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

NORMA MARCHESINI , MARIANA BOLLO , GABRIELA HERNANDEZ , MONICA N. GARRIDO , ESTELA E. MACHADO-DOMENECH.

MOLECULAR AND BIOCHEMICAL PARASITOLOGY

Elseiver

Lugar: Amsterdam; Año: 2002 vol. 120 p. 83 - 91

0166-6851

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 ([Ca2+]i) was transient, reaching the resting level at 87±8 s (n=48) of agonist addition. Myo-inositol 1,4,5-triphosphate (InsP3) production and [Ca2+]i mobilisation were carbachol dose-dependent. The maximally effective concentrations of agonist ranged between1×10−6 and 1×10−5 M. The increase in carbachol concentration resulted in an evident attenuation of [Ca2+]i mobilisation andin [3H]InsP3 levels. Pretreatment of the cells with 10 mM U73122, a phospholipase C (PLC) inhibitor, showed that both InsP3 peaks triggered by carbachol were completely abolished, whereas there was not substantial change on the maximum elevation in [Ca2+]i. The first peak of InsP3 and InsPs was completely abolished when the cells were incubated with phorbol 12-myristate13-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]PtdInsP2 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.  © 2002 Elsevier Science B.V. All rights reserved. Keywords: Calcium signalling; Lipid kinase; PtdIns cycle; Trypanosoma cruzi