Sensitization of purinergic P2X3 receptors (P2X3Rs) contributes to the production of exaggerated nociceptive responses following inflammatory injury. We showed previously that prostaglandin E2 (PGE2) potentiates P2X3R-mediated ATP currents in dorsal root ganglion neurons isolated from both control and complete Freund’s adjuvant-induced inflamed rats. PGE2 potentiation of ATP currents depends only on PKA signaling in control neurons, but it depends on both PKA and PKC signaling in inflamed neurons. We further found that inflammation evokes an increase in exchange proteins directly activated by cAMP (Epacs) in dorsal root ganglions. This increase promotes the activation of PKC to produce a much enhanced PGE2 effect on ATP currents and to elicit Epac-dependent flinch nocifensive behavioral responses in complete Freund’s adjuvant rats. The link between Epac-PKC signaling and P2X3R sensitization remains unexplored. Here, we show that the activation of Epacs promotes the expression of phosphorylated PKC and leads to an increase in the cytoskeleton, F-actin, expression at the cell perimeter. Depolymerization of F-actin blocks PGE2-enhanced ATP currents and inhibits P2X3R-mediated nocifensive responses after inflammation. Thus, F-actin is dynamically involved in the Epac-PKC-dependent P2X3R sensitization. Furthermore, Epacs induce a PKC-dependent increase in the membrane expression of P2X3Rs. This increase is abolished by F-actin depolymerization, suggesting that F-actin mediates Epac-PKC signaling of P2X3R membrane expression. Thus, after inflammation, an Epac-PKC dependent increase in F-actin in dorsal root ganglion neurons enhances the membrane expression of P2X3Rs to bring about sensitization of P2X3Rs and abnormal pain behaviors.

Here we investigated effects of intramuscular (i.m.) heating-needle stimulation on persistent muscle nociception evoked by i.m. injection of different doses (50-200 µl) of complete Freund's adjuvant (CFA) in rats. Paw withdrawal reflexes evoked by noxious mechanical and heat stimulation as well as hind limb swelling were determined prior to and two weeks after the CFA injection. The unilateral injection of CFA induced a dose-related and long-lasting (5-14 d), bilateral secondary mechanical hyperalgesia and heat hypoalgesia associated with long-term limb swelling. A period of 30-45 min 43 °C heating-needle stimulation significantly enhanced the i.m. CFA-induced bilateral heat hypoalgesia and alleviated hind limb swelling. In contrast, 30-45 min 46 °C heating-needle stimulation markedly enhanced both mechanical hyperalgesia and heat hypoalgesia, but failed to influence the CFA-induced hind limb swelling. Microinjection of P2X3 receptor antagonist A-317491 (0.5-4.5 nmol/0.5 µl) into the thalamic ventromedial (VM) nucleus dose-dependently inhibited the 43 °C and 46 °C heating-needle stimulation-induced heat hypoalgesia, whereas the 46 °C heating-needle stimulation-induced mechanical hyperalgesia was significantly prevented by microinjection of A-317491 into the thalamic mediodorsal (MD) nucleus. In contrast, the hind limb swelling was not affected by the microinjection of A-317491 into the thalamic VM or MD nucleus. The present study indicates that in the CFA-induced persistent muscle nociception condition, 43 °C heating-needle stimulation selectively increases descending inhibition, which effect is modulated by the thalamic VM nucleus. In addition to the antinociceptive role of P2X3 receptors in the thalamic VM nucleus, P2X3 receptors within the thalamic MD nucleus participate in the descending facilitation evoked by i.m. 46 °C heating-needle stimulation.

Diabetic neuropathy is a common neuropathy associated with paresthaesia and pain. The mechanisms underlying the painful conditions are not well understood. The aim of this study is to investigate the participation of purinergic P2X3 receptors in painful diabetic neuropathy.

P2 purinergic receptors are overexpressed in certain cancer tissues, but the pathophysiologic relevance of purinergic signaling in hepatocellular carcinoma (HCC) remains unknown. To examine the role of P2 purinergic signaling in the pathogenesis of HCC and characterize extracellular nucleotide effects on HCC cell proliferation, two independent HCC patient cohorts were analyzed for P2 purinergic receptor expression, and nucleotide treated HCC cell lines were evaluated for effects on proliferation and cell cycle progression. Our studies suggest that multiple P2 purinergic receptor isoforms are overexpressed in liver tumors, as compared to uninvolved liver, and dysregulation of P2 purinergic receptor expression is apparent in HCC cell lines, as compared to human primary hepatocytes. High P2X3 purinergic receptor expression is associated with poor recurrence-free survival (RFS), while high P2Y13 expression is associated with improved RFS. Extracellular nucleotide treatment alone is sufficient to induce cell cycle progression, via activation of JNK signaling, and extracellular ATP-mediated activation of P2X3 receptors promotes proliferation in HCC cells.

