A-803467, a potent and selective Nav1.8 sodium channel blocker, attenuates neuropathic and inflammatory pain in the rat

Activation of tetrodotoxin-resistant sodium channels contributes to action potential electrogenesis in neurons. Antisense oligonucleotide studies directed against Na(v)1.8 have shown that this channel contributes to experimental inflammatory and neuropathic pain. We report here the discovery of A-803467, a sodium channel blocker that potently blocks tetrodotoxin-resistant currents (IC(50) = 140 nM) and the generation of spontaneous and electrically evoked action potentials in vitro in rat dorsal root ganglion neurons. In recombinant cell lines, A-803467 potently blocked human Na(v)1.8 (IC(50) = 8 nM) and was >100-fold selective vs. human Na(v)1.2, Na(v)1.3, Na(v)1.5, and Na(v)1.7 (IC(50) values >or=1 microM). A-803467 (20 mg/kg, i.v.) blocked mechanically evoked firing of wide dynamic range neurons in the rat spinal dorsal horn. A-803467 also dose-dependently reduced mechanical allodynia in a variety of rat pain models including: spinal nerve ligation (ED(50) = 47 mg/kg, i.p.), sciatic nerve injury (ED(50) = 85 mg/kg, i.p.), capsaicin-induced secondary mechanical allodynia (ED(50) approximately 100 mg/kg, i.p.), and thermal hyperalgesia after intraplantar complete Freund's adjuvant injection (ED(50) = 41 mg/kg, i.p.). A-803467 was inactive against formalin-induced nociception and acute thermal and postoperative pain. These data demonstrate that acute and selective pharmacological blockade of Na(v)1.8 sodium channels in vivo produces significant antinociception in animal models of neuropathic and inflammatory pain.     

包含P2X3亚基的受体与内脏高敏感性

三磷酸腺苷(ATP)门控阳离子通道型受体P2X31995年克隆成功,特别在小直径伤害性感觉神经元中高表达。越来越多的研究表明,包含P2X3亚基的同聚体P2X3及异聚体P2X2/3在介导内脏伤害性信息传递过程中起重要作用,有可能成为功能性胃肠疾病新的治疗靶点。     

Activation of P2X7 receptors in glial satellite cells reduces pain through downregulation of P2X3 receptors in nociceptive neurons

Purinergic ionotropic P2X7 receptors (P2X7Rs) are closely associated with excitotoxicity and nociception. Inhibition of P2X7R activation has been considered as a potentially useful strategy to improve recovery from spinal cord injury and reduce inflammatory damage to trauma. The physiological functions of P2X7Rs, however, are poorly understood, even though such information is essential for making the P2X7R an effective therapeutic target. We show here that P2X7Rs in satellite cells of dorsal root ganglia tonically inhibit the expression of P2X3Rs in neurons. Reducing P2X7R expression using siRNA or blocking P2X7R activity by antagonists elicits P2X3R up-regulation, increases the activity of sensory neurons responding to painful stimuli, and evokes abnormal nociceptive behaviors in rats. Thus, contrary to the notion that P2X7R activation is cytotoxic, P2X7Rs in satellite cells play a crucial role in maintaining proper P2X3R expression in dorsal root ganglia. Studying the mechanism underlying the P2X7R-P2X3R control, we demonstrate that activation of P2X7Rs evokes ATP release from satellite cells. ATP in turn stimulates P2Y1 receptors in neurons. P2Y1 receptor activation appears to be necessary and sufficient for the inhibitory control of P2X3R expression. We further determine the roles of the P2X7R-P2Y1-P2X3R inhibitory control under injurious conditions. Activation of the inhibitory control effectively prevents the development of allodynia and increases the potency of systemically administered P2X7R agonists in inflamed rats. Thus, direct blocking P2X7Rs, as proposed before, may not be the best strategy for reducing pain or lessening neuronal degeneration because it also disrupts the protective function of P2X7Rs.     

