Translocation of neuronal nitric oxide synthase to the plasma membrane by ATP is mediated by P2X and P2Y receptors
© Ohnishi et al; licensee BioMed Central Ltd. 2009
Received: 24 December 2008
Accepted: 20 July 2009
Published: 20 July 2009
The translocation of neuronal nitric oxide synthase (nNOS) from the cytosol to the membrane is functionally coupled to the activation of N-methyl-D-aspartate (NMDA) receptors at synapses. Whereas there is abundant evidence indicating that ATP and nitric oxide are involved in nociceptive transmission, whether nNOS is activated by ATP remains unknown. We recently established a fluorescence imaging system for examining nNOS translocation in PC12 cells expressing a yellow fluorescence protein-tagged nNOS N-terminal mutant, nNOSNT-YFP, and examined the effect of ATP on nNOS translocation using the system.
The translocation of nNOS was induced by ATP in the presence of NMDA and forskolin, an adenylate cyclase activator. The purinergic P2X receptor agonist 2-MeSATP and the P2Y agonist UTP significantly enhanced nNOS translocation; and simultaneous stimulation with 2-MeSATP and UTP exhibited the same concentration-response curve for the translocation as obtained with ATP. ATP, 2-MeSATP, and UTP increased the intracellular Ca2+ concentration ([Ca2+]i) in PC12 cells. Conversely, whereas the P2X receptor antagonist PPADS and the P2Y antagonist reactive blue-2 partially inhibited increases in the translocation of nNOS and [Ca2+]i by ATP, the non-selective P2 receptor antagonist suramin completely blocked them. In addition, the increase in the nNOS translocation by ATP was blocked by NMDA receptor antagonists and inhibitors of protein kinase A, protein kinase C, and Src kinase. Consistent with the expression of P2X and P2Y receptors in the spinal cord, ATP and UTP increased the [Ca2+]i in primary cultured spinal neurons. ATP potentiated and prolonged the [Ca2+]i increase produced by NMDA in the dorsal horn of the spinal cord. Furthermore, the selective P2X3/P2X2/3 antagonist A-317491 inhibited nNOS activation assessed by NO formation in spinal slices prepared from neuropathic pain model mice.
ATP is involved in nNOS translocation mediated by protein kinase C via activation of P2X and P2Y receptors and nNOS translocation may be an action mechanism of ATP in nocieptive processing in the spinal cord.
Adenine and uridine nucleotides are present in tissues and released from all different types of cells in the nervous system as well as from damaged tissues in the periphery under pathophysiological conditions. The released nucleotides are implicated in diverse sensory processes including pain transmission via purinergic P2X and P2Y receptors [1, 2]. To date 7 ionotropic P2X receptors  and 8 G-protein-coupled metabotropic P2Y receptors  have been cloned, and most of them are expressed on primary afferent neurons or spinal dorsal horn neurons. Exogenous administration of ATP and P2X-receptor agonists into the hind paw caused short-lasting nocifensor behaviors and thermal hyperalgesia [5, 6], as well as relatively long-lasting mechanical allodynia , in rodents. On the other hand, P2 antagonists including A-317491, a selective P2X3/P2X2/3-receptor antagonist decreased various nociceptive behaviors, inflammatory hyperalgesia, and neuropathic pain [8–11]. P2X3-deficient mice have reduced pain-related behaviors in the formalin test . Tsuda et al. also reported that the increased expression of P2X4-receptors induced by nerve injury or ATP stimulation in the spinal microglia produced allodynia .
In the central nervous system, nitric oxide (NO) is produced by neuronal NO synthase (nNOS) following the influx of Ca2+ through N-methyl-D-aspartate (NMDA) receptors [14–16], and has been implicated in synaptic plasticity such as central sensitization in the spinal cord [17, 18]. Co-localization of nNOS with NMDA receptors at the postsynaptic density (PSD) suggests that NMDA-receptor activity may be coupled to nNOS activation by a close spatial interaction . We recently showed that the increase in nNOS activity in the superficial dorsal horn of the spinal cord reflects a neuropathic pain state even 1 week after nerve injury  and that this nNOS activation may be reversibly regulated by the translocation of nNOS from the cytosol to the plasma membrane in the presence of NMDA and the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) . Unlike endothelial and inducible NOSs that anchor to the membrane by lipid modification, nNOS is unique in having an ~ 250 a.a. N-terminal extension containing a PSD-95/disc large/zonula occludens-1 (PDZ) domain and is recruited to membranes via protein-protein interactions [15, 16]. We recently constructed a yellow fluorescence protein (YFP)-tagged nNOS N-terminal mutant encompassing amino acid residues 1–299 (nNOSNT-YFP) and succeeded in visualizing its translocation by co-stimulation with NMDA and PACAP in PC12 cells stably expressing it . Thereby we demonstrated that PACAP was involved in nNOS translocation through the activation of both protein kinase C (PKC) following calcium mobilization and protein kinase A (PKA) mediated by PACAP receptor 1. ATP acts as an excitatory neurotransmitter in the dorsal horn of the spinal cord . The activation of P2X receptors not only mediates but also facilitates excitatory transmission, releasing glutamate from primary afferent fibers in the spinal cord [24, 25]. In the present study, we demonstrated that ATP could translocate nNOS from the cytosol to the plasma membrane mediated by PKC via activation of P2X and P2Y receptors in the presence of NMDA and forskolin, an adenylate cyclase activator, by using a fluorescence imaging system.
