Neuroglia interactions are essential for the nervous system and in the retina Müller cells intera... more Neuroglia interactions are essential for the nervous system and in the retina Müller cells interact with most of the neurons in a symbiotic manner. Glutathione (GSH) is a low-molecular weight compound that undertakes major antioxidant roles in neurons and glia, however, whether this compound could act as a signaling molecule in neurons and/or glia is currently unknown. Here we used embryonic avian retina to obtain mixed retinal cells or purified Müller glia cells in culture to evaluate calcium shifts induced by GSH. A dose response curve (0.1-10mM) showed that 5-10mM GSH, induced calcium shifts exclusively in glial cells (later labeled and identified as 2M6 positive cells), while neurons responded to 50mM KCl (labeled as βIII tubulin positive cells). BBG 100nM, a P2X7 blocker, inhibited the effects of GSH on Müller glia. However, addition of DNQX 70μM and MK-801 20μM, non-NMDA and NMDA blockers, had no effect on GSH calcium induced shift. Oxidized glutathione (GSSG) at 5mM failed to...
The ATP-gated P2X7R (P2X7R) is a channel, which is involved in events, such as inflammation, cell... more The ATP-gated P2X7R (P2X7R) is a channel, which is involved in events, such as inflammation, cell death, and pain. The most intriguing event concerning P2X7R functions is the phenomenon of pore dilation. Once P2X7R is activated, the permeability of the plasma membrane becomes higher, leading to the permeation of 1000 Da-weight solutes. The mechanisms involved in this process remain unclear. Nevertheless, this event is not exclusively through P2X7R, as other proteins may form large pores in the plasma membrane. Recent evidence concerning pore formation reveals putative P2X7R and other pores-associated protein complexes, revealing cross-interactive pharmacological and biophysical issues. In this work, we showed results that corroborated with cross-interactive aspects with P2X7R and pores in astrocytes. These cells expressed most of the pores, including P2X7R. We discovered that different pore types open with peculiar characteristics, as both anionic and cationic charged solutes permeate the plasma membrane, following P2X7R activation. Moreover, we showed that both synergic and additive relationships are found within P2X7, cationic, and anionic large pores. Therefore, our data suggest that other protein-related pores are assembled following the formation of P2X7R pore.
ATP and other nucleotides are released from cells through regulated pathways or following the los... more 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.
Since the nineteenth century, a great amount of different biological structures and processes hav... more Since the nineteenth century, a great amount of different biological structures and processes have been assessed by fluorescent dyes. Along with the uses of these compounds as vital and histological dyes, some fluorescent dyes have become valuable tools for the study of the pore phenomenon in plasma membranes. Some ion channels capable of forming large conductance channels, such as P2X7, TRPV1, VDAC-1 and the maxi-anion channels transiently alter the plasma membrane permeability, producing pores, which permit the passage of molecules of up to 1,000 Da. In this review, we discuss the uses of the fluorescent dyes chosen in diverse studies of this topic up to now.
Neuroscience-Dealing with Frontiers 46 2. Proteins that form large channels in mammal cells These... more Neuroscience-Dealing with Frontiers 46 2. Proteins that form large channels in mammal cells These large channels are distinguished by their proteins which can be classified in accordance with their function, mechanism for membrane penetration, the size of their pores, its pore-forming subunits, and type of pore-forming proteins within the membrane plane (-or). By comparing the protein configuration in the plasma membrane it is possible to actually evolutionarily delineate the pore-forming protein of distinct organisms. Proteins that form large conductance channels in the plasma membrane have been described in virus (Madan et al, 2007), bacteria (Huffman et al, 2004), fungi (Ojcius, D. M. & Young, J. D., 1991) and plantae (Klüsener & Weiler, 1999). Mostly due to these proteins being functionally linked to the mechanism of defense (plantae) or the invasion of the hosts (virus, bacteria, etc). These proteins are often secreted into the extracellular environment as monomers which then oligomerize and form the channel in the host membrane. In humans and other mammals the secreted proteins, there have been found the antimicrobial peptide families of defensins (
Nucleotides and nucleosides act as potent extracellular messengers via the activation of the fami... more Nucleotides and nucleosides act as potent extracellular messengers via the activation of the family of cell-surface receptors termed purinergic receptors. These receptors are categorized into P1 and P2 receptors (P2Rs). P2Rs are further classified into two distinct families, P2X receptors (P2XRs) and P2Y receptors (P2YRs). These receptors display broad tissue distribution throughout the body and are involved in several biological events. Immune cells express various P2Rs, and purinergic signaling mechanisms have been shown to play key roles in the regulation of many aspects of immune responses. Researchers have elucidated the involvement of these receptors in the host response to infections. The evidences indicate a dual function of these receptors, depending on the microorganism and the cellular model involved. Three recent reports have examined the relationship between the level of extracellular ATP, the mechanisms underlying purinergic receptors participating in the infection mechanism of HIV-1 in the cell. Although preliminary, these results indicate that purinergic receptors are putative pharmacological targets that should be further explored in future studies.
