Skip to main content
Log in

Distribution and characterisation of [3H]α,β-methylene ATP binding sites in the rat

  • Original Articles
  • Published:
Naunyn-Schmiedeberg's Archives of Pharmacology Aims and scope Submit manuscript

Summary

Radioligand binding studies have been performed to study the distribution of the binding sites for the P2x purinoceptor selective agonist radioligand, [3H]α,β-methylene ATP ([3H]αβ-meATP), in membranes prepared from various peripheral organs and several brain regions of the rat. In agreement with previous studies in the rat vas deferens, [3H]αβ-meATP labelled two populations of sites. One site exhibited high affinity for the ligand (Kd = 0.7 nM; Bmax = 1012 fmol.mg−1 protein) while the other site exhibited lower affinity (Kd = 70.8 nM) and higher capacity (Bmax = 7470 fmol. mg−1 protein). In competition studies, using a low concentration of radioligand (1 nM), the high affinity αβ-meATP binding sites in vas deferens membranes could be preferentially labelled (84–91%). Under these conditions, the PZx purinoceptor agonists, αβ-meATP and β,γ-methylene ATP, had the highest affinity with pIC50 values of 8.3 and 7.3 respectively. The P2y purinoceptor agonist, 2-methyl-thio-ATP (2-me-S-ATP), had lower affinity (pIC50 = 6.7), while uridine triphosphate, adenosine diphosphate and adenosine, agonists at the P2u, P2t and P1 purinoceptors, respectively, possessed low affinity (pIC50 values < 5.6). In addition, the P2 purinoceptor antagonists, cibacron blue and suramin, inhibited binding over the same concentration range at which they behave as functional antagonists at the P2x purinoceptor.

High and low affinity binding sites for [3H]αβ-meATP were also identified in a range of other peripheral tissues (spleen, heart and liver) and in several brain regions (striatum, cerebral cortex, hippocampus). In the spleen, heart, cerebral cortex and liver the Kd values at both the high affinity binding sites (Kd = 1 – 1.2 nM) and the low affinity binding sites (Kd = 98 – 158 nM) were similar to the respective Kd values at the high and low affinity binding sites in the vas deferens. In competition studies performed using a low concentration of radioligand (1 nM) these sites exhibited a similar pharmacological profile to that seen in the vas deferens. Detailed analysis of competition curves to several of the ATP analogues in each of the tissues revealed that the binding profile of the radioligand was complex since several compounds, and in particular ATP and 2-meS-ATP, identified a lower proportion of sites with high affinity than did αβ-meATP The simplest interpretation of these data is that there are both high and low affinity sites for [3H]αβ-meATP in all tissues, but that the high affinity sites display heterogeneity with respect to various purinoceptor agonists.

We conclude that this study demonstrates the presence of high affinity binding sites for [3H]αβ-meATP in a range of peripheral tissues and in several brain regions. These sites display a pharmacological profile which suggests that they reflect binding to a P2x purinoceptor. The apparent heterogeneity of these high affinity sites may be a consequence of agonist-induced conformational changes which we speculate may be linked with desensitisation states of the receptor.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Bean BP (1992) Pharmacology and electrophysiology of ATP-activated ion channels. Trends Pharmacol Sci 13:87–90

    Google Scholar 

  • Benham CD, Tsien RW (1987) A novel receptor-operated Ca2+-permeable channel activated by ATP in smooth muscle. Nature 328: 275–278

    Google Scholar 

  • Bo X, Burnstock G (1990) High- and low-affinity binding sites for [3H]-α,β-methylene ATP in rat urinary bladder membranes. Br J Pharmacol 101:291–296

    Google Scholar 

  • Bo X, Simon J, Burnstock G, Barnard EA (1992) Solubilization and molecular size determination of the P2x purinoceptor from rat vas deferens. J Biol Chem 267:17581–17587

    Google Scholar 

  • Burnstock G (1978) A basis of distinguishing two types of purinergic receptors. In: Straub RW, Bolis L (eds) cell membrane receptors for drugs and hormones: a multidisciplinary approach. Raven Press, New York, pp 107–118

    Google Scholar 

  • Burnstock G (1990) Overview: Purinergic mechanisms. Ann NY Acad Sci 603:1–17

    Google Scholar 

  • Burnstock G, Kennedy C (1985) Is there a basis for distinguishing two types of P2-purinoceptor? Gen Pharmacol 16:433–440

    Google Scholar 

  • Cheng YC, Prusoff WH (1973) Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 percent inhibition (IC50) of an enzymatic reaction. Biochem Pharmacol 22:3099–3108

    Google Scholar 

  • Choo LK (1981) The effect of reactive blue, an antagonist of ATP, on the isolated urinary bladders of guinea-pig and rat. J Pharm Pharmacol 33:248–250

    Google Scholar 

  • Cooper CL, Mooris AJ, Harden TK (1989) Guanine nucleotide-sensitive interaction of a radiolabelled agonist with a phospholipase C-linked P2y-purinergic receptor. J Biol Chem 264:6202–6206

