2--3--o-(2-4-6-trinitrophenyl)adenosine-5--triphosphate and pyridoxal-phosphate-6-azophenyl-2--4--disulfonic-acid

Our previous study together with other investigations have reported that neonatal hypoxia or ischemia induces long-term cognitive impairment, at least in part through brain inflammation and hypomyelination. However, the detailed mechanisms are not fully understood. Here, we used a rodent model of neonatal hypoxia by subjecting postnatal day 0 (P0) rat pups to systemic hypoxia (3.5 h). We found that neonatal hypoxia increased the glutamate content and initiated inflammatory responses at 4 h and 1 day after hypoxia, caused hypomyelination in the corpus callosum, and impaired hippocampus-dependent learning and memory when assessed 30-60 days after hypoxia. Interestingly, much of the hypoxia-induced brain damage was ameliorated by treatment with the ATP analogue 2',3'-0-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP; blocks all ionotropic P2X1-7 receptors), whereas treatment with pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; inhibits P2X1-3 and P2X5-7 receptors) was less neuroprotective. Our data indicated that activation of ionotropic ATP receptors might be partially, if not fully, involved in glutamate deregulation, neuroinflammation, hypomyelination, and cognitive dysfunction after neonatal hypoxia.

Topics: Adenosine Triphosphate; Animals; Animals, Newborn; Brain; Cognition Disorders; Disease Models, Animal; Female; Glutamic Acid; Hypoxia-Ischemia, Brain; Immunoblotting; Male; Maze Learning; Myelin Sheath; Neurogenesis; Neuroprotective Agents; Pyridoxal Phosphate; Rats; Rats, Sprague-Dawley

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

This study explored purinergic signaling in lumbosacral (LS) and thoracolumbar (TL) dorsal root ganglion neurons innervating the urinary bladder. In naïve mice, a greater proportion of LS (93%) than that of TL (77%) bladder neurons responded to purinergic agonists. Three types of purinergic currents were identified: 'sustained' (homomeric P2X2) currents were detected only in LS neurons, rapidly activating, 'slow' deactivating (heteromeric P2X2/3) currents predominated in both LS and TL neurons, and 'fast' activating/de-activating (homomeric P2X3) currents were detected only in TL neurons. Relative to TL bladder neurons, slow current density was greater in LS neurons, which also had a more negative action potential threshold and generated more action potentials in response to purinergic agonists (suggesting greater excitability of LS neurons). Single cell nested PCR documented P2X2 and P2X3 subunit expression in both TL and LS bladder neurons. Relative to saline treatment, bladder wall thickness and weight increased after cyclophosphamide (CYP) treatment. Both LS and TL neuron excitability increased (rheobase was decreased and responses to purinergic agonists increased) after CYP treatment. The proportion of sustained currents in LS bladder neurons increased fourfold after CYP bladder inflammation. Although proportions of slow and fast purinergic currents in TL neurons were unchanged by CYP treatment, the fast current density was greater than in saline-treated mice. These results in mouse, as previously described in rat, reveal differential purinergic signaling in TL and LS bladder neurons. The predominant currents and significant changes after inflammation, however, occur in different ganglia/sensory pathways in mouse and rat.

Topics: Adenosine Triphosphate; Animals; Cells, Cultured; Cyclophosphamide; Disease Models, Animal; Dogs; Ganglia, Spinal; Immunosuppressive Agents; Inflammatory Bowel Diseases; Male; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Knockout; Patch-Clamp Techniques; Peroxidase; Platelet Aggregation Inhibitors; Pyridoxal Phosphate; Receptors, Purinergic P2; Receptors, Purinergic P2X2; Receptors, Purinergic P2X3; Sensory Receptor Cells; Signal Transduction

P2X(3) and P2X(2/3) receptors are localized on sensory afferents both peripherally and centrally and have been implicated in various sensory functions. However, the physiological role of these receptors expressed presynaptically in the spinal cord in regulating sensory transmission remains to be elucidated. Here, a novel selective P2X(3) and P2X(2/3) antagonist, AF-792 [5-(5-ethynyl-2-isopropyl-4-methoxy-phenoxy)-pyrimidine-2,4-diamine, previously known as RO-5], in addition to less selective purinoceptor ligands, was applied intrathecally in vivo. Cystometry recordings were made to assess changes in the micturition reflex contractions after drug treatments. We found that AF-792 inhibited micturition reflex activity significantly (300 nmol; from baseline contraction intervals of 1.18 +/- 0.07 to 9.33 +/- 2.50 min). Furthermore, inhibition of P2X(3) and P2X(2/3) receptors in the spinal cord significantly attenuated spinal activation of extracellular-signal regulated kinases induced by acute peripheral stimulation of the bladder with 1% acetic acid by 46.4 +/- 12.0% on average. Hence, the data suggest that afferent signals originating from the bladder are regulated by spinal P2X(3) and P2X(2/3) receptors and establish directly an endogenous central presynaptic purinergic mechanism to regulate visceral sensory transmission. Identification of this spinal purinergic control in visceral activities may help the development of P2X(3) and P2X(2/3) antagonist to treat urological dysfunction, such as overactive bladder, and possibly other debilitating sensory disorders, including chronic pain states.

Topics: Adenosine Triphosphate; Animals; Female; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Platelet Aggregation Inhibitors; Pressure; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Pyrimidines; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2X2; Receptors, Purinergic P2X3; Signal Transduction; Spinal Cord; Urinary Bladder

Although previous studies have provided evidence for the expression of P2X receptors in renal proximal tubule, only one cell line study has provided functional evidence. The current study investigated the pharmacological properties and physiological role of native P2X-like currents in single frog proximal tubule cells using the whole-cell patch-clamp technique. Extracellular ATP activated a cation conductance (P2X(f)) that was also Ca²+-permeable. The agonist sequence for activation was ATP = αβ-MeATP > BzATP = 2-MeSATP, and P2X(f) was inhibited by suramin, PPADS and TNP-ATP. Activation of P2X(f) attenuated the rundown of a quinidine-sensitive K+ conductance, suggesting that P2X(f) plays a role in K+ channel regulation. In addition, ATP/ADP apyrase and inhibitors of P2X(f) inhibited regulatory volume decrease (RVD). These data are consistent with the presence of a P2X receptor that plays a role in the regulation of cell volume and K+ channels in frog renal proximal tubule cells.