Natural products contribute significantly to available drug therapies and have been a rich source... more Natural products contribute significantly to available drug therapies and have been a rich source for scientific investigation. In general, due to their low cost and traditional use in some cultures, they are an object of growing interest as alternatives to synthetic drugs. With several diseases such as cancer, and inflammatory and neuropathic diseases having been linked to the participation of purinergic (P2) receptors, there has been a flurry of investigations on ligands within natural products. Thirty-four different sources of these compounds have been found so far, that have shown either agonistic or antagonistic effects on P2 receptors. Of those, nine different plant sources demonstrated effects on P2X2, P2X3, P2X7, and possibly P2Y12 receptor subtypes. Microorganisms, which represent the largest group, with 26 different sources, showed effects on both receptor subtypes, ranging from P2X1 to P2X4 and P2X7, and P2Y1, P2Y2, P2Y4, and P2Y6. In addition, there were seventeen animal sources that affected P2X7 and P2Y1 and P2Y12 receptors. Natural products have provided some fascinating new mechanisms and sources to better understand the P2 receptor antagonism. Moreover, current investigations should clarify further pharmacological mechanisms in order to consider these products as potential new medicines.
The P2X7 receptor (P2X7R) is a nonselective cation channel that is activated by extracellular ATP... more The P2X7 receptor (P2X7R) is a nonselective cation channel that is activated by extracellular ATP and triggers the secretion of several proinflammatory substances, such as IL-1β, IL-18, TNF-α, and nitric oxide. Recently, several preclinical studies have demonstrated that this receptor participates in inflammation and pain mechanisms. Taken together, these results indicate that P2X7R is a promising pharmacological target, and compounds that modulate the function of this receptor show potential as new anti-inflammatory medicines. In this review, we discuss aspects of P2X7R pharmacology and the participation of this protein in inflammation and pain and provide an overview of some promising compounds that have been tested as antagonists of P2X7R, with clinical applicability.
Naunyn-Schmiedeberg's Archives of Pharmacology, 2013
The P2X7 receptor (P2X7R), an ATP-gated cation channel, is expressed predominantly in leukocytes.... more 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.
Journal of bioenergetics and biomembranes, Aug 15, 2016
The second-generation photosensitizer methylene blue (MB) exhibits photochemical and photophysica... more The second-generation photosensitizer methylene blue (MB) exhibits photochemical and photophysical properties suitable for photodynamic therapy (PDT)-based cancer treatment. However, the clinical application of MB is limited because of its high hydrophilicity, which hinders its penetration into tumor tissues. Therefore, new methods to improve the entry of MB into the cytoplasm of target cells are necessary. Because MB has a mass of 319 Da, transient pores on the plasma membrane, such as the pore induced by the P2X7 receptor (P2X7R) that allows the passage of molecules up to 900 Da, could be used. Using MTT viability assays, flow cytometry experiments, and fluorescence microscopy, we evaluated the toxicity and phototoxicity of MB and potentiation effects of ATP and MB on cell death processes in the J774 cell line (via a P2X7-associated pore). We observed that treatment with 5 μM MB for 15 min promoted the rate of entry of MB into the cytoplasm to 4.7 %. However, treatment with 5 μM M...
