P2X3 receptor antagonism attenuates the progression of heart failure.

Despite advances in the treatment of heart failure, prognosis is poor, mortality high and there remains no cure. Heart failure is associated with reduced cardiac pump function, autonomic dysregulation, systemic inflammation and sleep-disordered breathing; these morbidities are exacerbated by peripheral chemoreceptor dysfunction. We reveal that in heart failure the carotid body generates spontaneous, episodic burst discharges coincident with the onset of disordered breathing in male rats. Purinergic (P2X3) receptors were upregulated two-fold in peripheral chemosensory afferents in heart failure, and when antagonized abolished these episodic discharges, normalized both peripheral chemoreceptor sensitivity and the breathing pattern, reinstated autonomic balance, improved cardiac function, and reduced both inflammation and biomarkers of cardiac failure. Aberrant ATP transmission in the carotid body triggers episodic discharges that via P2X3 receptors play a crucial role in the progression of heart failure and as such offer a distinct therapeutic angle to reverse multiple components of its pathogenesis.

TNF-α enhances sensory DRG neuron excitability through modulation of P2X3 receptors in an acute colitis model.

Previous studies have demonstrated that acute colonic inflammation leads to an increase in dorsal root ganglia (DRG) neuronal excitability. However, the signaling elements implicated in this hyperexcitability have yet to be fully unraveled. Extracellular adenosine 5'-triphosphate (ATP) is a well-recognized sensory signaling molecule that enhances the nociceptive response after inflammation through activation of P2X3 receptors, which are expressed mainly by peripheral sensory neurons. The aim of this study is to continue investigating how P2X3 affects neuronal hypersensitivity in an acute colitis animal model. To achieve this, DNBS (Dinitrobenzene sulfonic acid; 200 mg/kg) was intrarectally administered to C57BL/6 mice, and inflammation severity was assessed according to the following parameters: weight loss, macroscopic and microscopic scores. Perforated patch clamp technique was used to evaluate neuronal excitability via measuring changes in rheobase and action potential firing in T8-L1 DRG neurons. A-317491, a well-established potent and selective P2X3 receptor antagonist, served to dissect their contribution to recorded responses. Protein expression of P2X3 receptors in DRG was evaluated by western blotting and immunofluorescence. Four days post-DNBS administration, colons were processed for histological analyses of ulceration, crypt morphology, goblet cell density, and immune cell infiltration. DRG neurons from DNBS-treated mice were significantly more excitable compared with controls; these changes correlated with increased P2X3 receptor expression. Furthermore, TNF-α mRNA expression was also significantly higher in inflamed colons compared to controls. Incubation of control DRG neurons with TNF-α resulted in similar cell hyperexcitability as measured in DNBS-derived neurons. The selective P2X3 receptor antagonist, A-317491, blocked the TNF-α-induced effect. These results support the hypothesis that TNF-α enhances colon-innervating DRG neuron excitability via modulation of P2X3 receptor activity.

Photobiomodulation inhibits inflammation in the temporomandibular joint of rats.

Photobiomodulation (PBM) has been applied as a non-invasive technique for treating temporomandibular joint symptoms, especially on painful condition's relief, however the anti-inflammatory mechanism underlying the effect of PBM remains uncertain. This study aims to evaluate the mechanisms of action of PBM (808 nm) in a carrageenan-induced inflammation on temporomandibular joint (TMJ) of rats. In this study male Wistar rats were pre-treated with irradiation of a low-power diode laser for 15 s on TMJ (infra-red 808 nm, 100 mW, 50 J/cm2 and 1.5 J) 15 min prior an injection in the temporomandibular joint of carrageenan (100 µg/TMJ). 1 h after the TMJ treatments, the rats were terminally anesthetized for joint cavity wash and periarticular tissues collect. Samples analysis demonstrated that PBM inhibit leukocytes chemotaxis in the TMJ and significantly reduces amounts of TNF-α, IL-1ß and CINC-1. In addition, Western blotting analysis demonstrated that PBM significantly decreased the protein levels of P2X3 and P2X7 receptors in the periarticular tissues. On the other hand, PBM was able to increase protein level of IL-10 (anti-inflammatory cytokine). In summary, it is possible to suggest that PBM inhibit inflammatory chemotaxis, modulation the balance of the pro- and anti-inflammatory characteristics of inflammatory cells.

