Shinya Ueno

Cell type‐specific ATP‐activated responses in rat dorsal root ganglion neurons

The aim of our study is to clarify the relationship between expression pattern of P2X receptors and the cell type of male adult rat (Wistar) dorsal root ganglion (DRG) neurons. We identified the nociceptive cells of acutely dissociated DRG neurons from adult rats type using capsaicin sensitivity. Two types of ATP‐activated currents, one with fast, the other with slow desensitization, were found under voltage‐clamp conditions. In addition, cells with fast but not slow desensitization responded to capsaicin, indicating that there was a relationship between current kinetics and capsaicin‐sensitivity. Both types of neurons were responsive to ATP and α, β methylene‐ATP (α,βmeATP). The concentration of α,βmeATP producing half‐maximal activation (EC50) of neurons with fast desensitization was less (11 μM) than that of neurons with slow desensitization (63 μM), while the Hill coefficients were similar. Suramin and pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid tetrasodium (PPADS) antagonized α,βmeATP‐induced currents in both types of neurons. In situ hybridization revealed that small cells of the DRG predominantly expressed mRNAs of P2X3 and medium‐sized cells expressed mRNAs of P2X2 and P2X3. In contrast, both of mRNAs were not detected in large cells of the DRG. These results suggest that capsaicin‐sensitive, small‐sized DRG neurons expressed mainly the homomeric P2X3 subunit and that capsaicin‐insensitive, medium‐sized DRG neurons expressed the heteromultimeric receptor with P2X2 and P2X3.

In vivo pathway of thermal hyperalgesia by intrathecal administration of α,β-methylene ATP in mouse spinal cord: Involvement of the glutamate-NMDA receptor system

The aim of the present study is to characterize the role of the P2X receptor in spinal nociceptive processing in vivo. We investigated the mechanisms of the P2X receptor agonist α,β‐methylene ATP (α,βmeATP)‐induced modulation of acute nociceptive signalling in mouse spinal cord. Intrathecal administration of α,βmeATP produced a significant and dose‐dependent thermal hyperalgesic response. This response was completely blocked by intrathecal pretreatment with the non‐selective P2 receptor antagonist, pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulphonate (PPADS) and the selective P2X1, P2X3 and P2X2+3 receptor antagonist, 2′,3′‐O‐(2,4,6‐trinitrophenyl)adenosine 5′‐triphosphate (TNP‐ATP). Pretreatment with α,βmeATP 15, 30 and 60 min prior to administration of a second dose of α,βmeATP diminished the α,βmeATP‐induced thermal hyperalgesia. A potent agonist for the P2X1 receptor, β,γ‐methylene‐L‐ATP, did not show the hyperalgesic response, indicating that the P2X1 receptor is not involved in the spinal nociceptive pathway. In fura‐2 experiments using mouse dorsal root ganglion (DRG) neurons, α,βmeATP (100 μM) increased intracellular Ca2+ ([Ca2+]i). This was not produced by a second application of α,βmeATP. The same DRG neurons also showed a marked [Ca2+]i increase in response to capsaicin (3 μM). Intrathecal pretreatment with the Ca2+‐dependent exocytosis inhibitor, botulinum neurotoxin B, abolished the thermal hyperalgesia by α,βmeATP. Furthermore, thermal hyperalgesia was significantly inhibited by the N‐methyl‐D‐aspartate (NMDA) receptor antagonists, 2‐amino‐5‐phosphonopentanoate (APV), dizocilpine and ifenprodil. These findings suggest that α,βmeATP‐induced thermal hyperalgesia may be mediated by the spinal P2X3 receptor subtype that causes unresponsiveness by repetitive agonist applications, and that α,βmeATP (perhaps through P2X3 receptors) may evoke spinal glutamate release which, in turn, leads to the generation of thermal hyperalgesia via activation of NMDA receptors.

Evidence for the involvement of spinal endogenous ATP and P2X receptors in nociceptive responses caused by formalin and capsaicin in mice

The aim of the present study is to characterize the role of spinal endogenous ATP and P2X receptors in the generation of neurogenic and inflammatory pain. We examined the effects of intrathecal treatment with P2X receptor antagonists on the formalin‐ and capsaicin‐induced nociceptive behaviours in mice. Intrathecal pretreatment with the general P2 receptor antagonist, pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), significantly suppressed both the first and second phases of the formalin‐induced nociceptive behaviour. The second phase of the nociceptive response was also suppressed by intrathecal treatment with PPADS after the first phase. Furthermore, pretreatment with the selective antagonist for the P2X1, P2X3 and P2X2+3 receptors, 2′,3′‐O‐(2,4,6‐trinitrophenyl)adenosine 5′‐triphosphate (TNP‐ATP), significantly reduced the first phase, but not the second phase. The second phase was also not suppressed by intrathecal TNP‐ATP after the first phase. Capsaicin‐induced nociceptive behaviour that has been shown to be a model for neurogenic pain, was also significantly suppressed by intrathecal pretreatment with PPADS or TNP‐ATP. Nociceptive behaviour in the first phase of the formalin test and in the capsaicin test were significantly inhibited by intrathecal pretreatment with α,β‐methylene ATP (α,βmeATP: 5 μg mouse−1) 15 min prior to injection of formalin or capsaicin. This treatment has been previously shown to desensitize spinal P2X3 receptor subtypes in vivo. These findings suggest that spinal endogenous ATP may play a role in (1) the formalin‐ and capsaicin‐induced neurogenic pain via the PPADS‐ and TNP‐ATP‐sensitive P2X receptors which are also desensitized by α,βmeATP (perhaps the P2X3 receptor subtype) and (2) formalin‐induced inflammatory pain via PPADS‐sensitive, TNP‐ATP‐ and α,βmeATP‐insensitive P2X (and/or P2Y) receptors.

