Development of P2X receptor clusters on smooth muscle cells in relation to nerve varicosities in the rat urinary bladder

Postnatal development of the distribution of different isoforms of purinergic (P2X) receptors on smooth muscle cells in relation to the development of the innervation of the cells by nerve varicosities in the rat urinary bladder has been determined with immunofluorescence and confocal microscopy. Antibodies against the extracellular domains of the P2X₁ to P2X₆ receptors were used to detect the receptors in the bladder. Several other antibodies were used to identify sympathetic varicosities and Schwann cells. At one day postnatal (D1) there were few strings of varicosities denoting isolated axons, with most axons confined to large nerve trunks. Small size clusters of P2X₁ to P2X₆ receptor subtypes (about 0.4 µm diameter) were observed in the muscle which were independent of each other, and sometimes juxtaposed to the rare isolated varicosity strings. At D4 large numbers of strings of varicosities could be discerned throughout the detrusor. Most of these clouds of small P2X₁ to P2X₆ receptor clusters in their immediate vicinity. Some of these were colocalised with the varicosities, which were of parasympathetic origin as they failed to counter-stain with antibodies to tyrosine hydroxylase. Up to D14 there was a gradual coalescence of many of the isolated P2X_1–6 small receptor clusters so that they became colocalised, often at varicosities. Most of the varicosities in isolated strings possessed receptor clusters at this time. By D21 it was rare to find varicosity strings in the detrusor that were not either in close juxtaposition with P2X small receptor clusters or possessing such clusters in colocalisation. However, large numbers of small P2X receptor clusters, many of which consisted of a mixture of isoforms, could be found spatially unrelated to nerve varicosities throughout the detrusor muscle. In the adult, single axons were either coextensive with one or more isoforms of P2X receptor clusters or these were immediately juxtaposed to the axons so that is was rare to find a varicosity that did not possess a receptor cluster. However, different combinations of colocalised P2X receptor isoforms could still be discerned in small clusters unrelated to varicosities. These observations are discussed in relation to the mechanism of formation of the receptor clusters and their migration beneath parasympathetic varicosities during development.     

P2X receptors are differentially expressed on vasopressin- and oxytocin-containing neurons in the supraoptic and paraventricular nuclei of rat hypothalamus

In the present study, the distribution of P2X receptor protein and colocalization of P2X receptors with vasopressin and oxytocin in the supraoptic and paraventricular nuclei of rat hypothalamus was studied using double-labeling fluorescence immunohistochemistry. The results showed that vasopressin-containing neurons expressed P2X₂, P2X₄, P2X₅ and P2X₆ receptor and oxytocin-containing neurons expressed P2X₂, P2X₄ and P2X₅ receptors in the supraoptic nucleus. In the paraventricular nucleus, vasopressin-containing neurons expressed P2X₄, P2X₅ and P2X₆ receptors, while oxytocin-containing neurons expressed P2X₄ receptors. This study provides the first evidence that P2X receptor subunits are differentially expressed on vasopressin- and oxytocin-containing neurons in the supraoptic and paraventricular nuclei, and hence, provides a substantial neuroanatomical basis for possible functional interactions between the purinergic and vasopressinergic systems, and the purinergic and oxytocinergic systems in the rat hypothalamus. Wei Guo and Jihu Sun contributed equally to this work.     

Expression of P2X(2) and P2X (3) receptors in the rat carotid sinus, aortic arch, vena cava, and heart, as well as petrosal and nodose ganglia