Topics: Adenosine Triphosphate; Animals; Cells, Cultured; Kidney; Kidney Tubules, Proximal; Patch-Clamp Techniques; Purinergic Agonists; Purinergic Antagonists; Pyridoxal Phosphate; Rana temporaria; Receptors, Purinergic P2X; Thionucleotides

Many studies have shown that adenosine triphosphate (ATP), as a neurotransmitter, is involved in plastic changes of synaptic transmission in central nervous system. In the present study, we tested whether extracellular ATP can induce long-term potentiation (LTP) of C-fiber-evoked field potentials in spinal dorsal horn. The results showed the following: (1) ATP at a concentration of 0.3 mM induced spinal LTP of C-fiber-evoked field potentials, lasting for at least 5 h; (2) spinal application of 2',3'-O-(2,4,6-trinitrophenyl)adenosine-5-triphosphate (TNP-ATP; an antagonist of P2X(1-4) receptors), but not pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; an antagonist of P2X(1,2,3,5,7) receptors), 30 min before ATP blocked ATP-induced LTP, indicating that ATP may induce spinal LTP by activation of P2X(4) receptors; (3) at 60 min after LTP induction the level of phospho-p38 mitogen-activated protein kinase (p-p38 MAPK) was significantly elevated and at 180 min after LTP the number of P2X(4) receptors increased significantly; both p-p38 and P2X(4) receptors were exclusively co-located with the microglia marker, but not with neuronal or astrocyte marker; (4) spinal application of TNP-ATP but not PPADS prevented p38 activation; (5) spinal application of SB203580, a p38 MAPK inhibitor, prevented both spinal LTP and the upregulation of P2X(4) receptors. The results suggested that ATP may activate p38 MAPK by binding to intrinsic P2X(4) receptors in microglia, and subsequently enhance the expression of P2X(4) receptors, contributing to spinal LTP.

Topics: Adenosine Triphosphate; Animals; Astrocytes; Electric Stimulation; Evoked Potentials; Extracellular Space; Long-Term Potentiation; Male; Microglia; p38 Mitogen-Activated Protein Kinases; Posterior Horn Cells; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2X4; Sciatic Nerve; Spinal Cord; Up-Regulation

Opioids, although fundamental to the treatment of pain, are limited in efficacy by side effects including tolerance and hyperalgesia. Using an in vitro culture system, we report that morphine increased microglial migration via a novel interaction between mu-opioid and P2X(4) receptors, which is dependent upon PI3K/Akt pathway activation. Morphine at 100 nm enhanced migration of primary microglial cells toward adenosine diphosphate by 257, 247, 301, 394, and 345% following 2, 6, 12, 24, and 48 h of stimulation, respectively. This opioid-dependent migration effect was inhibited by naloxone and confirmed to be mu-opioid receptor-dependent through the use of selective agonists and antagonists. PPADS [pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid)], a P2X(1-3,5-7) antagonist, had no effect on microglial migration; however, TNP-ATP [2',3'-O-(2,4,6-trinitrophenyl)-ATP], a P2X(1-7) antagonist, inhibited morphine-induced migration, suggesting a P2X(4) receptor-mediated effect. The PI3K inhibitors wortmannin and LY294002 decreased morphine-induced microglial migration. Iba1 protein, a microglial marker, and P2X(4) receptor expression were significantly increased after 6, 12, 24, and 48 h of morphine stimulation. Together, these results provide evidence for two phases of morphine effects on microglia. The initial phase takes place in minutes, involves PI3K/Akt pathway activation and leads to acutely enhanced migration. The longer-term phase occurs on the order of hours and involves increased expression of Iba1 and P2X(4) receptor protein, which imparts a promigratory phenotype and is correlated with even greater migration. These data provide the first necessary step in supporting microglial migration as an attractive target for the prevention or attenuation of morphine-induced side effects including tolerance and hyperalgesia.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Animals; Benzeneacetamides; Calcium-Binding Proteins; Cell Movement; Cells, Cultured; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enzyme Inhibitors; Gene Expression Regulation; Microfilament Proteins; Microglia; Morphine; Naloxone; Narcotic Antagonists; Narcotics; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2X4; Signal Transduction; Time Factors

Increased sensitivity of the afferent innervation of the gastrointestinal tract reportedly underlies symptoms of discomfort and pain in functional bowel disorders. The present investigation aimed to examine whether the purinergic P2X(2) and P2X(3) receptor subunits contribute to the mechanosensitivity of small intestinal afferents in normal mice and in a murine model of postinfectious gut dysfunction. Mesenteric afferent nerve activity was recorded in a mouse jejunum preparation maintained in vitro. As has been shown previously, ramp distension of the jejunal segment evoked biphasic afferent discharge, reflecting activation of low and high threshold fibres. The average pressure-afferent response curve in mice deficient in both P2X(2) and P2X(3) subunits (n = 14) was not significantly different from that of the wild-type control preparations (n = 13). Application of pyridoxal 5-phosphate 6-azophenyl-2 ,4-disulphonic acid (PPADS) (30 micromol L(-1)), a P2X and P2Y antagonist, or 2,4,6-trinitrophenol-adenosine 5'-triphosphate (10 micromol L(-1)), an antagonist selective for homomeric P2X(3) and heteromeric P2X(2/3) receptors, had no effect on the averaged pressure-afferent response curve in wild-type animals. In Trichinella spiralis-infected mice, the magnitude of mesenteric afferent responses to jejunal distension was greater at day 21 and day 56 postinfection compared with the sham control preparations demonstrating the development of afferent hypersensitivity. PPADS had no significant effect upon mechanically evoked afferent discharge rates in sham treated preparations (n = 5), but significantly inhibited afferent sensitivity to jejunal distension in preparations from mice at day 21 (n = 6) and day 56 (n = 7) postinfection. These results suggest that purinergic mechanisms play no role in mechanosensory transduction in the normal small intestine but contribute significantly to postinfectious mechano-hypersensitivity.

Topics: Adenosine Triphosphate; Animals; Fluorescent Dyes; Hypersensitivity; Intestine, Small; Irritable Bowel Syndrome; Mesentery; Mice; Mice, Inbred C57BL; Mice, Knockout; Neurons, Afferent; Physical Stimulation; Purinergic Agonists; Purinergic Antagonists; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Receptors, Purinergic; Receptors, Purinergic P2; Receptors, Purinergic P2X2; Receptors, Purinergic P2X3; Signal Transduction; Trichinella spiralis; Trichinellosis

The styryl pyridinium dyes, FM1-43 and AM1-43, are fluorescent molecules that can permeate the mechanotransduction channels of hair cells, the sensory receptors of the inner ear. When these dyes are applied to hair cells, they enter the cytoplasm rapidly, resulting in a readily detectable intracellular fluorescence that is often used as a molecular indication of mechanotransduction channel activity. However, such dyes can also permeate the ATP receptor, P2X(2). Therefore, we explored the contribution of P2X receptors to the loading of hair cells with AM1-43. The chick inner ear was found to express P2X receptors and to release ATP, similar to the inner ear of mammals, allowing for the endogenous stimulation of P2X receptors. The involvement of these receptors was evaluated pharmacologically, by exposing the sensory epithelium of the chick inner ear to 5 microM AM1-43 under different experimental conditions and measuring the fluorescence in hair cells after fixation of the tissue. Pre-exposure of the tissue to 5 mM EGTA for 15 min, which should eliminate most of the gating "tip links" of the mechanotransduction channels, deceased fluorescence by only 44%. In contrast, P2X receptor antagonists (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid [PPADS], suramin, 2',3'-O-(2,4,6-trinitrophenyl) ATP [TNP-ATP], and d-tubocurarine) had greater effects on dye loading. PPADS, suramin, and TNP-ATP all decreased intracellular AM1-43 fluorescence in hair cells by at least 69% when applied at a concentration of 100 microM. The difference between d-tubocurarine-treated and control fluorescence was statistically insignificant when d-tubocurarine was applied at a concentration that blocks the mechanotransduction channel (200 microM). At a concentration that also blocks P2X(2) receptors (2 mM), d-tubocurarine decreased dye loading by 72%. From these experiments, it appears that AM1-43 can enter hair cells through endogenously activated P2X receptors. Thus, the contribution of P2X receptors to dye entry should be considered when using styryl pyridinium dyes to detect hair cell mechanotransduction channel activity, especially in the absence of explicit mechanical stimulation of stereocilia.

