Constitutive Activity of the Human 1-Adrenergic Receptor in 1-Receptor Transgenic Mice

We tested the hypothesis that the human 1-adrenergic receptor displays constitutive activity and that -adrenergic antagonists differ in their ability to modulate this constitutive activity. Transfection of the cDNAs of the human 1and 2-adrenergic receptors into COS-7 cells caused increases in basal cAMP that were proportional to the receptor levels, thus demonstrating constitutive activity for both subtypes. At comparable receptor levels, the increase in basal cAMP was about 5-fold higher for the 2than for the 1-subtype. As a model for enhanced -adrenergic signaling at the whole-organ level, we used transgenic mice with heart-specific overexpression of the human 1-adrenergic receptor. In this model, the 1-adrenergic receptor displayed constitutive activity as evidenced by a higher spontaneous beating rate of isolated right atria from 1-transgenic versus wild-type mice. This difference was abolished by the addition of CGP20712A, demonstrating inverse agonist properties of this compound. We then tested whether various -adrenergic antagonists currently in clinical use for the treatment of heart failure differ in their ability to modulate constitutive activity of the cardiac 1-adrenergic receptor. The 1-selective antagonists metoprolol and bisoprolol showed significant inverse agonist activity at the 1-adrenergic receptor. Carvedilol behaved as a neutral antagonist and xamoterol displayed marked partial agonist activity. We conclude that the human 1-adrenergic receptor displays constitutive activity that is considerably lower than that of the 2-subtype. -Adrenergic antagonists currently in clinical use differ in their ability to exert inverse agonist activity at the human 1-adrenergic receptor, which may contribute to their therapeutic effects. Activation of cardiac -adrenergic receptors plays a central role in regulating the physiological responses of the heart to an increased demand (Brodde and Michel, 1999). Chronic activation of cardiac -adrenergic receptors occurs in heart failure because of an increase in sympathetic activity and circulating catecholamine levels (Chidsey and Braunwald, 1966). Although this adaptive response to compensate for the heart’s inability to meet hemodynamic demands has traditionally been appreciated as positive inotropic support, the perception of this phenomenon has changed within the past decade. Several lines of evidence now indicate that the chronic sympathetic activation seen in heart failure is detrimental and indeed plays an important part in the progression of this disease. Myocardial toxicity of infused catecholamines has been demonstrated both in animal studies and in humans (Rona, 1985). Recently, chronic heart-specific activation of 1-adrenergic receptors in a transgenic animal model has been shown to cause myocyte hypertrophy, myocardial fibrosis, and eventually heart failure (Engelhardt et al., 1999; Bisognano et al., 2000). Several large clinical trials with -adrenergic receptor antagonists have demonstrated a significant benefit of this therapeutic principle (Australia/ New Zealand Heart Failure Research Collaborative Group, 1997; CIBIS-II, 1999; MERIT-HF, 1999). Thus, within the past decade a paradigm shift has occurred, from inotropic support toward pharmacological suppression of sympathetic activation (Bristow, 2000a). However, the substances among the available -adrenergic receptor antagonists that provide the greatest benefit in the setting of heart failure remain unclear (Bristow, 2000b) . One general property that contributes to the therapeutic effects of receptor antagonist drugs is their ability to modulate the activation state of the receptor. G-protein-coupled receptors display different degrees of constitutive activity (de Ligt et al., 2000). Among the adrenergic receptors, the 2-adrenergic receptor has been found to possess considerable constitutive activity and has been used as one of the prototypical receptors to develop the concept of inverse agonism. These experiments have been carried out in vitro with reconstituted systems (Freissmuth et al., 1991) and after transfection of receptors in cell culture (Adie and Milligan, 1994; Chidiac et al., 1994) and have more recently been extended to transgenic animals (Bond et al., 1995; Zhou et al., 1999a,b). Transgenic mice with 200-fold overexpression of the human 2adrenergic receptor displayed a marked increase in basal These studies were supported by grants from the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, and the European Union. ABBREVIATIONS: MHC, myosin heavy chain; HPLC, high-performance liquid chromatography. 0026-895X/01/6004-712–717$3.00 MOLECULAR PHARMACOLOGY Vol. 60, No. 4 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 857/930419 Mol Pharmacol 60:712–717, 2001 Printed in U.S.A. 712 at A PE T Jornals on M ay 2, 2017 m oharm .aspeurnals.org D ow nladed from tension and beating frequency of isolated atria compared with wild-type animals. This increase was inhibited by 70% upon the addition of ICI-118,551, acting as an inverse agonist at this receptor. In the present study, we assessed the intrinsic activity of both 1and 2-adrenergic receptors. We then determined the effects of propranolol, metoprolol, bisoprolol, CGP 20712A, carvedilol, and xamoterol on the activation level of the 1-adrenergic receptor in atria from transgenic mice with cardiac overexpression of human 1-adrenergic receptors. Materials and Methods Cell Culture and Transfection. COS-7 cells were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum and transfected with the cDNA for the human 1or 2-adrenergic receptors under the control of the cytomegalovirus promotor by the DEAE-dextran method (Ausubel et al., 1997). The total amount of transfected DNA varied from 0.001 to 5 g/plate. Twenty-four hours after transfection, cells were split, and assays were done 48 h after