Metabotropic P2Y receptors inhibit P2X3 receptor-channels via G protein-dependent facilitation of their desensitization

BACKGROUND AND PURPOSE: The aim of the present study was to investigate whether the endogenous metabotropic P2Y receptors modulate ionotropic P2X(3) receptor-channels. EXPERIMENTAL APPROACH: Whole-cell patch-clamp experiments were carried out on HEK293 cells permanently transfected with human P2X(3) receptors (HEK293-hP2X(3) cells) and rat dorsal root ganglion (DRG) neurons. KEY RESULTS: In both cell types, the P2Y(1,12,13) receptor agonist, ADP-beta-S, inhibited P2X(3) currents evoked by the selective agonist, alpha,beta-methylene ATP (alpha,beta-meATP). This inhibition could be markedly counteracted by replacing in the pipette solution the usual GTP with GDP-beta-S, a procedure known to block all G protein heterotrimers. P2X(3) currents evoked by ATP, activating both P2Y and P2X receptors, caused a smaller peak amplitude and desensitized faster than those currents evoked by the selective P2X(3) receptor agonist alpha,beta-meATP. In the presence of intracellular GDP-beta-S, ATP- and alpha,beta-meATP-induced currents were identical. Recovery from P2X(3) receptor desensitization induced by repetitive ATP application was slower than the recovery from alpha,beta-meATP-induced desensitization. When G proteins were blocked by intracellular GDP-beta-S, the recovery from the ATP- and alpha,beta-meATP-induced desensitization were of comparable speed. CONCLUSIONS AND IMPLICATIONS: Our results suggest that the activation of P2Y receptors G protein-dependently facilitates the desensitization of P2X(3) receptors and suppresses the recovery from the desensitized state. Hence, the concomitant stimulation of P2X(3) and P2Y receptors of DRG neurons by ATP may result both in an algesic effect and a partly counterbalancing analgesic activity.     

P2 receptor-types involved in astrogliosis in vivo.

1. In the nucleus accumbens (NAc) of rats, the involvement of P2X and P2Y receptors in the generation of astrogliosis in vivo, was investigated by local application of their respective ligands. The agonists used had selectivities for P2X1,3 (alpha,beta-methylene adenosine 5'-triphosphate; alpha,beta-meATP), P2Y1,12 (adenosine 5'-O-(2-thiodiphosphate; ADP-beta-S) and P2Y2,4,6 receptors (uridine 5'-O-(3-thiotriphosphate; UTP-gamma-S). Pyridoxalphosphate-6-azophenyl-2,4-disulphonic acid (PPADS) was used as a non-selective antagonist. The astroglial reaction was studied by means of immunocytochemical double-labelling with antibodies to glial fibrillary acidic protein (GFAP) and 5-bromo-2'-deoxyuridine (BrdU). 2. The agonist-induced changes in comparison to the artificial cerebrospinal fluid (aCSF)-treated control side reveal a strong mitogenic potency of ADP-beta-S and alpha,beta-meATP, whereas UTP-gamma-S was ineffective. The P2 receptor antagonist PPADS decreased the injury-induced proliferation when given alone and in addition inhibited all agonist effects. 3. The observed morphogenic changes included hypertrophy of astrocytes, elongation of astrocytic processes and up-regulation of GFAP. A significant increase of both GFAP-immunoreactivity (IR) and GFA-protein content (by using Western blotting) was found after microinfusion of alpha,beta-meATP or ADP-beta-S. In contrast, UTP-gamma-S failed to increase the GFAP-IR. The morphogenic effects were also inhibited by pre-treatment with PPADS. 4. A double immunofluorescence approach with confocal laser scanning microscopy showed the localisation of P2X3 and P2Y1 receptors on the GFAP-labelled astrocytes. 5. In conclusion, the data suggest that P2Y (P2Y1 or P2Y12) receptor subtypes are involved in the generation of astrogliosis in the NAc of rats, with a possible minor contribution of P2X receptor subtypes.     

Stimulation of P2Y1 receptors causes anxiolytic-like effects in the rat elevated plus-maze: implications for the involvement of P2Y1 receptor-mediated nitric oxide production.

