Convergence of peptidergic and non-peptidergic protein markers in the human dorsal root ganglion and spinal dorsal horn.

Peripheral sensory neurons are characterized by their size, molecular profiles, and physiological responses to specific stimuli. In mouse, the peptidergic and non-peptidergic subsets of nociceptors are distinct and innervate different lamina of the spinal dorsal horn. The unique molecular signature and neuroanatomical organization of these neurons supports a labeled line theory for certain types of nociceptive stimuli. However, long-standing evidence supports the polymodal nature of nociceptors in many species. We have recently shown that the peptidergic marker, CGRP, and the non-peptidergic marker, P2X3R, show largely overlapping expression at the mRNA level in human dorsal root ganglion (DRG). Herein, our aim was to assess the protein distribution of nociceptor markers, including their central projections, in the human DRG and spinal cord. Using DRGs obtained from organ donors, we observed that CGRP and P2X3R were co-expressed by approximately 33% of human DRG neurons and TrpV1 was expressed in ~60% of human DRG neurons. In the dorsal spinal cord, CGRP, P2X3R, TrpV1, and Nav1.7 proteins stained the entirety of lamina 1-2, with only P2XR3 showing a gradient of expression. This was confirmed by measuring the size of the substantia gelatinosa using Hematoxylin and Eosin staining of adjacent sections. Our findings are consistent with the known polymodal nature of most primate nociceptors and indicate that the central projection patterns of nociceptors are different between mice and humans. Elucidating how human nociceptors connect to subsets of dorsal horn neurons will be important for understanding the physiological consequences of these species differences.

Effects of resveratrol in the signaling of neuropathic pain involving P2X3 in the dorsal root ganglion of rats.

Neuropathic pain is a major public health problem because it has a considerable impact on life quality of patients. Neuropathic pain caused by a lesion or disease of the somatosensory nervous system, which causes unpleasant and abnormal sensation (dysesthesia), an increased response to painful stimuli (hyperalgesia), and pain in response to a stimulus that does not normally provoke pain (allodynia). P2X receptors from dorsal root ganglion (DRG) play a crucial role in facilitating pain transmission at peripheral and spinal sites. Resveratrol (Res) has neuroprotective effects and improves the pathological and behavioral outcomes of various types of nerve injury. The present study examined the effects of Res on neuropathic pain. Neuropathic pain animal model was created by partial sciatic nerve ligation (pSNL) surgery. We found that consecutive intraperitoneal administration of Res for 21 days reduced the mechanical and thermal nociceptive responses induced by pSNL in a dose-dependent manner. Moreover, Res administration reversed P2X3 expression and phosphorylation of ERK in DRG neurons after peripheral nerve injury. Our results suggested that Res may ameliorate neuropathic pain by suppressing P2X3 up-regulation and ERK phosphorylation in DRG of neuropathic pain rats. Therefore, we concluded that Res has a significant analgesic effect on alleviating neuropathic pain, and thus may serve as a therapeutic approach for neuropathic pain.

Resveratrol alleviates diabetic mechanical allodynia in rats by downregulating P2X3R.

Mechanical allodynia, which develops in patients of diabetes mellitus as a neuropathic manifestation, remains without an effective treatment. The aim of the present study was to investigate the effects and potential mechanisms underlying resveratrol (RES) in a rat model of streptozocin (STZ)­induced diabetic mechanical allodynia (DMA). The rat model of DMA was established by the administration of an intraperitoneal injection of STZ. From day 8 post­STZ injection, rats were administered with an intragastric injection of various doses of RES for 14 consecutive days. The von Frey filaments were applied to detect the paw withdrawal threshold and evaluate the analgesic effects of RES. Based on the dose­effect curve, the ED50 of RES was calculated. Immunofluorescence staining and western blotting were performed to detect the expression of purinergic receptor P2X3 (P2X3R) in the dorsal root ganglion (DRG) and spinal dorsal horn (SDH) following RESED50 treatment. The results indicated that RES significantly alleviated mechanical allodynia in DMA model rats in a dose­dependent manner. Compared with the control group, the expression of P2X3R in DRG neurons and SDH terminals was markedly decreased following the administration of RESED50 (P<0.05). Collectively, the results indicated that RES displayed a dose­dependent analgesic effect on DMA model rats. Furthermore, P2X3R expression downregulation in the DRG and SDH may be a mechanism underlying the analgesic effects of RES on DMA­related behaviors.

