Comparative studies on the affinities of ATP derivatives for P2X-purinoceptors in rat urinary bladder (original) (raw)
Adenosine triphosphate is full antagonist at human P2Y1 purinoceptors
Neuroscience Letters, 2000
Both agonistic and antagonistic effects have been reported for ATP at P2Y 1 purinoceptors at micromolar ligand concentrations. These con¯icting data hamper speci®cation of the true pharmacological pro®le as well as structural requirements for antagonistic ligands of this receptor. In this report the type of ATP activity at human P2Y 1 receptors in hP2Y 1 ±1321N1 cells was revisited. In parallel, kinetics of degradation of ATP in the assay mixture was analysed. It was found that transformation of this ligand to ADP was responsible for initiation of synthesis of inositol phosphates, observed in the presence of ATP in hP2Y 1 ±1321N1 cells. This agonistic effect was abolished in the presence of the triphosphate regeneration system (CP/CPK). On the other hand, if the agonistic effect caused by degradation product of ATP was taken into consideration, this ligand behaved as a full antagonist at P2Y 1 receptors and was characterized by the apparent inhibitory constant 5 mM. q
Role of ATP and related purines in inhibitory neurotransmission to the pig urinary bladder neck
British Journal of Pharmacology, 2009
As adenosine 5′-triphosphate (ATP) is one of the inhibitory mediators of the bladder outflow region, this study investigates the possible release of ATP or related purines in response to electrical field stimulation (EFS) and the purinoceptor(s) involved in nerve-mediated relaxations of the pig urinary bladder neck. Experimental approach: Urothelium-denuded and intact phenylephrine-precontracted strips were mounted in organ baths containing physiological saline solution at 37°C and gassed with 95% O2 and 5% CO2 for isometric force recordings. Key results: EFS, in the presence of atropine, guanethidine and N G -nitro-L-arginine, and exogenous purines, produced frequency-and concentration-dependent relaxations respectively. Adenosine 5′-diphosphate (ADP) and adenosine were more potent than ATP in producing relaxation, while uridine 5′-triphosphate, uridine 5′-diphosphate and a,b-methylene ATP were less effective. The non-selective P2 antagonist suramin, and the P2Y1 and P1 receptor blockers 2′-deoxy-N6-methyladenosine 3′,5′-bisphosphate tetrasodium and 8-(p-sulphophenyl)theophylline, respectively, inhibited the responses to EFS and ATP. The P1 agonist's potency was: 5′-N-ethylcarboxamidoadenosine (NECA)>4-2[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzene propanoic acid hydrochloride>2-chloro-N 6 -cyclopentyladenosine>-2-chloro-6-[[(3iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-b-D-ribofuranuronamide = adenosine. 4-(-[7-amino-2-(2-furyl) [1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl) phenol, an A2A antagonist, reduced the relaxations to EFS, adenosine and NECA.
The effect of the neuropeptide substance P on desensitization of ATP receptors of PC12 cells
British Journal of Pharmacology, 1997
1 Patch clamp recording (whole cell con®guration) was employed to investigate the modulatory action of substance P on inward currents elicited by adenosine 5'-triphosphate (ATP, focally applied via a pressure pipette) from phaechromocytoma (PC12) cells usually held at 770 mV. 2 Bath-applied substance P (0.2 ± 20 mM) had no eect on baseline membrane current but reversibly reduced ATP peak currents in a concentration-dependent fashion. The depressant eect was not associated with a change in the ATP current reversal potential.
