New heterocyclic ligands for the adenosine receptors P1 and for the ATP receptors P2

Extracellular adenosine and adenine nucleotides induce various cellular responses through activation of P1 and P2 receptors. P1 receptors preferentially recognize adenosine and four different G protein-coupled receptors (A(1), A(2A), A(2B), and A(3) subtypes) have been identified. On the other hand, P2 receptors are activated by adenine and/or uridine nucleotides and classified into two families: ionotropic P2X and G protein-coupled P2Y receptors. In this article, we summarize our studies which led to development of new potent and selective heterocyclic ligands for the adenosine receptors P1 and for the ATP receptors P2X(7).     

Vasoconstrictor and vasodilator effects of guanine nucleotides in the rat aorta

Although GTP, like ATP and UTP, is stored in platelet dense granules, little is known about its vascular effects. The present study was carried out in order to characterize the effects of GTP and related compounds in the rat aorta. Contractions were examined in aortic rings at resting tension. In rings with intact endothelium, GTP, GDP, guanosine 5’-O-(3-thiotriphosphate) (GTPγS) and guanosine 5’-O-(2-thiodiphosphate) (GDPβS) caused small contractions. In endothelium-denuded rings, the contractions were unchanged or increased and persisted after desensitization of P2X-receptors by α,β-methylene ATP. Relaxations were examined in aortic rings precontracted with noradrenaline. In rings with intact endothelium, GTP (EC₅₀ 131μM), GDP (no maximal effect obtained), GTPγS (EC₅₀ 6.8μM) and guanosine (EC₅₀ 822μM) caused prominent relaxation, whereas GDPβS caused further contraction. In endothelium-denuded rings, the relaxant effect of GTP was greatly reduced, that of GDP and guanosine was unchanged, and that of GTPγS was abolished. Relaxations by GTP and GTPγS in endo-thelium-intact rings were studied in more detail. The relaxation by GTP was slightly and the relaxation by GTPγS greatly reduced after treatment with N^G-nitro-L-arginine methyl ester. Pre-exposure to a high concentration of the P2Y-receptor agonist 2-methylthio ATP (MeSATP) did not attenuate the effects of GTP and GTPγS. Four compounds previously identified as antagonists at the P2Y- and P2U-receptors of rat aortic endothelium – suramin, reactive blue 2, pyridoxalphosphate-6-azophenyl-2’,5’-disulphonate (iso-PPADS) and 5,5’-(1,1’-biphenyl-4,4’-diylbisazo)-bis-7-amino-6-hydroxy-naphthalene-1,4-disulphonate (NH05) – were tested against GTP and GTPγS. Suramin, reactive blue 2 and iso-PPADS were much less potent against GTP and GTPγS than previously found against (the P2Y effect of) MeSATP. Suramin, iso-PPADS and NH05 were about as potent against GTP and GTPγS as previously found against (the P2U effect of) UTP and in particular ATP. It is concluded that guanine nucleotides can cause both contraction and relaxation of the rat aorta. The high concentrations of GTP and GDP required, and in the case of contraction the small size of the response, make a physiological role of the vascular effects of these nucleotides unlikely. GTP and GTPγS elicit endothelium-dependent relaxation through P2U-receptors. GTP in addition relaxes the aorta through smooth muscle receptors, possibly by way of its degradation product guanosine. The stable analog GTPγS is a relatively potent and selective agonist for the endothelial P2U-receptor. Received: 21 March 1997 / Accepted: 31 July 1997     

P2Y receptors of MDCK cells: epithelial cell regulation by extracellular nucleotides