1,8-cineole decreases neuropathic pain probably via a mechanism mediating P2X3 receptor in the dorsal root ganglion.

1,8-cineole is a natural monoterpene cyclic ether present in eucalyptus and has been reported to exhibit anti-inflammatory and antioxidant effects. The therapeutic effects of 1,8-cineole on neuropathic pain and the molecular mechanisms of its pharmacological actions remain largely unknown. In the present study, we investigated the analgesic mechanisms of orally administered 1,8-cineole in a rat model of chronic constriction injury (CCI) and examined the drug-induced modulation of P2X3 receptor expression in dorsal root ganglia. The mechanical withdrawal threshold and thermal withdrawal latency were measured in rats to assess behavioural changes 7 and 14 days after CCI surgery. Changes in P2X3 receptor mRNA expression of L4-5 dorsal root ganglia were analysed using quantitative real-time polymerase chain reaction at the 7th and 14th postoperative day. Additionally, we examined the expression of P2X3 receptor protein in L4-5 dorsal root ganglia 7 and 14 days after surgery using immunohistochemistry and western blots. We found that 1,8-cineole can alleviate pathological pain caused by P2X3 receptor stimulation and explored new methods for the prevention and treatment of neuropathic pain.

Parameter-specific analgesic effects of electroacupuncture mediated by degree of regulation TRPV1 and P2X3 in inflammatory pain in rats.

AIMS: Observing the parameter-specific anti-hyperalgesic effects of EA with different stimulation times and frequencies on painful hyperalgesia mediated by the level of TRPV1 and P2X3 expression in DRG after CFA injection. MAIN METHODS: The model was induced by the injection of CFA in each rat's right hind paw. EA treatment was applied to the bilateral ST36 and BL60. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were tested with Von Frey filaments and the radiant heat source of the test instrument, respectively. TRPV1 and P2X3 expressions were measured by immunofluorescence and western blot. αß-meATP and capsaicine combined with EA were further utilized to investigate the change in PWL. KEY FINDINGS: Different stimulation times (20, 30, 45 min) combined with different frequencies (2 Hz, 100 Hz, 2/100 Hz) of EA have analgesic effects on the PWT and PWL; however, the level of the hypoalgesic efficacy of EA was primarily associated with EA frequency. The analgesic effect of EA was better at 100 Hz than at 2 Hz. The level of regulation of 100 Hz EA on TRPV1 and P2X3 in DRG was greater than that of 2 Hz. Furthermore, both TRPV1 agonist and P2X3 agonist may impair the level of EA analgesia. SIGNIFICANCE: EA has a parameter-specific effect on chronic inflammatory pain relief, which primarily depend on the stimulation frequency and not on the stimulation time at a certain stimulation time. The parameter-specific analgesic effect of EA is at least partially related to mediation of the protein level of TRPV1 and P2X3 expression in DRG of CFA rats.

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.

ATP-Gated P2X3 Receptors Are Specialised Sensors of the Extracellular Environment.

P2X3 receptors are ion channels expressed by autonomic and sensory nerves and specialised in transducing extracellular ATP signals. Structural data, together with functional and biochemical studies, suggest that conformational changes of P2X3 receptors upon agonist binding influence downstream intracellular molecular mechanisms relevant for neuronal responses. Activity of P2X3 receptors is implicated in pain, itch, asthma, cardiovascular dysfunction and other pathologies. The study of these receptors has therefore a large potential in the field of drug development and interdisciplinary efforts could clarify molecular mechanisms controlling P2X3 receptor function in different physiological or pathological contexts.

Urothelial Functional Protein and Sensory Receptors in Patients With Interstitial Cystitis/Bladder Pain Syndrome With and Without Hunner's Lesion.

