Errors Introduced in Radioligand Binding Studies Due to Displaceable,Cation Dependent, [3H]prazosin Binding to Glass-Fibre Filters and Glass Surfaces

Abstract

[³H]prazosin not only specifically and homogeneously labels α₁-adrenoceptors, but also binds to glass surfaces and non-linearly to the glass-fibre filters, commonly used in radioligand binding experiments. Binding to filters can be modulated by unlabeled α-adrenergic compounds and cations. If no correction is applied for displaceable filter binding, analysis of [³H]prazosin binding experiments leads to erroneous results. Analysis of [³H]prazosin saturation experiments on guinea-pig cerebral cortex membranes with correction for filter binding before the non-linear fit procedure indicated that [³H]prazosin labels a homogeneous population of α₁-adrenoceptors (R_tot: 8.33 fmol˙mg^?1 wet tissue) with a dissociation constant of 1.28×10^?10 M. However, analysis of the same data after correction for non-specific binding, (determined in parallel experiments by adding 10 μM phentolamine to the incubation medium) resulted in a best fit to a model in which [³H]prazosin labels two α₁-adrenoceptor subpopulations (R₁: 15.0 fmol˙mg^?1 and R₂: 14.6 fmol˙mg^?1 wet tissue) with dissociation constants of respectively 1.78×10^?10 and 5.63×10^?9 M. The discrepancy between the two methods of analysis is due to displacement of the radioligand from the filters by phentolamine.

Prazosin and oxymetazoline are also able to displace filter-bound [³H]prazosin. The extent to which displaceable filter binding distorts the proper results depends on the actual magnitude of the error and also on the method of analysis.