Metabotropic P2Y Receptors Research Articles

The Neuropharmacological Study for ATP Receptors Especially in Pain

A growing body of evidence indicates that extracellular nucleotides released or leaked from non-excitable cells as well as neurons play important roles in the regulation of neuronal and glial functions in the whole body through ATP receptors. ATP receptors (ionotropic P2X and metabotropic P2Y receptors) are the most abundant receptor families in living organisms. In the central nervous system, these receptors participate in synaptic transmission and in intercellular communications between neurons and glia. Glia cells are classified into astrocytes, oligodendrocytes and microglia. There are many reports on the role of ATP receptors (P2X4, P2X7, P2Y6 and P2Y12 receptors) expressed in spinal microglia. We have reported that several molecules presumably activate microglia in neuropathic pain after peripheral nerve injury. P2X4 receptors expressed in microglia in particular play a critical role in neuropathic pain signaling. The expression and activity of P2X4 receptors are up-regulated and enhanced predominantly in activated microglia in the spinal cord where damaged sensory fibers project. These findings provide novel targets for developing new medicines to treat neuropathic pain.

Selective activation of KCa3.1 and CRAC channels by P2Y2 receptors promotes Ca(2+) signaling, store refilling and migration of rat microglial cells.

Microglial activation involves Ca2+ signaling, and numerous receptors can evoke elevation of intracellular Ca2+. ATP released from damaged brain cells can activate ionotropic and metabotropic purinergic receptors, and act as a chemoattractant for microglia. Metabotropic P2Y receptors evoke a Ca2+ rise through release from intracellular Ca2+ stores and store-operated Ca2+ entry, and some have been implicated in microglial migration. This Ca2+ rise is expected to activate small-conductance Ca2+-dependent K+ (SK) channels, if present. We previously found that SK3 (KCa2.3) and KCa3.1 (SK4/IK1) are expressed in rat microglia and contribute to LPS-mediated activation and neurotoxicity. However, neither current has been studied by elevating Ca2+ during whole-cell recordings. We hypothesized that, rather than responding only to Ca2+, each channel type might be coupled to different receptor-mediated pathways. Here, our objective was to determine whether the channels are differentially activated by P2Y receptors, and, if so, whether they play differing roles. We used primary rat microglia and a rat microglial cell line (MLS-9) in which riluzole robustly activates both SK3 and KCa3.1 currents. Using electrophysiological, Ca2+ imaging and pharmacological approaches, we show selective functional coupling of KCa3.1 to UTP-mediated P2Y2 receptor activation. KCa3.1 current is activated by Ca2+ entry through Ca2+-release-activated Ca2+ (CRAC/Orai1) channels, and both CRAC/Orai1 and KCa3.1 channels facilitate refilling of Ca2+ stores. The Ca2+ dependence of KCa3.1 channel activation was skewed to abnormally high concentrations, and we present evidence for a close physical association of the two channel types. Finally, migration of primary rat microglia was stimulated by UTP and inhibited by blocking either KCa3.1 or CRAC/Orai1 channels. This is the first report of selective coupling of one type of SK channel to purinergic stimulation of microglia, transactivation of KCa3.1 channels by CRAC/Orai1, and coordinated roles for both channels in store refilling, Ca2+ signaling and microglial migration.

Intracellular ATP modulates vascular distinctive P2X receptors in rat arterial smooth muscle

The extracellular nucleotide sensitive P2 receptor comprises two different groups: ionotropic P2X and metabotropic P2Y receptors. It is generally considered that the P2X receptor lacks intracellular ATP binding sites.In rat arterial myocytes, however, we identified that P2X1 and 5 receptors form a heteromultimer and found that its activation kinetics is modulated by cytosolic ATP, since a significant decrease was observed in desensitization of P2X receptor current evoked by repeated application of α,β‐methylene ATP (10μM) at 5 minute intervals with increasing intracellular ATP concentration (0, 2 and 5mM to 52.4±6.0%, 45.7±5.4% and 33.5±3.0%, respectively: n=5–13). In contrast, ADP and AMP did not modulate the current. Similar effects were observed in rat cerebral and mesenteric artery myocytes.Therefore, we performed a homology search for ATP‐binding motifs in P2X receptors, and found P2X1 and 5 receptors have the motifs at the pore‐forming transmembrane regions. In addition, we confirmed the co‐localization of P2X1 and 5 receptor proteins in each arterial tissue by RT‐PCR, western blot and immunostaining analyses.These results suggest that P2X1 and 5 receptors may play important roles in metabolic regulation in conditions such as hypoxia.

