Comparative studies on the affinities of ATP derivatives for P2X-purinoceptors in rat urinary bladder (original) (raw)
Related papers
Drug Development Research, 1994
The structure-activity relationships for a variety of adenine nucleotide analogues at Pzx-and P,,-purinoceptors were investigated. Compounds formed by structural modifications of the ATP molecule including substitutions of the purine ring (C2, C8, N1, and N6-substituents, and a uridine base instead of adenine), the ribose moiety (2' and 3'-positions), and the triphosphate group (lower phosphates, bridging oxygen substitution, and cyclization) were prepared. Pharmacological activity at P,,-purinoceptors was assayed in the guinea pig taenia coli, endothelial cells of the rabbit aorta, smooth muscle of the rabbit mesenteric artery, and turkey erythrocyte membranes. Activity at P,,-purinoceptors was assayed in the rabbit saphenous artery and the guinea-pig vas deferens and urinary bladder. Some of the analogues displayed selectivity, or even specificity, for either the PZx-or the P,,-purinoceptors.
British Journal of Pharmacology, 1996
The binding of [3H]-afmeATP, [35S]-ATPyS and [cx33P]-ATP to a human bladder P2X purinoceptor, transiently expressed in CHO-Kl cells using the Semliki Forest Virus (SFV) expression system, was examined. The characteristics of the binding sites were compared with results obtained in rat vas deferens, a tissue in which the radioligands are thought to label P2x purinoceptors and in which the endogenous P2X purinoceptor displays high homology with the human bladder P2X purinoceptor. 2 In non-infected CHO-Kl cells, 100 gM ATP evoked only small inward currents (40 pA) in approximately 30% of the cells when studied by the whole-cell voltage clamp technique. In membranes prepared from either these non-infected cells or cells infected with SFV containing the LacZ gene (SFV-LacZ), [3H]-aBmeATP bound with low affinity (pKd = 7.04; Bma, = 8.88 pmol ml-' protein) and there was only a low density of [35"]-ATPyS binding sites (pKd = 8.74; Bmax = 358 fmol ml-' protein). These binding sites differed from those present in rat vas deferens. Thus, pIC50 values for aBmeATP (6.5) and L-flymeATP (4.0) at the [3H]-a3meATP binding sites in non-infected CHO-KI cells were much lower than the respective pICso values of 8.3 and 7.7, determined in rat vas deferens. Similarly, affinity estimates (pICso values) for ATP (6.82), 2-meS-ATP (5.43), ATPyS (7.06) and aefmeATP (4.84) at the [35S]-ATPyS binding sites in non-infected CHO-Kl cells were up to 2291 fold lower than the respective values of 9.01, 8.79, 8.73 and 7.57, determined in rat vas deferens. 3 In CHO-KI cells infected using SFV containing the cDNA for the human bladder P2X purinoceptor (SFV-h.P2X), ATP, 2-meS-ATP and aBmeATP evoked large inward currents (2-7 nA) in whole cell voltage clamp studies. In membranes prepared from these SFV-h.P2X infected cells, [3H]-ameATP binding was increased, compared to that measured in the non infected or SFV-LacZ infected cells, with only high affinity [3H]-apmeATP binding sites being detected (pKd= 9.21; Bmax = 3.54 pmol mg'protein). The pIC50 values for aBmeATP (8.2) and L-flymeATP (7.2) in competing for these sites were the same or similar to the values determined in rat vas deferens. 4 A high density of [35S]-ATPyS binding sites (pd = 9.09; Bma, = 6.82 pmol mg-' protein) was also present in the membranes from CHO-Kl cells infected with SFV-h.P2x and affinity estimates (pIC50 values) for ATP (8.93), 2-meS-ATP (8.23), ATPyS (8.08), and apmeATP (7.17) at competing for these sites were as much as 631 fold higher than the respective values determined in non-infected CHO-Kl cells but were close to the values determined in rat vas deferens. Similar data were obtained with [aC33P]-ATP as radioligand. 5 These data suggest that [3H]-axmeATP, [35S]-ATPyS and [33P]-ATP label the human bladder recombinant P2X purinoceptor expressed in CHO-KI cells following infection with SFV-h.P2x and provide further corroborative evidence to support the contention that the high affinity binding sites for these radioligands in rat vas deferens are P2X purinoceptors.
