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The known seven mammalian receptor subunits (P2X1-7) form cationic channels gated by ATP. Three subunits compose a receptor channel. Each subunit is a polypeptide consisting of two transmembrane regions (TM1 and TM2), intracellular N- and C-termini, and a bulky extracellular loop. Crystallization allowed the identification of the 3D structure and gating cycle of P2X receptors. The agonist-binding pocket is located at the intersection of two neighbouring subunits. In addition to the mammalian P2X receptors, their primitive ligand-gated counterparts with little structural similarity have also been cloned. Selective agonists for P2X receptor subtypes are not available, but medicinal chemistry supplied a range of subtype-selective antagonists, as well as positive and negative allosteric modulators. Knockout mice and selective antagonists helped to identify pathological functions due to defective P2X receptors, such as male infertility (P2X1), hearing loss (P2X2), pain/cough (P2X3), neuropathic pain (P2X4), inflammatory bone loss (P2X5), and faulty immune reactions (P2X7). [ABSTRACT FROM AUTHOR]

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Background and Purpose: Presynaptic CB₁ cannabinoid receptors can be activated by endogenous cannabinoids (endocannabinoids) synthesized by postsynaptic neurones. The hypothesis of the present work was that activation of calcium-permeable transmitter-gated ion channels in postsynaptic neurones, specifically of P2X purine receptors, can lead to endocannabinoid production and retrograde synaptic signalling.Experimental Approach: GABAergic inhibitory postsynaptic currents (IPSCs) were recorded with patch-clamp techniques in Purkinje cells in mouse cerebellar slices. Purine receptors on Purkinje cells were activated by pressure ejection of ATP from a pipette.Key Results: ATP evoked an inward current in Purkinje cells, most likely due to P2X receptor activation. The ATP-evoked currents were accompanied by currents via voltage-gated calcium channels. ATP suppressed electrical stimulation-evoked IPSCs and miniature IPSCs (mIPSCs) recorded in the presence of tetrodotoxin, and these effects were prevented by the CB₁ antagonist rimonabant and the calcium chelator BAPTA (applied into the Purkinje cell). ATP also suppressed mIPSCs when voltage-gated calcium channels were blocked by cadmium, and intracellular calcium stores were depleted by thapsigargin. However, ATP failed to suppress mIPSCs when the extracellular calcium concentration was zero.Conclusions and Implications: ATP elicits CB₁ receptor-dependent retrograde synaptic suppression, which is probably mediated by an endocannabinod released by the postsynaptic neurone. An increase in intracellular calcium concentration in the postsynaptic neurone is necessary for this retrograde signalling. We propose that ATP increases the calcium concentration by two mechanisms: calcium enters into the neurone via the P2X receptor ion channel and the ATP-evoked depolarization triggers voltage-gated calcium channels. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2010) 112, 1261–1272. Intense neuronal activity in the sensory retina is associated with a volume increase of neuronal cells (Uckermann et al., J. Neurosci. 2004, 24:10149) and a decrease in the osmolarity of the extracellular space fluid (Dmitriev et al., Vis. Neurosci. 1999, 16:1157). Here, we show the existence of an endogenous purinergic mechanism that prevents hypoosmotic swelling of retinal glial (Müller) cells in mice. In contrast to the cells from wild-type mice, hypoosmotic stress induced rapid swelling of glial cell somata in retinal slices from mice deficient in P2Y1, adenosine A1 receptors, or ecto-5′-nucleotidase (CD73). Consistently, glial cell bodies in retinal slices from wild-type mice displayed osmotic swelling when P2Y1 or A1 receptors, or CD73, were pharmacologically blocked. Exogenous ATP, UTP, and UDP inhibited glial swelling in retinal slices, while the swelling of isolated glial cells was prevented by ATP but not by UTP or UDP, suggesting that uracil nucleotides indirectly regulate the glial cell volume via activation of neuronal P2Y4/6 and neuron-to-glia signaling. It is suggested that autocrine/paracrine activation of purinergic receptors and enzymes is crucially involved in the regulation of the glial cell volume. [ABSTRACT FROM AUTHOR]