Topics: Adenosine Triphosphate; Animals; Chelating Agents; Chickens; Connexins; Egtazic Acid; Epithelium; Fluorescence; Fluorescent Dyes; Hair Cells, Auditory; In Vitro Techniques; Mechanotransduction, Cellular; Purinergic P2 Receptor Antagonists; Pyridinium Compounds; Pyridoxal Phosphate; Quaternary Ammonium Compounds; Receptors, Purinergic P2; Suramin; Tubocurarine

Painful diabetic neuropathy causes hyperalgesia and does not respond to commonly used analgesics such as non-steroidal anti-inflammatory drugs or opioids at doses below those producing disruptive side effects. In the present study, we examined the effect of P2X receptor antagonists, which are known to modulate the pain pathway, on mechanical hyperalgesia in streptozotocin (STZ)-induced diabetic mice. The paw withdrawal frequency measured by von Frey filaments, began to significantly increase 5 days after STZ injection and was maintained for more than 14 days. Intrathecal administration of P2X receptor antagonists (PPADS and TNP-ATP) inhibited the mechanical allodynia in diabetic mice. The levels of P2X(2) and P2X(3) receptors mRNA were significantly increased in diabetic mice at 14 days after the intravenous injection of STZ. These results suggest that the upregulation of P2X(2), P2X(3) and/or P2X(2/3) receptor in DRG neurons is associated with mechanical allodynia in STZ-induced diabetic mice.

Topics: Adenosine Triphosphate; Animals; Diabetes Mellitus, Experimental; Diabetic Neuropathies; Disease Models, Animal; Ganglia, Spinal; Hyperalgesia; Male; Mice; Nociceptors; Pain Measurement; Physical Stimulation; Platelet Aggregation Inhibitors; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Receptors, Purinergic P2; Receptors, Purinergic P2X2; Receptors, Purinergic P2X3; RNA, Messenger; Sensory Receptor Cells; Up-Regulation

In this study we report the coupling of nucleotide receptors to GSK-3 signalling, a relevant survival pathway in cerebellar granule neurons. P2X(7) agonist BzATP induced a 3-4-fold increase in GSK-3 phosphorylation, which is reported to be associated with the catalytic activity inhibition. This effect was dependent on extracellular calcium and PKC, and independent of PI3-K (phosphatidyl-inositol-3-kinase)/Akt, the main survival route of neurotrophins. BzATP also prevented the apoptosis of granule neurons induced by the pharmacological inhibition of the PI3-K signalling. Both effects, BzATP-mediated GSK-3 phosphorylation and neuroprotection, were abolished by P2X(7) receptor antagonists, BBG, PPADS and A-438079. We found that BzATP prevented the progressive GSK-3 dephosphorylation and caspase-3 activation occurring under conditions of sustained PI3-K inhibition. These results reveal that P2X(7) receptor activation could provide a relevant survival route alternative to classical neurotrophic factors.

Topics: Adenosine Triphosphate; Animals; Animals, Newborn; Calcium; Cell Survival; Cells, Cultured; Cerebellum; Chromones; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gene Expression Regulation; Glycogen Synthase Kinase 3; In Situ Nick-End Labeling; Morpholines; Neurons; Phosphatidylinositol 3-Kinases; Phosphorylation; Platelet Aggregation Inhibitors; Protein Kinase C; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Rats; Receptors, Purinergic P2; Receptors, Purinergic P2X7; Time Factors

Microglia, the CNS resident macrophages responsible for the clearance of degenerating cellular fragments, are essential to tissue remodeling and repair after CNS injury. ATP can be released in large amounts after CNS injury and may mediate microglial activity through the ionotropic P2X and the metabotropic P2Y receptors. This study indicates that exposure to a high concentration of ATP for 30 min rapidly induces changes of the microglial cytoskeleton, and significantly attenuates microglial phagocytosis. A pharmacological approach showed that ATP-induced inhibition of microglial phagocytotic activity was due to P2X(7)R activation, rather than that of P2YR. Activation of P2X(7)R by its agonist, 2'-3'-O-(4-benzoyl)benzoyl-ATP (BzATP), produced a Ca(2+)-independent reduction in microglial phagocytotic activity. In addition, the knockdown of P2X(7)R expression by lentiviral-mediated shRNA interference or the blockade of P2X(7)R activation by the specific antagonists, oxidized ATP (oxATP) and brilliant blue G, has efficiently restored the phagocytotic activity of ATP and BzATP-treated microglia. Our results reveal that P2X(7)R activation may induce the formation of a Ca(2+)-independent signaling complex, which results in the reduction of microglial phagocytosis. This suggests that exposure to ATP for a short-term period may cause insufficient clearance of tissue debris by microglia through P2X(7)R activation after CNS injury, and that blockade of this receptor may preserve the phagocytosis of microglia and facilitate CNS tissue repair.

Topics: Actins; Adenosine Triphosphate; Animals; Animals, Newborn; Benzoxazoles; Calcium; Cells, Cultured; Cerebral Cortex; Cyclophilin A; Dose-Response Relationship, Drug; Flow Cytometry; Microglia; Phagocytosis; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Quinolinium Compounds; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2X2; RNA, Messenger; Time Factors; Transduction, Genetic