    Google Scholar 

  • Dubyak GR (1991) Signal transduction by P2 purinergic receptors for extracellular ATP. Am J Respir Cell Mol Biol 4:295–300

    Google Scholar 

  • Dunn PM, Blakeley AGH (1988) Suramin: a reversible P2-purinoceptor antagonist in the mouse vas deferens. Br J Pharmacol 93:243–245

    Google Scholar 

  • Edwards FA, Gibb AJ, Colquhoun D (1992) ATP receptor-mediated synaptic currents in the central nervous system. Nature 359: 144–147

    Google Scholar 

  • Gordon JL (1986) Extracellular ATP: effects, sources and fate. Biochem J 233:309–319

    Google Scholar 

  • Harden TK, Boyer JL, Brown HA, Cooper CL, Jeffs RA, Martin MW (1990) Biochemical properties of a P2y-purinergic receptor. Ann NY Acad Sci 603:256–266

    Google Scholar 

  • Hoyle CHV, Knight GE, Burnstock G (1990) Suramin anatogonizes responses to P2-purinoceptor agonists and purinergic nerve stimulation in the guinea-pig urinary bladder and taenia coli. Br J Pharmacol 99:617–621

    Google Scholar 

  • Humphrey PPA, Kennedy I, Michel AD, Morton RA (1993) Preliminary studies on purine P2 receptors in rat spleen. Br J Pharmacol 108:175P

    Google Scholar 

  • Kasakov L, Burnstock G (1983) The use of the slowly degradable analog, α,β-methylene ATP, to produce desensitisation of the P2-purinoceptor: effect on non-adrenergic, non-cholinergic responses of the guinea-pig urinary bladder. Eur J Pharmacol 86:291–294

    Google Scholar 

  • Kennedy C (1990) P1- and P2-purinoceptor subtypes — An update. Arch Int Pharmacodyn 303:30–50

    Google Scholar 

  • Lippiello PM, Sears SB, Fernandes KG (1987) Kinetics and mechanism of l-[3H]nicotine binding to putative high affinity receptor sites in rat brain. Mol Pharmacol 31:392–400

    Google Scholar 

  • Michel AD, Whiting RL (1984) Analysis of ligand binding data using a miercomputer. Br J Pharmacol 83:460P

    Google Scholar 

  • Michel AD, Brown HC, Sewter C, Kennedy I, Humphrey PPA (1993 a) Identification of [3H]αβ-methylene-ATP binding sites in rat brain and peripheral tissues. Br J Pharmacol 108:44P

  • Michel AD, Sewter CE, Brown H, Humphrey PPA (1993b) Heterogeneity of high affinity binding sites for [3H]αβ-methylene-ATP in rat tissues. Br J Pharmacol 109:110P

    Google Scholar 

  • Munson PJ, Rodbard D (1980) LIGAND: A versatile computerised approach for the characterisation of ligand binding systems. Anal Biochem 107:220–239

    Google Scholar 

  • O'Connor SE, Dainty IA, Leff P (1991) Further subclassification of ATP receptors based on agonist studies. Trends Pharmacol Sci 12:137–141

    Google Scholar 

  • Scatchard G (1949) The attractions of proteins for small molecules and ions. Ann NY Acad Sci 51, 660–672

    Google Scholar 

  • Seifert R, Schultz G (1989) Involvement of pyrimidinoceptors in the regulation of cell functions by uridine and by uracil nucleotides. Trends Pharmacol Sci 10: 365–369

    Google Scholar 

  • Tschöpl M, Harms L, Nörenberg W, Illes P (1992) Excitatory effects of adenosine 5′-triphosphate on rat locus coeruleus neurons. Eur J Pharmacol 213:71–77

    Google Scholar 

  • von Kügelgen I, Häussinger D, Starke K (1987) Evidence for a vasoconstriction-mediating receptor for UTP, distinct for the P2 purinoceptor, in rabbit ear artery. Naunyn-Schmiedebergs Arch Pharmacol 336:556–560

    Google Scholar 

  • Welford LA, Cusack NJ, Hourani SMO (1987) The structure-activity relationships of ectonucleotidases and of excitatory P2 purinoceptors: evidence that dephosphorylation of ATP analogues reduces pharmacological potency. Eur J Pharmacol 141:123–130

    Google Scholar 

  • Wonnacott S (1990) The paradox of nicotinic acetylcholine receptor upregulation by nicotine. Trends Pharmacol Sci 11:216–219

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Additional information

Correspondence to: A. D. Michel at the above address

Rights and permissions

Reprints and permissions

About this article

Cite this article

Michel, A.D., Humphrey, P.P.A. Distribution and characterisation of [3H]α,β-methylene ATP binding sites in the rat. Naunyn-Schmiedeberg's Arch Pharmacol 348, 608–617 (1993). https://doi.org/10.1007/BF00167237

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00167237

Key words

Navigation