Protean Agonism at a 2 A-Adrenoceptors

The coupling of the endogenously expressed a2A-adrenoceptors in human erythroleukemia cells (HEL 92.1.7) to Ca mobilization and inhibition of forskolin-stimulated cAMP production was investigated. The two enantiomers of medetomidine [(6)-[4-(1-[2,3-dimethylphenyl]ethyl)-1H-imidazole]HCl] produced opposite responses. Dexmedetomidine behaved as an agonist in both assays (i.e., it caused Ca mobilization and depressed forskolin-stimulated cAMP production). Levomedetomidine, which is a weak agonist in some test systems, reduced intracellular Ca levels and further increased forskolin-stimulated cAMP production and therefore can be classified as an inverse agonist. A neutral ligand, MPV-2088, antagonized responses to both ligands. Several other, chemically diverse a2-adrenergic ligands also were tested. Ligands that could promote increases in Ca levels and inhibition of cAMP production could be classified as full or partial agonists. Their effects could be blocked by the a2-adrenoceptor antagonist rauwolscine and by pertussis toxin treatment. Some typical antagonists such as rauwolscine, idazoxan, and atipamezole had inverse agonist activity like levomedetomidine. The results suggest that the a2A-adrenoceptors in HEL 92.1. 7 cells exist in a precoupled state with pertussis toxin-sensitive G proteins, resulting in a constitutive mobilization of intracellular Ca and inhibition of cAMP production in the absence of agonist. This constitutive activity can be antagonized by inverse agonists such as levomedetomidine and rauwolscine. Levomedetomidine can be termed a “protean agonist” because it is capable of activating uncoupled a2-adrenoceptors in other systems and inhibiting the constitutive activity of precoupled a2-adrenoceptors in HEL 92.1. 7 cells. With this class of compounds, the inherent receptor “tone” could be adjusted, which should provide a new therapeutic principle in receptor dysfunction. Many G protein-coupled receptors undergo spontaneous activation in the absence of agonist (Allen et al., 1991; Lefkowitz et al., 1993; Barker et al., 1994; Samama et al., 1994; Tian et al., 1994; Tiberi and Caron, 1994; Milligan et al., 1995). Basal activity has previously been detected in cells and in membrane preparations from cells endogenously expressing opioid receptors (Costa and Hertz, 1989). In experimental systems, spontaneous activity can be achieved through receptor mutation, overexpression, or G protein overexpression (Lefkowitz et al., 1993; Milligan et al., 1995) as has been shown for adrenergic, dopaminergic, and 5-hydroxytryptamine receptors (Allen et al., 1991; Ren et al., 1993; Barker et al., 1994; Samama et al., 1994; Tiberi and Caron, 1994; Chidiac et al., 1996). The spontaneous receptor activity can be demonstrated using antagonists that produce an inverse response. Two types of antagonists are found, neutral antagonists and negative antagonists (Costa and Hertz, 1989). Antagonists with negative activity have also been termed inverse agonists. A new class of G proteincoupled receptor ligands called “protean agonists” has been proposed on theoretical grounds. Depending on the basal This work was supported by the Borg Foundation, Magnus Ehrnrooth Foundation, Technology Development Center of Finland, Sigrid Jusélius Foundation, Swedish Medical Research Council, and Cancer Research Fund of Sweden. C.C. J. and J.P.K. contributed equally to this work. ABBREVIATIONS: [Ca]i, intracellular free Ca 21 concentration; IBMX, 3-isobuthyl-1-methyl-xanthine; TES, 2-([2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino)ethanesulfonic acid; TBM, TES-buffered medium; clonidine, 2-(2,6-dichloroaniline)-2-imidazoline HCl; guanabenz, 1-(2,6-dichlorobenzylideneamino)guanidine); guanfacine, N-(aminoiminomethyl)-2,6-dichlorobenzenacetamide; idazoxan, (6)-2-(1,4-benzodioxan-2-yl)-2-imidazoline HCl; oxymetazoline, (3-[4,5-dihydro-1H-imidazol-2-yl]-methyl)-6-(1,1-dimethylethyl)-2,4-dimethylphenol HCl; propranolol, (6)-1-(isopropylamino)-3-(1-naphthyloxy)-2-propanol HCl; naphazoline, 4,5-dihydro-2-(1-naphthalenylmethyl)-1H-imidazole; xylazine, N-(2,6-dimethylphenyl)-5,6-dihydro-4H-1,3-thiazin2-amine HCl; rauwolscine, 17a-hydroxy-20a-yohimban-16b-carboxylic acid methyl ester HCl; tizanidine, 5-chloro-4-(2-imidazolin-2-yl-amino)-2,1,3benzothiadiazole; atipamezole, 4(5)-(2-ethyl-indan-2-yl)imidazole HCl; detomidine, 4(5)-(2,3-dimethylbenzyl)imidazole]HCl; medetomidine, (6)-[4-(1-[2,3dimethylphenyl]ethyl)-1H-imidazole]HCl; dexmedetomidine, (1)-(S)-4-(1-[2,3-dimethylphenyl]ethyl)-1H-imidazole]HCl; levomedetomidine (2)-(R)-4-(1[2,3-dimethylphenyl]ethyl)-1H-imidazole HCl; EGTA, ethylene glycol bis(b-aminoethyl ether)-N,N,N9,N9-tetraacetic acid; HEPES, 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid. 0026-895X/98/050963-06$3.00/0 Copyright © by The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 53:963–968 (1998). 963 at A PE T Jornals on M ay 8, 2017 m oharm . aspeurnals.org D ow nladed from activity of the receptor, these should either act as agonists or inverse agonists (Kenakin, 1995). The rationale put forward by Kenakin (1995) has been that a protean agonist is a weak agonist (i.e., having a low efficacy). The basal receptor activity produces an active receptor state (R*) that competes with the ligand-induced active state (AR*) for the G protein. As AR* has a lower affinity for the G protein than R* a factual decrease (i.e., inverse agonism by the ligand) will be observed when there is a substantial basal formation of the R*. In the absence of a marked spontaneous production of R*, a protean agonist will act like a normal agonist. The results of Chidiac et al. (1996) have recently shown the existence of protean agonists in practice. The HEL 92.1. 7 cell line shows robust increases in [Ca]i (Michel et al., 1989; Musgrave and Seifert, 1995), as well as inhibition of adenylyl cyclase in response to agonist stimulation of the endogenous a2A-adrenoceptor (McKernan et al., 1987). Both responses are sensitive to pertussis toxin and hence mediated by Gi/o-type G protein. In this study, precoupling of the receptors was probed by measurement of changes in [Ca]i and cAMP levels in response to a variety of a2adrenoceptor ligands. Materials and Methods