Nociception, pain, and antinociception: current concepts

Summary The physiology of nociception involves a complex interaction of peripheral and central nervous system (CNS) structures, extending from the skin, the viscera and the musculoskeletal tissues to the cerebral cortex. The pathophysiology of chronic pain shows alterations of normal physiological pathways, giving rise to hyperalgesia or allodynia. After integration in the spinal cord, nociceptive information is transferred to thalamic structures before it reaches the somatosensory cortex. Each of these levels of the CNS contain modulatory mechanisms. The two most important systems in modulating nociception and antinociception, the N-methyl-D-aspartate (NMDA) and opioid receptor system, show a close distribution pattern in nearly all CNS regions, and activation of NMDA receptors has been found to contribute to the hyperalgesia associated with nerve injury or inflammation. Apart from substance P (SP), the major facilitatory effect in nociception is exerted by glutamate as the natural activator of NMDA receptors. Stimulation of ionotropic NMDA receptors causes intraneuronal elevation of Ca²⁺ which stimulates nitric oxide synthase (NOS) and the production of nitric oxide (NO). NO as a gaseous molecule diffuses out from the neuron and by action on guanylyl cyclase, NO stimulates in neighboring neurons the formation of cGMP. Depending on the expression of cGMP-controlled ion channels in target neurons, NO may act excitatory or inhibitory. NO has been implicated in the development of hyperexcitability, resulting in hyperalgesia or allodynia, by increasing nociceptive transmitters at their central terminals. Among the three subtypes of opioid receptors, μ- and δ-receptors either inhibit or potentiate NMDA receptor-mediated events, while κ opioids antagonize NMDA receptor-mediated activity. Recently, CRH has been found to act at all levels of the neuraxis to produce analgesia. Modulation of nociception occurs at all levels of the neuraxis, thus, eliciting the multidimensional experience of pain involving sensory-discriminative, affective-motivational, cognitive and locomotor components.      

Ethanol sensitivity in ATP-gated P2X receptors is subunit dependent

BACKGROUND: P2X receptors are ligand-gated cation channels that are gated by synaptically released extracellular adenosine 5'-triphosphate (ATP). P2X receptors are inhibited by ethanol; however, few investigations have focused on ethanol's effects in P2X receptors. Recently, recombinant homomeric P2X4 receptors were reported to be sensitive to ethanol's inhibitory action, whereas recombinant P2X3 receptors were insensitive to ethanol. The two recombinant studies were conducted in different expression systems by using different techniques; therefore, questions remain. The present study tests the hypothesis that ethanol sensitivity in P2X receptors is subunit dependent. METHODS: The effects of ethanol (25-200 mM +/- ATP) on rat recombinant homomeric P2X2 and P2X4 receptors expressed in Xenopus oocytes were tested by using two-electrode voltage clamp techniques. RESULTS: Ethanol inhibited EC10 ATP-gated currents significantly less in P2X2 versus P2X4 receptors. A second study found that ethanol right-shifted the ATP concentration-response curve in P2X2 receptors, which significantly increased the EC50 for ATP without altering the Hill slope or maximal current response to ATP. These latter characteristics of ethanol action in P2X2 receptors agree with previous work in P2X4 receptors. There was no effect of ethanol when tested in the absence of ATP. CONCLUSION: The findings are the first to show (1) ethanol inhibition of ATP-activated currents on P2X2 receptors, (2) differences in ethanol sensitivity between homomeric P2X receptors when tested under matched conditions, and (3) evidence that suggests similar mechanisms of ethanol action for P2X2 and P2X4 receptors. These findings provide the first direct support for the hypothesis that ethanol sensitivity in P2X receptors is subunit dependent.     

Bradykinin as a pain mediator: receptors are localized to sensory neurons, and antagonists have analgesic actions.

Autoradiographic studies localize [3H]bradykinin receptor binding sites to the substantia gelatinosa, dorsal root, and a subset of small cells in both the dorsal root and trigeminal ganglia of the guinea pig. [3H]Bradykinin labeling is also observed over myocardial/coronary visceral afferent fibers. The localization of [3H]bradykinin receptors to nociceptive pathways supports a role for bradykinin in pain mediation. Several bradykinin antagonists block bradykinin-induced acute vascular pain in the rat. The bradykinin antagonists also relieve bradykinin- and urate-induced hyperalgesia in the rat paw. These results indicate that bradykinin is a physiologic mediator of pain and that bradykinin antagonists have analgesic activity in both acute and chronic pain models.     