The purinergic P2X3 receptor (P2X3R) expressed in the dorsal root ganglion (DRG) sensory neuron and the P2X7 receptor (P2X7R) expressed in the surrounding satellite glial cell (SGC) are two major receptors participating in neuron-SGC communication in adult DRGs. Activation of P2X7Rs was found to tonically reduce the expression of P2X3Rs in DRGs, thus inhibiting the abnormal pain behaviors in adult rats. P2X receptors are also actively involved in sensory signaling in developing rodents. However, very little is known about the developmental change of P2X7Rs in DRGs and the interaction between P2X7Rs and P2X3Rs in those animals. We therefore examined the expression of P2X3Rs and P2X7Rs in postnatal rats and determined if P2X7R-P2X3R control exists in developing rats.

Recently, we reported that astrocytes in the trigeminal caudal nucleus (Vc) of the brain stem express a purinergic receptor P2X3, which is involved in the craniofacial pathologic pain. Although we observed protein expression of P2X3 receptors (P2X3 Rs) in the astrocyte of the Vc, it is still unclear that astrocyte has functional P2X3Rs in Vc. To address this issue, we recorded asrtocytic P2X3Rs by using whole cell voltage-clamp recording in the Vc of the GFAP-GFP mice, which was used as a guide to astrocytes with green fluorescence. While measuring voltage ramp-induced astrocytic membrane current, we found the amplitude of the current was increased when we applied P2-purinoreceptor agonist, α,β-meATP. This increase was blocked by co-application of A317491, P2X3R antagonist. These results demonstrate that astrocytes in the Vc express functional P2X3Rs, which might be critical in craniofacial pathologic pain.

Purinergic P2X and P2Y receptors are involved in mediating intercellular signalling via purines such as adenosine triphosphate (ATP). P2X and P2Y receptors have been implicated in numerous body functions including learning, memory and sleep. All of these body functions show time-of-day-dependent variations controlled by the master circadian oscillator located in the suprachiasmatic nucleus (SCN). Evidence exists for a role of purinergic signalling in intercellular coupling within SCN. However, few studies have been performed on the expression of purinergic receptors in SCN. Therefore, we analyse the expression of seven P2X (P2X1-7) and eight P2Y (P2Y1-2, 4, 6, 11-14) receptors in mouse SCN and address their time-of-day-dependent variation by using immunohistochemistry and real-time polymerase chain reaction. At the early light phase, P2X and P2Y receptors show a low to moderate, homogenously distributed immunoreaction throughout SCN. P2Y13 reveals strong immunoreaction in fibres within the core region of SCN. From the fifteen analysed P2 receptors, seven exhibit a time-of-day-dependent variation in SCN. P2X1 immunoreaction is very low in the early light phase with a minor increase at the end of the dark phase. P2X4 immunoreaction strongly increases during the dark phase in soma cells in the core region and in a dense network of fibres in the shell region of SCN. P2X3 immunoreaction is moderately elevated during the dark phase. Conversely, immunoreaction for P2Y2, P2Y12 and P2Y14 moderately increases at the early light phase and P2Y6 immunoreaction displays a moderate increase at the mid-light phase. Thus, this study demonstrates a time-of-day-dependent variation of P2 receptors in mouse SCN.

P2X3 and P2X2/3 purinergic receptor-channels, expressed in primary sensory neurons that mediate nociception, have been implicated in neuropathic and inflammatory pain responses. The phospholipids phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) are involved in functional modulation of several types of ion channels. We report here evidence that these phospholipids are able to modulate the function of homomeric P2X3 and heteromeric P2X2/3 purinoceptors expressed in dorsal root ganglion (DRG) nociceptors and in heterologous expression systems.

