Mathematical modelling of human P2X-mediated plasma membrane electrophysiology and calcium dynamics in microglia.

Regulation of cytosolic calcium (Ca2+) dynamics is fundamental to microglial function. Temporal and spatial Ca2+ fluxes are induced from a complicated signal transduction pathway linked to brain ionic homeostasis. In this paper, we develop a novel biophysical model of Ca2+ and sodium (Na+) dynamics in human microglia and evaluate the contribution of purinergic receptors (P2XRs) to both intracellular Ca2+ and Na+ levels in response to agonist/ATP binding. This is the first comprehensive model that integrates P2XRs to predict intricate Ca2+ and Na+ transient responses in microglia. Specifically, a novel compact biophysical model is proposed for the capture of whole-cell patch-clamp currents associated with P2X4 and P2X7 receptors, which is composed of only four state variables. The entire model shows that intricate intracellular ion dynamics arise from the coupled interaction between P2X4 and P2X7 receptors, the Na+/Ca2+ exchanger (NCX), Ca2+ extrusion by the plasma membrane Ca2+ ATPase (PMCA), and Ca2+ and Na+ leak channels. Both P2XRs are modelled as two separate adenosine triphosphate (ATP) gated Ca2+ and Na+ conductance channels, where the stoichiometry is the removal of one Ca2+ for the hydrolysis of one ATP molecule. Two unique sets of model parameters were determined using an evolutionary algorithm to optimise fitting to experimental data for each of the receptors. This allows the proposed model to capture both human P2X7 and P2X4 data (hP2X7 and hP2X4). The model architecture enables a high degree of simplicity, accuracy and predictability of Ca2+ and Na+ dynamics thus providing quantitative insights into different behaviours of intracellular Na+ and Ca2+ which will guide future experimental research. Understanding the interactions between these receptors and other membrane-bound transporters provides a step forward in resolving the qualitative link between purinergic receptors and microglial physiology and their contribution to brain pathology.

NMDAR-dependent long-term depression is associated with increased short term plasticity through autophagy mediated loss of PSD-95.

Long-term depression (LTD) of synaptic strength can take multiple forms and contribute to circuit remodeling, memory encoding or erasure. The generic term LTD encompasses various induction pathways, including activation of NMDA, mGlu or P2X receptors. However, the associated specific molecular mechanisms and effects on synaptic physiology are still unclear. We here compare how NMDAR- or P2XR-dependent LTD affect synaptic nanoscale organization and function in rodents. While both LTDs are associated with a loss and reorganization of synaptic AMPARs, only NMDAR-dependent LTD induction triggers a profound reorganization of PSD-95. This modification, which requires the autophagy machinery to remove the T19-phosphorylated form of PSD-95 from synapses, leads to an increase in AMPAR surface mobility. We demonstrate that these post-synaptic changes that occur specifically during NMDAR-dependent LTD result in an increased short-term plasticity improving neuronal responsiveness of depressed synapses. Our results establish that P2XR- and NMDAR-mediated LTD are associated to functionally distinct forms of LTD.

Purinergic Modulation of Activity in the Developing Auditory Pathway.

Purinergic P2 receptors, activated by endogenous ATP, are prominently expressed on neuronal and non-neuronal cells during development of the auditory periphery and central auditory neurons. In the mature cochlea, extracellular ATP contributes to ion homeostasis, and has a protective function against noise exposure. Here, we focus on the modulation of activity by extracellular ATP during early postnatal development of the lower auditory pathway. In mammals, spontaneous patterned activity is conveyed along afferent auditory pathways before the onset of acoustically evoked signal processing. During this critical developmental period, inner hair cells fire bursts of action potentials that are believed to provide a developmental code for synaptic maturation and refinement of auditory circuits, thereby establishing a precise tonotopic organization. Endogenous ATP-release triggers such patterned activity by raising the extracellular K+ concentration and contributes to firing by increasing the excitability of auditory nerve fibers, spiral ganglion neurons, and specific neuron types within the auditory brainstem, through the activation of diverse P2 receptors. We review recent studies that provide new models on the contribution of purinergic signaling to early development of the afferent auditory pathway. Further, we discuss potential future directions of purinergic research in the auditory system.

P2X Electrophysiology and Surface Trafficking in Xenopus Oocytes.

