Spontaneous Activation of  2 - but Not  1 -Adrenoceptors Expressed in Cardiac Myocytes from  1  2 Double Knockout Mice YING-YING ZHOU, 1 DONGMEI YANG, WEI-ZHONG ZHU, SHENG-JUN ZHANG, DING-JI WANG, DAN K. ROHRER, ERIC DEVIC, BRIAN K. KOBILKA, EDWARD G. LAKATTA, HEPING CHENG, and RUI-PING XIAO Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, Maryland (Y.-Y.Z., D.Y, W.-Z.Z., S.-J.Z, D.-J.W., E.G.L., H.C., R.-P.X.); National Laboratory of Biomembrane and Membrane Biotechnology, Peking University, Beijing, People’s Republic of China (D.Y., H.C.); and Howard Hughes Medical Institute, Stanford University Medical Center, Stanford, California (D.K.R., E.D., B.K.K.) Received March 24, 2000; accepted July 18, 2000 This paper is available online at http://www.molpharm.org ABSTRACT Although ligand-free, constitutive  2 -adrenergic receptor (AR) signaling has been demonstrated in naive cell lines and in transgenic mice overexpressing cardiac  2 -AR, it is unclear whether the dominant cardiac -AR subtype,  1 -AR, shares the ability of spontaneous activation. In the present study, we expressed human  1 - or  2 -AR via recombinant adenoviral infection in ventricular myocytes isolated from  1  2 -AR double knockout mice, creating pure  1 -AR and  2 -AR systems with variable receptor densities. A contractile response to a nonse- lective -AR agonist, isoproterenol, was absent in double knockout mouse myocytes but was fully restored after adeno- viral  1 -AR or adenoviral  2 -AR infection. Increasing the titer of adenoviral vectors (multiplicity of infection 10 –1000) led to a dose-dependent expression of  1 - or  2 -AR with a maximal density of 1207  173 (36-fold over the wild-type control value) and 821  38 fmol/mg protein (69-fold), respectively. Using confocal immunohistochemistry, we directly visualized the cel- lular distribution of  1 -AR and  2 -AR and found that both subtypes were distributed on the cell surface membrane and transverse tubules, resulting in a striated pattern. In the ab- sence of ligand,  2 -AR expression resulted in graded increases in baseline cAMP and contractility up to 428% and 233% of control, respectively, at the maximal  2 -AR density. These ef- fects were specifically reversed by a  2 -AR inverse agonist, ICI 118,551 (10 -7 M). In contrast, overexpression of  1 -AR, even at a greater density, failed to enhance either basal cAMP or con- tractility; the alleged  1 -AR inverse agonist, CGP 20712A (10 -6 M), had no significant effect on basal contraction in these cells. Thus, we conclude that acute  2 -AR overexpression in cardiac myocytes elicits significant physiological responses due to spontaneous receptor activation; however, this property is -AR subtype specific because  1 -AR does not exhibit agonist- independent spontaneous activation. G protein-coupled receptors (GPCRs) constitute the largest class of cell surface-signaling molecules, which are widely involved in regulating vital cellular processes. -Adrenergic receptor (-AR) is a prototypical GPCR. At least two -AR subtypes,  1 -AR and  2 -AR, coexist in the heart of many mammalian species, including human (Xiao and Lakatta, 1993; Xiao et al., 1994; Altschuld et al., 1995; for review see Xiao et al., 1999b). Stimulation of these receptors by cat- echolamines increases cardiac contractility and heart rate and accelerates cardiac relaxation via a G s -adenylyl cyclase- cAMP-protein kinase A-signaling cascade. Although there is a high degree of structural and functional similarity between these -AR subtypes, recent studies have shown that -AR subtypes play strikingly different functional roles via distinct signaling pathways in the heart. In particular,  2 -AR, but not  1 -AR, couples to pertussis toxin-sensitive G i proteins in addition to the well established G s -signaling pathway (Xiao et al., 1995, 1999a; Kuschel et al., 1999). A GPCR is proposed to exist in an equilibrium between two conformational states, an inactive form (R) and an active form (R*), that can interact with G proteins (Samama et al., 1993; Bond et al., 1995; Neilan et al. 1999). In addition to ligand-induced activation, a small percentage of receptors This work was supported by National Institutes of Health intramural (E.G.L., H.C., R.-P.X.) and extramural grants (B.K.K.), and by a grant from the National Science Foundation for Outstanding Youth of China (H.C.). 1 Present address: Pediatric Cardiology, New York University Medical Cen- ter, New York, NY 10016. ABBREVIATIONS: GPCR, G protein-coupled receptor; -AR, -adrenergic receptor; WT, wild type; ICI, ICI 118,551; CGP, CGP 20712A; DKO, double knockout; ISO, isoproterenol; FBS, fetal bovine serum; MEM, minimal essential medium; T peak , the time from stimulation to peak shortening; T 50 , the time from the peak to 50% relaxation; TA, cell twitch amplitude; ICYP, [ 125 I]cyanopindolol; m.o.i., multiplicity of infection; PBS, phosphate-buffered saline; HA, hemagglutinin; IBMX, 3-isobutyl-1-methylxanthine; PTX, pertussis toxin. 0026-895X/00/050887-08$3.00/0 MOLECULAR PHARMACOLOGY Vol. 58, No. 5 Copyright © 2000 The American Society for Pharmacology and Experimental Therapeutics 179/861098 Mol Pharmacol 58:887–894, 2000 Printed in U.S.A. 887 at ASPET Journals on July 20, 2018 molpharm.aspetjournals.org Downloaded from