Skip to main content
NCBI home page
British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 1996 Jan;117(1):63–70. doi: 10.1111/j.1476-5381.1996.tb15155.x

High affinity P2x-purinoceptor binding sites for [35S]-adenosine 5'-O-[3-thiotriphosphate] in rat vas deferens membranes.

A D Michel 1, P P Humphrey 1
PMCID: PMC1909358  PMID: 8825344

Abstract

1. The binding sites labelled by [35S]-adenosine 5'-O-[3-thiotriphosphate]([35S]-ATP gamma S) at 4 degrees C in rat vas deferens membranes were studied and compared to the sites labelled by [3H]-alpha,beta-methylene ATP ([3H]-alpha beta meATP) to ascertain whether [35S]-ATP gamma S can be used to label the P2x purinoceptor. 2. In the presence of 4 mM CaCl2, the binding of 0.2 nM [35S]-ATP gamma S to vas deferens membranes was increased 3.4 fold, when compared to studies performed in the absence of calcium. However, binding did not appear to be solely to P2x purinoceptors since [35S]-ATP gamma S labelled a heterogeneous population of sites and about 72% of the sites possessed high affinity (pIC50 = 7.5) for guanosine 5'-O-[3-thiotriphosphate] (GTP gamma S). Even in the presence of 1 microM GTP gamma S, to occlude the sites with high affinity for GTP gamma S, the binding of [35S]-ATP gamma S was heterogeneous and since there was also evidence of extensive metabolism of ATP in the presence of calcium, the binding of [35S]-ATP gamma S under these conditions was not studied further. 3. In the absence of calcium ions, [35S]-ATP gamma S bound to a single population of sites (pKD = 9.23; Bmax = 4270 fmol mg-1 protein). Binding reached steady state within 3 h (t1/2 = 38 min), was stable for a further 4 h and was readily reversible upon addition of 10 microM unlabelled ATP gamma S (t1/2 = 45 min). In competition studies the binding of 0.2 nM [35S]-ATP gamma S was inhibited by a number of P2x purinoceptor agonists and antagonists, but not by adenosine receptor agonists, staurosporine (1 microM) or several ATPase inhibitors. The rank order of agonist affinity estimates (pIC50 values) in competing for the [35S]-ATP gamma S binding sites was: ATP (9.01), 2-methylthio- ATP (8.79), ATP gamma S (8.73), alpha beta meATP (7.57), ADP (7.24), beta, gamma-methylene ATP (7.18), L-beta, gamma-methylene ATP (5.83), alpha, beta-methylene ADP (4.36). 4. Affinity estimates (pIC50 values) for the P2x purinoceptor antagonists, suramin (5.20), pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (4.23), pyridoxal 5-phosphate (3.42), cibacron blue (5.70) and Evan's blue (5.79) were broadly similar to those obtained at the [3H]-alpha beta meATP binding sites in vas deferens. However, ATP, 2-methylthio-ATP, ATP gamma S and ADP displayed 17-512 fold higher affinity for the [35S]-ATP gamma S, than for the [3H]-alpha beta meATP binding sites, whereas alpha beta meATP and L-beta, gamma-methylene ATP displayed 5 and 28 fold, respectively, higher affinity for the [3H]-alpha beta meATP than for the [35S]-ATP gamma S binding sites. 5. The differences in agonist affinity for the [35S]-ATP gamma S and [3H]-alpha beta meATP binding sites probably reflect the fact that the former sites were labelled in the absence of calcium, while the latter sites were labelled in its presence. This could differentially affect ionisation state and/or metabolism of the nucleotides when using the two radioligands. Since affinity estimates for ATP, 2-methylthio-ATP, ATP gamma S, alpha beta meATP and L-beta, gamma-methylene ATP were different when calcium ions were omitted in studies using [3H]-alpha beta meATP but similar to the affinity estimates obtained at the [35S]-ATP gamma S binding sites labelled in the absence of calcium, it is likely that [35S]-ATP gamma S and [3H]-alpha beta meATP label the same sites in rat vas deferens. 6. We conclude that, in the absence of divalent cations, [35S]-ATP gamma S labels P2x purinoceptors in rat vas deferens and as such may represent a new, high specific activity, radioligand for the study of such receptors.

