a-317491 and alpha-beta-methyleneadenosine-5--triphosphate

The aim of the present work was to clarify whether heterotrimeric P2X2/3 receptors have a fixed subunit stoichiometry consisting of one P2X2 and two P2X3 subunits as previously suggested, or a flexible stoichiometry containing also the inverse subunit composition. For this purpose we transfected HEK293 cells with P2X2 and P2X3 encoding cDNA at the ratios of 1:2 and 4:1, and analysed the biophysical and pharmacological properties of the generated receptors by means of the whole-cell patch-clamp technique. The concentration-response curves for the selective agonist α,β-meATP did not differ from each other under the two transfection ratios. However, co-expression of an inactive P2X2 mutant and the wild type P2X3 subunit and vice versa resulted in characteristic distortions of the α,β-meATP concentration-response relationships, depending on which subunit was expressed in excess, suggesting that HEK293 cells express mixtures of (P2X2)1/(P2X3)2 and (P2X2)2/(P2X3)1 receptors. Whereas the allosteric modulators H+ and Zn2+ failed to discriminate between the two possible heterotrimeric receptor variants, the α,β-meATP-induced responses were blocked more potently by the competitive antagonist A317491, when the P2X2 subunit was expressed in deficit of the P2X3 subunit. Furthermore, blue-native PAGE analysis of P2X2 and P2X3 subunits co-expressed in Xenopus laevis oocytes and HEK293 cells revealed that plasma membrane-bound P2X2/3 receptors appeared in two clearly distinct heterotrimeric complexes: a (P2X2-GFP)2/(P2X3)1 complex and a (P2X2-GFP)1/(P2X3)2 complex. These data strongly indicate that the stoichiometry of the heteromeric P2X2/3 receptor is not fixed, but determined in a permutational manner by the relative availability of P2X2 and P2X3 subunits.

Topics: Adenosine Triphosphate; Allosteric Regulation; Animals; Calcium; Cations, Divalent; Cell Membrane; Dose-Response Relationship, Drug; HEK293 Cells; Humans; Membrane Potentials; Mutation; Oocytes; Patch-Clamp Techniques; Phenols; Polycyclic Compounds; Purinergic P2X Receptor Agonists; Purinergic P2X Receptor Antagonists; Receptors, Purinergic P2X2; Receptors, Purinergic P2X3; Transfection; Xenopus laevis

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

P2X3 receptors (P2XRs), as members of the purine receptor family, are deeply involved in chronic pain sensation and therefore, specific, competitive antagonists are of great interest for perspective pain management. Heretofore, Schild plot analysis has been commonly used for studying the interaction of competitive antagonists and the corresponding receptor. Unfortunately, the steady-state between antagonist and agonist, as a precondition for this kind of analysis, cannot be reached at fast desensitizing receptors like P2X3R making Schild plot analysis inappropriate. The aim of this study was to establish a new method to analyze the interaction of antagonists with their binding sites at the rapidly desensitizing human P2X3R. The patch-clamp technique was used to investigate the structurally divergent, preferential antagonists A317491, TNP-ATP and PPADS. The P2X1,3-selective α,β-methylene ATP (α,β-meATP) was used as an agonist to induce current responses at the wild-type (wt) P2X3R and several agonist binding site mutants. Afterwards a Markov model combining sequential transitions of the receptor from the closed to the open and desensitized mode in the presence or absence of associated antagonist molecules was developed according to the measured data. The P2X3R-induced currents could be fitted correctly with the help of this Markov model allowing identification of amino acids within the binding site which are important for antagonist binding. In conclusion, Markov models are suitable to simulate agonist antagonist interactions at fast desensitizing receptors such as the P2X3R. Among the antagonists investigated, TNP-ATP and A317491 acted in a competitive manner, while PPADS was identified as a (pseudo)irreversible blocker.

