Evidence that 2-methylthioATP and 2-methylthioADP are both agonists at the rat hepatocyte P2Y(1) receptor

In the absence of selective antagonists, pharmacological characterization of P2Y receptor subtypes has relied heavily upon their distinct agonist profiles. 2-methylthioADP (2-MeSADP) is a selective agonist for the P2Y(1) receptor. The agonist action of 2-MeSATP at the P2Y(1) receptor has recently been questioned. The effects of both 2-MeSADP and 2-MeSATP have been studied on rat hepatocytes injected with the bioluminescent Ca(2+) indicator, aequorin. Single hepatocytes generate series of repetitive transients in cytosolic free calcium concentration ([Ca(2+)](i)) when stimulated with agonists acting through the phosphoinositide signalling pathway. The transients induced by 2-MeSADP and 2-MeSATP in the same cell were indistinguishable, indicating that they act at a common receptor. In contrast the transients evoked by ATP and UTP had very different profiles. Treatment of 2-MeSATP with an ATP-regenerating system to remove contaminating 2-MeSADP did not abolish its agonist activity. Application of the P2Y(1) antagonist, adenosine-3'-phosphate-5'-phosphate (A3P5P) inhibited the transients induced by both 2-MeSADP and 2-MeSATP. In contrast the transients induced by ATP and UTP were enhanced by the addition of A3P5P. These results indicate that both 2-MeSADP and 2-MeSATP are agonists at the rat hepatocyte P2Y(1) receptor.     

P2Y1 receptors mediate inhibitory neuromuscular transmission in the rat colon

Background and purpose:

Inhibitory junction potentials (IJP) are responsible for smooth muscle relaxation in the gastrointestinal tract. The aim of this study was to pharmacologically characterize the neurotransmitters [nitric oxide (NO) and adenosine triphosphate (ATP)] and receptors involved at the inhibitory neuromuscular junctions in the rat colon using newly available P2Y₁ antagonists.

Experimental approach:

Organ bath and microelectrode recordings were used to evaluate the effect of drugs on spontaneous mechanical activity and resting membrane potential. IJP and mechanical relaxation were studied using electrical field stimulation (EFS).

Key results:

N^ω-nitro-L-arginine (L-NNA) inhibited the slow component of the IJP and partially inhibited the mechanical relaxation induced by EFS. MRS2179, MRS2500 and MRS2279, all selective P2Y₁ receptor antagonists, inhibited the fast component of the IJP without having a major effect on the relaxation induced by EFS. The combination of both L-NNA and P2Y₁ antagonists inhibited the fast and the slow components of the IJP and completely blocked the mechanical relaxation induced by EFS. Sodium nitroprusside caused smooth muscle hyperpolarization and cessation of spontaneous motility that was prevented by oxadiazolo[4,3-α]quinoxalin-1-one. Adenosine 5′-O-2-thiodiphosphate, a preferential P2Y agonist, hyperpolarized smooth muscle cells and decreased spontaneous motility. This effect was inhibited by P2Y₁ antagonists.

Conclusions and implications:

The co-transmission process in the rat colon involves ATP and NO. P2Y₁ receptors mediate the fast IJP and NO the slow IJP. The rank order of potency of the P2Y₁ receptor antagonists is MRS2500 greater than MRS2279 greater than MRS2179. P2Y₁ receptors might be potential pharmacological targets for the regulation of gastrointestinal motility.     

Metabotropic P2Y1 receptors inhibit P2X3 receptor-channels in rat dorsal root ganglion neurons

Whole-cell patch-clamp recordings from cultured rat dorsal root ganglion neurons demonstrated that the P2Y1 receptor agonists adenosine 5'-O-2-thiodiphosphate (ADP-beta-S) and 2-methylthio adenosine 5'-diphosphate (2-MeSADP) inhibit the alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-meATP)-induced P2X3 receptor-currents. This effect could be antagonized by the wide-spectrum G protein blocker GDP-beta-S and the P2Y(1) receptor antagonist MRS 2179. The P2Y12,13 receptor antagonist AR-C6993MX and pertussis toxin, a blocker of Galphai/o, did not interact with the effect of ADP-beta-S. Hence, the results indicate that ADP-sensitive P2Y1 receptors of rat dorsal root ganglion neurons inhibit ionotropic P2X3 receptors via G protein-activation.     

