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

X Bo, G Burnstock

Index: Br. J. Urol. 76 , 297, (1995)

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Abstract

To characterize [3H] alpha, beta-methylene adenosine 5'-triphosphate ([3H] alpha, beta-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] alpha, beta-MeATP binding sites in human bladder.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] alpha, beta-MeATP and the bound and free radioligand separated by filtration. For autoradiography, cryostat sections were incubated with 10 nM [3H] alpha, beta-MeATP, washed, dried and exposed for 2 weeks to emulsion-coated coverslips. In both experiments, 100 microM beta, gamma-methylene ATP was used to determine non-specific binding.Six of 16 specimens in the binding assay and three of seven specimens in the localization study showed specific [3H] alpha, beta-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] alpha, beta-MeATP binding was alpha, beta-methylene ATP > beta, gamma-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] alpha, beta-MeATP binding sites were located only over the smooth muscle of the bladder.The results suggest that P2x-purinoceptors exist in human urinary bladder, although at a lower density than reported for rodent urinary bladder.