The expression of purinergic receptors (P2X) on rat vestibular ganglion neurons (VGNs) was examined using whole-cell patch-clamp recordings. An application of adenosine 5'-triphosphate (ATP; 100microM) evoked inward currents in VGNs at a holding potential of -60mV. The decay time constant of the ATP-evoked currents was 2-4s, which is in between the values for rapidly desensitizing subgroups (P2X1 and P2X3) and slowly desensitizing subgroups (P2X2, P2X4, etc.), suggesting the heterogeneous expression of P2X receptors. A dose-response experiment showed an EC(50) of 11.0microM and a Hill's coefficient of 0.82. Suramin (100microM) reversibly inhibited the ATP-evoked inward currents. Alpha, beta-methylene ATP (100microM), a P2X-specific agonist, also evoked inward currents but less extensively than ATP. An application of adenosine 5'-dihosphate (ADP; 100microM) evoked similar, but much smaller, currents. The current-voltage relationship of the ATP-evoked conductance showed pronounced inward rectification with a reversal potential more positive than 0mV, suggesting non-selective cation conductance. However, the channel was not permeable to a large cation (N-methyl-d-glucamine) and acidification (pH 6.3) had little effect on the ATP-evoked conductance. RT-PCR confirmed the expression of five subtypes (P2X2-P2X6) in VGNs. The physiological role of P2X receptors includes the modulation of excitability at the synapses between hair cells and dendrites and/or trophic support (or also neuromodulation) from supporting cells surrounding the VGNs.

Epigallocatechin gallate (EGCG) has exhibited antitumor properties against bladder cancer. However, its effects in interstitial cystitis (IC) have not been investigated.

To evaluate whether P2X receptors are involved in responses to noxious pulp stimulation, the P2X3 and P2X2/3 receptor agonist α,β-methyleneATP (α,β-meATP) was applied to the molar tooth pulp and nocifensive behavior and extracellular-signal regulated kinase (ERK) phosphorylation in trigeminal spinal subnucleus caudalis (Vc), trigeminal spinal subnucleus interpolaris (Vi), upper cervical spinal cord (C1/C2) and paratrigeminal nucleus (Pa5) neurons were analyzed in rats.

Aims Insular cortex is a brain region critical for processing of the sensation. Purinergic receptors are involved in the formation of chronic pain. The aim of the present study was to explore the role and mechanism of P2X3 receptors (P2X3Rs) in insular cortex in chronic visceral pain. Methods Chronic visceral pain in adult rats was induced by neonatal maternal deprivation and measured by detecting the threshold of colorectal distension. Western blotting, immunofluorescence, and real-time quantitative polymerase chain reaction techniques were used to detect the expression and distribution of P2X3Rs. Synaptic transmission in insular cortex was recorded in brain slices by patch clamp techniques. Results Expression of P2X3Rs both at mRNA and protein levels in right hemisphere of insular cortex was significantly increased in neonatal maternal deprivation rats. In addition, P2X3Rs were expressed with NeuN or synaptophysin but not with glial fibrillary acidic protein and CD11b. The co-localization of P2X3Rs with NeuN or synaptophysin was greatly enhanced in right hemisphere of insular cortex in neonatal maternal deprivation rats. Furthermore, neonatal maternal deprivation markedly increased both the frequency and amplitude of miniature excitatory postsynaptic current in right hemisphere of insular cortex. Incubation of A347091 significantly decreased the frequency of spontaneous excitatory postsynaptic current and miniature excitatory postsynaptic current of insular cortex neurons of neonatal maternal deprivation rats. Incubation of P2X3Rs agonists α,β-mATP remarkably increased the frequency of spontaneous excitatory postsynaptic current and miniature excitatory postsynaptic current of the right hemisphere of insular cortex neurons of healthy control rats. Importantly, injection of A317491 significantly enhanced the colorectal distension threshold of neonatal maternal deprivation rats, while injection of α,β-mATP into right but not left insular cortex markedly decreased the colorectal distension threshold in healthy control rats. Conclusions Overall, our data provide integrated pharmacological, biochemical, and functional evidence demonstrating that P2X3Rs are physically and functionally interconnected at the presynaptic level to control synaptic activities in the right insular cortex, thus contributing to visceral pain of neonatal maternal deprivation rats.

Experiments using P2X3 knock-out mice or more general P2X receptor antagonists suggest that P2X3 receptors contribute to visceral hypersensitivity. We aimed to investigate the effect of the selective P2X3 antagonist A-317491 on visceral sensitivity under physiological conditions, during acute colitis and in the post-inflammatory phase of colitis.