1. Madin-Darby canine kidney (MDCK) cells, a well- differentiated renal epithelial cell line derived from distal tubule/collecting duct, respond to extracellular nucleotides by altering ion flux and the production of arachidonic acid-derived products, in particular prostaglandin E2 (PGE2). Our work has defined the receptors and signalling events involved in such responses. 2. We have found evidence for expression of at least three P2Y receptor subtypes (P2Y1, P2Y2 and P2Y11) in MDCK-D1 cells, a subclone from parental MDCK. 3. These receptors appear to couple to increases in calcium and protein kinase C activity, probably via a Gq/G11-mediated activation of phospholipase C. 4. In addition, P2Y receptor activation can promote a prominent increase in cAMP. This includes both a P2Y2 receptor-mediated cyclo-oxygenase (COX)-dependent component and another COX-independent component mediated by other P2Y receptors. 5. We have documented that changing media in which cells are grown releases ATP and, in turn, activates P2Y receptors. Such release of ATP contributes in a major way to basal cAMP levels in these cells. 6. The data indicate that MDCK cells are a useful model to define the regulation of epithelial cells by extracellular nucleotides. Of particular note, spontaneous or stretch-induced release of ATP and subsequent activation of one or more P2Y receptors contributes to establishing the basal activity of signalling pathways.     

Response of the guinea-pig urinary bladder to purine and pyrimidine nucleotides

The ability of purines and pyrimidines to cause and inhibit contractile responses was examined in strips of guinea-pig urinary bladder. Adenosine 5′-triphosphate (ATP), β, γ-methylene ATP (APPCP), adenosine 5′-diphosphate (ADP), guanosine 5′-triphosphate (GTP) and cytidine 5′-triphosphate (CTP) caused concentration-dependent contractions of the bladder. The order of potency was APPCP > ATP > GTP=CTP > ADP. Adenosine 5′-monophosphatedenosine, guanosine 5′-diphosphate, guanosine, 5′-monophosphate, guanosine, cytidine 5′-disphosphate, cytidine 5′-monophosphate and cytidine had no apparent contractile activity up to 10⁻ M. Cumulative administrations of 5 × 10⁻⁴ M APPCP, ATP, ADP, GTP or CTP resulted in complete desensitization of the tissue to the contractile activity of the nucleotide. Tissues desensitized to GTP or CTP were non-responsive to ATP suggesting cross-tachyphylaxis. Nucleotides lacking contractile activity or nucleosides did not alter the response of the bladder to ATP; except AMP and adenosine which significantly reduced the contraction. These results suggest that the polyphosphate structure, and not the base, is the determining portion of the nucleotide for promoting contractile activity and the development of tachyphylaxis.     

P2X4 receptors interact with both P2X2 and P2X7 receptors in the form of homotrimers

BACKGROUND AND PURPOSE

The P2X receptor family consists of seven subunit types – P2X1–P2X7. All but P2X6 are able to assemble as homotrimers. In addition, various subunit permutations have been reported to form heterotrimers. Evidence for heterotrimer formation includes co-localization, co-immunoprecipitation and the generation of receptors with novel functional properties; however, direct structural evidence for heteromer formation, such as chemical cross-linking and single-molecule imaging, is available in only a few cases. Here we examined the nature of the interaction between two pairs of subunits – P2X2 and P2X4, and P2X4 and P2X7.

EXPERIMENTAL APPROACH

We used several experimental approaches, including in situ proximity ligation, co-immunoprecipitation, co-isolation on affinity beads, chemical cross-linking and atomic force microscopy (AFM) imaging.

KEY RESULTS

Both pairs of subunits co-localize upon co-transfection, interact intimately within cells, and can be co-immunoprecipitated and co-isolated from cell extracts. Despite this, chemical cross-linking failed to show evidence for heteromer formation. AFM imaging of isolated receptors showed that all three subunits had the propensity to form receptor dimers. This self-association is likely to account for the observed close interaction between the subunit pairs, in the absence of true heteromer formation.

CONCLUSIONS AND IMPLICATIONS

We conclude that both pairs of receptors interact in the form of distinct homomers. We urge caution in the interpretation of biochemical evidence indicating heteromer formation in other cases.     

