Identification of P2X receptors in cultured mouse and rat parasympathetic otic ganglion neurones including P2X knockout studies

We have used patch-clamp recording from cultured neurones, immunohistochemistry and gene deletion techniques to characterize the P2X receptors present in mouse otic ganglion neurones, and demonstrated the presence of similar receptors in rat neurones. All neurones from wild-type (WT) mice responded to ATP (EC(50) 109 microM), but only 38% also responded to alpha beta-meATP (EC(50) 39 microM). The response to alpha beta-meATP was blocked by TNP-ATP with an IC(50) of 38.6 nM. Lowering extracellular pH and co-application of Zn(2+) potentiated responses to ATP and alpha beta-meATP. In P2X(3)(-/-) mouse otic ganglion, all neurones tested responded to 100 microM ATP with a sustained current, but none responded to alpha beta-meATP. In P2X(2)(-/-) mice, no sustained currents were observed, but 36% of neurones responded to both ATP and alpha beta-meATP with transient currents. In P2X(2)/P2X(3)(Dbl-/-) mice, no responses to ATP or alpha beta-meATP were detected, suggesting that other P2X subunits were not involved. In rat otic ganglia, 96% of neurones responded to both ATP and alpha beta-meATP with sustained currents, suggesting a greater proportion of neurones expressing P2X(2/3) receptors. The maximum response to alpha beta-meATP was 40-60% of that evoked by ATP in the same cell. Immunohistochemistry revealed staining for P2X(2) and P2X(3) subunits in WT mouse otic ganglion neurones, which was absent in knockout animals. In conclusion, we have shown for the first time that at least two distinct P2X receptors are present in mouse and rat otic neurones, probably homomeric P2X(2) and heteromeric P2X(2/3) receptors.     

Trichloroethanol inhibits ATP-induced membrane currents in cultured HEK 293-hP2X3 cells

Membrane currents in response to the application of alpha, beta-methylene ATP (alpha,beta-meATP) were recorded by the whole-cell patch-clamp technique in human embryonic kidney 293 cells transfected with the human P2X3 receptor (HEK 293-hP2X3 cells). Trichloroethanol, the biologically active metabolite of chloral hydrate, but not ethanol itself concentration-dependently and reversibly inhibited the current responses. It was concluded that the reported analgesic effect of chloral hydrate may be due to the interruption of pain transmission in dorsal root ganglia expressing P2X3 receptors.     

Evidence for the involvement of bradykinin in chemically-evoked cystitis in anaesthetized rats

Summary The effect of Hoe 140, a potent bradykinin B2 receptor antagonist, on the micturition reflex and detrusor hyperreflexia induced by chemical cystitis has been investigated in anaesthetized rats. Hoe 140 (1–100 nmol/kg i. v.) produced a dose-dependent blockade of the contraction of the rat urinary bladder induced by i. v. administration of bradykinin (100 nmol/kg) without affecting the response produced by the selective tachykinin NK-1 receptor agonist, [Sar⁹] substance P (SP) sulfone (1 nmol/kg i. v.). At doses which produce selective and long-lasting blockade of bradykinin receptors in the urinary bladder, Hoe 140 did not modify urodynamic parameters in normal rats.Intravesical instillation of xylene in female rats decreased bladder capacity and increased micturition frequency. These effects also occurred in rats pretreated with capsaicin as adults. Hoe 140 did not modify xylene-induced cystitis. Intraperitoneal administration of cyclophosphamide (150 mg/kg, 48 h before) decreased bladder capacity and increased micturition frequency. These effects of cyclophosphamide were abolished in rats pretreated with capsaicin as adults. Hoe 140 increased bladder capacity and decreased micturition frequency in rats pretreated with cyclophosphamide.Addition of bradykinin (10 µmol/l) to the medium in the superfused rat urinary bladder preparation evoked a prompt increase in the outflow of calcitonin gene-related peptide like immunoreactivity (CGRP-LI). Hoe 140 (3 µmol/l) inhibited (by about 50%) the CGRP-LI outflow stimulated by bradykinin.These findings demonstrate the participation of bradykinin, through 132 receptors, in the genesis of detrusor hyperreflexia during cyclophosphamide-induced cystitis. Capsaicin-sensitive primary afferent neurons are a likely target for Hoe 140 action in this model of experimental cystitis, as exemplified by its ability to prevent CGRP-LI outflow by bradykinin.Correspondence to C. A. Maggi at the above address     

