Functional characterization of P2X3 receptors fused with fluorescent proteins

P2X receptor function in the CNS is poorly understood, and currently available data are partly inconsistent. In the presented study, we investigated P2X3 receptors stably expressed in HEK293 cells. Non-stationary noise analysis of whole cell currents and rapid ATP application through flash photolysis allowed for assessing the single channel conductance (6.6 pS) and the fast activation kinetics of the receptor (20 ms). The characteristics of channel desensitization and pharmacological properties matched previous findings. The properties of wild type receptors were compared with P2X3 constructs carrying a fluorescent tag (ECFP or DsRed2) at the C-terminus. These fluorescently labeled subunits formed functional receptors, with neither the affinity of the ligand binding site nor channel properties (ion selectivity, gating kinetics, single channel conductance) differing from wild type. We conclude that both fusion proteins tested here are suitable for generating transgenic mice, which can be expected to promote understanding of the physiological role of P2X3 receptors in CNS signaling.     

Gintonin, a ginseng-derived lysophosphatidic acid receptor ligand, potentiates ATP-gated P2X1 receptor channel currents

Ginseng, the root of Panax ginseng C.A. Meyer, is used as a general tonic. Recently, we isolated a novel ginsengderived lysophosphatidic acid (LPA) receptor ligand, gintonin. Gintonin activates G protein-coupled LPA receptors with high affinity in cells endogenously expressing LPA receptors, e.g., Xenopus oocytes. P2X receptors are ligandgated ion channels activated by extracellular ATP, and 7 receptor subtypes (P2X₁–P2X₇) have been identified. Most of the P2X₁ receptors are expressed in the smooth muscles of genitourinary organs involved in reproduction. A main characteristic of the P2X₁ receptor is rapid desensitization after repeated ATP treatment of cells or tissues expressing P2X₁ receptors. In the present study, we examined the effect of gintonin on P2X₁ receptor channel activity. P2X₁ receptors were heterologously expressed in Xenopus oocytes. ATP treatment of oocytes expressing P2X₁ receptors induced large inward currents (I _ATP ), but repetitive ATP treatments induced a rapid desensitization of I _ATP . Gintonin treatment after P2X₁ receptor desensitization potentiated I _ATP in a concentration-dependent manner. We further examined the signaling transduction pathways involved in gintonin-mediated potentiation of I _ATP . Gintoninmediated I _ATP potentiation was blocked by Ki16425, an LPA1/3 receptor antagonist, a PKC inhibitor, a PLC inhibitor, and a PI4-Kinase inhibitor but not by a calcium chelator. In addition, mutations of the phosphoinositide binding site of the P2X₁ receptor greatly attenuated the gintonin-mediated I _ATP potentiation. These results indicate that G protein-coupled LPA receptor activation by gintonin is coupled to the potentiation of the desensitized P2X₁ receptor through a phosphoinositide-dependent pathway.     

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.     

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.     

P2X<Subscript>2</Subscript> and P2X<Subscript>3</Subscript> purinoceptors in the rat enteric nervous system

Adenosine 5-triphosphate receptors are known to be involved in fast excitatory postsynaptic currents in myenteric neurons of the digestive tract. In the present study, the distribution of P2X₂ and P2X₃ receptor mRNA was examined by in situ hybridisation while P2X₂ and P2X₃ receptor protein was localised by immunohistochemical methods. In addition, P2X₂ and P2X₃ receptors were colocalised with calbindin and calretinin in the myenteric and submucosal plexus. P2X₂- and P2X₃-immunoreactive neurons were found in the myenteric and submucosal plexuses throughout the entire length of the rat digestive tract from the stomach to the colon. Approximately 60%, 70% and 50% of the ganglion cells in the myenteric plexus of the gastric corpus, ileum and distal colon, and 56% and 45% in the submucosal plexus of the ileum and distal colon, respectively, showed positive immunoreactivity to the P2X₂ receptor. Approximately 10%, 2% and 15% of the ganglion cells in the myenteric plexus of the gastric corpus, ileum and distal colon, and 62% and 40% in the submucosal plexus of the ileum and distal colon, respectively, showed positive immunoreactivity to the P2X₃ receptor. Double-labelling studies showed that about 10–25% of the neurons with P2X₂ immunoreactivity in myenteric plexus and 30–50% in the submucosal plexus were found to express calbindin or calretinin. About 80% of the neurons with P2X₃ receptor immunoreactivity in the myenteric plexus and about 40% in the submucosal plexus expressed calretinin. Approximately 30–75% of the neurons with P2X₃ receptor immunoreactivity in the submucosal plexus expressed calbindin, while none of them were found to express calbindin in the myenteric plexus.     