PC12 cells and PC12 cells stably expressing nNOSNT-YFP (PC12N cells) were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 5% fetal calf serum, 10% horse serum, 50 U/ml penicillin, and 50 μg/ml streptomycin at 37°C in a 5% CO2 atmosphere. The chemicals used and their sources were as follow: NMDA, MK-801, 2-amino-5-phosphonovaleric acid (APV), calphostin C, PP2, suramin, pyridoxal-phosphate-6-azophenyl-2',4'-sulfonic acid (PPADS), 2-(methylthio) adenosine 5'-triphosphate (2-MeSATP), 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran- 4-one (LY294002), 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059), reactive blue-2 (RB-2), A-317491, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580), cytosine β-D-arabinofuranoside (Ara-C), Dnase I, and 8-bromo-cAMP (8-Br-cAMP) from Sigma (St. Louis, MO, USA); 8-Br-cGMP from Calbiochem (La Jolla, CA, USA); nerve growth factor (NGF), roscovitine, and UTP from Wako Pure Chemicals (Osaka, Japan); ATP from Oriental Yeast Co. (Tokyo, Japan); 1-[N, O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) and N-[2-(4-bromocinnamylamino)-ethyl]-5-isoquinoline (H-89) from Seikagaku Kogyo (Tokyo, Japan); fura-2 acetoxymethyl ester and NG-nitro-L-arginine methyl ester (L-NAME) from Dojindo (Kumamoto, Japan); diaminorhodamine-4M acetoxymethyl ester (DAR-4M AM) from Daiichi Pure Chemicals (Tokyo, Japan); and PACAP from Peptide Institute (Osaka, Japan). Other chemicals were of reagent grade.
Male ddY mice were purchased from Shizuoka Laboratory Centre (Hamamatsu, Japan). The animals were housed under conditions of a 12-h light-darkness cycle, a constant temperature of 22 ± 2°C and 60 ± 10% humidity. They were allowed free access to food and water before testing. All animal experiments were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals and were approved by the Animal Experimentation Committee of Kansai Medical University.
nNOSNT-YFP translocation assay
PC12N cells were plated on poly-L-lysine-coated glass-bottomed 35-mm dishes at a density of 1 × 104 cells/cm2 and caused to differentiate by 5-day treatment with 50 ng/ml NGF. Translocation of nNOSNT-YFP was examined in the cells essentially as reported previously . Briefly, after a 30-min incubation with test agents, the cells were rinsed with phosphate-buffered saline, fixed with 4% paraformaldehyde in 0.12 M sodium phosphate buffer, pH 7.4, for 20 min at room temperature, and then rinsed with phosphate-buffered saline. Digital images were captured on a Zeiss LSM510 laser-scanning confocal microscope (Oberkochen, Germany). The intensity of nNOSNT-YFP fluorescence was quantified by using ImageJ. To evaluate the translocation of nNOSNT-YFP to the plasma membrane in PC12N cells, we counted the number of cells possessing foci of nNOSNT-YFP on their plasma membrane and expressed this number as a percentage of the total cells examined. More than 40 cells were observed for each datum point, and at least 4 experiments were carried out in each analysis.