P2X3 receptors contribute to transition from acute to chronic muscle pain.

This study aimed to evaluate whether the development and/or maintenance of chronic-latent muscle hyperalgesia is modulated by P2X3 receptors. We also evaluate the expression of P2X3 receptors and PKCε of dorsal root ganglions during these processes. A mouse model of chronic-latent muscle hyperalgesia, induced by carrageenan and evidenced by PGE2, was used. Mechanical muscle hyperalgesia was measured by Randall-Selitto analgesimeter. The involvement of P2X3 receptors was analyzed by using the selective P2X3 receptors antagonist A-317491 by intramuscular or intrathecal injections. Expression of P2X3 and PKCε in dorsal root ganglion (L4-S1) were evaluated by Western blotting. Intrathecal blockade of P2X3 receptors previously to carrageenan prevented the development and maintenance of acute and chronic-latent muscle hyperalgesia, while intramuscular blockade of P2X3 receptors previously to carrageenan only reduced the acute muscle hyperalgesia and had no effect on chronic-latent muscle hyperalgesia. Intrathecal, but not intramuscular, blockade of P2X3 receptors immediately before PGE2, in animals previously sensitized by carrageenan, reversed the chronic-latent muscle hyperalgesia. There was an increase in total and phosphorylated PKCε 48 h after the beginning of acute muscle hyperalgesia, and in P2X3 receptors at the period of chronic muscle hyperalgesia. P2X3 receptors expressed on spinal cord dorsal horn contribute to transition from acute to chronic muscle pain. We also suggest an interaction of PKCε and P2X3 receptors in this process. Therefore, we point out P2X3 receptors of the spinal cord dorsal horn as a pharmacological target to prevent the development or reverse the chronic muscle pain conditions.

P2X3 receptor antagonism reduces the occurrence of apnoeas in newborn rats.

Hyperreflexia of the peripheral chemoreceptors is a potential contributor of apnoeas of prematurity (AoP). Recently, it was shown that elevated P2X3 receptor expression was associated with elevated carotid body afferent sensitivity. Therefore, we tested whether P2X3 receptor antagonism would reduce AoP known to occur in newborn rats. Unrestrained whole-body plethysmography was used to record breathing and from this the frequency of apnoeas at baseline and following administration of either a P2X3 receptor antagonist - AF-454 (5 mg/kg or 10 mg/kg s.c.) or vehicle was derived. In a separate group, we tested the effects of AF-454 (10 mg/kg) on the hypoxic ventilatory response (10 % FiO2). Ten but not 5 mg/kg AF-454 reduced the frequency of AoP and improved breathing regularity significantly compared to vehicle. Neither AF-454 (both 5 and 10 mg/kg) nor vehicle affected baseline respiration. However, P2X3 receptor antagonism (10 mg/kg) powerfully blunted hypoxic ventilatory response to 10 % FiO2. These data suggest that P2X3 receptors contribute to AoP and the hypoxic ventilatory response in newborn rats but play no role in the drive to breathe at rest.

P2X3 receptors contribute to muscle pain induced by static contraction by a mechanism dependent on neutrophil migration.

P2X3 receptors are involved with several pain conditions. Muscle pain induced by static contraction has an important socioeconomic impact. Here, we evaluated the involvement of P2X3 receptors on mechanical muscle hyperalgesia and neutrophil migration induced by static contraction in rats. Also, we evaluated whether static contraction would be able to increase muscle levels of TNF-α and IL-1ß. Male Wistar rats were pretreated with the selective P2X3 receptor antagonist, A-317491, by intramuscular or intrathecal injection and the static contraction-induced mechanical muscle hyperalgesia was evaluated using the Randall-Selitto test. Neutrophil migration was evaluated by measurement of myeloperoxidase (MPO) kinetic-colorimetric assay and the cytokines TNF-α and IL-1ß by enzyme-linked immunosorbent assay. Intramuscular or intrathecal pretreatment with A-317491 prevented static contraction-induced mechanical muscle hyperalgesia. In addition, A-317491 reduced static contraction-induced mechanical muscle hyperalgesia when administered 30 and 60 min of the beginning of static contraction, but not after 30 and 60 min of the end of static contraction. Intramuscular A-317491 also prevented static contraction-induced neutrophil migration. In a period of 24 h, static contraction did not increase muscle levels of TNF-α and IL-1ß. These findings demonstrated that mechanical muscle hyperalgesia and neutrophil migration induced by static contraction are modulated by P2X3 receptors expressed on the gastrocnemius muscle and spinal cord dorsal horn. Also, we suggest that P2X3 receptors are important to the development but not to maintenance of muscle hyperalgesia. Therefore, P2X3 receptors can be pointed out as a target to musculoskeletal pain conditions induced by daily or work-related activities.

