Large-conductance channel formation mediated by P2X7 receptor activation is regulated through distinct intracellular signaling pathways in peritoneal macrophages and 2BH4 cells.

The P2X(7) receptor (P2X7R) is a ligand-gated ATP receptor that acts as a low- and large-conductance channel (pore) and is known to be coupled to several downstream effectors. Recently, we demonstrated that the formation of a large-conductance channel associated with the P2X(7) receptor is induced by increasing the intracellular Ca(2+) concentration (Faria et al., Am J Physiol Cell Physiol 297:C28-C42, 2005). Here, we investigated the intracellular signaling pathways associated with P2X(7) large-conductance channel formation using the patch clamp technique in conjunction with fluorescent imaging and flow cytometry assays in 2BH4 cells and peritoneal macrophages. Different antagonists were applied to investigate the following pathways: Ca(2+)-calmodulin, phospholipase A, phospholipase D, phospholipase C, protein kinase C (PKC), mitogen-activated protein kinase (MAPK), MAPK/extracellular signal-regulated kinase, phosphoinositide 3-kinase (PI3K), and cytoskeletal proteins. Macroscopic ionic currents induced by 1 mM ATP were reduced by 85% in the presence of PKC antagonists. The addition of antagonists for MAPK, PI3K, and the cytoskeleton (actin, intermediary filament, and microtubule) blocked 92%, 83%, and 95% of the ionic currents induced by 1 mM ATP, respectively. Our results show that PKC, MAPK, PI3K, and cytoskeletal components are involved in P2X(7) receptor large-channel formation in 2BH4 cells and peritoneal macrophages.

Naphthoimidazoles promote different death phenotypes in Trypanosoma cruzi.

SUMMARY: In a screening of 65 derivatives of natural quinones using bloodstream trypomastigotes of Trypanosoma cruzi, the 3 naphthoimidazoles derived from beta-lapachone - N1, N2 and N3--were selected as the most active. Investigation of their mode of action led to the characterization of mitochondrion, reservosomes and DNA as their main targets, and stimulated further studies on death pathways. Ultrastructural analysis revealed both autophagic (autophagosomes) and apoptotic-like (membrane blebbing) phenotypes. Flow cytometry analysis showed, in N2-treated trypomastigotes, a small increase of phosphatidylserine exposure, and a large increase in the percentage of necrosis, caused by N1 or N2. These death phenotypes were not detected in treated epimastigotes. The strong increase in labelling of monodansyl cadaverine, the inhibition of the death process by wortmannin or 3-methyladenine, the overexpression of ATG genes in treated epimastigotes, together with ultrastructural evidence point to autophagy as the predominant phenotype induced by the naphthoimidazoles. However, there are other pathways occurring concomitantly with variable intensities, justifying the need to detail the molecular features involved.

P2X7 modulatory web in Trypanosoma cruzi infection.

P2X7 is a member of the purinergic receptors family, with extracellular adenosine triphosphate (ATP) as the main agonist, promoting cations influx and membrane permeabilization that can lead to cell death. We previously proposed that extracellular ATP is involved in thymus atrophy induced by Trypanosoma cruzi infection through the induction of CD4+/CD8+ double-positive cell death and that P2X7 could be involved in this process. To further elucidate this possibility raised by in vitro assays, in this study, we used P2X7-/- mice and observed no difference in thymus atrophy or parasitemia when compared to C57Bl/6. We then decided to investigate other aspects of purinergic receptor interplay that could be better evidenced by the infection and observed that (1) thymocytes from infected and noninfected C57Bl/6 mice express P2X4 and P2X7 receptors (Western blotting), but ATP-induced membrane permeabilization only occurs in thymocytes from infected mice; (2) peritoneal macrophages from noninfected C57Bl/6 mice (P2X4+ and P2X7+) are permeabilized by ATP. Although macrophages from infected C57Bl/6 mice are P2X7- but P2X4+, they are resistant to ATP, either through permeabilization or Ca++ influx (fluorimetry); (3) using noninfected P2X7-/- mice, C57Bl/6 infected mice, and different agonistic stimuli, we observed interesting cross-talks among P2X and P2Y receptors (flow cytometry).