a-317491 and Pain

Acute systemic administration of a novel and highly selective non-nucleotide P2X(3)/P2X(2/3) receptor antagonist, A-317491, has been shown to reduce chronic hyperalgesia and allodynia in several animal models of pathological pain in the absence of cardiovascular and CNS side effects. Furthermore, these studies have also outlined the antinociceptive profile for a P2X(3)/P2X(2/3) receptor antagonist, as A-317491 was effective in models of chronic inflammatory and neuropathic pain, but not in models of acute, acute inflammatory or visceral pain. The development of A-317491 has also added to the current understanding of P2X(3)/P2X(2/3) receptor pharmacology and its contributions to nociceptive transmission and modulation. To this end, recent studies have demonstrated that both spinal and peripheral P2X(3)/P2X(2/3) receptors have significant but differential contributions to nociception in animal models of nerve or tissue injury, and that antagonism of spinal P2X(3)/P2X(2/3) receptors results in an indirect activation of the opioid system to alleviate inflammatory thermal hyperalgesia and chemogenic nociception. Thus, preclinical data have shown considerable promise for the utility of a P2X(3)/P2X(2/3) receptor antagonist to alleviate various forms of chronic pain. Furthermore, the discovery of this selective and metabolic stable antagonist for P2X(3)/P2X(2/3) receptors has also been useful in defining the contributions of these receptors to states of pathological pain.

Topics: Analgesics; Animals; Drug Evaluation, Preclinical; Hyperalgesia; Pain; Phenols; Polycyclic Compounds; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2X2; Receptors, Purinergic P2X3

ATP released from damaged or inflamed tissues can act at P2X receptors expressed on primary afferent neurones. The resulting depolarization can initiate action potentials that are interpreted centrally as pain. P2X(3) subunits are found in a subset of small-diameter, primary afferent neurones, some of which are also sensitive to capsaicin. They can form homo-oligomeric channels, or they can assemble with P2X(2) subunits into hetero-oligomers. Studies with antagonists selective for P2X(3)-containing receptors, experiments with antisense oligonucleotides to reduce P2X(3) subunit levels, and behavioural testing of P2X(3) knock-out mice, all suggest a role for the P2X(2/3) receptor in the signalling of chronic inflammatory pain and some features of neuropathic pain. The availability of such tools and experimental approaches promises to accelerate our understanding of the other physiological roles for P2X receptors on primary afferent neurones.

Topics: Adenosine Triphosphate; Animals; Dose-Response Relationship, Drug; Humans; Pain; Peripheral Nerves; Phenols; Polycyclic Compounds; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2; Receptors, Purinergic P2X3

Our recent studies demonstrated that regional bone loss in the unloaded hind limbs of tail-suspended mice triggered pain-like behaviors due to the acidic environment in the bone induced by osteoclast activation. The aims of the present study were to examine whether TRPV1, ASIC and P2X (known as nociceptors) are expressed in bone, and whether the antagonists to those receptors affect the expression of osteoblast and osteoclast regulators, and prevent the triggering of not only pain-like behaviors but also high bone turnover conditions in tail-suspension model mice. The hind limb-unloaded mice were subjected to tail suspension with the hind limbs elevated for 14days. The effects of the TRPV1, ASIC3, P2X2/3 antagonists on pain-like behaviors as assessed by the von Frey test, paw flick test and spontaneous pain scale; the expressions of TRPV1, ASICs, and P2X2 in the bone; and the effects of those antagonists on osteoblast and osteoclast regulators were examined. In addition, we evaluated the preventive effect of continuous treatment with a TRPV1 antagonist on the trigger for pain-like behavior and bone loss in tail-suspended mice. Pain-like behaviors were significantly improved by the treatment with TRPV1, ASIC, P2X antagonists; TRPV1, ASICs and P2X were expressed in the bone tissues; and the antagonists to these receptors down-regulated the expression of osteoblast and osteoclast regulators in tail-suspended mice. In addition, continuous treatment with a TRPV1 antagonist during tail-suspension prevented the induction of pain-like behaviors and regional bone loss in the unloaded hind limbs. We, therefore, believe that those receptor antagonists have a potential role in preventing the triggering of skeletal pain with associated regional bone metabolic disorder.