OBJECTIVE: To investigate the urothelium function and sensory receptors difference between interstitial cystitis/bladder pain syndrome (IC/BPS) patients with or without Hunner's lesion. METHODS: Fourteen female IC/BPS patients with Hunner's lesion (Hunner IC) and 14 age-matched IC/BPS patients without Hunner's lesions (non-Hunner IC) were enrolled. Bladder mucosa biopsies were obtained. Bladder inflammation, eosinophil infiltration, and urothelial denudation were graded on a 4-point scale after staining with hematoxylin and eosin. Adhesive protein E-cadherin, tryptase, and zonula occuldens-1 in the bladder tissues were assessed with immunofluorescence staining. Urothelial muscarinic receptors M2, M3, endothelial nitric oxide synthase (eNOS), and purinergic receptor P2X3 were evaluated by Western blotting. RESULTS: Hunner IC patients had a significantly higher mean visual analog scale pain score and smaller cystometric bladder capacity than non-Hunner IC patients. The Hunner IC bladder specimens showed more severe or moderate eosinophilic infiltration and urothelial denudation than the non-Hunner IC bladder specimens did. The E-cadherin expression was significantly lower, and eNOS expression was significantly higher in the Hunner IC bladder samples than in the non-Hunner IC samples. The other functional proteins or sensory receptors did not differ between groups. CONCLUSION: Bladder inflammation and urothelial cell adhesion defects were more severe in the Hunner IC than that in the non-Hunner IC patients. eNOS was significantly higher in the Hunner IC than in the non-Hunner IC bladder samples, suggesting that eNOS expression difference may implicate different pathogenesis in 2 types of IC.

TRPV1, ASICs and P2X2/3 expressed in bone cells simultaneously regulate bone metabolic markers in ovariectomized mice.

OBJECTIVES: Nociceptors are expressed at peripheral terminals of neurons. Recent studies have shown that TRPV1, a nociceptor, is expressed in bone tissue and regulates bone metabolism. We have demonstrated that a TRPV1 antagonist improved pain-like behavior in ovariectomized (OVX) mice. The aim of this study was to determine whether nociceptors, including TRPV1, acid-sensing ion channel (ASIC) and P2X2/3 are expressed in bone cells, and to examine the effects of nociceptor antagonists on bone metabolism. METHODS: The expression of nociceptors in femoral bone tissue and cultured bone marrow cells in OVX and sham-operated mice were examined. The effects of nociceptor antagonists on the up-regulated expression of bone metabolic markers, Runx2, Osterix, osteocalcin and RANKL, were also examined. RESULTS: TRPV1, ASIC 2 and 3, and P2X2 and 3, were expressed in bone tissue and bone marrow cells, and the expression levels of ASIC1 and 2, and P2X2 were significantly increased in OVX mice in comparison with those in sham mice. Treatment with nociceptor antagonists significantly inhibited the expression of bone metabolic markers in OVX mice. CONCLUSION: An array of nociceptors, TRPV1, ASICs and P2X2/3, could simultaneously regulate not only increases in skeletal pain but also bone turnover in OVX mice.

Upregulated P2X3 Receptor Expression in Patients with Intractable Temporal Lobe Epilepsy and in a Rat Model of Epilepsy.

Purinergic P2X3 receptors (P2X3Rs) play extensive roles in nerve cells in the central nervous system, particularly in hyperexcitability and calcium (Ca(2+)) influx. However, the role of P2X3Rs in epilepsy has not been previously investigated. To determine the relationship between P2X3Rs and epilepsy, the expression and cellular location of P2X3Rs in patients with intractable temporal lobe epilepsy (TLE) and in a lithium chloride-pilocarpine-induced chronic rat model of epilepsy were assessed. Furthermore, the function of P2X3Rs was assessed in vitro. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis were used to evaluate the expression levels of P2X3Rs in brain tissues from TLE patients and an epileptic rat model, whereas immunofluorescence labeling was applied to determine the distribution of target proteins. Whole-cell recording was subsequently performed to identify the influence of P2X3Rs on seizure-like discharges. P2X3Rs were located at the cell bodies and dendrites of neurons with significantly increased expression in the TLE patients and epileptic rat model. In vitro, P2X3R activation accelerated sustained repetitive firing, whereas P2X3R inhibition led to relatively low-frequency discharges. To the best of our knowledge, this is the first study provide evidence that upregulated P2X3R expression exists in both epileptic humans and rats and may aggravate the epileptic state in vitro. Thus, P2X3Rs may represent a novel therapeutic target for antiepileptic drugs.

