Errors introduced in radioligand binding studies due to displaceable, cation dependent, [3H]prazosin binding to glass-fibre filters and glass surfaces.

[3H]prazosin not only specifically and homogeneously labels alpha 1-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 alpha-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 alpha 1-adrenoceptors (Rtot: 8.33 fmol.mg-1 wet tissue) with a dissociation constant of 1.28 x 10(-10) M. However, analysis of the same data after correction for non-specific binding, (determined in parallel experiments by adding 10 microM phentolamine to the incubation medium) resulted in a best fit to a model in which [3H]prazosin labels two alpha 1-adrenoceptor subpopulations (R1: 15.0 fmol.mg-1 and R2: 14.6 fmol.mg-1 wet tissue) with dissociation constants of respectively 1.78 x 10(-10) and 5.63 x 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.

Determination of alpha 1-adrenoceptor subtype selectivity by [3H]-prazosin displacement studies in guinea-pig cerebral cortex and rat spleen membranes.

1. [3H]-prazosin homogeneously labels alpha 1-adrenoceptors in guinea-pig cerebral cortex and rat spleen membranes with dissociation constants of 1.28 and 1.49 x 10(-10) M respectively. 2. Phentolamine and WB 4101 displacement studies show that guinea-pig cerebral cortex contains 30% alpha 1A- and 70% alpha 1B-adrenoceptor subtypes, whereas rat spleen contains a virtually homogeneous alpha 1B-adrenoceptor subtype population. The alpha 1-adrenoceptor population of rat thoracic aorta is predominantly of the alpha 1A-adrenoceptor subtype, and in guinea-pig thoracic aorta it is mainly of the alpha 1B-adrenoceptor subtype. 3. Half of the compounds displacing [3H]-prazosin bound to guinea-pig cerebral cortex membranes display alpha 1A-adrenoceptor selectivity. Among these compounds, WB 4101 and methoxamine are most selective, displaying selectivity ratios of approximately 38 and approximately 26 respectively. 4. The affinity constants of the non-selective compounds for the alpha 1-adrenoceptor in guinea-pig cerebral cortex membranes correlate well with the affinity constants obtained for alpha 1B-adrenoceptors in rat spleen membranes. The affinities of selective compounds for the alpha 1B-adrenoceptor subtype in guinea-pig cerebral cortex correlate very well with their affinity for alpha 1B-adrenoceptor in the rat spleen homogenate. Both regression lines coincide with the line of identity. The affinity constants of selective compounds for the alpha 1A-adrenoceptors in guinea-pig cerebral cortex only apparently correlate with the affinity for either the alpha 1B-adrenoceptors in guinea-pig cerebral cortex or in the rat spleen. Regression analyses indicate a straight line relationship (r2>0.9) between pKEA and Pk1B but the regression lines deviate from the line of identity.

Mechanism of action of H2-antagonists on histamine- or dimaprit-stimulated H2-receptors of spontaneously beating guinea-pig atrium.

Cimetidine, ranitidine and famotidine are antagonists of the histamine H2-receptors on the spontaneously beating right atrium of the guinea pig. When analyzed by the classical Schild method their pA2-values are respectively: 6.3, 6.8 and 7.7 with dimaprit as agonist and 5.8, 6.5 and 7.7 with histamine as agonist. Radioligand-displacement studies with [3H]-tiotidine as radioligand resulted in pKd values for cimetidine, ranitidine and famotidine of 6.3; 6.9 and 8.2 respectively. In dimaprit-stimulated atria all antagonists added at concentrations above their Kd values depressed the maximal increase in frequency. In the presence of histamine this effect was much less pronounced and only visible at concentrations of ranitidine and famotidine around 10.Kd. The rightward shift of the curves as well as the decrease in Emax are reversible but the dissociation constants of the antagonists are small (less than 10(-3) s-1). The spontaneously beating right atrium showed receptor reserve for histamine and virtually no receptor reserve for dimaprit. The results have been interpreted in a model in which H2-antagonists act mainly by competing with the agonist for the histamine receptor site but have in addition a distinct affinity for a secondary site on the receptor. Occupation of this site by the antagonist prevents building up of the stimulus elicited by the agonist and thus decreases the Emax. In systems with receptor reserve (histamine) the effect of antagonist binding to the secondary binding site is seen only at high concentration of antagonist while in absence of receptor reserve (dimaprit) the depression of Emax is directly visible. Simulations of the model show that the affinity of this secondary binding site is 50-(famotidine) to 100-(cimetidine and ranitidine) fold lower than for the agonist binding site.