Topics: Acid Sensing Ion Channels; Anilides; Animals; Bone Diseases, Metabolic; Cinnamates; Cnidarian Venoms; Femur; Hindlimb Suspension; Humerus; Male; Mice; Mice, Inbred C57BL; Osteoblasts; Osteoclasts; Pain; Phenols; Polycyclic Compounds; Purinergic P2X Receptor Antagonists; Receptors, Purinergic P2X2; Receptors, Purinergic P2X3; TRPV Cation Channels

The cerebrospinal fluid (CSF)-contacting nucleus is implicated in the descending inhibitory pathway in pain processing, whereas the cellular and molecular mechanisms underpinning CSF-contacting nucleus regulating pain signals remains largely elusive. ATP is evidenced to inhibit pain transmission at supraspinal level by the mediation of the receptor P2X, wherein its subtype P2X3 is identified as the most potent. Our present experiment investigated the functionality of P2X3 receptors in CSF-contacting nucleus in the formalin-evoked inflammatory pain. Immunofluorescence and western blot revealed the expression of P2X3 receptors in the CSF-contacting nucleus and their upregulated expression subsequent to administration of formalin in rat model. ATP (a P2X3 receptor agonist, 100nmol/5µl) by intracerebroventricular (i.c.v.) administration ameliorated pain behaviors and enhanced c-Fos immunoreactivity in the neurons of the periaqueductal gray (PAG), both of which were discounted by pre-administration of A-317491 (a selective P2X3 receptor antagonist, 25nmol/5µl). After the CSF-contacting nucleus was ablated by cholera toxin subunit B-saporin, ATP failed to induce analgesia, with the c-Fos immunoreactivity in the PAG neurons remaining intact. Our results validated that P2X3 receptors in the CSF-contacting nucleus are pivotal in inflammatory pain processing via the activation of PAG neurons.

Topics: Adenosine Triphosphate; Animals; Cerebrospinal Fluid; Cholera Toxin; Disease Models, Animal; Disinfectants; Formaldehyde; Injections, Intraventricular; Male; Neurons; Pain; Pain Measurement; Pain Threshold; Periaqueductal Gray; Phenols; Polycyclic Compounds; Purinergic P2X Receptor Agonists; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2X3; Ribosome Inactivating Proteins, Type 1; Saporins

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.. 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.. 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).. 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.

Topics: Animals; Dose-Response Relationship, Drug; Freund's Adjuvant; Hyperalgesia; Inflammation; Male; Masseter Muscle; Neurons; Pain; Phenols; Polycyclic Compounds; Rats; Rats, Wistar; Receptors, Purinergic P2X3; Trigeminal Ganglion

In recent years, studies have substantiated the view that P2X3 receptors play a part in the generation and transmission of purinergic signals in inflammatory and chronic neuropathic pain. Data have also been presented to suggest that the process of P2X3 receptor antagonism inhibits inflammatory hyperalgesia, involving the spinal opioid system. The aim of this study was to investigate the effect of the selective P2X3 receptor antagonist A-317491 on the development of antinociceptive tolerance to chronic morphine administration in mice. Daily systemic injection of A-317491 attenuated the morphine-induced antinociceptive tolerance to von Frey and thermal stimuli. Repeated morphine injections alone led to a significant rightward shift in the morphine dose-response curve compared with that with A-317491. A single dose of A-317491 also showed a reversal effect in morphine-tolerant mice. In a withdrawal test, co-administration of A-317491 and morphine also reduced the naloxone-induced withdrawal symptoms compared with the morphine-alone group. Thus, we propose that the P2X3 receptor is involved in the process of morphine antinociceptive tolerance and may be a new therapeutic target in the prevention of tolerance to morphine-induced antinociception.

Topics: Analgesics, Opioid; Animals; Dose-Response Relationship, Drug; Drug Tolerance; Male; Mice; Mice, Inbred BALB C; Morphine; Naloxone; Narcotic Antagonists; Pain; Phenols; Polycyclic Compounds; Purinergic P2X Receptor Antagonists; Receptors, Purinergic P2X3; Spinal Cord; Substance Withdrawal Syndrome

