Anael Viana Pinto Alberto

Pharmacological properties of a pore induced by raising intracellular Ca2

Recent studies on the P2X(7) receptor in 2BH4 cells and peritoneal macrophages have demonstrated that the raise in intracellular Ca(2+) 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(2+) 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 (HEK-293 and IT45-RI cells). In 2BH4 cells, the stimulation with ionomycin (5-10 microM) increased intracellular free Ca(2+) concentration and induced pore formation with conductance of 421 +/- 14 pS, half-time (t(1/2)) for ethidium bromide uptake of 118 +/- 17 s, and t(1/2) for Lucifer yellow of 122 +/- 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(2+) 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(2+) at the doses used by using lactate dehydrogenase release assay. In conclusion, increased concentration in intracellular Ca(+2) induces a novel membrane pore pharmacologically different from the P2X(7) associated pore and hemigap-junction pore.

Role of P2 Receptors as Modulators of Rat Eosinophil Recruitment in Allergic Inflammation.

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.

PHARMAVIRTUA: Educational Software for Teaching and Learning Basic Pharmacology.

information and communication technologies have become important tools for teaching scientific subjects such as anatomy and histology as well as other, nondescriptive subjects like physiology and pharmacology ([8][1]–[10][2]). Software has been used to facilitate the learning of specific concepts

An Improved Method for P2X7R Antagonist Screening

ATP physiologically activates the P2X7 receptor (P2X7R), a member of the P2X ionotropic receptor family. When activated by high concentrations of ATP (i.e., at inflammation sites), this receptor is capable of forming a pore that allows molecules of up to 900 Da to pass through. This receptor is upregulated in several diseases, particularly leukemia, rheumatoid arthritis and Alzheimers disease. A selective antagonist of this receptor could be useful in the treatment of P2X7R activation-related diseases. In the present study, we have evaluated several parameters using in vitro protocols to validate a high-throughput screening (HTS) method to identify P2X7R antagonists. We generated dose-response curves to determine the EC50 value of the known agonist ATP and the ICs50 values for the known antagonists Brilliant Blue G (BBG) and oxidized ATP (OATP). The values obtained were consistent with those found in the literature (0.7 ± 0.07 mM, 1.3-2.6 mM and 173-285 μM for ATP, BBG and OATP, respectively). The Z-factor, an important statistical tool that can be used to validate the robustness and suitability of an HTS assay, was 0.635 for PI uptake and 0.867 for LY uptake. No inter-operator variation was observed, and the results obtained using our improved method were reproducible. Our data indicate that our assay is suitable for the selective and reliable evaluation of P2X7 activity in multiwell plates using spectrophotometry-based methodology. This method might improve the high-throughput screening of conventional chemical or natural product libraries for possible candidate P2X7R antagonist or agonist

Single-cell Microinjection for Cell Communication Analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

Correction: An Improved Method for P2X7R Antagonist Screening.

There are errors in the seventh sentence of the Abstract. Specifically, the values provided for ATP, BBG and OATP are incorrect. The correct sentence is: The values obtained were consistent with those found in the literature (0.7 ± 0.07 mM, 1.3–2.6 μM and 173–285 μM for ATP, BBG and OATP, respectively). Additionally, there are errors in the units of Table 1. The correct units for columns BBG (IC50) and OATP (IC50) are μM. Please see the corrected Table 1 here. Table 1 IC50 or EC50 values for P2X7R agonists or antagonists using different methods.

Johrei Effects on Water: A Pilot Study by Counting Drops.

