Skip to main content
NCBI home page
British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 1996 Mar;117(6):1254–1260. doi: 10.1111/j.1476-5381.1996.tb16723.x

The binding characteristics of a human bladder recombinant P2X purinoceptor, labelled with [3H]-alpha beta meATP, [35S]-ATP gamma S or [33P]-ATP.

A D Michel 1, K Lundström 1, G N Buell 1, A Surprenant 1, S Valera 1, P P Humphrey 1
PMCID: PMC1909787  PMID: 8882623

Abstract

1. The binding of [3H]-alpha beta meATP, [35s]-ATP gamma S and [alpha 33P]-ATP to a human bladder P2X purinoceptor, transiently expressed in CHO-K1 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-K1 cells, 100 microM 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]-alpha beta meATP bound with low affinity (pKd = 7.04; Bmax = 8.88 pmol ml-1 protein) and there was only a low density of [35S]-ATP gamma S binding sites (pKd = 8.74; Bmax = 358 fmol ml-1 protein). These binding sites differed from those present in rat vas deferens. Thus, pIC50 values for alpha beta meATP (6.5) and L-beta gamma meATP (4.0) at the [3H]-alpha beta meATP binding sites in non-infected CHO-K1 cells were much lower than the respective pIC50 values of 8.3 and 7.7, determined in rat vas deferens. Similarly, affinity estimates (pIC50 values) for ATP (6.82), 2-meS-ATP (5.43), ATP gamma S (7.06) and alpha beta meATP (4.84) at the [35S]-ATP gamma S binding sites in non-infected CHO-K1 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-K1 cells infected using SFV containing the cDNA for the human bladder P2X purinoceptor (SFV-h.P2X), ATP, 2-meS-ATP and alpha beta meATP evoked large inward currents (2-7 nA) in whole cell voltage clamp studies. In membranes prepared from these SFV-h.P2X infected cells, [3H]-alpha beta meATP binding was increased, compared to that measured in the non infected or SFV-LacZ infected cells, with only high affinity [3H]-alpha beta meATP binding sites being detected (pKd = 9.21; Bmax = 3.54 pmol mg-1 protein). The pIC50 values for alpha beta meATP (8.2) and L-beta gamma meATP (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 [35H]-ATP gamma S binding sites (pKd = 9.09; Bmax = 6.82 pmol mg-1 protein) was also present in the membranes from CHO-K1 cells infected with SFV-h.P2X and affinity estimates (pIC50 values) for ATP (8.93), 2-meS-ATP (8.23), ATP gamma S (8.08), and alpha beta meATP (7.17) at competing for these sites were as much as 631 fold higher than the respective values determined in non-infected CHO-K1 cells but were close to the values determined in rat vas deferens. Similar data were obtained with [alpha 33P]-ATP as radioligand. 5. These data suggest that [3H]-alpha beta meATP, [35S]-ATP gamma S and [33P]-ATP label the human bladder recombinant P2X purinoceptor expressed in CHO-K1 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.

Full text

PDF
1254

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. Bean B. P. Pharmacology and electrophysiology of ATP-activated ion channels. Trends Pharmacol Sci. 1992 Mar;13(3):87–90. doi: 10.1016/0165-6147(92)90032-2. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. 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]
  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. 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]
  8. Evans R. J., Lewis C., Buell G., Valera S., North R. A., Surprenant A. Pharmacological characterization of heterologously expressed ATP-gated cation channels (P2x purinoceptors). Mol Pharmacol. 1995 Aug;48(2):178–183. [PubMed] [Google Scholar]
  9. 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]
  10. Iredale P. A., Hill S. J. Increases in intracellular calcium via activation of an endogenous P2-purinoceptor in cultured CHO-K1 cells. Br J Pharmacol. 1993 Dec;110(4):1305–1310. doi: 10.1111/j.1476-5381.1993.tb13960.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Khakh B. S., Humphrey P. P., Surprenant A. Electrophysiological properties of P2X-purinoceptors in rat superior cervical, nodose and guinea-pig coeliac neurones. J Physiol. 1995 Apr 15;484(Pt 2):385–395. doi: 10.1113/jphysiol.1995.sp020672. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Liljeström P., Garoff H. A new generation of animal cell expression vectors based on the Semliki Forest virus replicon. Biotechnology (N Y) 1991 Dec;9(12):1356–1361. doi: 10.1038/nbt1291-1356. [DOI] [PubMed] [Google Scholar]
  15. Lundström K., Mills A., Buell G., Allet E., Adami N., Liljeström P. High-level expression of the human neurokinin-1 receptor in mammalian cell lines using the Semliki Forest virus expression system. Eur J Biochem. 1994 Sep 15;224(3):917–921. doi: 10.1111/j.1432-1033.1994.00917.x. [DOI] [PubMed] [Google Scholar]
  16. Michel A. D., Chau N. M., Fan T. P., Frost E. E., Humphrey P. P. Evidence that [3H]-alpha,beta-methylene ATP may label an endothelial-derived cell line 5'-nucleotidase with high affinity. Br J Pharmacol. 1995 Jul;115(5):767–774. doi: 10.1111/j.1476-5381.1995.tb14999.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. Michel A. D., Humphrey P. P. High affinity P2x-purinoceptor binding sites for [35S]-adenosine 5'-O-[3-thiotriphosphate] in rat vas deferens membranes. Br J Pharmacol. 1996 Jan;117(1):63–70. doi: 10.1111/j.1476-5381.1996.tb15155.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Trezise D. J., Bell N. J., Kennedy I., Humphrey P. P. Effects of divalent cations on the potency of ATP and related agonists in the rat isolated vagus nerve: implications for P2 purinoceptor classification. Br J Pharmacol. 1994 Oct;113(2):463–470. doi: 10.1111/j.1476-5381.1994.tb17012.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. 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]

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

RESOURCES