Evidence for functional P2X receptors in a mouse cortical collecting duct cell line

P2 receptors are potent modulators of key solute and water transport mechanisms in the renal collecting duct (CD) [1]. Controversy exists over the P2 receptor subtype(s) responsible; most studies focus on metabotropic P2Y receptors and arguably ionotropic P2X receptors are largely overlooked.Using an M1 cell line derived from mouse CD and the excised outside‐out configuration of the patch‐clamp technique we have sought functional evidence for extracellular ATP‐gated P2X receptor ion channels. External bath solution contained 145 mM NaCl and the pipette solution contained 145 mM K+ (70 mM Cl−).In outside‐out patches, application of external ATP (100 μM) transiently activated flickery single channel currents (tact ~3s, tdes ~20s). At −100 mV, the mean inward single channel current amplitude (i), open probability (Po) and chord conductance of channels were 0.71±0.12 pA, 0.66±0.12, and 7.7±0.8 pS (n=5), respectively.We demonstrate that ATP directly activates receptors with singlechannel properties similar to those of P2X receptors. Consequently P2X receptors may regulate CD solute and water transport.Research supported by the Wellcome Trust, BBSRC and DiscoveryBiomed.

Control of P2X3 Channel Function by Metabotropic P2Y2 UTP Receptors in Primary Sensory Neurons

Purinergic signaling contributes significantly to pain mechanisms, and the nociceptor-specific P2X3 ATP receptor channel is considered a target in pain therapeutics. Recent findings suggesting the coexpression of metabotropic P2Y receptors with P2X3 implies that ATP release triggers the activation of both ionotropic and metabotropic purinoceptors, with strong potential for functional interaction. Modulation of native P2X3 function by P2Y receptor activation was investigated in rat dorsal root ganglia (DRG) neurons using whole cell patch-clamp recordings. Application of the selective P2Y receptor agonist UTP decreased peak amplitudes of α,β-meATP-evoked homomeric P2X3-mediated currents, but had no effect on heteromeric P2X2/3-mediated currents. Treatment with phospholipase C inhibitor U73122 significantly reversed P2X3 current inhibition induced by UTP-sensitive P2Y receptor activation. We previously reported the modulation of P2X receptors by phospholipids in DRG neurons and injection of exogenous phosphatidylinositol-4,5-bisphosphate (PIP(2)) fully reverses UTP-mediated regulation of P2X3 channel activity. Pharmacological as well as functional screening of P2Y receptor subtypes indicates the predominant involvement of P2Y2 receptor in P2X3 inhibition, and immunolocalization confirms a significant cellular coexpression of P2X3 and P2Y2 in rat DRG neurons. In summary, the function of P2X3 ATP receptor can be inhibited by P2Y2-mediated depletion of PIP(2). We propose that expression of P2Y2 purinoceptor in nociceptive sensory neurons provides an homeostatic mechanism to prevent excessive ATP signaling through P2X3 receptor channels.

Chronic Arsenic Exposure in Nanomolar Concentrations Compromises Wound Response and Intercellular Signaling in Airway Epithelial Cells