Nociception, pain, and antinociception: current concepts

The physiology of nociception involves a complex interaction of peripheral and central nervous system (CNS) structures, extending from the skin, the viscera and the musculoskeletal tissues to the cerebral cortex. The pathophysiology of chronic pain shows alterations of normal physiological pathways, giving rise to hyperalgesia or allodynia. After integration in the spinal cord, nociceptive information is transferred to thalamic structures before it reaches the somatosensory cortex. Each of these levels of the CNS contain modulatory mechanisms. The two most important systems in modulating nociception and antinociception, the N-methyl-D-aspartate (NMDA) and opioid receptor system, show a close distribution pattern in nearly all CNS regions, and activation of NMDA receptors has been found to contribute to the hyperalgesia associated with nerve injury or inflammation. Apart from substance P (SP), the major facilitatory effect in nociception is exerted by glutamate as the natural activator of NMDA receptors. Stimulation of ionotropic NMDA receptors causes intraneuronal elevation of Ca²⁺ which stimulates nitric oxide synthase (NOS) and the production of nitric oxide (NO). NO as a gaseous molecule diffuses out from the neuron and by action on guanylyl cyclase, NO stimulates in neighboring neurons the formation of cGMP. Depending on the expression of cGMP-controlled ion channels in target neurons, NO may act excitatory or inhibitory. NO has been implicated in the development of hyperexcitability, resulting in hyperalgesia or allodynia, by increasing nociceptive transmitters at their central terminals. Among the three subtypes of opioid receptors, μ- and δ-receptors either inhibit or potentiate NMDA receptor-mediated events, while κ opioids antagonize NMDA receptor-mediated activity. Recently, CRH has been found to act at all levels of the neuraxis to produce analgesia. Modulation of nociception occurs at all levels of the neuraxis, thus, eliciting the multidimensional experience of pain involving sensory-discriminative, affective-motivational, cognitive and locomotor components.     

Pharmacological properties of a potent and selective nonpeptide substance P antagonist.

We describe here the pharmacological properties of RP 67580 [(3aR,7aR)-7,7-diphenyl-2-[1-imino-2-(2-methoxyphenyl)ethyl] perhydroisoindol-4-one], a nonpeptide antagonist of substance P (SP). In vitro, the compound was found to inhibit in a competitive manner (Ki = 4.16 +/- 0.59 nM) [3H]SP binding to neurokinin receptors type 1 (NK1 receptors) in rat brain membranes. Contractions induced by SP and septide (a selective NK1 agonist) in guinea pig ileum were competitively inhibited by RP 67580 (pA2 = 7.16 and 7.59, respectively). Moreover, RP 67580 displayed the profile of a specific antagonist of NK1 receptors: it was not active on NK2 and NK3 receptors as seen in binding assays and in isolated preparations of rabbit pulmonary artery and rat portal vein. In the rat, low intravenous doses of RP 67580 totally inhibited the plasma extravasation induced by SP in the urinary bladder (ED50 = 0.04 mg/kg i.v.) and by antidromic electrical stimulation of the saphenous nerve in the hind paw skin (ED50 = 0.15 mg/kg i.v.). This compound was also active in two classical analgesic tests in mice: phenylbenzoquinone-induced writhing (ED50 = 0.07 mg/kg s.c.) and the formalin test (ED50 = 3.7 mg/kg s.c.). Its potency was of the same order as that of morphine. Thus we conclude that RP 67580, a SP antagonist, belongs to a class of drugs that may be useful in the management of various clinical pathologies where pain and neurogenic inflammation are involved.     

P2X3受体在心血管疾病中的研究进展

P2X3受体属于嘌呤能受体P2X受体家族中的一员,主要表达于初级感觉神经元中.ATP作为P2X3受体的天然配体,在病理状态如神经损伤、心肌缺血时释放显著增加.与ATP结合后,P2X3受体将外周信号传入神经中枢,进一步引起病理生理变化.研究证实P2X3受体与各种病理性疼痛,尤其是炎症性疼痛有关.近年来发现P2X3受体在高...     

Interplay between P2Y(1), P2Y(12), and P2X(1) receptors in the activation of megakaryocyte cation influx currents by ADP: evidence that the primary megakaryocyte represents a fully functional model of platelet P2 receptor signaling

The difficulty of conducting electrophysiologic recordings from the platelet has restricted investigations into the role of ion channels in thrombosis and hemostasis. We now demonstrate that the well-established synergy between P2Y(1) and P2Y(12) receptors during adenosine diphosphate (ADP)-dependent activation of the platelet alpha(IIb)beta(3) integrin also exists in murine marrow megakaryocytes, further supporting the progenitor cell as a bona fide model of platelet P2 receptor signaling. In patch clamp recordings, ADP (30 microM) stimulated a transient inward current at -70 mV, which was carried by Na(+) and Ca(2+) and was amplified by phenylarsine oxide, a potentiator of certain transient receptor potential (TRP) ion channels by phosphatidylinositol 4,5-bisphosphate depletion. This initial current decayed to a sustained phase, upon which repetitive transient inward cation currents with pre-dominantly P2X(1)-like kinetics were super-imposed. Abolishing P2X(1)-receptor activity prevented most of the repetitive currents, consistent with their activation by secreted adenosine triphosphate (ATP). Recordings in P2Y(1)-receptor-deficient megakaryocytes demonstrated an essential requirement of this receptor for activation of all ADP-evoked inward currents. However, P2Y(12) receptors, through the activation of PI3-kinase, played a synergistic role in both P2Y(1) and P2X(1)-receptor-dependent currents. Thus, direct stimulation of P2Y(1) and P2Y(12) receptors, together with autocrine P2X(1) activation, is responsible for the activation of nonselective cation currents by the platelet agonist ADP.     