Metabotropic P2Y1 receptors inhibit P2X3 receptor-channels in rat dorsal root ganglion neurons

Whole-cell patch-clamp recordings from cultured rat dorsal root ganglion neurons demonstrated that the P2Y1 receptor agonists adenosine 5'-O-2-thiodiphosphate (ADP-beta-S) and 2-methylthio adenosine 5'-diphosphate (2-MeSADP) inhibit the alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-meATP)-induced P2X3 receptor-currents. This effect could be antagonized by the wide-spectrum G protein blocker GDP-beta-S and the P2Y(1) receptor antagonist MRS 2179. The P2Y12,13 receptor antagonist AR-C6993MX and pertussis toxin, a blocker of Galphai/o, did not interact with the effect of ADP-beta-S. Hence, the results indicate that ADP-sensitive P2Y1 receptors of rat dorsal root ganglion neurons inhibit ionotropic P2X3 receptors via G protein-activation.     

Diinosine pentaphosphate: an antagonist which discriminates between recombinant P2X(3) and P2X(2/3) receptors and between two P2X receptors in rat sensory neurones

1. We have compared the antagonist activity of trinitrophenyl-ATP (TNP-ATP) and diinosine pentaphosphate (Ip(5)I) on recombinant P2X receptors expressed in XENOPUS: oocytes with their actions at native P2X receptors in sensory neurones from dorsal root and nodose ganglia. 2. Slowly-desensitizing responses to alpha,beta-methylene ATP (alpha,beta-meATP) recorded from oocytes expressing P2X(2/3) receptors were inhibited by TNP-ATP at sub-micromolar concentrations. However, Ip(5)I at concentrations up to 30 microM was without effect. 3. Nodose ganglion neurones responded to alpha,beta-meATP with slowly-desensitizing inward currents. These were inhibited by TNP-ATP (IC(50), 20 nM), but not by Ip(5)I at concentrations up to 30 microM. 4. In DRG neurones that responded to ATP with a rapidly-desensitizing inward current, the response was inhibited by TNP-ATP with an IC(50) of 0.8 nM. These responses were also inhibited by Ip(5)I with an IC(50) of 0.1 microM. Both antagonists are known to inhibit homomeric P2X(3) receptors. 5. Some DRG neurones responded to alpha,beta-meATP with a biphasic inward current, consisting of transient and sustained components. While the transient current was abolished by 1 microM Ip(5)I, the sustained component remained unaffected. 6. In conclusion, Ip(5)I is a potent antagonist at homomeric P2X(3) receptors but not at heteromeric P2X(2/3) receptors, and therefore should be a useful tool for elucidating the subunit composition of native P2X receptors.     

In vivo pathway of thermal hyperalgesia by intrathecal administration of alpha,beta-methylene ATP in mouse spinal cord: involvement of the glutamate-NMDA receptor system.

1. 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 alpha,beta-methylene ATP (alpha,betameATP)-induced modulation of acute nociceptive signalling in mouse spinal cord. 2. Intrathecal administration of alpha,betameATP 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 alpha,betameATP 15, 30 and 60 min prior to administration of a second dose of alpha,betameATP diminished the alpha,betameATP-induced thermal hyperalgesia. 3. A potent agonist for the P2X1 receptor, beta,gamma-methylene-L-ATP, did not show the hyperalgesic response, indicating that the P2X1 receptor is not involved in the spinal nociceptive pathway. 4. In fura-2 experiments using mouse dorsal root ganglion (DRG) neurons, alpha,betameATP (100 microM) increased intracellular Ca2+ ([Ca2+]i). This was not produced by a second application of alpha,betameATP. The same DRG neurons also showed a marked [Ca2+]i increase in response to capsaicin (3 microM). 5. Intrathecal pretreatment with the Ca2+-dependent exocytosis inhibitor, botulinum neurotoxin B, abolished the thermal hyperalgesia by alpha,betameATP. Furthermore, thermal hyperalgesia was significantly inhibited by the N-methyl-D-aspartate (NMDA) receptor antagonists, 2-amino-5-phosphonopentanoate (APV), dizocilpine and ifenprodil. 6. These findings suggest that alpha,betameATP-induced thermal hyperalgesia may be mediated by the spinal P2X3 receptor subtype that causes unresponsiveness by repetitive agonist applications, and that alpha,betameATP (perhaps through P2X3 receptors) may evoke spinal glutamate release which, in turn, leads to the generation of thermal hyperalgesia via activation of NMDA receptors.     

