Cellular signalling in Trypanosoma cruzi: biphasic behaviour of inositol phosphate cycle components evoked by carbachol.
Stimulation of epimastigote forms of Trypanosma cruzi with carbachol resulted in a long-lasting response. The earlier phase for inositol phosphates was rapid and transient, peaking at 1 min with a return to basal levels by 6 min. In a second phase, these metabolite levels reached maximal values at 10-12 min, with a later declination to basal values at about 20 min. The inositol phosphate response was quenched by parasite treatment with atropine. The elevation in intracellular free calcium ([Ca(2+)]i) was transient, reaching the resting level at 87+/-8 s (n=48) of agonist addition. Myo-inositol 1,4,5-triphosphate (InsP(3)) production and [Ca(2+)]i mobilisation were carbachol dose-dependent. The maximally effective concentrations of agonist ranged between 1x10(-6) and 1x10(-5) M. The increase in carbachol concentration resulted in an evident attenuation of [Ca(2+)]i mobilisation and in [3H]InsP(3) levels. Pretreatment of the cells with 10 microM U73122, a phospholipase C (PLC) inhibitor, showed that both InsP(3) peaks triggered by carbachol were completely abolished, whereas there was not substantial change on the maximum elevation in [Ca(2+)]i. The first peak of InsP(3) and InsP(s) was completely abolished when the cells were incubated with phorbol 12-myristate 13-acetate ester (TPA) for 30 min before carbachol stimulation. A biphasic behaviour for PtdIns 4-kinase activity was demonstrated by changes in [32P]PtdInsP levels. The time-course of PtdIns4P 5-kinase activity showed a rapid, significant and transient decrease of [32P]PtdInsP(2) from 0 time to the third min. At the end of this time the polyphosphoinositide began to return towards control levels but, interestingly, after 5-6 min of stimulation there was a subsequent more important increase over control levels which peaked at 10 min. There was also a detectable increment of DAG at 1 min with a maximum at 3 min, this level remaining elevated until at least 10 min. Subsequently, these levels returned to the base line or even below it.
Marchesini
Norma
N
Department of Veterinary Pathobiology, University of Illinois, 2001 S. Lincoln Ave., Urbana, IL 61801, USA.
Bollo
Mariana
M
Hernandez
Gabriela
G
Garrido
Monica N
MN
Machado-Domenech
Estela E
EE
D019870:1-Phosphatidylinositol 4-Kinase; D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D002217:Carbachol / Q000494:pharmacology*; D019852:Diacylglycerol Kinase / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism*; D014349:Trypanosoma cruzi / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism
D007295:Inositol Phosphates; D002217:Carbachol; D019852:Diacylglycerol Kinase; D019870:1-Phosphatidylinositol 4-Kinase; D010738:Type C Phospholipases; D002118:Calcium