Skip to main content
NCBI home page
British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 1994 Oct;113(2):614–620. doi: 10.1111/j.1476-5381.1994.tb17034.x

Differential effects of P2-purinoceptor antagonists on phospholipase C- and adenylyl cyclase-coupled P2Y-purinoceptors.

J L Boyer 1, I E Zohn 1, K A Jacobson 1, T K Harden 1
PMCID: PMC1510147  PMID: 7834215

Abstract

1. Stimulation of P2Y-purinoceptors on turkey erythrocytes and many other cell types results in activation of phospholipase C. In contrast, we have observed recently that P2Y-purinoceptors on C6 rat glioma cells are not coupled to phospholipase C, but rather, inhibit adenylyl cyclase. 2. In this study we investigated the pharmacological selectivity of the P2-purinoceptor antagonists, suramin, reactive blue 2, and pyridoxal phosphate 6-azophenyl 2',4'-disulphonic acid (PPADS) for phospholipase C- and adenylyl cyclase-coupled P2Y-purinoceptors. 3. In C6 glioma cells, suramin and reactive blue 2 competitively antagonized the inhibitory effect of 2MeSATP on adenylyl cyclase (pKB = 5.4 +/- 0.2 and 7.6 +/- 0.1, respectively), whereas PPADS at concentrations up to 100 microM had no effect. 4. In contrast, in the turkey erythrocyte preparation, PPADS at concentrations up to 30 microM was a competitive antagonist of P2Y-purinoceptor-stimulated phospholipase C activity (pKB = 5.9 +/- 0.1). Suramin and reactive blue 2 produced both a shift to the right of the concentration-effect of 2MeSATP for the activation of phospholipase C and a significant decrease in the maximal inositol phosphate response. 5. Turkey erythrocytes also express a phospholipase C-coupled beta-adrenoceptor. Concentrations of PPADS that competitively inhibited the P2Y-purinoceptor-mediated response had only minimal effects on the activation of phospholipase C by beta-adrenoceptors. In contrast, suramin and reactive blue 2 produced a non-competitive inhibition, characterized by decreases in the maximal response to isoprenaline with no change in the potency of this beta-adrenoceptor agonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Full text