Purinergic modulation of pacemaker Ca2+ activity in interstitial cells of Cajal

Purinoceptors are widely distributed throughout the body, and are thought to have important contributions to numerous functions. In this study, we characterised the contribution of purinoceptors to the mechanisms underlying spontaneous rhythmicity of the gastro-intestinal tracts. Using cell cluster preparations (100-200 microm diameter) obtained from murine ileum, we measured spontaneous intracellular Ca2+([Ca2+]i) oscillations in the presence of nifedipine, as an index of pacemaker [Ca2+]i activity in interstitial cells of Cajal (ICCs, c-Kit-immunopositive cells), the pacemaker cells for gastrointestinal motility. This small preparation also contained smooth muscle and enteric neurones. Using various purinoceptor agonists and an antagonist, we characterised both TTX-sensitive and insensitive modulations of pacemaker [Ca2+]i activity in ICCs. Continuous application of either ATP, ATPgammaS, suramin or alpha,beta-methylene ATP (alpha,beta-meATP) suppressed pacemaker [Ca2+]i activity. The inhibitory effect of alpha,beta-meATP was completely abolished by a prior application of TTX. On the other hand, even in the presence of TTX, continuous application of 2-methylthio ATP (2-MeSATP) at concentrations greater than 30 microM caused a prompt rise followed by a slow decline of the baseline [Ca2+]i, and pacemaker [Ca2+]i oscillations were gradually suppressed during the decline. Neither UTP nor alpha,beta-meATP at high concentrations (30-100 microM) produced a similar [Ca2+]i response. These results suggest that the TTX-resistant, direct purinergic modulation of pacemaker [Ca2+]i activity in ICCs is mediated via P2X purinoceptors distinct from those involved in TTX-sensitive modulation. The slow decline may be attributed to desensitisation of these purinoceptors. The possible involvement of other purinoceptors is also discussed.     

P2X2 characteristics of the ATP receptor coupled to [Ca2+]i increases in cultured Purkinje neurons from neonatal rat cerebellum.

P2X receptors present in cerebellar Purkinje cells have been studied by recording ATP-elicited [Ca2+]i signals from immuno-identified (calbindin+) cells in culture using fura-2 microfluorescence. The [Ca2+]i increases evoked by ATP were mimicked by 2MeSATP but not by alpha, beta-meATP and other purinoceptor agonists. The selective P2X1 antagonist diinosine pentaphosphate failed to inhibit ATP-elicited [Ca2+]i transients, but suramin and PPADS rapidly and reversibly blocked the [Ca2+]i responses to ATP and 2MeSATP. The IC50 values for suramin and PPADS inhibition were 48.7 +/- 4.4 and 5.9 +/- 0.3 microM, respectively. Both antagonists blocked completely the signal elicited by ATP, revealing that there was not a separate antagonist-insensitive P2X receptor population in Purkinje cells. The effect of ATP was potentiated by Zn2+ and H+ ions. A one unit acidification from pH 7.4 to 6.4 enhanced by 172% the [Ca2+]i transient elicited by an intermediate concentration of ATP. Conversely, alkalinization of the medium to pH 8.4 reduced the ATP response by 88%. This combination of pharmacological and modulatory properties indicates that endogenous P2X receptors present in Purkinje neurons are formed by P2X2 subunits, rather than the more abundantly expressed P2X4 purinoceptor subunits.     

Bradykinin-induced modulation of calcium signals in rat dorsal root ganglion neurons in vitro

