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The binding characteristics of a human bladder recombinant P 2X purinoceptor, labelled with [ 3 H]‐αβmeATP, [ 35 S]‐ATPγS or 33 [P]‐ATP
Author(s) -
Michel Anton D.,
Lundstrom Kenneth,
Buell Gary N.,
Surprenant Annmarie,
Valera Soledad,
Humphrey Patrick P. A.
Publication year - 1996
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1996.tb16723.x
Subject(s) - vas deferens , purinergic receptor , microbiology and biotechnology , biology , binding site , semliki forest virus , medicine , receptor , endocrinology , biochemistry , gene , rna
1 The binding of [ 3 H]‐αβmeATP, [ 35 S]‐ATPγS and [α 33 P]‐ATP to a human bladder P 2X 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 P 2x purinoceptors and in which the endogenous P 2X purinoceptor displays high homology with the human bladder P 2X purinoceptor. 2 In non‐infected CHO‐K1 cells, 100 μ m 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), [ 3 H]‐αβmeATP bound with low affinity (p K d = 7.04; B max = 8.88 pmol ml −1 protein) and there was only a low density of [ 35 S]‐ATPγS binding sites (p K d = 8.74; B max = 358 fmol ml −1 protein). These binding sites differed from those present in rat vas deferens. Thus, pIC 50 values for αβmeATP (6.5) and L‐βγmeATP (4.0) at the [ 3 H]‐αβmeATP binding sites in non‐infected CHO‐K1 cells were much lower than the respective pIC 50 values of 8.3 and 7.7, determined in rat vas deferens. Similarly, affinity estimates (pIC 50 values) for ATP (6.82), 2‐meS‐ATP (5.43), ATPγS (7.06) and αγmeATP (4.84) at the [ 35 S]‐ATPγS 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 P 2X purinoceptor (SFV‐h.P 2X ), ATP, 2‐meS‐ATP and αβmeATP evoked large inward currents (2–7 nA) in whole cell voltage clamp studies. In membranes prepared from these SFV‐h.P 2X infected cells, [ 3 H]‐αβmeATP binding was increased, compared to that measured in the non infected or SFV‐LacZ infected cells, with only high affinity [ 3 H]‐αβmeATP binding sites being detected (p K d = 9.21; B max = 3.54 pmol mg −1 protein). The pIC 50 values for αβmeATP (8.2) and L‐βγmeATP (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 [ 35 S]‐ATPγS binding sites (p K d = 9.09; B max = 6.82 pmol mg −1 protein) was also present in the membranes from CHO‐K1 cells infected with SFV‐h.P 2X and affinity estimates (pIC 50 values) for ATP (8.93), 2‐meS‐ATP (8.23), ATPγS (8.08), and αβmeATP (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 [α 33 P]‐ATP as radioligand. 5 These data suggest that [ 3 H]‐αβmeATP, [ 35 S]‐ATPγS and [ 33 P]‐ATP label the human bladder recombinant P 2X purinoceptor expressed in CHO‐K1 cells following infection with SFV‐h.P 2X and provide further corroborative evidence to support the contention that the high affinity binding sites for these radioligands in rat vas deferens are P 2X purinoceptors.