Pyridoxalphosphate-6-azophenyl-2',4'-disulfonate (7a, PPADS), a nonselective P2X receptor antagonist, was extensively modified to develop more stable, potent, and selective P2X₃ receptor antagonists as potential antinociceptive agents. Based on the results of our previous report, all strong anionic groups in PPADS including phosphate and sulfonate groups were changed to carboxylic acids or deleted. The unstable azo (-NN-) linkage of 7a was transformed to more stable carbon-carbon, ether or amide linkages through the synthesis of the 5-hydroxyl-pyridine moieties with substituents at 2 position via a Diels-Alder reaction. This resulted in the retention of antagonistic activity (IC50 = 400 ∼ 700 nM) at the hP2X₃ receptor in the two-electrode voltage clamp (TEVC) assay system on the Xenopus oocytes. Introduction of bulky aromatic groups at the carbon linker, as in compounds 13 h-n, dramatically improved the selectivity profiles of hP2X₃ when compared with mP2X₁ and hP2X₇ receptors. Among the substituents tested at the 2-position, the m-phenoxybenzyl group showed optimum selectivity and potency at the hP2X₃ receptor. In searching for effective substituents at the 4- and 3-positions, we found that compound 36j, with 4-carboxaldehyde, 3-propenoic acid and 2-(m-phenoxy)benzyl groups, was the most potent and selective hP2X₃ receptor antagonist with an IC50 of 60 nM at hP2X₃ and marginal antagonistic activities of 10 μM at mP2X₁ and hP2X₇. Furthermore, using an ex-vivo assay system, we found that compound 36j potently inhibited pain signaling in the rat dorsal horn with 20 μM 36j displaying 65% inhibition while 20 μM pregabalin, a clinically available drug, showed only 31% inhibition.

Topics: Animals; Dose-Response Relationship, Drug; Drug Design; Humans; Molecular Structure; Oocytes; Pain; Pyridoxal Phosphate; Receptors, Purinergic P2X3; Structure-Activity Relationship; Xenopus

Primary and metastatic cancers that affect bone are frequently associated with severe and intractable pain. The mechanisms underlying the development of bone cancer pain are largely unknown. In this study, we first demonstrated that a functional upregulation of P2X3 receptors in dorsal root ganglion (DRG) neurons is closely associated with the neuronal hyperexcitability and the cancer-induced bone pain in MRMT-1 tumor cell-inoculated rats. Second, we revealed that visinin-like protein 1 (VILIP-1), a member of visinin-like proteins that belong to the family of neuronal calcium sensor proteins is responsible for the observed upregulation of P2X3 receptors in DRG neurons. The interaction between the amino terminus of VLIP-1 and the carboxyl terminus of the P2X3 receptor is critical for the surface expression and functional enhancement of the receptor. Finally, overexpression of VILIP-1 increases the expression of functional P2X3 receptors and enhances the neuronal excitability in naive rat DRG neurons. In contrast, knockdown of VILIP-1 inhibits the development of bone cancer pain via downregulation of P2X3 receptors and repression of DRG excitability in MRMT-1 rats. Taken together, these results suggest that functional upregulation of P2X3 receptors by VILIP-1 in DRG neurons contributes to the development of cancer-induced bone pain in MRMT-1 rats. Hence, P2X3 receptors and VILIP-1 could serve as potential targets for therapeutic interventions in cancer patients for pain management. Pharmacological blockade of P2X3 receptors or knockdown of VILIP-1 in DRGs would be used as innovative strategies for the treatment of bone cancer pain.

Topics: Adenosine Triphosphate; Animals; Antineoplastic Agents; Bone Neoplasms; Cells, Cultured; Disease Models, Animal; Ganglia, Spinal; Green Fluorescent Proteins; Humans; Hyperalgesia; Membrane Potentials; Neurocalcin; Neurofilament Proteins; Neurons; Pain; Phenols; Polycyclic Compounds; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2X3; Transfection; Up-Regulation

The purinergic P2X3 and P2X2/3 receptors are in the peripheral nervous system almost exclusively confined to afferent sensory neurons, where they are found both at peripheral and central synapses. The P2X3 receptor is implicated in both neuropathic and inflammatory pain. However, the role of the P2X3 receptor in chronic cancer-induced bone pain is less known. Here we investigated the effect of systemic acute and chronic administration of the selective P2X3, P2X2/3 receptor antagonist (5-[[[(3-Phenoxyphenyl)methyl][(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]carbonyl]-1,2,4-benzenetricarboxylic acid sodium salt hydrate) (A-317491) in a murine model of cancer-induced bone pain. Chronic administration of A-317491 (30 μmol/kgs.c., b.i.d.) resulted in a transient attenuation of pain related behaviours in the early stage of the bone cancer model, but had no effect in the late and more progressed stage of bone cancer. Also, acute administration of A-317491 (100 μmol/kgs.c.) had no effect in the progressed stage of the bone cancer pain model. Thus, systemically administered A-317491 did not demonstrate a robust effect in the present mouse model of cancer-induced bone pain.