Review: Ca2+-mobilizing receptors for ATP and UTP
Cell Calcium, 1995
Extracellular nucleotides are potent Ca2+ mobilizing agents. A variety of receptors for extracellular ATP are recognised. Some are involved in fast neuronal transmission and operate as ligand-gated ion channels. Others are involved in the paracrine or autocrine modulation of cell function. Many receptors of this type are coupled to phosphoinositide-specific phospholipase C and, in some cases, other phospholipases. One of these receptors (P24, however, also appears to operate, at least in part, as a ligand-gated ion channel. Pharmacological data suggest that one nucleotide receptor subtype (currently designated P2u) responds selectively to either a purine nucleotide, ATP, or a pyrimidine nucleotide, UTP. According to an alternative view, ATP and UTP recognise distinct receptors. Because of the diversity of receptors for extracellular nucleotides this may be the case in some cells. Nevertheless, a G-protein coupled receptor that confers both ATP and UTP sensitivity has been cloned, expressed in cultured cell lines and sequenced. This receptor appears to have two ligand binding domains that may partially overlap. The nature of this overlap is discussed and a simple model presented. Activation of the receptor protein via one or other ligand binding domain may underlie some of the more subtle differences between the effects of ATP and UTP. Extracellular nucleotides Nucleotides are generally viewed as intracellular metabolites. They have long been recognised as im-Ahbreviarions: PI-PLC (phosphoinositide-specific phospholipase C); Ins 1,4,5 P.1 (inositol 1,4,5 trisphosphate); BzATP (3'-0-(4-benzoyl)benzoylATP); 2-MeSATP (2-methyithioATP); AMPCPP (a$-methylene ATP); GTP (guanosine 5'-triphosphate); ITP (inosine S-triphosphate); CTP (cytidine 5'-triphosphate), TTP (thymidine 5'-triphosphate.
A comparison of the binding characteristics of recombinant P2×1 and P2×2 purinoceptors
British Journal of Pharmacology, 1996
We have recently provided evidence that [35S]-adenosine 5'-O-[3-thiotriphosphate] ([35S]-ATPyS) can label the human bladder recombinant P2XI purinoceptor (human P2x1 purinoceptor). In this study we have characterized the binding of [35S]-ATPyS to a second P2x purinoceptor subtype, the rat PC12 phaeochromocytoma cell recombinant P2X2 purinoceptor (rat P2x2 purinoceptor), and compared its binding properties with those of both endogenous and recombinant P2X1 purinoceptors. 2 Infection of CHO-KI cells with the rat P2X2 purinoceptor using Semliki forest virus (SFV) resulted in the expression of high affinity (pKd = 9.3; Bm,,, = 18.1 pmol mg-1 protein) binding sites for [35S]-ATPyS but not for [3H]-a,fi-methylene ATP ([3H]-acfmeATP). Since functional P2X purinoceptors could be detected electrophysiologically in these cells, but not in non-infected or CHO-KI cells infected with SFV containing the LacZ gene, these results suggest that the rat P2X2 purinoceptor can be labelled using [35S]-ATPyS. 3 The binding characteristics of the rat P2X2 purinoceptor were compared with those of the human P2x1 purinoceptor, which was also expressed in the CHO-Kl cells using SFV. A major difference between the two recombinant P2X purinoceptor types was in the binding characteristics of a,#-methylene ATP (aBmeATP). Thus, in the absence of divalent cations, ameATP possessed low affinity for both the human P2X1 purinoceptor (pIC50 = 7.2) and rat P2x2 purinoceptor (pIC5o = 7.1) labelled using [35S]-ATPyS. However, when the recombinant P2X purinoceptors were labelled with [3H]-afimeATP in the presence of 4 mM CaCl2, the affinity of afmeATP for the human P2xI purinoceptor increased (pIC5m for ameATP = 8.2), while the affinity of the rat P2X2 purinoceptor for cxfimeATP did not change (pIC50 for axfmeATP = 6.8). 