Paracrine ATP signaling in the lung epithelium participates in a variety of innate immune functions, including mucociliary clearance, bactericide production, and as an initiating signal in wound repair. We evaluated the effects of chronic low-dose arsenic relevant to U.S. drinking water standards (i.e., 10 ppb [130nM]) on airway epithelial cells. Immortalized human bronchial epithelial cells (16HBE14o-) were exposed to 0, 130, or 330nM arsenic (as Na-arsenite) for 4-5 weeks and examined for wound repair efficiency and ATP-mediated Ca(2+) signaling. We found that chronic arsenic exposure at these low doses slows wound repair and reduces ATP-mediated Ca(2+) signaling. We further show that arsenic compromises ATP-mediated Ca(2+) signaling by altering both Ca(2+) release from intracellular stores (via metabotropic P2Y receptors) and Ca(2+) influx mechanisms (via ionotropic P2X receptors). To better model the effects of arsenic on ATP-mediated Ca(2+) signaling under conditions of natural exposure, we cultured tracheal epithelial cells obtained from mice exposed to control or 50 ppb Na-arsenite supplemented drinking water for 4 weeks. Tracheal epithelial cells from arsenic-exposed mice displayed reduced ATP-mediated Ca(2+) signaling dynamics similar to our in vitro chronic exposure. Our findings demonstrate that chronic arsenic exposure at levels that are commonly found in drinking water (i.e., 10-50 ppb) alters cellular mechanisms critical to airway innate immunity.

Expression of P2X1 and P2X4 receptors in rat trigeminal ganglion neurons

Extracellular ATP, an essential pain mediator, is received by cell-surface ionotropic P2X and/or metabotropic P2Y receptors. Although the contribution of P2X₃ and/or P2X(2/3) receptors toward the pain mechanism is well described in trigeminal ganglion neurons, the expression of other subtypes of P2X receptor remains to be clarified. We examined expression of P2X receptor mRNA and measured intracellular free Ca²⁺ concentration ([Ca²⁺]i) by the activation of these receptors by fura-2 fluorescence in primary cultured rat trigeminal ganglion neurons. Real-time reverse transcription-PCR analysis revealed mRNA expression of P2X receptor subtype P2X₁, P2X₃, and P2X₄ in trigeminal ganglion neurons. In the presence of extracellular Ca²⁺, the application of P2X receptors agonists, ATP, α,β-methylene ATP or β,γ-methylene ATP induced Ca²⁺ influx significantly. The ATP-induced increase in [Ca²⁺]i was inhibited by a series of selective antagonists for P2X₁, P2X₃, or P2X₄ receptors. These results indicate that trigeminal ganglion neurons functionally express P2X₁, P2X₃, and P2X₄ receptors and that these receptors are involved in the mediation of not only nociceptive but also neuropathic pain in the orofacial area.

Multiple P2Y subtypes in spinal microglia are involved in neuropathic pain after peripheral nerve injury

A prominent signaling pathway in the development of neuropathic pain involves ATP acting on microglial purinergic receptors. Among the P2Y metabotropic receptors, we reported before that the P2Y12 receptor is upregulated in microglia following nerve injury and involved in the phosphorylation of p38 MAPK, and in the development of pain behavior. In this study, we examined the expression of P2Y6, P2Y13, and P2Y14 receptors in the spinal cord and whether these receptors are involved in the pathogenesis of neuropathic pain following peripheral nerve injury. We found that spared nerve injury induced a dramatic increase of not only P2Y12, but also P2Y6, 13, and 14 receptor mRNA expression in spinal microglia. The increase continued for at least 2 weeks after injury. To determine whether p38 MAPK can induce the expression of P2Y receptors, we administered intrathecally the p38 MAPK inhibitor SB203580 and found that it significantly suppressed P2Y6, P2Y13, and P2Y14 but not P2Y12 mRNAs. Intrathecal injection of the specific P2Y6 antagonist MRS2578, specific P2Y13 antagonist MRS2211 or P2Y14 antisense LNA, attenuated mechanical pain hypersensitivity. The mixture of three antagonists for P2Y6, 12, and 13 showed a longer suppressive effect on pain behavior than the individual treatments. Our data demonstrate that ATP and other nucleotides may stimulate activated microglia with the upregulation of P2Y6, P2Y12, P2Y13, and P2Y14 receptors following nerve injury and these receptors are involved in the development of neuropathic pain.