Variable transcriptional responsiveness of the P2X3 receptor gene during CFA-induced inflammatory hyperalgesia.

The purinergic receptor P2X3 (P2X3-R) plays important roles in molecular pathways of pain, and reduction of its activity or expression effectively reduces chronic inflammatory and neuropathic pain sensation. Inflammation, nerve injury, and cancer-induced pain can increase P2X3-R mRNA and/or protein levels in dorsal root ganglia (DRG). However, P2X3-R expression is unaltered or even reduced in other pain studies. The reasons for these discrepancies are unknown and might depend on the applied traumatic intervention or on intrinsic factors such as age, gender, genetic background, and/or epigenetics. In this study, we sought to get insights into the molecular mechanisms responsible for inflammatory hyperalgesia by determining P2X3-R expression in DRG neurons of juvenile male rats that received a Complete Freund's Adjuvant (CFA) bilateral paw injection. We demonstrate that all CFA-treated rats showed inflammatory hyperalgesia, however, only a fraction (14-20%) displayed increased P2X3-R mRNA levels, reproducible across both sides. Immunostaining assays did not reveal significant increases in the percentage of P2X3-positive neurons, indicating that increased P2X3-R at DRG somas is not critical for inducing inflammatory hyperalgesia in CFA-treated rats. Chromatin immunoprecipitation (ChIP) assays showed a correlated (R2 = 0.671) enrichment of the transcription factor Runx1 and the epigenetic active mark histone H3 acetylation (H3Ac) at the P2X3-R gene promoter in a fraction of the CFA-treated rats. These results suggest that animal-specific increases in P2X3-R mRNA levels are likely associated with the genetic/epigenetic context of the P2X3-R locus that controls P2X3-R gene transcription by recruiting Runx1 and epigenetic co-regulators that mediate histone acetylation.

Cyclin-dependent kinase 5 modulates the P2X2a receptor channel gating through phosphorylation of C-terminal threonine 372.

The purinergic P2X2 receptor (P2X2R) is an adenosine triphosphate-gated ion channel widely expressed in the nervous system. Here, we identified a putative cyclin-dependent kinase 5 (Cdk5) phosphorylation site in the full-size variant P2X2aR (TPKH), which is absent in the splice variant P2X2bR. We therefore investigated the effects of Cdk5 and its neuronal activator, p35, on P2X2aR function. We found an interaction between P2X2aR and Cdk5/p35 by co-immunofluorescence and co-immunoprecipitation in HEK293 cells. We also found that threonine phosphorylation was significantly increased in HEK293 cells co-expressing P2X2aR and p35 as compared to cells expressing only P2X2aR. Moreover, P2X2aR-derived peptides encompassing the Cdk5 consensus motif were phosphorylated by Cdk5/p35. Whole-cell patch-clamp recordings indicated a delay in development of use-dependent desensitization (UDD) of P2X2aR but not of P2X2bR in HEK293 cells co-expressing P2X2aR and p35. In Xenopus oocytes, P2X2aRs showed a slower UDD than in HEK293 cells and Cdk5 activation prevented this effect. A similar effect was found in P2X2a/3R heteromeric currents in HEK293 cells. The P2X2aR-T372A mutant was resistant to UDD. In endogenous cells, we observed similar distribution between P2X2R and Cdk5/p35 by co-localization using immunofluorescence in primary culture of nociceptive neurons. Moreover, co-immunoprecipitation experiments showed an interaction between Cdk5 and P2X2R in mouse trigeminal ganglia. Finally, endogenous P2X2aR-mediated currents in PC12 cells and P2X2/3R mediated increases of intracellular Ca in trigeminal neurons were Cdk5 dependent, since inhibition with roscovitine accelerated the desensitization kinetics of these responses. These results indicate that the P2X2aR is a novel target for Cdk5-mediated phosphorylation, which might play important physiological roles including pain signaling.