4 Affinity estimates of 15 other nucleotide analogues for the [35S]-ATPyS binding sites on the two recombinant P2X purinoceptor subtypes were surprisingly similar (less than 5 fold difference), the only exception being 2'-deoxy ATP which possessed 8 fold higher affinity for rat P2x2 than for human P2XI purinoceptors. In contrast dextran sulphate and the P2 purinoceptor antagonists, pyridoxalphosphate-6azophenyl-2',4'-disulphonic acid and 4,4'-diisothiocyanatostilbene-2,2'disulphonic acid, possessed 7 to 33 fold higher affinity for the human P2XI than for the rat P2X2 purinoceptor. These data provide a correlation coefficient (r) of 0.894. 5 There was some evidence for species differences in the P2x1 purinoceptor. Thus, most nucleotides possessed slightly greater (up to 9-10 fold), while the P2 purinoceptor antagonists possessed slightly lower (up to 7-16 fold), affinity for the endogenous rat vas deferens and rat bladder P2XI purinoceptors than for the human recombinant P2Xl purinoceptor. These differences were reflected in a slightly lower correlation coefficient, when comparing across species between the human recombinant P2X1 purinoceptor and the endogenous P2XI purinoceptors labelled in either the rat deferens (r = 0.915) or the rat bladder (r = 0.932), than when comparing within species between the endogenous rat vas deferens and rat bladder P2Xl purinoceptors (r = 0.995). 6 In summary, [35S]-ATPyS can be used to label the recombinant P2X1 and P2x2 purinoceptors. Despite the marked differences reported between these two forms of P2X purinoceptor in functional studies, the differences in binding studies were more limited. However, a number of antagonists could discriminate between the P2X purinoceptor subtypes in the binding studies raising expectations that selective antagonists for these receptors can be developed.
P2X-purinoceptors in the heart: Actions of ATP and UTP
Life Sciences, 1997
Positive inotropic effects of ATP and UTP (1 pM -In&Q were studied in isolated rat and guinea pig cardiac tissues. The potency order obtained was ATPWTE' in both species, suggesting possible interaction with P2Xpurinoceptors. Binding studies using [3H]a$-methylene A'I'P as marker of P2X-purinoceptors revealed two receptor sites: one high-, the other low-affinity, in atria and ventricles from rat and guinea pig. Both ATP and UTP were found to bind high&inity sites of [3H]a,pmethylene ATP. The effects of various calcium inhibitors such as nifdipine, dantrolene, ryanodine and TMB-8 on positive inotropic effects induced by ATP and UTP were also studied. The results suggest that ATP and UTP may increase inotropism by interaction with PZX-purinoceptors by means of a calcium-dependent mechanism.
British Journal of Pharmacology, 1996
A series of chain-extended 2-thioether derivatives of adenosine monophosphate were synthesized and tested as agonists for activation of the phospholipase C-linked P2Y-purinoceptor of turkey erythrocyte membranes, the adenylyl cyclase-linked P2Y-purinoceptor of C6 rat glioma cells, and the cloned human P2u-receptor stably expressed in 132INI human astrocytoma cells. 2 Although adenosine monophosphate itself was not an agonist in the two P2Y-purinoceptor test systems, eleven different 2-thioether-substituted adenosine monophosphate analogues were full agonists. The most potent of these agonists, 2-hexylthio AMP, exhibited an EC50 value of 0.2 nM for activation of the C6 cell receptor. This potency was 16,000 fold greater than that of ATP and was only 10 fold less than the potency of 2-hexylthio ATP in the same system. 2-hexylthio adenosine was inactive. 3 Monophosphate analogues that were the most potent activators of the C6 cell P2y-purinoceptor were also the most potent activators of the turkey erythrocyte P2y-purinoceptor. However, agonists were in general more potent at the C6 cell receptor, and potency differences varied between 10 fold and 300 fold between the two receptors. 4 Although 2-thioether derivatives of adenosine monophosphate were potent P2Y-purinoceptor agonists no effect of these analogues on the human P2u-purinoceptor were observed. 5 These results support the view that a single monophosphate is sufficient and necessary for full agonist activity at P2Y-purinoceptors, and provide insight for strategies for development of novel P2Y- purinoceptor agonists of high potency and selectivity.