Interaction of P2 purinergic receptors with cellular macromolecules

Ionotropic P2X and metabotropic P2Y receptors interact with a number of macromolecules in the cell membrane which may contribute to their functional plasticity. P2X receptors are homomeric or heteromeric assemblies of three subunits. P2Y receptors may form oligomeric complexes either with the same or with other P2Y receptor types. Although the signalling mechanism of P2X receptor channels is fast (within milliseconds) and relatively simple, by originating from the opening of an ion channel permeable to mono- and divalent cations, various macromolecules may modify the trafficking of these receptors to and from the cell membrane, as well as their activation and desensitization kinetics, and the possible opening of membrane pores induced by long-lasting exposure to agonists. P2X and Cys-loop receptors may physically interact with each other, resulting in mutual current occlusion. Heteromeric P2Y receptors may, via G_s, G_q/11 or G_i/o protein-coupling and activation of the respective transduction mechanisms, mediate responses in the range of a few seconds. However, P2Y receptors may also interact with the signalling cascade of, e.g. receptor tyrosine kinases, and thereby mediate responses on a much slower time scale (within hours to days). In addition, P2Y receptors may interact with small, homomeric G proteins, integrins, and PDZ proteins. Eventually, P2Y receptors may cross-talk via Gα-dependent signalling with other G protein-coupled receptors and via Gβγ (or indirectly Gα)-dependent signalling with various ion channels. Thus, the activation of P2X and P2Y receptors by extracellular adenosine triphosphate/adenosine diphosphate or uridine triphosphate/uridine diphosphate may trigger specific chains of events which interact at the level of the individual elements both with each other and with the transduction mechanisms of other receptors, creating a huge diversity of the possible effects.     

P2Y receptors and atherosclerosis in apolipoprotein E-deficient mice

Background and purpose:

P2Y nucleotide receptors are involved in the regulation of vascular tone, smooth muscle cell (SMC) proliferation and inflammatory responses. The present study investigated whether they are involved in atherosclerosis.

Experimental approach:

mRNA of P2Y receptors was quantified (RT-PCR) in atherosclerotic and plaque-free aorta segments of apolipoprotein E-deficient (apoE^–/–) mice. Macrophage activation was assessed in J774 macrophages, and effects of non-selective purinoceptor antagonists on atherosclerosis were evaluated in cholesterol-fed apoE^–/– mice.

Key results:

P2Y₆ receptor mRNA was consistently elevated in segments with atherosclerosis, whereas P2Y₂ receptor expression remained unchanged. Expression of P2Y₁ or P2Y₄ receptor mRNA was low or undetectable, and not influenced by atherosclerosis. P2Y₆ mRNA expression was higher in cultured J774 macrophages than in cultured aortic SMCs. Furthermore, immunohistochemical staining of plaques demonstrated P2Y₆-positive macrophages, but few SMCs, suggesting that macrophage recruitment accounted for the increase in P2Y₆ receptor mRNA during atherosclerosis. In contrast to ATP, the P2Y₆-selective agonist UDP increased mRNA expression and activity of inducible nitric oxide synthase and interleukin-6 in J774 macrophages; this effect was blocked by suramin (100–300 µM) or pyridoxal-phosphate-6-azophenyl-2′-4′-disulphonic acid (PPADS, 10–30 µM). Finally, 4-week treatment of cholesterol-fed apoE^–/– mice with suramin or PPADS (50 and 25 mg·kg⁻¹·day⁻¹ respectively) reduced plaque size, without changing plaque composition (relative SMC and macrophage content) or cell replication.

Conclusions and implications:

These results suggest involvement of nucleotide receptors, particularly P2Y₆ receptors, during atherosclerosis, and warrant further research with selective purinoceptor antagonists or P2Y₆ receptor-deficient mice.     

