EVIDENCE THAT [H-3] ALPHA,BETA-METHYLENE ATP MAY LABEL AN ENDOTHELIAL-DERIVED CELL-LINE 5'-NUCLEOTIDASE WITH HIGH-AFFINITY

1 In membranes prepared from a permanent cell line of endothelial orig in (WEC cells), [H-3]]-alpha,beta-methylene ATP ([H-3]-alpha,beta-meAT P) labelled high (pK(d)=9.5; B-max=3.75 pmol mg(-1) protein): and low (pK(d)=7.2; B-max=23.3 pmol mg(-1) protein) affinity binding sites. Th e high affinity [H-3]-alpha,beta-meATP binding sites in the WEC cell m embranes 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 (pI C(50)=8.26) for alpha,beta-meATP. 2 The high affinity [H-3]-alpha,beta -meATP binding sites appeared heterogeneous since in competition Studi es a number of nucleotide analogues (alpha,beta-meADP, ATP, ADP, AMP, GTP, GppNHp, GMP) and adenosine identified two populations of the site s labelled by 1 nM [H-3]-alpha,beta-meATP. The proportion of sites wit h high affinity for these compounds was found to vary between 42 and 6 9%. 3 Approximately 60-69% of the binding sites labelled with 1 nM [H- 3]-alpha,beta-meATP possessed high affinity for alpha,beta-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 (pIC(50)=7.35) and adenosine (pIC(50)-5.45 ). The sites at which these compounds possessed high affinity were pro bably the same, since, in the presence of GMP at a concentration. (10 mu M) sufficient to inhibit selectively the binding of [H-3]-alpha,bet a-meATP, the [3H]-alpha,beta-meATP binding sites with high affinity fo r AMP, UTP, alpha,beta-meADP, GTP, GppNHp and adenosine were also occl uded. 4 WEC cell membranes were able to metabolize a trace concentrati on (6 nM) of [H-3]-AMP to [H-3]-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 [H-3]-AMPase activity wer e almost identical to their high affinity pIC(50) estimates obtained i n the binding assay. Although alpha,beta-meADP, alpha,beta-meATP, beta ,gamma-meATP, ATP, ADP and GppNHp identified heterogeneity in the [H-3 ]=AMPase activity of the WEC cells, their pIC(50) values for inhibitin g the major portion of the [H-3]-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 po ssesses high affinity for both alpha,beta-meADP and alpha,beta-meATP a nd that this enzyme can be labelled by [H-3]-alpha,beta-meATP. 5 In th e presence of 10 mu M GMP, the affinity estimates for alpha,beta-meADP , AMP, GMP, GTP,GppNHp, ADP and adenosine at the high affinity [H-3]-a lpha,beta-meATP binding sites that remained available, were low and si milar to their affinity estimates at the high affinity [H-3]-alpha,bet a-meATP binding sites of rat vas deferens. Since the high affinity [H- 3]-alpha,beta-meATP binding sites in rat vas deferens are thought to b e P-2x purinoceptors it is possible that the high affinity [H-3]-alpha ,beta-meATP binding sites in the WEC which possess low affinity for al pha,beta-meADP are also P-2x purinoceptors.