Xenopus oocytes serve as a standard heterologous expression system for the study of various ligand-gated ion channels including ATP P2X receptors. Here we describe the whole-cell two-electrode voltage clamp and biotinylation/Western blotting techniques to investigate the functional properties and surface trafficking from P2X-expressing oocytes.

P2X receptors and acupuncture analgesia.

Purinergic signaling has recently been suggested to constitute the cellular mechanism underlying acupuncture-induced analgesia (AA). By extending the original hypothesis on endogenous opioids being released during AA, Geoffrey Burnstock and Maiken Nedergaard supplied evidence for the involvement of purinoceptors (P2 and P1/A1 receptors) in the beneficial effects of AA. In view of certain pain states (e.g. neuropathic pain) which respond only poorly to therapy with standard analgesics, as well as with respect to the numerous unwanted effects of opioids and non-steroidal anti-inflammatory drugs, it is of great significance to search for alternative therapeutic options. Because clinical studies on AA yielded sometimes heterogeneous results, it is of eminent importance to relay on experiments carried out on laboratory animals, by evaluating the data with stringent statistical methods including comparison with a sufficient number of control groups. In this review, we summarize the state of the art situation with respect to the participation of P2 receptors in AA and try to forecast how the field is likely to move forward in the future.

P2 receptor interaction and signalling cascades in neuroprotection.

Nucleotides can contribute to the survival of different glial and neuronal models at the nervous system via activation of purinergic P2X and P2Y receptors. Their activation counteracts different proapoptotic events, such as excitotoxicity, mitochondrial impairment, oxidative stress and DNA damage, which concur to elicit cell loss in different processes of neurodegeneration and brain injury. Thus, it is frequent to find that different neuroprotective mediators converge in the activation of the same intracellular survival pathways to protect cells from death. The present review focuses on the role of P2Y1 and P2Y13 metabotropic receptors, and P2X7 ionotropic receptors to regulate the balance between survival and apoptosis. In particular, we analyze the intracellular pathways involved in the signaling of these nucleotide receptors to elicit survival, including calcium/PLC, PI3K/Akt/GSK3, MAPK cascades, and the expression of antioxidant and antiapoptotic genes. This review emphasizes the novel contribution of nucleotide receptors to maintain cell homeostasis through the regulation of MAP kinases and phosphatases. Unraveling the different roles found for nucleotide receptors in different models and cellular contexts may be crucial to delineate future therapeutic applications based on targeting nucleotide receptors for neuroprotection.

Characterization of the antagonist actions of 5-BDBD at the rat P2X4 receptor.

P2X receptors (P2XRs) are a family of ATP-gated ionic channels that are expressed in numerous excitable and non-excitable cells. Despite the great advance on the structure and function of these receptors in the last decades, there is still lack of specific and potent antagonists for P2XRs subtypes, especially for the P2X4R. Here, we studied in detail the effect of the P2X4R antagonist 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) on ATP-induced currents mediated by the rat P2X4R and compared its specificity among another rat P2XRs. We found that 5-BDBD is a potent P2X4R antagonist, with an IC50 of 0.75 µM when applied for 2 min prior and during ATP stimulation. Moreover, at 10 µM concentration, 5-BDBD did not affect the ATP-induced P2X2aR, P2X2bR, and P2X7R current amplitude or the pattern of receptor desensitization. However, at 10 µM concentration but not 0.75 µM 5-BDBD inhibited the P2X1R and P2X3R-gated currents by 13 and 35% respectively. Moreover, we studied the effects of 5-BDBD in long-term potentiation experiments performed in rat hippocampal slices, finding this antagonist can partially decrease LTP, a response that is believed to be mediated in part by endogenous P2X4Rs. These results indicate that 5-BDBD could be used to study the endogenous effects of the P2X4R in the central nervous system and this antagonist can discriminate between P2X4R and other P2XRs, when they are co-expressed in the same tissue.

Cholecystokinin Switches the Plasticity of GABA Synapses in the Dorsomedial Hypothalamus via Astrocytic ATP Release.

