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 studied, except for EGTA, affected cytosolic Ca²⁺ levels. Control assays with 11 μM thapsigargin, which mobilizes noncytoplasmic Ca²⁺ stores by inhibiting endoplasmic reticulum Ca²⁺-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 Pent 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.