Topics: Adenosine Triphosphate; Binding Sites; Binding, Competitive; Drug Interactions; HEK293 Cells; Humans; Kinetics; Markov Chains; Membrane Potentials; Models, Biological; Mutation; Patch-Clamp Techniques; Phenols; Polycyclic Compounds; Protein Binding; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Receptors, Purinergic P2X3

Extracellular ATP facilitates pain transmission at peripheral and spinal sites via the P2X receptors and the P2X3 subtype is an important candidate for this effect. Electroacupuncture (EA) has been clinically utilized to manage chronic pain. In this study, with neuropathic pain model of Sprague-Dawley (SD) rats, the P2X3 receptor protein level and expression location in the midbrain periaqueductal gray (PAG), a crucial site in endogenous pain modulatory system, were evaluated by Western blotting and immunohistochemistry. The results showed (1) pain thresholds were decreased while P2X3 receptor expression was up-regulated in the lateral PAG (lPAG) when neuropathic pain occurred. When the lPAG was pretreated with P2X3 receptors, antagonist A-317491 attenuated the antinociceptive effect produced by intra-lPAG injection of alpha,beta-methylene-ATP (alpha, beta-meATP), an agonist for P2X3 receptor. (2) Multiple EA treatments begot enhanced pain thresholds and increased P2X3 receptor immunoreactivity in the lPAG in neuropathic pain rats. Conversely, the down-regulated P2X3 receptor expression in the lPAG with antisense oligodeoxynucleotide (ODN) for P2X3 gene significantly attenuated the antinociceptive effect of EA treatment. These results suggest that P2X3 receptors in the lPAG play an inhibitory role in pain modulation and EA exerts a marked therapeutic effect in relieving neuropathic pain in CCI rats, which may be related to its regulative effect on the expression of P2X3 receptors in the lPAG. In conclusion, P2X3 receptors in the lPAG are involved in the supraspinal antiociception effect of EA treatment.

Topics: Adenosine Triphosphate; Animals; Central Nervous System Agents; Disease Models, Animal; Electroacupuncture; Male; Neuralgia; Oligodeoxyribonucleotides, Antisense; Pain Threshold; Periaqueductal Gray; Phenols; Polycyclic Compounds; Random Allocation; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2X3; Up-Regulation

To clarify the basic mechanism involved in the pathophysiology of cystitis by characterizing the urodynamic parameters, pharmacologically relevant (muscarinic and purinergic) receptors, and the in vivo release of adenosine triphosphate (ATP) in the bladder of hydrochloric acid (HCl)-treated rats.. The muscarinic and purinergic receptors in rat tissue were measured by radioreceptor assays using (N-methyl-³H) scopolamine methyl chloride ([³H]NMS) and αβ-methylene-ATP (2,8-³H) tetrasodium salt ([³H]αβ-MeATP), respectively. The urodynamic parameters and ATP levels were measured using a cystometric method and the luciferin-luciferase assay, respectively.. In the HCl-treated rats, the micturition interval and micturition volume were significantly (48% and 55%, respectively, P <.05) decreased and the number of micturitions was significantly (3.2-fold, P <.05) increased compared with those of the control rats. The maximal number of binding sites for [³H]NMS and [³H]αβ-MeATP was significantly (55% and 72%, respectively, P <.001) decreased in the bladder of HCl-treated rats, suggesting downregulation of both muscarinic and purinergic receptors. In the HCl-treated rats, the inhibition constant, K(i), values for oxybutynin, solifenacin, and darifenacin were significantly (1.3-1.4-fold, P <.05) increased, but those for tolterodine and AF-DX116 were unchanged. Similarly, the inhibition constant for A-317491, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid tetrasodium, and MRS2273 was significantly (5.5, 11, and 7.6-fold, respectively, P <.001) increased. Furthermore, the in vivo release of ATP was significantly (P <.05) enhanced in the HCl-treated rat bladder.. Both muscarinic and purinergic mechanisms might be, at least in part, associated with the urinary dysfunction due to cystitis.

Topics: Adenosine Triphosphate; Animals; Benzhydryl Compounds; Benzofurans; Cresols; Cystitis; Disease Models, Animal; Down-Regulation; Female; Hydrochloric Acid; Mandelic Acids; N-Methylscopolamine; Organophosphonates; Phenols; Phenylpropanolamine; Pirenzepine; Polycyclic Compounds; Pyridoxal Phosphate; Pyrrolidines; Quinuclidines; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Receptors, Purinergic; Solifenacin Succinate; Tetrahydroisoquinolines; Tolterodine Tartrate; Urinary Bladder; Urination; Urodynamics