BACKGROUND Water is a key ingredient in the creation and sustainment of life. Moreover, water may be a key vehicle in the processes of energy healing, such as in the preparation of homeopathic remedies and spiritual treatments. Given these properties, the purpose of this study was to investigate whether the application of Johrei to water could lead to significant changes in the hydrodynamic behaviour of the fluid. METHODS Four regular Johrei practitioners (P1, P2, P3 and P4) were selected for this study. Dripping water produced at the tip of a capillary was used as the hydrodynamic behaviour model. This behaviour was modelled mathematically, and tuning parameters φ4 and τ were used to assess significant differences in the dripping water samples that were subjected to Johrei compared with the samples that were not so treated. The tuning parameters were obtained using the Levenberg-Marquardt fitting algorithm. The data sets for each Johrei practitioner and the control experiment were analysed using ANOVA and a paired t-test. RESULTS The mathematical model exhibited an excellent fit to our data, generating correlation coefficients (r) greater than or equal to 0.999. Significant differences were observed in both τ (P1 and P2, P < 0.05 and P < 0.01, respectively) and φ4 (P2, P < 0.01). As expected, no significant difference for the control experiment (without Johrei) was observed. CONCLUSIONS Our results indicated a statistically significant change in the hydrodynamic behaviour of water correlated with Johrei treatment for 50% of the participating Johrei practitioners.

P2R in Eosinophils and Possible Role in Migration

ATP and other nucleotides can be released from cells through regulated pathways or following the loss of plasma membrane integrity. These nucleotides act on P2 family of receptors that are divided in P2X ionotropic receptors and G protein-coupled P2Y receptors. Such receptors have been characterized in many rat immune cells, one exception are eosinophils which are involved in several pathological and physiological processes.The eosinophils were obtained from peritoneal lavage of wistar rats followed by a purification step of Metrizamide density-gradient centrifugation. Firstly, we have performed an immunofluorescence characterization using antibodies against P2XR and P2YR. The cells were positives for P2X 1,2,4 and 7 and [[Unsupported Character - Codename s]]P2Y 1,2 and 4. Our next step was to verify whether those receptors were functional using patch clamp recording which showed that ATP (1504±283 pA/pF)and ATPγS (1231±164 pA/pF) were the most potent agonists where the others elicited little (α,β me ATP, ADP, BzATP, β,γ me ATP, 2me SATP) or no response (UDP, cAMP, adenosine). After that we have tested the participation of these receptors in eosinophils migration in vitro (1 or 2h) using a transwell chamber in order to investigate their possible physiological role. ATP and other agonists were able to increase migration, an effect which could be blocked by suramin, a general blocker of P2R. In keeping with this idea, we tested whether they are implicated in the migration of eosinophils using an inflammation model of rat allergic pleurisy. Our results suggest an increase of eosinophils migration induced by ATP. Corroborating with the transwell results, suramin also blocked migration.As far as we are concerned, this study was the first to demonstrate that rat eosinophils express P2X and P2Y which can increase migration of eosinophils in vitro and in vivo.

Pharmacological Properties of a Pore Induced by Rising in Intracellular Ca2

AbstractRecent studies on the P2X7 receptor in 2BH4 cells and peritoneal macrophages have demonstrated that a rise in intracellular Ca2+ concentration induces a pore opening similar to P2X7 receptor pore. Herein, we have investigated whether the pore activated by rising in intracellular Ca2+ concentration is associated to P2X7 receptor. Using patch clamp in cell attached, whole cell configuration and dye uptake, we measured the pore opening in cell types that express the P2X7 receptor (2BH4 cells and peritoneal macrophages), and in cells that do not express this receptor (HEK-293 and IT45-RI cells). In 2BH4 cells, the stimulation with ionomycin (5-10μM) increased intracellular free Ca2+ concentration and induced pore formation with conductance of 421 ± 14 pS, t1/2 for ethidium bromide (EB) uptake of 118 ± 17 s, and t1/2 for Lucifer yellow (LY) of 122 ± 11 s. P2X7 receptor antagonists did not block this effect. Stimulation of HEK-293 and IT45-RI cells resulted in pore formation with properties similar to those found for 2BH4 cells. Connexin hemichannels inhibitors (carbenoxolone and heptanol) also did not inhibit the pore induced effect following rise in intracellular Ca2+ concentration. However, 5-(N,N-hexamethylene)- amiloride (HMA), a P2X7 receptor pore blocker, inhibited the induced pore. Moreover, intracellular signalling enzymes, such as calmodulin, phospholipase-C (PLC), mitogen-activated protein kinase (MAPK), 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 rise of intracellular Ca2+, at the doses used by using lactate dehydrogenase (LDH) release assay. In conclusion, increased mobilization of intracellular Ca+2 induces a novel membrane pore pharmacologically different from the P2X7 associated pore and hemichannel pore.