Topics: Animals; Bone Neoplasms; Cell Line, Tumor; Cell Survival; Clone Cells; Male; Mice; Pain; Phenols; Polycyclic Compounds; Purinergic P2X Receptor Antagonists; Receptors, Purinergic P2X2; Receptors, Purinergic P2X3; Time Factors

Extracellular ATP acts on purinergic receptors as a potent agonist for a variety of different cell types, including cardiomyocytes and nodose ganglia. P2X3 receptor is the most abundant P2X-receptor subtype in heart and nodose ganglia. This study wants to observe the role of P2X3 receptor in myocardial ischemic injury and nociceptive transmission via nodose ganglia. The serum lactate dehydrogenase (LDH), creatine kinase (CK) and CK isoform MB (CK-MB) activities were measured by automatic biochemistry analyzer. The electrocardiogram (ECG) recorded ST-segment changes and cardiac arrhythmia. The expression of P2X3 immunoreactivity, mRNA and protein were analyzed by immunohistochemistry, in situ hybridization and western blotting. Myocardial ischemia enhanced the serum LDH, CK and CK-MB activities and caused premature beats. P2X3 receptor antagonist A-317491 decreased the serum enzyme activities and improved premature beats in myocardial ischemic rats. The expression of P2X3 mRNA and protein in the ischemic injury heart were higher than that in the naive heart as control. A-317491 reduced the expression of P2X3 mRNA and protein in the myocardial ischemic injury. The myocardial ischemic injury increased the expression of P2X3 immunoreactivity and mRNA in nodose ganglia. In rats treated with A-317491, the expression of P2X3 immunoreactivity and mRNA in nodose ganglia was reduced. Blocking the nociceptive transmission mediated by P2X3 receptor may protect the cardiac function. According to these results, P2X3 receptor could be thought of as a new target for treating myocardial ischemic injury and cardiac arrhythmia and inhibiting nociceptive transmission of myocardial ischemic injury.

Topics: Animals; Creatine Kinase; Creatine Kinase, MB Form; Disease Models, Animal; Electrocardiography; Female; Gene Expression Regulation; L-Lactate Dehydrogenase; Male; Myocardial Ischemia; Nodose Ganglion; Pain; Phenols; Polycyclic Compounds; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2X3; Synaptic Transmission

A recent report from our laboratory shows that subcutaneous (s.c.) injection of melittin could induce persistent spontaneous nociception (PSN) and primary thermal or mechanical hyperalgesia. However, the exact peripheral mechanisms underlying melittin-induced multiple pain-related behaviors remain unclear. In this study, behavioral tests combined with pharmacological manipulations were used to explore potential roles of local P2X and P2Y receptors in melittin-induced inflammatory pain and hyperalgesia. Post-treatment of the primary injury site with s.c. injection of A-317491 (a potent P2X(3)/P2X(2/3) receptor antagonist) and Reactive Blue 2 (a potent P2Y receptor antagonist) could significantly suppress the development of melittin-evoked PSN and hypersensitivity (thermal and mechanical). Our control experiments demonstrated that local administration of either antagonist into the contralateral hindpaw produced no significant effect on any kind of pain-associated behaviors. Taken together, these data indicate that activation of P2X and P2Y receptors might be essential to the maintenance of melittin-induced primary thermal and mechanical hyperalgesia as well as on-going pain.

Topics: Animals; Hypersensitivity; Male; Melitten; Nociceptors; Pain; Phenols; Polycyclic Compounds; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2X

In recent months, a series of chemically diverse antagonists has been identified for the ATP-gated P2X(7) receptor. In particular, two classes of highly-selective competitive P2X(7) antagonists have been developed by Michael Jarvis and his colleagues at Abbott Laboratories. These di-substituted tetrazole and cyanoguanidine derivatives are outstanding for a number of reasons (not least their stability, selectivity, potency and, of course, reversibility); most exciting is their near equal potency at human and rodent P2X(7) isoforms. Armed with drugs such as A740003 and newer A438079, Jarvis and colleagues have explored the role of P2X(7) receptors in the onset and persistence of chronic pain in animal models. Their findings - and applicability to the human condition - are reviewed in this current issue of British Journal of Pharmacology. This accompanying Commentary describes the progress made by Jarvis and others in developing novel P2X(7) antagonists for pain relief.

