Characterization of [Ca2+]i responses in primary cultures of mouse cardiomyocytes induced by Trypanosoma cruzi trypomastigotes.

Trypanosoma cruzi, the protozoan responsible for Chagas disease, employs distinct strategies to invade mammalian host cells. In the present work we investigated the participation of calcium ions on the invasion process using primary cultures of embryonic mice cardiomyocytes which exhibit spontaneous contraction in vitro. Using Fura 2-AM we found that T. cruzi was able to induce a sustained increase in basal intracellular Ca2+ level in heart muscle cells (HMC), the response being associated or not with Ca2+ transient peaks. Assays performed with both Y and CL strains indicated that the changes in intracellular Ca2+ started after parasites contacted with the cardiomyocytes and the evoked response was higher than the Ca2+ signal associated to the spontaneous contractions. The possible role of the extracellular and intracellular Ca2+ levels on T. cruzi invasion process was evaluated using the extracellular Ca2+ chelator EGTA alone or in association with the calcium ionophore A23187. Significant dose dependent inhibition of the invasion levels were found when intracellular calcium release was prevented by the association of EGTA +A23187 in calcium free medium. Dose response experiments indicated that EGTA 2.5 mM to 5 mM decreased the invasion level by 15.2 to 35.1% while A23187 (0.5 M) alone did not induce significant effects (17%); treatment of the cultures with the protease inhibitor leupeptin did not affect the endocytic index, thus arguing against the involvement of leupeptin sensitive proteases in the invasion of HMC.

Attenuation of parasite cAMP levels in T. cruzi-host cell membrane interactions in vitro.

Previous investigations have shown that the adhesion of T. cruzi plasma membrane vesicles (PMV) to monolayers of host cell myoblasts and to immobilized heart muscle sarcolemma membranes (PAM) on polyacrylamide beads is mediated by the interaction of T. cruzi attachment sites with the muscarinic cholinergic and beta-adrenergic receptors of the host cell membrane. It has also been shown that this interaction is blunted by the specific antagonists of the mammalian receptors atropine and propranol, respectively. In the studies reported here, PAM also rapidly attached to swimming T. cruzi trypomastigotes in a complex, concentration-dependent fashion and binding isotherms showed that the equilibrium between free and bound PAM is rapidly reached within 2 minutes of incubation in physiologically balanced salt solutions. In this time frame, trypomastigote cAMP levels are significantly reduced from steady state values within 30 seconds of the addition of PAM in a buffer system containing a diesterase inhibitor. Maximal attenuation of cAMP levels was measured between 1 and 2 minutes of the addition of PAM to T. cruzi trypomastigotes. The degree of cAMP level attenuation was reduced by blocking PAM attachment with either atropine or propranol. On the basis of these results we propose that a likely pathway for the negative parasite signal generated upon adhesion of host muscle cell membranes to the surface of the flagellates is from the parasite's surface attachment sites directly to a Pertussis toxin sensitive inhibitory protein Gi, thereby blunting adenyl cyclase activity and cAMP formation.

Modulation of Trypanosoma cruzi adhesion to host muscle cell membranes by ligands of muscarinic cholinergic and beta adrenergic receptors.

Plasma membrane vesicles (PMVs) of Trypanosoma cruzi adhered to L6 myoblast host cells as a function of time and concentration in saturation phenomena in a similar fashion to that reported in a previous publication. The initial adhesion rate (A0) of T. cruzi PMVs to L6 myoblasts in tissue culture was inhibited by acetylcholine (10(-5) M), isoproterenol (10(-5) M) and norepinephrine (10(-8) M) (range 29.1-50.3% of control). Atropine, the antagonist of muscarinic cholinergic receptors (10(-5) M), and propranolol or pindolol, the antagonists of beta-adrenergic receptors (10(-5) M), were also equal inhibitors of the T. cruzi PMV to L6 myoblast adhesion rate (range 26.1 to 55.5% of control). The alpha-adrenergic receptor ligands yohimbine and phentolamine (10(-5) M) showed no A0 inhibitory activity in similar assays. The interaction of T. cruzi PMVs with type I host muscle sarcolemma receptors was clearly defined in assays which used porcine heart atrial membranes (PAMs) immobilized on cationic polyacrylamide beads. In this parasite membrane-host cell membrane assay system, 10(-10) M atropine and 5 x 10(-9) M propranolol produced a shift of an S-shaped T. cruzi PMV to PAM saturation isotherm to the right, suggesting that a negative cooperative interaction was produced between the intramembrane ligand binding site and another, surface heterotropic T. cruzi PMV adhesion site. Atropine and propranolol were equieffective inhibitors of the T. cruzi striated muscle sarcolemma recognition process, raising the possibility that T. cruzi attachment molecules annexed pairs of muscarinic cholinergic and beta-adrenergic receptors to effect adhesion of the two membrane surfaces.