With single- and double-labeling immunofluorescence techniques, the distribution patterns and morphological characteristics of P2X₂- and P2X₃-immunoreactive nerve fiber terminals and neuronal bodies have been studied in the main circulatory system baroreceptors and the nodose and petrosal ganglia of rats. A high density of P2X₂- and P2X₃-immunoreactive nerve fiber terminals was detected in the carotid sinus. P2X₂- and P2X₃-immunoreactive nerve fiber terminals were also distributed widely in the aortic arch, atrium, vena cava, and ventricles. Almost all the P2X₂-immunoreactive nerve fiber terminals were immunoreactive for P2X₃ receptors. P2X₂- and P2X₃-immunoreactive neuronal bodies were also detected in the nodose and petrosal ganglia, which are the sources of the P2X₂- and P2X₃-immunoreactive nerve terminals. P2X₂ and P2X₃ receptors were expressed in the same ganglionic neurons. These data indicate that extracellular ATP, via the homomeric P2X₂ and P2X₃ receptors, and heteromeric P2X_2/3 receptor in the sensory receptors of carotid sinus, aortic arch, atrium, and vena cava, may be involved in the regulation of systematic circulation blood pressure.     

Mouse Leydig cells express multiple P2X receptor subunits

ATP acts on cellular membranes by interacting with P2X (ionotropic) and P2Y (metabotropic) receptors. Seven homomeric P2X receptors (P2X₁–P2X₇) and seven heteromeric receptors (P2X_1/2, P2X_1/4, P2X_1/5, P2X_2/3, P2X_2/6, P2X_4/6, P2X_4/7) have been described. ATP treatment of Leydig cells leads to an increase in [Ca²⁺]_i and testosterone secretion, supporting the hypothesis that Ca²⁺ signaling through purinergic receptors contributes to the process of testosterone secretion in these cells. Mouse Leydig cells have P2X receptors with a pharmacological and biophysical profile resembling P2X₂. In this work, we describe the presence of several P2X receptor subunits in mouse Leydig cells. Western blot experiments showed the presence of P2X₂, P2X₄, P2X₆, and P2X₇ subunits. These results were confirmed by immunofluorescence. Functional results support the hypothesis that heteromeric receptors are present in these cells since 0.5 μM ivermectin induced an increase (131.2 ± 5.9%) and 3 μM ivermectin a decrease (64.2 ± 4.8%) in the whole-cell currents evoked by ATP. These results indicate the presence of functional P2X₄ subunits. P2X₇ receptors were also present, but they were non-functional under the present conditions because dye uptake experiments with Lucifer yellow and ethidium bromide were negative. We conclude that a heteromeric channel, possibly P2X_2/4/6, is present in Leydig cells, but with an electrophysiological and pharmacological phenotype characteristic of the P2X₂ subunit.     

Distribution of purinergic P2X receptors in the equine digit, cervical spinal cord and dorsal root ganglia

Purinergic pathways are considered important in pain transmission, and P2X receptors are a key part of this system which has received little attention in the horse. The aim of this study was to identify and characterise the distribution of P2X receptor subtypes in the equine digit and associated vasculature and nervous tissue, including peripheral nerves, dorsal root ganglia and cervical spinal cord, using PCR, Western blot analysis and immunohistochemistry. mRNA signal for most of the tested P2X receptor subunits (P2X_{1–5, 7}) was detected in all sampled equine tissues, whereas P2X₆ receptor subunit was predominantly expressed in the dorsal root ganglia and spinal cord. Western blot analysis validated the specificity of P2X_{1–3, 7} antibodies, and these were used in immunohistochemistry studies. P2X_{1–3, 7} receptor subunits were found in smooth muscle cells in the palmar digital artery and vein with the exception of the P2X₃ subunit that was present only in the vein. However, endothelial cells in the palmar digital artery and vein were positive only for P2X₂ and P2X₃ receptor subunits. Neurons and nerve fibres in the peripheral and central nervous system were positive for P2X_1–3 receptor subunits, whereas glial cells were positive for P2X₇ and P2X_{1 and 2} receptor subunits. This previously unreported distribution of P2X subtypes may suggest important tissue specific roles in physiological and pathological processes.     

Nav 1.8-null mice show stimulus-dependent deficits in spinal neuronal activity

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 P2X_2/3 receptors, attenuated the antinociceptive effect produced by i.c.v. administered α,β-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 α,β-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/P2X_2/3 receptors play an inhibitory role in pain transmission.     

