Blackwell Publishing Journal Backfiles 1879-2005
. 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 Ca2+-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.
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