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

Abstract

1 The chemoreactive ligands 5(2-(((1'-(4'-isothiocyanatophenylamino)thiocarbonyl)-amino)-2-methylpropyl)amino-2-hydroxypropoxy)-3,4-dihydrocarbostyril (DCITC) and 8-hydroxy-5(2-(((1'-(4'-isothiocyanatophenylamino)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 b-adrenoceptor in DDT 1 MF-2 (DDT) cells and the rat isolated aorta. 3 In the rat isolated aorta, DCITC (0.1 mM) did not a ect either the phenylephrine-mediated tissue contraction or the acetylcholine-mediated relaxation. DCITC attenuated the maximal (7)-isoprenalinemediated relaxation of a phenylephrine contracted aorta in a concentration-dependent manner and shifted the dose-response curves for (7)-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 (7)-isoprenaline ws 286 nM and the estimated receptor reserve for this agonist was 23% at the maximal response. 4 HCITC and (7)-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 (7)-isoprenaline was 7.03. However, HCITC produced a greater maximal relaxation of the tissue than (7)-isoprenaline. The HCITC-mediated stimulation of cyclic AMP accumulation and relaxation of the isolated tissue were blocked when the bantagonist 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 b-agonist.

Structure-affinity profile of 8-hydroxycarbostyril-based agonists that dissociate slowly from the β2-adrenoceptor

“…Using the irreversible antagonist DCITC, the agonists' receptor reserve for the maximal stimulation of cAMP accumulation varied from 18% to 42%. Therefore, to achieve a more proportional relationship between receptor occupancy and response and thereby improve the k off estimate, the receptor content (receptor reserve) was reduced by pretreating the cells with the irreversible β-antagonist DCITC (Deyrup et al 1998). Desensitization of the β 2 AR in DDT cells has been reported (Scarpace et al 1985;Strasser et al 1986) and may have occurred during the time course that cells were exposed to agonists (10 min).…”

“…Attached cells were rinsed twice with 2×10 ml HBSS and incubated for 20 min at 37°C in 10 ml HBSS containing 1 nM 5 [2-[[[1'-(4'-isothiocyanatophenyl)thiocarbonyl]-amino]-2-methylpropyl]amino-2-hydroxypropoxy]-3,4-dihydrocarbostyril (DCITC), a potent irreversible antagonist for the βAR (Deyrup et al 1998). At the end of the incubation, the cells were washed 15 times by the addition of icecold HBSS (10 ml) followed 5 min later by aspiration to remove the buffer.…”

GS-5759, a Bifunctionalβ2-Adrenoceptor Agonist and Phosphodiesterase 4 Inhibitor for Chronic Obstructive Pulmonary Disease with a Unique Mode of Action: Effects on Gene Expression in Human Airway Epithelial Cells

“…Agonist affinities were estimated in 6ÂCRE BEAS-2B reporter cells by fractional, irreversible, b 2 -adrenoceptor inactivation (Furchgott, 1966). Agonist E/[A] curves were generated in cells pretreated (60 minutes) with vehicle or the alkylating agent 5(2-(((19-(49-isothiocyanatephenylamino)thiocarbonyl)amino)-2-methylpropyl) amino-2-hydroxypropoxy)-3,4-dihydrocarbostyril (DCITC; 100nM) and then washed in SFM (Deyrup et al, 1998). Each pair of E/[A] curves was fit simultaneously to the operational model of agonism (eq.…”

An estimation of β2‐adrenoceptor reserve on human bronchial smooth muscle for some sympathomimetic bronchodilators

“…Specifically, the aim of the study was to establish if a large receptor density could provide a plausible explanation for the relative resistance of HASM cells to functional desensitization. To this end, a carbostyril‐based irreversible β‐adrenoceptor antagonist, DCITC [5(2‐(((1′‐(4′‐isothiocyanatophenylamino)thiocarbonyl)‐amino)‐2‐methyl‐propyl)amino‐2‐hydroxypropoxy)‐3,4‐dihydrocarbostyril; Figure 1; Deyrup et al. , 1998], was employed to inactivate a fraction of functional β 2 ‐adrenoceptors on HASM to a level where the upper asymptote of agonist concentration–response ( E /[A]) curves was significantly depressed.…”