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Irreversible binding of a carbostyril‐based agonist and antagonist to the β‐adrenoceptor in DDT 1 MF‐2 cells and rat aorta
Author(s) -
Deyrup Malgorzata D,
Greco Phillip G,
Otero Deborah H,
Dennis Donn M,
Gelband Craig H,
Baker Stephen P
Publication year - 1998
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1038/sj.bjp.0701801
Subject(s) - isoprenaline , phenylephrine , agonist , chemistry , dissociation constant , medicine , endocrinology , receptor , antagonist , intrinsic activity , forskolin , fenoterol , biophysics , stereochemistry , biochemistry , biology , stimulation , blood pressure , asthma
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 DDT 1 MF‐2 (DDT) cells and the rat isolated aorta. In DDT cell membranes DCITC and HCITC inhibited (−)[ 125 I]‐iodocyanopindolol (CYP) binding to the β‐adrenoceptor with IC 50 values of 1.1 and 18 n M , respectively. (−)‐Isoprenaline inhibited [ 125 I]‐CYP binding with an IC 50 of 355 n M . 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 μ M 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 μ M ) 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 n M 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 pD 2 values (negative logarithm to base 10 of EC 50 ) 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 pD 2 value of 6.62, whereas the pD 2 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‐agonist.