Inter-subunit disulfide cross-linking in homomeric and heteromeric P2X receptors

P2X receptors are ATP-gated cation channels and assembled as homotrimers or heterotrimers from seven cloned subunits. Each subunit contains two transmembrane domains connected by a large extracellular loop. We have previously shown that replacement of two conserved residues, K68 and F291, by cysteine residues leads to disulfide cross-linking between neighbouring P2X₁ subunits. Since mutation of these residues results in a reduced ATP potency and cysteine cross-linking is prevented in the presence of ATP, we suggested an inter-subunit ATP binding site. To investigate whether the proximity of these residues is preserved in other P2X subtypes, we tested for spontaneous cystine formation between the corresponding P2X₂ (K69C, F289C), P2X₃ (K63C, F280C), and P2X₄ (K67C, F294C) mutants upon pairwise expression in Xenopus laevis oocytes. Non-reducing SDS-PAGE analysis of the purified receptors revealed a specific dimer formation between P2X₂K69C and P2X₂F289C mutants. Likewise, co-expression of P2X₁K68C and P2X₂F289C, but not P2X₁F291C and P2X₂K69C, mutants resulted in dimer formation between the respective subunits. Cross-linked P2X_1/2 heteromers showed strongly reduced or absent function that was selectively recovered upon treatment with DTT. Cross-linking was less efficient between P2X₃ or P2X₄ mutants but could be enhanced by the short cysteine-reactive cross-linker MTS-2-MTS. These results show that the spatial proximity and/or orientation of residues analogous to positions K68 and F291 in P2X₁ are preserved in P2X₂ receptors and at one of two possible interfaces in heteromeric P2X_1/2 receptors but appears to be redundant for P2X₃ and P2X₄ receptor function. EBSA Satellite Meeting: Ion channels, Leeds, July 2007.     

ATP receptors in pain sensation: Involvement of spinal microglia and P2X<Subscript>4</Subscript> receptors

There is abundant evidence that extracellular ATP and other nucleotides have an important role in pain signaling at both the periphery and in the CNS. At first, it was thought that ATP was simply involved in acute pain, since ATP is released from damaged cells and excites directly primary sensory neurons by activating their receptors. However, neither blocking P2X/Y receptors pharmacologically nor suppressing the expression of P2X/Y receptors molecularly in sensory neurons or in the spinal cord had an effect on acute physiological pain. The focus of attention now is on the possibility that endogenous ATP and its receptor system might be activated in pathological pain states, particularly in neuropathic pain. Neuropathic pain is often a consequence of nerve injury through surgery, bone compression, diabetes or infection. This type of pain can be so severe that even light touching can be intensely painful; unfortunately, this state is generally resistant to currently available treatments. An important advance in our understanding of the mechanisms involved in neuropathic pain has been made by a recent work demonstrating the crucial role of ATP receptors (i.e., P2X₃ and P2X₄ receptors). In this review, we summarize the role of ATP receptors, particularly the P2X₄ receptor, in neuropathic pain. The expression of P2X₄ receptors in the spinal cord is enhanced in spinal microglia after peripheral nerve injury, and blocking pharmacologically and suppressing molecularly P2X₄ receptors produce a reduction of the neuropathic pain behaviour. Understanding the key roles of ATP receptors including P2X₄ receptors may lead to new strategies for the management of neuropathic pain.     

