Interactions of putatively irreversible antagonists with β1 − and β2 − adrenergic receptors

Abstract

Three compounds that have been suggested to irreversibly inactivate β-adrenergic receptors were studied: NHNPNBE [ N-(2-hydroxy-3-[1-napthoxy]-propyl)- N-bromoacetylethylenediamine], BAAM (bromoacetylalprenololmenthane), and Ro 3-7894 [1-(5-chloracetylaminobenzfuran-2-yl)-2-isopropylaminoethanol]. Membranes of rat cerebral cortex were used as a source of predominantly β 1 − adrenergic receptors and membranes of rat cerebellum were used as a source of predominantly β 2 − adrenergic receptors. β− Adrenergic receptor binding sites were studied by Scatchard analysis of saturation isotherms of specific [ 125I]-pindolol ([ 125I]PIN) binding. NHNPNBE added to the incubation medium competitively inhibited specific [125 I]PIN binding in both cerebellum and cerebral cortex with K l values of 1–2 μM. in each tissue. After washout of membranes pretreated with NHNPNBE for 30 min at 37°, no loss of specific [ 125I]PIN binding sites was observed in either cerebellum or cortex except at very high concentrations (30–100 μM). Ro 3-7894 caused a simple competitive inhibition of specific [ 125I]PIN binding in rat cerebellar membranes with a K I , of approximately 14 μM, an effect which was reversed completely by washing. In cerebral cortex, Ro 3-7894 added to the incubation medium apparently decreased the density of [ 125I]PIN binding sites with an IC 50 around 1 μM. This effect was reversed after washing the membranes twice. However, in the presence of Ro 3-7894 some Scatchard plots showed a slight curvature. Further saturation of the [ 125I]PIN binding sites in cerebral cortex showed that the inhibition by Ro 3-7894 was competitive but with a high- and low-affinity component, consistent with Ro 3-7894 being a β 1-selective competitive antagonist. Ro 3-7894 was also β 1ft-selective in other tissues. BAAM added to the incubation medium competitively inhibited specific [ 125I]PIN binding in both cerebellum and cortex with K l values of 0.006 to 0.03 μM, but was about 5-fold more potent in cerebellum. After treatment of membranes with higher concentrations of BAAM for 30 min at 37° and washing twice, there was a dose-dependent decrease in the density of specific [ 125I]PIN binding sites with ic 50 values of approximately 0.3 μM in both tissues. Similar effects were observed in rat heart. These data suggest that NHNPNBE is a simple competitive antagonist at both β 1 − and β 2-adrenergic receptors except at very high concentrations. Ro 3-7894 is a β 1-selective competitive antagonist with no apparent irreversible effects. BAAM is a slightly β 2-selective competitive antagonist which can also irreversibly decrease the density of both β 1- and β 2-adrenergic receptor binding sites at relatively low concentrations.