Antihyperalgesic effect of CB1 receptor activation involves the modulation of P2X3 receptor in the primary afferent neuron.

Cannabinoid system is a potential target for pain control. Cannabinoid receptor 1 (CB1) activation play a role in the analgesic effect of cannabinoids once it is expressed in primary afferent neurons. This study investigates whether the anti-hyperalgesic effect of CB1 receptor activation involves P2X3 receptor in primary afferent neurons. Mechanical hyperalgesia was evaluated by electronic von Frey test. Cannabinoid effect was evaluated using anandamide or ACEA, a non-selective or a selective CB1 receptor agonists, respectively; AM251, a CB1 receptor antagonist, and antisense ODN for CB1 receptor. Calcium imaging assay was performed to evaluated α,ß-meATP-responsive cultured DRG neurons pretreated with ACEA. Anandamide or ACEA administered in peripheral tissue reduced the carrageenan-induced mechanical hyperalgesia. The reduction in the carrageenan-induced hyperalgesia induced by ACEA was completely reversed by administration of AM251 as well as by the intrathecal treatment with antisense ODN for CB1 receptor. Also, ACEA reduced the mechanical hyperalgesia induced by bradykinin and by α,ß-meATP, a P2X3 receptor non-selective agonist, but not by tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1ß) and chemokine-induced chemoattractant-1 (CINC-1). Finally, CB1 receptors are co-localized with P2X3 receptors in DRG small-diameter neurons and the treatment with ACEA reduced the number of α,ß-meATP-responsive cultured DRG neurons. Our data suggest that the analgesic effect of CB1 receptor activation is mediated by a negative modulation of the P2X3 receptor in the primary afferent neurons.

P2X3 and P2X2/3 Receptors Play a Crucial Role in Articular Hyperalgesia Development Through Inflammatory Mechanisms in the Knee Joint Experimental Synovitis.

Osteoarthritis (OA) is a degenerative and progressive disease characterized by cartilage breakdown and by synovial membrane inflammation, which results in disability, joint swelling, and pain. The purinergic P2X3 and P2X2/3 receptors contribute to development of inflammatory hyperalgesia, participate in arthritis processes in the knee joint, and are expressed in chondrocytes and nociceptive afferent fibers innervating the knee joint. In this study, we hypothesized that P2X3 and P2X2/3 receptors activation by endogenous ATP (adenosine 5'-triphosphate) induces articular hyperalgesia in the knee joint of male and female rats through an indirect sensitization of primary afferent nociceptors dependent on the previous release of pro-inflammatory cytokines and/or on neutrophil migration. We found that the blockade of articular P2X3 and P2X2/3 receptors significantly attenuated carrageenan-induced hyperalgesia in the knee joint of male and estrus female rats in a similar manner. The carrageenan-induced knee joint inflammation increased the expression of P2X3 receptors in chondrocytes of articular cartilage. Further, the blockade of articular P2X3 and P2X2/3 receptors significantly reduced the increased concentration of TNF-α, IL-6, and CINC-1 and the neutrophil migration induced by carrageenan. These findings indicate that P2X3 and P2X2/3 receptors activation by endogenous ATP is essential to hyperalgesia development in the knee joint through an indirect sensitization of primary afferent nociceptors dependent on the previous release of pro-inflammatory cytokines and/or on neutrophil migration.

Muscle hyperalgesia induced by peripheral P2X3 receptors is modulated by inflammatory mediators.