The isolation and primary culture of neurons were prepared from the spinal cord of E13-E15 ddY mice. Briefly, after the pregnant animals had been anesthetized with isoflurane, the spinal cords below the cervical segment were collected from embryos under sterile technique and placed in ice-cold phosphate-buffered saline containing 0.1% glucose. The spinal cords were minced with scissors in 5 ml of DMEM containing 0.5 mg/ml of trypsin and kept at 37°C for 30 min. After incubation, 5 ml of DMEM containing 10% fetal calf serum, 40 ng/ml gentamicin, and 50 μl DNase I were added. The cell pellet was suspended in 5 ml of DMEM containing 10% fetal calf serum and 40 ng/ml gentamicin, filtrated and centrifuged for 5 min at 1000 rpm. After washing the cell pellet twice, the spinal neurons were plated on 35-mm dishes at 1 × 105 cells/ml. The cells were kept in DMEM containing 10% fetal calf serum, 20 ng/ml NGF, 40 ng/ml gentamicin and 10 μM Ara-C at 37°C in a CO2 incubator for 24 h, replaced with fresh media without Ara-C, and cultured for 3–7 days until use.
Measurement of intracellular free Ca2+ concentration ([Ca2+]i) and cAMP content
Changes in [Ca2+]i in NGF-differentiated PC12 cells, spinal neurons and spinal slices were measured as described previously [21, 22]. After the cells had been incubated with 5 μM fura-2acetoxymethyl ester for 30 min in DMEM containing 5% fetal calf serum and 10% horse serum, the fura-2-loaded cells on an inverted fluorescence microscope (Olympus IX-70, Tokyo, Japan) were stimulated with ATP, 2-MeSATP or UTP in HEPES-buffered saline solution (HBS) or in Ca2+-free HBS supplemented with 6 mM EGTA at 2–3 ml/min. The cells were excited at 340 and 380 nm and the fluorescence emission signal was monitored by using an Aquacosmos-Ratio imaging system (Hamamatsu Photonics, Hamamatsu, Japan) with a cooled charge-coupled device camera. [Ca2+]i was expressed as a ratio of fluorescence emission intensity at 340 and 380 nm. For the measurement of [Ca2+]i changes in spinal slices, lumbosacral segments prepared from 7-14-day-old ddY mice were cut using a vibrating blade microtome (VT-1000S; Leica, Nussloch, Germany), and slices were incubated for 2 h in artificial cerebrospinal fluid bubbled with 95% O2 and 5% CO2 at 37°C. The slices (350-μm thick) thus obtained from lumbar segments L4-L6 were used for [Ca2+]i measurements.
For measurement of cAMP contents, NGF-differentiated PC12 cells were incubated for 15 min with test agents in HBS containing 0.5 mM 3-iso-butyl-1-methylxanthine, and intracellular cAMP levels were determined by use of a cAMP radioimmunoassay kit (GE Healthcare, Piscataway, NJ, USA).
Measurement of NO in spinal slices
Neuropathic pain mode was prepared by left L5 spinal nerve transection of 3-week-old ddY mice and slices were prepared from the lumbar spinal cord of the neuropathic pain model mice 7 days after operation as described previously . After a 2-h incubation of the slices in artificial cerebrospinal fluid containing 1 mM L-NAME bubbled with 95% O2/5% CO2 at 37°C, slices were incubated in artificial cerebrospinal fluid containing 10 μM DAR-4M AM and 1 mM L-NAME for 2 h at room temperature. The slices were kept in a Krebs solution containing 1 mM L-NAME for more than 1 h after loading, and then were placed in the recording chamber. NO generation in spinal slices was started by replacement of 1 mM L-NAME with 1 mM arginine in a Krebs superfusion buffer equilibrated with 95% O2/5% CO2. The slices were excited at 540 ± 10 nm and the fluorescence emission signal with a >590-nm long-pass filter was monitored by using the Aquacosmos-Ratio imaging system. Increase in NO formation in the spinal cord was expressed as the ratio of fluorescence intensity of DAR-4M to that prior to treatment.
Reverse transcriptase-polymerase chain reaction (RT-PCR)
Primer sequences employed for RT-PCR analysis
Primer sequences (5' to 3')
Product length (bp)
TGGCG TTCTG GGTAT TAAGA TCGG
CAGTG GCCTG GTCAC TGGCG A
GAGGC ATCAT GGGTA TCCAG ATCAA G
GAGCG GGGTG GAAAT GTAAC TTTAG
CGATT CACTC TCCAG TCCG
GGTCC TCCAG TAGAA ACCG
CGCTT CAACG AGGAC TTCA
CCATG AGCAC GTAAC AGAC
GTTGC CTATG AGCTA TGCAG
ACCAT GACTG CCGAA CTGAA
GGAGA CCTTG CCTGC CGCCT GGTA
TACCA CGACA GCCAT ACGGG CCGC
TTCTA CAATG AGCTG CGTGT GGC
CTC(A/G)T AGCTC TTCTC CAGGG AGGA
All data were presented as the mean ± SEM. Statistical analyses of the results were made by using Student's t test or the Mann-Whitney U test. EC50 values were calculated by use of the computer program for graded responses (ver 1.2) http://chiryo.phar.nagoya-cu.ac.jp/javastat/Graded50-j.htm.