PDF
614

Selected References

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

  1. ARUNLAKSHANA O., SCHILD H. O. Some quantitative uses of drug antagonists. Br J Pharmacol Chemother. 1959 Mar;14(1):48–58. doi: 10.1111/j.1476-5381.1959.tb00928.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berrie C. P., Hawkins P. T., Stephens L. R., Harden T. K., Downes C. P. Phosphatidylinositol 4,5-bisphosphate hydrolysis in turkey erythrocytes is regulated by P2y purinoceptors. Mol Pharmacol. 1989 Apr;35(4):526–532. [PubMed] [Google Scholar]
  3. Boyer J. L., Downes C. P., Harden T. K. Kinetics of activation of phospholipase C by P2Y purinergic receptor agonists and guanine nucleotides. J Biol Chem. 1989 Jan 15;264(2):884–890. [PubMed] [Google Scholar]
  4. Boyer J. L., Lazarowski E. R., Chen X. H., Harden T. K. Identification of a P2Y-purinergic receptor that inhibits adenylyl cyclase. J Pharmacol Exp Ther. 1993 Dec;267(3):1140–1146. [PubMed] [Google Scholar]
  5. Brown H. A., Lazarowski E. R., Boucher R. C., Harden T. K. Evidence that UTP and ATP regulate phospholipase C through a common extracellular 5'-nucleotide receptor in human airway epithelial cells. Mol Pharmacol. 1991 Nov;40(5):648–655. [PubMed] [Google Scholar]
  6. 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]
  7. Charest R., Blackmore P. F., Exton J. H. Characterization of responses of isolated rat hepatocytes to ATP and ADP. J Biol Chem. 1985 Dec 15;260(29):15789–15794. [PubMed] [Google Scholar]
  8. Choo L. K. The effect of reactive blue, an antagonist of ATP, on the isolated urinary bladders of guinea-pig and rat. J Pharm Pharmacol. 1981 Apr;33(4):248–250. doi: 10.1111/j.2042-7158.1981.tb13770.x. [DOI] [PubMed] [Google Scholar]
  9. Crema A., Frigo G. M., Lecchini S., Manzo L., Onori L., Tonini M. Purine receptors in the guinea-pig internal anal sphincter. Br J Pharmacol. 1983 Mar;78(3):599–603. doi: 10.1111/j.1476-5381.1983.tb08820.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dubyak G. R., Cowen D. S., Meuller L. M. Activation of inositol phospholipid breakdown in HL60 cells by P2-purinergic receptors for extracellular ATP. Evidence for mediation by both pertussis toxin-sensitive and pertussis toxin-insensitive mechanisms. J Biol Chem. 1988 Dec 5;263(34):18108–18117. [PubMed] [Google Scholar]
  11. Dubyak G. R., el-Moatassim C. Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides. Am J Physiol. 1993 Sep;265(3 Pt 1):C577–C606. doi: 10.1152/ajpcell.1993.265.3.C577. [DOI] [PubMed] [Google Scholar]
  12. Dunn P. M., Blakeley A. G. Suramin: a reversible P2-purinoceptor antagonist in the mouse vas deferens. Br J Pharmacol. 1988 Feb;93(2):243–245. doi: 10.1111/j.1476-5381.1988.tb11427.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fedan J. S., Lamport S. J. P2-purinoceptor antagonists. Ann N Y Acad Sci. 1990;603:182–197. doi: 10.1111/j.1749-6632.1990.tb37672.x. [DOI] [PubMed] [Google Scholar]
  14. Filtz T. M., Li Q., Boyer J. L., Nicholas R. A., Harden T. K. Expression of a cloned P2Y purinergic receptor that couples to phospholipase C. Mol Pharmacol. 1994 Jul;46(1):8–14. [PubMed] [Google Scholar]
  15. 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]
  16. Fischer B., Boyer J. L., Hoyle C. H., Ziganshin A. U., Brizzolara A. L., Knight G. E., Zimmet J., Burnstock G., Harden T. K., Jacobson K. A. Identification of potent, selective P2Y-purinoceptor agonists: structure-activity relationships for 2-thioether derivatives of adenosine 5'-triphosphate. J Med Chem. 1993 Nov 26;36(24):3937–3946. doi: 10.1021/jm00076a023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Forsberg E. J., Feuerstein G., Shohami E., Pollard H. B. Adenosine triphosphate stimulates inositol phospholipid metabolism and prostacyclin formation in adrenal medullary endothelial cells by means of P2-purinergic receptors. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5630–5634. doi: 10.1073/pnas.84.16.5630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Frederich R. C., Jr, Waldo G. L., Harden T. K., Perkins J. P. Characterization of agonist-induced beta-adrenergic receptor-specific desensitization in C62B glioma cells. J Cyclic Nucleotide Protein Phosphor Res. 1983;9(2):103–118. [PubMed] [Google Scholar]