Purinergic P2X7 Receptors Mediate ATP-induced Saliva Secretion by the Mouse Submandibular Gland

Salivary glands express multiple isoforms of P2X and P2Y nucleotide receptors, but their in vivo physiological roles are unclear. P2 receptor agonists induced salivation in an ex vivo submandibular gland preparation. The nucleotide selectivity sequence of the secretion response was BzATP ≫ ATP > ADP ≫ UTP, and removal of external Ca²⁺ dramatically suppressed the initial ATP-induced fluid secretion (∼85%). Together, these results suggested that P2X receptors are the major purinergic receptor subfamily involved in the fluid secretion process. Mice with targeted disruption of the P2X₇ gene were used to evaluate the role of the P2X₇ receptor in nucleotide-evoked fluid secretion. P2X₇ receptor protein and BzATP-activated inward cation currents were absent, and importantly, purinergic receptor agonist-stimulated salivation was suppressed by more than 70% in submandibular glands from P2X₇-null mice. Consistent with these observations, the ATP-induced increases in [Ca²⁺]_i were nearly abolished in P2X₇^–/– submandibular acinar and duct cells. ATP appeared to also act through the P2X₇ receptor to inhibit muscarinic-induced fluid secretion. These results demonstrate that the ATP-sensitive P2X₇ receptor regulates fluid secretion in the mouse submandibular gland.Salivation is a Ca²⁺-dependent process (1, 2) primarily associated with the neurotransmitters norepinephrine and acetylcholine, release of which stimulates α-adrenergic and muscarinic receptors, respectively. Both types of receptors are coupled to G proteins that activate phospholipase Cβ (PLCβ) during salivary gland stimulation. PLCβ activation cleaves phosphatidylinositol 1,4-bisphosphate resulting in diacylglycerol and inositol 1,4,5-trisphosphate (InsP₃) production. Activation of Ca²⁺-selective InsP₃ receptor channels localized to the endoplasmic reticulum of salivary acinar cells increases the intracellular free calcium concentration ([Ca²⁺]_i).⁴ Depletion of the endoplasmic reticulum Ca²⁺ pool triggers extracellular Ca²⁺ influx and a sustained elevation in [Ca²⁺]_i. This increase in [Ca²⁺]_i activates Ca²⁺-dependent K⁺ and Cl^– channels promoting Cl^– secretion across the apical membrane and a lumen negative, electrochemical gradient that supports Na⁺ efflux into the lumen. The accumulation of NaCl creates an osmotic gradient which drives water movement into the lumen, thus generating isotonic primary saliva. This primary fluid is then modified by the ductal system, which reabsorbs NaCl and secretes KHCO₃ producing a final saliva that is hypotonic (1, 2).Salivation also has a non-cholinergic, non-adrenergic component, the origin of which is unclear (3). In addition to muscarinic and α-adrenergic receptors, salivary acinar cells express other receptors that are coupled to an increase in [Ca²⁺]_i such as purinergic P2 and substance P receptors. Like muscarinic and α-adrenergic receptors, P2 receptor activation leads to a sustained increase in [Ca²⁺]_i in salivary acinar cells (4). In contrast, substance P receptor activation rapidly desensitizes and therefore generates only a relatively transient increase in [Ca²⁺]_i (5) that is unlikely to appreciably contribute to fluid secretion. The purinergic P2 receptor family is comprised of G protein-coupled P2Y and ionotropic P2X receptors activated by extracellular di- and triphosphate nucleotides. Activation of both subfamilies of P2 receptors causes an increase in [Ca²⁺]_i. P2Y receptors increase [Ca²⁺]_i via InsP₃-induced Ca²⁺ mobilization from intracellular stores (similar to α-adrenergic and muscarinic receptors) while P2X receptors act as ligand-gated, non-selective cation channels that mediate extracellular Ca²⁺ influx (6). Salivary gland tissues express at least four isoforms of P2X (P2X₄ and P2X₇) and P2Y (P2Y₁ and P2Y₂) subtypes; however, their in vivo physiological significance has yet to be characterized (7–11).Our results revealed that ATP acts in isolation to stimulate fluid secretion from the mouse submandibular gland, but secretion is inhibited when ATP is simultaneously presented with a muscarinic receptor agonist. Ablation of the P2X₇ gene had no effect on the salivary flow rate evoked by muscarinic receptor activation, but markedly reduced ATP-mediated fluid secretion and rescued the inhibitory effects of ATP on muscarinic receptor activation. Submandibular gland acinar cells from P2X₇^–/– animals had dramatically impaired ATP-activated Ca²⁺ signaling, consistent with this being the mechanism responsible for the reduction in ATP-mediated fluid secretion in these mice. Together, these results demonstrated that ATP regulates salivation, acting mainly through the P2X₇ receptor. Activation of the P2X₇ receptor may play a major role in non-adrenergic, non-cholinergic stimulated fluid secretion.     