The human (h) P2X3 receptor and its mutants deficient in one out of four N-glycosylation sites were expressed in HEK293 cells. Concentration–response curves were generated by whole-cell recordings of α,β-methylene ATP (α,β-meATP)-induced currents. A gradual change of external pH from the alkaline 8.0 to the acidic 5.0 successively decreased the maximum current amplitude ( Emax) without affecting the EC50 value. The replacement of Asn-139 and -170 by Asp (N139D, N170D) abolished the pH sensitivity of the wild-type (WT) hP2X3 receptor. In the case of N194D, the Emax was again the highest at the alkaline pH value with no change from 7.4 to 6.5, whereas in the case of N290D, there was an inverse pH sensitivity, with an increase of Emax in the acidic range. However, this effect appeared to be due to enhanced protonation by the insertion of Asp into the receptor, because replacement of Asn by the neutral Thr resulted in a comparable potency of α,β-meATP at any of the pH values investigated. In accordance with the reported finding that His-206 is involved in the modulation of WT P2X3 receptors by protons, we showed that the normal change of Emax by an acidic, but not alkaline pH was abolished after substitution of this His by Ala. However, the double mutant H206A + N290D did not react to acidification or alkalinization with any change in Emax. In conclusion, only fully N-glycosylated P2X3 receptors recognize external pH with a modified sensitivity towards α,β-meATP. [ABSTRACT FROM AUTHOR]

Mixed striatal cell cultures containing neurons and glial cells were grown either in neurobasal medium (NBM) or Dulbecco's modified Eagle's medium (DMEM). Whole-cell patch-clamp recordings indicated that, if at all, only a single, low amplitude spike was evoked shortly after starting the injection of a depolarizing current pulse into NBM neurons. In contrast, DMEM neurons fired series of high amplitude action potentials, without apparent spike frequency adaptation. The possible reason for the observed action potential failure in NBM neurons was a low density of Na+ channels per unit of membrane surface area. However, both in NBM and DMEM neurons, ATP did not induce inward current responses via P2X receptor-channels, although GABAA and N-methyl-D-aspartate (NMDA) receptor-channels could be activated by muscimol and NMDA, respectively. Ca2+ imaging experiments by means of the Fura-2 method were utilized to measure intracellular Ca2+ ([Ca2+]i) in neurons and glial cells. NBM, but not DMEM neurons responded to ATP with [Ca2+]i transients; glial cells grown in either culture medium were equally sensitive to ATP. ATP caused an increase of [Ca2+]i by a mechanism only partly dependent on external Ca2+; the residual ATP effect was blocked by cyclopiazonic acid (CPA) and was therefore due to the release of Ca2+ from its intracellular pools. The receptor involved was characterized by P2 receptor antagonists (PPADS, MRS 2179, AR-C69931MX) and was found to belong to the P2Y1 subtype. CPA caused an early [Ca2+]i response due to release from intracellular storage sites, followed by a late [Ca2+]i response due to the influx of this cation from the extracellular space, probably triggered by the opening of store-operated channels (SOCs) in the plasma membrane. It is concluded that in partial analogy with the effect of CPA, ATP releases [Ca2+]i via the Gq/phospholipase C/inositoltrisphosphate (IP3) pathway, thereby opening SOCs. It is hypothesized that this effect of ATP may have an important role for the proliferation and migration of striatal neuronal progenitors. J. Cell. Physiol. 209: 81–93, 2006. © 2006 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The present study was aimed to clarify the role of purinergic signalling in the regulation of ingestion behaviour. The ATP/ADP analogues 2-methylthioATP (2-MeSATP) and adenosine 5′-O-(2-thiodiphosphate) (ADPβS) increased the food intake after intracerebroventricular infusion in 18-h food-deprived rats. This effect was abolished by pretreatment with the non-selective P2X/P2Y receptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) or the selective P2Y1 receptor antagonist MRS 2179, respectively. The stimulation of food intake mediated by ADPβS was also blocked by pretreatment with the nitric oxide synthase (NOS) inhibitor Nw-nitro-l-arginine methylester (L-NAME), as well as with the inhibitor of the soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), suggesting that the orexigenic effect seems to be closely related with the ensuing formation of nitric oxide. The immunohistochemical staining indicating a co-localization of P2Y1 receptor- and nNOS-immunoreactivities in a population of neurons in the ventromedial hypothalamic nucleus (VMH) agrees with this assumption. Further experiments with the direct local application of these compounds into the VMH and lateral hypothalamic nucleus (LH) show that the stimulation of P2Y1 receptors in these functionally antagonistic brain regions exerts an increased food intake. Hence, different signal transduction mechanisms may operate in the VMH and LH. Our assumption is supported by distinct effects of the NOS inhibitor L-NAME in these two hypothalamic nuclei. The present data suggest that ATP/ADP, acting as extracellular signal molecules in the rat brain, are involved in the regulation of food intake, possibly depending on P2Y1-receptor-mediated nitric oxide production. [ABSTRACT FROM AUTHOR]