We used combined patch-clamp-microfluorimetric recordings to examine the effects of bradykinin on [Ca2+]i transients and the Ca2+ current (ICa) in rat dorsal root ganglion neurons in vitro. Bradykinin increased [Ca2+]i in approximately 20% of dorsal root ganglion cells examined and inhibited the ICa in approximately 65% of dorsal root ganglion cells. Bradykinin also inhibited the ICa when [Ca2+]i was buffered with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or when Ba2+ was the charge carrier. When ICa's of increasing duration were elicited in these neurons, [Ca2+]i transients were produced that increased in amplitude but eventually approached an asymptote at longer voltage steps. Similarly, the amplitude of the [Ca2+]i transient also approached an asymptote in current-clamp recordings when cells were induced to fire a large number of action potentials. The bradykinin-induced inhibition of the amplitude of the [Ca2+]i transient was more pronounced at shorter voltage steps. At pulse durations that produced asymptotic [Ca2+]i signals, bradykinin no longer decreased the amplitude of the rise in [Ca2+]i, although it still reduced the ICa. In current-clamp recordings, bradykinin also reduced the [Ca2+]i signal that accompanied the generation of action potentials, but again bradykinin was more effective for shorter spike trains. Bradykinin also depolarized the majority of neurons (65%). The reduction in [Ca2+]i produced by bradykinin in sensory neurons may be an important factor contributing to bradykinin-induced excitation of primary sensory afferents.     

The effect of capsaicin on voltage-gated calcium currents and calcium signals in cultured dorsal root ganglion cells.

1. The effects of capsaicin on voltage-gated Ca2+ currents (ICa), and intracellular Ca2+ concentrations [( Ca2+]i) in cultured dorsal root ganglion (DRG) neurones of the rat were examined in vitro by use of combined patch clamp-microfluorometric recordings. 2. Under voltage-clamp conditions, capsaicin (0.1-10 microM) caused a concentration-dependent decrease in the magnitude of the ICa, an elevation in the holding current (Ih) and a concomitant rise in the [Ca2+]i in most cells examined. Repeated application of capsaicin produced marked desensitization. 3. Some decrease in the ICa produced by capsaicin was also observed when the rise in [Ca2+]i was buffered with EGTA or BAPTA and when Ba2+ was used as the charge carrier; under these conditions the desensitization previously observed was smaller. 4. The decrement in voltage-gated current was smaller in Ba2+ containing solutions than in Ca2+ containing solutions suggesting that the capsaicin-induced influx of Ca2+ partially mediated the observed decrease in the voltage-gated current. In cells which showed a marked response to capsaicin an outward (positive) current was sometimes observed upon depolarization from -80 to 0 mV. This effect was consistent with an outward movement of cations through the capsaicin conductance pathway which may also account, in part, for the apparent reduction in ICa by capsaicin. 5. The effects of capsaicin under voltage-clamp conditions were prevented by ruthenium red (1 microM). 6. Under current clamp conditions, capsaicin depolarized and caused a rise in [Ca2+]i in the majority of DRG cells examined. Both of these effects could be prevented by ruthenium red (500 nM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Agonist-dependence of recovery from desensitization of P2X(3) receptors provides a novel and sensitive approach for their rapid up or downregulation

1. Fast-desensitizing P2X(3) receptors of nociceptive dorsol root ganglion (DRG) neurons are thought to mediate pain sensation. Since P2X(3) receptor efficiency is powerfully modulated by desensitization, its underlying properties were studied with patch-clamp recording. 2. On rat cultured DRG neurons, 2 s application of ATP (EC(50)=1.52 microm), ADP (EC(50)=1.1 microm) or alpha,beta-meATP (EC(50)=1.78 microm) produced similar inward currents that fully desensitized, at the same rate, back to baseline. Recovery from desensitization was much slower after ATP and ADP than after alpha,beta-meATP and, in all cases, it had sigmoidal time course. 3. By alternating the application of ATP and alpha,beta-meATP, we observed complete cross-desensitization indicating that these agonists activated the same receptors. This notion was confirmed by the similar antagonism induced by 2', 3'-O-(2,4,6,trinitrophenyl)-adenosine triphosphate (TNP-ATP). 4. Recovery from desensitization elicited by ATP was unexpectedly shaped by transient application of alpha,beta-methylene-adenosine triphosphate (alpha,beta-meATP), and vice versa. Thus, short-lasting, full desensitization produced by alpha,beta-meATP protected receptors from long-lasting desensitization induced by subsequent ATP applications. ATP and ADP had similar properties of recovery from desensitization. 5. Low nm concentrations of alpha,beta-meATP (unable to evoke membrane currents) could speed up recovery from ATP-induced desensitization, while low nm concentrations of ATP enhanced it. Ambient ATP levels were found to be in the pm range (52+/-3 pm). 6. The phenomenon of cross-desensitization and protection was reproduced by rP2X(3) receptors expressed by rat osteoblastic cell 17/2.8 or human embryonic kidney cell 293 cells, indicating P2X(3) receptor specificity. 7. It is suggested that transient application of an agonist that generates rapid recovery from desensitization, is a novel, powerful tool to modulate P2X(3) receptor responsiveness to the natural agonist ATP.     