Full text

PDF
63

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abbracchio M. P., Burnstock G. Purinoceptors: are there families of P2X and P2Y purinoceptors? Pharmacol Ther. 1994;64(3):445–475. doi: 10.1016/0163-7258(94)00048-4. [DOI] [PubMed] [Google Scholar]
  2. Barnard E. A., Burnstock G., Webb T. E. G protein-coupled receptors for ATP and other nucleotides: a new receptor family. Trends Pharmacol Sci. 1994 Mar;15(3):67–70. doi: 10.1016/0165-6147(94)90280-1. [DOI] [PubMed] [Google Scholar]
  3. Beukers M. W., Pirovano I. M., van Weert A., Kerkhof C. J., IJzerman A. P., Soudijn W. Characterization of ecto-ATPase on human blood cells. A physiological role in platelet aggregation? Biochem Pharmacol. 1993 Dec 3;46(11):1959–1966. doi: 10.1016/0006-2952(93)90637-c. [DOI] [PubMed] [Google Scholar]
  4. Bo X. N., Burnstock G. High- and low-affinity binding sites for [3H]-alpha, beta-methylene ATP in rat urinary bladder membranes. Br J Pharmacol. 1990 Oct;101(2):291–296. doi: 10.1111/j.1476-5381.1990.tb12703.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bo X. N., Burnstock G. [3H]-alpha, beta-methylene ATP, a radioligand labelling P2-purinoceptors. J Auton Nerv Syst. 1989 Oct;28(1):85–88. doi: 10.1016/0165-1838(89)90010-6. [DOI] [PubMed] [Google Scholar]
  6. Bo X., Fischer B., Maillard M., Jacobson K. A., Burnstock G. Comparative studies on the affinities of ATP derivatives for P2x-purinoceptors in rat urinary bladder. Br J Pharmacol. 1994 Aug;112(4):1151–1159. doi: 10.1111/j.1476-5381.1994.tb13204.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bo X., Simon J., Burnstock G., Barnard E. A. Solubilization and molecular size determination of the P2x purinoceptor from rat vas deferens. J Biol Chem. 1992 Sep 5;267(25):17581–17587. [PubMed] [Google Scholar]
  8. Brake A. J., Wagenbach M. J., Julius D. New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor. Nature. 1994 Oct 6;371(6497):519–523. doi: 10.1038/371519a0. [DOI] [PubMed] [Google Scholar]
  9. Burnstock G., Kennedy C. Is there a basis for distinguishing two types of P2-purinoceptor? Gen Pharmacol. 1985;16(5):433–440. doi: 10.1016/0306-3623(85)90001-1. [DOI] [PubMed] [Google Scholar]
  10. Cascalheira J. F., Sebastião A. M. Adenine nucleotide analogues, including gamma-phosphate-substituted analogues, are metabolised extracellularly in innervated frog sartorius muscle. Eur J Pharmacol. 1992 Nov 3;222(1):49–59. doi: 10.1016/0014-2999(92)90462-d. [DOI] [PubMed] [Google Scholar]
  11. Cheng Y., Prusoff W. H. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol. 1973 Dec 1;22(23):3099–3108. doi: 10.1016/0006-2952(73)90196-2. [DOI] [PubMed] [Google Scholar]
  12. Cooper C. L., Morris A. J., Harden T. K. Guanine nucleotide-sensitive interaction of a radiolabeled agonist with a phospholipase C-linked P2y-purinergic receptor. J Biol Chem. 1989 Apr 15;264(11):6202–6206. [PubMed] [Google Scholar]