Subtype-specific control of P2X receptor channel signaling by ATP and Mg2+

The identity and forms of activating ligands for ion channels are fundamental to their physiological roles in rapid electrical signaling. P2X receptor channels are ATP-activated cation channels that serve important roles in sensory signaling and inflammation, yet the active forms of the nucleotide are unknown. In physiological solutions, ATP is ionized and primarily found in complex with Mg²⁺. Here we investigated the active forms of ATP and found that the action of MgATP²⁻ and ATP⁴⁻ differs between subtypes of P2X receptors. The slowly desensitizing P2X2 receptor can be activated by free ATP, but MgATP²⁻ promotes opening with very low efficacy. In contrast, both free ATP and MgATP²⁻ robustly open the rapidly desensitizing P2X3 subtype. A further distinction between these two subtypes is the ability of Mg²⁺ to regulate P2X3 through a distinct allosteric mechanism. Importantly, heteromeric P2X2/3 channels present in sensory neurons exhibit a hybrid phenotype, characterized by robust activation by MgATP²⁻ and weak regulation by Mg²⁺. These results reveal the existence of two classes of homomeric P2X receptors with differential sensitivity to MgATP²⁻ and regulation by Mg²⁺, and demonstrate that both restraining mechanisms can be disengaged in heteromeric channels to form fast and sensitive ATP signaling pathways in sensory neurons.Seven subtypes of P2X receptors have been identified in mammals that can form either homomeric (P2X1, P2X2, P2X3, P2X4, P2X5, P2X7) or heteromeric (P2X1/2, P2X1/4, P2X1/5, P2X2/3, P2X2/5, P2X2/6, P2X4/6, and possibly, P2X4/7) channels (1–8). These subtypes of P2X receptors have distinct gating properties, pharmacology, and cellular distributions. P2X1 and P2X3 receptors desensitize within a few hundred milliseconds when opened by ATP, and their distributions are restricted to either smooth muscle cells and platelets (P2X1) or a subset of sensory neurons (P2X3) (1, 9–14). P2X2 and P2X4 receptors exhibit slow desensitization during prolonged ATP application, and these receptors are the most abundant subtypes in the central nervous system (15). P2X2 subunits also express in a subset of sensory neurons; however, in these cells they only form heteromeric channels with P2X3 subunits (12, 16, 17). In sensory neurons, P2X3 homomeric channels together with P2X2/3 heteromeric channels play important roles in mediating the primary sensory effects of ATP, and knock-out animals with either P2X3 deletion or P2X2 and P2X3 double-deletions have revealed critical roles in taste, pain, oxygen sensing, and bladder filling (17–20).A long-standing conundrum in P2X receptor-mediated signaling concerns the forms of ATP that activate these channels. In neutral solutions, ATP is ionized and exists mostly as free ATP (ATP⁴⁻), an efficient chelator of divalent cations such as Mg²⁺, and to a lesser extent Ca²⁺ (21). In extracellular biological compartments, such as the synaptic cleft, Ca²⁺ and Mg²⁺ are present in the millimolar range, and therefore only a relatively small fraction of ATP released from vesicles is present in the free form. Although a range of important studies have explored the regulatory effects of Ca²⁺ and Mg²⁺ on P2X receptor channels (22–30), the essential question of which forms of ATP serve as agonists remains unresolved. Several previous studies have reported that P2X2, P2X7, and the native P2X receptors in cilia are activated by ATP in solutions containing low concentrations of divalent cations, and that the addition of divalent cations shifts the concentration dependence for activation of the channels to higher ATP concentrations, suggesting that either ATP⁴⁻ is the most active form of ATP or that divalent cations regulate those subtypes through allosteric mechanisms (27, 30–36). In the present study, we investigated the form(s) of ATP that serve as agonists for a range of subtypes of P2X receptor channels. Our primary focus was to determine whether ATP⁴⁻ or MgATP²⁻ are the principal agonists and to explore whether Mg²⁺ might serve specific regulatory roles. Our results demonstrate that the action of MgATP²⁻ and ATP⁴⁻ differ between subtypes of P2X receptors, and reveal that heteromeric channels can have unique hybrid phenotypes, findings that will be crucial for understanding the physiological functions of these channels in both the peripheral and central nervous systems.     

