High‐ and low‐affinity binding sites for [3H]‐α,β‐methylene ATP in rat urinary bladder membranes

Open Access
Publisher Website
Google Scholar
Abstract
1 The characteristics of [3H]-α,β-methylene adenosine 5′-triphosphate ([3H]-α,β-MeATP) binding to membrane preparations of rat urinary bladder detrusor were studied. 2 The rat bladder membrane preparation was obtained by multiple centrifugation. [3H]-quinuclidinyl benzilate ([3H]-QNB) binding to this preparation demonstrated that the muscarinic receptor density was 4.32 times higher than that in the homogenate. [3H]-α,β-MeATP binding was increased 3.88 times. 3 Saturation analysis revealed that the rat bladder membrane contained a high density of [3H]-α,β-MeATP binding sites, which could be divided into a high-affinity component (Kd = 8.1–8.9 nm) and a low-affinity component (Kd = 67.0–119.8 nm). 4 Magnesium ions inhibited the maximum binding in a concentration-dependent manner. The maximum high-affinity binding was reduced from 10.32 pmol mg−1 protein in magnesium-free buffer to 4.62 pmol mg−1 protein with 25 mm MgCl2, while the maximum low-affinity binding was reduced from 58.84 pmol mg−1 protein to 14.24 pmol mg−1 protein. Kd values were not greatly affected. 5 The binding was a rapid reversible process. The association rate constants were 7.64 × 107 m−1 min−1 for high-affinity binding, and 7.31 × 106 m−1 min−1 for low-affinity binding. The dissociation rate constants were 0.2896 min−1 for high-affinity binding, and 0.6348 min−1 for the low-affinity binding. 6 Displacement experiments with unlabelled purinoceptor ligands confirmed that [3H]-α,β-MeATP mainly binds to P2X-purinoceptors. The potency order was: α,β-methylene ATP > β,γ-methylene ATP > suramin > ATP > ADP > 2-methylthio ATP ≫ adenosine. 7 The results indicate that [3H]-α,β-MeATP is a radioligand for the P2X-purinoceptor, which satisfies the basic criteria for use in radioligand binding assay.