Inhibitory GABAergic and glycinergic neurotransmission to cardioinhibitory cardiac vagal neurons (CVNs) increase during inspiratory activity and likely mediate respiratory sinus arrhythmia, while the frequency of excitatory postsynaptic currents (EPSCs) in CVNs are unaltered during the different phases of respiration. However, following hypoxia and hypercapnia (H/H), the parasympathetic activity to the heart increases and thus far, identification of the pathways and neurotransmitters that are responsible for exciting CVNs post H/H are unclear. This study identifies different excitatory pathways to CVNs recruited post H/H. Spontaneous and inspiratory-related EPSCs were recorded in CVNs before, during, and after 10 min of H/H in an in vitro slice preparation that retains rhythmic respiratory activity. Before and during H/H, EPSCs in CVNs were completely blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and d(-)-2-amino-5-phosphonopentanoic acid (AP5), selective AMPA/kainate and N-methyl-d-apartate (NMDA) receptor blockers, respectively. However, after H/H, there was a significant increase in EPSCs during each inspiratory burst. While some of the inspiratory-related EPSCs were blocked by the broad purinergic receptor antagonist pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid (PPADS) and the specific P2X receptor antagonist 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate monolithium trisodium salt (TNP-ATP) a P2X receptor blocker, most of the recruited excitatory neurotransmission to CVNs is serotonergic because odansetron, a selective 5-HT3 antagonist, abolished the majority of the spontaneous and inspiratory-related EPSCs evoked during recovery from H/H. The results from this study suggest that following episodes of H/H, two nonglutamatergic excitatory pathways, purinergic and serotonergic, activating P2X and 5-HT3 receptors, respectively, are recruited to excite CVNs in the post H/H recovery period.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Adenosine Triphosphate; Animals; Animals, Newborn; Drug Interactions; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Hypercapnia; Hypoxia; In Vitro Techniques; Neurons; Nucleus Accumbens; Ondansetron; Patch-Clamp Techniques; Platelet Aggregation Inhibitors; Pyridoxal Phosphate; Rats; Rats, Sprague-Dawley; Respiration; Serotonin; Serotonin Antagonists; Vagus Nerve; Valine

Recent work has shown that adenosine 5'-triphosphate (ATP) plays an important role in modulating the activity of parasympathetic cardiac vagal neurons that dominate the neural control of heart rate. This study examined the mechanisms by which activation of ATP receptors modulates excitatory neurotransmission to cardiac vagal neurons. Glutamatergic activity to cardiac vagal neurons was isolated and examined using whole-cell patch-clamp recordings in an in vitro brain slice preparation in rats. ATP (100 microM) evoked increases in the frequency of glutamatergic miniature excitatory postsynaptic currents (mEPSCs) in cardiac vagal neurons which were blocked by the broad P2 receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 100 microM). Application of the selective P2X receptor agonist, alpha, beta-methylene ATP (100 microM), also increased glutamatergic mEPSCs neurotransmission to cardiac vagal neurons indicating P2X receptors enhance glutamatergic release to cardiac vagal neurons. The evoked increase in glutamatergic mEPSC was unaltered by the voltage-gated calcium channel blocker cadmium, and was abolished by the selective P2X receptor antagonist 2',3'-O-(2,4,6-Trinitrophenyl) adenosine 5'-triphosphate, TNP-ATP (100 microM). This work demonstrates that the ATP evoked facilitation of excitatory neurotransmission to cardiac vagal neurons is dependent upon activation of P2X receptors on glutamatergic presynaptic terminals.

Topics: Adenosine Triphosphate; Animals; Excitatory Postsynaptic Potentials; Ganglia, Parasympathetic; Glutamic Acid; Heart; Medulla Oblongata; Neurons; Organ Culture Techniques; Parasympathetic Nervous System; Patch-Clamp Techniques; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Rats; Rats, Sprague-Dawley; Receptors, Glutamate; Receptors, Purinergic P2; Receptors, Purinergic P2X; Synaptic Transmission; Vagus Nerve

ATP plays an important role in signal transduction between neuronal and glial circuits and within glial networks. Here we describe currents activated by ATP in astrocytes acutely isolated from cortical brain slices by non-enzymatic mechanical dissociation. Brain slices were prepared from transgenic mice that express enhanced green fluorescent protein under the control of the human glial fibrillary acidic protein promoter. Astrocytes were studied by whole-cell voltage clamp. Exogenous ATP evoked inward currents in 75 of 81 astrocytes. In the majority ( approximately 65%) of cells, ATP-induced responses comprising a fast and delayed component; in the remaining subpopulation of astrocytes, ATP triggered a smoother response with rapid peak and slowly decaying plateau phase. The fast component of the response was sensitive to low concentrations of ATP (with EC(50) of approximately 40 nm). All ATP-induced currents were blocked by pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS); they were insensitive to ivermectin. Quantitative real-time PCR demonstrated strong expression of P2X(1) and P2X(5) receptor subunits and some expression of P2X(2) subunit mRNAs. The main properties of the ATP-induced response in cortical astrocytes (high sensitivity to ATP, biphasic kinetics, and sensitivity to PPADS) were very similar to those reported for P2X(1/5) heteromeric receptors studied previously in heterologous expression systems.

Topics: Adenosine Triphosphate; Animals; Animals, Newborn; Astrocytes; Calcium; Cerebral Cortex; Dose-Response Relationship, Drug; Dose-Response Relationship, Radiation; Flow Cytometry; Glial Fibrillary Acidic Protein; Green Fluorescent Proteins; Humans; Membrane Potentials; Mice; Mice, Transgenic; Patch-Clamp Techniques; Platelet Aggregation Inhibitors; Pyridoxal Phosphate; Receptors, Purinergic P2; Receptors, Purinergic P2X; Receptors, Purinergic P2X2; Receptors, Purinergic P2X5

The present study was undertaken to determine the role of P2X3 receptor (P2X3R) on heat hyperalgesia in a newly developed rat model of trigeminal neuropathic pain. The unilateral infraorbital nerve (IoN) was partially ligated by 6-0 silk. To assess heat sensitivity, a vibrissal pad (VP) was placed on a hot plate and the latency until the rats withdrew their head was measured. Mechanical sensitivity of VP was also assessed by the use of von Frey filament. Both heat and mechanical hyperalgesia were observed at the VP ipsilateral to the IoN ligation. The latency to heat stimuli was prolonged after subcutaneous administration of pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, P2X1,2,3,5,7,1/5,2/3R antagonist) and 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP, P2X1,3,2/3,1/5R antagonist). The latency was shortened after administration of alpha,beta-methylene ATP (alpha,beta-meATP, P2X1,3,2/3R agonist), although no changes appeared after administration of beta,gamma-methylene-L-ATP (beta,gamma-me-L-ATP, P2X1R agonist). The protein gene product-9.5 and calcitonin gene-related peptide immunoreactive nerve fibers significantly decreased in the VP skin of ipsilateral to the IoN ligation. In the ipsilateral trigeminal ganglion, the number of P2X3-immunoreactive neurons significantly increased in the small cell group. In this study, we developed an experimental model of trigeminal neuropathic pain by partial ligation of IoN, which produced heat and mechanical hyperalgesia in the VP. Pharmacological and immunohistochemical studies revealed that the P2X3R plays an important role in the heat hyperalgesia observed in this model.. The study describes the development of a novel model of trigeminal neuropathic pain. Heat hyperalgesia in this model was inhibited by peripheral injection of P2XR antagonists. The results suggest that P2X3R is a potential target for development of a novel therapy for trigeminal neuropathic pain.