Translocation of nNOS from the cytosol to the plasma membrane by ATP
Involvement of P2 and NMDA receptors in nNOS translocation by ATP
To examine whether the activation of NMDA receptors was involved in the nNOSNT-YFP translocation with ATP in the presence of NMDA and forskolin, we next examined the effect of NMDA receptor antagonists on nNOS translocation in PC12N cells (Figure 2). The increase in the translocation was completely blocked by NMDA receptor antagonists MK-801 and APV. These results demonstrate that the activation of both P2 and NMDA receptors was required for the nNOS translocation by ATP in the presence of NMDA and forskolin.
Involvement of P2X receptors or P2Y receptors in nNOS translocation by ATP
Signal transduction coupled to ATP and involved in nNOS translocation
Signaling pathways involved in nNOS translocation by ATP in PC12N cells
To further clarify the signal pathways involved in the translocation in PC12N cells, we examined the effect of other kinase inhibitors, LY294002 (phosphatidylinositol 3-kinase), PD98059 (extracellular signal-regulated kinase), SB203580 (p38 mitogen-activated protein kinase), and roscovitine (cyclin-dependent kinase 5) on the translocation of nNOSNT-YFP induced by ATP, NMDA, and forskolin (Figure 6B). None of them significantly attenuated the translocation. These results confirmed our recent findings , demonstrating that nNOS translocation is mediated by activation of PKA, PKC, and Src kinase in PC12 cells.
Expression of P2X and P2Y receptors in PC12N cells and spinal cord
Characterization of [Ca2+]i responses to ATP and UTP in cultured spinal neurons
Effect on P2X3 antagonist on NO formation in the spinal cord of neuropathic pain model mice
nNOS translocation by ATP
NO is well known to be implicated in nociceptive processing in the spinal cord . Based on the results of a study using mice lacking the PACAP gene , we previously suggested that PACAP is a key molecule of pain hypersensitivity that acts by promoting the functional coupling of nNOS to NMDA receptors. To address this suggestion, we recently established a fluorescence imaging system for examining nNOS translocation in PC12N cells and demonstrated that the synergism of PACAP and NMDA was critical for the translocation and activation of nNOS through PKA, PKC, and Src kinase via PACAP receptor 1 and NMDA receptors . Purinergic signaling is involved in long-term inflammatory and neuropathic pain as well as in acute pain [1, 2] and ATP increases the intracellular Ca2+ level in differentiated PC12 cells . Different from the synergism of PACAP and NMDA, ATP and NMDA could not translocate nNOSNT-YFP in PC12N cells (Figure 1). This can be explained by the difference in the ability of cAMP formation between PACAP and ATP (Figure 5A). Therefore nNOS was translocated by ATP and NMDA in the presence of forskolin (Figure 1B) or the membrane-permeable cAMP analogue 8-Br-cAMP (Figure 5B). As observed in our recent study , nNOSNT-YFP was localized to the membrane in 10–12% of the cells before stimulation and maximally in 25–30% of the cells after the stimulation with 100 μM NMDA and 1 nM PACAP. Since nNOSNT-YFP was over-expressed in PC12N cells, the reason that the maximum translocation was at most 25–30% of the cells might be ascribed to the nature of nNOS translocation by forming a tertiary complex with endogenous PSD-95 and NMDA receptors on the membrane. In fact, a nNOSNT-YFP mutant lacking the β-finger of the PDZ domain of nNOS was scarcely localized to the membrane before stimulation; and the translocation to the membrane was not increased by stimulation with NMDA and PACAP. Furthermore, NO formation by PACAP and NMDA was markedly attenuated in PC12N cells, probably because over-expressed nNOSNT-YFP competed with endogenous nNOS for forming the tertiary complex on the membrane. Consistent with our recent findings , the inhibitors of PKA, PKC, and Src kinase blocked the enhancement of nNOSNT-YFP translocation by ATP, forskolin, and NMDA (Figure 6A). Although P2X and P2Y receptors activate several signaling pathways, the inhibitors of other kinases failed to block the translocation of nNOSNT-YFP (Figure 6B). The present study extended our recent studies by use of the fluorescence imaging system and confirmed that the activation of Ca2+/PKC and cAMP/PKA pathways is necessary and sufficient for nNOS translocation in the presence of NMDA.