The widespread and abundant distribution of P2Y receptors in the mammalian brain suggests important functions for these receptors in the CNS. To study a possible involvement of the P2Y receptors in the regulation of fear and anxiety, the influences of the P2Y(1,11,12) receptor-specific agonist adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS), the P2X(1,3) receptor agonist alpha,beta-methylene ATP (alpha,betameATP), the unspecific P2 receptor antagonist pyridoxalphosphate-6-azopheny l-2',4'-disulfonic acid (PPADS), and the specific P2Y(1) receptor antagonist N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS 2179) on the elevated plus-maze behavior of the rat were investigated. All tested compounds were given intracerebroventricularly (0.5 microl). ADPbetaS (50 and 500 fmol) produced an anxiolytic-like behavioral profile reflected by an increase of the open arm exploration. The anxiolytic-like effects were antagonized by pretreatment with PPADS (5 pmol) or MRS 2179 (5 pmol). Both compounds caused anxiogenic-like effects when given alone. Furthermore, the anxiolytic-like effects of ADPbetaS could be antagonized by pretreatment with the nitric oxide synthase (NOS) inhibitor N(w)-nitro-L-arginine methyl ester (L-NAME). In addition, the anxiogenic-like effects of PPADS were reversed by the pretreatment with L-arginine (500 pmol), which is the natural substrate for NOS, but not by D-arginine (500 pmol), which is not. Immunofluorescence staining revealed the presence of P2Y(1) receptors on neurons in different brain regions such as hypothalamus, amygdala, hippocampus and the periaqueductal gray. Furthermore, the colocalization of P2Y(1) receptors and neuronal NOS (nNOS) on some neurons in these regions could be demonstrated. The highest density of P2Y(1)- and nNOS-immunoreactivity was detected in the dorsomedial hypothalamic nucleus. Taken together, the present results suggest that P2Y(1) receptors are involved in the modulation of anxiety in the rat. The anxiolytic-like effects after stimulation of P2Y(1) receptors seem to be in close connection with the related nitric oxide production.     

P2Y receptor-mediated Ca2+ signalling in cultured rat aortic smooth muscle cells.

1. ATP, UTP, ADP and ADP-beta-S elicited Ca2+ -signals in cultured aortic smooth muscle cells although ADP, UDP and ADP-beta-S gave approximately 40% of the maximal response seen with ATP and UTP. Adenosine, AMP or alpha,beta-methylene-ATP had no effect. These responses were attributed to P2Y2/4 and P2Y1 receptors, which we assumed could be selectively activated by UTP and ADP-beta-S respectively. 2. The response to UTP was reduced (approximately 50%) by pertussis toxin, whilst this toxin had no effect upon the response to ADP-beta-S. This suggests P2Y2/4 receptors simultaneously couple to pertussis toxin-sensitive and -resistant G proteins whilst P2Y1 receptors couple to only the toxin-resistant proteins. 3. Repeated stimulation with UTP or ADP-beta-S caused desensitization which was potentiated by 12-O-tetradecanoyl phorbol-13-acetate (TPA) and attenuated by staurosporine. 4. TPA completely abolished sensitivity to ADP-beta-S but the response to UTP had a TPA-resistant component. In pertussis toxin-treated cells, however, TPA could completely abolish sensitivity to UTP and so the TPA-resistant part of this response seems to be mediated by pertussis toxin-sensitive G proteins. 5. Loss of sensitivity to UTP did not occur when pertussis toxin-treated cells were repeatedly stimulated with this nucleotide, suggesting that pertussis toxin-sensitive G proteins mediate this effect. The toxin did not, however affect desensitization to ADP-beta-S.     

Inhibitory purinergic P2 receptor characterisation in rat distal colon

The aim of this study was to characterise the P2 receptors involved in purinergic relaxant responses in rat distal colon circular muscle. Concentration-response curves for purinergic agonists were constructed on methacholine-precontracted circular muscle strips of rat distal colon in the absence and presence of the nerve blocker TTX and the ecto-nucleotidase inhibitor ARL67156. The effects of the P2 receptor antagonists RB2, PPADS, suramin, MRS2179 and NF279, the NO-synthase inhibitor L-NAME and the small conductance K(+) channel blocker apamin were investigated. The localisation of the different P2 receptors was examined immunocytochemically. Immunocytochemistry demonstrated the expression of P2Y(1), P2Y(6) and P2X(1) receptors on smooth muscle cells and P2Y(2), P2Y(12), P2X(2) and P2X(3) receptors in the myenteric plexus; almost a quarter of the P2Y(2)-immunopositive neurons co-expressed nNOS. The P2X-selective agonist alphabetameATP and the P2Y-selective agonist ADPbetaS were the most potent relaxants; their effects were abolished by apamin. The effect of ADPbetaS was antagonised by the P2Y(1)-selective antagonist MRS2179 pointing to interaction with the muscular P2Y(1)-receptors. The relaxant effect of alphabetameATP was partially reduced by TTX and concentration-dependently antagonised by PPADS, suramin, RB2 and the P2X(1)-selective antagonist NF279; this correlates with an interaction with neuronal P2X(3) and muscular P2X(1) receptors. UTP was the least potent agonist; its effect was markedly increased by ARL67156, nearly abolished by TTX and reduced by L-NAME. This points to interaction with the neuronal P2Y(2)-receptors inducing relaxation, at least partially, by NO release.     