Whether synapses in appetite-regulatory brain regions undergo long-term changes in strength in response to satiety peptides is poorly understood. Here we show that following bursts of afferent activity, the neuromodulator and satiety peptide cholecystokinin (CCK) shifts the plasticity of GABA synapses in the dorsomedial nucleus of the hypothalamus of male Sprague Dawley rats from long-term depression to long-term potentiation (LTP). This LTP requires the activation of both type 2 CCK receptors and group 5 metabotropic glutamate receptors, resulting in a rise in astrocytic intracellular calcium and subsequent ATP release. ATP then acts on presynaptic P2X receptors to trigger a prolonged increase in GABA release. Our observations demonstrate a novel form of CCK-mediated plasticity that requires astrocytic ATP release, and could serve as a mechanism for appetite regulation.SIGNIFICANCE STATEMENT Satiety peptides, like cholecystokinin, play an important role in the central regulation of appetite, but their effect on synaptic plasticity is not well understood. The current data provide novel evidence that cholecystokinin shifts the plasticity from long-term depression to long-term potentiation at GABA synapses in the rat dorsomedial nucleus of the hypothalamus. We also demonstrate that this plasticity requires the concerted action of cholecystokinin and glutamate on astrocytes, triggering the release of the gliotransmitter ATP, which subsequently increases GABA release from neighboring inhibitory terminals. This research reveals a novel neuropeptide-induced switch in the direction of synaptic plasticity that requires astrocytes, and could represent a new mechanism by which cholecystokinin regulates appetite.

Genistein Suppressing the ROS-Induced Hypersensitivity of Rat Vagal Lung C-Fiber Afferents through an ERα-Mediated Mechanism.

Reactive oxygen species (ROS) may induce hypersensitivity of vagal lung C-fibers (VLCFs) through the interaction of transient receptor potential ankyirn 1 (TRPA1) and P2X receptors. Genistein is a soy-derived isoflavone that exerts antioxidant effects by binding to estrogen receptors (ERs), ERα and ERß. We investigated whether ER activation by genistein can suppress H2O2-mediated VLCF hypersensitivity and identified the types of ERs involved. Results revealed that subcutaneous injection of genistein or 4,4',4"-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT, a selective ERα agonist) can attenuate H2O2-induced VLCF hypersensitivity. The suppressive effects of genistein and PPT were inhibited by an additional treatment with ICI182780 (a nonselective ER antagonist) or 1,3-bis(4- hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP, a selective ERα antagonist). Treatment with a combination of PPT, HC030031 (a TRPA1 receptor antagonist), and iso-pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (iso-PPADS, a P2X receptor antagonist) did not further inhibit H2O2-induced VLCF hypersensitivity as compared with combined HC030031 and iso-PPADS treatment. In conclusion, ERα activation by genistein can suppress H2O2- induced VLCF hypersensitivity through its functional interaction with TRPA1 and P2X receptors.

P2X-Receptor Antagonists Inhibit the Interaction of S. aureus Hemolysin A with Membranes.

The pore forming hemolysin A, Hla, is a major virulence factor of Staphylococcus aureus. Apparently, 1-2 pore(s) per cell suffice(s) to cause cell death. Accumulated experimental evidence points towards a major role of ATP-gated purinergic receptors (P2XR) for hemolysis caused by Hla, complement and other pore forming proteins, presumably by increasing membrane permeability. Indeed, in experiments employing rabbit erythrocytes, inhibitory concentrations of frequently employed P2XR-antagonists were in a similar range as previously reported for erythrocytes of other species and other toxins. However, Hla-dependent hemolysis was not enhanced by extracellular ATP, and oxidized adenosinetriphosphate (oxATP) had only a minor inhibitory effect. Unexpectedly, P2XR-inhibitors also prevented Hla-induced lysis of pure lipid membranes, demonstrating that the inhibition did not even depend on the presence of P2XR. Fluorescence microscopy and gel-electrophoresis clearly revealed that P2XR-inhibitors interfere with binding and subsequent oligomerisation of Hla with membranes. Similar results were obtained employing HaCaT-cells. Furthermore, calorimetric data and hemolysis experiments with Hla pre-treated with pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) showed that this compound directly binds to Hla. Our results call for a critical re-assessment of the appealing concept, which suggests that P2XR are general amplifiers of damage by pore-forming proteins.

Participation of Purinergic P2X Receptors in the Thermoregulatory Response to Cooling.