Evidence for functional P2X4/P2X7 heteromeric receptors

The cytolytic ionotropic ATP receptor P2X7 has several important roles in immune cell regulation, such as cytokine release, apoptosis, and microbial killing. Although P2X7 receptors are frequently coexpressed with another subtype of P2X receptor, P2X4, they are believed not to form heteromeric assemblies but to function only as homomers. Both receptors play a role in neuropathic pain; therefore, understanding how they coordinate the cellular response to ATP is important for the development of effective pain therapies. Here, we provide biochemical and electrophysiological evidence for an association between P2X4 and P2X7 that increases the diversity of receptor currents mediated via these two subtypes. The heterologously expressed receptors were coimmunoprecipitated from human embryonic kidney (HEK) 293 cells, and the endogenous P2X4 and P2X7 receptors were similarly coimmunoprecipitated from bone marrow-derived macrophages. In HEK293 cells, the fraction of P2X4 receptors biotinylated at the plasma membrane increased 2-fold in the presence of P2X7 although there was no change in overall expression. Coexpression of a dominant-negative P2X4 mutant (C353W) with P2X7, inhibited P2X7 receptor mediated currents by greater than 2-fold, whereas a nonfunctional but non-dominant-negative mutant (S341W) did not. Coexpression of P2X4S341W with P2X7 produced a current that was potentiated by ivermectin and inhibited by 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5-triphosphate (TNP-ATP), whereas expression of P2X7 alone produced a current that was insensitive to both of these compounds at the concentrations used. These results demonstrate a structural and functional interaction between P2X4 and P2X7, which suggests that they associate to form heteromeric receptors.     

Evidence for P2Y1, P2Y2, P2Y6 and atypical UTP-sensitive receptors coupled to rises in intracellular calcium in mouse cultured superior cervical ganglion neurons and glia

1 P2Y receptors are expressed in the nervous system and are involved in calcium signalling in neurons and glia. In the superior cervical ganglion (SCG), RT-PCR analysis indicated the presence of P2Y(1,2&6) receptors. Rises in intracellular calcium in response to P2Y receptor stimulation were determined from adult mouse cultured SCG neurons and glia. 2 ADP evoked suramin (100 microM)- and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 30 microM)-sensitive rises in intracellular calcium in approximately 80% of SCG neurons (EC50 approximately 20 microM). ADP-evoked responses were abolished in neurons from P2Y1 receptor-deficient mice (responses to UTP were unaffected). 3 The pyrimidines UTP (EC50 approximately 85 microM) and UDP (EC50>90 microM) evoked PPADS- and suramin-sensitive responses in approximately 70 and approximately 20% of SCG neurons, respectively. 4 In SCG glial cells, ADP (EC50 approximately 30 microM) evoked calcium responses in approximately 50% of glia. These were suramin and PPADS sensitive and essentially abolished in SCG glial cells cultured from adult P2Y1 receptor-deficient mice. 5 UTP (EC50 approximately 25 microM) and UDP (EC50>200 microM) evoked suramin- and pyridoxalphosphate-6-azophenyl-2',5'-disulphonate-sensitive rises in calcium in approximately 60 and 20% SCG glial cells, respectively. 6 These results indicate the presence of several P2Y receptors coupled to an increase in intracellular calcium in the SCG: ADP-sensitive P2Y1 receptors and UDP-sensitive P2Y6 receptors in SCG neurons and glial cells, a novel UTP-sensitive P2Y receptor in SCG neurons and UTP- and ATP-sensitive P2Y2 receptors in SCG glia.     

Presynaptic adenosine and P2Y receptors

Adenine-based purines, such as adenosine and ATP, are ubiquitous molecules that, in addition to their roles in metabolism, act as modulators of neurotransmitter release through activation of presynaptic P1 purinoceptors or adenosine receptors (activated by adenosine) and P2 receptors (activated by nucleotides). Of the latter, the P2Y receptors are G protein-coupled, whereas the P2X receptors are ligand-gated ion channels and not covered in this review.     