The role of P2X3 receptors in bilateral masseter muscle allodynia in rats.

AIM: To determine the relationship between bilateral allodynia induced by masseter inflammation and P2X3 receptor expression changes in trigeminal ganglia (TRG) and the influence of intramasseteric P2X3 antagonist administration on bilateral masseter allodynia. METHODS: To induce bilateral allodynia, rats received a unilateral injection of complete Freund's adjuvant (CFA) into the masseter muscle. Bilateral head withdrawal threshold (HWT) was measured 4 days later. Behavioral measurements were followed by bilateral masseter muscle and TRG dissection. Masseter tissue was evaluated histopathologically and TRG tissue was analyzed for P2X3 receptor mRNA expression by using quantitative real-time polymerase chain reaction (PCR) analysis. To assess the P2X3 receptor involvement in nocifensive behavior, two doses (6 and 60 µg/50 µL) of selective P2X3 antagonist A-317491 were administrated into the inflamed masseter muscle 4 days after the CFA injection. Bilateral HWT was measured at 15-, 30-, 60-, and 120-minute time points. RESULTS: HWT was bilaterally reduced after the CFA injection (P<0.001). Intramasseteric inflammation was confirmed ipsilaterally to the CFA injection. Quantitative real-time PCR analysis demonstrated enhanced P2X3 expression in TRG ipsilaterally to CFA administration (P<0.01). In comparison with controls, the dose of 6 µg of A-317491 significantly increased bilateral HWT at 15-, 30-, and 60-minute time points after the A-317491 administration (P<0.001), whereas the dose of 60 µg of A-317491 was efficient at all time points ipsilaterally (P=0.004) and at 15-, 30-, and 60-minute time points contralaterally (P<0.001). CONCLUSION: Unilateral masseter inflammation can induce bilateral allodynia in rats. The study provided evidence that P2X3 receptors can functionally influence masseter muscle allodynia and suggested that P2X3 receptors expressed in TRG neurons are involved in masseter inflammatory pain conditions.

Effect of electroacupuncture on P2X3 receptor regulation in the peripheral and central nervous systems of rats with visceral pain caused by irritable bowel syndrome.

The aim of this study is to investigate the role of the purinergic receptor P2X3 in the peripheral and central nervous systems during acupuncture treatment for the visceral pain of irritable bowel syndrome (IBS). A total of 24 8-day-old Sprague-Dawley (SD) neonatal male rats (SPF grade) were stimulated using colorectal distention (CRD) when the rats were awake. The modeling lasted for 2 weeks with one stimulation per day. After 6 weeks, the rats were randomly divided into three groups of eight each: (1) the normal group (NG, n = 8); (2) the model group (MG, n = 8); and (3) the model + electroacupuncture group (EA, n = 8) that received electroacupuncture at a needling depth of 5 mm at the Shangjuxu (ST37, bilateral) and Tianshu (ST25, bilateral) acupoints. The parameters of the Han's acupoint nerve stimulator (HANS) were as follows: sparse-dense wave with a frequency of 2/100 Hz, current of 2 mA, 20 min/stimulation, and one stimulation per day; the treatment was provided for seven consecutive days. At the sixth week after the treatment, the abdominal withdrawal reflex (AWR) score was determined; immunofluorescence and immunohistochemistry were used to measure the expression of the P2X3 receptor in myenteric plexus neurons, prefrontal cortex, and anterior cingulate cortex; and, a real-time PCR assay was performed to measure the expression of P2X3 messenger RNA (mRNA) in the dorsal root ganglion (DRG) and spinal cord. After stimulation with CRD, the expression levels of the P2X3 receptor in the inter-colonic myenteric plexus, DRG, spinal cord, prefrontal cortex, and anterior cingulate cortex were upregulated, and the sensitivity of the rats to IBS visceral pain was increased. Electroacupuncture (EA) could downregulate the expression of the P2X3 receptor and ease the sensitivity to visceral pain. The P2X3 receptor plays an important role in IBS visceral pain. The different levels of P2X3 in the peripheral enteric nervous system and central nervous system mediate the effects of the EA treatment of the visceral hyperalgesia of IBS.