嘌呤P2受体的分子生物学研究进展

嘌呤P2受体家族是目前发现的最复杂的受体家族之一,在体内分布广泛,功能复杂,目前已有7种P2X受体和8种P2Y受体被克隆。P2受体的分类和研究历史非常复杂,随着分子生物技术的发展,P2受体的研究取得了明显的进展,特别是新克隆出的P2Y_(12),P2Y_(13)和P2Y_(14)受体,丰富了P2受体家族,使其结构特征、药理学特性不断被揭示。本文对P2受体的研究历史、分类方法、分子结构特征和药理学特性做一概述,并对最新报道的P2受体的相互作用以及关于P2Y_(15)受体的争论进行综述。     

Characterization by antagonists of P2-receptors mediating endothelium-dependent relaxation in the rat aorta

The receptors through which 2-methylthio ATP (MeSATP), adenosine 5′-O-(2-thiodiphosphate) (ADPβS), UTP and ATP elicit endothelium-dependent relaxation of noradrenaline-precontracted rings of the rat aorta were characterized by means of a series of antagonists. The acetylcholine-induced relaxation and the degradation of MeSATP, UTP and ATP were also studied. The potency of the nucleotides at producing relaxation decreased in the order MeSATP (EC₅₀ 0.24 μM) > ADPβS (0.43 μM) > UTP (1.09 μM) > ATP (3.53 μM). MeSATP, ADPβS and UTP did not cause relaxation when the endothelium had been destroyed; high concentrations of ATP still caused some relaxation. The relaxation by MeSATP, ADPβS and UTP became very small after treatment of the rings with N^G-nitro-l-arginine methyl ester; the relaxation by ATP was less affected. Pre-exposure to MeSATP (100 μM) abolished or almost abolished the relaxation normally elicited by MeSATP and ADPβS, did not change that elicited by UTP and slightly enhanced the relaxation elicited by ATP. Of nine compounds examined as antagonists, six attenuated selectively the effect of some or all of the nucleotides (as compared to acetylcholine): suramin, reactive blue 2, pyridoxalphosphate-6-azophenyl-2′,5′-disulphonate (iso-PPADS), pyridoxalphosphate-6-azophenyl-2′,4′-disulphonate (PPADS), reactive red 2 and 5,5′-(1,1′-biphenyl-4,4′-diylbisazo)-bis-7-amino-6-hydroxy-naphthalene-1,4-disulphonate (NH05). Decreases of maximal relaxations and slopes different from unity in Schild plots often indicated non-competitive kinetics of the antagonism. For each of the six ‘selective’ antagonist, the apparent K _d values against MeSATP and against ADPβS were similar: none of the six differentiated between MeSATP and ADPβS. Also, for each of four ‘selective’ antagonists, the apparent K _d values against UTP and against ATP were similar: none of the four differentiated between these two nucleotides (two antagonists did not act against UTP and ATP in the ‘selective’ concentration range). On the other hand, for five of the six ‘selective’ antagonists (the exception being NH05), the apparent K _d values against MeSATP and ADPβS were considerably lower than those against UTP and ATP. At the highest concentrations tested against agonist-evoked relaxations, the antagonists did not alter the removal from the incubation medium, by pieces of rat aorta, of MeSATP, UTP and ATP. It is concluded that nucleotides cause endothelium-dependent relaxation of the rat aorta through two sites: a P2Y-receptor and a P2U-receptor. The receptors may be pharmacologically similar to a bovine endothelial P2Y (P2Y₁) and a cloned rat P2U (P2Y₂) receptor, respectively. ATP acts mainly through the P2U-receptor. Suramin, reactive blue 2, iso-PPADS, PPADS and reactive red 2 are more potent at the P2Y- than the P2U-receptor. NH05 does not discriminate between the two receptors but is the most potent P2U antagonist so far described. Received: 21 March 1997 / Accepted: 31 July 1997     

Evidence for two separate vasoconstriction-mediating nucleotide receptors,both distinct from the P2X-receptor,in rabbit basilar artery: a receptor for pyrimidine nucleotides and a receptor for purine nucleotides