High Throughput Functional Assays for P2X Receptors

Adenosine triphosphate (ATP) activates two receptor superfamilies, metabotropic P2Y and ionotropic P2X receptors. The P2X receptors are nonselective cation channels that are widely expressed on excitable cells including neurons, glia, and smooth muscle cells. The protocols in this unit are useful for evaluating ligands that interact with P2X receptors on native cells or that are cloned and expressed in recombinant heterologous cell systems. Calcium imaging methods are described for the pharmacological characterization of fast or slowly desensitizing P2X receptors. While these methods are readily applicable to a wide variety of ligand-gated ion channels, the protocols provided herein detail how they can be used to measure activation of homomeric P2X3 (fast desensitizing) and heteromeric P2X2/3 (slowly desensitizing) receptors. Appropriate agonists and/or calcium dyes can be substituted to assess activity at other P2X receptor subtypes. An additional protocol is provided for measuring P2X7 receptor-mediated pore formation in THP-1, a native human acute monocytic leukemia cell line that can be used to study homomeric P2X7 (non-desensitizing) receptors that are expressed on macrophages and microglial cells.

P2X4R and P2X7R in neuropathic pain

AbstractNeuropathic pain is often a consequence of nerve injury or of diseases such as diabetes, infection, autoimmune disease, or cancer. Neuropathic pain can be agonizing, can persist over long periods, and, unfortunately, is often resistant to known painkillers. There is a rapidly growing body of evidence indicating that signaling by extracellular nucleotides through purinergic P2 receptors [ionotropic P2X receptors (P2XRs) and metabotropic P2Y receptors (P2YRs)] play crucial roles in neuropathic pain. Following peripheral nerve injury, the expression of purinergic receptors (e.g., P2X4R) is markedly upregulated specifically in microglia, a type of glial cells known as resident macrophages in the central nervous system. Activation of P2X4R and P2X7R in microglia causes release of diffusible factors (such as brain‐derived neurotrophic factor, interleukin‐1β, interleukin‐6, and tumor necrosis factor‐α) involved in nerve‐injury‐induced pain behaviors and hyperexcitability of dorsal horn neurons. Suppression of the function or expression of these purinergic receptors strongly suppresses neuropathic pain. Recent advances further our understanding of mechanisms by which microglial purinergic receptors contribute to the pathogenesis of neuropathic pain. WIREs Membr Transp Signal 2012, 1:513–521. doi: 10.1002/wmts.47For further resources related to this article, please visit the WIREs website.