Optical control of trimeric P2X receptors and acid-sensing ion channels

P2X receptors are trimeric membrane proteins that function as ion channels gated by extracellular ATP. We have engineered a P2X2 receptor that opens within milliseconds by irradiation at 440 nm, and rapidly closes at 360 nm. This requires bridging receptor subunits via covalent attachment of 4,4''-bis(maleimido)azobenzene to a cysteine residue (P329C) introduced into each second transmembrane domain. The cis–trans isomerization of the azobenzene pushes apart the outer ends of the transmembrane helices and opens the channel in a light-dependent manner. Light-activated channels exhibited similar unitary currents, rectification, calcium permeability, and dye uptake as P2X2 receptors activated by ATP. P2X3 receptors with an equivalent mutation (P320C) were also light sensitive after chemical modification. They showed typical rapid desensitization, and they could coassemble with native P2X2 subunits in pheochromocytoma cells to form light-activated heteromeric P2X2/3 receptors. A similar approach was used to open and close human acid-sensing ion channels (ASICs), which are also trimers but are unrelated in sequence to P2X receptors. The experiments indicate that the opening of the permeation pathway requires similar and substantial movements of the transmembrane helices in both P2X receptors and ASICs, and the method will allow precise optical control of P2X receptors or ASICs in intact tissues.P2X receptors and acid-sensing ion channels (ASICs) are trimeric membrane ion channels gated by binding extracellular ligands. P2X receptors are gated by extracellular ATP, and their physiological roles include neuroeffector transmission, primary afferent transmission (e.g., taste, hearing, chemoreception), central control of respiration, and neuroinflammation (1–3). ASICs are gated by protons and are involved in pain sensation (4, 5). The experimental study of ligand-gated channels in intact tissues is often hampered by difficulties in application of the appropriate ligand while recording ion channel activity in the millisecond time domain, and there are advantages to controlling channel activation by surrogate optical methods. The increase in our knowledge of molecular and atomic structure of ligand-gated channels over the past 10 years has allowed one such approach (photoswitchable tethered ligands) to become much more sophisticated, because cysteines can be introduced into the channel protein exactly where required to form an attachment point. The method has been applied to pentameric nicotinic receptors (6) and tetrameric glutamate receptors (7, 8). Although attaching ligands through photoswitchable tethers is proving extremely valuable, an intimate structural knowledge of a closed and open state of a channel also allows for optical control of conformation at parts of the protein that are remote from the agonist binding site (9–11).High-resolution structures are available for P2X receptors (closed: ref. 12; open: ref. 13) and ASICs (closed: refs. 14 and 15; open: ref. 16). In both these trimeric channels the second of the two transmembrane domains (TM2) of each subunit lines the permeation pathway (12–14, 16, 17), and the outermost ends of the TM2s undergo substantial lateral displacement when the channel opens (Fig. 1A) (13, 16). We therefore reasoned that the energy provided by an azobenzene molecule undergoing cis to trans isomerization should be sufficient to force apart the TM2 domains and open the permeation pathway.Open in a separate windowFig. 1.Light activation of P2X2 receptors. (A) Models of rat P2X2 receptor showing closed (Left) and open (Right) conformations. (Upper) Space-fill of trimeric holoprotein. (Lower) Ribbon representation of TM domains, from extracellular side. The positions of P329 are indicated in green. (B) BMA, in its cis state (Left) and trans state (Right). (C) Aligned sequences of second transmembrane domains of rat P2X2 and P2X3 receptors, and human ASIC1a. (D) Light-activated P2X2[P329C] receptors. Currents were evoked by illumination at 440 nm (2 s, blue bar) and turned off by illumination at 360 nm (2 s, violet bar). Light-induced currents were 35% ± 4% (n = 11) of the amplitude of maximum currents evoked by ATP. There was no effect of 440-nm or 360-nm illumination at wild-type P2X2 receptors or at P2X2[P329S] receptors (middle traces), but normal responses to ATP. When the P329C mutation was combined with K69A mutation, ATP (100 μM, 2 s) had no effect (right trace), whereas light-induced currents were present. Preincubated for 10–12 min with BMA (10 μM), in each case. ATP was 3, 10, 10, and 100 μM (left to right). Currents normalized to the peak amplitude evoked by ATP, except that P329C/K69A uses the same scale as P329C. Actual peak amplitudes were as follows: P329C 1879 pA, wild type 1937 pA, P329S 1954 pA, and P329C/K69A 1360 pA.We synthesized a bis(maleimido)azobenzene (Fig. 1B) of molecular dimensions appropriate to react with two cysteines in different subunits: this produced a P2X2 receptor that was opened and closed by different wavelengths of light. We extended the studies to a second member of the P2X family (P2X3) and thus produced an optically controlled heteromeric P2X2/3 receptor. Despite the lack of primary similarity, including the TM2 domain (Fig. 1C), there are similarities between the overall conformational changes that underlie channel opening in P2X receptors and in ASICs (18). We anticipate that the introduction of these engineered channels into cells and animals, when combined with the covalent modification with bis(maleimido)azobenzene, will facilitate the further investigation of the physiological roles of P2X receptors and ASICs.     