ATP, via activation of P2X3 receptors, has been highlighted as a key target in inflammatory hyperalgesia. Therefore, the aim of this study was to confirm whether the activation of P2X3 receptors in the gastrocnemius muscle of rats induces mechanical muscle hyperalgesia and, if so, to analyze the involvement of the classical inflammatory mediators (bradykinin, prostaglandins, sympathetic amines, pro-inflammatory cytokines and neutrophil migration) in this response. Intramuscular administration of the non-selective P2X3 receptor agonist α,ß-meATP in the gastrocnemius muscle of rats induced mechanical muscle hyperalgesia, which, in turn, was prevented by the selective P2X3 and P2X2/3 receptors antagonist A-317491, the selective bradykinin B1-receptor antagonist Des-Arg9-[Leu8]-BK (DALBK), the cyclooxygenase inhibitor indomethacin, the ß1- or ß2-adrenoceptor antagonist atenolol and ICI 118,551, respectively. Also, the nonspecific selectin inhibitor fucoidan. α,ß-meATP induced increases in the local concentration of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin 1ß (IL-1ß), which were reduced by bradykinin antagonist. Finally, α,ß-meATP also induced neutrophil migration. Together, these findings suggest that α,ß-meATP induced mechanical hyperalgesia in the gastrocnemius muscle of rats via activation of peripheral P2X3 receptors, which involves bradykinin, prostaglandins, sympathetic amines, pro-inflammatory cytokines release and neutrophil migration. It is also indicated that bradykinin is the key modulator of the mechanical muscle hyperalgesia induced by P2X3 receptors. Therefore, we suggest that P2X3 receptors are important targets to control muscle inflammatory pain.

Peripheral P2X7 receptor-induced mechanical hyperalgesia is mediated by bradykinin.

P2X7 receptors play an important role in inflammatory hyperalgesia, but the mechanisms involved in their hyperalgesic role are not completely understood. In this study, we hypothesized that P2X7 receptor activation induces mechanical hyperalgesia via the inflammatory mediators bradykinin, sympathomimetic amines, prostaglandin E2 (PGE2), and pro-inflammatory cytokines and via neutrophil migration in rats. We found that 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate triethylammonium salt (BzATP), the most potent P2X7 receptor agonist available, induced a dose-dependent mechanical hyperalgesia that was blocked by the P2X7 receptor-selective antagonist A-438079 but unaffected by the P2X1,3,2/3 receptor antagonist TNP-ATP. These findings confirm that, although BzATP also acts at both P2X1 and P2X3 receptors, BzATP-induced hyperalgesia was mediated only by P2X7 receptor activation. Co-administration of selective antagonists of bradykinin B1 (Des-Arg(8)-Leu(9)-BK (DALBK)) or B2 receptors (bradyzide), ß1 (atenolol) or ß2 adrenoceptors (ICI 118,551), or local pre-treatment with the cyclooxygenase inhibitor indomethacin or the nonspecific selectin inhibitor fucoidan each significantly reduced BzATP-induced mechanical hyperalgesia in the rat hind paw. BzATP also induced the release of the pro-inflammatory cytokines tumor necrosis factor α (TNF-α), interleukin (IL)-1ß, IL-6 and cytokine-induced neutrophil chemoattractant-1 (CINC-1), an effect that was significantly reduced by A-438079. Co-administration of DALBK or bradyzide with BzATP significantly reduced BzATP-induced IL-1ß and CINC-1 release. These results indicate that peripheral P2X7 receptor activation induces mechanical hyperalgesia via inflammatory mediators, especially bradykinin, which may contribute to pro-inflammatory cytokine release. These pro-inflammatory cytokines in turn may mediate the contributions of PGE2, sympathomimetic amines and neutrophil migration to the mechanical hyperalgesia induced by local P2X7 receptor activation.

Expression of P2X3 and P2X 5 myenteric receptors varies during the intestinal postnatal development in the guinea pig.

P2X3 receptor expression in various tissues appears to be modulated by age. In the present study, we used single cell RT-PCR to determine the number of P2X3 positive myenteric neurons at different stages of guinea pig postnatal development, and we tested if similar changes also occur to other myenteric P2X receptors. Moreover, we carried out whole-cell recordings using Patch Clamp techniques to determine possible changes in P2X receptors sensitivity to ATP and α,ß-methylene ATP (α,ß-meATP) between newborn and adult animals. Our data indicate that P2X3 subunit transcripts are present in a larger number of myenteric neurons from newborn guinea pigs whereas P2X5 mRNA is found more frequently in adults. Expression of P2X2 and P2X4 transcripts does not change during postnatal development. In newborn animals, virtually all neurons expressing P2X3 also expressed P2X2 transcripts. This is important because these two subunits are known to form heteromeric channels. ATP potency to activate P2X receptors in neurons of both newborn and adult animals was the same. α,ß-meATP, a known P2X3 receptor agonist, induces only a marginal current despite the fact of the higher presence of P2X3 subunits in newborns. These findings imply that P2X3 subunits are mainly forming heteromeric, α,ß-meATP insensitive channels perhaps because P2X3 contributes with only one subunit to the heterotrimers while the other subunits could be P2X2, P2X4, or P2X5.

