Absence of transitory [Ca2+]i flux during early in vitro metacyclogenesis of Trypanosoma cruzi.
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.
Krassner
S M
SM
Department of Developmental and Cell Biology, University of California, Irvine 92717.
Chang
J
J
Pak
S
S
Luc
K O
KO
Granger
B
B
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D012701:Serotonin / Q000494:pharmacology; D015398:Signal Transduction; D013729:Terpenes / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin; D014349:Trypanosoma cruzi / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*
D013729:Terpenes; D012701:Serotonin; D019284:Thapsigargin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D004121:Dimethyl Sulfoxide