The exercise pressor reflex is due to activation of thin fiber afferents within contracting muscle. These afferents are in part stimulated by ATP activation of purinergic 2X (P2X) receptors during contraction. Which of the P2X receptors contribute to the reflex is unknown; however, P2X2/3 and P2X3 receptor subtypes are good candidates because they are located on thin fiber afferents and are involved in sensory neurotransmission. To determine if P2X2/3 and P2X3 receptors evoke the metabolic component of the exercise pressor reflex, we examined the effect of two P2X2/3 and P2X3 antagonists, A-317491 (10 mg/kg) and RO-3 (10 mg/kg), on the pressor response to injections of α,β-methylene ATP (α,β-MeATP; 50 μg/kg), freely perfused static contraction, contraction of the triceps surae muscles while the circulation was occluded, and postcontraction circulatory occlusion in decerebrate cats. We found that the antagonists reduced the pressor response to α,β-MeATP injection (before Δ 20 ± 3 mmHg; drug Δ 11 ± 3 mmHg; P < 0.05), suggesting the antagonists were effective in blocking P2X2/3 and P2X3 receptors. P2X2/3 and P2X3 receptor blockade reduced the pressor response to freely perfused contraction (before Δ 33 ± 5 mmHg; drug Δ 15 ± 5 mmHg; P < 0.05), contraction with the circulation occluded (before Δ 52 ± 7 mmHg; drug Δ 20 ± 4 mmHg; P < 0.05), and during postcontraction circulatory occlusion (before Δ 15 ± 1 mmHg; drug Δ 5 ± 1 mmHg; P < 0.05). Our findings suggest that P2X2/3 and P2X3 receptors contribute to the metabolic component of the exercise pressor reflex in decerebrate cats.

Topics: Adenosine Triphosphate; Animals; Blood Pressure; Cats; Decerebrate State; Electric Stimulation; Female; Muscle Contraction; Muscle, Skeletal; Neurons, Afferent; Phenols; Physical Exertion; Polycyclic Compounds; Purinergic P2X Receptor Antagonists; Receptors, Purinergic P2X2; Receptors, Purinergic P2X3; Reflex; Time Factors

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

Despite the rapid progress made in understanding the significant role played by signalling via extracellular ATP in physiology and pathology, there has been no clear information generated on its involvement in the emetic response.. In the present study, the emetogenic potential of extracellular ATP signalling in mammalian species was examined using ferrets and Suncus murinus (house musk shrews). A slowly degradable ATP analogue, alpha,beta-methyleneATP (alpha,beta-meATP), was used to activate the P2X receptors, and either the non-selective P2 receptor antagonist, pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), or the specific P2X(3) homomer and P2X(2/3) heteromer antagonist, A-317491, were tested against the agonist-induced response.. Intraperitoneal injection of alpha,beta-meATP produced significant emetic responses in ferrets (1 - 30 mg kg(-1)) and in Suncus murinus (5 - 50 mg kg(-1)). The responses occurred frequently within the first 10 min after administration, much less frequently from 11 to 60 min and no responses occurred later than 60 min. The emetic responses were completely inhibited by intraperitoneal pre-treatment with PPADS (100 mg kg(-1)) or A-317491 (100 mg kg(-1)). Abdominal surgical vagotomy did not reduce the emetic response in Suncus murinus significantly.. These results for the first time indicate that the activation of P2X receptors evokes emetic responses in mammalian species. The P2X(3) homomer and.or P2X(2/3) heteromer in the area postrema could be responsible for the emetic response. This finding contributes to the elucidation of the roles played by extracellular ATP signalling in various emetic symptoms.

Topics: Adenosine Triphosphate; Animals; Antineoplastic Agents; Dose-Response Relationship, Drug; Ferrets; Injections, Intraperitoneal; Male; Models, Animal; Nausea; Phenols; Polycyclic Compounds; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Receptors, Purinergic P2; Shrews; Species Specificity; Time Factors; Vagotomy; Vomiting