P2X3 receptors induced inflammatory nociception modulated by TRPA1, 5-HT3 and 5-HT1A receptors.

It has been described that endogenous ATP via activation of P2X3 and P2X2/3 receptors contributes to inflammatory nociception in different models, including the formalin injected in subcutaneous tissue of the rat's hind paw. In this study, we have evaluated whether TRPA1, 5-HT3 and 5-HT1A receptors, whose activation is essential to formalin-induced inflammatory nociception, are involved in the nociception induced by activation of P2X3 receptors on subcutaneous tissue of the rat's hind paw. We have also evaluated whether the activation of P2X3 receptors increases the susceptibility of primary afferent neurons to formalin action modulated by activation of TRPA1, 5-HT3 or 5-HT1A receptors. Nociceptive response intensity was measured by observing the rat's behavior and considering the number of times the animal reflexively raised its hind paw (flinches) in 60min. Local subcutaneous administration of the selective TRPA1, 5-HT3 or 5-HT1A receptor antagonists HC 030031, tropisetron and WAY 100,135, respectively, prevented the nociceptive responses induced by the administration in the same site of the non-selective P2X3 receptor agonist αßmeATP. Administration of the selective P2X3 and P2X2/3 receptor antagonist A-317491 or pretreatment with oligonucleotides antisense against P2X3 receptor prevented the formalin-induced behavioral nociceptive responses during the first and second phases. Also, the co-administration of a subthreshold dose of αßmeATP with a subthreshold dose of formalin induced nociceptive behavior, which was prevented by local administration of tropisetron, HC 030031 or WAY 100, 135. These findings have demonstrated that the activation of P2X3 receptors induces inflammatory nociception modulated by TRPA1, 5-HT3 and 5-HT1A receptors. Also, they suggest that inflammatory nociception is modulated by the release of endogenous ATP and P2X3 receptor activation, which in turn, increases primary afferent nociceptor susceptibility to the action of inflammatory mediators via interaction with TRPA1, 5-HT3 and 5-HT1A receptors in the peripheral tissue.

Neuronal P2X3 receptor activation is essential to the hyperalgesia induced by prostaglandins and sympathomimetic amines released during inflammation.

We have demonstrated that the activation of P2X3 receptor on peripheral afferent neurons is critical to development of inflammatory hyperalgesia in peripheral tissue, although pharmacological administration of prostaglandin E(2) or sympathomimetic amines is enough to sensitize primary afferent neurons by acting directly in neuronal receptors. Therefore, to clarify this ambiguity this study verifies whether P2X3 receptor activation on primary afferent neurons enables the sensitization induced by prostaglandin E(2) or sympathomimetic amine. Initially, this study confirmed that co-administration of A317491 (60 µg/paw), a selective P2X3 receptor antagonist, or pre-treatment with dexamethasone (1 mg/mL/kg) prevents the mechanical hyperalgesia induced by carrageenan (300 µg/paw) in the rat's hind paw. Sub-threshold doses of PGE(2) (4 ng/paw) or dopamine (0.4 µg/paw), that do not induce hyperalgesia by themselves, when injected just following αßmeATP or carrageenan in rats treated with dexamethasone induced hyperalgesia, which is prevented by A317491 or treatment with periganglionar (DRG-L5) injections of ODN-antisense, against P2X3 receptor. Furthermore, because PKCɛ translocation induces an increase of neuronal susceptibility to inflammatory mediators, this study demonstrates that αßmeATP in peripheral tissue increases the expression of PKCɛ in cell membranes of DRG-L5, and in contrast, the administration of PKCɛ translocation inhibitor (1 µg/paw) in peripheral tissue 45 min before αßmeATP, prevented the hyperalgesia induced by sub-threshold dose of PGE(2) (4 ng/paw). In conclusion, this study suggests that neuronal P2X3 receptor activation and the consequent PKCɛ translocation increase the susceptibility of nociceptor to inflammatory mediators allowing the development of inflammatory hyperalgesia.

