Cellular signalling in Trypanosoma cruzi: biphasic behaviour of inositol phosphate cycle components evoked by carbachol

Abstract

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 (Ins P 3) production and [Ca 2+]i mobilisation were carbachol dose-dependent. The maximally effective concentrations of agonist ranged between 1×10 −6 and 1×10 −5 M. The increase in carbachol concentration resulted in an evident attenuation of [Ca 2+]i mobilisation and in [ 3H]Ins P 3 levels. Pretreatment of the cells with 10 μM U73122, a phospholipase C (PLC) inhibitor, showed that both Ins P 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 Ins P 3 and Ins P 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]PtdIns P levels. The time-course of PtdIns4 P 5-kinase activity showed a rapid, significant and transient decrease of [ 32P]PtdIns P 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.