Identification and quantification of β-adrenoceptor sites in red blood cells from rats

Abstract

For the direct characterization of β-adrenoceptors in membrane preparations from the reticulocyte rich blood of rats treated with acetyl phenylhydrazide the β-adrenergic antagonist ligand (3H)(-)dihydroalprenolol (DHAP) was used. 1. Specific binding of DHAP demonstrated one type of binding site in these membrane preparations. There was no evidence for negative co-operativity between the sites. A mean KD-value amounting to 6.51\+-0.8 nM (n=15) was calculated from equilibrium experiments; a similar KD-value (8.25 nM) was evaluated from the ratio of the rate constants of the dissociation and association reactions. In preparations of reticulocyte rich blood (53% reticulocytes) a mean density of DHAP binding sites of 0.602\+-0.05 pmoles/mg protein (n=15) was determined. The respective value in membrane preparations from reticulocyte poor blood, i.e. from untreated animals (2% reticulocytes), amounted to only 0.224\+-0.03 pmoles/mg protein (n=5) whereas the mean KD-value remained unaltered (KD=6.84\+-2.2 nM; n=5). 2. Specific binding sites for DHAP in membranes from reticulocyte rich blood can be looked at as true \gb-adrenoceptors: Specific binding of DHAP was competitively inhibited by β-adrenoceptor agonists and antagonists according to the structural specificity and stereospecificity of these compounds. The KD-values for agonists increased in the order isoprenaline < adrenaline < noradrenaline