Runx1 and C/EBPß transcription factors directly up-regulate P2X3 gene transcription.

Recent evidence indicates that transcription factor Runx1 modulates the expression of several phenotypic markers in dorsal root ganglia (DRGs) neurons, including the pain-related P2X3 receptor. In several cell lineages C/EBP transcription factors interact with the Runx factor family members to jointly bind and activate transcription of target genes. Here, we examine whether these two transcription factors directly regulate P2X3 gene expression. Through in silico analyses of the first 2 kb of the P2X3 gene promoter we identified putative consensus-binding sites for both Runx1 and C/EBPß transcription factors. Transient over-expression in PC12 cells of either Runx1 or C/EBPß increases P2X3 gene promoter activity and co-expression of both factors results in an additive stimulatory effect on the promoter function. Accordingly, chromatin immunoprecipitation assays demonstrate that both Runx1 and C/EBPß bind to the P2X3 promoter in PC12 cells expressing this gene. Site-directed mutagenesis of the proximal Runx1 and C/EBPß consensus elements in the P2X3 promoter decrease Runx1- and C/EBPß-mediated transcriptional activity. Moreover, C/EBPß-mediated enhancement of the P2X3 promoter requires a functional Runx1 binding site. Altogether our results support a functional and coordinated role for Runx1 and C/EBPß transcription factors during activation of P2X3 gene transcription.

Chronic intermittent hypoxia augments sympatho-excitatory response to ATP but not to L-glutamate in the RVLM of rats.

The development of sympathetic overactivity and hypertension in rats submitted to chronic intermittent hypoxia (CIH) involve alterations in the central mechanisms controlling respiratory and autonomic functions. Herein, we assessed whether CIH alters glutamatergic and/or purinergic signaling in the ventrolateral medulla (VLM), a region that encompasses the pre-sympathetic neurons and respiratory neurons of the ventral respiratory column. Groups of juvenile rats were exposed for 10 days to CIH (6% O(2) for 40s, every 9min, 8h/day) or normoxia (controls). Following treatment, in situ working heart-brainstem preparations were performed to record simultaneously respiratory and sympathetic motor outputs. In separate CIH and control groups, the VLM was dissected for western-blot analyses of ionotropic glutamatergic and P2 receptors. l-glutamate microinjections (1, 3 or 10mM) into VLM of control (n=6) and CIH groups (n=10) produced similar increases of sympathetic and abdominal activities associated with phrenic nerve inhibition; immunoreactive NMDAR1 and GluR2/3 densities at the VLM were also alike between groups (n=4). In contrast, VLM microinjections of ATP (1, 10 or 50mM) evoked larger sympatho-excitatory responses in CIH (n=8) than in control rats (n=7, P<0.05) whilst the abdominal increase and phrenic nerve inhibition were of comparable magnitudes. The immunoreactive densities of P2X3 and P2X4 receptors, but not P2X1 and P2Y2, were 20% higher in VLM of CIH (n=8; P<0.05) than controls (n=8). Altogether, our findings suggest that CIH augments purinergic signaling in the VLM, supporting the concept that nucleotides play a role in the dynamic central control of the sympathetic autonomic function.

P2X3 and P2X2/3 receptors mediate mechanical hyperalgesia induced by bradykinin, but not by pro-inflammatory cytokines, PGE2 or dopamine.