Characterization of purinergic receptors involved in nNOS translocation
ATP acts via various subtypes of ionotropic P2X receptors and/or metabotropic P2Y receptors. The translocation of nNOSNT-YFP by ATP was completely blocked by the non-selective P2 receptor antagonist suramin and significantly reduced by the P2X antagonist PPADS or the P2Y antagonist RB-2 (Figure 2). Conversely, 2-MeSATP, a P2X receptor agonist or UTP, a P2Y2 and P2Y4 receptor agonist, stimulated the translocation of nNOSNT-YFP to the plasma membrane (Figure 3A). However, the increase in the translocation of nNOSNT-YFP by 2-MeSATP or UTP was not as great as that obtained with ATP itself. Co-stimulation with 2-MeSATP and UTP increased the translocation of nNOSNT-YFP in a concentration-dependent manner, with the increase being comparable to that obtained with ATP and with similar EC50 values (Figure 3B). ATP, 2-MeSATP, and UTP induced the increase in [Ca2+]i with EC50 values of 3.5, 10.8, and 2.84 μM, respectively, in PC12N cells (Figure 4A); and ATP could increase the [Ca2+]i in the absence of extracellular Ca2+(Figure 4B), demonstrating that P2Y receptors as well as P2X receptors were involved in the increase in the [Ca2+]i of PC12N cells. Furthermore, it is intriguing that, whereas the non-selective P2 receptor antagonist suramin fully suppressed the ATP-induced increase in [Ca2+]i, PPADS and RB-2 reduced it only partially (Figure 4C). Consistent with the results of a previous study , P2X1, P2X3, and P2X4 mRNAs were detected by RT-PCR in both undifferentiated and NGF-differentiated PC12 cells; and P2X6 mRNA was detected in NGF-differentiated PC12 cells (Figure 7). Since P2X2 receptors were cloned from NGF-differentiated PC12 cells , P2X receptors except for P2X7 are present in NGF-differentiated PC12 cells. As for metabotropic P2Y receptors, besides P2Y12 receptors that couple to Gi/o, the other P2Y receptors P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 are coupled to the Gq/11 proteins, leading to stimulation of phosphoinositide metabolism, release of intracellular Ca2+ stores and activation of PKC. P2Y2, P2Y4, and P2Y6 mRNAs were found in both undifferentiated and NGF-differentiated PC12 cells. Since the P2Y6 receptor was not activated by UTP, ATP and UTP may act on P2Y2 and P2Y4 receptors in NGF-differentiated PC12N cells. Arslan et al. extensively characterized Ca2+ responses in PC12 cells and suggested that P2X2/P2X2b, P2X4, and P2Y2 receptors contribute to the ATP-induced [Ca2+]i increase in NGF-differentiated PC12 cells . Thus the presence of multiple P2X and P2Y receptors in PC12 cells suggests that several P2 receptor subtypes are involved in the increase in [Ca2+]i and nNOS translocation and that the effects of ATP on them seem to be additive.
Like PC12 cells, many P2X and P2Y receptors mRNAs were detected by RT-PCR in the spinal cord (Figure 7). To clarify the involvement of P2 receptors in nNOS translocation in the spinal cord, we examined the effect of ATP and UTP on [Ca2+]i in primary cultured spinal neurons. While ATP and UTP increased the magnitude of [Ca2+]i levels in a concentration-dependent manner, ATP was more potent than UTP (Figure 9A), suggesting that both P2X and P2Y receptors are involved in [Ca2+]i changes by ATP in the spinal cord. This notion was supported by the effect of antagonists on the ATP-evoked [Ca2+]i responses (Figure 9B). While the P2Y receptor antagonist RB-2 inhibited 41%, the non-selective P2 receptor antagonist suramin reduced the increase in 78% of the cells responsive to 1 μM ATP. Interestingly, A-317491, a selective P2X3 antagonist, reduced only 6.8% of the ATP-responsive cells. These results are consistent with the previous studies that P2X3 receptors are expressed in DRG neurons and are transported to the central process in the spinal cord [28, 29].
