IRREVERSIBLE BINDING OF A CARBOSTYRIL-BASED AGONIST AND ANTAGONIST TOTHE BETA-ADRENOCEPTOR IN DDT1 MF-2 CELLS AND RAT AORTA

1 The chemoreactive ligands pyl)amino-2-hydroxypropoxy)-3,4-dihydrocar bostyril (DCITC) and -methylprop-2-yl)amino-1-hydroxyethyl)-carbostyri l (HCITC) were synthesized and shown to be potent irreversible antagon ist and agonist ligands, respectively, for the beta-adrenoceptor in DD T1 MF-2 (DDT) cells and the rat isolated aorta. 2 In DDT cell membrane s DCITC and HCITC inhibited (-)[I-125]-iodocyanopindolol (CYP) binding to the beta-adrenoceptor with IC50 values of 1.1 and 18 nM, respectiv ely. (-)-Isoprenaline inhibited [I-125]-CYP binding with an IC50 of 35 5 nM. Pretreatment of membranes with either chemoreactive ligand produ ced a time-and concentration-dependent decrease in the beta-adrenocept or content, indicating irreversible receptor binding. DCITC at concent rations up to 10 mu M did not stimulate cyclic AMP accumulation in DDT cells nor did it amplify forskolin-stimulated cyclic AMP accumulation . 3 In the rat isolated aorta, DCITC (0.1 mu M) did not affect either the phenylephrine-mediated tissue contraction or the acetylcholine-med iated relaxation. DCITC attenuated the maximal (-)-isoprenaline-mediat ed relaxation of a phenylephrine contracted aorta in a concentration-d ependent manner and shifted the dose-response curves for (-)-isoprenal ine to the right. The DCITC-induced decrease in maximal response was n ot reversed by extensive tissue washing. By use of the operational mod el of agonism, the calculated dissociation constant for (-)-isoprenali ne ws 286 nM and the estimated receptor reserve for this agonist was 2 3% at the maximal response. 4 HCITC and (-)-isoprenaline stimulated cy clic AMP accumulation in DDT cells with pD(2) values (negative logarit hm to base 10 of EC50) of 7.95 and 7.97, respectively, and both mediat ed the same maximal stimulation. In the rat isolated aorta, HCITC prod uced a concentration-dependent relaxation of the tissue with a pD(2) v alue of 6.62, whereas the pD(2) for (-)-isoprenaline was 7.03. However , HCITC produced a greater maximal relaxation of the tissue than (-)-i soprenaline. The HCITC-mediated stimulation of cyclic AMP accumulation and relaxation of the isolated tissue were blocked when the beta-anta gonist 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 beta-agonist.