Absence of Transitory [Ca²⁺]_I Flux During Early In Vitro Metacyclogenesis of Trypanosoma cruzi¹

Abstract

The phorbol ester TPA (phorbol 12-myristate 13-acetate) substitutes for CO2 as an agonist for transforming Trypanosoma cruzi epimastigotes to the metacyclic trypomastigote stage in a starvation medium consisting of phosphate buffered saline + 10 mM proline, 10 mM sodium acetate and 0.035% NaHCO3. Since TPA is thought to stimulate protein kinase C by mimicking the activity of the secondary messenger diacylglycerol, the above result suggested that T. cruzi metacyclogenesis could be activated by a Ca(2+)-dependent protein kinase C signal induction pathway. Accordingly, cytosolic calcium flux ([Ca2+]i) in epimastigotes, activated with 5% CO2 or TPA (10(-7) M), was measured with the Ca2+ molecular probe, fluo-3AM. In addition, [Ca2+]i was measured in cells incubated with putative metacyclogenic agonists (e.g. proline, glutamate, bioamines, ionophores and catecholamines). None of the compounds studies, except for EGTA, affected cytosolic Ca2+ levels. Control assays with 11 microM thapsigargin, which mobilizes noncytoplasmic Ca2+ stores by inhibiting endoplasmic reticulum Ca(2+)-ATPase, validated our fluorometric assay procedure. Although thapsigargin significantly increases cytoplasmic Ca2+ fluorescence, it has no effect on transformation. The protein kinase C inhibitors staurosporine, H-7 and HA 1004 were tested for their effect on T. cruzi metacyclogenesis. Low concentrations of staurosporine and HA 1004 significantly elevated Peru strain transformation while H-7 had no effect on Peru strain metacyclogenesis. Inhibitor H-7 did significantly depress CL transformation. The results indicate that induction of T. cruzi metacyclic trypomastigote formation by CO2 and TPA is not accompanied by changes in cytosolic Ca2+ and do not provide supporting evidence for participation of a protein kinase C-mediated phosphoinositide cascade in metacyclogenesis.

Sympathetic Resonance Technology™: Scientific Foundation and Summary of Biologic and Clinical Studies

“…A quantitative bioassay, the T. cruzi Metacyclogenesis Assay was used. The purpose of this pilot study was to measure the effects of SRT on an in vitro model system for cell growth and differentiation (Krassner et al, 1991(Krassner et al, , 1993.…”