Measurement of dorsal root ganglion P2X mRNA by SYBR Green fluorescence

The P2X receptor is a receptor-gated cationic channel that responds to ATP. The quantification of P2X mRNA expression in dorsal root ganglion (DRG) provides important information for neuropathic pain studies. We developed a rapid and sensitive external-standard-based real-time quantitative PCR assay for the quantification of mRNA of P2X receptors in mouse tissue samples. The assay uses a double-stranded DNA fluorescent dye, SYBR Green I, to continuously monitor product formation with a GeneAmp 5700 Sequence Detection System (PE Applied Biosystems). To establish the quantitative PCR amplification in a wide range of target transcripts, optimum parameters of primer sequences, concentrations of primers and/or templates, and PCR thermal protocols were experimentally determined. We also tested the reliability of this method in established experimental murine models, which were made by ligation or cutting down of the sciatic nerve. The parameters defined in this assay should be applicable to the quantification of other types of pain models and other tissue samples of mouse.

Downregulation of P2X3 receptor-dependent sensory functions in A/J inbred mouse strain.

There is large variability in the various pain responses including those to tissue injury among inbred mouse strains. However, the determinant factors for the strain‐specific differences remain unknown. The P2X3 sensory‐specific ATP‐gated channel has been implicated as a damage‐sensing molecule that evokes a pain sensation by receiving endogenous ATP from injured tissue. In this study, to clarify the contribution of the sensory P2X3 signalling to strain‐specific differences in tissue injury pain, we examined whether the P2X3‐mediated in vivo and in vitro responses in dorsal root ganglion (DRG) neurons are changed in the A/J inbred mouse strain, which is known to be resistant to tissue injury pain caused by formalin. Here we found that A/J mice exhibited a low magnitude of nocifensive behaviour induced by the P2X agonist α,β‐methylene ATP (αβmeATP) into the hindpaw compared with C57BL/6 J mice. This behaviour was blocked by P2X3 antisense oligodeoxynucleotides. The low magnitude of the in vivo pain sensation could be observed similarly in the in vitro response; the increase in the intracellular Ca2+ increase by αβmeATP in capsaicin‐sensitive DRG neurons from A/J mice was significantly lower than that from C57BL/6 J mice. In A/J DRG neurons the P2X3 protein level was significantly lower compared with C57BL/6 J DRG neurons. The change in P2X3 protein was selective because P2X2 protein was expressed equally in both strains. The present study suggests that the downregulation of sensory P2X3 could be one of the molecular predispositions to low sensitivity to tissue injury pain in the A/J inbred mouse strain.

Bidirectional modulation of P2X receptor-mediated response by divalent cations in rat dorsal motor nucleus of the vagus neurons

The modulatory effects of Zn2+ and other divalent cations on the ATP‐induced responses of preganglionic neurons acutely dissociated from the rat dorsal motor nucleus of the vagus (DMV) were examined using a nystatin‐perforated patch technique under voltage‐clamp. DMV neurons were identified by back‐filling of DiI placed on the vagal bundle at the neck. Zn2+ exerts a concentration‐dependent effect on P2X receptor‐mediated current (IATP): a potentiation by low concentrations of Zn2+ (≤ 50 µm) and an inhibition by high concentrations (> 50 µm). Inhibition of the ATP response was associated with a prolongation of the rising phase of IATP. Cu2+ mimicked Zn2+ regarding the biphasic modulation of IATP. On the other hand, Ni2+ potentiated, but failed to inhibit, the ATP response even at a concentration of 3 mm. Quantitative RT‐PCR revealed the similarity of P2X2 mRNA expression between the DMV and superior cervical ganglion (SCG) but not in the dorsal root ganglion (DRG) and hypoglossal nucleus (XII). The results from the electrophysiological and molecular approaches suggest that functional P2X receptors expressed in DMV neurons are characterized mainly by the P2X2 and P2X2/6 subtype. DMV neurons possess similar P2X receptor characteristics to SCG neurons.

Propofol potentiates ATP-activated currents of recombinant P2X4 receptor channels expressed in human embryonic kidney 293 cells

We examined the effects of a general anesthetic 2, 6-diisopropylphenol (propofol) on ATP- and alpha,beta-methylene ATP (alphabetameATP)-activated currents in the human embryonic kidney 293 (HEK 293) cells expressing recombinant P2X receptor channels, using the whole-cell patch-clamp method. Propofol at clinical relevant concentrations ( approximately 56 microM) potentiated the current responses through the P2X(4) receptor in a dose-dependent manner, whereas propofol did not affect the responses through the P2X(2) receptor or through the heterologous complex of the P2X(2) and P2X(3) (P2X(2+3)) receptor. These results suggest that activation of P2X(4) subtype in the brain and the motor neurons of the spinal anterior horn might be involved in the excitatory effect by propofol such as convulsion and unexpected movements.