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5-Isothiocyanato-2-benzofuranyl-2-imidazoline (BU99006) is an irreversible ligand based on the highly selective I2 binding site ligand 2BFI. In competition binding assays it has been shown to have high affinity and selectivity for the I2 binding site and to irreversibly inhibit the binding of [3H]2BFI. In this present study we have sought to confirm and expand on these findings both in vitro and in vivo. In vitro pre-incubation of rat whole brain membranes with BU99006 (10 μM) was shown to reduce the specific binding of [3H]2BFI to 10% of the control values, an effect not seen using 2BFI or BU224. Pre-treatment of rat whole brain membranes by BU99006, or by the α2-adrenoceptor antagonists RX821002 or rauwolscine had no effect on the specific binding of [3H]RX821002. In vivo pre-treatment of rats with BU99006 (15 mg kg−1, i.v.) caused a substantial loss of [3H]2BFI specific binding in subsequent in vitro saturation analysis and autoradiography; this loss was shown to be dose dependent. These data indicate that BU99006 is selectively and irreversibly affecting I2 binding sites both in vitro and in vivo and that it represents an invaluable tool in the further understanding of the I2 binding site. [Copyright &y& Elsevier]

Fluphenazine-N-mustard (FNM) has been shown to irreversibly block dopaminergic receptor sites and inhibit certain dopaminergically-mediated behaviors. In this study we measured whether FNM has any differential effects on D and D dopaminergic events. Accordingly, we examined the relative effects of FNM on rotational behavior induced by SKF 38393 (D agonist) and Ly 171555 (D agonist) in mice with unilateral, 6-hydroxydopamine-induced lesions of the striatum and the effects of FNM on the binding of [H]Sch 23390 (D ligand) and [H]spiroperidol (D ligand) to mouse striatal membranes. FNM inhibited rotational behavior induced by Ly 171555 at doses 10-fold lower than those required to block rotations induced by SKF 38393 (ID values: Ly 171555=1.8 μmole/kg, IP; SKF 38393=16 μmole/kg, IP). The inhibitory effect of high doses of FNM (20 μmole/kg) on rotational behavior was overcome by increasing the dose of SKF 38393 and apomorphine, a nonselective dopaminergic agonist. By contrast, the inhibitory effect of FNM was not overcome by Ly 171555, even when given in doses more than 100 times its ED. Using striatal homogenates in vitro, FNM inhibited the specific binding of [H]spiroperidol at concentrations about 10-fold lower than those required to inhibit the binding of [H]Sch 23390 (IC values: [H]spiroperidol=90 nM; [H]Sch 23390=840 nM). Considerably higher concentrations of FNM were needed to irreversibly inhibit calmodulin activity in striatal homogenates (IC=10 μM). In vivo, FNM inhibited the binding of [H]spiroperidol measured ex vivo (ID=4 μmole/kg), but did not inhibit the binding of [H]Sch 23390, even when given in doses as high as 100 μmole/kg. These studies indicate that FNM was approximately 10 times more potent at inhibiting D-than D-mediated behavior and at displacing D versus D ligands and suggest that FNM may be useful for studying and differentiating D- and D-mediated events. [ABSTRACT FROM AUTHOR]

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]