Caged agonist of P2Y1 and P2Y12 receptors for light-directed facilitation of platelet aggregation

We have prepared a caged form (MRS2703) of a potent dual agonist of the P2Y(1) and P2Y(12) nucleotide receptors, 2-MeSADP, by blocking the beta-phosphate group with a 1-(3,4-dimethyloxyphenyl)eth-1-yl phosphoester. Although MRS2703 is itself inactive at human P2Y(1) and P2Y(12) receptors expressed heterologously in 1321N1 astrocytoma cells or in washed human platelets, this derivative readily regenerates the parent agonist upon mild irradiation with long-wave UV light (360 nm). The functional effect of the regenerated agonist was demonstrated by a rise in intracellular calcium mediated by either P2Y(1) or P2Y(12) receptors in transfected cells. Washed human platelets exposed to a solution of MRS2703 were induced to aggregate upon UV irradiation. At 1.0 microM MRS2703, full aggregation was achieved within 1 min of irradiation. Thus, this caged nucleotide promises to be a useful probe for potent P2Y receptor activation with light-directed spatial and temporal control.     

Regulation of death and survival in astrocytes by ADP activating P2Y1 and P2Y12 receptors

ADP is the endogenous agonist for both P2Y(1) and P2Y(12) receptors, which are important therapeutic targets. It was previously demonstrated that ADP and a synthetic agonist, 2-methylthioadenosine 5'-diphosphate (2MeSADP), can induce apoptosis by activating the human P2Y(1) receptor heterologously expressed in astrocytoma cells. However, it was not known whether the P2Y(12) receptor behaved similarly. We demonstrated here that, unlike with the G(q)-coupled P2Y(1) receptor, activation of the G(i)-coupled P2Y(12) receptor does not induce apoptosis. Furthermore, activation of the P2Y(12) receptor by either ADP or 2MeSADP significantly attenuates the tumor necrosis factor alpha (TNFalpha)-induced apoptosis in 1321N1 human astrocytoma cells. This protective effect was blocked by the P2Y(12) receptor antagonist 2-methylthioAMP and by inhibitors of phospholipase C (U73122) and protein kinase C (chelerythrin), but not by the P2Y(1) receptor antagonist MRS2179. Toward a greater mechanistic understanding, we showed that hP2Y(12) receptor activation by 10nM 2MeSADP, activates Erk1/2, Akt, and JNK by phosphorylation. However, at a lower protective concentration of 100pM 2MeSADP, activation of the hP2Y(12) receptor involves only phosphorylated Erk1/2, but not Akt or JNK. This activation is hypothesized as the major mechanism for the protective effect induced by P2Y(12) receptor activation. Apyrase did not affect the ability of TNFalpha to induce apoptosis in hP2Y(12)-1321N1 cells, suggesting that the endogenous nucleotides are not involved. These results may have important implications for understanding the signaling cascades that follow activation of P2Y(1) and P2Y(12) receptors and their opposing effects on cell death pathways.     

Extended pharmacological profiles of rat P2Y2 and rat P2Y4 receptors and their sensitivity to extracellular H+ and Zn2+ ions

1. Two molecularly distinct rat P2Y receptors activated equally by adenosine-5'-triphosphate (ATP) and uridine-5'-triphosphate (UTP) (rP2Y2 and rP2Y4 receptors) were expressed in Xenopus oocytes and studied extensively to find ways to pharmacologically distinguish one from the other. 2. Both P2Y subtypes were activated fully by a number of nucleotides. Tested nucleotides were equipotent at rP2Y4 (ATP=UTP=CTP=GTP=ITP), but not at rP2Y2 (ATP=UTP>CTP>GTP>ITP). For dinucleotides (ApnA, n=2-6), rP2Y4 was only fully activated by Ap4A, which was as potent as ATP. All tested dinucleotides, except for Ap2A, fully activated rP2Y2, but none were as potent as ATP. ATP gamma S and BzATP fully activated rP2Y2, whereas ATP gamma S was a weak agonist and BzATP was inactive (as an agonist) at rP2Y4 receptors. 3. Each P2Y subtype showed different sensitivities to known P2 receptor antagonists. For rP2Y2, the potency order was suramin>PPADS= RB-2>TNP-ATP and suramin was a competitive antagonist (pA2, 5.40). For rP2Y4, the order was RB-2>suramin>PPADS> TNP-ATP and RB-2 was a competitive antagonist (pA2, 6.43). Also, BzATP was an antagonist at rP2Y4 receptors. 4. Extracellular acidification (from pH 8.0 to pH 5.5) enhanced the potency of ATP and UTP by 8-10-fold at rP2Y4 but did not affect agonist responses at rP2Y2 receptors. 5. Extracellular Zn2+ ions (0.1-300 microM) coapplied with ATP inhibited agonist responses at rP2Y4 but not at rP2Y2 receptors. 6. These two P2Y receptors differ significantly in terms of agonist and antagonist profiles, and the modulatory activities of extracellular H+ and Zn2+ ions. These pharmacological differences will help to distinguish between rP2Y2 and rP2Y4 receptors, in vivo.     