Distribution of P2Y<Subscript>2</Subscript> receptors in the guinea pig enteric nervous system and its coexistence with P2X<Subscript>2</Subscript> and P2X<Subscript>3</Subscript> receptors,neuropeptide Y,nitric oxide synthase and calretinin

The distribution of P2Y₂ receptor-immunoreactive (ir) neurons and fibers and coexistence of P2Y₂ with P2X₂ and P2X₃ receptors, neuropeptide Y (NPY), calretinin (CR), calbindin (CB) and nitric oxide synthase (NOS) was investigated with immunostaining methods. The results showed that P2Y₂-ir neurons and fibers were distributed widely in myenteric and submucous plexuses of the guinea pig stomach corpus, jejunum, ileum and colon. The typical morphology of P2Y₂-ir neurons was a long process with strong positive staining on the same side of the cell body. The P2Y₂-ir neurons could be Dogiel type 1. About 40–60% P2X₃-ir neurons were immunoreactive for P2Y₂ in the myenteric plexus and all the P2X₃-ir neurons expressed the P2Y₂ receptor in the submucosal plexus; almost all the NPY-ir neurons and the majority of CR-ir neurons were also immunoreactive for P2Y₂, especially in the myenteric plexus of the small intestine; no P2Y₂-ir neurons were immunoreactive for P2X₂ receptors, CB and NOS. It is shown for the first time that S type/Dogiel type 1 neurons with fast P2X and slow P2Y receptor-mediated depolarizations could be those neurons expressing both P2Y₂-ir and P2X₃-ir and that they are widely distributed in myenteric and submucosal plexuses of guinea pig gut.     

Development of nerves expressing P2X<Subscript>3</Subscript> receptors in the myenteric plexus of rat stomach

Development of neurones and fibres expressing P2X₃ receptors in the myenteric plexus of rat stomach and coexistence of the P2X₃ receptor with calbindin, calretinin and NOS during postnatal development, were investigated with immunostaining methods. Extrinsic nerves expressing P2X₃ receptors appeared as early as E12 and were localised in the trunk and branches of the vagus nerve, which extended rapidly onto the whole rat stomach from E12 to E14. Intrinsic neurone cell bodies with P2X₃-immunoreactivity in the myenteric ganglia were first demonstrated postnatally at P1, and at P14, when the number of neurones expressing the P2X₃ receptor peaked at 45%. P2X₃ receptor-immunoreactivity decreased subsequently, and at P60 only about 11% were P2X₃-immunoreactive. Intraganglionic laminar nerve endings and intramuscular arrays were first demonstrated postnatally at P1 and P7, respectively. In the early postnatal days, there were many growth cone-like structures with strong P2X₃ immunostaining associated with these endings and arrays. Double-immunostaining showed that 9–15% of P2X₃-immunoreactive neurones in the gastric myenteric plexus expressed calbindin D-28 k only in the early postnatal days, while 14–21% of neurones from P1 to P60 increasingly expressed calretinin. About 20% of neurones with P2X₃ immunoreactivity coexpressed NOS throughout perinatal development.     

