Kinetic study of the plasma-membrane potential in procyclic and bloodstream forms of Trypanosoma brucei brucei using the fluorescent probe bisoxonol

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Abstract
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 °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.