Evidence that [3H]-α,β-methylene ATP may label an endothelial-derived cell line 5′-nucleotidase with high affinity
Article first published online: 19 JUL 2012
1995 British Pharmacological Society
British Journal of Pharmacology
Volume 115, Issue 5, pages 767–774, July 1995
How to Cite
Michel, A.D., Chau, N.-M., Fan, T.-P.D., Frost, E.E. and Humphrey, P.P.A. (1995), Evidence that [3H]-α,β-methylene ATP may label an endothelial-derived cell line 5′-nucleotidase with high affinity. British Journal of Pharmacology, 115: 767–774. doi: 10.1111/j.1476-5381.1995.tb14999.x
- Issue published online: 19 JUL 2012
- Article first published online: 19 JUL 2012
- Received December 13, 1994 Revised February 28, 1995 Accepted March 16, 1995
- [3H]-α,β-methylene ATP;
- α,β-methylene ADP;
- endothelial cell;
- 1In membranes prepared from a permanent cell line of endothelial origin (WEC cells), [3H]-α,β-methylene ATP ([3H]-α,β-meATP) labelled high (pKd = 9.5; Bmax = 3.75 pmol mg−1 protein) and low (pKd = 7.2; Bmax = 23.3 pmol mg−1 protein) affinity binding sites. The high affinity [3H]-α,β-meATP binding sites in the WEC cell membranes could be selectively labelled with a low concentration of the radioligand (1 nM). In competition studies performed at a radioligand concentration of 1 nM, 88.6% of the sites possessed high affinity (pIC50 = 8.26) for α,β-meATP.
- 2The high affinity [3H]-α,β-meATP binding sites appeared heterogeneous since in competition studies a number of nucleotide analogues (α,β-meADP, ATP, ADP, AMP, GTP, GppNHp, GMP) and adenosine identified two populations of the sites labelled by 1 nM [3H]-α,β-meATP. The proportion of sites with high affinity for these compounds was found to vary between 42 and 69°.
- 3Approximately 60–69% of the binding sites labelled with 1 nM [3H]-α,β-meATP possessed high affinity for α,β-meADP (pIC50 = 8.87), AMP (pIC50 = 7.12), GMP (pIC50 = 7.34), UTP (pIC50 = 6.12), GTP (pIC50 = 7.59), GppNHp (plC50 = 735) and adenosine (pIC50 = 5.45). The sites at which these compounds possessed high affinity were probably the same, since, in the presence of GMP at a concentration (10 μm) sufficient to inhibit selectively the binding of [3H]-α,β-meATP, the [3H]-α,β-meATP binding sites with high affinity for AMP, UTP, α,β-meADP, GTP, GppNHp and adenosine were also occluded.
- 4WEC cell membranes were able to metabolize a trace concentration (6 nM) of [3H]-AMP to [3H]-adenosine under the conditions of the binding assay. The pIC50 values of adenosine (5.99), GMP (7.55) and the substrate AMP (7.19) for inhibiting this [3H]-AMPase activity were almost identical to their high affinity pIC50 estimates obtained in the binding assay. Although α,β-meADP, α,β-meATP, β,γ-meATP, ATP, ADP and GppNHp identified heterogeneity in the [3H]-AMPase activity of the WEC cells, their pIC50 values for inhibiting the major portion of the [3H]-AMPase activity were similar to their respective high affinity pIC50 values in the binding assay. It thus seems likely that WEC cells express a form of 5′-nucleotidase that possesses high affinity for both α,β-meADP and α,β-meATP and that this enzyme can be labelled by [3H]-α,β-meATP.
- 5In the presence of 10 μm GMP, the affinity estimates for α,β-meADP, AMP, GMP, GTP, GppNHp, ADP and adenosine at the high affinity [3H]-α,β-meATP binding sites that remained available, were low and similar to their affinity estimates at the high affinity [3H]-α,β-meATP binding sites of rat vas deferens. Since the high affinity [3H]-α,β-meATP binding sites in rat vas deferens are thought to be P2x purinoceptors it is possible that the high affinity [3H]-α,β-meATP binding sites in the WEC which possess low affinity for α,β-meADP are also P2x purinoceptors.