We have characterized the mouse carotid body (CB) with special attention to nicotinic, purinergic and dopaminergic receptors as well as the TASK-1 K(+)-channel. Mouse CB sections were stained immunohistochemically and visualized using fluorescent and confocal microscopy. The CB type 1 cells contained the alpha3 (n=8), alpha4 (n=7), alpha7 (n=4) and beta2 (n=3) nicotinic acetylcholine receptor (nAChR) subunits, the ATP-receptors P2X(2) (n=15) and P2X(3) (n=9), the dopamine D(2) receptor (n=9) and the TASK-1 K(+)-channel (n=7). Here we report the presence of alpha3, alpha4, alpha7 and beta2 nAChR subunits, the D(2) receptor and the TASK-1 K(+)-channel in the mouse CB. Also, we confirm the presence of the P2X(2) and P2X(3) receptors in mouse CB. Thus, we have localized nicotinergic, purinergic and dopaminergic receptors and the TASK-1 K(+)-channel on a protein level in one species. Our data are in line with the theory that the CB chemoreceptor cell hosts an orchestra of receptor systems that ultimately modulate the response to hypoxia.

Extracellular agonists linked to inositol-1,4,5-trisphosphate (IP3) formation elicit cytosolic Ca2+ oscillations in many cell types, but despite a common signaling pathway, distinct agonist-specific Ca2+ spike patterns are observed. Using qPCR, we show that rat hepatocytes express multiple purinergic P2Y and P2X receptors (R). ADP acting through P2Y1R elicits narrow Ca2+ oscillations, whereas UTP acting through P2Y2R elicits broad Ca2+ oscillations, with composite patterns observed for ATP. P2XRs do not play a role at physiological agonist levels. The discrete Ca2+ signatures reflect differential effects of protein kinase C (PKC), which selectively modifies the falling phase of the Ca2+ spikes. Negative feedback by PKC limits the duration of P2Y1R-induced Ca2+ spikes in a manner that requires extracellular Ca2+. By contrast, P2Y2R is resistant to PKC negative feedback. Thus, the PKC leg of the bifurcated IP3 signaling pathway shapes unique Ca2+ oscillation patterns that allows for distinct cellular responses to different agonists.

We have tested the hypothesis that neuropathic pain acting as a stressor drives functional plasticity in the sympathoadrenal system. The relation between neuropathic pain and adrenal medulla function was studied with behavioral, immunohistochemical and electrophysiological techniques in rats subjected to chronic constriction injury of the sciatic nerve. In slices of the adrenal gland from neuropathic animals, we have evidenced increased cholinergic innervation and spontaneous synaptic activity at the splanchnic nerve⁻chromaffin cell junction. Likewise, adrenomedullary chromaffin cells displayed enlarged acetylcholine-evoked currents with greater sensitivity to α-conotoxin RgIA, a selective blocker of α9 subunit-containing nicotinic acetylcholine receptors, as well as increased exocytosis triggered by voltage-activated Ca2+ entry. Altogether, these adaptations are expected to facilitate catecholamine output into the bloodstream. Last, but most intriguing, functional and immunohistochemical data indicate that P2X3 and P2X7 purinergic receptors and transient receptor potential vanilloid-1 (TRPV1) channels are overexpressed in chromaffin cells from neuropathic animals. These latter observations are reminiscent of molecular changes characteristic of peripheral sensitization of nociceptors following the lesion of a peripheral nerve, and suggest that similar phenomena can occur in other tissues, potentially contributing to behavioral manifestations of neuropathic pain.

We have previously shown that endogenous adenosine 5'-triphosphate (ATP), via P2X3 and P2X2/3 receptors, plays an essential role in carrageenan-induced articular hyperalgesia model in rats' knee joint. In the present study, we used the rat knee joint incapacitation test, Enzyme-Linked Immunosorbent Assay (ELISA), and myeloperoxidase enzyme activity assay, to test the hypothesis that the activation of P2X3 and P2X2/3 receptors by their agonist induces articular hyperalgesia mediated by the inflammatory mediators bradykinin, prostaglandin, sympathomimetic amines, pro-inflammatory cytokines and by neutrophil migration. We also tested the hypothesis that the activation of P2X3 and P2X2/3 receptors contributes to the articular hyperalgesia induced by the inflammatory mediators belonging to carrageenan inflammatory cascade. The non-selective P2X3 and P2X2/3 receptors agonist αβ-meATP induced a dose-dependent articular hyperalgesia, which was significantly reduced by the selective antagonists for P2X3 and P2X2/3 receptors (A-317491), bradykinin B1- (DALBK) or B2-receptors (bradyzide), β1-(atenolol) or β2-adrenoceptors (ICI-118,551), by the pre-treatment with cyclooxygenase inhibitor (indomethacin) or with the nonspecific selectin inhibitor (Fucoidan). αβ-meATP induced the release of pro-inflammatory cytokines TNFα, IL-1β, IL-6, and CINC-1, as well as the neutrophil migration. Moreover, the co-administration of A-317491 significantly reduced the articular hyperalgesia induced by bradykinin, prostaglandin E2 (PGE2), and dopamine. These findings suggest that peripheral P2X3 and P2X2/3 receptors activation induces articular hyperalgesia by an indirect sensitization of the primary afferent nociceptor of rats' knee joint through the release of inflammatory mediators. Further, they also indicate that the activation of these purinergic receptors by endogenous ATP mediates the bradykinin-, PGE2-, and dopamine-induced articular hyperalgesia.