British Journal of Pharmacology, 1987
1 Isopolar methylene phosphonate analogues ofadenosine triphosphate (ATP) were synthesized and tested on the guinea-pig isolated taenia coli (where ATP causes relaxation) and urinary bladder (where ATP causes contraction), to see if restoration of the electronegativity of the methylene linkage would enhance pharmacological potency. The compounds used were the dichloromethylene and difluoromethylene analogues of adenosine 5'-(P,y-methylene)triphosphonate (AMP-PCP), L-adenosine 5'-(P,y-methylene)triphosphonate (L-AMP-PCP) and 2-methylthioadenosine 5'-(f,y-methylene)triphosphonate (2-methylthio-AMP-PCP). 2 The order ofpotency of the analogues depended on the tissue, and was independent of the nature of the purine or ribose moieties. None of the analogues was degraded by ectonucleotidases on either tissue. 3 In the taenia coli the order of potency for relaxation was difluoromethylene > dichloromethylene > methylene, and this reflected the order ofelectronegativity of the analogues. The isopolar analogues of L-AMP-PCP were inactive in the taenia coli. 4 In the bladder the order of potency for contraction was difluoromethylene> methylene> dichloromethylene, suggesting that electronegativity is of lesser importance here. The isopolar analogues of L-AMP-PCP were active in this tissue. 5 The differences between the two tissues in the order of potency for these non-degradable analogues supports suggestions that P2-purinoceptors in the taenia coli (P2y) are different from those in the bladder (P2X). The isopolar analogues of L-AMP-PCP, like L-AMP-PCP itself, were selective agonists at the P2x-purinoceptor.
British Journal of Pharmacology, 2003
Heteromeric P2X2/3 receptors are much more sensitive than homomeric P2X2 receptors to αβ-methylene-ATP, and this ATP analogue is widely used to discriminate the two receptors on sensory neurons and other cells.We sought to determine the structural basis for this selectivity by synthesising ADP and ATP analogues in which the αβ and/or βγ oxygen atoms were replaced by other moieties (including –CH2–, –CHF–, –CHCl–, –CHBr–, –CF2–, –CCl2–, –CBr2–, –CHSO3–, –CHPO3–, –CFPO3–, –CClPO3–, –CH2–CH2–, –C≡C–, –NH–, –CHCOOH–).We tested their actions as agonists or antagonists by whole-cell recording from human embryonic kidney cells expressing P2X2 subunits alone (homomeric P2X2 receptors), or cells expressing both P2X2 and P2X3 subunits, in which the current through heteromeric P2X2/3 receptors was isolated.ADP analogues had no agonist or antagonist effect at either P2X2 or P2X2/3 receptors. All the ATP analogues tested were without agonist or antagonist activity at homomeric P2X2 receptors, except βγ-difluoromethylene-ATP, which was a weak agonist.At P2X2/3 receptors, βγ-imido-ATP, βγ-methylene-ATP, and βγ-acetylene-ATP were weak agonists, whereas αβ,βγ- and βγ,γδ-bismethylene-AP4 were potent full agonists. βγ-Carboxymethylene-ATP and βγ-chlorophosphonomethylene-ATP were weak antagonists at P2X2/3 receptors (IC50 about 10 μM).The results indicate (a) that the homomeric P2X2 receptor presents very stringent structural requirements with respect to its activation by ATP; (b) that the heteromeric P2X2/3 receptor is much more tolerant of αβ and βγ substitution; and (c) that a P2X2/3-selective antagonist can be obtained by introduction of additional negativity at the βγ-methylene.Heteromeric P2X2/3 receptors are much more sensitive than homomeric P2X2 receptors to αβ-methylene-ATP, and this ATP analogue is widely used to discriminate the two receptors on sensory neurons and other cells.We sought to determine the structural basis for this selectivity by synthesising ADP and ATP analogues in which the αβ and/or βγ oxygen atoms were replaced by other moieties (including –CH2–, –CHF–, –CHCl–, –CHBr–, –CF2–, –CCl2–, –CBr2–, –CHSO3–, –CHPO3–, –CFPO3–, –CClPO3–, –CH2–CH2–, –C≡C–, –NH–, –CHCOOH–).We tested their actions as agonists or antagonists by whole-cell recording from human embryonic kidney cells expressing P2X2 subunits alone (homomeric P2X2 receptors), or cells expressing both P2X2 and P2X3 subunits, in which the current through heteromeric P2X2/3 receptors was isolated.ADP analogues had no agonist or antagonist effect at either P2X2 or P2X2/3 receptors. All the ATP analogues tested were without agonist or antagonist activity at homomeric P2X2 receptors, except βγ-difluoromethylene-ATP, which was a weak agonist.At P2X2/3 receptors, βγ-imido-ATP, βγ-methylene-ATP, and βγ-acetylene-ATP were weak agonists, whereas αβ,βγ- and βγ,γδ-bismethylene-AP4 were potent full agonists. βγ-Carboxymethylene-ATP and βγ-chlorophosphonomethylene-ATP were weak antagonists at P2X2/3 receptors (IC50 about 10 μM).The results indicate (a) that the homomeric P2X2 receptor presents very stringent structural requirements with respect to its activation by ATP; (b) that the heteromeric P2X2/3 receptor is much more tolerant of αβ and βγ substitution; and (c) that a P2X2/3-selective antagonist can be obtained by introduction of additional negativity at the βγ-methylene.British Journal of Pharmacology (2003) 140, 1027–1034. doi:10.1038/sj.bjp.0705531
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
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.
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.
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.