Topics: Acetamides; Analgesics, Non-Narcotic; Animals; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Humans; Magnesium; Nerve Endings; Neurons, Afferent; Pain; Phenols; Polycyclic Compounds; Purinergic P2 Receptor Antagonists; Pyridines; Quinolines; Receptors, Purinergic P2; Receptors, Purinergic P2X7; Tetrazoles

Several lines of evidence suggest that extracellular ATP plays a role in pain signaling through the activation of ionotropic P2X-receptors, especially homomeric P2X3- and heteromeric P2X2/3-receptors on capsaicin-sensitive and -insensitive primary afferent neurons, respectively, at peripheral and spinal sites. We investigated the mechanisms of the induction and maintenance of mechanical allodynia produced by a single intrathecal (i.t.) administration of ATP in rats. We found that i.t. administration of ATP and the P2X-receptor agonist alpha,beta-methylene-ATP produced tactile allodynia which lasted more than 1 week. The i.t. ATP- and alpha,beta-methylene-ATP-produced long-lasting allodynia remained in neonatal capsaicin-treated adult rats. I.t. administration of a P2X3/P2X2/3-receptor selective antagonist completely prevented the induction (co-administration on day 0) and partially attenuated the early phase (day 1 post-ATP administration), but not the late phase (day 7 post-ATP administration) of maintenance of allodynia. The N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 completely prevented the induction phase, but not the early and late phases of maintenance of allodynia. Immunohistochemical and immunoblotting studies for microglial and astrocytic markers revealed that i.t. ATP administration caused spinal microglial activation within 1 day, and astrocytic activation which peaked at 1-3 days after ATP administration. Furthermore, minocycline, a microglial inhibitor, attenuated the induction but not the early and late phases of maintenance, while fluorocitrate, a glial metabolic inhibitor, attenuated the induction and the early phase but not the late phase of maintenance. Taken together, these results suggest that the activation of P2X-receptors, most likely spinal P2X2/3-receptors on capsaicin-insensitive primary afferent neurons, triggers the induction of long-lasting allodynia through NMDA receptors, and the induction and early maintenance phase, but not the late phase, is mediated through the functions of spinal glial cells.

Topics: Adenosine Triphosphate; Animals; Astrocytes; Behavior, Animal; Blotting, Western; Capsaicin; Citrates; Immunohistochemistry; Injections, Spinal; Ligation; Male; Microglia; Minocycline; Neuroglia; Pain; Pain Measurement; Phenols; Physical Stimulation; Polycyclic Compounds; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Receptors, Purinergic P2; Receptors, Purinergic P2X2; Sciatic Nerve; Spinal Cord

Extracellular ATP is known to mediate synaptic transmission as a neurotransmitter or a neuromodulator via ionotropic P2X and metabotropic P2Y receptors. Several lines of evidence have suggested that ATP facilitates pain transmission at peripheral and spinal sites via the P2X receptors, in which the P2X3 subtype is considered as an important candidate for the effect. Conversely, we previously found that the activation of supraspinal P2X receptors evoked antinociception. However, the subtypes responsible for the antinociception via supraspinal P2X receptors remain unclear. In the present study, we showed that intracerebroventricular (i.c.v.) pretreatment with A-317491 (1 nmol), the novel non-nucleotide antagonist selective for P2X3 and P2X2/3 receptors, attenuated the antinociceptive effect produced by i.c.v. administered alpha,beta-methylene-ATP (10 nmol), the P2X receptor agonist, in rats. Similarly, the abolishment of the P2X3 receptor mRNA in the brainstem by repeated i.c.v. pretreatments with antisense oligodeoxynucleotide for P2X3 gene once a day for 5 consecutive days diminished the antinociceptive effect of alpha,beta-methylene-ATP. Furthermore, i.c.v. administration of A-317491 (1 and 10 nmol) significantly enhanced the inflammatory nociceptive behaviors induced by the intraplantar injection of formalin and intraperitoneal injection of acetic acid. Taken together, these results suggest that supraspinal P2X3/P2X2/3 receptors play an inhibitory role in pain transmission.