Diinosine pentaphosphate: an antagonist which discriminates between recombinant P2X(3) and P2X(2/3) receptors and between two P2X receptors in rat sensory neurones

1. We have compared the antagonist activity of trinitrophenyl-ATP (TNP-ATP) and diinosine pentaphosphate (Ip(5)I) on recombinant P2X receptors expressed in XENOPUS: oocytes with their actions at native P2X receptors in sensory neurones from dorsal root and nodose ganglia. 2. Slowly-desensitizing responses to alpha,beta-methylene ATP (alpha,beta-meATP) recorded from oocytes expressing P2X(2/3) receptors were inhibited by TNP-ATP at sub-micromolar concentrations. However, Ip(5)I at concentrations up to 30 microM was without effect. 3. Nodose ganglion neurones responded to alpha,beta-meATP with slowly-desensitizing inward currents. These were inhibited by TNP-ATP (IC(50), 20 nM), but not by Ip(5)I at concentrations up to 30 microM. 4. In DRG neurones that responded to ATP with a rapidly-desensitizing inward current, the response was inhibited by TNP-ATP with an IC(50) of 0.8 nM. These responses were also inhibited by Ip(5)I with an IC(50) of 0.1 microM. Both antagonists are known to inhibit homomeric P2X(3) receptors. 5. Some DRG neurones responded to alpha,beta-meATP with a biphasic inward current, consisting of transient and sustained components. While the transient current was abolished by 1 microM Ip(5)I, the sustained component remained unaffected. 6. In conclusion, Ip(5)I is a potent antagonist at homomeric P2X(3) receptors but not at heteromeric P2X(2/3) receptors, and therefore should be a useful tool for elucidating the subunit composition of native P2X receptors.     

P2 receptors in the murine gastrointestinal tract

The actions of adenosine, adenosine 5'-triphosphate (ATP), 2-methylthio adenosine diphosphate ADP (2-MeSADP), 2-methylthio ATP (2-MeSATP), alpha,beta-methylene ATP (alpha,beta-meATP) and uridine triphosphate (UTP) on isolated segments of mouse stomach (fundus), duodenum, ileum and colon were investigated. The localization of P2Y(1), P2Y(2), P2Y(4), P2X(1) and P2X(2) receptors and neuronal nitric oxide synthase (NOS) were examined immunohistochemically, and P2Y(1) mRNA was examined with in situ hybridization. The order of potency for relaxation of longitudinal muscle of all regions was: 2-MeSADP>/=2-MeSATP>alpha,beta-meATP>ATP=UTP=adenosine. This is suggestive of P2Y(1)-mediated relaxation and perhaps a further P2Y receptor subtype sensitive to alpha,beta-meATP. As ATP and UTP are equipotent, the presence of a P2Y(2) receptor is indicated. ATP responses were inhibited by the P2Y(1)-selective antagonist MRS 2179, and suramin. P2Y(1) receptors were visualized immunohistochemically in the smooth muscle of the ileum and in a subpopulation for myenteric neurones, which also stained for NOS. P2Y(1) mRNA was localized in neurones in both myenteric and submucosal ganglia in the ileum. Taken together, these results suggest that ATP was acting on non-adrenergic, non-cholinergic inhibitory neurons, which release both nitric oxide (NO) and ATP. Reduced relaxations to 2-MeSADP by tetrodotoxin and N(omega)-nitro-L-arginine methyl ester, are consistent with this possibility. Adenosine acts via P1 receptors to relax smooth muscle of the mouse gut. Segments of mouse colon (in contrast to the stomach and small intestine) were contracted by nucleotides with the potency order: 2-MeSATP>alpha,betameATP>ATP; the contractions showed no desensitization and were antagonized by suramin and PPADS, consistent with responses mediated by P2X(2) receptors. Immunoreactivity to P2X(2) receptors was demonstrated on both longitudinal and circular muscle of the colon, but not in the other regions of the gut, except for a small subpopulation of myenteric neurones. In summary, neuronal P2Y(1) receptors appear to mediate relaxation, largely through NO in all regions of the mouse gut, and to a lesser extent by P2Y(1), P2Y(2) and a novel P2Y receptor subtype responsive to alpha,beta-meATP in smooth muscle, while P2X(2) receptors mediate contraction of colonic smooth muscle.     