Corpora amylacea (CA) have an affinity to nucleic acids as shown by in situ hybridization experiments. However, little is known about the specificity of this interaction, as well as the mechanism involved. We investigated the ability of different probes of digoxigenin-labeled oligonucleotides corresponding to some specific neuronal receptors, both sense and antisense, to bind to CA from human autopsy brain tissue. The bound nucleotides were detected with antidigoxigenin antibody and the signal was further amplified using the tyramide signal amplification system. The affinity of binding varies with the sequence of nucleotides. The most intense signal is produced by the adenosine-2A receptor antisense probe and the least intense signal is produced by the N-methyl-D-aspartate receptor sense probe. The affinity of binding for the same probe does not depend on the localization of CA in the central nervous system. Complete staining loss by proteinase K pretreatment in higher concentrations shows that the binding motif is partially proteinaceous. The circumferential but not the punctate internal staining is diminished by mild amylglucosidase pretreatment, suggesting a process of progressive apposition and condensation. [ABSTRACT FROM AUTHOR]

Abstract: Extracellular purines act as neuromodulators on transmitter release and may exert toxic effects at higher concentrations. In microdialysis studies, endogenous ATP facilitated the extracellular concentration of glutamate in the nucleus accumbens (NAc) of rats. Additionally, P2 receptors are involved in astrogliosis in vivo after a stab wound injury in the same region, suggesting that these receptors, preferentially the metabotropic P2Y1 receptor subtype, mediate also trophic responses. Two sets of experimental findings support the involvement of purinergic and glutamatergic mechanisms in the response of brain to mechanical damage. First, in the present studies, the initial time course of extracellular ATP and glutamate was analyzed after a mechanical injury. The concentration of ATP in microdialysates was elevated only in the first 15-min sample whereas glutamate returned to a basal concentration not before a 90-min period had elapsed. We suggest, that the acute injury-evoked stimulation of P2 receptors contributes to glutamate-mediated excitotoxicity. Second, the expression of P2Y1 receptors and their possible relation to glutamatergic structures, identified by neuronal vesicular glutamate transporters (VGLUTs), were elucidated in non-treated and mechanically injured animals after 4 days. The number of P2Y1-positive cells was significantly increased after injury. Furthermore, P2Y1 receptor-labeled cells do not exhibit immunoreactivity for VGLUT1 and VGLUT2 without and after injury. However, after injury, a co-expression of the P2Y1 receptor on VGLUT3-immunopositive cells in the NAc was observed. No VGLUT1-, 2- and 3-immunoreactivity was found on P2Y1-positive glial fibrillary acidic protein-immunopositive astrocytes at both conditions. Our data suggest that the expression of P2Y1 receptors at neurons and astrocytes is modulated in response to cerebral injury. It can be assumed, that the enhanced sensitivity of neurons to purinergic signaling may be related directly or indirectly to changes of the glutamatergic transmission. [Copyright &y& Elsevier]