Topics: Adenosine Triphosphate; Animals; Calcitonin Gene-Related Peptide; Disease Models, Animal; Hyperalgesia; Immunohistochemistry; Ligation; Male; Neurons, Afferent; Nociceptors; Pain Measurement; Pain Threshold; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Rats; Rats, Inbred Lew; Reaction Time; Receptors, Purinergic P2; Receptors, Purinergic P2X3; Trigeminal Ganglion; Trigeminal Nerve; Trigeminal Neuralgia; Ubiquitin Thiolesterase

Central sensitization represents a sustained hypersensitive state of dorsal horn nociceptive neurons that can be evoked by peripheral inflammation or injury to nerves and tissues. It reflects neuroplastic changes such as increases in neuronal spontaneous activity, receptive field size, and responses to suprathreshold stimuli and a decrease in activation threshold. We recently demonstrated that purinergic receptor mechanisms in trigeminal subnucleus caudalis (Vc; medullary dorsal horn) are also involved in the initiation and maintenance of central sensitization in brain stem nociceptive neurons of trigeminal subnucleus oralis. The aim of the present study was to investigate whether endogenous ATP is involved in the development of central sensitization in Vc itself. The experiments were carried out on urethan/alpha-chloralose anesthetized and immobilized rats. Single neurons were recorded and identified as nociceptive-specific (NS) in the deep laminae of Vc. During continuous saline superfusion (0.6 ml/h it) over the caudal medulla, Vc neuronal central sensitization was readily induced by mustard oil application to the tooth pulp. However, this mustard-oil-induced central sensitization could be completely blocked by continuous intrathecal superfusion of the wide-spectrum P2X receptor antagonist pyridoxal-phosphate-6-azophenyl-2, 4-disulphonic acid tetra-sodium (33-100 microM) and by apyrase (an ectonucleotidase enzyme, 30 units/ml). Superfusion of the selective P2X1, P2X3 and P2X(2/3) receptor antagonist 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (6-638 microM) partially blocked the Vc central sensitization. The two P2X receptor antagonists did not significantly affect the baseline nociceptive properties of the Vc neurons. These findings implicate endogenous ATP as an important mediator contributing to the development of central sensitization in nociceptive neurons of the deep laminae of the dorsal horn.

Topics: Action Potentials; Adenosine Triphosphate; Analysis of Variance; Animals; Apyrase; Brain Mapping; Drug Interactions; Male; Mustard Plant; Neurons; Nociceptors; Physical Stimulation; Plant Extracts; Plant Oils; Platelet Aggregation Inhibitors; Pyridoxal Phosphate; Rats; Rats, Sprague-Dawley; Sensory Thresholds; Stimulation, Chemical; Trigeminal Caudal Nucleus

The pathophysiological mechanisms of orofacial deep-tissue pain is still unclear. Previously, P2X receptors (P2XR) in sensory neurons have been shown to play a role in the signal transduction of cutaneous pain. We investigated the functional significance of P2X3R in relation to orofacial deep-tissue pain caused by monoarthritis of the temporomandibular joint (TMJ). Monoarthritis was induced by the injection of complete Freund's adjuvant (CFA) into the unilateral TMJ of the rat. The pain associated with monoarthritis was assessed by the pressure pain threshold (PPT), which was defined as the amount of pressure required to induce vocalization. Fifteen days after CFA-treatment, changes in PPT were examined after injection of P2XR agonists or antagonists into the TMJ. The number of cells expressing P2X3R in trigeminal ganglia (TG) was investigated by immunohistochemistry. Inflamed TMJ showed a continuous decline in PPT during the experimental period (P<0.001). Injection of alpha,beta-meATP, an agonist of P2X1,3,2/3R, dramatically reduced the bilateral PPTs of both inflamed and non-inflamed TMJs (P<0.01) although beta,gamma-me-l-ATP, a selective agonist of P2X1R, did not. The decreased PPTs of inflamed TMJ were reversed either by PPADS, an antagonist of P2X1,2,3,5,1/5,4/5R, or by TNP-ATP, an antagonist of P2X1,3,2/3,1/5R. Immunohistochemically, the number of P2X3R-positive cells increased in the small cell group in TG (P<0.01), whereas there was no change in medium or large cell groups after the CFA-injection. Retrograde tracing confirmed that TMJ neurons in the TG exhibited P2X3R immunoreactivity. Our results suggested that P2X3R plays an important role in orofacial pressure pain caused by monoarthritis of TMJ.

Topics: Adenosine Triphosphate; Analysis of Variance; Animals; Arthritis; Cell Count; Drug Interactions; Facial Pain; Freund's Adjuvant; Functional Laterality; Immunohistochemistry; Male; Neurons; Pain Threshold; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Rats; Rats, Inbred Lew; Receptors, Purinergic P2; Receptors, Purinergic P2X3; Stilbamidines; Temporomandibular Joint Disorders; Time Factors; Trigeminal Ganglion

ATP is released in a vesicular manner from nerve terminals mainly at higher stimulation frequencies. There is a robust expression of ATP (P2) receptors in the brain, but their role is primarily unknown. We report that ATP analogs biphasically modulate the evoked release of glutamate from purified nerve terminals of the rat hippocampus, the facilitation being mediated by P2X1, P2X2/3, and P2X3 [antagonized by 8-(benzamido)naphthalene-1,3,5-trisulfonate and 2',3'-O-(2,4,6-trinitrophenyl)-ATP] and the inhibition by P2Y1, P2Y2, and/or P2Y4 [antagonized by reactive blue 2 and 2'deoxy-N6-methyladenosine-3',5'-bisphosphate and mimicked by P1-(urinine 5'-),P4-(inosine 5'-) tetraphosphate and 2-methylthio-ADP] receptors. The combination of single-cell PCR analysis of rat hippocampal pyramidal neurons, Western blot analysis of purified presynaptic active zone fraction, and immunocytochemical analysis of hippocampal glutamatergic terminals revealed that the P2 receptors expressed in glutamatergic neurons, located in the active zone and in glutamatergic terminals, were precisely P2X1, P2X2, and P2X3 subunits and P2Y1, P2Y2, and P2Y4 receptors. This provides coincident functional and molecular evidence that P2 receptors are present and act presynaptically as a modulatory system controlling hippocampal glutamate release.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Adenylyl Imidodiphosphate; Animals; Astrocytoma; Calcium; Cell Line; Cell Line, Tumor; Glutamic Acid; Hippocampus; Kidney; Male; Potassium; Pyramidal Cells; Pyridoxal Phosphate; Rats; Rats, Wistar; Receptors, Presynaptic; Receptors, Purinergic P2; Receptors, Purinergic P2X; Receptors, Purinergic P2X2; Receptors, Purinergic P2X3; Receptors, Purinergic P2Y1; Receptors, Purinergic P2Y2; Recombinant Fusion Proteins; RNA, Messenger; Subcellular Fractions; Suramin; Synaptosomes; Transfection; Triazines; Triazoles; Xanthines