Metabotropic P2Y receptors inhibit P2X3 receptor-channels via G protein-dependent facilitation of their desensitization

BACKGROUND AND PURPOSE: The aim of the present study was to investigate whether the endogenous metabotropic P2Y receptors modulate ionotropic P2X(3) receptor-channels. EXPERIMENTAL APPROACH: Whole-cell patch-clamp experiments were carried out on HEK293 cells permanently transfected with human P2X(3) receptors (HEK293-hP2X(3) cells) and rat dorsal root ganglion (DRG) neurons. KEY RESULTS: In both cell types, the P2Y(1,12,13) receptor agonist, ADP-beta-S, inhibited P2X(3) currents evoked by the selective agonist, alpha,beta-methylene ATP (alpha,beta-meATP). This inhibition could be markedly counteracted by replacing in the pipette solution the usual GTP with GDP-beta-S, a procedure known to block all G protein heterotrimers. P2X(3) currents evoked by ATP, activating both P2Y and P2X receptors, caused a smaller peak amplitude and desensitized faster than those currents evoked by the selective P2X(3) receptor agonist alpha,beta-meATP. In the presence of intracellular GDP-beta-S, ATP- and alpha,beta-meATP-induced currents were identical. Recovery from P2X(3) receptor desensitization induced by repetitive ATP application was slower than the recovery from alpha,beta-meATP-induced desensitization. When G proteins were blocked by intracellular GDP-beta-S, the recovery from the ATP- and alpha,beta-meATP-induced desensitization were of comparable speed. CONCLUSIONS AND IMPLICATIONS: Our results suggest that the activation of P2Y receptors G protein-dependently facilitates the desensitization of P2X(3) receptors and suppresses the recovery from the desensitized state. Hence, the concomitant stimulation of P2X(3) and P2Y receptors of DRG neurons by ATP may result both in an algesic effect and a partly counterbalancing analgesic activity.     

Pharmacology of vanilloids at recombinant and endogenous rat vanilloid receptors

This study compared the actions of members of five different chemical classes of vanilloid agonists at the recombinant rat vanilloid VR1 receptor expressed in HEK293 cells, and at endogenous vanilloid receptors on dorsal root ganglion cells and sensory nerves in the rat isolated mesenteric arterial bed. In mesenteric beds, vanilloids elicited dose-dependent vasorelaxation with the rank order of potency: resiniferatoxin>capsaicin=olvanil>phorbol 12-phenyl-acetate 13-acetate 20-homovanillate (PPAHV)>isovelleral. Scutigeral was inactive. Responses were abolished by capsaicin pretreatment and inhibited by ruthenium red. In VR1-HEK293 cells and dorsal root ganglion neurones, Ca(2+) responses were induced by resiniferatoxin>capsaicin=olvanil>PPAHV; all four were full agonists. Isovelleral and scutigeral were inactive. The resiniferatoxin-induced Ca(2+) response had a distinct kinetic profile. Olvanil had a Hill coefficient of approximately 1 whilst capsaicin, resiniferatoxin and PPAHV had Hill coefficients of approximately 2 in VR1-HEK293 cells. The capsaicin-induced Ca(2+) response was inhibited in a concentration-dependent manner by ruthenium red>capsazepine>isovelleral. These data show that resiniferatoxin, capsaicin, olvanil and PPAHV, but not scutigeral and isovelleral, are agonists at recombinant rat VR1 receptors and endogenous vanilloid receptors on dorsal root ganglion neurones and in the rat mesenteric arterial bed. The vanilloids display the same relative potencies (resiniferatoxin>capsaicin=olvanil>PPAHV) in all of the bioassays.     