Expression of P2X5 receptors in the rat, cat, mouse and guinea pig dorsal root ganglion

P2X receptors are ATP-gated cationic channels composed of seven cloned subunits (P2X_{1 –7}). P2X₃ homomultimer and P2X_2/3 heteromultimer receptors expressed by primary afferent dorsal root ganglion (DRG) neurons are involved in pain processing. The aim of the study was to investigate the expression of the P2X₅ receptor subunit in DRG in different species including mouse, rat, cat and guinea pig. Immunohistochemistry showed that P2X₅ receptors exhibited low levels of immunostaining in rat DRG, but high levels in mouse and guinea pig. Only a few neurons were immunoreactive for P2X₅ receptors in cat. In mouse DRG, the P2X₅ receptor was expressed largely by medium-diameter neurons (42.9 %), less in small (29.3 %) and large (27.8 %) neurons. In contrast, in the guinea pig DRG, P2X₅ receptor expression was greatest in small-diameter (42.6 %), less in medium- (36.3 %) and large-diameter (21.1 %) neurons. Colocalization experiments revealed that, in mouse DRG, 65.5, 10.9 and 27.1 % of P2X₅ receptors were immunoreactive for NF-200, CGRP and calbindin, while only a few P2X₅-immunoreactive (IR) neurons were coexpressed with IB4 or with NOS. In guinea pig DRG, a total of 60.5 and 40.5 % of P2X₅-IR neurons were coexpressed with IB4 or with CGRP, while 20.3 and 24.5 % of P2X₅ receptors were coexpressed with NF-200 or with NOS. Only a few P2X₅-IR neurons were coexpressed with calbindin in guinea pig DRG. It will be of great interest to clarify the relative physiological and pathophysiological roles of P2X₅ receptors.     

Lack of evidence for direct phosphorylation of recombinantly expressed P2X2 and P2X3 receptors by protein kinase C

P2X₃ and P2X₂₊₃ receptors are present on sensory neurons, where they contribute not only to transient nociceptive responses, but also to hypersensitivity underlying pathological pain states elicited by nerve injuries. Increased signalling through P2X₃ and P2X₂₊₃ receptors may arise from an increased routing to the plasma membrane and/or gain of function of pre-existing receptors. An obvious effector mechanism for functional modulation is protein kinase C (PKC)-mediated phosphorylation, since all P2X family members share a conserved consensus sequence for PKC, TXR/K, within the intracellularly located N-terminal domain. Contradictory reports have been published regarding the exact role of this motif. In the present study, we confirm that site-directed elimination of the potential phosphor-acceptor threonine or the basic residue in the P+2 position of the TXR/K sequence accelerates desensitization of P2X₂ receptors and abolishes P2X₃ receptor function. Moreover, the PKC activator phorbol 12-myristate 13-acetate increased P2X₃ (but not P2X₂) receptor-mediated currents. Biochemically, however, we were unable to demonstrate by various experimental approaches a direct phosphorylation of wild-type P2X₂ and P2X₃ receptors expressed in both Xenopus laevis oocytes and HEK293 cells. In conclusion, our data support the view that the TXR/K motif plays an important role in P2X function and that phorbol 12-myristate 13-acetate is capable of modulating some P2X receptor subtypes. The underlying mechanism, however, is unlikely to involve direct PKC-mediated P2X receptor phosphorylation.     