P2 receptors and platelet function

Following vessel wall injury, platelets adhere to the exposed subendothelium, become activated and release mediators such as TXA₂ and nucleotides stored at very high concentration in the so-called dense granules. Released nucleotides and other soluble agents act in a positive feedback mechanism to cause further platelet activation and amplify platelet responses induced by agents such as thrombin or collagen. Adenine nucleotides act on platelets through three distinct P2 receptors: two are G protein-coupled ADP receptors, namely the P2Y₁ and P2Y₁₂ receptor subtypes, while the P2X₁ receptor ligand-gated cation channel is activated by ATP. The P2Y₁ receptor initiates platelet aggregation but is not sufficient for a full platelet aggregation in response to ADP, while the P2Y₁₂ receptor is responsible for completion of the aggregation to ADP. The latter receptor, the molecular target of the antithrombotic drugs clopidogrel, prasugrel and ticagrelor, is responsible for most of the potentiating effects of ADP when platelets are stimulated by agents such as thrombin, collagen or immune complexes. The P2X₁ receptor is involved in platelet shape change and in activation by collagen under shear conditions. Each of these receptors is coupled to specific signal transduction pathways in response to ADP or ATP and is differentially involved in all the sequential events involved in platelet function and haemostasis. As such, they represent potential targets for antithrombotic drugs.     

The P2X7 receptor in retinal ganglion cells: A neuronal model of pressure-induced damage and protection by a shifting purinergic balance

Retinal ganglion cells process the visual signal and transmit it along their axons in the optic nerve to the brain. Molecular, immunohistochemical, and functional analyses indicate that the majority of retinal ganglion cells express the ionotropic P2X₇ receptor. Stimulation of the receptor can lead to a rise in intracellular calcium and cell death, although death does not involve the opening of a large diameter pore. Adenosine acting at A₃ receptors can attenuate the rise in calcium and death accompanying P2X₇ receptor activation, suggesting that dephosphorylation of ATP into adenosine is neuroprotective and that the balance of extracellular purines can influence neuronal survival. Increased intraocular pressure can lead to release of excessive extracellular ATP in the retina and damage ganglion cells by acting on P2X₇ receptors, implicating a role for the receptor in the loss of ganglion cell activity in glaucoma. In summary, the activation of P2X₇ receptors has both physiologic and pathophysiologic implications for ganglion cell function. These characteristics may also provide an insight into the contributions the P2X₇ receptor makes to neurons elsewhere.     

5-Hydroxytryptamine Inhibits P2X2 Receptor Channel Pore Mutants

1.5-Hydroxytryptamine (10 M) enhanced ionic current mediated through the wild-type P2X₂ receptor/channel expressed in Xenopus oocytes.2.5-Hydroxytryptamine (10 M) inhibited a current mediated through P2X₂ receptor/channel mutants when Thr³³⁰ or Asn³³³ was replaced with Ile (T330I and N333I).3.Our results suggest that neutralization of Thr³³⁰ or Asn³³³ exposes a high-affinity, inhibitory binding site for 5-hydroxytryptamine. This implies that 5-hydroxytryptamine interacts with the P2X₂ receptor/channel at their channel pores.     

Changes in P2X<Subscript>3</Subscript> purinoceptors in sensory ganglia of the mouse during embryonic and postnatal development

The expression of the P2X₃ nucleotide receptor in embryonic day 14–18, postnatal day 1–14 and adult mouse sensory ganglia was examined using immunohistochemistry. Nearly all sensory neurons in dorsal root ganglia, trigeminal ganglia and nodose ganglia in embryos at embryonic day 14 expressed P2X₃ receptors, but after birth there was a gradual decline to about 50% of neurons showing positive immunostaining for P2X₃. In embryos there were only small neurons, while from postnatal day 7 both large and small neurons were present. Isolectin B₄ (IB₄)-positive neurons in dorsal, trigeminal and nodose ganglia did not appear until birth, but the numbers increased to about 50% by postnatal day 14 when a high proportion of IB₄-positive neurons were also positively labelled for the P2X₃ receptor. About 10% of neurons in dorsal, trigeminal and nodose ganglia were positive for calcitonin gene-related peptide in embryos, nearly all of which stained for P2X₃ receptors. This increased postnatally to about 35–40% in adults, although only a few colocalised with P2X₃ receptors. Neurofilament 200 was expressed in about 50% of neurons in trigeminal ganglia in the embryo, and this level persisted postnatally. All neurofilament 200-positive neurons stained for P2X₃ in embryonic dorsal root ganglia, trigeminal ganglia and nodose ganglia, but by adulthood this was significantly reduced. The neurons that were positive for calbindin in embryonic dorsal, trigeminal and nodose ganglia showed colocalisation with P2X₃ receptors, but few showed colocalisation postnatally.     