Involvement of the endogenous hydrogen sulfide/Ca(v) 3.2 T-type Ca(2+) channel pathway in cystitis-related bladder pain in mice

BACKGROUND AND PURPOSE Hydrogen sulfide (H(2) S), generated by enzymes such as cystathionine-γ-lyase (CSE) from L-cysteine, facilitates pain signals by activating the Ca(v) 3.2 T-type Ca(2+) channels. Here, we assessed the involvement of the CSE/H(2) S/Ca(v) 3.2 pathway in cystitis-related bladder pain. EXPERIMENTAL APPROACH Cystitis was induced by i.p. administration of cyclophosphamide in mice. Bladder pain-like nociceptive behaviour was observed and referred hyperalgesia was evaluated using von Frey filaments. Phosphorylation of ERK in the spinal dorsal horn was determined immunohistochemically following intravesical administration of NaHS, an H(2) S donor. KEY RESULTS Cyclophosphamide caused cystitis-related symptoms including increased bladder weight, accompanied by nociceptive changes (bladder pain-like nociceptive behaviour and referred hyperalgesia). Pretreatment with DL-propargylglycine, an inhibitor of CSE, abolished the nociceptive changes and partly prevented the increased bladder weight. CSE protein in the bladder was markedly up-regulated during development of cystitis. Mibefradil or NNC 55-0396, blockers of T-type Ca(2+) channels, administered after the symptoms of cystitis appeared, reversed the nociceptive changes. Further, silencing of Ca(v) 3.2 protein by repeated intrathecal administration of mouse Ca(v) 3.2-targeting antisense oligodeoxynucleotides also significantly attenuated the nociceptive changes, but not the increased bladder weight. Finally, the number of cells staining positive for phospho-ERK was increased in the superficial layer of the L6 spinal cord after intravesical administration of NaHS, an effect inhibited by NNC 55-0396. CONCLUSION AND IMPLICATIONS Endogenous H(2) S, generated by up-regulated CSE, caused bladder pain and referred hyperalgesia through the activation of Ca(v) 3.2 channels, one of the T-type Ca(2+) channels, in mice with cyclophosphamide-induced cystitis.     

A study of P2X1 receptor function in murine megakaryocytes and human platelets reveals synergy with P2Y receptors

We have examined the role of ATP-dependent P2X(1) receptors in megakaryocytes (MKs) and platelets using receptor-deficient mice and selective agonists. Alpha,beta-meATP- and ATP- evoked ionotropic inward currents were absent in whole-cell recordings from MKs of P2X(1)(-/-) mice, demonstrating that the P2X receptor phenotype in MKs, and by inference, platelets, is due to expression of homomeric P2X(1) receptors. P2X(1) receptor deficiency had no effect on MK (CD 41) numbers or size distribution, showing that it is not essential for normal MK development. P2Y receptor-stimulated [Ca(2+)](i) responses were unaffected in MKs from P2X(1)(-/-) mice, however the inward cation current associated with Ca(2+) release was reduced by approximately 50%, suggesting an interaction between the membrane conductances activated by P2X(1) and P2Y receptors. Interaction between P2X(1) and P2Y receptors in human platelets was also examined using [Ca(2+)](i) recordings from cell suspensions. Alpha,beta-meATP (10 microM) evoked a rapid transient P2X(1) receptor-mediated increase in [Ca(2+)](i), whereas ADP-(10 microM) evoked P2Y receptor responses were slower, peaked at a higher level and remained elevated for longer periods. Co-application of alpha, beta-meATP and ADP resulted in marked acceleration and amplification of the peak [Ca(2+)](i) response. We conclude that ionotropic P2X(1) receptors may play a priming role in the subsequent activation of metabotropic P2Y receptors during platelet stimulation.     

Molecular cloning, functional characterization of the porcine transient receptor potential V1 (pTRPV1) and pharmacological comparison with endogenous pTRPV1

In the present study, we cloned a porcine orthologue of transient receptor potential V1 (pTRPV1) and heterologously expressed it in human embryonic kidney (HEK) 293 cells to characterize its pharmacological properties. At the amino acid level, pTRPV1 was highly homologous (83-90%) to other orthologues of TRPV1. The expression of receptors was examined with current and [Ca2+]i responses to capsaicin using whole-cell patch-clamp and fura-2 ratio imaging techniques, respectively, and by immunostaining with an anti-TRPV1 antibody. The receptors were characterized by changes in [Ca2+]i in response to various vanilloid agonists, low pH and heat and by the effects of TRPV1 antagonists on them. The various TRPV1 agonists activated pTRPV1 in a dose-dependent manner in the order of potency of resiniferatoxin (RTX) > olvanil > capsaicin > phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV), phorbol 12,13-dinonanoate 20-homovanillate (PDNHV). Isovelleral and scutigeral had no effect. Endogenous vanilloids (anandamide > 15 (s)-HPETE > NADA), low pH and noxious heat (>42 degrees C) activated pTRPV1. Comparison of amino acid sequences with various mammalian TRPV1 homologues suggested some novel putative vanilloid recognition sites. TRPV1 antagonists, iodoRTX, ruthenium red and capsazepine suppressed capsaicin-induced responses. Similar to human TRPV1, but not rodent TRPV1, capsazepine was effective in blocking pH- and heat-induced responses. Similar pharmacological profiles were observed in cultured porcine dorsal root ganglion neurons. We discuss putative amino acid residues related to pharmacological differences among mammalian TRPV1 homologues.     