Neuronally enriched primary cerebrocortical cultures were exposed to glucose-free medium saturated with argon ( in vitro ischemia) instead of oxygen (normoxia). Ischemia did not alter P2X7 receptor mRNA, although serum deprivation clearly increased it. Accordingly, P2X7 receptor immunoreactivity (IR) of microtubuline-associated protein 2 (MAP2)-IR neurons or of glial fibrillary acidic protein (GFAP)-IR astrocytes was not affected; serum deprivation augmented the P2X7 receptor IR only in the astrocytic, but not the neuronal cell population. However, ischemia markedly increased the ATP- and 2′-3′- O-(4-benzoylbenzoyl)-adenosine 5′-triphosphate (BzATP)-induced release of previously incorporated [3H]GABA. Both Brilliant Blue G and oxidized ATP inhibited the release of [3H]GABA caused by ATP application; the Brilliant Blue G-sensitive, P2X7 receptor-mediated fraction, was much larger after ischemia than after normoxia. Whereas ischemic stimulation failed to alter the amplitude of ATP- and BzATP-induced small inward currents recorded from a subset of non-pyramidal neurons, BzATP caused a more pronounced increase in the frequency of miniature inhibitory postsynaptic currents (mIPSCs) after ischemia than after normoxia. Brilliant Blue G almost abolished the effect of BzATP in normoxic neurons. Since neither the amplitude of mIPSCs nor that of the muscimol-induced inward currents was affected by BzATP, it is assumed that BzATP acts at presynaptic P2X7 receptors. Finally, P2X7 receptors did not enhance the intracellular free Ca2+ concentration either in proximal dendrites or in astrocytes, irrespective of the normoxic or ischemic pre-incubation conditions. Hence, facilitatory P2X7 receptors may be situated at the axon terminals of GABAergic non-pyramidal neurons. When compared with normoxia, ischemia appears to markedly increase P2X7 receptor-mediated GABA release, which may limit the severity of the ischemic damage. At the same time we did not find an accompanying enhancement of P2X7 mRNA or protein expression, suggesting that receptors may become hypersensitive because of an increased efficiency of their transduction pathways. [ABSTRACT FROM AUTHOR]

1 Vasoactive intestinal peptide (VIP) is present in the hippocampus in three subtypes of GABAergic interneurones, two of which innervate preferentially other interneurones, responsible for pyramidal cell inhibition. We investigated how pre- and postsynaptic modulation of GABAergic transmission (to both pyramidal cells and interneurones) by VIP could influence excitatory synaptic transmission in the CA1 area of the hippocampus. 2 VIP (0.1-100 nM) increased [³H]GABA release from hippocampal synaptosomes (maximum effect at 1 nM VIP; 6.3.8 ± 4.0%) but did not change [³H]glulamate release. 3 VIP (0.3-30nM) enhanced synaptic transmission in hippocampal slices (maximum effect at 1 nM VIP; field excitatory postsynaptic potentials (epsp) slope: 23 7 ± 1.1%; population spike amplitude: 20.3 ± 1.7%). The action on field epsp slope was fully dependent on GABAergic transmission since it was absent in the presence of picrotoxin (50 μM) plus CGP55845 (1 μM). 4 VIP (1 nM) did not change paired-pulse facilitation but increased paired-pulse inhibition in CA1 pyramidal cells (16.0 ± 0.9%), reinforcing the involvement of GABAergic transmission in the action of VIP. 5 VIP (1 nM) increased muscimol-evoked inhibitory currents by 36.4 ± 8.7% in eight out often CA1 interneurones in the siraiuin radianini. This suggests that VIP promotes increased inhibition of interneurones that control pyramidal cells, leading to disinhibition of synapiic transmission to pyramidal cell dendrites. 6 In conclusion, concerted pre- and postsynaptic actions of VIP lead to disinhibition of pyramidal cell dendrites causing an enhancement of synaptic transmission. [ABSTRACT FROM AUTHOR]