P2X7 Receptors in Rat Brain: Presence in Synaptic Terminals and Granule Cells

ATP stimulates [Ca²⁺]_i increases in midbrain synaptosomes via specific ionotropic receptors (P2X receptors). Previous studies have demonstrated the implication of P2X₃ subunits in these responses, but additional P2X subunits must be involved. In the present study, ATP and BzATP proved to be able to induce intrasynaptosomal calcium transients in the midbrain synaptosomes, their effects being potentiated when assayed in a Mg₂₊-free medium. Indeed, BzATP was shown to be more potent than ATP, and their effects could be inhibited by PPADS and KN-62, but not by suramin. This activity profile is consistent with the presence of functional P2X₇ receptors in the midbrain terminals. The existence of presynaptic responses to selective P2X7 agonists could be confirmed by means of a microfluorimetric technique allowing [Ca²⁺]_i measurements in single synaptic terminals. Additionally, the P2X₇ receptor protein could be identified in the midbrain synaptosomes and in axodendritic prolongations of cerebellar granule cells by immunochemical staining.     

Estrogen altered visceromotor reflex and P2X3 mRNA expression in a rat model of colitis

P2X₃ and P2X_2/3 receptors are expressed in peripheral tissues and dorsal root ganglia (DRG) and participate in peripheral pain. However, the mechanisms underlying P2X receptor-mediated nociception at different ovarial hormone levels has not been examined. In this study, 24 female rats were randomly divided into sham-operated (sham), ovariectomized (OVX), estrogen-treated, and estrogen–progesterone-treated groups with colitis. In each group, the visceromotor reflex (VMR) to colorectal distension was tested and the DRG were harvested for a real-time PCR analysis of P2X₃ and P2X₂ receptor mRNA. In OVX rats with colitis we found that the VMR to colorectal distension and P2X₃ receptor mRNA in DRG were both significantly decreased. Estrogen replacement reversed the decrease. However, neither the VMR nor the P2X₃ mRNA level in DRG from OVX colitis rats was reversed by the complex of estrogen and progesterone. Patch-clamp recording showed that in colitis rats, estradiol rapidly potentiated the sustained and transient currents evoked by ATP to 336 ± 49% and 122 ± 12% of controls, respectively, in a subpopulation of DRG neurons, which were blocked by ICI 182, 780, an antagonist of the estrogen receptor. Whereas progesterone rapidly inhibited the transient currents induced by ATP to 67 ± 10% of control and had no effect on the sustained currents evoked by the same agonist. These results indicate that P2X₃ receptors are likely to be an important contributor to the altered colonic functions in colitis rats, where the underlying mechanisms are closely related to endogenous estrogen modulation.     

<Emphasis Type="Italic">N</Emphasis>-Arylpiperazine modified analogues of the P2X<Subscript>7</Subscript> receptor KN-62 antagonist are potent inducers of apoptosis of human primary osteoclasts

Summary The P2X₇ nucleotide receptor is an ATP-gated ion channel that plays an important role in bone cell function. Here, we investigated the effects of L-tyrosine derivatives 1–3 as potent P2X₇ antagonists on human primary osteoclasts. We found that the level of expression of P2X₇ receptor increased after treatment with the derivatives 1–3, together with the induction of high levels of apoptosis. This effect is associated with activation of caspase-3 and inhibition of expression of IL-6. Interestingly, no pro-apoptotic effect of compounds 1–3 was found on human osteoblasts. Our results suggest that the development of specific P2X₇ receptor antagonists may be considered a useful tool to modulate apoptosis of human osteoclasts. Since bone loss due to osteoclast-mediated resorption represents one of the major unsolved problem in osteopenic disorders, the identification of molecules able to induce apoptosis of osteoclasts is of great interest for the development of novel therapeutic strategies.     