NF449: a subnanomolar potency antagonist at recombinant rat P2X1 receptors

Antagonistic effects of the novel suramin analogue 4,4',4",4"'-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid (NF449) were studied on contractions of the rat vas deferens elicited by alpha,beta-methylene ATP (alphabetameATP; mediated by P2X1 receptors), contractions of the guinea-pig ileal longitudinal smooth muscle elicited by alphabetameATP (mediated by P2X3 receptors) or adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS; mediated by P2Y1 receptors), ATP-induced increases of [Ca2+]i in human embryonic kidney (HEK) 293 cells (mediated by P2Y2 receptors), inward currents evoked by ATP in follicle cell-free Xenopus laevis oocytes expressing rP2X1 or rP2X3 receptors and degradation of ATP by ecto-nucleotidases in folliculated Xenopus laevis oocytes. In addition, NF449 was examined for its P2 receptor specificity in rat vas deferens (alpha1A-adrenoceptors) and guinea-pig ileum (histamine H1 and muscarinic M3 receptors). At native (pIC50=7.15) and recombinant (pIC50=9.54) P2X1 receptors, NF449 was a highly potent antagonist. The P2X3 receptors present in guinea-pig ileum (pIC50=5.04) or expressed in oocytes (pIC50 approximately 5.6) were much less sensitive for NF449. It also was a very weak antagonist at P2Y1 receptors in guinea-pig ileum (pIC50=4.85) and P2Y2 receptors in HEK 293 cells (pIC50=3.86), and showed very low inhibitory potency on ecto-nucleotidases (pIC50<3.5). NF449 (100 microM) did not interact with alpha1A-adrenoceptors or histamine H1 and muscarinic M3 receptors. Thus, the antagonism by NF449 is highly specific for P2 receptors. In conclusion, the subnanomolar potency at rP2X1 receptors and the rank order of potency, P2X1 > P2X3 > P2Y1 > P2Y2 > ecto-nucleotidases, make NF449 unique among the P2 receptor antagonists reported to date. NF449 may fill the long-standing need for a P2X1-selective radioligand.     

Methyllycaconitine, alpha-bungarotoxin and (+)-tubocurarine block fast ATP-gated currents in rat dorsal root ganglion cells

The effects of nicotinic acetylcholine receptor antagonists were studied on currents evoked by application of ATP to rat isolated dorsal root ganglion cells, and human embryonic kidney 293 cells expressing rat P2X(3) and P2X(2/3) receptors. The rapidly desensitising (within 100 ms) current in dorsal root ganglion cells was inhibited by methyllycaconitine, alpha-bungarotoxin and (+)-tubocurarine (concentrations giving half-maximal inhibition were approximately 40, 60 and 800 nm, respectively), but not by hexamethonium (100 microm) or mecamylamine (100 microm). The sustained (>250 ms) current in dorsal root ganglion cells was inhibited by (+)-tubocurarine (80% by 10 microm), but not by methyllycaconitine (200 nm), alpha-bungarotoxin (200 nm), mecamylamine (100 microm) or hexamethonium (100 microm). Rapidly desensitising currents evoked by alpha,betamethylene-ATP in human embryonic kidney cells expressing P2X(3) receptors were inhibited by methyllycaconitine and alpha-bungarotoxin, at concentrations similar to those effective in dorsal root ganglion cells. The results indicate that some nicotinic acetylcholine receptor antagonists are potent blockers of P2X receptors on neurons, particularly the homo-oligomeric P2X(3) receptor. This finding suggests that these drugs should be used with care to discriminate between P2X and neuronal acetylcholine receptor types.     

