Irreversible binding of a carbostyril‐based agonist and antagonist to the β‐adrenoceptor in DDT1 MF‐2 cells and rat aorta

The chemoreactive ligands 5(2‐(((1′‐(4′‐isothiocyanatophenylamino)thiocarbonyl)‐amino)‐2‐methyl‐propyl)amino‐2‐hydroxypropoxy)‐3,4‐dihydrocarbostyril (DCITC) and 8‐hydroxy‐5(2‐(((1′‐(4′‐isothiocya‐natophenylamino)thiocarbonyl)amino)‐2‐methylprop‐2‐yl)amino‐1‐hydroxyethyl)‐carbostyril (HCITC)were synthesized and shown to be potent irreversible antagonist and agonist ligands, respectively, for the β‐adrenoceptor in DDT1 MF‐2 (DDT) cells and the rat isolated aorta. In DDT cell membranes DCITC and HCITC inhibited (−)[125I]‐iodocyanopindolol (CYP) binding to the β‐adrenoceptor with IC50 values of 1.1 and 18 nM, respectively. (−)‐Isoprenaline inhibited [125I]‐CYP binding with an IC50 of 355 nM. Pretreatment of membranes with either chemoreactive ligand produced a time‐ and concentration‐dependent decrease in the β‐adrenoceptor content, indicating irreversible receptor binding. DCITC at concentrations up to 10 μM did not stimulate cyclic AMP accumulation in DDT cells nor did it amplify forskolin‐stimulated cyclic AMP accumulation. In the rat isolated aorta, DCITC (0.1 μM) did not affect either the phenylephrine‐mediated tissue contraction or the acetylcholine‐mediated relaxation. DCITC attenuated the maximal (−)‐isoprenaline‐mediated relaxation of a phenylephrine contracted aorta in a concentration‐dependent manner and shifted the dose‐response curves for (−)‐isoprenaline to the right. The DCITC‐induced decrease in maximal response was not reversed by extensive tissue washing. By use of the operational model of agonism, the calculated dissociation constant for (−)‐isoprenaline ws 286 nM and the estimated receptor reserve for this agonist was 23% at the maximal response. HCITC and (−)‐isoprenaline stimulated cyclic AMP accumulation in DDT cells with pD2 values (negative logarithm to base 10 of EC50) of 7.95 and 7.97, respectively, and both mediated the same maximal stimulation. In the rat isolated aorta, HCITC produced a concentration‐dependent relaxation of the tissue with a pD2 value of 6.62, whereas the pD2 for (−)‐isoprenaline was 7.03. However, HCITC produced a greater maximal relaxation of the tissue than (−)‐isoprenaline. The HCITC‐mediated stimulation of cyclic AMP accumulation and relaxation of the isolated tissue were blocked when the β‐antagonist propranolol was added concurrently. In contrast, once the HCITC‐mediated responses were established, the addition of propranolol did not result in any attenuation indicating that HCITC is an irreversible β‐agon