The P2X7 receptor is an ATP-sensitive ligand-gated cation channel, expressed predominantly in cells with immune origin. Recent studies have demonstrated that P2X7 may play an important role in pain signaling. In the present study, the expression of P2X7 receptors in non-neuronal cells and neurons isolated from dorsal root ganglia was characterized using patch clamp, pharmacological and confocal microscopy approaches. In small diameter DRG neurons, 100 microM 2', 3'-O-(4-benzoylbenzoyl)-ATP (BzATP) evoked an inward current, which was inhibited completely by 1 microM A-317491, a potent and selective P2X3 receptor antagonist. In contrast, BzATP evoked concentration-dependent increases in inward currents in non-neuronal DRG cells with an EC50 value of 26 +/- 0.14 microM, which were resistant to the blockade by A-317491. The activity to evoke cationic currents by P2X receptor agonists in non-neuronal cells showed a rank order of BzATP > ATP > alpha,beta-meATP. Pyridoxal-phosphate-6-azophenyl-,2',4'-disulphonic acid (PPADS) and Mg2+ produced concentration-dependent inhibition of BzATP-evoked currents in non-neuronal cells. Confocal microscopy revealed positive immunoreactivity of anti-P2X7 receptor antibodies on non-neuronal cells. No anti-P2X7 immunoreactivity was observed on DRG neurons. Further electrophysiological studies showed that prolonged agonist activation of P2X7 receptors in non-neuronal cells did not lead to cytolytic pore formation. Taken together, the present study demonstrated functional expression of P2X7 receptors in non-neuronal but not in small diameter neurons from rat DRG. Modulation of P2X7 receptors in non-neuronal cells might have impact on peripheral sensory transduction under normal and pathological states.

Topics: Adenosine Triphosphate; Animals; Cells, Cultured; Dose-Response Relationship, Drug; Drug Interactions; Electric Stimulation; Ganglia, Spinal; Immunohistochemistry; Membrane Potentials; Microscopy, Confocal; Neuroglia; Patch-Clamp Techniques; Phenols; Phosphopyruvate Hydratase; Polycyclic Compounds; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Rats; Receptors, Purinergic P2; Receptors, Purinergic P2X7; Sodium

The mechanism of mechanical hyperalgesia in inflammation might involve a 'mechanochemical' process whereby stretch evokes the release of adenosine 5'-triphosphate (ATP) from the damaged tissue that then excites nearby primary sensory nerve terminals. In the present study, phosphorylated extracellular signal-regulated protein kinase (pERK) immunoreactivity was used as a marker indicating functional activation of primary afferent neurons to examine the P2X receptor-mediated noxious response in DRG neurons in a rat model of peripheral inflammation. We found that very few pERK-labeled DRG neurons were detected in normal rats after alpha, beta methylene-ATP (alphabetame-ATP) intraplantar injection. However, a number of DRG neurons were labeled for pERK after alphabetame-ATP injection to the complete Freund's adjuvant (CFA) induced inflamed paw. Seventy-three percent of pERK-labeled DRG neurons co-expressed the P2X3 receptor. After mechanical noxious stimulation to the hind paw of CFA-inflamed rats, we found many more pERK-labeled neurons compared to those in the normal rats. Administration of the P2X3 receptor antagonists, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid or 2'- (or 3')-O-(trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP), significantly decreased the mechanical stimulation-evoked pERK labeling in CFA-inflamed rats, but not in normal rats. We also found the recruitment of neurons with myelinated A fibers labeled for pERK in CFA-inflamed rats, which was reversed by P2X3 receptor antagonists. Moreover, TNP-ATP dose dependently reduced the mechanical hypersensitivity of CFA rats. These data suggest that the P2X receptors in primary afferent neurons increase their activity with enhanced sensitivity of the intracellular ERK signaling pathway during inflammation and then contribute to the hypersensitivity to mechanical noxious stimulation in the inflammatory state.

Topics: Adenosine Triphosphate; Animals; Cell Count; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Freund's Adjuvant; Functional Laterality; Ganglia, Spinal; Immunohistochemistry; Inflammation; Male; Mitogen-Activated Protein Kinases; Neurofilament Proteins; Neurons; Phosphorylation; Physical Stimulation; Platelet Aggregation Inhibitors; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2X

P2X receptors have been suggested to be expressed on the central terminals of A delta-afferent fibers innervating dorsal horn lamina V and play a role in modulating sensory synaptic transmission. These P2X receptors have been widely thought to be P2X2+3 receptors. However, we have recently found that P2X receptor-mediated modulation of sensory transmission in lamina V is not inhibited by trinitrophenyl-adenosine triphosphate (TNP-ATP), a potent antagonist of P2X1, P2X3 homomers, and P2X2+3 heteromers. To provide direct evidence for the presence of TNP-ATP-resistant P2X receptors on primary afferent fibers, we examined alpha,beta-methylene-ATP (alpha beta meATP)-evoked currents and their sensitivity to TNP-ATP in rat dorsal root ganglion (DRG) neurons. alpha beta meATP evoked fast currents, slow currents, and mixed currents that contained both fast and slow current-components. Fast currents and fast current components in the mixed currents were both completely inhibited by 0.1 microM TNP-ATP (n = 14). Both slow currents and slow-current components in the mixed currents showed broad spectrum of sensitivity to 1 microM TNP-ATP, ranging from complete block (TNP-ATP-sensitive) to little block (TNP-ATP-resistant). TNP-ATP-resistant currents evoked by 10 microM alpha beta meATP could be largely inhibited by 10 microM iso-pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid. Cells with P2X currents that were highly resistant to TNP-ATP were found to be insensitive to capsaicin. These results suggest that TNP-ATP-resistant P2X receptor subtypes are expressed on capsaicin-insensitive A delta-afferent fibers and play a role in modulating sensory transmission to lamina V neurons.

Topics: Adenosine Triphosphate; Afferent Pathways; Animals; Capsaicin; Ganglia, Spinal; Nerve Fibers; Neurons; Patch-Clamp Techniques; Pyridoxal Phosphate; Rats; Rats, Sprague-Dawley; Receptors, Purinergic P2; Receptors, Purinergic P2X; Spinal Cord; Synaptic Transmission

Pain after nerve damage is an expression of pathological operation of the nervous system, one hallmark of which is tactile allodynia-pain hypersensitivity evoked by innocuous stimuli. Effective therapy for this pain is lacking, and the underlying mechanisms are poorly understood. Here we report that pharmacological blockade of spinal P2X4 receptors (P2X4Rs), a subtype of ionotropic ATP receptor, reversed tactile allodynia caused by peripheral nerve injury without affecting acute pain behaviours in naive animals. After nerve injury, P2X4R expression increased strikingly in the ipsilateral spinal cord, and P2X4Rs were induced in hyperactive microglia but not in neurons or astrocytes. Intraspinal administration of P2X4R antisense oligodeoxynucleotide decreased the induction of P2X4Rs and suppressed tactile allodynia after nerve injury. Conversely, intraspinal administration of microglia in which P2X4Rs had been induced and stimulated, produced tactile allodynia in naive rats. Taken together, our results demonstrate that activation of P2X4Rs in hyperactive microglia is necessary for tactile allodynia after nerve injury and is sufficient to produce tactile allodynia in normal animals. Thus, blocking P2X4Rs in microglia might be a new therapeutic strategy for pain induced by nerve injury.