In vivo pathway of thermal hyperalgesia by intrathecal administration of alpha,beta-methylene ATP in mouse spinal cord: involvement of the glutamate-NMDA receptor system.

1. The aim of the present study is to characterize the role of the P2X receptor in spinal nociceptive processing in vivo. We investigated the mechanisms of the P2X receptor agonist alpha,beta-methylene ATP (alpha,betameATP)-induced modulation of acute nociceptive signalling in mouse spinal cord. 2. Intrathecal administration of alpha,betameATP produced a significant and dose-dependent thermal hyperalgesic response. This response was completely blocked by intrathecal pretreatment with the non-selective P2 receptor antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonate (PPADS) and the selective P2X1, P2X3 and P2X2-3 receptor antagonist, 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP). Pretreatment with alpha,betameATP 15, 30 and 60 min prior to administration of a second dose of alpha,betameATP diminished the alpha,betameATP-induced thermal hyperalgesia. 3. A potent agonist for the P2X1 receptor, beta,gamma-methylene-L-ATP, did not show the hyperalgesic response, indicating that the P2X1 receptor is not involved in the spinal nociceptive pathway. 4. In fura-2 experiments using mouse dorsal root ganglion (DRG) neurons, alpha,betameATP (100 microM) increased intracellular Ca2+ ([Ca2+]i). This was not produced by a second application of alpha,betameATP. The same DRG neurons also showed a marked [Ca2+]i increase in response to capsaicin (3 microM). 5. Intrathecal pretreatment with the Ca2+-dependent exocytosis inhibitor, botulinum neurotoxin B, abolished the thermal hyperalgesia by alpha,betameATP. Furthermore, thermal hyperalgesia was significantly inhibited by the N-methyl-D-aspartate (NMDA) receptor antagonists, 2-amino-5-phosphonopentanoate (APV), dizocilpine and ifenprodil. 6. These findings suggest that alpha,betameATP-induced thermal hyperalgesia may be mediated by the spinal P2X3 receptor subtype that causes unresponsiveness by repetitive agonist applications, and that alpha,betameATP (perhaps through P2X3 receptors) may evoke spinal glutamate release which, in turn, leads to the generation of thermal hyperalgesia via activation of NMDA receptors.     

Activation of adenosine and P2Y receptors by ATP in human peripheral nerve

Receptors for ATP in the peripheral nervous system may contribute to the transduction of sensory, including nociceptive, stimuli and are candidates in the pathogenesis of neuropathic pain. In a complex neural tissue, such as the human peripheral nerve trunk, ATP may activate P2X, P2Y, and adenosine receptors present on various cell types. Experiments were performed on segments of isolated human sural nerves. The experimental set-up enabled simultaneous recording of C fiber excitability, intracellular Ca(2+) ([Ca(2+)](i)) and extracellular K(+) activity (aK(e)). The increase in excitability of unmyelinated fibers seen during bath application of both ATP and adenosine was reversed to a reduction in axonal excitability in the presence of 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolol[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385), an antagonist of adenosine A2 receptors. The pharmacological profile of the axonal subexcitability indicates the presence and activation of adenosine A1 receptors. Intracellular Ca(2+) transients were observed during bath application of ATP but not of adenosine and were blocked by 2'-deoxy- N(6)-methyladenosine 3',5'-bisphosphate (MRS 2179), an antagonist at P2Y(1) receptors. K(+)-sensitive microelectrodes were used to search for a possible activation of P2X receptors by ATP. In isolated rat vagus nerve, activation of P2X receptors by alpha,beta-methylene-adenosine 5'-triphosphate (alpha,beta-meATP) and by diadenosine pentaphosphate (Ap5A) resulted in a rapid, transient rise in the extracellular K(+) activity. In contrast, in human nerve, application of P2X receptor agonists did not result in a detectable elevation of aK(e). The data suggest that ATP-induced changes in axonal excitability and of [Ca(2+)](i) result from activation of adenosine A2, A1 and P2Y nucleotide receptors in human nerve; a contribution of P2X receptors was not found with the methods used. It is suggested that antagonists of A2 receptors might suppress enhanced activity in human nociceptive afferent nerve fibers under conditions in which ATP and/or adenosine is released into the trunk of a human peripheral nerve.     