Ectonucleotidases in Cancer and Inflammation

In 1970, Geoffrey Burnstock described the release of extracellular adenosine triphosphate (ATP) as a transmitter substance by nonadrenergic inhibitory nerves, and later in 1972, he formulated the hypothesis of purinergic neurotransmission [1]. Together with Che Su and John Bevan, Geoffrey Burnstock built the foundation to what was to become an entirely new field of research in biology [2]. While early studies focused on the role of purinergic receptors in neurotransmission, it soon became obvious that extracellular ATP and its hydrolyzed derivative adenosine had important roles in immune regulation. It is now recognized that purinergic signaling not only regulates neurotransmission and inflammation, but also influences diverse biological pathways, such as cell survival, proliferation, differentiation, lipid synthesis, and cell motility. In 1978, Burnstock proposed two types of purinergic receptors: P1 receptors selective for adenosine and P2 receptors selective for ATP and ADP. In 1985, a pharmacological approach was proposed to distinguish between two types of P2 receptors: ionotropic P2X and metabotropic P2Y receptors [3]. Nucleotides, such as ATP, are released by a variety of cell types especially under stress conditions. Cancer cells, for instance, are known to release ATP in the tumor microenvironment. Nucleotides that are released in response to a stress signal can be hydrolyzed by membrane-bound enzymes called ectonucleotidases. By regulating the levels of extracellular nucleotides and nucleosides, ectonucleotidases are thus involved in numerous physiological and pathological conditions. In this special issue, P. Chernogorova and R. Zeiser discuss the role of ectonucleotidases, in particular CD39 (NTPDase1) and CD73 (ecto-5′-nucleotidase) on allograft rejection, acute graft-versus-host disease (GvHD), and graft-versus-leukemia (GvL) effect. Interestingly, recent evidence suggests that purinergic signaling influences the severity of alloimmune responses. As highlighted by P. Chernogorova and R. Zeiser, these studies suggest potential clinical use of recombinant ectonucleotidases or adenosine receptor agonists for regulation of alloimmune responses which can be tailored according to the clinical situation. F. Ghiringhelli et al. review the current literature on the role of ectonucleotidases in tumor immune escape, with a focus on the immune regulatory function of Th17 cells. Th17 cells have emerged as key participants in a wide range of immune disorders and cancers. While the role of Th17 cells in cancer immunity remains controversial, F. Ghiringhelli et al. describe their recent seminal observation that ectonucleotidases are expressed on Th17 cells and that this is relevant in the context of tumor growth. A. Battastini and colleagues present an overview of the various roles of purinergic signaling in gliomas. In gliomas, the presence of an inflammatory infiltrate is directly correlated with tumor malignancy, and this appears to be regulated in part by purinergic signaling. The authors discuss the observation that ATP triggers glioma cells to release proinflammatory factors important for the recruitment of monocytes and neutrophils, thereby favoring tumor growth. The authors discuss their own work that aims to establish whether ectonucleotidases are involved in macrophage polarization in gliomas. A. Czopik and colleagues present a research article describing the protective role of CD73 in a model of colitis induced by administration of dextran sulfate sodium salt (DSS). The authors demonstrate that compared to wild-type mice, CD73-deficient gene-targeted mice are highly susceptible to DSS-induced colitis. The authors conclude that CD73 expression in the colon is critical for regulating the magnitude and the resolution of colonic immune responses. K. M. Dwyer and colleagues discuss the role of the CD39-adenosinergic axis in the pathogenesis of type 1 and type 2 diabetes. They discuss recent work suggesting that CD39 is involved in the pathophysiology of pancreatic dysfunction. This has importance clinical consequences, as drug development targeting different components of the pathway may be of relevance in the treatment of both type 1 and type 2 diabetes. The authors also discuss unanswered questions, such as what is the source of ecto-5′-nucleotidase activity given the lack of CD73 expression within the pancreas. J. Stagg and colleagues describe the role of CD73 and extracellular adenosine signaling in promoting tumor growth through paracrine and autocrine action. The authors discuss their recent work on the role of CD73 in spontaneous tumor growth and on the role of CD73 and adenosine on endothelial and immune cells. In conclusion, this special issue on ectonucleotidases aims to acquaint investigators not familiar with this field with important recent advances with the hope of attracting scientists to join the effort of exploiting modulation of purinergic signaling for therapeutic purposes to treat cancer and inflammatory diseases. John Stagg Linda F. Thompson Karen M. Dwyer

Coupling of P2Y Mediated Store Depletion and Ca2+ Entry (SOCE) to KCa3.1 (KCnn4/SK4/Ik) Channel Activation in Microglia

We recently published that SK3/KCa2.3 and SK4/KCa3.1 channels contribute to microglial activation and neurotoxicity via regulation of p38 MAPK activation; and were intrigued that two SK channels are involved in the same outcomes. Cloned SK channels are activated by similar rises in Ca2+; thus, we asked whether they are functionally coupled to different Ca2+ sources. Damaged neurons release ATP, a purinergic receptor ligand that activates microglia by raising intracellular Ca2+. Purinergic responses are mediated by ionotropic P2X or metabotropic P2Y receptors. Here, we stimulated MLS-9 cells (a microglia cell line that expresses both SK3 and SK4) with UTP to trigger P2Y-mediated responses; i.e., depletion of intracellular Ca2+ stores, and subsequent store operated Ca2+ entry (SOCE). In Fura-2 loaded cells, UTP evoked a rapid Ca2+ transient, followed by a second, more sustained rise due to SOCE. The second phase was decreased by the inhibitor 2-APB, which we recently showed blocks Ca2+ release-activated Ca2+ (CRAC) channels in microglia. In perforated-patch recordings, UTP activated a robust current, which was blocked by the SK4 inhibitor, TRAM-34 (but not by the SK3 blocker, apamin), and was therefore, SK4/KCa3.1. SK4 activation by UTP was also inhibited by 2-APB, suggesting that SOCE is required for, and coupled to, SK4 channel activation. SK4 activity was apparently needed for Ca2+ entry and store re-filling; the SK4 blocker, TRAM-34, accelerated the decay in internal Ca2+ after UTP, and reduced the amplitude of a second Ca2+ release from stores evoked by the SERCA pump inhibitor, thapsigargin. Our data support selective coupling of SK4 channels (but not SK3) to Ca2+ entry through SOCE/CRAC. Our current model is that SK4 activation maintains a hyperpolarized membrane potential that helps drive Ca2+ entry through CRAC, facilitating store refilling.

