CHARACTERIZATION AND AUTORADIOGRAPHIC LOCALIZATION OF [H-3] ALPHA,BETA-METHYLENE ADENOSINE 5'-TRIPHOSPHATE BINDING-SITES IN HUMAN URINARY-BLADDER

Objectives To characterize [H-3]alpha,beta-methylene adenosine 5'-triphosphate ([H-3]alpha,beta-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 [H-3]alpha,beta-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 [H-3]alpha,beta-MeATP and the bound and free radioligand separated by filtration. For autoradiography, cryostat sections were incubated with 10 nM [H-3]alpha,beta-MeATP, washed, dried and exposed for 2 weeks to emulsion-coated coverslips. In both experiment s, 100 mu M beta,gamma-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 [H-3]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 [H-3]alpha,beta-MeATP binding was alpha,beta-methylene ATP > beta,gamma-methylene ATP> suramin > 2-methylthio ATP > ATP > ADP much greater than adenosine, which indicates that the binding sites are, or are linked to, P-2x-purinoceptors. Autoradiography showed that the specific [H-3]alpha,beta-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.