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A potent irreversible β-adrenergic derivative of pindolol possessing a chemically reactive group (Br-AAM-pindolol) was synthesized. This compound devoid of agonist properties, competed for all (H)-dihydroalprenolol (H-DHA) binding sites in C glioma cell and rat cerebellum membranes. Pretreatment of C glioma cell membranes with Br-AAM-pindolol and subsequent washing resulted in a time- and dose-dependent blockade of β-adrenergic receptors. A 50% blockade was achieved in the presence of 1.6 nM Br-AAM-pindolol. This blockade occurs specifically at the β-adrenergic receptor level, as: 1) it induced a decrease of maximal isoproterenol stimulated adenylate cyclase activity with no modification of basal and sodium fluoride stimulated activity and 2) decreases of (H)-DHA binding and stimulation of adenylate cyclase activity by the agonist were suppressed in the presence of isoproterenol, a β-adrenergic agonist. Furthermore, Br-AAM-pindolol treatment did not affect (H)-diazepam binding in C glioma cell membranes. Pretreatment of C glioma cells with Br-AAM-pindolol also reduced the response of adenylate cyclase to isoproterenol and the number of β-adrenergic receptors. The blockade of β-adrenergic receptors of C glioma cells by Br-AAM-pindolol was non-competitive, whereas the blockade obtained with AM-pindolol, a derivative of pindolol devoid of alkylating properties, was competitive. The irreversible blockade of β-adrenergic receptors by Br-AAM-pindolol in rat erythrocyte membranes was substantiated by the demonstration that no recovery of β-adrenergic receptors occurred during long term incubation of the membranes (48 h) following Br-AAM-pindolol treatment and subsequent washing. Double reciprocal plotting of equiactive isoproterenol concentrations in dose-response curves of adenylate cyclase from membranes of control and Br-AAM-pindolol treated C glioma cells permitted calculation of the dissociation constant for isoproterenol from its binding sites (1.5±0.2×10 M, n=11). This is very close to the dissociation constant of the agonist derived from binding experiments (1.7±0.5×10 M, n=13). These results suggest that Br-AAM-pindolol is a potent irreversible β-adrenergic antagonist and may be useful for pharmacological and physiological studies of σ-adrenergic receptors. [ABSTRACT FROM AUTHOR]