  13. Crack B. E., Beukers M. W., McKechnie K. C., Ijzerman A. P., Leff P. Pharmacological analysis of ecto-ATPase inhibition: evidence for combined enzyme inhibition and receptor antagonism in P2X-purinoceptor ligands. Br J Pharmacol. 1994 Dec;113(4):1432–1438. doi: 10.1111/j.1476-5381.1994.tb17157.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Crack B. E., Pollard C. E., Beukers M. W., Roberts S. M., Hunt S. F., Ingall A. H., McKechnie K. C., IJzerman A. P., Leff P. Pharmacological and biochemical analysis of FPL 67156, a novel, selective inhibitor of ecto-ATPase. Br J Pharmacol. 1995 Jan;114(2):475–481. doi: 10.1111/j.1476-5381.1995.tb13251.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Evans R. J., Kennedy C. Characterization of P2-purinoceptors in the smooth muscle of the rat tail artery: a comparison between contractile and electrophysiological responses. Br J Pharmacol. 1994 Nov;113(3):853–860. doi: 10.1111/j.1476-5381.1994.tb17071.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fedan J. S., Dagirmanjian J. P., Attfield M. D., Chideckel E. W. Evidence that the P2x purinoceptor of the smooth muscle of the guinea pig vas deferens is an ATP4- receptor. J Pharmacol Exp Ther. 1990 Oct;255(1):46–51. [PubMed] [Google Scholar]
  17. Fine J., Cole P., Davidson J. S. Extracellular nucleotides stimulate receptor-mediated calcium mobilization and inositol phosphate production in human fibroblasts. Biochem J. 1989 Oct 15;263(2):371–376. doi: 10.1042/bj2630371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fredholm B. B., Abbracchio M. P., Burnstock G., Daly J. W., Harden T. K., Jacobson K. A., Leff P., Williams M. Nomenclature and classification of purinoceptors. Pharmacol Rev. 1994 Jun;46(2):143–156. [PMC free article] [PubMed] [Google Scholar]
  19. Kajekar R., Gupta P., Shepperson N. B., Brain S. D. Effect of a 5-HT1 receptor agonist, CP-122,288, on oedema formation induced by stimulation of the rat saphenous nerve. Br J Pharmacol. 1995 May;115(1):1–2. doi: 10.1111/j.1476-5381.1995.tb16310.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Khakh B. S., Michel A., Humphrey P. P. Estimates of antagonist affinities at P2X purinoceptors in rat vas deferens. Eur J Pharmacol. 1994 Oct 3;263(3):301–309. doi: 10.1016/0014-2999(94)90726-9. [DOI] [PubMed] [Google Scholar]
  21. Khakh B. S., Surprenant A., Humphrey P. P. A study on P2X purinoceptors mediating the electrophysiological and contractile effects of purine nucleotides in rat vas deferens. Br J Pharmacol. 1995 May;115(1):177–185. doi: 10.1111/j.1476-5381.1995.tb16336.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lustig K. D., Shiau A. K., Brake A. J., Julius D. Expression cloning of an ATP receptor from mouse neuroblastoma cells. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5113–5117. doi: 10.1073/pnas.90.11.5113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lustig K. D., Sportiello M. G., Erb L., Weisman G. A. A nucleotide receptor in vascular endothelial cells is specifically activated by the fully ionized forms of ATP and UTP. Biochem J. 1992 Jun 15;284(Pt 3):733–739. doi: 10.1042/bj2840733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Michel A. D., Humphrey P. P. Distribution and characterisation of [3H]alpha,beta-methylene ATP binding sites in the rat. Naunyn Schmiedebergs Arch Pharmacol. 1993 Dec;348(6):608–617. doi: 10.1007/BF00167237. [DOI] [PubMed] [Google Scholar]
  25. Michel A. D., Humphrey P. P. Effects of metal cations on [3H]alpha,beta-methylene ATP binding in rat vas deferens. Naunyn Schmiedebergs Arch Pharmacol. 1994 Aug;350(2):113–122. doi: 10.1007/BF00241084. [DOI] [PubMed] [Google Scholar]