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.

Sensory hyperinnervation and increase in NGF, TRPV1 and P2X3 expression in the epidermis following cast immobilization in rats.

BACKGROUND: Cast immobilization is known to induce pain in humans and experimental animal models; however, the detailed mechanisms underlying this pain have yet to be elucidated. Recently, several lines of evidence have indicated that morphological changes in sensory innervation and changes in the expression of pain-related molecules in the epidermis are related to certain painful conditions. The aim of the present study was to temporally investigate the histological changes in the glabrous skin of the rat hind paw after 1, 2 and 4 weeks of ankle joint immobilization by casting. METHODS: The von Frey test and the plantar test were performed to examine noxious sensitivity of the skin. Immunohistochemical methods were used to assess sensory nerve fibre profiles and to examine the expression of the nerve growth factor (NGF), transient receptor potential vanilloid 1 (TRPV1) and P2X3 in the epidermis. RESULTS: Cast immobilization produced a time-dependent increase in mechanical and thermal sensitivity. In the plantar skin of immobilized rats, both myelinated A fibres and unmyelinated C fibres were increased. NGF, TRPV1 and P2X3 expression levels in the epidermis were also increased. Although the level of NGF expression did not display a meaningful change throughout the immobilization period, other changes became remarkable, depending on the period of immobilization. CONCLUSIONS: The time course of the increase in peripheral nerve fibres and in the expression of TRPV1 and P2X3 paralleled the development of hypersensitivity, which suggests that histological changes of the skin following cast immobilization may have some relation to the resulting hypersensitivity.

Botulinum toxin A detrusor injections reduce postsynaptic muscular M2, M3, P2X2, and P2X3 receptors in children and adolescents who have neurogenic detrusor overactivity: a single-blind study.

OBJECTIVE: To analyze the effect of OnabotulinumtoxinA detrusor injections on postsynaptic muscular receptors in children and adolescents with neurogenic detrusor overactivity. MATERIALS AND METHODS: A bladder augmentation became necessary in 10 children and adolescents (7 males, 3 females; median age, 12 years) who had neurogenic detrusor overactivity. Seven had previously received 1 to 8 (average 3.86) OnabotulinumtoxinA detrusor injections, but their detrusor pressure could not be maintained at tolerable levels because of low-compliance bladder. The last injection session had been completed an average of 3 months (range, 1.5-3.5 months) previously. Three patients had never received that therapy and were considered controls. On the bladder dome resections, a specific receptor analysis (muscarinic M2 and M3 and purinergic P2X1, P2X2, and P2X3) was performed with confocal immunofluorescence, and nerve fiber density was analyzed with light-microscopic 3,3'-diaminobenzidine-immunohistochemical staining. RESULTS: Receptor analysis showed a downregulation of all examined receptors after OnabotulinumtoxinA injections; the reductions in M2, M3, P2X2, and P2X3 receptors reached a significance level of P <.05 (Mann-Whitney test). The ratios of means (OnabotulinumtoxinA-to-control) were 0.26 for M2, 0.33 for M3, 0.35 for P2X1, 0.19 for P2X2, and 0.37 for P2X3. CONCLUSION: OnabotulinumtoxinA detrusor injections led to significant reductions in muscular M2, M3, P2X2, and P2X3 receptors. The reductions probably affect the generated force in the urinary bladder and could contribute to the clinically observed increase in residual urine.

Expression and electrophysiological characteristics of P2X3 receptors in interstitial cells of Cajal in rats with partial bladder outlet obstruction.