Interaction between P2Y and NMDA receptors in layer V pyramidal neurons of the rat prefrontal cortex

In the first part of this study, monosynaptic excitatory postsynaptic potentials (EPSPs) in layer V of the rat prefrontal cortex were evoked by electrical stimulation of layer I. Recordings by intracellular sharp microelectrodes showed that EPSPs were concentration-dependently facilitated by the P2 receptor antagonistic ATP analogue 2-methylthio ATP (2-MeSATP), while ATP itself depressed the synaptic potentials. The inhibitory effect of ATP turned into facilitation in the presence of the adenosine A(1) receptor antagonist DPCPX. The 2-MeSATP-induced potentiation of EPSP amplitudes were prevented by the P2 receptor antagonists PPADS and Suramin. The EPSP was almost abolished by coapplication of the NMDA receptor antagonist AP-5 and the AMPA/kainate receptor antagonist CNQX. After blockade of the NMDA receptor-mediated part of the EPSP by AP-5, the stimulatory effect of 2-MeSATP disappeared. When NMDA or AMPA were pressure-applied onto pyramidal cells, only the NMDA-induced depolarization was potentiated by 2-MeSATP. In the second part of the study, NMDA-induced currents were measured by whole-cell patch-clamp pipettes. ATP, 2-MeSATP, UDP and UTP potentiated the response to NMDA, while ADP-beta-S was inactive. PPADS antagonized the effect of ATP. Synaptic isolation of pyramidal neurons by a Ca(2+)-free medium or tetrodotoxin did not alter the effect of ATP which, however, was markedly depressed when GTP in the micropipette was replaced by GDP-beta-S. These observations suggest that in layer V pyramidal neurons of the prefrontal cortex postsynaptically localized P2Y receptors interact with NMDA receptor-channels.     

Ethanol differentially affects ATP-gated P2X(3) and P2X(4) receptor subtypes expressed in Xenopus oocytes

P2X receptors are cation-selective, ligand-gated ion channels activated by synaptically released, extracellular adenosine 5'-triphosphate (ATP). ATP-gated currents are inhibited by ethanol when tested in dorsal root ganglion and CA1 neurons. Recently, we reported differences in sensitivity to ethanol inhibition between homomeric P2X(2) and P2X(4) receptors expressed in Xenopus oocytes, which suggested that subunit composition of native P2X receptors determines their ethanol sensitivity. The present study extended the investigation to P2X(3) receptors. The effects of ethanol and zinc ions (Zn(2+)) were tested on homomeric P2X(3) and P2X(4) receptors expressed in Xenopus oocytes using two-electrode voltage clamp. Ethanol potentiated ATP-gated P2X(3) receptor currents in a concentration dependent manner. In contrast, ethanol inhibited P2X(4) receptor function. Ethanol did not directly alter receptor function, nor did it alter the Hill coefficient or maximal ATP response (E(max)) in either P2X(3) or P2X(4) receptors. Ethanol increased the maximal response to Zn(2+) ATP-gated currents in P2X3 receptors which suggests that ethanol and Zn(2+) act on different sites. The differences in ethanol response of P2X(3) and P2X(4) receptors set the stage for future investigations that will use chimeric P2X receptors or other molecular manipulations of P2X structure to investigate the molecular sites and mechanisms of action of ethanol.     

The novel pyridoxal-5'-phosphate derivative PPNDS potently antagonizes activation of P2X(1) receptors

Pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonat e) (PPNDS) potently antagonized P2X(1) receptor-mediated responses in rat vas deferens (pK(B)=7.43) and Xenopus laevis oocytes (pIC(50)=7. 84). It showed lower (up to 20,000-fold) inhibitory potency on ecto-nucleotidase in Xenopus oocytes and on P2Y(1) receptors in guinea-pig ileum (pA(2)=6.13). PPNDS did not interact with alpha(1A)-adrenoceptors, adenosine A(1) and A(2B), histamine H(1) and muscarinic M(3) receptors. Thus, PPNDS is a novel, specific P2 receptor antagonist and represents the pyridoxal-5'-phosphate derivative with the highest potency at P2X(1) receptors.     

