The P2X7 receptor (P2X7R), an ATP-gated cation channel, is expressed predominantly in leukocytes. Activation of P2X7R has been implicated in the formation of a cytolytic pore (i.e., a large conductance channel) that allows the passage of molecules up to 900 Da in macrophages. At least two hypotheses have been presented to explain the conversion of a nonselective cation channel to a cytolytic pore. One hypothesis suggests that the pore is a separate molecular structure activated by P2X7R, and the second asserts that this is an intrinsic property of P2X7R (pore dilation). Based on connexin knockout and hemichannel antagonist studies, some groups have concluded that connexins and pannexins, the hemichannel-forming proteins in vertebrates, are fundamental components of the large conductance channel associated with P2X7R. Dye uptake and electrophysiology experiments were used to evaluate the efficacy and specificity of some hemichannel antagonists under conditions known to open the large conductance channel associated with P2X7R. Hemichannel antagonists and interference RNA (RNAi) targeting pannexin-1 did not affect P2X7R macroscopic currents [ATP, 1,570±189 pA; ATP+100 μM carbenoxolone (CBX), 1,498±100 pA; ATP+1 mM probenecid (Prob), 1,522±9 pA] or dye uptake in a FACS assay (ATP, 63±5%; ATP+100 μM CBX, 51.51±8.4%; ATP+1 mM Prob, 57.7±4.3%) in mouse macrophages. These findings strongly suggest that the high-permeability pore evident after prolonged P2X7R activation does not occur through connexin or pannexin hemichannels in murine macrophages. Another membrane protein may be involved in P2X7R pore formation.

Photodynamic therapy (PDT) combines light, a photosensitizing chemical substance, and molecular oxygen to elicit cell death and is employed in the treatment of a variety of diseases, including cancer. The development of PDT treatment strategies requires in vitro assays to develop new photosensitizers. One such assay is the MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide developed in 1983 and widely used in PDT studies. Despite the exponential growth in the number of publications, a uniform MTT protocol for use in the PDT area is lacking. Herein, we list and standardize the conditions to evaluate the photosensitizer methylene blue (MB) in glioblastoma and neuroblastoma cell lines. In addition, we review technical pitfalls and identify several variables that must be taken into consideration in order to provide accurate results with MTT. We conclude that for each cell line we must have a dose-response curve using the MTT assay and good controls for the standardization. Additionally, the optimal values of the time and cell density must be in the linear range of the curve to avoid errors. We describe all relevant points and outline the best normalization techniques to observe the differences between treatments.

-Recent studies on the P2X 7 receptor in 2BH4 cells and peritoneal macrophages have demonstrated that the raise in intracellular Ca 2ϩ concentration induces a pore opening similar to P2X 7 receptor pore. Herein, we have investigated whether the pore activated by the elevation of intracellular Ca 2ϩ concentration is associated to P2X 7 receptor. Using patch clamp in cell attached, whole cell configuration, and dye uptake, we measured the pore opening in cell types that express the P2X 7 receptor (2BH4 cells and peritoneal macrophages) and in cells that do not express this receptor . In 2BH4 cells, the stimulation with ionomycin (5-10 M) increased intracellular free Ca 2ϩ concentration and induced pore formation with conductance of 421 Ϯ 14 pS, half-time (t 1 ⁄2) for ethidium bromide uptake of 118 Ϯ 17 s, and t 1 ⁄2 for Lucifer yellow of 122 Ϯ 11 s. P2X 7 receptor antagonists did not block these effects. Stimulation of HEK-293 and IT45-RI cells resulted in pore formation with properties similar to those found for 2BH4 cells. Connexin hemichannel inhibitors (carbenoxolone and heptanol) also did not inhibit the pore-induced effect following the increase in intracellular Ca 2ϩ concentration. However, 5-(N,N-hexamethylene)-amiloride, a P2X 7 receptor pore blocker, inhibited the induced pore. Moreover, intracellular signaling modulators, such as calmodulin, phospholipase C, mitogen-activated protein kinase, and cytoskeleton components were important for the pore formation. Additionally, we confirmed the results obtained for electrophysiology by using the flow cytometry, and we discarded the possibility of cellular death induced by raising intracellular Ca 2ϩ at the doses used by using lactate dehydrogenase release assay. In conclusion, increased concentration in intracellular Ca ϩ2 induces a novel membrane pore pharmacologically different from the P2X 7 associated pore and hemigap-junction pore.P2X 7 receptor; pore formation; second messenger P2X RECEPTORS are members of a family of P2 receptors with seven subtypes (P2X 1 -P2X 7 ) that induce an increase in intracellular Ca 2ϩ concentration when activated. Among the P2X receptors, P2X 7 subtype is the most divergent component of this family in terms of pharmacological properties, molecular structure, and function (54). This receptor forms a nonselective cation channel upon low ATP concentration, whereas at high ATP concentration, this receptor induces a pore formation that allows the flux of molecules of up to 900 Da and may lead to cell death. Although P2X 7 receptor is associated with expression of both functional channels and large pores, the structure of the large pore-forming subunits and whether the channel and the large pore are the same structure are still not known.From a physiological point of view, P2X 7 is mainly expressed in the immune system found in all cells of this system studied so far. The activation of P2X 7 receptor has several effects in the immune system such as release of mature interleukin-␤ and other pro-inflammatory cytokines (interleukin-18,...

ATP and other nucleotides are released from cells through regulated pathways or following the loss of plasma membrane integrity. Once outside the cell, these compounds can activate P2 receptors: P2X ionotropic receptors and G protein-coupled P2Y receptors. Eosinophils represent major effector cells in the allergic inflammatory response and they are, in fact, associated with several physiological and pathological processes. Here we investigate the expression of P2 receptors and roles of those receptors in murine eosinophils. In this context, our first step was to investigate the expression and functionality of the P2X receptors by patch clamping, our results showed a potency ranking order of ATP>ATPγS> 2meSATP> ADP> αβmeATP> βγmeATP>BzATP> UTP> UDP>cAMP. This data suggest the presence of P2X1, P2X2 and P2X7. Next we evaluate by microfluorimetry the expression of P2Y receptors, our results based in the ranking order of potency (UTP>ATPγS> ATP > UDP> ADP >2meSATP > αβmeATP) suggests the presence of P2Y2, P2Y4, P2Y6 and P2Y11. Moreover, we confirmed our findings by immunofluorescence assays. We also did chemotaxis assays to verify whether nucleotides could induce migration. After 1 or 2 hours of incubation, ATP increased migration of eosinophils, as well as ATPγS, a less hydrolysable analogue of ATP, while suramin a P2 blocker abolished migration. In keeping with this idea, we tested whether these receptors are implicated in the migration of eosinophils to an inflammation site in vivo, using a model of rat allergic pleurisy. In fact, migration of eosinophils has increased when ATP or ATPγS were applied in the pleural cavity, and once more suramin blocked this effect. We have demonstrated that rat eosinophils express P2X and P2Y receptors. In addition, the activation of P2 receptors can increase migration of eosinophils in vitro and in vivo, an effect blocked by suramin.

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