UTP affects the Schwannoma cell line proteome through P2Y receptors leading to cytoskeletal reorganisation

Glial cells in the peripheral nervous system, such as Schwann cells, respond to nucleotides, which play an important role in axonal regeneration and myelination. Metabotropic P2Y receptor agonists are promising therapeutic molecules for peripheral neuropathies. Nevertheless, the proteomic mechanisms involved in nucleotide action on Schwann cells remain unknown. Here, we studied intracellular protein changes in RT4-D6P2T Schwann cells after treatment with nucleotides and Nucleo CMP Forte (CMPF), a nucleotide-based drug. After treatment with CMPF, 2-D DIGE revealed 11 differential gel spots, which were all upregulated. Among these, six different proteins were identified by MS. Some of these proteins are involved in actin remodelling (actin-related protein, Arp3), membrane vesicle transport (Rab GDP dissociation inhibitor β, Rab GDI), and the endoplasmic reticulum stress response (protein disulfide isomerase A3, PDI), which are hallmarks of a possible P2Y receptor signalling pathway. Expression of P2Y receptors in RT4-D6P2T cells was demonstrated by RT-PCR and a transient elevation of intracellular calcium measured in response to UTP. Actin reorganisation was visualized after UTP treatment using phalloidin-FITC staining and was blocked by the P2Y antagonist suramin, which also inhibited Arp3, Rab GDI, and PDI protein upregulation. Our data indicate that extracellular UTP interacts with Schwann P2Y receptors and activates molecular machinery that induces changes in the glial cell cytoskeleton.

Purinergic signalling mobilizes mitochondrial Ca2+ in mouse Sertoli cells

Intimate bidirectional communication between Sertoli cells and developing germ cells ensures the integrity and efficiency of spermatogenesis. Yet, a conceptual mechanistic understanding of the physiological principles that underlie Sertoli cell autocrine and paracrine signalling is lacking. Here, we characterize a purinergic Ca(2+) signalling network in immature mouse Sertoli cells that consists of both P2X2 and P2Y2 purinoceptor subtypes, the endoplasmic reticulum and, notably, mitochondria. By combining a transgenic mouse model with a dedicated bioluminescence imaging device, we describe a novel method to monitor mitochondrial Ca(2+) mobilization in Sertoli cells at subcellular spatial and millisecond temporal resolution. Our data identify mitochondria as essential components of the Sertoli cell signalling 'toolkit' that control the shape of purinergic Ca(2+) responses, and probably several other paracrine Ca(2+)-dependent signals.

P2X Receptors and Their Roles in Astroglia in the Central and Peripheral Nervous System

Astrocytes are a class of neural cells that control homeostasis at all levels of the central and peripheral nervous system. There is a bidirectional neuron–glia interaction via a number of extracellular signaling molecules, glutamate and ATP being the most widespread. ATP activates ionotropic P2X and metabotropic P2Y receptors, which operate in both neurons and astrocytes. Morphological, biochemical, and functional evidence indicates the expression of astroglial P2X1/5 heteromeric and P2X7 homomeric receptors, which mediate physiological and pathophysiological responses. Activation of P2X1/5 receptors triggers rapid increase of intracellular Na+ that initiates immediate cellular reactions, such as the depression of the glutamate transporter to keep high glutamate concentrations in the synaptic cleft, the activation of the local lactate shuttle to supply energy substrate to pre- and postsynaptic neuronal structures, and the reversal of the Na+/Ca2+ exchange resulting in additional Ca2+ entry. The consequences of P2X7 receptor activation are mostly but not exclusively mediated by the entry of Ca2+ and result in reorganization of the cytoskeleton, inflammation, apoptosis/necrosis, and proliferation, usually at a prolonged time scale. Thus, astroglia detect by P2X1/5 and P2X7 receptors both physiological concentrations of ATP secreted from presynaptic nerve terminals and also much higher concentrations of ATP attained under pathological conditions.