P2X7 receptors stimulate AKT phosphorylation in astrocytes

1. Emerging evidence indicates that nucleotide receptors are widely expressed in the nervous system. Here, we present evidence that P2Y and P2X receptors, particularly the P2X(7) subtype, are coupled to the phosphoinositide 3-kinase (PI3K)/Akt pathway in astrocytes. 2. P2Y and P2X receptor agonists ATP, uridine 5'-triphosphate (UTP) and 2',3'-O-(4-benzoyl)-benzoyl ATP (BzATP) stimulated Akt phosphorylation in primary cultures of rat cortical astrocytes. BzATP induced Akt phosphorylation in a concentration- and time-dependent manner, similar to the effect of BzATP on Akt phosphorylation in 1321N1 astrocytoma cells stably transfected with the rat P2X(7) receptor. Activation was maximal at 5 - 10 min and was sustained for 60 min; the EC(50) for BzATP was approximately 50 microM. In rat cortical astrocytes, the positive effect of BzATP on Akt phosphorylation was independent of glutamate release. 3. The effect of BzATP on Akt phosphorylation in rat cortical astrocytes was significantly reduced by the P2X(7) receptor antagonist Brilliant Blue G and the P2X receptor antagonist iso-pyridoxal-5'-phosphate-6-azophenyl-2',4'-disulfonic acid, but was unaffected by trinitrophenyl-ATP, oxidized ATP, suramin and reactive blue 2. 4. Results with specific inhibitors of signal transduction pathways suggest that extracellular and intracellular calcium, PI3K and a Src family kinase are involved in the BzATP-induced Akt phosphorylation pathway. 5. In conclusion, our data indicate that stimulation of astrocytic P2X(7) receptors, as well as other P2 receptors, leads to Akt activation. Thus, signaling by nucleotide receptors in astrocytes may be important in several cellular downstream effects related to the Akt pathway, such as cell cycle and apoptosis regulation, protein synthesis, differentiation and glucose metabolism.     

Minor contribution of ATP P2 receptors to electrically-evoked electrographic seizure activity in hippocampal slices: Evidence from purine biosensors and P2 receptor agonists and antagonists

While the position of adenosine as an endogenous anticonvulsant is well established, it is unclear to what extent its precursor, ATP, contributes to seizure activity via P2 receptors. In this study we have addressed this issue through the use of ATP biosensors and agonists and antagonists of ATP P2 receptors to detect the release and role of ATP, respectively, during electrically-evoked electrographic seizure-like events (eSLEs) in rat hippocampal slices. The broad-spectrum P2 receptor antagonists RB-2 and PPADS (10μM) caused a small ～30% inhibition of eSLE duration, and a reduction in intensity. This inhibition of eSLEs was partially reproduced with the P2X(1,2/3,3) antagonist NF023 (10μM), but not the P2X(7) antagonist BBG (10μM). However, the P2X receptor agonist α,β-meATP did not enhance eSLEs, but instead reduced their duration. Furthermore, we could discern no role for P2Y(1) receptors in electrically-evoked eSLEs: both the P2Y(1) antagonist MRS2179 (10μM) and the P2Y(1) receptor agonist 2-methylthioADP (10μM) were without effect on eSLEs. Consistent with a minor role for ATP P2 receptors on eSLEs we could detect no ATP release during eSLEs, although appreciable quantities of adenosine were detected, which had a pronounced inhibitory action on eSLEs via A(1) receptors. We conclude that the role of ATP P2 receptors in modulating electrographic seizure activity is limited, at least in models such as this one requiring electrical stimulation of afferent fibres. We further conclude that the presence and action of adenosine under these conditions may primarily reflect direct release of this purine.     