Activation kinetics of single P2X receptors

After the primary structure of P2X receptors had been identified, their function had to be characterized on the molecular level. Since these ligand-gated ion channels become activated very quickly after binding of ATP, methods with adequate time resolution have to be applied to investigate the early events induced by the agonist. Single-channel recordings were performed to describe conformational changes on P2X₂, P2X₄, and P2X₇ receptors induced by ATP and also by allosteric receptor modifiers. The main results of these studies and the models of P2X receptor kinetics derived from these observations are reviewed here. The investigation of purinoceptors by means of the patch clamp technique following site-directed mutagenesis will probably reveal more details of P2X receptor function at the molecular level.     

Expression of P2X receptors on immune cells in the rat liver during postnatal development

Single and double-labeling immunofluorescence and RT-PCR expression of P2X receptor proteins and mRNAs were used in a study of the liver of postnatal rats. OX62 and ED1 were used as markers for dendritic and macrophage (Kupffer) cells respectively. The results showed that the P2X₆ receptor subunit was up-regulated by 15-fold on hepatic sinusoid cells during postnatal days P1 to P60. Subpopulations of Kupffer cells co-expressed P2X₄ and P2X₆ receptor subunits and dendritic cells co-expressed P2X₄ and P2X₇ receptor subunits. Lipopolysaccharide (endotoxin) injected into the peritoneal cavity led to increased expression of the P2X₆ receptor on Kupffer cells, suggesting that the P2X₆ receptor subunit may be up-regulated by endotoxin. This study presents the first evidence that P2X receptors are widely distributed in the rat liver immune system and that activation of Kupffer and dendritic cells in the rat liver might be regulated by extracellular ATP.     

Involvement of P2X receptors in the NAD+-induced rise in [Ca2+]i in human monocytes

In the present study, we show that the extracellular addition of nicotinamide adenine dinucleotide (NAD⁺) induces a transient rise in [Ca²⁺]_i in human monocytes caused by an influx of extracellular calcium. The NAD⁺-induced Ca²⁺ response was prevented by adenosine triphosphate (ATP), suggesting the involvement of ATP receptors. Of the two subtypes of ATP receptors (P2X and P2Y), the P2X receptors were considered the most likely candidates. By the use of subtype preferential agonists and antagonists, we identified P2X₁, P2X₄, and P2X₇ receptors being engaged in the NAD⁺-induced rise in [Ca²⁺]_i. Among the P2X receptor subtypes, the P2X₇ receptor is unique in facilitating the induction of nonselective pores that allow entry of ethidium upon stimulation with ATP. In monocytes, opening of P2X₇ receptor-dependent pores strongly depends on specific ionic conditions. Measuring pore formation in response to NAD⁺, we found that NAD⁺ unlike ATP lacks the ability to induce this pore-forming response. Whereas as little as 100 μM ATP was sufficient to activate the nonselective pore, NAD⁺ at concentrations up to 2 mM had no effect. Taken together, these data indicate that despite similarities in the action of extracellular NAD⁺ and ATP there are nucleotide-specific variations. So far, common and distinct features of the two nucleotides are only beginning to be understood.     

Blockade of murine T cell activation by antagonists of P2Y6 and P2X7 receptors

Extracellular nucleotides and their metabolites activate ionotropic P2X and metabotropic P2Y receptors on the surface of various types of cells. Here, we investigated the involvement of P2X and P2Y receptor-mediated signaling in TCR-dependent T cell activation. Murine T cells were activated by stimulation of TCR, and both CD25 expression and interleukin (IL)-2 production were observed in activated T cells. Ecto-nucleotidase apyrase and P2Y₆ antagonist MRS2578 significantly blocked the increases of both CD25 expression and IL-2 production, and P2X₇ antagonists A438079 and oxidized ATP inhibited IL-2 production rather than CD25 expression, suggesting the involvement of P2Y₆ and P2X₇ receptors in different processes of T cell activation. MRS2578 also blocked TCR-dependent elevation of cytosolic Ca²⁺ in T cells. The P2X₇ and P2Y₆ receptors were expressed in murine CD4 T cells. In conclusion, our results indicate that activation of P2Y₆ and P2X₇ receptors contributes to T cell activation via TCR.     