Methanocarba modification of uracil and adenine nucleotides: high potency of Northern ring conformation at P2Y1, P2Y2, P2Y4, and P2Y11 but not P2Y6 receptors.

The potency of nucleotide antagonists at P2Y1 receptors was enhanced by replacing the ribose moiety with a constrained carbocyclic ring (Nandanan, et al. J. Med. Chem. 2000, 43, 829-842). We have now synthesized ring-constrained methanocarba analogues (in which a fused cyclopropane moiety constrains the pseudosugar ring) of adenine and uracil nucleotides, the endogenous activators of P2Y receptors. Methanocarba-adenosine 5'-triphosphate (ATP) was fixed in either a Northern (N) or a Southern (S) conformation, as defined in the pseudorotational cycle. (N)-Methanocarba-uridine was prepared from the 1-amino-pseudosugar ring by treatment with beta-ethoxyacryloyl cyanate and cyclization to form the uracil ring. Phosphorylation was carried out at the 5'-hydroxyl group through a multistep process: Reaction with phosphoramidite followed by oxidation provided the 5'-monophosphates, which then were treated with 1,1'-carbonyldiimidazole for condensation with additional phosphate groups. The ability of the analogues to stimulate phospholipase C through activation of turkey P2Y1 or human P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors stably expressed in astrocytoma cells was measured. At recombinant human P2Y1 and P2Y2 receptors, (N)-methanocarba-ATP was 138- and 41-fold, respectively, more potent than racemic (S)-methanocarba-ATP as an agonist. (N)-methanocarba-ATP activated P2Y11 receptors with a potency similar to ATP. (N)-Methanocarba-uridine 5'-triphosphate (UTP) was equipotent to UTP as an agonist at human P2Y2 receptors and also activated P2Y4 receptors with an EC(50) of 85 nM. (N)-Methanocarba-uridine 5'-diphosphate (UDP) was inactive at the hP2Y6 receptor. The vascular effects of (N)-methanocarba-UTP and (N)-methanocarba-UDP were studied in a model of the rat mesenteric artery. The triphosphate was more potent than UTP in inducing a dilatory P2Y4 response (pEC(50) = 6.1 +/- 0.2), while the diphosphate was inactive as either an agonist or antagonist in a P2Y6 receptor-mediated contractile response. Our results suggest that new nucleotide agonists may be designed on the basis of the (N) conformation that favors selectivity for P2Y1, P2Y2, P2Y4, and P2Y11 receptors.     

Novel consequences of voltage-dependence to G-protein-coupled P2Y1 receptors

BACKGROUND AND PURPOSE: Emerging evidence suggests that activation of G-protein-coupled receptors (GPCRs) can be directly regulated by membrane voltage. However, the physiological and pharmacological relevance of this effect remains unclear. We have further examined this phenomenon for P2Y1 receptors in the non-excitable megakaryocyte using a range of agonists and antagonists. EXPERIMENTAL APPROACH: Simultaneous whole-cell patch clamp and fura-2 fluorescence recordings of rat megakaryocytes, which lack voltage-gated Ca2+ influx, were used to examine the voltage-dependence of P2Y1 receptor-evoked IP3-dependent Ca2+ mobilization. RESULTS: Depolarization transiently and repeatedly enhanced P2Y1 receptor-evoked Ca2+ mobilization across a wide concentration range of both weak, partial and full, potent agonists. Moreover, the amplitude of the depolarization-evoked [Ca2+]i increase displayed an inverse relationship with agonist concentration, such that the greatest potentiating effect of voltage was observed at near-threshold levels of agonist. Unexpectedly, depolarization also stimulated an [Ca2+]i increase in the absence of agonist during exposure to the competitive antagonists A3P5PS and MRS2179, or the allosteric enhancer 2,2'-pyridylisatogen tosylate. A further effect of some antagonists, particularly suramin, was to enhance the depolarization-evoked Ca2+ responses during co-application of an agonist. Of several P2Y1 receptor inhibitors, only SCH202676, which has a proposed allosteric mechanism of action, could block ADP-induced voltage-dependent Ca2+ release. CONCLUSIONS AND IMPLICATIONS: The ability of depolarization to potentiate GPCRs at near-threshold agonist concentrations represents a novel mechanism for coincidence detection. Furthermore, the induction and enhancement of voltage-dependent GPCR responses by antagonists has implications for the design of therapeutic compounds.     