Topics: Acetic Acid; Adenosine Triphosphate; Analgesics; Animals; Dose-Response Relationship, Drug; Formaldehyde; Male; Pain; Pain Measurement; Phenols; Polycyclic Compounds; Purinergic P2 Receptor Antagonists; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2X2; Receptors, Purinergic P2X3

P2X3/P2X2/3 receptors have emerged as important components of nociception. However, there is limited information regarding the neurochemical systems that are affected by antagonism of the P2X3/P2X2/3 receptor and that ultimately contribute to the ensuing antinociception. In order to determine if the endogenous opioid system is involved in this antinociception, naloxone was administered just prior to the injection of a selective P2X3/P2X2/3 receptor antagonist, A-317491, in rat models of neuropathic, chemogenic, and inflammatory pain. Naloxone (1-10 mg kg(-1), i.p.), dose-dependently reduced the antinociceptive effects of A-317491 (1-300 micromol kg(-1), s.c.) in the CFA model of thermal hyperalgesia and the formalin model of chemogenic pain (2nd phase), but not in the L5-L6 spinal nerve ligation model of neuropathic allodynia. In comparison experiments, the same doses of naloxone blocked or attenuated the actions of morphine (2 or 8 mg kg(-1), s.c.) in each of these behavioral models. Injection of a peripheral opioid antagonist, naloxone methiodide (10 mg kg(-1), i.p.), did not affect A-317491-induced antinociception in the CFA and formalin assays, suggesting that the opioid component of this antinociception occurred within the CNS. Furthermore, this utilization of the central opioid system could be initiated by antagonism of spinal P2X3/P2X2/3 receptors since the antinociceptive actions of intrathecally delivered A-317491 (30 nmol) in the formalin model were reduced by both intrathecally (10-50 nmol) and systemically (10 mg kg(-1), i.p.) administered naloxone. This utilization of the opioid system was not specific to A-317491 since suramin-, a nonselective P2X receptor antagonist, induced antinociception was also attenuated by naloxone. In in vitro studies, A-317491 (3-100 microM) did not produce any agonist response at delta opioid receptors expressed in NG108-15 cells. A-317491 had been previously shown to be inactive at the kappa and mu opioid receptors. Furthermore, naloxone, at concentrations up to 1 mM, did not compete for [3H] A-317491 binding in 1321N1 cells expressing human P2X3 receptors. Taken together, these results indicate that antagonism of spinal P2X3/P2X2/3 receptors results in an indirect activation of the opioid system to alleviate inflammatory hyperalgesia and chemogenic nociception.

Topics: Analgesia; Animals; Arthritis, Experimental; Dose-Response Relationship, Drug; Endorphins; Formaldehyde; Freund's Adjuvant; Inflammation; Injections, Spinal; Ligation; Male; Naloxone; Narcotic Antagonists; Pain; Peripheral Nervous System Diseases; Phenols; Polycyclic Compounds; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2X2; Receptors, Purinergic P2X3; Suramin

We have recently reported that systemic delivery of A-317491, the first non-nucleotide antagonist that has high affinity and selectivity for blocking P2X3 homomeric and P2X2/3 heteromeric channels, is antinociceptive in rat models of chronic inflammatory and neuropathic pain. In an effort to further evaluate the role of P2X3/P2X2/3 receptors in nociceptive transmission, A-317491 was administered either intrathecally or into the hindpaw of a rat in several models of acute and chronic nociception. Intraplantar (ED50=300 nmol) and intrathecal (ED50=30 nmol) injections of A-317491 produced dose-related antinociception in the CFA model of chronic thermal hyperalgesia. Administration of A-317491 by either route was much less effective to reduce thermal hyperalgesia in the carrageenan model of acute inflammatory hyperalgesia. Intrathecal, but not intraplantar, delivery of A-317491 attenuated mechanical allodynia in both the chronic constriction injury and L5-L6 nerve ligation models of neuropathy (ED50=10 nmol for both models). Intrathecal injections of A-317491 did not impede locomotor performance. Both routes of injection were effective in reducing the number of nocifensive events triggered by the injection of formalin into a hindpaw. Nocifensive behaviors were significantly reduced in both the first and second phases of the formalin assay (intrathecal ED50=10 nmol, intraplantar ED50>300 nmol). Nocifensive behaviors induced by the P2X receptor agonist alpha,beta-meATP were also significantly reduced by intraplantar injection of A-317491. These data indicate that both spinal and peripheral P2X3/P2X2/3 receptors have significant contributions to nociception in several animal models of nerve or tissue injury. Intrathecal administration of A-317491 appears to be more effective than intraplantar administration to reduce tactile allodynia following peripheral nerve injury.

Topics: Acute Disease; Adenosine Triphosphate; Analgesics, Non-Narcotic; Animals; Carrageenan; Chronic Disease; Hindlimb; Hot Temperature; Inflammation; Injections, Spinal; Male; Motor Activity; Neuropeptides; Pain; Pain Measurement; Peripheral Nervous System Diseases; Phenols; Polycyclic Compounds; Purinergic P2 Receptor Antagonists; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2X2; Receptors, Purinergic P2X3