Spontaneous Activation of b2- but Not b1-Adrenoceptors Expressed in Cardiac Myocytes from b1b2 Double Knockout Mice

Although ligand-free, constitutive b2-adrenergic receptor (AR) signaling has been demonstrated in naive cell lines and in transgenic mice overexpressing cardiac b2-AR, it is unclear whether the dominant cardiac b-AR subtype, b1-AR, shares the ability of spontaneous activation. In the present study, we expressed human b1or b2-AR via recombinant adenoviral infection in ventricular myocytes isolated from b1b2-AR double knockout mice, creating pure b1-AR and b2-AR systems with variable receptor densities. A contractile response to a nonselective b-AR agonist, isoproterenol, was absent in double knockout mouse myocytes but was fully restored after adenoviral b1-AR or adenoviral b2-AR infection. Increasing the titer of adenoviral vectors (multiplicity of infection 10–1000) led to a dose-dependent expression of b1or b2-AR with a maximal density of 1207 6 173 (36-fold over the wild-type control value) and 821 6 38 fmol/mg protein (69-fold), respectively. Using confocal immunohistochemistry, we directly visualized the cellular distribution of b1-AR and b2-AR and found that both subtypes were distributed on the cell surface membrane and transverse tubules, resulting in a striated pattern. In the absence of ligand, b2-AR expression resulted in graded increases in baseline cAMP and contractility up to 428% and 233% of control, respectively, at the maximal b2-AR density. These effects were specifically reversed by a b2-AR inverse agonist, ICI 118,551 (10 M). In contrast, overexpression of b1-AR, even at a greater density, failed to enhance either basal cAMP or contractility; the alleged b1-AR inverse agonist, CGP 20712A (10 26 M), had no significant effect on basal contraction in these cells. Thus, we conclude that acute b2-AR overexpression in cardiac myocytes elicits significant physiological responses due to spontaneous receptor activation; however, this property is b-AR subtype specific because b1-AR does not exhibit agonistindependent 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. b-Adrenergic receptor (b-AR) is a prototypical GPCR. At least two b-AR subtypes, b1-AR and b2-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 catecholamines increases cardiac contractility and heart rate and accelerates cardiac relaxation via a Gs-adenylyl cyclasecAMP-protein kinase A-signaling cascade. Although there is a high degree of structural and functional similarity between these b-AR subtypes, recent studies have shown that b-AR subtypes play strikingly different functional roles via distinct signaling pathways in the heart. In particular, b2-AR, but not b1-AR, couples to pertussis toxin-sensitive Gi proteins in addition to the well established Gs-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 Center, New York, NY 10016. ABBREVIATIONS: GPCR, G protein-coupled receptor; b-AR, b-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; Tpeak, the time from stimulation to peak shortening; T50, the time from the peak to 50% relaxation; TA, cell twitch amplitude; ICYP, [ 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 A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from are active in the absence of ligand. The ligand-independent activation of b2-AR has been elegantly demonstrated in several systems overexpressing wild-type (WT) b2-AR (Chidiac et al., 1994; Milano et al., 1994; Bond et al., 1995) or expressing the constitutively active mutant of b2-AR (Samama et al., 1993). For example, ligand-free cardiac b2-AR activation is evidenced by significantly increased basal adenylyl cyclase activity, cAMP accumulation, or contractility in the absence of any ligand, and the b2-AR inverse agonist, ICI 118,511 (ICI), reverses these augmentations (Milano et al., 1994; Bond et al., 1995; Xiao et al., 1999a; Zhou et al., 1999a). However, whether b1-AR shares the ability of spontaneous activation is still controversial. In transgenic mice overexpressing b1-AR, the basal adenylyl cyclase activity and cardiac function remain unchanged compared with WT controls (Bertin et