  26. Motte S., Pirotton S., Boeynaems J. M. Evidence that a form of ATP uncomplexed with divalent cations is the ligand of P2y and nucleotide/P2u receptors on aortic endothelial cells. Br J Pharmacol. 1993 Aug;109(4):967–971. doi: 10.1111/j.1476-5381.1993.tb13715.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Munson P. J., Rodbard D. Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem. 1980 Sep 1;107(1):220–239. doi: 10.1016/0003-2697(80)90515-1. [DOI] [PubMed] [Google Scholar]
  28. Trezise D. J., Bell N. J., Khakh B. S., Michel A. D., Humphrey P. A. P2 purinoceptor antagonist properties of pyridoxal-5-phosphate. Eur J Pharmacol. 1994 Jul 11;259(3):295–300. doi: 10.1016/0014-2999(94)90656-4. [DOI] [PubMed] [Google Scholar]
  29. Trezise D. J., Kennedy I., Humphrey P. P. Characterization of purinoceptors mediating depolarization of rat isolated vagus nerve. Br J Pharmacol. 1993 Nov;110(3):1055–1060. doi: 10.1111/j.1476-5381.1993.tb13920.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Trezise D. J., Michel A. D., Grahames C. B., Khakh B. S., Surprenant A., Humphrey P. P. The selective P2X purinoceptor agonist, beta,gamma-methylene-L-adenosine 5'-triphosphate, discriminates between smooth muscle and neuronal P2X purinoceptors. Naunyn Schmiedebergs Arch Pharmacol. 1995 Jun;351(6):603–609. doi: 10.1007/BF00170159. [DOI] [PubMed] [Google Scholar]
  31. Valera S., Hussy N., Evans R. J., Adami N., North R. A., Surprenant A., Buell G. A new class of ligand-gated ion channel defined by P2x receptor for extracellular ATP. Nature. 1994 Oct 6;371(6497):516–519. doi: 10.1038/371516a0. [DOI] [PubMed] [Google Scholar]
  32. Webb T. E., Simon J., Krishek B. J., Bateson A. N., Smart T. G., King B. F., Burnstock G., Barnard E. A. Cloning and functional expression of a brain G-protein-coupled ATP receptor. FEBS Lett. 1993 Jun 14;324(2):219–225. doi: 10.1016/0014-5793(93)81397-i. [DOI] [PubMed] [Google Scholar]
  33. Welford L. A., Cusack N. J., Hourani S. M. ATP analogues and the guinea-pig taenia coli: a comparison of the structure-activity relationships of ectonucleotidases with those of the P2-purinoceptor. Eur J Pharmacol. 1986 Oct 7;129(3):217–224. doi: 10.1016/0014-2999(86)90431-0. [DOI] [PubMed] [Google Scholar]
  34. Welford L. A., Cusack N. J., Hourani S. M. The structure-activity relationships of ectonucleotidases and of excitatory P2-purinoceptors: evidence that dephosphorylation of ATP analogues reduces pharmacological potency. Eur J Pharmacol. 1987 Sep 2;141(1):123–130. doi: 10.1016/0014-2999(87)90418-3. [DOI] [PubMed] [Google Scholar]
  35. Yount R. G. ATP analogs. Adv Enzymol Relat Areas Mol Biol. 1975;43:1–56. doi: 10.1002/9780470122884.ch1. [DOI] [PubMed] [Google Scholar]
  36. Ziganshin A. U., Hoyle C. H., Ziganshina L. E., Burnstock G. Effects of cyclopiazonic acid on contractility and ecto-ATPase activity in guinea-pig urinary bladder and vas deferens. Br J Pharmacol. 1994 Nov;113(3):669–674. doi: 10.1111/j.1476-5381.1994.tb17044.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from British Journal of Pharmacology are provided here courtesy of The British Pharmacological Society

RESOURCES