  19. Gordon J. L. Extracellular ATP: effects, sources and fate. Biochem J. 1986 Jan 15;233(2):309–319. doi: 10.1042/bj2330309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Harden T. K., Hawkins P. T., Stephens L., Boyer J. L., Downes C. P. Phosphoinositide hydrolysis by guanosine 5'-[gamma-thio]triphosphate-activated phospholipase C of turkey erythrocyte membranes. Biochem J. 1988 Jun 1;252(2):583–593. doi: 10.1042/bj2520583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Harden T. K., Scheer A. G., Smith M. M. Differential modification of the interaction of cardiac muscarinic cholinergic and beta-adrenergic receptors with a guanine nucleotide binding component(s). Mol Pharmacol. 1982 May;21(3):570–580. [PubMed] [Google Scholar]
  22. Hoyle C. H., Knight G. E., Burnstock G. Suramin antagonizes responses to P2-purinoceptor agonists and purinergic nerve stimulation in the guinea-pig urinary bladder and taenia coli. Br J Pharmacol. 1990 Mar;99(3):617–621. doi: 10.1111/j.1476-5381.1990.tb12979.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lambrecht G., Friebe T., Grimm U., Windscheif U., Bungardt E., Hildebrandt C., Bäumert H. G., Spatz-Kümbel G., Mutschler E. PPADS, a novel functionally selective antagonist of P2 purinoceptor-mediated responses. Eur J Pharmacol. 1992 Jul 7;217(2-3):217–219. doi: 10.1016/0014-2999(92)90877-7. [DOI] [PubMed] [Google Scholar]
  24. Leff P., Wood B. E., O'Connor S. E. Suramin is a slowly-equilibrating but competitive antagonist at P2x-receptors in the rabbit isolated ear artery. Br J Pharmacol. 1990 Nov;101(3):645–649. doi: 10.1111/j.1476-5381.1990.tb14134.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lin W. W., Chuang D. M. Endothelin- and ATP-induced inhibition of adenylyl cyclase activity in C6 glioma cells: role of Gi and calcium. Mol Pharmacol. 1993 Jul;44(1):158–165. [PubMed] [Google Scholar]
  26. Londos C., Cooper D. M., Wolff J. Subclasses of external adenosine receptors. Proc Natl Acad Sci U S A. 1980 May;77(5):2551–2554. doi: 10.1073/pnas.77.5.2551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Manzini S., Hoyle C. H., Burnstock G. An electrophysiological analysis of the effect of reactive blue 2, a putative P2-purinoceptor antagonist, on inhibitory junction potentials of rat caecum. Eur J Pharmacol. 1986 Aug 15;127(3):197–204. doi: 10.1016/0014-2999(86)90364-x. [DOI] [PubMed] [Google Scholar]
  28. Meeker R. B., Harden T. K. Muscarinic cholinergic receptor-mediated activation of phosphodiesterase. Mol Pharmacol. 1982 Sep;22(2):310–319. [PubMed] [Google Scholar]
  29. Meyerhof W., Müller-Brechlin R., Richter D. Molecular cloning of a novel putative G-protein coupled receptor expressed during rat spermiogenesis. FEBS Lett. 1991 Jun 24;284(2):155–160. doi: 10.1016/0014-5793(91)80674-r. [DOI] [PubMed] [Google Scholar]
  30. O'Connor S. E., Dainty I. A., Leff P. Further subclassification of ATP receptors based on agonist studies. Trends Pharmacol Sci. 1991 Apr;12(4):137–141. doi: 10.1016/0165-6147(91)90530-6. [DOI] [PubMed] [Google Scholar]
  31. Okajima F., Sato K., Nazarea M., Sho K., Kondo Y. A permissive role of pertussis toxin substrate G-protein in P2-purinergic stimulation of phosphoinositide turnover and arachidonate release in FRTL-5 thyroid cells. Cooperative mechanism of signal transduction systems. J Biol Chem. 1989 Aug 5;264(22):13029–13037. [PubMed] [Google Scholar]
  32. Okajima F., Tokumitsu Y., Kondo Y., Ui M. P2-purinergic receptors are coupled to two signal transduction systems leading to inhibition of cAMP generation and to production of inositol trisphosphate in rat hepatocytes. J Biol Chem. 1987 Oct 5;262(28):13483–13490. [PubMed] [Google Scholar]
  33. Pianet I., Merle M., Labouesse J. ADP and, indirectly, ATP are potent inhibitors of cAMP production in intact isoproterenol-stimulated C6 glioma cells. Biochem Biophys Res Commun. 1989 Sep 15;163(2):1150–1157. doi: 10.1016/0006-291x(89)92341-3. [DOI] [PubMed] [Google Scholar]