Significance of NO formation by ATP in nociceptive transmission
There is growing evidence that purinergic receptors in the central and peripheral nervous systems are involved in modification of pain sensation [1, 2]. NO production following activation of P2 receptors has been shown to exhibit both anti-nociceptive and pro-nociceptive effects. The present study showed that ATP increased the translocation of nNOSNT-YFP in PC12N cells in the presence of NMDA and forskolin, suggesting the generalization that the simultaneous activation of PKA, PKC, and Src-family kinase is essential for the translocation of nNOSNT-YFP to the membrane. Although mechanisms of synergism of ATP and NMDA for nNOS translocation (Figure 2) remain to be clarified, the [Ca2+]i increase was enhanced and prolonged by simultaneous stimulation of NMDA and ATP both in the superficial layer and deeper layers of the spinal cord in situ (Figure 10B–D) In behavioral studies, platelet-activating factor-evoked tactile allodynia is mediated by ATP and the subsequent NMDA and NO cascade through capsaicin-sensitive fibers . PPADS reduces pain-related behaviors by inhibiting the increased activity of the NO/NOS system in a neuropathic pain model  Inoue's group recently showed that mice lacking all isoforms of NOS displayed a reduction in nerve injury-induced neuropathic pain and exhibited a reduction in microglia activation in the spinal cord . We previously showed that the increase in nNOS activity in the superficial dorsal horn of the spinal cord reflects a neuropathic pain state even 1 week after nerve injury  and that this nNOS activation may be reversibly regulated by the translocation of nNOS from the cytosol to the plasma membrane in the presence of NMDA and PACAP . Consistent with our previous study , NO formation in the dorsal horn was more prominent in the ipsilateral side to L5-spinal nerve transection 7 days after operation and this NO formation was markedly blocked by A-317491 (Figure 11). Thus the present study also demonstrates that ATP might be involved in long-term neuropathic pain by promoting the functional coupling of nNOS to NMDA receptors under conditions in which neuromodulators such as prostaglandin E2 that couple to the cAMP/PKA pathway are released by continuous noxious inputs from the periphery. Since the stimulation of a single neuron or glia may activate multiple networks, a concomitant stimulation of facilitatory and inhibitory circuits as a result of ATP release is possible.
In the present study, we demonstrated that ATP could translocate nNOS from the cytosol to the plasma membrane in the presence of NMDA and forskolin by using a fluorescence imaging system. Moreover, the translocation of nNOSNT-YFP to the plasma membrane by ATP was additively mediated by PKC via activation of P2X and P2Y receptors. In a neuropathic pain model, nNOS was in an activated state, which was blocked by the P2X3/P2X2/3antagonist. The present study suggests that nNOS translocation may be an action mechanism of ATP in nocieptive processing in the spinal cord.
This work was supported in part by grants from the programs Grants-in-Aid for COE Research and Scientific Research on Priority Areas from the Ministry Education, Culture, Sports, Science and Technology of Japan, and Grants-in-Aid for Scientific Research (S) and (C) from Japan Society for the Promotion of Science and Japan Foundation of Applied Enzymology.
- Kennedy C, Assis TS, Currie AJ, Rowan EG: Crossing the pain barrier: P2 receptors as targets for novel analgesics. J Physiol 2003, 553: 683–694. 10.1113/jphysiol.2003.049114PubMed CentralPubMedView ArticleGoogle Scholar
- Burnstock G: Purinergic P2 receptors as targets for novel analgesics. Pharmacol Ther 2006, 110: 433–454. 10.1016/j.pharmthera.2005.08.013PubMedView ArticleGoogle Scholar
- North RA, Surprenant A: Pharmacology of cloned P2X receptors. Annu Rev Pharmacol Toxicol 2000, 40: 563–580. 10.1146/annurev.pharmtox.40.1.563PubMedView ArticleGoogle Scholar