Topics: Adenosine Triphosphate; Animals; Astrocytes; Cells, Cultured; Disease Models, Animal; Male; Microglia; Neurons; Pain; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Rats; Rats, Wistar; Receptors, Purinergic P2; Receptors, Purinergic P2X4; Spinal Cord; Spinal Nerves; Touch

The effects of ATP receptor agonists/antagonists on skin barrier recovery rate were evaluated in hairless mice. Topical application of ATP and alpha,beta-methylene ATP (agonist of P2X receptor) delayed barrier recovery. Topical application of suramin (nonspecific ATP receptor antagonist), pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (P2X receptor antagonist), and 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) (P2X1, P2X3, P2X2/3 antagonist) after barrier disruption accelerated the barrier repair. The P2Y type receptor antagonist Reactive Blue 2 did not affect the barrier repair process. Moreover, topical application of TNP-ATP prevented epidermal hyperplasia induced by barrier insult under low environmental humidity. ATP was secreted immediately after tape stripping on skin in organ culture. alpha,beta-Methylene ATP increased intercellular calcium in cultured keratinocytes and the increase was blocked by TNP-ATP. Both reverse transcription polymerase chain reaction assay and immunohistochemical study showed the existence of protein that had a structure similar to P2X3 on hairless mouse epidermis. These results suggest that cutaneous barrier homeostasis can be regulated by cation flux through a P2X3-like ATP receptor.

Topics: Adenosine Triphosphate; Animals; Antineoplastic Agents; Calcium; Cells, Cultured; Epidermis; Fluorescent Dyes; Hyperplasia; Immunohistochemistry; Keratinocytes; Male; Mice; Mice, Hairless; Platelet Aggregation Inhibitors; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Reverse Transcriptase Polymerase Chain Reaction; Suramin; Water; Wound Healing

1. Currents through heteromeric P2X(2/3) receptors were evoked by applying alpha,beta-methylene-ATP to human embryonic kidney cells transfected with cDNAs encoding the P2X(2) and P2X(3) subunits. The concentration of alpha,beta-methylene-ATP were < or =30 microM because higher concentrations can activate homomeric P2X(2) receptors. The kinetics of action of three structurally unrelated antagonists were studied; these were 2', 3'-O-(2,4,6,trinitrophenyl)-ATP (TNP-ATP), pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonate (PPADS) and suramin. The association and dissociation rate constants were determined by pre-applying the antagonist for various periods prior to the co-application of agonist and antagonist, or by changing the solution from one containing only the agonist to one containing both agonist and antagonist. The high affinity of TNP-ATP for the P2X(2/3) receptor (K(D) approximately 2 nM) results from fast binding (k(+1) approximately 100 microM(-1) s(-1)) rather than slow unbinding (k(-1) approximately 0.3 s(-1)). For suramin (K(D) approximately 1 microM) the association rate constant ( approximately 1 microM(-1) s(-1)) was 100 times slower than that of TNP-ATP but the dissociation rate constant was similar (k(-1) approximately 1 s(-1)). PPADS (K(D) approximately 0.1 microM) associated and dissociated some 100 - 10,000 times more slowly than the other antagonists.

Topics: Adenosine Triphosphate; Animals; Antineoplastic Agents; Cell Line; Dose-Response Relationship, Drug; Fluorescent Dyes; Humans; Platelet Aggregation Inhibitors; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Rats; Receptors, Purinergic P2; Receptors, Purinergic P2X2; Receptors, Purinergic P2X3; Suramin; Transfection

Exogenous ATP has been shown to be algogenic in both animal and humans. Research has focused on the P2X3 ligand-gated ion channel, as it is preferentially expressed on nociceptive C-fibers. In addition, P2X3 receptor gene disrupted mice show decreased responses to somatic painful stimuli. However, the potential role of P2X receptor activation in visceral pain has not yet been evaluated. In the present study, the systemic administration of suramin, and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid, PPADS, both non-selective P2X receptor antagonists, dose-dependently reduced acetic acid-induced abdominal constrictions in mice (ED(50)=34.5 micromol/kg and ED50=70 micromol/kg, respectively). Furthermore, 2'-(or-3')-O-(trinitrophenyl)adenosine 5'- tri-phosphate (TNP-ATP) potently (IC50=10 nM) blocked the functional activation of P2X3 receptors in vitro and attenuated acetic acid-induced visceral pain. In the abdominal constriction assay, TNP-ATP (ED(50)=6.35 micromol/kg, i.p.) was 6-10 fold more potent than suramin and PPADS to reduce nociceptive behavior. In addition, TNP-ATP was 10 fold more potent than TNP-AMP (2'-(or-3')-O-(trinitrophenyl)adenosine 5'-mono-phosphate) (ED50=63.5 micromol/kg, i.p.) at reducing acetic acid-induced nociception. At the highest dose, TNP-ATP completely abolished nociceptive behavior, as did morphine (ED50=3 micromol/kg, i.p.). While TNP-ATP is also a potent antagonist of P2X1 receptors, P2X1 receptor mediated responses have not been shown in dorsal root ganglia and diinosine pentaphosphate, IP5I, a potent and selective P2X1 receptor antagonist, was ineffective at reducing abdominal constrictions. Thus, the antinociceptive effects of TNP-ATP appear to be mediated through activation of homomeric P2X3and/or heteromeric P2X2/3 receptors. Together, these results show that activation of P2X3 containing receptors plays a role in the transmission of inflammatory visceral pain.

Topics: Abdomen; Acetic Acid; Adenosine Triphosphate; Analgesics; Animals; Antineoplastic Agents; Behavior, Animal; Calcium; Dose-Response Relationship, Drug; Male; Mice; Mice, Inbred Strains; Nociceptors; Pain; Platelet Aggregation Inhibitors; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Receptors, Purinergic P2X3; Suramin

The present study explores the possible involvement of a purinergic mechanism in mechanosensory transduction in the bladder using P2X(3) receptor knock-out (P2X(3)-/-) and wild-type control (P2X(3)+/+) mice. Immunohistochemistry revealed abundant nerve fibers in a suburothelial plexus in the mouse bladder that are immunoreactive to anti-P2X(3). P2X(3)-positive staining was completely absent in the subepithelial plexus of the P2X(3)-/- mice, whereas staining for calcitonin gene-related peptide and vanilloid receptor 1 receptors remained. Using a novel superfused mouse bladder-pelvic nerve preparation, we detected a release of ATP proportional to the extent of bladder distension in both P2X(3)+/+ and P2X(3)-/- mice, although P2X(3)-/- bladder had an increased capacity compared with that of the P2X(3)+/+ bladder. The activity of multifiber pelvic nerve afferents increased progressively during gradual bladder distension (at a rate of 0.1 ml/min). However, the bladder afferents from P2X(3)-/- mice showed an attenuated response to bladder distension. Mouse bladder afferents of P2X(3)+/+, but not P2X(3)-/-, were rapidly activated by intravesical injections of P2X agonists (ATP or alpha,beta-methylene ATP) and subsequently showed an augmented response to bladder distension. By contrast, P2X antagonists [2',3'-O-(2,4,6-trinitrophenyl)-ATP and pyridoxal 5-phosphate 6-azophenyl-2',4'-disulfonic acid] and capsaicin attenuated distension-induced discharges in bladder afferents. These data strongly suggest a major sensory role for urothelially released ATP acting via P2X(3) receptors on a subpopulation of pelvic afferent fibers.