Puerarin alleviates burn-related procedural pain mediated by P2X3 receptors

Pain is a major problem after burns. Procedural pain evoked by burn dressing changes is common in patients, and its management is a critical part of treatment in acute burn injuries. Burn pain is very likely the most difficult form of acute pain to treat. ATP contributes to inflammation, and ATP is implicated in peripheral pain signaling via actions upon P2X₃ receptors. Puerarin is extracted from a traditional Chinese medicine and may act on P2X₃ receptor mechanisms. The Visual Analogue Scale (VAS) has been shown to be a sensitive indicator of pain intensity and treatment effects. Peripheral blood mononuclear cells (PBMCs) are involved in nociception or pain after burn injury. Burn patients were randomly divided into normal saline (NS) group (salt solution is saline) and puerarin-treated group and pain (Visual Analogue Scale scores) and inflammation (PBMCs) measured. Burn pain produces a stress response, so blood glucose, insulin, and cortisol levels in burn patients were determined. Furthermore, the expression of P2X₃ protein and mRNA in PBMCs was detected. The VAS scores in the puerarin-treated group were lower than those in NS group. The blood glucose, insulin, and cortisol levels in the puerarin-treated group at post-dressing changes were significantly decreased in comparison with those in NS group. The expression levels of P2X₃ protein and mRNA in PBMCs of burn patients in NS group were significantly increased in comparison with those in the puerarin-treated group. Puerarin can antagonize inflammatory factors (such as ATP) and decrease the upregulated expressions of P2X₃ protein and mRNA in PBMCs after burns to decrease VAS. Thus, puerarin had an analgesic effect on procedural pain in dressing changes of burn patients related to P2X₃ receptors.     

Optimization of the physicochemical and pharmacokinetic attributes in a 6-azauracil series of P2X7 receptor antagonists leading to the discovery of the clinical candidate CE-224,535

High throughput screening (HTS) of our compound file provided an attractive lead compound with modest P2X₇ receptor antagonist potency and high selectivity against a panel of receptors and channels, but also with high human plasma protein binding and a predicted short half-life in humans. Multi-parameter optimization was used to address the potency, physicochemical and pharmacokinetic properties which led to potent P2X₇R antagonists with good disposition properties. Compound 33 (CE-224,535) was advanced to clinical studies for the treatment of rheumatoid arthritis.     

Different properties of P2X7 receptor in hippocampal and cortical astrocytes

P2X₇ receptor is a ligand-gated ion channel, which can induce the opening of large membrane pores. Here, we provide evidence that the receptor induces pore formation in astrocytes cultured from cortex, but not from the hippocampus. Furthermore, P2X₇ receptor activation promptly induces p38 mitogen-activated protein kinase (MAPK) phosphorylation in cortical but not in hippocampal astrocytes. Given the role of p38 MAPK activation in pore opening, these data suggest that defective coupling of the receptor to the enzyme could occur in hippocampal cultures. The different capabilities of the receptor to open membrane pores cause relevant functional consequences. Upon pore formation, caspase-1 is activated and pro-IL1-β is cleaved and released extracellularly. The receptor stimulation does not result in interleukin-1beta secretion from hippocampal astrocytes, although the pro-cytokine is present in the cytosol of lipopolysaccharide-primed cultures. These results open the possibility that activation of P2X₇ receptors differently influences the neuroinflammatory processes in distinct brain regions.     

2′, 3′-<Emphasis Type="Italic">O</Emphasis>-(4-benzoylbenzoyl)–ATP-mediated calcium signaling in rat glioma C6 cells: role of the P2Y<Subscript>2</Subscript> nucleotide receptor

In this study, we examined the response of glioma C6 cells to 2′,3′-O-(4-benzoylbenzoyl)-ATP (BzATP) and showed that the BzATP-induced calcium signaling does not involve the P2X₇ receptor activity. We show here that in the absence of extracellular Ca²⁺, BzATP-generated increase in [Ca²⁺]_i via Ca²⁺ release from intracellular stores. In the presence of calcium ions, BzATP established a biphasic Ca²⁺ response, in a manner typical for P2Y receptors. Brilliant Blue G, a selective antagonist of the rat P2X₇ receptor, did not reduce any of the two components of the Ca²⁺ response elicited by BzATP. Periodate-oxidized ATP blocked not only BzATP- but also UTP-induced Ca²⁺ elevation. Moreover, BzATP did not open large transmembrane pores. What is more, a cross-desensitization between UTP and BzATP occurred, which clearly shows that in glioma C6 cells BzATP activates most likely the P2Y₂ but not the P2X₇ receptors.