Lumbar disc herniation (LDH) is a major cause of discogenic low back pain and sciatica, but the underlying mechanisms remain largely unknown. Hydrogen sulfide (H2S) is becoming recognized for its involvement in a wide variety of processes including inflammation and nociception. The present study was designed to investigate the roles of the H2S signaling pathway in the regulation of expression and function of purinergic receptors (P2XRs) in dorsal root ganglion (DRG) neurons from rats with LDH. LDH was induced by implantation of autologous nucleus pulposus (NP), harvested from rat tail, in lumbar 5 and 6 spinal nerve roots. Implantation of autologous NP induced persistent pain hypersensitivity, which was partially reversed by an intrathecal injection of A317491, a potent inhibitor of P2X3Rs and P2X2/3Rs. The NP induced persistent pain hypersensitivity was associated with the increased expression of P2X3Rs, but not P2X1Rs and P2X2Rs, receptors in L5-6 DRGs. NP implantation also produced a 2-fold increase in ATP-induced intracellular calcium signals in DRG neurons when compared to those of controls (P < 0.05). Interestingly, NP implantation significantly enhanced expression of the endogenous hydrogen sulfide producing enzyme, cystathionine-β-synthetase (CBS). Systematic administration of O-(Carboxymethyl) hydroxylamine hemihydrochloride (AOAA), an inhibitor of CBS, suppressed the upregulation of P2X3R expression and the potentiation of ATP-induced intracellular calcium signals in DRG neurons (P < 0.05). Intrathecal injection of AOAA markedly attenuated NP induced- persistent pain hypersensitivity. Our results suggest that sensitization of P2X3Rs, which is likely mediated by CBS-H2S signaling in primary sensory neurons, contributes to discogenic pain. Targeting CBS/H2S-P2X3R signaling may represent a potential treatment for neuropathic pain caused by LDH.

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.

BACKGROUND Epilepsy is a complex neurologic disorder with abnormal electrical impulses in the brain. A crucial role of purinergic signalling in the proper working of the nervous system has been reported but much less is known about the modulation of P2X3 purinergic receptors in epilepsy. This study investigated the effect of NF110, a potent P2X3 receptor antagonist, in the rat epilepsy model of pentylenetetrazole (PTZ)-induced kindling. MATERIAL AND METHODS The mean kindling score, motor activity, locomotion, emotional tension, anxiety, discrimination ability, learning, memory, serum neuron-specific enolase (sNSE), hippocampal IL-1β and TNF-α, thiobarbituric acid-reactive substance (TBARS), catalase (CAT) and reduced glutathione (GSH), and mitochondrial complex I, II, and IV levels of PTZ-kindling animals were assessed. RESULTS The PTZ-kindling animals have shown impaired motor activity, locomotion, discrimination ability, learning, and memory, along with increased emotional tension, anxiety, neuronal damage (increased sNSE), hippocampal pro-inflammatory mediators (increased IL-1β and TNF-α), oxidative stress (increased TBARS, decreased GSH and CAT), and mitochondrial dysfunction. The administration of NF110 in 3 different doses has significantly and dose-dependently corrected PTZ-kindling-induced impaired behavior, learning, memory, locomotion, motor activity, discrimination ability, neuronal damage, hippocampal inflammation, oxidative stress, and mitochondrial dysfunction. These beneficial effects of NF110 in PTZ-kindling animals were significantly abolished by the administration of the P2X agonist α, β methylene-ATP. CONCLUSIONS P2X3 receptors play a very important role in kindling epilepsy and further research should be done to design P2X3 modulators for their possible therapeutic benefits in epileptic disorders.