Functional evidence for interaction between prostaglandin EP3 and kappa-opioid receptor pathways in tactile pain induced by human immunodeficiency virus type-1 (HIV-1) glycoprotein gp120

HIV-1 glycoprotein gp120 administered intrathecally induces tactile pain (allodynia) in animals. In the present study, we investigated the mechanism of gp120-induced allodynia and possible functional connections with factors modulating pain transmission at the spinal level. Gp120 evoked allodynia in a dose-dependent manner with the maximum effect at 1 pg/mouse, and stimulated a rapid increase in intracellular free Ca2+ concentration ([Ca2+]i) in the dorsal horn cells of the spinal cord. These responses evoked by gp120 were blocked by galactocerebroside. The gp120-induced allodynia was also attenuated by the non-steroidal anti-inflammatory drug indomethacin, which inhibits prostaglandin synthesis, and did not develop in mice lacking the EP3 prostaglandin E receptor subtype (EP3(-/-)). Pretreatment of spinal slices with indomethacin dose-dependently decreased the percentage of the cells that showed increased [Ca2+]i in response to gp120, and the decrease was reversed by addition of the selective EP3 agonist ONO-AE-248. The kappa-opioid agonist U-50,488 significantly enhanced the gp120-stimulated increase in [Ca2+]i in spinal slices prepared from EP3(-/-) mice, and the simultaneous addition of U-50,488 with gp120 reproduced the gp120-induced allodynia in EP3(-/-) mice. These results suggest that gp120 induced allodynia by increasing [Ca2+]i, concomitant with activation of prostanoid EP3 and kappa-opioid receptors in the spinal cord.     

Targeting the visceral purinergic system for pain control

Experimental evidence is presented to support the hypothesis that purinergic mechanosensory transduction can initiate visceral pain in urinary bladder, ureter, gut and uterus. In general, physiological reflexes are mediated via P2X3 and P2X2/3 receptors on low threshold sensory fibres, while these receptors on high threshold sensory fibres mediate pain. Potential therapeutic strategies are considered for the treatment of visceral pain in such conditions as renal colic, interstitial cystitis and inflammatory bowel disease by purinergic agents, including P2X3 and P2X2/3 receptor antagonists that are orally bioavailable and stable in vivo and agents that modulate ATP release and breakdown.     

Methyllycaconitine, alpha-bungarotoxin and (+)-tubocurarine block fast ATP-gated currents in rat dorsal root ganglion cells

The effects of nicotinic acetylcholine receptor antagonists were studied on currents evoked by application of ATP to rat isolated dorsal root ganglion cells, and human embryonic kidney 293 cells expressing rat P2X(3) and P2X(2/3) receptors. The rapidly desensitising (within 100 ms) current in dorsal root ganglion cells was inhibited by methyllycaconitine, alpha-bungarotoxin and (+)-tubocurarine (concentrations giving half-maximal inhibition were approximately 40, 60 and 800 nm, respectively), but not by hexamethonium (100 microm) or mecamylamine (100 microm). The sustained (>250 ms) current in dorsal root ganglion cells was inhibited by (+)-tubocurarine (80% by 10 microm), but not by methyllycaconitine (200 nm), alpha-bungarotoxin (200 nm), mecamylamine (100 microm) or hexamethonium (100 microm). Rapidly desensitising currents evoked by alpha,betamethylene-ATP in human embryonic kidney cells expressing P2X(3) receptors were inhibited by methyllycaconitine and alpha-bungarotoxin, at concentrations similar to those effective in dorsal root ganglion cells. The results indicate that some nicotinic acetylcholine receptor antagonists are potent blockers of P2X receptors on neurons, particularly the homo-oligomeric P2X(3) receptor. This finding suggests that these drugs should be used with care to discriminate between P2X and neuronal acetylcholine receptor types.