Characterization and autoradiographic localization of [3H]α,β-methylene adenosine 5'-triphosphate binding sites in human urinary bladder.

Objectives To characterize [³H]α,β-methylene adenosine 5′-triphosphate ([³H]α,β-MeATP, a radioligand for P_2X-purinoceptors) binding sites in the washed homogenates and membrane preparations of human urinary bladder and, using autoradiography, to localize [³H]α,β-MeATP binding sites in human bladder. Materials and methods Specimens were obtained from the fundus of the urinary bladder of male patients aged 56-79 years. The washed homogenates or membrane preparations of the bladder specimens were incubated with [³H]α,β-MeATP and the bound and free radioligand separated by filtration. For autoradiography, cryostat sections were incubated with 10 nM [³H]α,β-MeATP, washed, dried and exposed for 2 weeks to emulsion-coated coverslips. In both experiments, 100 μ mβ,γ-methylene ATP was used to determine non-specific binding. Results Six of 16 specimens in the binding assay and three of seven specimens in the localization study showed specific [³H]α,β-MeATP binding. The binding process was saturable and the specific binding sites were composed of a high-and low-affinity component. The specific binding to membrane preparations was reduced in the presence of Mg²⁺ in the incubation medium. Competitive displacement experiments showed that the order of potency of the unlabelled ligands to displace the [³H]α,β-MeATP binding was α,β-methylene ATP < β,γ-methylene ATP < suramin < 2-methylthio ATP < ATP > ADP ≫ adenosine, which indicates that the binding sites are, or are linked to, P_2X-purinoceptors. Autoradiography showed that the specific [³H]α,β-MeATP binding sites were located only over the smooth muscle of the bladder. Conclusions The results suggest that P_2X-purinoceptors exist in human urinary bladder, although at a lower density than reported for rodent urinary bladder. [ABSTRACT FROM AUTHOR]