Proteinases and proteinase-activated receptor 2: a possible role to promote visceral hyperalgesia in rats

BACKGROUND & AIMS: PAR-2s are highly expressed throughout the gastrointestinal tract. These receptors are cleaved by trypsin and mast cell tryptase and can be activated by peptides corresponding to the tethered ligand of the receptor (SLIGRL-NH2 for rat). The aim of this study was to determine whether colonic administration of PAR-2 agonists affects visceral sensitivity to rectal distention in conscious rats. METHODS: Abdominal contractions (a criteria of visceral pain) were recorded in rats equipped with intramuscular electrodes. Rectal distention was performed at various times after intracolonic infusion of SLIGRL-NH2 and trypsin. Inflammation parameters and permeability were followed in the colon after the intracolonic injections. Fos expression at a spinal level (L4-L6) was also studied 2 hours after intracolonic injection of SLIGRL-NH2. RESULTS: Rectal distention significantly increased abdominal contractions starting at the RD volume of 0.8 mL. Intracolonic injection of SLIGRL-NH2 (200 microg/rat) and trypsin (200 U/rat), but not vehicle, LRGILS-NH2 (control peptide), boiled trypsin, or SLIGRL-NH2 injected IP, significantly increased (P < 0.05) abdominal contractions for high volumes of distention, 10- and 24-hour postinfusion. SLIGRL-NH2-induced hyperalgesia was inhibited by a NK1 receptor antagonist (SR 140333) but not by indomethacin. Intracolonic injection of SLIGRL-NH2 elevated spinal Fos expression and caused increased intestinal permeability but did not cause detectable inflammation. CONCLUSIONS: Intracolonic infusion of subinflammatory doses of PAR-2 agonists activated spinal afferent neurons and produced a delayed rectal hyperalgesia that involves changes in intestinal permeability and the activation of NK1 receptors. These results identify a possible role for proteinases and PAR-2 in the genesis of visceral hyperalgesia.     

P2X receptor immunoreactivity in different arteries from the femoral, pulmonary, cerebral, coronary and renal circulations.

The expression of the seven P2X receptor subunits (P2X1-7) in the rat vascular system was determined using subtype-selective antibodies. Arteries of different sizes (from arterioles to conduit vessels) from a range of vascular beds were used to give an overview of receptor expression. P2X1 receptor immunoreactivity was detected in the smooth muscle layer of arteries. The relative level of P2X1 receptor immunoreactivity was dependent on the size of the artery and the vascular bed; expression was highest in small and medium arteries. P2X4 receptors were detected in all arteries; once again, the relative level of expression was dependent on the size of the artery and the vascular bed. P2X5 receptor immunoreactivity was barely detectable in most arteries studied. P2X7 receptor immunoreactivity was generally punctate and associated with the outer adventitial layer. Immunoreactivity for P2X2, P2X3 and P2X6 receptors was not detected in arteries. These results demonstrate that arteries express multiple P2X receptor subunits and that there is a heterogeneity in the level of expression. The properties of artery P2X receptors correspond to homomeric P2X1 receptors, and the function of P2X4 and P2X5 receptor subunits in arteries is unclear.     