Postnatal development of P2 receptors in the murine gastrointestinal tract

The actions of purine and pyrimidine compounds on isolated segments of the mouse intestine were investigated during postnatal development. The localization of P2Y(1), P2Y(2), P2Y(4), P2X(1,) P2X(2) and P2X(3) receptors were examined immunohistochemically, and levels of expression of P2Y(1), P2X(1) and P2X(2) were studied by Western immunoblot. From day 12 onwards, the order of potency for relaxation of longitudinal muscle of all regions was 2-MeSADP>or=alpha,beta-meATP>or=ATP=UTP=adenosine, suggesting P2Y(1) receptors. This was supported by the sensitivity of responses to 2-MeSADP to the selective antagonist MRS 2179 and P2Y(1) receptor immunoreactivity on longitudinal muscle and a subpopulation of myenteric neurons. A further alpha,beta-meATP-sensitive P2Y receptor subtype was also indicated. ATP and UTP were equipotent suggesting a P2Y(2) and/or P2Y(4) receptor. Adenosine relaxed the longitudinal muscle in all regions via P1 receptors. The efficacy of all agonists to induce relaxation of raised tone preparations increased with age, being comparable to adult by day 20, the weaning age. During postnatal development the contractile response of the ileum and colon was via P2Y(1) receptors, while the relaxant response mediated by P2Y(1) receptors gradually appeared along the mouse gastrointestinal tract, being detectable in the stomach from day 3 and in the duodenum from day 6. In the ileum and colon relaxant responses to 2-MeSADP were not detected until days 8 and 12, respectively. 2-MeSADP induced contractions on basal tone preparations from day 3, but decreased significantly at day 12 and disappeared by day 20. At day 8, contractions of colonic longitudinal muscle to ATP showed no desensitisation suggesting the involvement of P2X(2) receptors. Immunoreactivity to P2X(2) receptors only was observed on the longitudinal muscle of the colon and ileum from day 1 and on a subpopulation of myenteric neurons from day 3. These data suggest that P2Y(1) receptors undergo postnatal developmental changes in the mouse gut, with a shift from contraction to relaxation. Such changes occur 1 week before weaning and may contribute to the changes that take place in the gut when the food composition changes from maternal milk to solid food.     

Subsensitivity of P2X but not vanilloid 1 receptors in dorsal root ganglia of rats caused by cyclophosphamide cystitis

The application of cyclophosphamide to rats was used to induce interstitial cystitis. Behavioural studies indicated a strong pain reaction that developed within 2 h and levelled off thereafter causing a constant pain during the following 18 h. Neurons prepared from L6/S1 dorsal root ganglia innervating the urinary bladder responded to the application of capsaicin or alpha,beta-methylene ATP (alpha,beta-meATP) with an increase of intracellular Ca2+ ([Ca2+]i). The [Ca2+]i responses to capsaicin were identical in the dorsal root ganglion cells of cyclophosphamide- and saline-treated rats, whereas alpha,beta-meATP induced less increase in [Ca2+]i in the cyclophosphamide-treated animals than in their saline-treated counterparts. Hence, alpha,beta-meATP-sensitive P2X3 and/or P2X2/3 receptors of L6/S1 dorsal root ganglion neurons were functionally downregulated during subacute pain caused by experimental cystitis. In contrast, capsaicin-sensitive vanilloid 1 receptors did not react to the same procedure. Thoracal dorsal root ganglia, not innervating the urinary bladder, were also unaltered in their responsiveness to alpha,beta-meATP by cyclophosphamide treatment.     

Purine- and pyrimidine-induced responses and P2Y receptor characterization in the hamster proximal urethra

1. Purine and pyrimidine compounds were investigated on hamster proximal urethral circular smooth muscle preparations. In situ hybridization studies were carried out to localize P2Y(1), P2Y(2), P2Y(4) and P2Y(6) mRNA. Protein expression was studied using Western blotting analysis with antibodies against P2Y(1) and P2Y(2) receptors. 2. The hamster urethra relaxed with an agonist potency order of: 2-MeSADP>beta,gamma-meATP=ATP=adenosine=ADP>2-MeSATP>alpha,beta-meATP>TTP>CTP=UTP>GTP=UDP. The high potency of 2-MeSADP is suggestive of an action via P2Y(1) receptors. Although the order is not characteristic for any known single P2Y receptor subtype, it may represent a combination of P2Y receptor subtypes. 4. The selective P2Y(1) receptor antagonist MRS2179 inhibited ATP-, 2-MeSADP-, 2-MeSATP-, beta,gamma-meATP-, and to a lesser degree alpha,beta-meATP-induced responses. 3. Adenosine, but not ATP, was inhibited by the adenosine receptor antagonist 8-phenyltheophylline, indicating that ATP was not acting via adenosine following enzymatic breakdown. 5. Western blotting analysis showed the expression of both P2Y(1) and P2Y(2) receptors, confirming the results obtained with in situ hybridization that showed the expression of both P2Y(1) and P2Y(2), but not P2Y(4) or P2Y(6) mRNA, in smooth muscle layers of the hamster proximal urethra. 6. It is proposed that the relaxant response of the urethra to ATP may be evoked through the activation of the combination of receptors for P2Y(1) and to a lesser extent P2Y(2) receptors, which may mediate a trophic effect in addition. A P2Y subtype responsive to alpha,beta-meATP and P1 receptors may contribute to urethral smooth muscle relaxation.