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Absence of Transitory [Ca 2+ ] I Flux During Early In Vitro Metacyclogenesis of Trypanosoma cruzi 1
Author(s) -
KRASSNER STUART M.,
CHANG JOHNNY,
PAK SUNG,
LUC KIMOANH,
GRANGER BARBARA
Publication year - 1993
Publication title -
journal of eukaryotic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.067
H-Index - 77
eISSN - 1550-7408
pISSN - 1066-5234
DOI - 10.1111/j.1550-7408.1993.tb04907.x
Subject(s) - thapsigargin , biology , protein kinase c , egta , endoplasmic reticulum , staurosporine , phorbol , microbiology and biotechnology , biochemistry , diacylglycerol kinase , protein kinase a , calcium , kinase , chemistry , organic chemistry
. The phorbol ester TPA (phorbol 12‐myristate 13‐acetate) substitutes for CO 2 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% NaHCO 3 . 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 ([Ca 2+ ] i ) in epimastigotes, activated with 5% CO 2 or TPA (10 ‐7 M), was measured with the Ca 2+ molecular probe, fluo‐3AM. In addition, [Ca 2+ ] 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 Ca 2+ levels. Control assays with 11 μM thapsigargin, which mobilizes noncytoplasmic Ca 2+ stores by inhibiting endoplasmic reticulum Ca 2+ ‐ATPase. validated our fluorometric assay procedure. Although thapsigargin significantly increases cytoplasmic Ca 2+ 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 CO 2 and TPA is not accompanied by changes in cytosolic Ca 2+ and do not provide supporting evidence for participation of a protein kinase C‐mediated phosphoinositide cascade in metacyclogenesis.