2-Alkylthio-substituted platelet P2Y12 receptor antagonists reveal pharmacological identity between the rat brain Gi-linked ADP receptors and P2Y12

The Gi-linked platelet ADP receptor, now designated as P2Y12, accounts for ADP-induced inhibition of adenylyl cyclase in platelets and certain clonal rat cell lines. The pharmacology of this receptor is well characterized. Based on the functional approach of [35S]GTPgammaS autoradiography, we recently disclosed the widespread presence of Gi-linked ADP receptors in the rat nervous system. Based on initial pharmacological analysis, these receptors were strikingly similar with P2Y12. Here, we extend this analysis by comparing the potencies of six 2-alkylthio-substituted ATP analogues, including the adenosine-aspartate conjugate 2-hexylthio-AdoOC(O)Asp2 and five AR-C compounds (AR-C67085, AR-C69931, AR-C78511, AR-C69581, AR-C70300) with wide range of affinities towards P2Y12, in reversing 2-methylthio-ADP stimulated G protein activity in rat brain sections and human platelet membranes. Closely matching pIC50 values (r2=0.99) revealed pharmacological similarity between the two receptors with one exception: AR-C67085 more avidly recognized the platelet P2Y12. Further analysis of the rat brain pIC50 data against those available for three of the AR-C compounds in reversing P2Y12-mediated adenylyl cyclase inhibition in rat platelets (r2=0.96) and rat C6 glioma cells (r2=1.00) demonstrated that the three P2Y receptors are pharmacologically indistinguishable. We conclude that the rat brain Gi-linked ADP receptors, as revealed using [35S]GTPgammaS autoradiography, correspond to P2Y12.     

P2Y(1) and P2Y(2) receptors are coupled to the NO/cGMP pathway to vasodilate the rat arterial mesenteric bed

1. To assess the role of nucleotide receptors in endothelial-smooth muscle signalling, changes in perfusion pressure of the rat arterial mesenteric bed, the luminal output of nitric oxide (NO) and guanosine 3',5' cyclic monophosphate (cGMP) accumulation were measured after the perfusion of nucleotides. 2. The rank order of potency of ATP and analogues in causing relaxation of precontracted mesenteries was: 2-MeSADP=2-MeSATP>ADP>ATP=UDP=UTP>adenosine. The vasodilatation was coupled to a concentration-dependent rise in NO and cGMP production. MRS 2179 selectively blocked the 2-MeSATP-induced vasodilatation, the NO surge and the cGMP accumulation, but not the UTP or ATP vasorelaxation. 3. mRNA encoding for P2Y(1), P2Y(2) and P2Y(6) receptors, but not the P2Y(4) receptor, was detected in intact mesenteries by RT-PCR. After endothelium removal, only P2Y(6) mRNA was found. 4. Endothelium removal or blockade of NO synthase obliterated the nucleotides-induced dilatation, the NO rise and cGMP accumulation. Furthermore, 2-MeSATP, ATP, UTP and UDP contracted endothelium-denuded mesenteries, revealing additional muscular P2Y and P2X receptors. 5. Blockade of soluble guanylyl cyclase reduced the 2-MeSATP and UTP-induced vasodilatation and the accumulation of cGMP without interfering with NO production. 6. Blockade of phosphodiesterases with IBMX increased 15-20 fold the 2-MeSATP and UTP-induced rise in cGMP; sildenafil only doubled the cGMP accumulation. A linear correlation between the rise in NO and cGMP was found. 7. Endothelial P2Y(1) and P2Y(2) receptors coupled to the NO/cGMP cascade suggest that extracellular nucleotides are involved in endothelial-smooth muscle signalling. Additional muscular P2Y and P2X receptors highlight the physiology of nucleotides in vascular regulation.     