Purinergic signaling in the cerebellum: Bergmann glial cells express functional ionotropic P2X7 receptors

Astrocytes constitute active networks of intercommunicating cells that support the metabolism and the development of neurons and affect synaptic functions via multiple pathways. ATP is one of the major neurotransmitters mediating signaling between neurons and astrocytes. Potentially acting through both purinergic metabotropic P2Y receptors (P2YRs) and ionotropic P2X receptors (P2XRs), up until now ATP has only been shown to activate P2YRs in Bergmann cells, the radial glia of the cerebellar cortex that envelopes Purkinje cell afferent synapses. In this study, using multiple experimental approaches in acute cerebellar slices we demonstrate the existence of functional P2XRs on Bergmann cells. In particular, we show here that Bergmann cells express uniquely P2X7R subtypes: (i) immunohistochemical analysis revealed the presence of P2X7Rs on Bergmann cell processes, (ii) in whole cell recordings P2XR pharmacological agonists induced depolarizing currents that were blocked by specific antagonists of P2X7Rs, and could not be elicited in slices from P2X₇R-deficient mice and finally, (iii) calcium imaging experiments revealed two distinct calcium signals triggered by application of exogenous ATP: a transient signal deriving from release of calcium from intracellular stores, and a persistent one following activation of P2X7Rs. Our data thus reveal a new pathway by which extracellular ATP may affect glial cell function, thus broadening our knowledge on purinergic signaling in the cerebellum.

Purine P2Y receptors in ATP-mediated regulation of non-quantal acetylcholine release from motor nerve endings of rat diaphragm

We established the effect of ATP, which is released together with acetylcholine (ACh), on the non-quantal ACh release (NQR) in rat diaphragm endplates and checked what kind of purine receptors are involved. NQR was estimated by the amplitude of endplate hyperpolarization (the H-effect) following the blockade of postsynaptic nicotinic receptors and cholinesterase. 100 μM ATP reduced the H-effect to 66% of the control. The action of ATP remained unchanged after the inhibition of ionotropic P2X receptors by Evans blue and PPADS, but disappeared after the application of the broad spectrum P2 receptor antagonist suramin, metabotropic P2Y receptor blocker reactive blue 2 and U73122, an inhibitor of phospholipase C. P2Y-mediated regulation is not coupled to presynaptic voltage-dependent Ca 2+ channels. During the simultaneous application of ATP and glutamate (which is another ACh cotransmitter reducing non-quantal release), the additive depressant effect led to a disappearance of the H-effect. This can be explained by the independence of the action of ATP and glutamate. Unlike the effects of purines on the spontaneous quantal secretion of ACh, its non-quantal release is regulated via P2Y receptors coupled to G q/11 and PLC. ATP thus regulates the neuromuscular synapse by two different pathways.

Distribution of ecto-nucleotidases in mouse sensory circuits suggests roles for nucleoside triphosphate diphosphohydrolase-3 in nociception and mechanoreception

Nucleotide-activated P2X channels and P2Y metabotropic receptors participate in nociceptive signaling. Agonist availability is regulated by nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), -2, -3, and -8, a family of enzymes that hydrolyze extracellular ATP to generate ADP (a P2Y agonist) and AMP. They provide a major source of extracellular AMP, the substrate for adenosine production by ecto-5'-nucleotidase (NT5E), and thereby regulate adenosine (P1) receptor signaling. NTPDases vary in their efficiency of tri- and diphosphate hydrolysis; therefore, which family members are expressed impacts nucleotide availability and half-life. This study employed enzyme activity histochemistry to examine the distribution of ATPase activity and immunohistochemistry for NTPDase1, 2, 3, and 8 in dorsal root ganglion (DRG) and spinal cord. Nucleotidase activity was robust in spinal dorsal horn, confirming that nociceptive pathways are a major site of nucleotide transmission. In DRG, extensive staining revealed ATPase activity in a subset of neurons and in non-neuronal cells. mRNA for NTPDase1-3, but not NTPDase8, was detected in lumbar DRG and spinal cord. Immunoreactivity for NTPDase3 closely matched the distribution of ATPase activity, labeling DRG central projections in the dorsal root and superficial dorsal horn, as well as intrinsic spinal neurons concentrated in lamina II. In DRG, NTPDase3 co-localized with markers of nociceptors and with NT5E. In addition, labeling of a subset of larger-diameter neurons in DRG was consistent with intense staining of Meissner corpuscle afferents in glabrous skin. Merkel cells and terminal Schwann cells of hair follicle afferents were also labeled, but the axons themselves were negative. We propose that NTPDase3 is a key regulator of nociceptive signaling that also makes an unexpected contribution to innocuous tactile sensation.