In the present study, the P2 receptor-mediated modulation of the extracellular glutamate concentration was investigated by microdialysis in the nucleus accumbens (NAc) of freely moving rats. Because of the known interference of dopaminergic and glutamatergic mechanisms in this area the experiments were performed with animals intra-accumbally treated with 6-hydroxydopamine (6-OHDA) to deplete dopamine pools. Perfusion of the NAc with the prototypic P2 receptor agonist 2-methylthioadenosine 5′-triphosphate (2-MeSATP, 0.1, 1 and 10mM) concentration-dependently increased the extracellular level of glutamate in this area. Pretreatment with the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS, 0.1mM) decreased the basal extracellular glutamate concentration and inhibited the 2-MeSATP-induced outflow of glutamate. In rats treated with 6-OHDA, 2-MeSATP increased the total extracellular glutamate to an extent about fivefold larger than in sham-lesioned rats. The perfusion of the dopamine-depleted NAc with the D2/D3 dopamine receptor agonist quinpirole (0.1mM) diminished the basal concentration of glutamate and reduced the effect of 2-MeSATP on the extracellular glutamate.These results provide evidence that the stimulation of P2 receptors is involved in the increase of accumbal extracellular glutamate in vivo. This behaviourally relevant mechanism depends on a dopamine D2 receptor-mediated tone in the nucleus accumbens. Furthermore, the inhibition of P2 receptors may prevent, at least partly, glutamate-mediated neurodegeneration. [Copyright &y& Elsevier]

1. The presence of ionotropic P2X receptors, targets of ATP in fast synaptic transmission, as well as metabotropic P2Y receptors, known to activate K+ currents in cultured neostriatal neurons, was invistifated in medium-spiny neutrons and cholinergic interneurones contained in neostriatal brain slices from 5-26-day-old rats. 2. In these cells, adenosine-5'-triphosphate (ATP) (100-1000 µM), 2-methylthioadenosine-5'-triphosphate (2MeSATP), α,β-methuleneadenosine-5'-triphosphate (α,βmeATP, 30-300 µM, each) and adenosine-5'-O-(3-thiotriphosphate (ATPγS) (100 µM) failed to evoke P2X receptor currents even when 8-cyclopentyl-1,3,-dipropylxanthine (DPCPX, 0.1 µM), apyrase (10 U ml-1) or intracellular Cs+ was used to prevent occluding effects of the ATP breakdown product adenosine, desensitization of P2X receptors by endogenous ATP and an interference with the activation of K+ channels, respectively. P2X receptor agonists were also ineffective in outside-out patches withdrawn from the brain slice tissue. Muscimol (10 µM) evoked GABAA receptor-mediated currents under all these conditions. 3. When used as a control, locus coeruleus neurons responded with P2X receptor-mediated currents for ATP (300 µM) each. 4. ATP and adenosine-5'-diphosphate (ADP) (100 µM, each) did not activate K+ currents in the neostriatal neutrons. 5. Despite the observed lack of function, P2X2 and P2Y1 immunofluoroscence was found in roughly 50% of the medium-spiny neurons and cholinergic interneutrones. 6. A role of ATP in synaptic transmission to striatal medium-spiny neutrons and cholinergic interneurones appears unlikely, however, the otherwise silent P2X and P2Y receptors may gain functionality under-certain yet unknown conditions. [ABSTRACT FROM AUTHOR]

1: N-type Ca2+ channel modulation by an endogenous P2Y receptor was investigated by the whole-cell patch-clamp method in HEK 293 cells transfected with the functional rabbit N-type calcium channel. 2: The current responses (ICa(N)) to depolarizing voltage steps were depressed by ATP in a concentration-dependent manner. Inclusion of either guanosine 5'-O-(3-thiodiphosphate) or pertussis toxin into the pipette solution as well as a strongly depolarizing prepulse abolished the inhibitory action of ATP. 3: In order to identify the P2Y receptor subtype responsible for this effect, several preferential agonists and antagonists were studied. Whereas the concentration-response curves of ADP and adenosine 5'-O-(2-thiodiphosphate) indicated a higher potency of these agonists than that of ATP, a,ß-methylene ATP, UTP and UDP were considerably less active. The effect of ATP was abolished by the P2Y receptor antagonists suramin and N6-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-ß,?-dichloromethylene-ATP, but not by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, 2'deoxy-N6-methyladenosine-3',5'-diphosphate or 2-methylthio AMP. 4: Using reverse transcription and polymerase chain reaction, mRNA for the P2Y1, P2Y4, P2Y6, P2Y11 and P2Y13 receptor subtypes, but not the P2Y2, and P2Y12 subtypes, was detected in HEK 293 cells. 5: Immunocytochemistry confirmed the presence of P2Y1, and to a minor extent that of P2Y4, but not of P2Y2 receptors. 6: Hence, it is tempting to speculate that P2Y13 receptors may inhibit N-type Ca2+ channels via the ß? subunits of the activated Gi protein.British Journal of Pharmacology (2004) 141, 141-151. doi:10.1038/sj.bjp.0705588 [ABSTRACT FROM AUTHOR]