Ribose Modified Nucleosides and Nucleotides as Ligands for Purine Receptors
Nucleosides, Nucleotides and Nucleic Acids, 2001
Molecular modeling of receptors for adenosine and nucleotide (P2) receptors with docked ligand, based on mutagenesis, was carried out. Adenosine 3 ′ ,5 ′-bisphosphate derivatives act as selective P2Y 1 antagonists/partial agonists. The ribose moiety was replaced with carbocyclics, smaller and larger rings, conformationally constrained rings, and acyclics, producing compounds that retained receptor affinity. Conformational constraints were built into the ribose rings of nucleoside and nucleotide ligands using the methanocarba approach, i.e. fused cyclopropane and cyclopentane rings in place of ribose, suggesting a preference for the Northern (N) conformation among ligands for P2Y 1 and A 1 and A 3 ARs. Modulation of adenosine receptors (P1) and nucleotide (P2) receptors by selective agonists and antagonists (1,2) has the potential for the treatment of wide range of diseases, including those of the cardiovascular, inflammatory, and central nervous systems. There are four subtypes of adenosine receptors (A 1 , A 2A , A 2B , and A 3), all of which are G protein-coupled receptors (GPCRs) generally coupled to adenylate cyclase. Extracellular nucleotides, principally ATP, ADP, UTP, and UDP, act through two families of membrane-bound P2 receptors: P2Y subtypes, GPCRs which are activated by both adenine and uracil nucleotides and generally coupled to phospholipase C; and P2X subtypes, ligand-gated ion channels which are activated principally by adenine nucleotides (2). As many as seven subtypes have been cloned within each family. Agonists of adenosine and P2 receptors are almost exclusively nucleosides and nucleotides, respectively, while antagonists of these receptors are structurally more diverse (1). In comparison to the adenosine receptors, much less is known about the specific effects of P2 receptors, largely due to the lack of selective ligands. We are currently designing and synthesizing novel ligands for both adenosine and P2 receptors. Recent methods utilized in these investigations include: conformationally constraining the ribose, or ribose-like, moiety of nucleosides and nucleotides to freeze a conformation that may provide favorable affinity and/or selectivity at P1 and P2 receptors (3,4); modifying known receptor antagonists (5-7); use of a template approach based on the
European Journal of Pharmacology, 2009
We characterized the pharmacological properties of P2 receptors expressed in G292 osteoblastic cells by studying the responses or changes in intracellular Ca 2+ level to P2 receptor agonists, antagonists and modulators. ATP induced robust responses in a concentration-dependent manner with EC 50 of 0.5 ± 0.07 μM. While α,β-methylene-ATP (αβmeATP) and 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP) were ineffective, ADP mimicked the action of ATP with EC 50 of 0.7 ± 0.2 μM. UTP and UDP also evoked responses with EC 50 of 2.0 ± 0.4 μM and 0.5 ± 0.1 μM respectively, but their responses were much smaller, resulting in an order of the response magnitude: ATP~ADP NN UTP~UDP. The responses evoked by ATP and ADP were blocked by pyridoxal-5'-phosphate-6-azophenyl-2,4,-disulfonate (PPADS) with IC 50 of 3.0 ± 0.05 μM and 5.0 ± 0.4 μM respectively, but not by suramin up to 30 μM. ATP-evoked responses were insensitive to inhibition by trinitrophenyl-ATP (TNP-ATP) and brilliant blue G. ADP-evoked responses were significantly inhibited by 2'deoxy-N 6 -methyladenosine-3',5'-biphosphate (MRS2179) and 2-chloro-N 6 -methyl-(N)-methanocarba-2'deoxyadenosine-3',5'-bisphosphate (MRS2279) with IC 50 of 48 ± 1.9 μM and 7.7 ± 0.9 μM respectively. Taken together, these results provide strong evidence for functional expression of ATP-sensitive P2Y receptors and particularly P2Y 1 -like receptor in G292 cells.