Inverse agonism and neutral antagonism at a constitutively active alpha‐1a adrenoceptor

Abstract

1 We have studied the antagonist action of prazosin and KMD-3213 in a constitutively active mutant of the human alpha-1a adrenoceptor in which Ala 271 was substituted to Thr and was expressed in CHO cells. Inverse agonism was characterized by up-regulation of receptor density, a decrease in basal GTPgS binding, and a reduction in basal inositol-1,4,5-trisphosphate (IP 3 ) level. 2 According to the above criteria, prazosin acted as an inverse agonist, whilst KMD-3213 behaved as a neutral antagonist. 3 Compared with the wild-type receptor, mutant receptor exhibited single a nity sites for [ 3 H]-prazosin, [ 3 H]-KMD and the non-radioactive ligands tested, and displayed signi®cantly higher a nities for several agonists but not for the two antagonists. 4 Administration of KMD-3213 to prazosin-treated CHO cells expressing the mutant receptor reversed the inverse agonism of prazosin resulting in rapid increases in cellular IP 3 , in intracellular [Ca 2+ ] and in the rate of extracellular acidi®cation. 5 These results indicated that a neutral antagonist can reverse the action of an inverse agonist at the receptor site. The distinct properties of inverse agonist and neutral antagonist in a ecting receptor function may be important for the clinical use of such antagonists.

Inverse Agonist Activity of Selected Ligands of the Cysteinyl-Leukotriene Receptor 1

“…It was shown that inverse agonists could induce the internalization of the CCK A receptor (Roettger et al, 1997), although increased cell surface expression has been reported for other receptors after inverse agonist treatment (Smit et al, 1996;Lee et al, 1997;Zhu et al, 2000). Our results showed that none of the three more potent inverse agonists (Montelukast, MK571, and Zafirlukast) induced receptor sequestration; rather, they all promoted cell surface expression.…”

“…Our results showed that none of the three more potent inverse agonists (Montelukast, MK571, and Zafirlukast) induced receptor sequestration; rather, they all promoted cell surface expression. Increased receptor expression has usually been seen after prolonged treatment of cells with the inverse agonists and possibly implicated the activation of cellular pathways, resulting in resensitization and up-regulation of receptor expression (Smit et al, 1996;Lee et al, 1997;Zhu et al, 2000). However, this would not apply 6.…”

Pharmacological Characterization and Cross Talk of α1A- and α1B-Adrenoceptors Coexpressed in Human Embryonic Kidney 293 Cells

“…Prazosin has been shown to be an inverse agonist of ␣ 1A -and ␣ 1B -ARs (Rossier et al, 1999;Zhu et al, 2000), whereas KMD-3213 is an ␣ 1A -AR-selective neutral antagonist (Zhu et al, 2000;Zhang et al, 2002). Therefore, it is likely that ␣ 1A -and ␣ 1B -ARs and their combined expression in HEK 293 cells have little constitutive activity.…”

Detection of receptor ligands by monitoring selective stabilization of aRenillaluciferase‐tagged, constitutively active mutant, G‐protein‐coupled receptor

“…Moreover, they reported a lack of upregulation of this CAM form when using KMD-3213, which they indicated to be a neutral antagonist (Zhu et al, 2000), suggesting a possible means to discriminate between neutral and inverse agonist ligands. Although intriguing, the basis for these di erences were not established and the e cacy of KMD-3213 was very similar to that of BMY7378, which did produce strong upregulation (Zhu et al, 2000).…”

“…Interestingly, Zhu et al (2000) have recently observed that sustained treatment with a variety of ligands which function as inverse agonist at a CAM form of the human a 1a -adrenoceptor produces substantial upregulation of the mutant but not the wild-type receptor. Moreover, they reported a lack of upregulation of this CAM form when using KMD-3213, which they indicated to be a neutral antagonist (Zhu et al, 2000), suggesting a possible means to discriminate between neutral and inverse agonist ligands. Although intriguing, the basis for these di erences were not established and the e cacy of KMD-3213 was very similar to that of BMY7378, which did produce strong upregulation (Zhu et al, 2000).…”