Neuroglia interactions are essential for the nervous system and in the retina Müller cells intera... more Neuroglia interactions are essential for the nervous system and in the retina Müller cells interact with most of the neurons in a symbiotic manner. Glutathione (GSH) is a low-molecular weight compound that undertakes major antioxidant roles in neurons and glia, however, whether this compound could act as a signaling molecule in neurons and/or glia is currently unknown. Here we used embryonic avian retina to obtain mixed retinal cells or purified Müller glia cells in culture to evaluate calcium shifts induced by GSH. A dose response curve (0.1-10mM) showed that 5-10mM GSH, induced calcium shifts exclusively in glial cells (later labeled and identified as 2M6 positive cells), while neurons responded to 50mM KCl (labeled as βIII tubulin positive cells). BBG 100nM, a P2X7 blocker, inhibited the effects of GSH on Müller glia. However, addition of DNQX 70μM and MK-801 20μM, non-NMDA and NMDA blockers, had no effect on GSH calcium induced shift. Oxidized glutathione (GSSG) at 5mM failed to...
The ATP-gated P2X7R (P2X7R) is a channel, which is involved in events, such as inflammation, cell... more The ATP-gated P2X7R (P2X7R) is a channel, which is involved in events, such as inflammation, cell death, and pain. The most intriguing event concerning P2X7R functions is the phenomenon of pore dilation. Once P2X7R is activated, the permeability of the plasma membrane becomes higher, leading to the permeation of 1000 Da-weight solutes. The mechanisms involved in this process remain unclear. Nevertheless, this event is not exclusively through P2X7R, as other proteins may form large pores in the plasma membrane. Recent evidence concerning pore formation reveals putative P2X7R and other pores-associated protein complexes, revealing cross-interactive pharmacological and biophysical issues. In this work, we showed results that corroborated with cross-interactive aspects with P2X7R and pores in astrocytes. These cells expressed most of the pores, including P2X7R. We discovered that different pore types open with peculiar characteristics, as both anionic and cationic charged solutes permeate the plasma membrane, following P2X7R activation. Moreover, we showed that both synergic and additive relationships are found within P2X7, cationic, and anionic large pores. Therefore, our data suggest that other protein-related pores are assembled following the formation of P2X7R pore.
ATP and other nucleotides are released from cells through regulated pathways or following the los... more 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.
Since the nineteenth century, a great amount of different biological structures and processes hav... more Since the nineteenth century, a great amount of different biological structures and processes have been assessed by fluorescent dyes. Along with the uses of these compounds as vital and histological dyes, some fluorescent dyes have become valuable tools for the study of the pore phenomenon in plasma membranes. Some ion channels capable of forming large conductance channels, such as P2X7, TRPV1, VDAC-1 and the maxi-anion channels transiently alter the plasma membrane permeability, producing pores, which permit the passage of molecules of up to 1,000 Da. In this review, we discuss the uses of the fluorescent dyes chosen in diverse studies of this topic up to now.
Neuroscience-Dealing with Frontiers 46 2. Proteins that form large channels in mammal cells These... more Neuroscience-Dealing with Frontiers 46 2. Proteins that form large channels in mammal cells These large channels are distinguished by their proteins which can be classified in accordance with their function, mechanism for membrane penetration, the size of their pores, its pore-forming subunits, and type of pore-forming proteins within the membrane plane (-or). By comparing the protein configuration in the plasma membrane it is possible to actually evolutionarily delineate the pore-forming protein of distinct organisms. Proteins that form large conductance channels in the plasma membrane have been described in virus (Madan et al, 2007), bacteria (Huffman et al, 2004), fungi (Ojcius, D. M. & Young, J. D., 1991) and plantae (Klüsener & Weiler, 1999). Mostly due to these proteins being functionally linked to the mechanism of defense (plantae) or the invasion of the hosts (virus, bacteria, etc). These proteins are often secreted into the extracellular environment as monomers which then oligomerize and form the channel in the host membrane. In humans and other mammals the secreted proteins, there have been found the antimicrobial peptide families of defensins (
Nucleotides and nucleosides act as potent extracellular messengers via the activation of the fami... more Nucleotides and nucleosides act as potent extracellular messengers via the activation of the family of cell-surface receptors termed purinergic receptors. These receptors are categorized into P1 and P2 receptors (P2Rs). P2Rs are further classified into two distinct families, P2X receptors (P2XRs) and P2Y receptors (P2YRs). These receptors display broad tissue distribution throughout the body and are involved in several biological events. Immune cells express various P2Rs, and purinergic signaling mechanisms have been shown to play key roles in the regulation of many aspects of immune responses. Researchers have elucidated the involvement of these receptors in the host response to infections. The evidences indicate a dual function of these receptors, depending on the microorganism and the cellular model involved. Three recent reports have examined the relationship between the level of extracellular ATP, the mechanisms underlying purinergic receptors participating in the infection mechanism of HIV-1 in the cell. Although preliminary, these results indicate that purinergic receptors are putative pharmacological targets that should be further explored in future studies.