We studied the role of purinergic P2X receptors in the body response to cooling. In experiments on rats, P2X receptor antagonist PPADS was administered in different modes, which resulted in changes of different characteristics of the thermoregulatory response to cold. Iontophoresis of P2X antagonist into the skin decreased the thermal thresholds of all thermoregulatory responses to cooling, which can attest to a modulating effect of P2X receptors on peripheral thermosensitive afferents. Intraperitoneal administration of P2X antagonist suppressed thermoregulatory activity of skeletal muscles (shivering) developing during cooling without changing the thresholds of thermoregulatory responses. The findings suggest that ATP and P2X receptors play an important role in the formation of the response to cooling.

Interaction of αß-Methylene ATP with the Cholinergic Twitch Response in the Guinea-Pig Ileum.

In this study, direct effects of the P2X purinoceptor agonist αß-methylene ATP (αß-meATP) and effects on the cholinergic twitch response of the electrically stimulated longitudinal muscle-myenteric plexus (LMMP) strip of the guinea-pig ileum, were investigated. αß-meATP (1, 3, and 10 µmol/l) induced short-lasting contractions on its own, followed by an inhibition of the twitch response during its presence in the organ bath. The inhibitor of small conductance Ca2+-activated K+ (SK) channels, apamin (100 nmol/l), prevented the inhibitory effect of αß-meATP on the twitch response, whereas tetraethylammonium (300 µmol/l), a blocker of voltage-gated K+ channels and an inhibitor at nicotinic acetylcholine receptors, augmented the inhibitory effect of αß-meATP on the twitch response. It is concluded, that there is a functional interaction between P2X receptors and nicotinic receptors in the LMMP strip, and that a major part of the excitatory input to the cholinergic motor neuron evoking the twitch response is purinergic and not nicotinergic.

Role of peripheral sigma-1 receptors in ischaemic pain: Potential interactions with ASIC and P2X receptors.

BACKGROUND: The role of peripheral sigma-1 receptors (Sig-1Rs) in normal nociception and in pathologically induced pain conditions has not been thoroughly investigated. Since there is mounting evidence that Sig-1Rs modulate ischaemia-induced pathological conditions, we investigated the role of Sig-1Rs in ischaemia-induced mechanical allodynia (MA) and addressed their possible interaction with acid-sensing ion channels (ASICs) and P2X receptors at the ischaemic site. METHODS: We used a rodent model of hindlimb thrombus-induced ischaemic pain (TIIP) to investigate their role. Western blot was performed to observe changes in Sig-1R expression in peripheral nervous tissues. MA was measured after intraplantar (i.pl.) injections of antagonists for the Sig-1, ASIC and P2X receptors in TIIP rats or agonists of each receptor in naïve rats. RESULTS: Sig-1R expression significantly increased in skin, sciatic nerve and dorsal root ganglia at 3 days post-TIIP surgery. I.pl. injections of the Sig-1R antagonist, BD-1047 on post-operative days 0-3 significantly attenuated the development of MA during the induction phase, but had no effect on MA when given during the maintenance phase (days 3-6 post-surgery). BD-1047 synergistically increased amiloride (an ASICs blocker)- and TNP-ATP (a P2X antagonist)-induced analgesic effects in TIIP rats. In naïve rats, i.pl. injection of Sig-1R agonist PRE-084 alone did not produce MA; but it did induce MA when co-administered with either an acidic pH solution or a sub-effective dose of αßmeATP. CONCLUSION: Peripheral Sig-1Rs contribute to the induction of ischaemia-induced MA via facilitation of ASICs and P2X receptors. Thus, peripheral Sig-1Rs represent a novel therapeutic target for the treatment of ischaemic pain.

Enhancement of Muscle T Regulatory Cells and Improvement of Muscular Dystrophic Process in mdx Mice by Blockade of Extracellular ATP/P2X Axis.

Infiltration of immune cells and chronic inflammation substantially affect skeletal and cardiac muscle degeneration in Duchenne muscular dystrophy. In the immune system, extracellular adenosine triphosphate (ATP) released by dying cells is sensed as a danger associated molecular pattern through P2 purinergic receptors. Specifically, the P2X7 subtype has a prominent role in regulating immune system physiology and contributes to inflammasome activation also in muscle cells. Here, we show that in vivo blockade of the extracellular ATP/P2X purinergic signaling pathway by periodate-oxidized ATP delayed the progression of the dystrophic phenotype and dampened the local inflammatory response in mdx mice, a spontaneous mouse model of dystrophin deficiency. Reduced infiltration of leukocytes and macrophages and decreased expression of IL-6 were revealed in the muscles of periodate-oxidized ATP-treated mdx mice. Concomitantly, an increase in Foxp3(+) immunosuppressive regulatory T cells was observed and correlated with enhanced myofiber regeneration. Moreover, we detected reduced concentrations of profibrotic cytokines, including transforming growth factor-ß and connective tissue growth factor, in muscles of periodate-oxidized ATP-treated mdx mice. The improvement of inflammatory features was associated with increased strength and reduced necrosis, thus suggesting that pharmacologic purinergic antagonism altering the adaptive immune component in the muscle infiltrates might represent a promising therapeutic approach in Duchenne muscular dystrophy.