- Abbracchio MP, Burnstock G, Boeynaems JM, Barnard EA, Boyer JL, Kennedy C, Knight GE, Fumagalli M, Gachet C, Jacobson KA, Weisman GA: International Union of Pharmacology LVIII: update on the P2Y G protein-coupled nucleotide receptors: from molecular mechanisms and pathophysiology to therapy. Pharmacol Rev 2006, 58: 281–341. 10.1124/pr.58.3.3PubMed CentralPubMedView ArticleGoogle Scholar
- Bland-Ward PA, Humphrey PPA: Acute nociception mediated by hindpaw P2X receptor activation in the rat. Br J Pharmacol 1997, 122: 365–371. 10.1038/sj.bjp.0701371PubMed CentralPubMedView ArticleGoogle Scholar
- Wismer CT, Faltynek CR, Jarvis MF, McGaraughty S: Distinct neurochemical mechanisms are activated following administration of different P2X receptor agonists into the hindpaw of a rat. Brain Res 2003, 965: 187–193. 10.1016/S0006-8993(02)04193-8PubMedView ArticleGoogle Scholar
- Tsuda M, Koizumi S, Kita A, Shigemoto Y, Ueno S, Inoue K: Mechanical allodynia caused by intraplantar injection of P2X receptor agonist in rats: involvement of heteromeric P2X 2/3 receptor signaling in capsaicin-insensitive primary afferent neurons. J Neurosci 2000, 20: RC90.PubMedGoogle Scholar
- Tsuda M, Ueno S, Inoue K: Evidence for the involvement of spinal endogenous ATP and P2X receptors in nociceptive responses caused by formalin and capsaicin in mice. Br J Pharmacol 1999, 128: 1497–1504. 10.1038/sj.bjp.0702960PubMed CentralPubMedView ArticleGoogle Scholar
- Jarvis MF, Burgard EC, McGaraughty S, Honore P, Lynch K, Brennan TJ, Subieta A, van Biesen T, Cartmell J, Bianchi B, Niforatos W, Kage K, Yu H, Mikusa J, Wismer CT, Zhu CZ, Chu K, Lee CH, Stewart AO, Polakowski J, Cox BF, Kowaluk E, Williams M, Sullivan J, Faltynek C: A-31 a novel potent and selective non-nucleotide antagonist of P2X 3 and P2X 2/3 receptors, reduces chronic inflammatory and neuropathic pain in the rat. Proc Natl Acad Sci USA 7491, 99: 17179–17184. 10.1073/pnas.252537299View ArticleGoogle Scholar
- McGaraughty S, Wismer CT, Zhu CZ, Mikusa J, Honore P, Chu KL, Lee CH, Faltynek CR, Jarvis MF: Effects of A-31 a novel and selective P2X 3 /P2X 2/3 receptor antagonist, on neuropathic, inflammatory and chemogenic nociception following intrathecal and intraplantar administration. Br J Pharmacol 7491, 140: 1381–1388. 10.1038/sj.bjp.0705574View ArticleGoogle Scholar
- Chen Y, Li GW, Wang C, Gu Y, Huang LYM: Mechanisms underlying enhanced P2X receptor-mediated responses in the neuropathic pain state. Pain 2005, 119: 38–48. 10.1016/j.pain.2005.09.007PubMedView ArticleGoogle Scholar
- Cockayne DA, Hamilton SG, Zhu QM, Dunn PM, Zhong Y, Novakovic S, Malmberg AB, Cain G, Berson A, Kassotakis L, Hedley L, Lachnit WG, Burnstock G, McMahon SB, Ford APWD: Urinary bladder hyporeflexia and reduced pain-related behaviour in P2X 3 -deficient mice. Nature 2000, 407: 1011–1015. 10.1038/35039519PubMedView ArticleGoogle Scholar
- Tsuda M, Shigemoto-Mogami Y, Koizumi S, Mizokoshi A, Kohsaka S, Salter MW, Inoue K: P2X 4 receptors induced in spinal microglia gate tactile allodynia after nerve injury. Nature 2003, 424: 778–783. 10.1038/nature01786PubMedView ArticleGoogle Scholar
- Garthwaite J, Charles SL, Chess-Williams R: Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as intercellular messenger in the brain. Nature 1988, 336: 385–388. 10.1038/336385a0PubMedView ArticleGoogle Scholar
- Sattler R, Xiong Z, Lu WY, Hafner M, MacDonald JF, Tymianski M: Specific coupling of NMDA receptor activation to nitric oxide neurotoxicity by PSD-95 protein. Science 1999, 284: 1845–1848. 10.1126/science.284.5421.1845PubMedView ArticleGoogle Scholar
- Oess S, Icking A, Fulton D, Govers R, Müller-Esterl W: Subcellular targeting and trafficking of nitric oxide synthases. Biochem J 2006, 396: 401–409. 10.1042/BJ20060321PubMed CentralPubMedView ArticleGoogle Scholar
- Meller ST, Gebhart GF: Nitric oxide (NO) and nociceptive processing in the spinal cord. Pain 1993, 52: 127–136. 10.1016/0304-3959(93)90124-8PubMedView ArticleGoogle Scholar
