintain their Na+-diffusion gradient. However, most of the ATPase inhibitors tested had little or no effect on the plasma-membrane potential of procyclics suggesting that this form of trypanosome may rely on several regulation mechanisms.

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