  34. Pirotton S., Raspe E., Demolle D., Erneux C., Boeynaems J. M. Involvement of inositol 1,4,5-trisphosphate and calcium in the action of adenine nucleotides on aortic endothelial cells. J Biol Chem. 1987 Dec 25;262(36):17461–17466. [PubMed] [Google Scholar]
  35. Reilly W. M., Saville V. L., Burnstock G. An assessment of the antagonistic activity of reactive blue 2 at P1- and P2-purinoceptors: supporting evidence for purinergic innervation of the rabbit portal vein. Eur J Pharmacol. 1987 Aug 4;140(1):47–53. doi: 10.1016/0014-2999(87)90632-7. [DOI] [PubMed] [Google Scholar]
  36. Rice W. R., Singleton F. M. Reactive blue 2 selectively inhibits P2y-purinoceptor-stimulated surfactant phospholipid secretion from rat isolated alveolar type II cells. Br J Pharmacol. 1989 May;97(1):158–162. doi: 10.1111/j.1476-5381.1989.tb11937.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rooney T. A., Hager R., Thomas A. P. Beta-adrenergic receptor-mediated phospholipase C activation independent of cAMP formation in turkey erythrocyte membranes. J Biol Chem. 1991 Aug 15;266(23):15068–15074. [PubMed] [Google Scholar]
  38. Sato K., Okajima F., Kondo Y. Extracellular ATP stimulates three different receptor-signal transduction systems in FRTL-5 thyroid cells. Activation of phospholipase C, and inhibition and activation of adenylate cyclase. Biochem J. 1992 Apr 1;283(Pt 1):281–287. doi: 10.1042/bj2830281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Soltoff S. P., McMillian M. K., Talamo B. R. Coomassie Brilliant Blue G is a more potent antagonist of P2 purinergic responses than Reactive Blue 2 (Cibacron Blue 3GA) in rat parotid acinar cells. Biochem Biophys Res Commun. 1989 Dec 29;165(3):1279–1285. doi: 10.1016/0006-291x(89)92741-1. [DOI] [PubMed] [Google Scholar]
  40. Stutchfield J., Cockcroft S. Undifferentiated HL60 cells respond to extracellular ATP and UTP by stimulating phospholipase C activation and exocytosis. FEBS Lett. 1990 Mar 26;262(2):256–258. doi: 10.1016/0014-5793(90)80204-v. [DOI] [PubMed] [Google Scholar]
  41. Valeins H., Merle M., Labouesse J. Pre-steady state study of beta-adrenergic and purinergic receptor interaction in C6 cell membranes: undelayed balance between positive and negative coupling to adenylyl cyclase. Mol Pharmacol. 1992 Dec;42(6):1033–1041. [PubMed] [Google Scholar]
  42. Vaziri C., Downes C. P. G-protein-mediated activation of turkey erythrocyte phospholipase C by beta-adrenergic and P2y-purinergic receptors. Biochem J. 1992 Jun 15;284(Pt 3):917–922. doi: 10.1042/bj2840917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Voogd T. E., Vansterkenburg E. L., Wilting J., Janssen L. H. Recent research on the biological activity of suramin. Pharmacol Rev. 1993 Jun;45(2):177–203. [PubMed] [Google Scholar]
  44. 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]
  45. Wilkinson G. F., Purkiss J. R., Boarder M. R. The regulation of aortic endothelial cells by purines and pyrimidines involves co-existing P2y-purinoceptors and nucleotide receptors linked to phospholipase C. Br J Pharmacol. 1993 Mar;108(3):689–693. doi: 10.1111/j.1476-5381.1993.tb12862.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zhou Q. Y., Li C., Olah M. E., Johnson R. A., Stiles G. L., Civelli O. Molecular cloning and characterization of an adenosine receptor: the A3 adenosine receptor. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7432–7436. doi: 10.1073/pnas.89.16.7432. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Ziganshin A. U., Hoyle C. H., Bo X., Lambrecht G., Mutschler E., Bäumert H. G., Burnstock G. PPADS selectively antagonizes P2X-purinoceptor-mediated responses in the rabbit urinary bladder. Br J Pharmacol. 1993 Dec;110(4):1491–1495. doi: 10.1111/j.1476-5381.1993.tb13990.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. van Calker D., Müller M., Hamprecht B. Adenosine regulates via two different types of receptors, the accumulation of cyclic AMP in cultured brain cells. J Neurochem. 1979 Nov;33(5):999–1005. doi: 10.1111/j.1471-4159.1979.tb05236.x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES