Inhibition Of P2X Receptors Research Articles

Therapeutic potentials and structure-activity relationship of 1,3-benzodioxole N-carbamothioyl carboxamide derivatives as selective and potent antagonists of P2X4 and P2X7 receptors

P2X receptors (P2XRs) are ligand-gated membrane ion channels. ATP binds to open these ion channels and results in membrane depolarization. Hyperactivities and overexpression of P2XRs are related to various pathophysiological conditions such as chronic pain, inflammatory diseases, rheumatoid arthritis, various neurological disorders, and cancer. Inhibition of P2XRs is a potential drug target that is an emerging therapeutic tool for disease management. In the present study, 17 new compounds were synthesized based on 1,3-benzodioxole-5-carboxylic acid and were investigated for P2XRs inhibition. Ca2+ influx assay was performed on P2XRs expressed h-1321N1 astrocytoma cell lines. The synthesized compounds exhibited high potency and selectivity towards h-P2X4R and h-P2X7R. In silico studies were carried out that began with the development of a homology model for h-P2X7R with subsequent molecular docking studies of the most potent P2XRs antagonists. 9o (N-((2-bromo-4-isopropylphenyl)carbamothioyl)benzo[d] [1,3]dioxole-5-carboxamide) was found to have significant inhibitory potential and selectivity for h-P2X4R with an IC50 ± SEM of 0.039 ± 0.07 μM. Whereas, 9q (N-(quinolin-8-ylcarbamothioyl)benzo[d] [1,3]dioxole-5-carboxamide) was selective and most potent antagonist for h-P2X7R with an IC50 ± SEM of 0.018 ± 0.06 μM. Both antagonists, 9o and 9q, exhibited a non-competitive negative allosteric mode of antagonism.

Connexin-43-Hemichannel-Mediated ATP Efflux Triggers Arrhythmogenic CA2+ Waves via P2X Purinoceptor Current in Atrial Myocytes

Myocardium is subjected to mechanical stresses and adapts to them. Atrial myocytes are exposed to high shear stress during hemodynamic overload and blood regurgitation. We have previously shown evidence that shear stress (∼16 dyn/cm2)-induced atrial global Ca2+ waves are abolished by the blockades of ATP release, gap junction hemichannel or P2 purinergic signaling. In this study, we assessed activation of gap junction hemichannels by shear stress, and its role in induction of arrhythmogenic currents and in proarrhythmic Ca2+ waves in rat atrial myocytes. Calcein dye efflux, but not oregon flux, was accelerated by shear application. The shear-induced calcein efflux was enhanced by zero external Ca2+, and was suppressed by La3+, but not by probenecid, suggesting activation of connexins by shear stress. Shear stress produced inward cation (Cs+) currents at resting potentials, and this current was completely suppressed by La3+ or carbenoxolone, and was enhanced by quinine. The current was also partly suppressed by P2X receptor inhibition (by ∼50%) or by blockade of P2Y1 receptor/transient receptor potential melastatin subfamily 4 (by ∼30%). Shear-induced NMDG+ current was one-fifth of the Cs+ current, showed linear voltage-dependence with a reversal at 0 mV and was eliminated by introduction of anti-connexin-43 antibodies. Shear-induced ATP release from a monolayer of atrial cells, assessed by chemiluninescence, was abolished by either connexin-43 hemichannel inhibitor Gap 19 or connexin-43 knock-down. Simultaneous measurement of ATP release and Ca2+ images using a sniffer patch clamp and two-dimensional confocal microscopy, respectively, further revealed that ATP releases occurred at 200-300-ms prior to the onset of the Ca2+ waves under shear stress and were sustained under prolonged shear stimulus. Our data suggest that shear stress induces connexin-43-hemichannel-mediated ATP release, thereby initiating P2X purinoceptor-dependent triggered Ca2+ waves in atrial myocytes.

P2X1, P2X4, and P2X7 Receptor Knock Out Mice Expose Differential Outcome of Sepsis Induced by α-Haemolysin Producing Escherichia coli.

α-haemolysin (HlyA)-producing Escherichia coli commonly inflict severe urinary tract infections, including pyelonephritis, which comprises substantial risk for sepsis. In vitro, the cytolytic effect of HlyA is mainly mediated by ATP release through the HlyA pore and subsequent P2X1/P2X7 receptor activation. This amplification of the lytic process is not unique to HlyA but is observed by many other pore-forming proteins including complement-induced haemolysis. Since free hemoglobin in the blood is known to be associated with a worse outcome in sepsis one could speculate that inhibition of P2X receptors would ameliorate the course of sepsis. Surprisingly, this study demonstrates that and mice are exceedingly sensitive to sepsis with uropathogenic E. coli. These mice have markedly lower survival, higher cytokine levels and activated intravascular coagulation. Quite the reverse is seen in mice, which had markedly lower cytokine levels and less coagulation activation compared to controls after exposure to uropathogenic E. coli. The high cytokine levels in the mouse are unexpected, since P2X7 is implicated in caspase-1-dependent IL-1β production. Here, we demonstrate that IL-1β production during sepsis with uropathogenic E. coli is mediated by caspase-8, since caspase-8 and RIPK3 double knock out mice show substantially lower cytokine during sepsis and increased survival after injection of TNFα. These data support that P2X7 and P2X4 receptor activation has a protective effect during severe E. coli infection.

Effect of type II diabetes on male rat bladder contractility.

Type II diabetes is the most prevalent form of diabetes. One of the primary complications of diabetes that significantly affects quality of life is bladder dysfunction. Many studies on diabetic bladder dysfunction have been performed in models of type I diabetes; however, few have been performed in animal models of type II diabetes. Using the Zucker Diabetic Fatty (ZDF) rat model of type II diabetes, we examined the contractility and sensitivity of bladder smooth muscle in response to mediators of depolarization-induced contraction, muscarinic receptor-mediated contraction, ATP-induced contraction, and neurogenic contraction. Studies were performed at 16 and 27 wk of age to monitor the progression of diabetic bladder dysfunction. Voiding behavior was also quantified. The entire bladder walls of diabetic rats were hypertrophied compared with that of control rats. Contractility and sensitivity to carbachol and ATP were increased at 27 wk in bladder smooth muscle strips from diabetic rats, suggesting a compensated state of diabetic bladder dysfunction. Purinergic signaling was increased in response to exogenous ATP in bladders from diabetic animals; however, the purinergic component of neurogenic contractions was decreased. The purinergic component of neurogenic contraction was reduced by P2X receptor desensitization, but was unchanged by P2X receptor inhibition in diabetic rats. Residual and tetrodotoxin-resistant components of neurogenic contraction were increased in bladder strips from diabetic animals. Overall, our results suggest that in the male ZDF rat model, the bladder reaches the compensated stage of function by 27 wk and has increased responsiveness to ATP.

P2X receptor‐dependent erythrocyte damage by α‐hemolysin from Escherichia coli triggers phagocytosis by THP‐1 cells

The pore‐forming exotoxin α‐hemolysin from E. coli causes a significant volume reduction of human erythrocytes that precedes the ultimate swelling and lysis. This shrinkage results from activation of Ca2+‐sensitive K+ (KCa3.1) and Cl− channels (TMEM16A) and reduced functions of both of these channels potentiate the HlyA‐induced haemolysis. This means that Ca2+‐ dependent activation of KCa3.1 and TMEM16A protects the cells against early haemolysis. Simultaneous to the HlyA‐induced shrinkage the erythrocytes show increased exposure of phosphatidyl‐serine (PS) in the outer plasma membrane leaflet, which is known to be a keen trigger for phagocytosis.We hypothesize that exposure to HlyA elicits removal of the damaged erythrocytes by phagocytic cells. Cultured THP‐1 cells as a model for erythrocytal phagocytosis was verified by a variety of methods including live cell imaging. We consistently found the HlyA to very potently trigger phagocytosis of erythrocytes by THP‐1 cells. The HlyA‐induced phagocytosis was prevented by inhibition of KCa3.1, which is known to reduce PS‐exposure in human erythrocytes exposed to both ionomycin and HlyA. Moreover, we show that the P2X receptor inhibition, which prevents the cell damages caused by HlyA, also reduced that HlyA‐induced PS‐exposure and phagocytosis. Based on these results, we propose that erythrocytes, damaged by HlyA‐insertion, are effectively cleared from the blood stream. This mechanism will potentially reduce the risk of intravascular hemolysis

P2X Receptor-Dependent Erythrocyte Damage by α-Hemolysin from Escherichia coli Triggers Phagocytosis by THP-1 Cells

The pore-forming exotoxin α-hemolysin from E. coli causes a significant volume reduction of human erythrocytes that precedes the ultimate swelling and lysis. This shrinkage results from activation of Ca2+-sensitive K+ (KCa3.1) and Cl− channels (TMEM16A) and reduced functions of either of these channels potentiate the HlyA-induced hemolysis. This means that Ca2+-dependent activation of KCa3.1 and TMEM16A protects the cells against early hemolysis. Simultaneous to the HlyA-induced shrinkage, the erythrocytes show increased exposure of phosphatidylserine (PS) in the outer plasma membrane leaflet, which is known to be a keen trigger for phagocytosis. We hypothesize that exposure to HlyA elicits removal of the damaged erythrocytes by phagocytic cells. Cultured THP-1 cells as a model for erythrocytal phagocytosis was verified by a variety of methods, including live cell imaging. We consistently found the HlyA to very potently trigger phagocytosis of erythrocytes by THP-1 cells. The HlyA-induced phagocytosis was prevented by inhibition of KCa3.1, which is known to reduce PS-exposure in human erythrocytes subjected to both ionomycin and HlyA. Moreover, we show that P2X receptor inhibition, which prevents the cell damages caused by HlyA, also reduced that HlyA-induced PS-exposure and phagocytosis. Based on these results, we propose that erythrocytes, damaged by HlyA-insertion, are effectively cleared from the blood stream. This mechanism will potentially reduce the risk of intravascular hemolysis.

Leukotoxin fromAggregatibacter actinomycetemcomitanscauses shrinkage and P2X receptor-dependent lysis of human erythrocytes

Leukotoxin (LtxA) is a virulence factor secreted by the bacterium Aggregatibacter actinomycetemcomitans, which can cause localized aggressive periodontitis and endocarditis. LtxA belongs to the repeat-in-toxin (RTX) family of exotoxins of which other members inflict lysis by formation of membrane pores. Recently, we documented that the haemolytic process induced by another RTX toxin [α-haemolysin (HlyA) from Escherichia coli] requires P2X receptor activation and consists of sequential cell shrinkage and swelling. In contrast, the cellular and molecular mechanisms of LtxA-mediated haemolysis are not fully understood. Here, we investigate the effect of LtxA on erythrocyte volume and whether P2 receptors also play a part in LtxA-mediated haemolysis. We observed that LtxA initially decreases the cell size, followed by a gradual rise in volume until the cell finally lyses. Moreover, LtxA triggers phosphatidylserine (PS) exposure in the erythrocyte membrane and both the shrinkage and the PS-exposure is preceded by increments in the intracellular Ca(2+) concentration ([Ca(2+)](i)). Interestingly, LtxA-mediated haemolysis is significantly potentiated by ATP release and P2X receptor activation in human erythrocytes. Furthermore, the LtxA-induced [Ca(2+)](i) increase and following volume changes partially depend on P2 receptor activation. Theseobservations imply that intervention against local P2-mediated auto- and paracrine signalling may prevent LtxA-mediated cell damage.

492 CAVEOLAE-MEDIATED REGULATION OF PURINERGIC SIGNALING IN THE BLADDER SUPPRESSES SPONTANEOUS ACTIVITY

You have accessJournal of UrologyBladder and Urethra: Anatomy, Physiology and Pharmacology II1 Apr 2012492 CAVEOLAE-MEDIATED REGULATION OF PURINERGIC SIGNALING IN THE BLADDER SUPPRESSES SPONTANEOUS ACTIVITY Vivian Cristofaro, Subbarao Yalla, and Maryrose Sullivan Vivian CristofaroVivian Cristofaro Boston, MA More articles by this author , Subbarao YallaSubbarao Yalla Boston, MA More articles by this author , and Maryrose SullivanMaryrose Sullivan Boston, MA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.562AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bladder smooth muscle (BSM) caveolae, membrane domains that modulate responses to physiologic stimuli, are reduced in animal models of DO. These animals exhibit increased bladder spontaneous activity (SA) and altered purinergic signaling. In this study, we investigated whether caveolae-mediated regulation of the purinergic system plays a role in maintaining the level of SA in the bladder. METHODS Rat bladder strips without mucosa were mounted in organ bath. SA and responses induced by αβmeATP were measured before and after caveolae depletion (achieved by methyl-β-cyclodextrin, mβCD). Changes in SA were also determined after P2X receptor inhibition (NF449, NF279, PPADS, or after αβmeATP-desensitization) and in the presence of ivermectin (IVC, a P2X4 positive modulator) or BDBD (P2X4 antagonist), or after the connexin (Cx) hemichannel blocker, carbenoxolone (CBX). Differences among these treatments in levels of ATP released either spontaneously or upon stimulation (induced by KCl in BSM tissue and hypotonic Krebs exposure in BSM cells) were determined by luciferase assay. P2X4 receptor expression in BSM tissue and the interaction of caveolin proteins with P2X4 receptor and Cx43 were determined by western blot and co-immunoprecipitation. RESULTS Depletion of caveolae inhibited P2X-mediated contractile responses and augmented SA in BSM tissue, and increased basal ATP release from BSM cells. SA and ATP levels in BSM tissue were also increased by inhibiting P2X receptors and by exposure to IVC. BDBD prevented the augmentation in SA caused by P2X receptor inhibition and the KCl-induced ATP release from BSM tissue. Similarly, CBX attenuated the increase in ATP induced by mβCD or by hypotonic Krebs in BSM cells, and prevented the KCl-induced ATP release from BSM tissue. P2X4 and Cx43 were expressed in BSM tissue and co-precipitated with caveolin proteins. CONCLUSIONS The impaired regulation of purinergic signaling and the ATP-induced increase in SA after mβCD is consistent with enhanced SA in animal models of DO in which caveolae are reduced, suggesting a role for caveolae in restraining SA. Loss of caveolae exposed an excitatory component, potentially mediated by P2X4 receptor activation, which was prevented by CBX, suggesting that SA levels are modulated by the effects of endogenous ATP released from BSM cells through Cx hemichannels. The molecular interaction of Cx43 with caveolin proteins supports the concept that hemichannel-mediated ATP release is suppressed by caveolae. Therefore, loss of BSM caveolae may cause dysregulation of this mechanism and lead to DO. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e201 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Vivian Cristofaro Boston, MA More articles by this author Subbarao Yalla Boston, MA More articles by this author Maryrose Sullivan Boston, MA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Neuroprotective effects of inhibiting N-methyl- d-aspartate receptors, P2X receptors and the mitogen-activated protein kinase cascade: A quantitative analysis in organotypical hippocampal slice cultures subjected to oxygen and glucose deprivation

Cell death was assessed by quantitative analysis of propidium iodide uptake in rat hippocampal slice cultures transiently exposed to oxygen and glucose deprivation, an in vitro model of brain ischemia. The hippocampal subfields CA1 and CA3, and fascia dentata were analyzed at different stages from 0 to 48 h after the insult. Cell death appeared at 3 h and increased steeply toward 12 h. Only a slight additional increase in propidium iodide uptake was seen at later intervals. The mitogen-activated protein kinases extracellular signal-regulated kinase 1 and extracellular signal-regulated kinase 2 were activated immediately after oxygen and glucose deprivation both in CA1 and in CA3/fascia dentata. Inhibition of the specific mitogen-activated protein kinase activator mitogen-activated protein kinase kinase by PD98059 or U0126 offered partial protection against oxygen and glucose deprivation-induced cell damage. The non-selective P2X receptor antagonist suramin gave neuroprotection of the same magnitude as the N-methyl- d-aspartate channel blocker MK-801 (∼70%). Neuroprotection was also observed with the P2 receptor blocker PPADS. Immunogold data indicated that hippocampal slice cultures (like intact hippocampi) express several isoforms of P2X receptors at the synaptic level, consistent with the idea that the effects of suramin and PPADS are mediated by P2X receptors. Virtually complete neuroprotection was obtained by combined blockade of N-methyl- d-aspartate receptors, P2X receptors, and mitogen-activated protein kinase kinase. Both P2X receptors and N-methyl- d-aspartate receptors mediate influx of calcium. Our results suggest that inhibition of P2X receptors has a neuroprotective potential similar to that of inhibition of N-methyl- d-aspartate receptors. In contrast, our comparative analysis shows that only partial protection can be achieved by inhibiting the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase cascade, one of the downstream pathways activated by intracellular calcium overload.