Activation of peripheral P2X3 and P2X2/3 receptors by endogenous ATP is essential to the development of inflammatory hyperalgesia. We have previously demonstrated that this essential role of P2X3 and P2X2/3 receptors in the development of mechanical hyperalgesia induced by the inflammatory agent carrageenan is mediated by an indirect sensitization of the primary afferent nociceptors dependent on the previous release of tumor necrosis factor alpha (TNF-α) and by a direct sensitization of the primary afferent nociceptors. Therefore, in this study we asked whether activation of P2X3 and P2X2/3 receptors contribute to the mechanical hyperalgesia induced by the inflammatory mediators involved in carrageenan-induced mechanical hyperalgesia, such as bradykinin, tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1ß), interleukin-6 (IL-6), chemokine-induced chemoattractant-1 (CINC-1), prostaglandin E2 (PGE2) and dopamine. Co-administration of the non-selective P2X3 receptor antagonist TNP-ATP or the selective P2X3 and P2X2/3 receptor antagonist A-317491 with bradykinin, but not with TNF-α, IL-1ß, IL-6, CINC-1, PGE2 or dopamine, prevented in a dose-dependent manner the mechanical hyperalgesia. We also verified whether the activation of P2X3 and P2X2/3 receptors by endogenous ATP contributes to bradykinin-induced mechanical hyperalgesia via neutrophil migration and/or cytokine release. Co-administration of TNP-ATP or A-317491 did not affect either neutrophil migration or the increased concentration of TNF-α, IL-1ß, IL-6 and CINC-1 induced by bradykinin. These findings demonstrate that the activation of P2X3 and P2X2/3 receptors by endogenous ATP mediates bradykinin-induced mechanical hyperalgesia by a mechanism that does not depend on neutrophil migration or cytokines release.

Involvement of temporomandibular joint P2X3 and P2X2/3 receptors in carrageenan-induced inflammatory hyperalgesia in rats.

The aim of this study was to investigate the role of P2X3, P2X2/3 and P2X7 receptors in the development of TMJ hyperalgesia induced by carrageenan. We also investigated the expression of mRNA of P2X7 receptors in the trigeminal ganglia and the existence of functional P2X7 receptors in the rat's TMJ. The P2X1, P2X3 and P2X2/3 receptor antagonist TNP-ATP, but not the selective P2X7 receptor antagonist A-438079, significantly reduced carrageenan-induced TMJ inflammatory hyperalgesia. The qPCR assay showed that mRNA of P2X7 receptors are expressed in the trigeminal ganglia but this expression is not increased by the inflammation induced by carrageenan in the TMJ region. The P2X7 receptor agonist BzATP induced TMJ inflammatory hyperalgesia that was significantly reduced by pretreatment with dexamethasone. These results indicate that P2X3 and P2X2/3 but not P2X7 receptors are involved in carrageenan-induced TMJ inflammatory hyperalgesia. However, functional P2X7 receptors are expressed in the TMJ region. The activation of these receptors by BzATP sensitizes the primary afferent nociceptors in the TMJ through the previous release of inflammatory mediators. The findings of this study point out P2X3 and P2X2/3 receptors, but not P2X7 receptors, as potential targets for the development of new analgesic drugs to control TMJ inflammatory pain.

Peripheral mechanisms underlying the essential role of P2X3,2/3 receptors in the development of inflammatory hyperalgesia.

Activation of P2X3,2/3 receptors by endogenous ATP contributes to the development of inflammatory hyperalgesia. Given the clinical importance of mechanical hyperalgesia in inflammatory states, we hypothesized that the activation of P2X3,2/3 receptors by endogenous ATP contributes to carrageenan-induced mechanical hyperalgesia and that this contribution is mediated by an indirect and/or a direct sensitization of the primary afferent nociceptors. Co-administration of the selective P2X3,2/3 receptors antagonist A-317491, or the non-selective P2X3 receptor antagonist, TNP-ATP, with carrageenan blocked the mechanical hyperalgesia induced by carrageenan, and significantly reduced the increased concentration of tumor necrosis factor alpha (TNF-alpha) and chemokine-induced chemoattractant-1 (CINC-1) but not of interleukin-1 beta (IL-1 beta) induced by carrageenan. Co-administration of the selective P2X3,2/3 receptors antagonist A-317491 with carrageenan did not affect the neutrophil migration induced by carrageenan. Intrathecal administration of oligonucleotides antisense against P2X3 receptors for seven days significantly reduced the expression of P2X3 receptors in the saphenous nerve and significantly reduced the mechanical hyperalgesia induced by carrageenan. We concluded that the activation of P2X3,2/3 receptors by endogenous ATP is essential to the development of the mechanical hyperalgesia induced by carrageenan. Furthermore, we showed that this essential role of P2X3,2/3 receptors in the development of carrageenan-induced mechanical hyperalgesia is mediated by an indirect sensitization of the primary afferent nociceptors dependent on the previous release of TNF-alpha and by a direct sensitization of the primary afferent nociceptors.