Emerging roles for P2X1 receptors in platelet activation

The platelet surface membrane possesses three P2 receptors activated by extracellular adenosine nucleotides; one member of the ionotropic receptor family (P2X(1)) and two members of the G-protein-coupled receptor family (P2Y(1) and P2Y(12)). P2Y(1) and P2Y(12) receptors have firmly established roles in platelet activation during thrombosis and haemostasis, whereas the importance of the P2X(1) receptor has been more controversial. However, recent studies have demonstrated that P2X(1) receptors can generate significant functional platelet responses alone and in synergy with other receptor pathways. In addition, studies in transgenic animals indicate an important role for P2X(1) receptors in platelet activation, particularly under conditions of shear stress and thus during arterial thrombosis. This review discusses the background behind discovery of P2X(1) receptors in platelets and their precursor cell, the megakaryocyte, and how signalling via these ion channels may participate in platelet activation.     

Platelet Ca(2+) responses coupled to glycoprotein VI and Toll-like receptors persist in the presence of endothelial-derived inhibitors: roles for secondary activation of P2X1 receptors and release from intracellular Ca(2+) stores

Inhibition of Ca(2+) mobilization by cyclic nucleotides is central to the mechanism whereby endothelial-derived prostacyclin and nitric oxide limit platelet activation in the intact circulation. However, we show that ～ 50% of the Ca(2+) response after stimulation of glycoprotein VI (GPVI) by collagen, or of Toll-like 2/1 receptors by Pam(3)Cys-Ser-(Lys)(4) (Pam(3)CSK(4)), is resistant to prostacyclin. At low agonist concentrations, the prostacyclin-resistant Ca(2+) response was predominantly because of P2X1 receptors activated by ATP release via a phospholipase-C-coupled secretory pathway requiring both protein kinase C and cytosolic Ca(2+) elevation. At higher agonist concentrations, an additional pathway was observed because of intracellular Ca(2+) release that also depended on activation of phospholipase C and, for TLR 2/1, PI3-kinase. Secondary activation of P2X1-dependent Ca(2+) influx also persisted in the presence of nitric oxide, delivered from spermine NONOate, or increased ectonucleotidase levels (apyrase). Surprisingly, apyrase was more effective than prostacyclin and NO at limiting secondary P2X1 activation. Dilution of platelets reduced the average extracellular ATP level without affecting the percentage contribution of P2X1 receptors to collagen-evoked Ca(2+) responses, indicating a highly efficient activation mechanism by local ATP. In conclusion, platelets possess inhibitor-resistant Ca(2+) mobilization pathways, including P2X1 receptors, that may be particularly important during early thrombotic or immune-dependent platelet activation.     

Adherent-Invasive E. coli enhances colonic hypersensitivity and P2X receptors expression during post-infectious period

Irritable Bowel Syndrome (IBS) and Inflammatory Bowel Disease (IBD) are related gastrointestinal disorders characterized by abdominal pain associated with colonic hypersensitivity (CHS). Studies in humans have reported an abnormal colonization of Adherent-Invasive E. coli (AIEC) in the ileum of Crohn's disease (CD) patients associated with overexpression of the bacterial colonizing receptor CEACAM6. The aim of the present study was to investigate whether AIEC reference strain LF82 could induce intestinal impairment during infectious and/or post-infectious periods and subsequently the development of CHS. Transgenic mice overexpressing human CEACAM6 protein (TG) and their wild-type littermates were gavaged by CD-associated AIEC bacteria (reference strain LF82) or PBS for 3 d. Colonic hypersensitivity was assessed by colorectal distension (CRD) test during infectious (D4) and post-infectious periods (D21). Several markers of intestinal inflammation were monitored and the colonic expression of purinergic P2X receptors was quantified. At D4, an increased visceromotor response (VMR) to the CRD test was observed in TG mice infected with CD-associated AIEC LF82 in comparison with non-infected TG mice and persisted in a subgroup of infected animals at D21 after bacteria clearance. Increased VMR was associated with low-grade intestinal inflammation, increased intestinal permeability and expression of P2X 3, 4 and 7. This study shows that certain susceptible hosts infected with CD-associated AIEC bacteria can develop persistent CHS associated with low-grade inflammation and increased P2X receptors expression. Thus, CD-associated AIEC infection in CEACAM6 transgenic mice could be used as a novel post-infectious mouse model mimicking quiescent IBD with IBS-like symptoms such as visceral pain.     