UNLABELLED: WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: In the urinary bladder, histological studies suggest a network of functionally connected interstitial cells of Cajal (ICCs) are located between the urothelium and sensory nerve endings, which might transfer signals directly between them. Purinergic P2X3 receptors may play roles in the micturition reflex pathway, and its expression profiles in ICCs could be altered in urinary bladder dysfunction. The present experiments showed a novel time-dependent P2X3 receptor up-regulation in ICCs in an experimental rat model of partial bladder outlet obstruction. OBJECTIVE: To investigate the expression and electrophysiological characteristics of purinergic P2X3 receptors in interstitial cells of Cajal (ICCs) at different time points after partial bladder outlet obstruction (PBOO) in rats. MATERIALS AND METHODS: In all, 48 female Sprague-Dawley rats were randomly divided into four groups: 4, 6 and 8 week PBOO groups and sham-operated controls. At 4 weeks after surgery, cystometry was performed to investigate bladder function in vivo. Subsequently, the rats were humanely killed at 4, 6 or 8 weeks and the bladders were harvested for measurements. P2X3 expression in ICCs of bladder was investigated by immunofluorescent staining. The level of P2X3 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). Inward currents in corresponding ICCs after PBOO were investigated electrophysiologically. RESULTS: Cystometrography showed a valid increase in maximum detrusor pressure in rats subjected to PBOO. The bladder weight in the PBOO group was significantly higher than that in the control group. In contrast to sham rats, there was a significant increase in the intensity of P2X3 staining in the ICCs in all PBOO groups. C-kit labelled isolated ICCs were strongly stained with the P2X3 antibody. RT-PCR showed that the expression of P2X3 mRNA was significantly up-regulated at 4, 6 and 8 weeks in the ICCs from the PBOO rats. In the ICCs, the mean peak amplitude of inward currents was significantly increased in the PBOO groups compared with the control group. CONCLUSIONS: The expression of P2X3 receptors showed a time dependent up-regulation in the ICCs of the bladder in rats with PBOO. PBOO induced the potentiation of P2X3 receptors function, as evidenced by α, ß-methylene ATP-enhanced inward currents in the ICCs of the rat bladder.

The effects of electroacupuncture on the extracellular signal-regulated kinase 1/2/P2X3 signal pathway in the spinal cord of rats with chronic constriction injury.

BACKGROUND: Electroacupuncture (EA), as a traditional clinical method, is widely accepted in pain clinics, but the analgesic effect of EA has not been fully demonstrated. In the present study, we investigated the effect of EA on chronic pain and expression of P2X3 receptors in the spinal cord of rats with chronic constriction injury (CCI). METHODS: The study was conducted in 2 parts. In part 1, Sprague Dawley rats were divided into 6 groups (n = 10): sham-CCI, CCI, LEA; CCI + 2 Hz EA at acupoints), HEA; CCI + 15 Hz EA at acupoints), NA-LEA (CCI + 2 Hz EA at nonacupoints), and NA-HEA (CCI + 15 Hz EA at nonacupoints). EA treatment was performed once a day on days 4 to 9 after CCI. Nociception was assessed using von Frey filaments and a hotplate apparatus. The protein and the messenger RNA (mRNA) levels of P2X3 receptors in the spinal cord were assayed by Western blotting and real-time polymerase chain reaction, respectively. In part 2, rats were divided into 5 groups (n = 10): sham-CCI, CCI, EA (CCI + EA at acupoints), NA-EA (CCI + EA at nonacupoints), and U0126 (CCI + intrathecal injection of U0126). EA treatment was conducted similar to part 1. Rats were given 5 µg U0126 in the U0126 group and 5% dimethyl sulfoxide intrathecally. Ten microliters was used as a vehicle for the other 4 groups twice a day on days 4 to 9 after CCI. Extracellular signal-regulated kinase 1/2 (ERK1/2) and ERK1/2 phosphorylation in the spinal cord were also assayed by Western blotting. RESULTS: EA treatment exhibited significant antinociceptive effects and reduced the CCI-induced increase of both protein and mRNA expression of P2X3 receptors in the spinal cord. Furthermore, 2 Hz EA had a better analgesic effect than 15 Hz EA, and the protein and mRNA level of P2X3 receptor in spinal cord were lower in rats treated with 2 Hz EA at acupoints than 15 Hz EA at acupoints. Either EA at acupoints or intrathecal injection of U0126 relieved allodynia and hyperalgesia and reduced the expression of P2X3 receptors and ERK1/2 phosphorylation in the spinal cord. CONCLUSIONS: The data demonstrated that EA alleviates neuropathic pain behavior, at least in part, by reducing P2X3 receptor expression in spinal cord via the ERK1/2 signaling pathway. Low frequency EA has a better analgesic effect than high frequency HEA on neuropathic pain.