Regulation of renal NaCl and water transport by the ATP/UTP/P2Y2receptor system

Extracellular nucleotides (e.g., ATP) activate ionotropic P2X and metabotropic P2Y receptors in the plasma membrane to regulate and maintain cell function and integrity. This includes the renal tubular and collecting duct system, where the locally released nucleotides act in a paracrine and autocrine way to regulate transport of electrolytes and water and maintain cell volume. A prominent role has been assigned to Gq-coupled P2Y(2) receptors, which are typically activated by both ATP and UTP. Studies in gene knockout mice revealed an antihypertensive activity of P2Y(2) receptors that is linked to vasodilation and an inhibitory influence on renal salt reabsorption. Flow induces apical ATP release in the thick ascending limb, and first evidence indicates an inhibitory influence of P2Y(2) receptor tone on the expression and activity of the Na-K-2Cl cotransporter NKCC2 in this segment. The apical ATP/UTP/P2Y(2) receptor system in the connecting tubule/cortical collecting duct mediates the inhibitory effect of dietary salt on the open probability of the epithelial sodium channel ENaC and inhibits ENaC activity during aldosterone escape. Connexin 30 has been implicated in the luminal release of the ATP involved in the regulation of ENaC. An increase in collecting duct cell volume in response to manipulating water homeostasis increases ATP release. The subsequent activation of P2Y(2) receptors inhibits vasopressin-induced cAMP formation and water reabsorption, which facilitates water excretion and stabilizes cell volume. Thus recent studies have established the ATP/UTP/P2Y(2) receptor system as a relevant regulator of renal salt and water homeostasis and blood pressure regulation. The pathophysiological relevance and therapeutic potential remains to be determined, but dual effects of P2Y(2) receptor activation on both the vasculature and renal salt reabsorption implicate these receptors as potential therapeutic targets in hypertension.

Purinergic signaling involved in Müller cell function in the mammalian retina

Purines (in particular, ATP and adenosine) act as neuro- and gliotransmitters in the sensory retina where they are involved in bidirectional neuron-glia signaling. This review summarizes the present knowledge about the expression and functional importance of P1 (adenosine) and P2 (nucleotide) receptors in Müller glial cells of the mammalian retina. Mammalian Müller cells express various subtypes of adenosine receptors and metabotropic P2Y receptors. Human Müller cells also express ionotropic P2X(7) receptors. Müller cells release ATP upon activation of metabotropic glutamate receptors and/or osmotic membrane stretching. The osmotic mechanism is abrogated under conditions associated with ischemia-hypoxia and inflammation, resulting in swelling of the Müller cells when the extracellular milieu is hypoosmotic. However, exogenous glutamate, which induces the release of ATP and adenosine, and thus activates P2Y(1) and A(1) adenosine receptors, respectively, prevents such osmotic swelling under pathological conditions, suggesting unimpaired receptor-induced release of ATP. In addition to the inhibition of swelling, which is implicated in regulating the volume of the extracellular space, purinergic signaling is involved in mediating neurovascular coupling. Furthermore, purinergic signals stimulate the proliferation of retinal precursor cells and Müller cells. In normal retinal information processing, Müller cells regulate the synaptic activity by the release of ATP and adenosine. In retinopathies, abrogation of the osmotic release of ATP, and the upregulation of ecto-apyrase (NTPDase1), may have neuroprotective effects by preventing the overactivation of neuronal P2X receptors that are implicated in apoptotic cell death. Pharmacological modulation of purinergic receptors of Müller cells may have clinical importance, e.g., for the clearance of retinal edema and for the inhibition of dysregulated cell proliferation in proliferative retinopathies.