Overview of the P2 receptors

The release of nucleotides in extracellular fluids can result from cell necrosis, exocytosis of secretory granules (such as platelet dense granules), or efflux through membrane channels. In addition, recent evidence suggests that vesicular trafficking is an important pathway of nucleotide release. Once in the extracellular fluids, they are rapidly degraded by ectonucleotidases, such as CD39, that play a key role in neutralizing the platelet aggregatory action of adenosine diphosphate (ADP), and act on two families of receptors: the ionotropic P2X receptors and the G-protein-coupled P2Y receptors. The family of P2X receptors encompasses seven genes. Currently, there are eight genuine P2Y receptors that can be subdivided into two structurally distinct subfamilies. Whereas P2X receptors are receptors of ATP, the different P2Y receptors are activated by distinct nucleotides, diphosphates or triphosphates, or purines or pyrimidines, some of them being conjugated to sugars. The study of knockout mice has demonstrated that P2X receptors play important roles in the neurogenic control of smooth muscle contraction, in pain and visceral perception, and in macrophage functions. The phenotype of P2Y null mice so far is more restricted: inhibition of platelet aggregation to ADP and increased bleeding time in P2Y (1)(-/-) and P2Y (12)(-/-) mice and lack of epithelial responsiveness to nucleotides in airways (P2Y (2)(-/-)) and intestine (P2Y (4)(0/-)).     

P2X(7) receptor antagonism attenuates the hypertension and renal injury in Dahl salt-sensitive rats

The P2X(7) receptor is a ligand-gated ion channel activated by extracellular ATP, and a common genetic variation in the P2X(7) gene significantly affects blood pressure. P2X(7) receptor expression is associated with renal injury and some inflammatory diseases. Brilliant blue G (BBG) is a selective rat P2X(7) receptor antagonist. In this study, to test whether BBG has protective effects on salt-sensitive hypertension and renal injury, Dahl salt-sensitive (DS) rats fed an 8% NaCl diet were i.p. injected with BBG (50?mg?kg(-1) per day) for 4 weeks. We also tested another P2X(7) receptor antagonist, namely A-438079 (100?mg?kg(-1) per day), for 7 days. We found that P2X(7) antagonism markedly attenuated salt-sensitive hypertension, urinary protein or albumin excretion, renal interstitial fibrosis and macrophage and T-cell infiltration in the DS rats, and significantly improved creatinine clearance. In an in vitro experiment using macrophages, we showed that lipopolysaccharide (LPS)-primed macrophages from the DS rats released more interleukin-1 beta in response to BzATP, a P2X(7) receptor agonist, than the macrophages from Lewis rats, possibly due to higher P2X(7) expression in the DS rats. In conclusion, in vivo blockade of P2X(7) receptors attenuated salt-sensitive hypertension and renal injury in the DS rats. Thus, P2X(7) appears to be responsible for a vicious cycle of salt-sensitive hypertension and renal injury in the DS rats, through higher expression in the immune cells. Furthermore, P2X(7) antagonists can prevent the development of salt-sensitive hypertension and renal injury, thus confirming that the P2X(7) receptor is an important therapeutic target.     

ATP-activated P2X2 current in mouse spermatozoa

Sperm cells acquire hyperactivated motility as they ascend the female reproductive tract, which enables them to overcome barriers and penetrate the cumulus and zona pellucida surrounding the egg. This enhanced motility requires Ca(2+) entry via cation channel of sperm (CatSper) Ca(2+)-selective ion channels in the sperm tail. Ca(2+) entry via CatSper is enhanced by the membrane hyperpolarization mediated by Slo3, a K(+) channel also present in the sperm tail. To date, no transmitter-mediated currents have been reported in sperm and no currents have been detected in the head or midpiece of mature spermatozoa. We screened a number of neurotransmitters and biomolecules to examine their ability to induce ion channel currents in the whole spermatozoa. Surprisingly, we find that none of the previously reported neurotransmitter receptors detected by antibodies alone are functional in mouse spermatozoa. Instead, we find that mouse spermatozoa have a cation-nonselective current in the midpiece of spermatozoa that is activated by external ATP, consistent with an ATP-mediated increase in intracellular Ca(2+) as previously reported. The ATP-dependent current is not detected in mice lacking the P2X2 receptor gene (P2rx2(-/-)). Furthermore, the slowly desensitizing and strongly outwardly rectifying ATP-gated current has the biophysical and pharmacological properties that mimic heterologously expressed mouse P2X2. We conclude that the ATP-induced current on mouse spermatozoa is mediated by the P2X2 purinergic receptor/channel. Despite the loss of ATP-gated current, P2rx2(-/-) spermatozoa have normal progressive motility, hyperactivated motility, and acrosome reactions. However, fertility of P2rx2(-/-) males declines with frequent mating over days, suggesting that P2X2 receptor adds a selection advantage under these conditions.