Targeted taste cell-specific overexpression of brain-derived neurotrophic factor in adult taste buds elevates phosphorylated TrkB protein levels in taste cells, increases taste bud size, and promotes gustatory innervation.

Brain-derived neurotrophic factor (BDNF) is the most potent neurotrophic factor in the peripheral taste system during embryonic development. It is also expressed in adult taste buds. There is a lack of understanding of the role of BDNF in the adult taste system. To address this, we generated novel transgenic mice in which transgene expression was driven by an α-gustducin promoter coupling BDNF expression to the postnatal expression of gustducin in taste cells. Immunohistochemistry revealed significantly stronger BDNF labeling in taste cells of high BDNF-expressing mouse lines compared with controls. We show that taste buds in these mice are significantly larger and have a larger number of taste cells compared with controls. To examine whether innervation was affected in Gust-BDNF mice, we used antibodies to neural cell adhesion molecule (NCAM) and ATP receptor P2X3. The total density of general innervation and specifically the gustatory innervation was markedly increased in high BDNF-expressing mice compared with controls. TrkB and NCAM gene expression in laser capture microdissected taste epithelia were significantly up-regulated in these mice. Up-regulation of TrkB transcripts in taste buds and elevated taste cell-specific TrkB phosphorylation in response to increased BDNF levels indicate that BDNF controls the expression and activation of its high affinity receptor in taste cells. This demonstrates a direct taste cell function for BDNF. BDNF also orchestrates and maintains taste bud innervation. We propose that the Gust-BDNF transgenic mouse models can be employed to further dissect the specific roles of BDNF in the adult taste system.

Prenatal expression of purinergic receptor P2X3 in human dorsal root ganglion.

The dorsal root ganglion (DRG) is consisted of neurons that relay multiple types of spinal sensory stimuli to the central nervous system. Several neuroactive molecules may be involved in sensory modulation especially pain processing at the DRG, including the purinergic receptor P2X3 and calcitonin-gene-related peptide (CGRP). P2X3 receptor has been considered a promising pharmaceutical target for the development of new pain medicine. Currently, litter is known about the expression of P2X3 in the human DRG. The present study characterized the localization of P2X3 in prenatal human DRG obtained from fetuses at 4-8 gestational months, by comparing to CGRP expression as well as binding pattern of isolectin-B4 (IB4), a marker of small DRG neurons presumably relevant to nociception. P2X3 immunoreactivity (IR) appeared in most neuron-like perikarya, with their numerical density reduced during the gestational period studied. P2X3 IR was co-labeled very commonly with IB4 binding and infrequently with CGRP IR and was not colocalized with IR for the gliocyte marker glutamine synthetase. Together, the data show an early and broad expression of P2X3 in prenatal human DRG neurons, pointing to a biological role of purinergic signaling during the development of spinal sensory system.

P2X3 receptor expression by HEK cells conditions their survival.

On nociceptive neurons, one important mechanism to generate pain signals is the activation of P2X3 receptors, which are membrane proteins gated by extracellular ATP. In this work, we have studied the recovery of recombinant P2X3 receptor expression in human embryonic kidney (HEK) cells. Our data demonstrated that HEK cells were not permissive for stable P2X3 expression, since the significant time-dependent cell loss. In vivo treatment with P2X3 receptor antagonist limited the effect. The expression of a single P2X3 point mutant Y393A, also largely accelerated cell death. We suggest the requirements of a permissive intracellular molecular machinery for appropriate receptor expression. The present report suggests that despite HEK cells are often used as recombinant expression system for the study a variety of receptors function, they represent a limiting permissive environment for P2X3 receptors.