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Evidence that [ 3 H]‐α,β‐methylene ATP may label an endothelial‐derived cell line 5′‐nucleotidase with high affinity

1995; Wiley; Volume: 115; Issue: 5 Linguagem: Inglês

10.1111/j.1476-5381.1995.tb14999.x

1476-5381

A.D. Michel, Ngoc Chau, Tai-Ping Fan, Emma E. Frost, P.P.A. Humphrey,

Neonatal Health and Biochemistry

In membranes prepared from a permanent cell line of endothelial origin (WEC cells), [ 3 H]‐α,β‐methylene ATP ([ 3 H]‐α,β‐meATP) labelled high (p K d = 9.5; B max = 3.75 pmol mg −1 protein) and low (p K d = 7.2; B max = 23.3 pmol mg −1 protein) affinity binding sites. The high affinity [ 3 H]‐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 (pIC 50 = 8.26) for α,β‐meATP. The high affinity [ 3 H]‐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 [ 3 H]‐α,β‐meATP. The proportion of sites with high affinity for these compounds was found to vary between 42 and 69°. Approximately 60–69% of the binding sites labelled with 1 nM [ 3 H]‐α,β‐meATP possessed high affinity for α,β‐meADP (pIC 50 = 8.87), AMP (pIC 50 = 7.12), GMP (pIC 50 = 7.34), UTP (pIC 50 = 6.12), GTP (pIC 50 = 7.59), GppNHp (plC 50 = 735) and adenosine (pIC 50 = 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 μ m ) sufficient to inhibit selectively the binding of [ 3 H]‐α,β‐meATP, the [ 3 H]‐α,β‐meATP binding sites with high affinity for AMP, UTP, α,β‐meADP, GTP, GppNHp and adenosine were also occluded. WEC cell membranes were able to metabolize a trace concentration (6 nM) of [ 3 H]‐AMP to [ 3 H]‐adenosine under the conditions of the binding assay. The pIC 50 values of adenosine (5.99), GMP (7.55) and the substrate AMP (7.19) for inhibiting this [ 3 H]‐AMPase activity were almost identical to their high affinity pIC 50 estimates obtained in the binding assay. Although α,β‐meADP, α,β‐meATP, β,γ‐meATP, ATP, ADP and GppNHp identified heterogeneity in the [ 3 H]‐AMPase activity of the WEC cells, their pIC 50 values for inhibiting the major portion of the [ 3 H]‐AMPase activity were similar to their respective high affinity pIC 50 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 [ 3 H]‐α,β‐meATP. In the presence of 10 μ m GMP, the affinity estimates for α,β‐meADP, AMP, GMP, GTP, GppNHp, ADP and adenosine at the high affinity [ 3 H]‐α,β‐meATP binding sites that remained available, were low and similar to their affinity estimates at the high affinity [ 3 H]‐α,β‐meATP binding sites of rat vas deferens. Since the high affinity [ 3 H]‐α,β‐meATP binding sites in rat vas deferens are thought to be P 2x purinoceptors it is possible that the high affinity [ 3 H]‐α,β‐meATP binding sites in the WEC which possess low affinity for α,β‐meADP are also P 2x purinoceptors.