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

The present study explored differences in sensitivity to purinergic agonists, protons, and capsaicin in lumbosacral (LS) and thoracolumbar (TL) sensory neurons that innervate the rat urinary bladder. The majority of LS neurons (93%) were sensitive to alpha,beta-methyleneATP (alpha,beta-metATP) compared with 50% of TL neurons. Based on inactivation kinetics, a slowly desensitizing current evoked by alpha,beta-metATP predominated in LS neurons (86%) compared with mixed components that characterized TL neuron responses (58%). The density of the slowly desensitizing current was greater in LS than in TL neurons (LS, 34.4 +/- 5.3 pA/pF; TL, 2.5 +/- 0.8 pA/pF). Almost all neurons in both ganglia responded to protons and to capsaicin (LS, 100%; TL, 98%). Proton-activated currents in bladder sensory neurons exhibited distinct inactivation kinetics as fast, intermediate, slowly desensitizing, and sustained components. More than one component was expressed in every cell. Although there was no difference in the percentage of neurons expressing more than one component, the density of the sustained current was significantly greater in LS than in TL neurons (LS, 86.1 +/- 16 pA/pF; TL, 30.3 +/- 7 pA/pF). Similarly, the capsaicin-evoked current was greater in LS than in TL neurons (LS, 129.6 +/- 17 pA/pF; TL, 86.9 +/- 11 pA/pF). Finally, a greater percentage of TL neurons bound isolectin B4 than LS neurons (LS, 61%; TL, 85%). The greater degree of alpha,beta-metATP, proton, and capsaicin responsiveness, in addition to differences in current type and current densities, in LS and TL neurons suggests that bladder pelvic and hypogastric/lumbar splanchnic afferents are functionally distinct and likely mediate different sensations arising from the urinary bladder.

Topics: Adenosine Triphosphate; Amiloride; Amino Acids; Animals; Capsaicin; Cells, Cultured; Dose-Response Relationship, Drug; Drug Interactions; Electric Stimulation; Enzyme Inhibitors; Ganglia, Spinal; Hydrogen-Ion Concentration; Immunohistochemistry; Lectins; Lumbosacral Region; Male; Membrane Potentials; Neurons, Afferent; Patch-Clamp Techniques; Phenols; Polycyclic Compounds; Protons; Purinergic Agonists; Purinergic Antagonists; Rats; Rats, Sprague-Dawley; Receptors, Purinergic; Sodium Channel Blockers; Thoracic Vertebrae; Time Factors; TRPV Cation Channels; Urinary Bladder

Motoneurons of the compact division of the nucleus ambiguus (cNA) are the final output neurons of the swallowing pattern generator. Thus, their normal function is critical to neonatal survival. To explore the role of purinergic signaling in modulating the excitability of these motoneurons during development, immunohistochemical and whole-cell recording techniques were used to characterize expression patterns of ionotropic P2X receptors and the effects of ATP on cNA motoneurons. Medullary slices containing the cNA were prepared from neonatal (P0-4) and juvenile (P15-21) rats. In neonatal cNA motoneurons, local application of 1 mM ATP produced a large (-133 +/- 17 pA; n = 78), desensitizing, inward current that was mimicked by 1 mM alpha,beta meATP and 2meSATP, and inhibited by the P2 antagonist, PPADS (5 microM), and the P2X3 antagonist, A-317481 (0.1-1 mM). In juvenile cNA motoneurons, 1 mM ATP produced negligible currents, while 10 mM ATP produced small (-59 +/- 14 pA; n = 42), primarily non-desensitizing currents. Immunohistochemistry demonstrated that in the neonate, the expression of P2X3 was robust, P2X2 and P2X5 moderate, P2X4 and P2X6 weak, and P2X1 absent. In the juvenile cNA, only low levels of P2X5 and P2X6 labeling were detected. These data indicate that P2X receptors in cNA motoneurons are profoundly downregulated during the first two postnatal weeks, and suggest a role for the purinoceptor system, particularly P2X3 receptors, in the control of esophageal motor networks during early postnatal periods.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Age Factors; Animals; Animals, Newborn; Cell Count; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Drug Interactions; Electric Stimulation; Enzyme Inhibitors; Gene Expression Regulation, Developmental; Glutamic Acid; Immunohistochemistry; In Vitro Techniques; Membrane Potentials; Motor Neurons; Nucleus Accumbens; Patch-Clamp Techniques; Phenols; Polycyclic Compounds; Pyridoxal Phosphate; Rats; Rats, Wistar; Receptors, Purinergic P2; Receptors, Purinergic P2X3; Tetrodotoxin; Time Factors

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