Kinetic study of the plasma-membrane potential in procyclic and bloodstream forms of Trypanosoma brucei brucei using the fluorescent probe bisoxonol.
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.
Defrise-Quertain
F
F
Department of Biochemisty, University of Geneva, Switzerland.
Fraser-L'Hostis
C
C
Coral
D
D
Deshusses
J
J
D000818:Animals; D001463:Barbiturates / Q000737:chemistry*; D002021:Buffers; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D005033:Ethylmaleimide / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000737:chemistry*; D007555:Isoxazoles / Q000737:chemistry*; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D014346:Trypanosoma brucei brucei / Q000502:physiology*; D014638:Vanadates / Q000494:pharmacology
D001463:Barbiturates; D002021:Buffers; D005456:Fluorescent Dyes; D007555:Isoxazoles; C052795:bis(1,3-dibutylbarbiturate)trimethine oxonol; D014638:Vanadates; D005033:Ethylmaleimide