al., 1993; Zolk et al., 1998; Engelhardt et al., 1999). Nevertheless, in guinea pig and human cardiomyocytes, in the presence of forskolin, the mixed b1and b2-AR antagonist, but not b2-AR antagonists, induces a marked decrease in basal L-type Ca current in the absence of ligand (Mewes et al., 1993). More recently, it has been shown that, in canine cardiac myocytes, a b1-AR antagonist, CGP 20712A (CGP), inhibited the basal ICa by 27% (Nagykaldi et al., 1999). These results were interpreted to indicate spontaneous activation of b1-AR. The overall goal of the present study was to examine whether b-AR subtypes are different in terms of their propensity of spontaneous activation. To avoid complicated interactions between b-AR subtypes, because of the lack of absolutely selective b-AR subtype ligands, we took advantage of a recently developed b1and b2-AR double knockout (DKO) mouse model (Rohrer et al., 1999) and recent advances in methods to culture adult mouse ventricular myocytes (Zhou et al., 2000). We expressed either b-AR subtype over a wide range of receptor density in cultured b1b2-AR DKO ventricular myocytes and investigated the physiological or biochemical responses in the absence or presence of b-AR subtype inverse agonists. Our results indicate that overexpression of b2-AR is associated with a robust increase in basal cAMP accumulation and contractility, which can be specifically reversed by the b2-AR inverse agonist, ICI. Surprisingly, overexpression of b1-AR to the same or even greater density has no effect on basal cAMP and contractility. These results indicate that b1-AR, unlike b2-AR, is not able to undergo ligand-independent constitutive activation in intact mouse cardiac myocytes. Experimental Procedures Myocyte Isolation, Culture, and Adenoviral Infection. The investigation conforms to National Institutes of Health guiding principles in the care and use of animals. Single mouse cardiac myocytes were isolated from the hearts of 2to 3-month-old mice with an enzymatic technique and then were cultured and infected with adenoviral vectors, as described previously (Zhou et al., 2000). Before culture, myocytes were washed three times with minimal essential medium (MEM) containing 1.2 mM Ca, 2.5% fetal bovine serum (FBS), and 1% penicillin-streptomycin and then plated at 0.5 to ;1 3 10/cm with the same medium in the culture dishes precoated with 10 mg/ml mouse laminin. After 1 h of culture (to achieve attachment), the culture medium was aspirated along with unattached cells. Adenovirus-mediated gene transfer was implemented by adding a minimal volume of the FBS-free MEM containing an appropriate titer of gene-carrying adenovirus. The full volume of FBS-free MEM was supplied after culture for another 1 to 2 h. All experiments were performed after 24 h of adenoviral infection. Measurement of Cell Contraction. Cells were placed on the stage of an inverted microscope (Zeiss, model IM-35, Zeiss, Thornwood, NY) and superfused with HEPES-buffered solution consisting of (in mM): CaCl2 1, NaCl 137, KCl 5.4, dextrose 15, MgSO4 1.3, NaH2PO4 1.2, and HEPES 20, pH 7.4 adjusted with NaOH. Each cell was illuminated with red (650–750 nm) light through the normal bright-field path of the microscope and electrically stimulated at 0.5 Hz at 23°C. Cell length was monitored from the bright-field image by an optical edge-tracking method using a photodiode array (model 1024 SAQ, Reticon, Boston, MA) with a 3-ms time resolution (Spurgeon et al., 1990). Tpeak was measured as the time from stimulation to peak shortening; T50 was measured as the time from the peak to 50% relaxation. Riadioligand-Binding Assay. Twenty-four hours after adenoviral infection, cardiac myocytes were harvested in lysis buffer (5 mM Tris-HCl, pH 7.4, with 5 mM EGTA) and homogenized with 15 strokes on ice. Samples were centrifuged at 30,000g for 15 min to pellet membranes. Membranes were resuspended in binding buffer (75 mM TrisHCl, pH 7.4, 12.5 mM MgCl2, 2 mM EDTA) and stored in aliquots at 280°C. Binding assays were performed on 25 mg of membrane protein using saturating amounts of the b-AR-specific ligand [I]cyanopindolol (ICYP). Nonspecific binding was determined in the presence of 10 mM propranolol. Reactions were conducted in 250 ml of binding buffer at 37°C for 1 h. The binding reaction was terminated by addition of ice-cold 10 mM Tris-HCl (pH 7.4) to the membrane suspension, followed by rapid vacuum filtration through glass-fiber filters (Whatman GF/C). Each filter was washed three times with an additional 7 ml of ice-cold 10 mM Tris-HCl. The radioactivity of the wet filters was determined in a gamma counter. All assays were performed in duplicate, and receptor density was normalized to milligrams of membrane protein. Kd and the maximal number of binding sites (Bmax) for ICYP were determined by Scatchard analysis of saturation binding isotherms. Immunocytochemical Staining and Confocal Imaging. b1b2DKO cells were infected by either adeno-b1-AR tagged with hemagglutinin (HA) or adeno-b2-AR [multiplicity of infection (m.o.i.) 100] for 24 h. Cells were washed twice with phosphate-buffered saline (PBS) and fixed with cold methanol plus acetone (7:3) for 10 min and rinsed twice with PBS containing 0.2% Triton. Nonspecific binding was reduced by a 30-min incubation with Blotto solution (5% BSA, 2% horse serum, 0.2% Triton, and 0.01% NaN3 in PBS, pH 7.4). Then, cells were incubated for 60 min at room temperature with primary antibodies for HA-tagged b1-AR (anti-HA monoclonal antibody diluted by 1:500) or for b2-AR (b2-AR polyclonal antibodies diluted by 1:100). After the cells were rinsed four times with PBS, including 0.2% Triton, they were stained with Texas Red-conjugated secondary antibodies (1:100, Vector Laboratories, Burlingame, CA) for another 60 min in the dark: horse anti-mouse IgG secondary antibodies were used for b1-AR, whereas goat anti-rabbit IgG secondary antibodies were used for b2-AR staining. As a negative control, cells were incubated with secondary antibodies in the absence of primary antibodies. As an additional negative control, another subset of DKO cells cultured without viral infection was treated with the same protocol. Immunofluorescence was then detected by a laser scanning confocal microscope (LSM-410, Zeiss) with optical section thickness of 1.0 mm. Measurement of cAMP Accumulation. After cells were treated with the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX, 1 mM), for 30 min at 37°C in a CO2 incubator, they were incubated with either b-AR agonists or inverse agonists for 10 min. Cells were then harvested, and cAMP levels were assayed as previously described (Xiao et al., 1999a) with minor modifications. Briefly, 10 ml of membrane vesicles (20 mg of total protein) were added to a 40-ml reaction solution to make a final concentration of 4 mM TrisEDTA and 1 mM IBMX. The reaction was performed at 37°C for 15 888 Zhou et al. at A PE T Jornals on Jne 0, 2017 m oharm .aspeurnals.org D ow nladed from min, and 25 ml of supernatant were assayed using a cAMP H assay kit obtained from Amersham (Arlington Heights, IL). Protein content was measured using the Bradford method (Bio-Rad, Richmond, CA) with bovine serum albumin as the standard. Materials. Forskolin, isoproterenol hydrochloride, propranolol, alprenolol, IBMX, and minimal essential medium were purchased from Sigma (St. Louis, MO). ICI 118,551 was kindly supplied by ICI Pharmaceutic Group (Wilmington, DE). CGP 20712A was kindly supplied by CIBA-GEIGY Corp. (East Hanover, NJ). Fetal bovine serum, penicillin-streptomycin, and mouse laminin were purchased from Life Technologies (Gaithersburg, MD). The cAMP assay kit was purchased from Amersham. [I]Cyanopindolol was purchased from NEN Life Science Products, Inc. (Boston, MA). Anti-HA monoclonal antibody and b2-AR polyclonal antibody were purchased from Berkeley Antibody Co. (Berkeley, CA) and Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), respectively. The secondary antibodies were purchased from Vector Laboratories. Data Analysis. Data are reported as means 6 S.E.M. Student’s t test, paired t test, or ANOVA were used, when appropriate, to test for differences among the means. A value of P , .05 was considered to be statistically significant.