Membrane currents and changes in the intracellular Ca[SUP2+] concentration ([Ca[SUP2+]][SUBi])were measured in HEK293 cells transfected with the human P2X[SUB3] receptor (HEK293-hP2X[SUB3]). RT-PCR and immunocytochemistry indicated the additional presence of endogenous P2Y[SUB1] and to some extent P2Y[SUB4] receptors. P2 receptor agonists induced inward currents in (HEK293-hP2X[SUB3] cells with the rank order of potency &alpha,&beta-meATP &asym; ATP > ADP-β-S > UTP. A comparable rise in [Ca[SUP2+]][SUBi] was observed after the slow superfusion of ATP, ADP-β-S and UTP; &alpha,&beta-meATP was ineffective. These data, in conjunction with results obtained by using the P2 receptor antagonists TNP-ATP, PPADS and MRS2179 indicate that the current response to &alpha,&beta-meATP is due to P2X[SUB3] receptor activation, while the ATP-induced rise in [Ca[SUP2+]][SUBi] is evoked by P2Y[SUB1] and P2Y[SUB4] receptor activation. TCE depressed the &alpha,&beta-meATP current in a manner compatible with a non-competitive antagonism. The ATP-induced increase of [Ca[SUP2+]][SUBi] was much less sensitive to the inhibitory effect of TCE than the current response to &alpha,&beta-meATP. The present study indicates that in HEK293-hP2X[SUB3] cells, TCE, but not ethanol, potently inhibits ligand-gated P2X[SUB3] receptors and, in addition, moderately interferes with G protein-coupled P2Y[SUB1] and P2Y[SUB4] receptors. Such an effect may be relevant for the interruption of pain transmission in dorsal root ganglion neurons following ingestion of chloral hydrate or trichloroethylene. [ABSTRACT FROM AUTHOR]

The present study was aimed to clarify the role of purinergic signalling in the regulation of ingestion behaviour. The ATP/ADP analogues 2-methylthioATP (2-MeSATP) and adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS) increased the food intake after intracerebroventricular infusion in 18-h food-deprived rats. This effect was abolished by pretreatment with the non-selective P2X/P2Y receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) or the selective P2Y1 receptor antagonist MRS 2179, respectively. The stimulation of food intake mediated by ADPbetaS was also blocked by pretreatment with the nitric oxide synthase (NOS) inhibitor Nw-nitro-L-arginine methylester (L-NAME), as well as with the inhibitor of the soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), suggesting that the orexigenic effect seems to be closely related with the ensuing formation of nitric oxide. The immunohistochemical staining indicating a co-localization of P2Y1 receptor- and nNOS-immunoreactivities in a population of neurons in the ventromedial hypothalamic nucleus (VMH) agrees with this assumption. Further experiments with the direct local application of these compounds into the VMH and lateral hypothalamic nucleus (LH) show that the stimulation of P2Y1 receptors in these functionally antagonistic brain regions exerts an increased food intake. Hence, different signal transduction mechanisms may operate in the VMH and LH. Our assumption is supported by distinct effects of the NOS inhibitor L-NAME in these two hypothalamic nuclei. The present data suggest that ATP/ADP, acting as extracellular signal molecules in the rat brain, are involved in the regulation of food intake, possibly depending on P2Y1-receptor-mediated nitric oxide production.