Topics: Adenosine Triphosphate; Animals; Capsaicin; Dilatation; Electrophysiology; Immunohistochemistry; In Vitro Techniques; Male; Mechanoreceptors; Mice; Mice, Knockout; Neurons, Afferent; Pelvis; Peripheral Nerves; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Receptors, Purinergic P2; Receptors, Purinergic P2X3; Urinary Bladder; Urothelium

1. In mechanically dissociated rat spinal cord substantia gelatinosa (SG) neurones attached with native presynaptic nerve endings, glycinergic miniature inhibitory postsynaptic currents (mIPSCs) were recorded using nystatin perforated patch recording mode under voltage-clamp conditions. Under these conditions, it was tested whether the changes in P2X receptor subtype on the glycinergic presynaptic nerve terminals occur during postnatal development. 2. ATP facilitated glycinergic mIPSC frequency in a concentration-dependent manner through all developmental stages tested, whereas alphabeta-methylene-ATP (alphabeta-me-ATP) was only effective at later developmental stages. 3. alphabeta-me-ATP-elicited mIPSC frequency facilitation was completely occluded in the Ca2+-free external solution, but it was not affected by adding 10(-4) M Cd2+. 4. alphabeta-me-ATP still facilitated mIPSC frequency even in the presence of 10(-6) M thapsigargin, a Ca2+ pump blocker. 5. In later developmental stages, ATP-elicited presynaptic or postsynaptic responses were reversibly blocked by 10(-5) M pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), but only partially blocked by 10(-7) M 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP). However, alphabeta-me-ATP-elicited presynaptic or postsynaptic responses were completely and reversibly blocked by either 10(-5) M PPADS or 10(-7) M TNP-ATP. 6. alphabeta-me-ATP significantly reduced the evoked glycinergic IPSC amplitude in postnatal 28-30 day neurones, whereas it had no effect in 10-12 day neurones. 7. It was concluded that alphabeta-me-ATP-sensitive P2X receptors were functionally expressed on the glycinergic presynaptic nerve terminals projecting to SG neurones in later developmental stages. Such developmental changes of presynaptic P2X receptor subtypes might contribute to synaptic plasticity such as the regulation of neuronal excitability and the fine controlling of the pain signal in spinal dorsal horn neurones.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Adenosine Triphosphate; Animals; Bicuculline; Excitatory Amino Acid Antagonists; Fluorescent Dyes; GABA Antagonists; Glycine; Membrane Potentials; Neural Inhibition; Neural Pathways; Neurons; Organ Culture Techniques; Patch-Clamp Techniques; Platelet Aggregation Inhibitors; Presynaptic Terminals; Pyridoxal Phosphate; Rats; Rats, Wistar; Receptors, Purinergic P2; Receptors, Purinergic P2X2; Receptors, Purinergic P2X4; Substantia Gelatinosa

P2X receptors for adenosine 5'-triphosphate (ATP) comprise a family of ligand-gated cation channels with distinct characteristics which are dependent on the receptor subunits (P2X1-7) expressed, and the homomeric or heteromeric assembly of protein subunits in individual cells. We describe the properties of P2X receptors expressed by cultured adult rat dorsal root ganglion cells on the basis of the time course of responses to ATP, alpha, beta-methylene adenosine 5'-triphosphate (alpha, beta-meATP) and 2-methyl-thioadenosine 5'-triphosphate (2-meSATP), and using the antagonists 2',3'-O-(2,4,6-trinitrophenyl) ATP (TNP-ATP), a novel and highly selective purinoceptor antagonist, suramin and iso-pyridocalphosphate-6-azophenyl-2',5' disulphonic acid (PPADS). ATP (10 microM) evoked inward currents in approximately 95% of neurons tested and > 80% responded with a fast transient inward current that rapidly inactivated during the continued presence of ATP. Of the remaining neurons, approximately 4% showed a sustained response and approximately 10% showed a combination of transient and sustained components. Rapid application of ATP, alpha,beta-meATP and 2meSATP demonstrated these to be full agonists of the rapidly inactivating P2X receptor (pA50 values = 5.83, 5.86 and 5.55, respectively), whilst uridine triphosphate (UTP) and 1-beta,gamma-methyleneadenosine 5'-triphosphate (1-beta,gamma-meATP) were ineffective as agonists. These rapidly inactivating responses could be inhibited by TNP-ATP, suramin and PPADS (pIC50 = 9.5, 6.5, 6.4, respectively). Using inactivation protocols, we demonstrate the presence of homomeric P2X3-like receptors and non-inactivating P2X receptors, which indicates that individual subsets of adult dorsal root ganglion neurons have distinct P2X receptor phenotypes, and that individual DRG neurons may express multiple P2X receptor subtypes.

Topics: Adenosine Triphosphate; Animals; Antineoplastic Agents; Fluorescent Dyes; Ganglia, Spinal; Gene Expression; Ion Channel Gating; Male; Membrane Potentials; Neurons; Patch-Clamp Techniques; Phenotype; Platelet Aggregation Inhibitors; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Rats; Rats, Wistar; Receptors, Purinergic P2; Receptors, Purinergic P2X; Receptors, Purinergic P2X2; Receptors, Purinergic P2X3; Receptors, Purinergic P2X4; Receptors, Purinergic P2X5; Receptors, Purinergic P2X7; Suramin; Thionucleotides; Uridine Triphosphate

1. 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. 2. 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. 3. 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. 4. Nociceptive behaviour in the first phase of the formalin test and in the capsaicin test were significantly inhibited by intrathecal pretreatment with alpha, beta-methylene ATP (alpha,betameATP: 5 microg 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. 5. 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 alpha,betameATP (perhaps the P2X3 receptor subtype) and (2) formalin-induced inflammatory pain via PPADS-sensitive, TNP-ATP- and alpha,betameATP-insensitive P2X (and/or P2Y) receptors.

Topics: Adenosine Triphosphate; Animals; Behavior, Animal; Capsaicin; Formaldehyde; Male; Mice; Motor Activity; Nociceptors; Pain; Pain Measurement; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Receptors, Purinergic P2; Spinal Cord