Summary Uridine 5-triphosphate- (UTP-) and adenosine 5-triphosphate-(ATP) induced vasoconstriction was studied in the rabbit basilar artery. The arteries were incubated and perfused at a constant rate of flow. Vasoconstriction was measured as an increase in perfusion pressure.Serotonin, histamine and noradrenaline caused concentration-dependent vasoconstriction, with potency decreasing in that order. Of the nucleotides tested, UTP, UDP, UMP, CTP, ATP, ADP, adenosine 5-O-(3-thio)triphosphate (ATPS), and ,-imido adenosine 5-triphosphate (AMP-PNP) elicited concentration-dependent vasoconstriction, whereas AMP, 2-methylthio-ATP, , -methylene-ATP and ,-methylene-ATP up to 10^–3 mol/l caused no or only a very small increase in perfusion pressure. The order of potency of the pyrimidine nucleotides was: UTP = UDP UMP = CTP; that of the purine nucleotides was: ATPS > AMP-PNP > ATP > ADP > 2-methylthio-ATP = , -methylene-ATP = ,-methylene-ATP. The vasoconstrictor effects of UTP and ATP were not or only to a minor degree influenced by: phentolamine; a mixture of atropine, diphenhydramine and methysergide; indometacin; nordihydroguaiaretic acid; denervation by 6-hydroxydopamine; or mechanical removal of endothelium. Prolonged exposure to ,-methylene-ATP elicited only a very small vasoconstriction and did not change the constrictor effects of UTP or ATP. Prolonged exposure to ATPS elicited marked vasoconstriction; subsequently, responses to ATP were reduced whereas those to UTP were, if anything, slightly enhanced. Reactive blue 2 reduced neither the UTP- nor the ATP-induced vasoconstriction. ATP 10^–3 mol/l elicited marked additional vasoconstriction after precontraction with UTP 10^–3 mol/l, whereas UTP elicited only a very small additional vasoconstriction when its concentration was doubled from 10^–3 to 2 × 10^–3 mol/l.It is concluded that, in the rabbit basilar artery, the vasoconstrictor response to UTP is mediated by a pyrimidine nucleotide receptor which is distinct from the P₂-purinoceptor, and that the vasoconstrictor response to ATP is mediated by a P₂-receptor which is distinct from the known P₂-subtypes.Send offprint requests to I. v. Kügelgen at the above address     

神经病理痛动物模型及其在P2X受体介导痛觉研究中的应用

神经病理痛是临床上常见病症,对人身心健康危害较大,其发病机制尚不清楚,目前无有效的治疗手段,加之慢性神经病理痛持续时间长,其研究成为疼痛领域的热点和重点。该文综述的多种神经病理痛的动物模型,复制了人类神经病理痛的各种症状,是研究神经病理痛的有效手段。三磷酸腺苷(ATP)是一种重要的疼痛信号物质,ATP可作用于P2X受体产生效应。应用神经病理痛动物模型,观察到P2X受体在神经病理痛的痛觉形成、传导和调节中有着重要作用,有望成为神经病理痛治疗的新作用位点。     

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.     

Roles of ectodomain and transmembrane regions in ethanol and agonist action in purinergic P2X2 and P2X3 receptors

The present work investigated sites of ethanol action in ATP-gated P2X receptors (P2XRs) using chimeric strategies that exploited the differences in ethanol response between P2X2R (inhibition) and P2X3R (potentiation). We tested ethanol (10–200 mM) effects on ATP- and α,β-methylene-ATP (α,β-meATP)-induced currents in wildtype P2X2, P2X3 and chimeric P2X2/P2X3Rs expressed in Xenopus oocytes using two-electrode voltage-clamp (−70 mV). Exchanging ectodomain regions of P2X2 and P2X3Rs reversed wildtype ethanol responses. Substituting back portions of the P2X2R ectodomain at TM interfaces in chimeras that contained the P2X3R ectodomain restored wildtype P2X2R-like ethanol response. Point mutations that replaced non-conserved ectodomain residues at TM interfaces of P2X3Rs with homologous P2X2R residues identified positions that reversed the direction (304) or changed the magnitude (53, 55 and 313) of ethanol response. Homologous substitutions in P2X2Rs did not significantly alter wildtype P2X2R-like ethanol responses. These findings suggest that ectodomain segments at TM interfaces play key roles in determining qualitative and quantitative responses to ethanol of P2X2 and P2X3Rs. Studies that substituted TM regions of P2X3R with respective P2X2R TMs indicate that the TM1, but not the TM2, region plays a role in determining the magnitude of ethanol response. Studies with ATP and α,β-meATP support prior indications that TM regions are important in agonist desensitization and suggest that both ectodomain and TM regions play roles in determining agonist potency and selectivity. Overall, these findings are the first to identify potential targets for ethanol in P2X2 and P2X3Rs and should provide insight into the sites of ethanol action in other P2XRs.     