Expression of P2X6 receptors in the enteric nervous system of the rat gastrointestinal tract

Expression of P2X₄ and P2X₆ receptor subunits in the gastrointestinal tract of the rat was studied with double-labeling fluorescence immunohistochemistry. The results showed that P2X₆ receptors were expressed widely in the submucosal and myenteric plexuses. In the myenteric plexus, P2X₆ receptors were expressed mainly in large size neurons which resembled Dogiel type II neurons. These P2X₆ receptor-immunoreactive (ir) neurons also expressed calbindin 28K, calretinin and neuronal nuclei (NeuN), proteins that are markers of intrinsic sensory neurons. In the submucosal plexus, all the calbindin 28K, calretinin and NeuN-ir cells were immunoreactive for P2X₆ receptors. P2X₆ receptors do not form homomultimers, but rather heteromultimers with either P2X₂ or P2X₄ receptors. P2X₄ receptors were not expressed in neurons, but were expressed in macrophages of the rat gastrointestinal tract. These data indicate that P2X₆ receptors are mainly expressed on intrinsic sensory neurons and that ATP, via P2X₆ receptors probably in heteromeric combination with P2X₂ receptors, may be involved in regulating the physiological functions of these neurons.     

P2X receptors in the rat uterine cervix,lumbosacral dorsal root ganglia,and spinal cord during pregnancy

ATP, an intracellular energy source, is released from cells during tissue stress, damage, or inflammation. The P2X subtype of the ATP receptor is expressed in rat dorsal root ganglion (DRG) cells, spinal cord dorsal horn, and axons in peripheral tissues. ATP binding to P2X receptors on nociceptors generates signals that can be interpreted as pain from damaged tissue. We have hypothesized that tissue stress or damage in the uterine cervix during late pregnancy and parturition can lead to ATP release and sensory signaling via P2X receptors. Consequently, we have examined sensory pathways from the cervix in nonpregnant and pregnant rats for the presence of purinoceptors. Antiserum against the P2X₃-receptor subtype showed P2X₃- receptor immunoreactivity in axon-like structures of the cervix, in small and medium-sized neurons in the L6/S1 DRG, and in lamina II of the L6/S1 spinal cord segments. Retrograde tracing confirmed the projections of axons of P2X₃-receptor-immunoreactive DRG neurons to the cervix. Some P2X₃-receptor-positive DRG neurons also expressed estrogen receptor- immunoreactivity and expressed the phosphorylated form of cyclic AMP response-element-binding protein at parturition. Western blots showed a trend toward increases of P2X₃-receptor protein between pregnancy (day 10) and parturition (day 22–23) in the cervix, but no significant changes in the DRG or spinal cord. Since serum estrogen rises over pregnancy, estrogen may influence purinoceptors in these DRG neurons. We suggest that receptors responsive to ATP are expressed in uterine cervical afferent nerves that transmit sensory information to the spinal cord at parturition.     

P2X<Subscript>3</Subscript> Receptor Involvement in Pain States

The understanding of how pain is processed at each stage in the peripheral and central nervous system is the precondition to develop new therapies for the selective treatment of pain. In the periphery, ATP can be released from various cells as a consequence of tissue injury or visceral distension and may stimulate the local nociceptors. The highly selective distribution of P2X₃ and P2X_2/3 receptors within the nociceptive system has inspired a variety of approaches to elucidate the potential role of ATP as a pain mediator. Depolarization by ATP of neurons in pain–relevant neuronal structures such as trigeminal ganglion, dorsal root ganglion, and spinal cord dorsal horn neurons are well investigated. P2X receptor-mediated afferent activation appears to have been implicated in visceral and neuropathic pain and even in migraine and cancer pain. This article reviews recently published research describing the role that ATP and P2X receptors may play in pain perception, highlighting the importance of the P2X₃ receptor in different states of pain.