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

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Abstract
Objectives To characterize [3H]α,β‐methylene adenosine 5′‐triphosphate ([3H]α,β‐MeATP, a radioligand for P2X‐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 μmβ,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, P2X‐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 P2X‐purinoceptors exist in human urinary bladder, although at a lower density than reported for rodent urinary bladder.