Natural products contribute significantly to available drug therapies and have been a rich source... more Natural products contribute significantly to available drug therapies and have been a rich source for scientific investigation. In general, due to their low cost and traditional use in some cultures, they are an object of growing interest as alternatives to synthetic drugs. With several diseases such as cancer, and inflammatory and neuropathic diseases having been linked to the participation of purinergic (P2) receptors, there has been a flurry of investigations on ligands within natural products. Thirty-four different sources of these compounds have been found so far, that have shown either agonistic or antagonistic effects on P2 receptors. Of those, nine different plant sources demonstrated effects on P2X2, P2X3, P2X7, and possibly P2Y12 receptor subtypes. Microorganisms, which represent the largest group, with 26 different sources, showed effects on both receptor subtypes, ranging from P2X1 to P2X4 and P2X7, and P2Y1, P2Y2, P2Y4, and P2Y6. In addition, there were seventeen animal sources that affected P2X7 and P2Y1 and P2Y12 receptors. Natural products have provided some fascinating new mechanisms and sources to better understand the P2 receptor antagonism. Moreover, current investigations should clarify further pharmacological mechanisms in order to consider these products as potential new medicines.
The P2X7 receptor (P2X7R) is a nonselective cation channel that is activated by extracellular ATP... more The P2X7 receptor (P2X7R) is a nonselective cation channel that is activated by extracellular ATP and triggers the secretion of several proinflammatory substances, such as IL-1β, IL-18, TNF-α, and nitric oxide. Recently, several preclinical studies have demonstrated that this receptor participates in inflammation and pain mechanisms. Taken together, these results indicate that P2X7R is a promising pharmacological target, and compounds that modulate the function of this receptor show potential as new anti-inflammatory medicines. In this review, we discuss aspects of P2X7R pharmacology and the participation of this protein in inflammation and pain and provide an overview of some promising compounds that have been tested as antagonists of P2X7R, with clinical applicability.
Naunyn-Schmiedeberg's Archives of Pharmacology, 2013
The P2X7 receptor (P2X7R), an ATP-gated cation channel, is expressed predominantly in leukocytes.... more 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.
Journal of bioenergetics and biomembranes, Aug 15, 2016
The second-generation photosensitizer methylene blue (MB) exhibits photochemical and photophysica... more The second-generation photosensitizer methylene blue (MB) exhibits photochemical and photophysical properties suitable for photodynamic therapy (PDT)-based cancer treatment. However, the clinical application of MB is limited because of its high hydrophilicity, which hinders its penetration into tumor tissues. Therefore, new methods to improve the entry of MB into the cytoplasm of target cells are necessary. Because MB has a mass of 319 Da, transient pores on the plasma membrane, such as the pore induced by the P2X7 receptor (P2X7R) that allows the passage of molecules up to 900 Da, could be used. Using MTT viability assays, flow cytometry experiments, and fluorescence microscopy, we evaluated the toxicity and phototoxicity of MB and potentiation effects of ATP and MB on cell death processes in the J774 cell line (via a P2X7-associated pore). We observed that treatment with 5 μM MB for 15 min promoted the rate of entry of MB into the cytoplasm to 4.7 %. However, treatment with 5 μM M...
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Papers by Leonardo Braga