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.     

Enhancement of the response to purinergic agonists in P2Y1 transfected 1321N1 cells by antagonists suramin and PPADS

1. We have previously shown that both suramin and pyridoxal-phosphate-6-azophenyl-2′, 4′ disulphonic acid (PPADS) act as antagonists at transfected P2Y₁ receptors. Here we show that under certain experimental conditions these two P2 antagonists can enhance the response to agonists acting at these receptors.
2. The expression of either P2Y₁ or P2Y₂ receptors in 1321N1 human astrocytoma cells results, on a change of medium, in an elevation of basal (no added agonist) accumulation of [³H]-inositol(poly)phosphates([³H]-InsP_x) compared to cells not expressing these receptors. This elevation is much greater in P2Y₁ transfectants than in P2Y₂ transfectants.
3. Both PPADS and suramin reduced this basal level of [³H]-InsP_x accumulation in the P2Y₁ expressing cells.
4. When a protocol was used which required changing the culture medium, antagonists were added at a concentration which reduced the basal accumulation by about 50%, there was a significant stimulation in response to increasing concentrations of 2-methylthioadenosine 5′-triphosphate (2MeSATP), in the absence of antagonists there was no significant effect of the agonist.
5. However, when 2MeSATP was added in the absence of a change of medium and with no antagonist present, there was a several fold increase in [³H]-InsP_x accumulation. These results show that a release of endogenous agonist activity (possibly ATP/ADP) from the P2Y₁ expressing cells can create conditions in which a response to an agonist such as 2MeSATP can only be seen in the presence of a competitive antagonist.

     

P2X7 receptors contribute to the currents induced by ATP in guinea pig intestinal myenteric neurons

The whole-cell configuration, several pharmacological tools, and single-cell RT-PCR were used to investigate the contribution of P2X7 subunits to the ATP-induced currents (I_ATP) in guinea pig myenteric neurons. I_ATP was recorded in the great majority of tested neurons. ATP concentration-response curve (0.01–10 mM) showed two phases, the first mediated by high-sensitive P2X receptors (hsP2X receptors), observed between 0.01–0.3 mM and the second mediated by low-sensitive P2X receptors (lsP2X receptors). The calculated EC₅₀ values of these phases were 38 and 1759 μM, respectively. 2′-3′-O-(4-benzoylbenzoyl)-ATP (BzATP) concentration-response curve was monophasic (0.01–1 mM), and less potent (EC₅₀ 142 μM) than ATP to activate hsP2X receptors. A strong inward rectification was noticed when hsP2X receptors were activated with ATP (0.1 mM) and for BzATP-induced currents (0.1 mM; I_BzATP) but a significant lower rectification was noticed when lsP2X receptors were activated (5 mM). Brilliant blue G (BBG) at a concentration of 0.3 μM (known to inhibit only P2X7 receptors) reduced I_ATP when lsP2X receptors contributed to it but neither affect hsP2X receptors nor I_BzATP. However, hsP2X receptors and I_BzATP were both inhibited by concentrations ≥ 1 μM of this antagonist. BzATP inhibited hsP2X receptors and therefore, it behaves as partial agonist on these receptors. Using the single-cell RT-PCR technique P2X7 mRNA was detectable in 7 out of 13 myenteric neurons exhibiting P2X2 mRNA. Altogether, our results show that low-sensitive P2X receptors are likely P2X7, whereas, the high-sensitive P2X channels are probably constituted, at least in part, by P2X2 subunits.