Subtype specific internalization of P2Y1 and P2Y2 receptors induced by novel adenosine 5'-O-(1-boranotriphosphate) derivatives

P2Y-nucleotide receptors represent important targets for drug development. The lack of stable and receptor specific agonists, however, has prevented successful therapeutic applications. A novel series of P-boronated ATP derivatives (ATP-alpha-B) were synthesized by substitution of a nonbridging O at P(alpha) with a BH(3) group. This introduces a chiral center, thus resulting in diastereoisomers. In addition, at C2 of the adenine ring a further substitution was made (Cl- or methylthio-). The pairs of diastereoisomers were denoted here as A and B isomers. Here, we tested the receptor subtype specificity of these analogs on HEK 293 cells stably expressing rat P2Y(1) and rat P2Y(2) receptors, respectively, both attached to the fluorescent marker protein GFP (rP2Y(1)-GFP, rP2Y(2)-GFP). We investigated agonist-induced receptor endocytosis, [Ca(2+)](i) rise and arachidonic acid (AA) release. Agonist-induced endocytosis of rP2Y(1)-GFP was more pronounced for the A isomers than the corresponding B counterparts for all ATP-alpha-B analogs. Both 2-MeS-substituted diastereoisomers induced a greater degree of agonist-induced receptor endocytosis as compared to the 2-Cl-substituted derivatives. Endocytosis results are in accordance with the potency to induce Ca(2+) release by these compounds in HEK 293 cells stably transfected with rP2Y(1). In case of rP2Y(2)-GFP, the borano-nucleotides were very weak agonists in comparison to UTP and ATP in terms of Ca(2+) release, AA release and in inducing receptor endocytosis. The different ATP-alpha-B derivatives and also the diastereoisomers were equally ineffective. Thus, the new agonists may be considered as potent and highly specific agonist drug candidates for P2Y(1) receptors. The difference in activity of the ATP analogs at P2Y receptors could be used as a tool to investigate structural differences between P2Y receptor subtypes.     

Desensitization of endothelial P2Y1 receptors by PKC-dependent mechanisms in pressurized rat small mesenteric arteries

Background and purpose:

Extracellular nucleotides play a crucial role in the regulation of vascular tone and blood flow. Stimulation of endothelial cell P2Y1 receptors evokes concentration-dependent full dilatation of resistance arteries. However, this GPCR can desensitize upon prolonged exposure to the agonist. Our aim was to determine the extent and nature of P2Y1 desensitization in isolated and pressurized rat small mesenteric arteries.

Experimental approach:

The non-hydrolyzable selective P2Y1 agonist ADPβS (3 µM) was perfused through the lumen of arteries pressurized to 70 mmHg. Changes in arterial diameter and endothelial cell [Ca²⁺]_i were obtained in the presence and absence of inhibitors of protein kinase C (PKC).

Key results:

ADPβS evoked rapid dilatation to the maximum arterial diameter but faded over time to a much-reduced plateau closer to 35% dilatation. This appeared to be due to desensitization of the P2Y1 receptor, as subsequent endothelium-dependent dilatation to acetylcholine (1 µM) remained unaffected. Luminal treatment with the PKC inhibitors BIS-I (1 µM) or BIS-VIII (1 µM) tended to augment concentration-dependent dilatation to ADPβS (0.1–3 µM) and prevented desensitization. Another PKC inhibitor, Gö 6976 (1 µM), was less effective in preventing desensitization. Measurements of endothelial cell [Ca²⁺]_i in pressurized arteries confirmed the P2Y1 receptor but not M₃ muscarinic receptor desensitization.

Conclusions and implications:

These data demonstrate for the first time the involvement of PKC in the desensitization of endothelial P2Y1 receptors in pressurized rat mesenteric arteries, which may have important implications in the control of blood flow by circulating nucleotides.