- Ji RR, Kohno T, Moore KA, Woolf CJ: Central sensitization and LTP: do pain and memory share similar mechanisms? Trends Neurosci 2003, 26: 696–705. 10.1016/j.tins.2003.09.017PubMedView ArticleGoogle Scholar
- Valtschanoff JG, Weinberg RJ: Laminar organization of the NMDA receptor complex within the postsynaptic density. J Neurosci 2001, 21: 1211–1217.PubMedGoogle Scholar
- Mabuchi T, Matsumura S, Okuda-Ashitaka E, Kitano T, Kojima H, Nagano T, Minami T, Ito S: Attenuation of neuropathic pain by the nociceptin/orphanin FQ antagonist JTC-801 is mediated by inhibition of nitric oxide production. Eur J Neurosci 2003, 17: 1384–1392. 10.1046/j.1460-9568.2003.02575.xPubMedView ArticleGoogle Scholar
- Mabuchi T, Shintani N, Matsumura S, Okuda-Ashitaka E, Hashimoto H, Muratani T, Minami T, Baba A, Ito S: Pituitary adenylate cyclase-activating polypeptide is required for the development of spinal sensitization and induction of neuropathic pain. J Neurosci 2004, 24: 7283–7291. 10.1523/JNEUROSCI.0983-04.2004PubMedView ArticleGoogle Scholar
- Ohnishi T, Okuda-Ashitaka E, Matsumura S, Katano T, Nishizawa M, Ito S: Characterization of signaling pathway for the translocation of neuronal nitric oxide synthase to the plasma membrane by PACAP. J Neurochem 2008, 105: 2271–2285. 10.1111/j.1471-4159.2008.05325.xPubMedView ArticleGoogle Scholar
- Bardoni R, Goldstein PA, Lee CJ, Gu JG, MacDermott AB: ATP P 2X receptors mediate fast synaptic transmission in the dorsal horn of the rat spinal cord. J Neurosci 1997, 17: 5297–5304.PubMedGoogle Scholar
- Gu JG, MacDermott AB: Activation of ATP P2X receptors elicits glutamate release from sensory neuron synapses. Nature 1997, 389: 749–753. 10.1038/39639PubMedView ArticleGoogle Scholar
- Nakatsuka T, Gu JG: ATP P2X receptor-mediated enhancement of glutamate release and evoked EPSCs in dorsal horn neurons of the rat spinal cord. J Neurosci 2001, 21: 6522–6531.PubMedGoogle Scholar
- Arslan G, Filipeanu CM, Irenius E, Kull B, Clementi E, Allgaier C, Erlinge D, Fredholm BB: P2Y receptors contribute to ATP-induced increases in intracellular calcium in differentiated but not undifferentiated PC12 cells. Neuropharmacology 2000, 39: 482–496. 10.1016/S0028-3908(99)00141-0PubMedView ArticleGoogle Scholar
- Brake AJ, Wagenbach MJ, Julius D: New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor. Nature 1994, 371: 519–523. 10.1038/371519a0PubMedView ArticleGoogle Scholar
- Vulchanova L, Riedl MS, Shuster SJ, Stone LS, Hargreaves KM, Buell G, Surprenant A, North RA: P2X 3 is expressed by DRG neurons that terminate in inner lamina II. Eur J Neurosci 1998, 10: 3470–3478. 10.1046/j.1460-9568.1998.00355.xPubMedView ArticleGoogle Scholar
- Kobayashi K, Fukuoka T, Yamanaka H, Dai Y, Obata K, Tokunaga A, Noguchi K: Differential expression patterns of mRNAs for P2X receptor subunits in neurochemically characterized dorsal root ganglion neurons in the rat. J Comp Neurol 2005, 481: 377–390. 10.1002/cne.20393PubMedView ArticleGoogle Scholar
- Morita K, Morioka N, Abdin J, Kitayama S, Nakata Y, Dohi T: Development of tactile allodynia and thermal hyperalgesia by intrathecally administered platelet-activating factor in mice. Pain 2004, 111: 351–359. 10.1016/j.pain.2004.07.016PubMedView ArticleGoogle Scholar
- Martucci C, Trovato AE, Costa B, Borsani E, Franchi S, Magnaghi V, Panerai AE, Rodella LF, Valsecchi AE, Sacerdote P, Colleoni M: The purinergic antagonist PPADS reduces pain related behaviours and interleukin-1β, interleukin-6, iNOS and nNOS overproduction in central and peripheral nervous system after peripheral neuropathy in mice. Pain 2008, 137: 81–95. 10.1016/j.pain.2007.08.017PubMedView ArticleGoogle Scholar
- Kuboyama K, Tsuda M, Tsutsui M, Toyohira Y, Tozaki-Saitoh H, Shimokawa H, Yanagihara N, Inoue K: Reduced spinal microglia activation and neuropathic pain after nerve injury in mice lacking all three nitric oxide synthases. [abstract]. The 3rd Asian Pain Symposium 2008, P33.Google Scholar
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