HIGH-AFFINITY AND LOW-AFFINITY BINDING-SITES FOR [H-3] ALPHA,BETA-METHYLENE ATP IN RAT URINARY-BLADDER MEMBRANES

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]-quinuclidnyl 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 (K(d) = 8.1-8.9 nM) and a low-affinity component (K(d) = 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. K(d) values were not greatly affected. 5. The binding was a rapid reversible process. The association rate constants were 7.64 x 107 M-1 min-1 for high-affinity binding, and 7.31 x 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 P(2x)-purinoceptors. The potency order was: α,β-methylene ATP > β,γ-methylene ATP > suramin > ATP > ADP > 2-methylthio AATP >> adenosine. 7. The results indicate that [3H]-α,β-MeATP is a radioligand for the P(2x)-purinoceptor, which satisfies the basic criteria for use in radioligand binding assay.