Control of bone development by P2X and P2Y receptors expressed in mesenchymal and hematopoietic cells.

Bone development and homeostasis require the interplay between several cell types, including mesenchymal osteoblasts and osteocytes, as well as hematopoietic osteoclasts. Recent evidence suggests that cell proliferation, differentiation and apoptosis of both mesenchymal and hematopoietic stem cells, which are fundamental for tissue regeneration and treatment of degenerative diseases, are controlled by P2 receptors (i.e., P2X and P2Y receptors). Both types of P2 receptors are versatile transducers of diverse signals activated by extracellular nucleotides like ATP that are released in response to tissue injury, infection or shear stress. The P2X family of receptors has been shown to mediate multiple signaling events including the influx of calcium, activation of mitogen activated protein kinases (MAPKs) and induction of AP-1 family members known to regulate bone development. Support for the significance of P2X7 in regulating bone development and homeostasis has been provided by several studies focusing on animal models and single nucleotide polymorphisms. P2 receptors are functionally expressed in both bone forming osteoblasts and bone resorbing osteoclasts, while recent findings also suggest that these receptors translate mechanical stimuli in osteocytes. Their ability to respond to external nucleotide analogs renders these cell surface proteins excellent targets for skeletal regenerative therapies. This overview summarizes mechanisms by which nucleotide receptors control skeletal cells and contribute to bone tissue development remodeling and repair.

Purinergic Modulation of Spinal Neuroglial Maladaptive Plasticity Following Peripheral Nerve Injury.

Modulation of spinal reactive gliosis following peripheral nerve injury (PNI) is a promising strategy to restore synaptic homeostasis. Oxidized ATP (OxATP), a nonselective antagonist of purinergic P2X receptors, was found to recover a neuropathic behavior following PNI. We investigated the role of intraperitoneal (i.p.) OxATP treatment in restoring the expression of neuronal and glial markers in the mouse spinal cord after sciatic spared nerve injury (SNI). Using in vivo two-photon microscopy, we imaged Ca(2+) transients in neurons and astrocytes of the dorsal horn of spinal cord at rest and upon right hind paw electrical stimulation in sham, SNI, and OxATP-treated mice. Neuropathic behavior was investigated by von Frey and thermal plantar test. Glial [glial fibrillary acidic protein (GFAP), ionized calcium-binding adaptor molecule 1 (Iba1)] and GABAergic [vesicular GABA transporter (vGAT) and glutamic acid decarboxylase 65/76 (GAD65/67)] markers and glial [glutamate transporter (GLT1) and GLAST] and neuronal amino acid [EAAC1, vesicular glutamate transporter 1 (vGLUT1)] transporters have been evaluated. In SNI mice, we found (i) increased glial response, (ii) decreased glial amino acid transporters, and (iii) increased levels of neuronal amino acid transporters, and (iv) in vivo analysis of spinal neurons and astrocytes showed a persistent increase of Ca(2+) levels. OxATP administration reduced glial activation, modulated the expression of glial and neuronal glutamate/GABA transporters, restored neuronal and astrocytic Ca(2+) levels, and prevented neuropathic behavior. In vitro studies validated that OxATP (i) reduced levels of reactive oxygen species (ROS), (ii) reduced astrocytic proliferation, (iii) increase vGLUT expression. All together, these data support the correlation between reactive gliosis and perturbation of the spinal synaptic homeostasis and the role played by the purinergic system in modulating spinal plasticity following PNI.

Architectural and functional similarities between trimeric ATP-gated P2X receptors and acid-sensing ion channels.

