Trypanosoma evansi: a convenient model for studying intracellular Ca(2+) homeostasis using fluorometric ratio imaging from single parasites.
The aim of this work was to measure, for the first time, the basal cytosolic Ca(2+) levels of Trypanosoma evansi and to explore the possibility of observing changes in the intracellular Ca(2+) concentration ([Ca(2+)](i)) using fluorescence ratio imaging techniques in single isolated parasites of this species. Under appropriate loading conditions, the high intracellular levels of the Ca(2+) fluorescence probe Fura-2 permits resolution, in real time, of single parasite [Ca(2+)](i) signals. Measurements of the basal [Ca(2+)](i) indicate that homeostatic mechanisms maintain [Ca(2+)](i) at 106 +/- 38 (n = 32) nM in the presence of 2 mM extracellular calcium. The resting [Ca(2+)](i) was unaffected by changes in extracellular Ca(2+) in the range from 0 to 10 mM. The Ca(2+) ionophore A23187 induced a large increase in [Ca(2+)](i) which (i) reached a steady state value even in the simultaneous presence of both external calcium and ionophore and (ii) returned to base line upon removal of extracellular Ca(2+). A dose-response curve of the protonophore nigericin shows that T. evansi contains an important pH-sensitive intracellular pool which may be released by this drug with a K(1/2) of 8 microM. These data demonstrate that this parasite contains highly efficient systems to control [Ca(2+)](i). Finally, our results, with the use of sera as source of an antibody-complement to induce Ca(2+) entry, demonstrate that it is possible to resolve fast [Ca(2+)](i) signals in single parasites from T. evansi.
Mendoza
M
M
Centro de Estudios Biomédicos y Veterinarios, Caracas, Venezuela.
Mijares
A
A
Rojas
H
H
Ramos
M
M
DiPolo
R
R
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D003165:Complement System Proteins / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004950:Esterases / Q000378:metabolism; D005456:Fluorescent Dyes; D005470:Fluorometry; D016257:Fura-2; D006706:Homeostasis; D007106:Immune Sera / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008297:Male; D008954:Models, Biological; D009550:Nigericin / Q000494:pharmacology; D051381:Rats; D014345:Trypanosoma / Q000187:drug effects / Q000378:metabolism*
D005456:Fluorescent Dyes; D007106:Immune Sera; D007476:Ionophores; D000001:Calcimycin; D003165:Complement System Proteins; D004950:Esterases; D009550:Nigericin; D002118:Calcium; D016257:Fura-2