ATP-gated P2X receptors and acid-sensing ion channels are two distinct ligand-gated ion channels that assemble into trimers. They are involved in many important physiological functions such as pain sensation and are recognized as important therapeutic targets. They have unrelated primary structures and respond to different ligands (ATP and protons) and are thus considered as two different ion channels. As a consequence, comparisons of the biophysical properties and underlying mechanisms have only been rarely made between these two channels. However, the recent determination of their molecular structures by X-ray crystallography has revealed unexpected parallels in the architecture of the two pores, providing a basis for possible functional analogies. In this review, we analyze the structural and functional similarities that are shared by these trimeric ion channels, and we outline key unanswered questions that, if addressed experimentally, may help us to elucidate how two unrelated ion channels have adopted a similar fold of the pore.

The role of P2X receptors in bone biology.

Bone is a highly dynamic organ, being constantly modeled and remodeled in order to adapt to the changing need throughout life. Bone turnover involves the coordinated actions of bone formation and bone degradation. Over the past decade great effort has been put into the examination of how P2X receptors regulate bone metabolism and especially for the P2X7 receptor an impressive amount of evidence has now documented its expression in osteoblasts, osteoclasts, and osteocytes as well as important functional roles in proliferation, differentiation, and function of the cells of bone. Key evidence has come from studies on murine knockout models and from pharmacologic studies on cells and animals. More recently, the role of P2X receptors in human bone diseases has been documented. Loss-of-functions polymorphisms in the P2X7 receptorare associated with bone loss and increased fracture risk. Very recently a report from a genetic study in multiple myeloma demonstrated that decreased P2X7 receptor function was associated with increased risk of developing multiple myeloma. In contrast, the risk of developing myeloma bone disease and subsequent vertebral fractures was increased in subjects carrying P2X7 receptor gain-of-function alleles as compared to subjects only carrying loss-of-function or normal functioning alleles. It is evident that P2X receptors are important in regulating bone turnover and maintaining bone mass, and thereby holding great potential as novel drug targets for treatment of bone diseases. However, further research is needed before we fully understand the roles and effects of P2X receptors in bone.

Purinergic component in the coronary vasodilatation to acetylcholine after ischemia-reperfusion in perfused rat hearts.

To determine the involvement of purinergic receptors in coronary endothelium-dependent relaxation, the response to acetylcholine (1 × 10(-8) to 3 × 10(-7)M) was recorded in isolated rat hearts perfused according to the Langendorff procedure before and after 30 min of ischemia and 15 min of reperfusion and after the inhibition of nitric oxide synthesis with L-NAME (10(-4)M), in the absence and presence of the antagonist of purinergic P2X receptors, PPADS (3 × 10(-6)M), and of the antagonist of purinergic P2Y receptors, Reactive Blue 2 (3 × 10(-7)M). In control conditions, the relaxation to acetylcholine was not altered by PPADS or Reactive Blue 2. The relaxation to acetylcholine was reduced after ischemia-reperfusion, and, in this condition, it was further reduced by treatment with PPADS or Reactive Blue 2. Likewise, the relaxation to acetylcholine was reduced by L-NAME, and reduced further by Reactive Blue 2 but not by PPADS. These results suggest that the relaxation to acetylcholine may be partly mediated by purinergic receptors after ischemia-reperfusion, due to the reduction of nitric oxide release in this condition.

Role of peripheral purinoceptors in the development of bee venom-induced nociception: a behavioural and electrophysiological study in rats.

Colocalization of purinergic P2X and P2Y receptors in dorsal root ganglion sensory neurons implies that these receptors play an integrative role in the nociceptive transmission process under inflammatory conditions. In the present study, behavioural and in vivo electrophysiological methods were used to examine the peripheral role of P2 receptors in the persistent nociceptive responses induced by subcutaneous bee venom injection (2 mg/mL) in. Sprague-Dawley rats Local pretreatment with the wide-spectrum P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 1 mmol/L, 50 µL) 10 min prior to s.c. bee venom injection significantly suppressed the duration of spontaneous nociceptive lifting/licking behaviour, inhibited mechanical hyperalgesia and decreased the firing of spinal dorsal horn wide dynamic range neurons in response to bee venom, without affecting primary thermal and mirror-image hyperalgesia. The localized antinociceptive action of PPADS was not due to a systemic effect, because application of the same dose of PPADS to the contralateral side was not effective. The results suggest that activation of peripheral P2 receptors is involved in the induction of nociceptive responses, mechanical hyperalgesia and the excitation of sensory spinal neurons.