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Propranolol promotes Egr1 gene expression in cardiomyocytes via β-adrenoceptors
European Journal of Pharmacology 587 (2008) 85–89
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / e j p h a r
Short communication
Propranolol promotes Egr1 gene expression in cardiomyocytes via β-adrenoceptors Mario Patrizio a,1, Marco Musumeci a,1, Tonino Stati a, Katia Fecchi a, Elisabetta Mattei c, Liviana Catalano b, Giuseppe Marano a,⁎ a b c
Dipartimento del Farmaco, Istituto Superiore di Sanità, Rome, Italy Centro Nazionale Sangue, Rome, Italy Istituto di Neurobiologia e Medicina Molecolare, Consiglio Nazionale delle Ricerche, Rome, Italy
A R T I C L E
I N F O
Article history: Received 14 September 2007 Received in revised form 17 March 2008 Accepted 2 April 2008 Available online 10 April 2008 Keywords: Adrenoceptors Propranolol Gene expression Egr1 Cardiomyocytes
A B S T R A C T Recent research has revealed that propranolol, a β-adrenoceptor antagonist, causes extracellular signalregulated kinase (ERK) cascade activation, nuclear translocation of phospho-ERK and increased transcriptional activity in cultured cell lines. Given the importance of β-adrenoceptor antagonists in the treatment of heart failure, we evaluated the capability of propranolol of promoting the ERK-dependent gene expression at the cardiomyocyte level. To this end, the gene expression of the early growth response factor 1 (Egr1), a wellrecognized indicator of nuclear extracellular signal-regulated kinase 1/2 (ERK1/2) activation, was assessed by quantitative real-time RT-PCR in vivo as well as in vitro experiments. Propranolol, administered at the dose of 10 mg/kg/day in C57BL/6 mice, caused a ≈19-fold increase of Egr1 mRNA expression in left ventricular myocardium along with a ≈2.1-fold increase of Egr1 protein expression. Isoproterenol, a nonselective β-adrenoceptor agonist, also increased Egr1 mRNA and protein expression but to a lesser degree. Remarkably, isoproterenol administration was associated with the development of cardiac hypertrophy, whereas propranolol-treated mice showed a completely normal cardiac morphology. The effect of propranolol on Egr1 mRNA expression was abrogated in mice lacking β1- and β2-adrenoceptors indicating that propranolol increases Egr1 mRNA expression in a β-adrenoceptor-dependent manner. The role of β-adrenoceptors was further confirmed by showing that propranolol was able to increase Egr1 mRNA and protein levels in cultured neonatal cardiomyocytes. Collectively, these results indicate that propranolol promotes Egr1 gene expression in cardiomyocytes via β-adrenoceptors with a mechanism which is independent of its ability to antagonize the effects of catecholamines. It is also suggested that cardiomyocyte growth and Egr1 gene overexpression are not obligate processes. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Over the past several years, much attention has been focused on β-adrenoceptor antagonists because of their efficacy in the treatment of common cardiovascular pathologies. Propranolol, a nonselective β-adrenoceptor antagonist, remains the prototype of this class of drugs and that with which all others are compared. Propranolol has been generally reported to be a pure antagonist, unable to activate β-adrenoceptors, or to be an inverse agonist (Azzi et al., 2001), that is, it reduces the basal accumulation of cAMP. However, recent research has revealed that propranolol can also act as an agonist for the extracellular signal-regulated kinase 1/2 (ERK1/2). Indeed, propranolol has been found to cause ERK cascade activation (Baker et al., 2003; Galandrin and Bouvier, 2006), nuclear translocation of phospho-ERK and increased gene transcription in a Gs/i-independent
⁎ Corresponding author. Dipartimento del Farmaco, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. Tel.: +39 0649903412; fax: +39 0649387104. E-mail address: [email protected] (G. Marano). 1 These authors contributed equally to the work. 0014-2999/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.04.017
manner in cultured cell lines (Azzi et al., 2003). However, whether propranolol is able to promote ERK-induced gene expression in cardiomyocytes remains to be ascertained. Given the importance of β-adrenoceptor antagonists in the treatment of heart failure, elucidation of molecular mechanisms underlying pharmacological action of propranolol could provide important clues for the identification of additional therapeutic targets. ERKs are primarily cytoplasmic in most quiescent cells, but activation of ERK1/2 promotes their translocation from the cytoplasm into the nucleus, where they phosphorylate and activate several transcription factors, including Egr1, a prototypical member of zincfinger family of transcription factors, which is a well-recognized indicator of nuclear ERK1/2 activation in a variety of cells (Guillemot et al., 2001; Wei et al., 2004). In the present study, propranolol was chronically administered to mice by osmotic minipumps for 2 weeks and the resulting effect on cardiac Egr1 gene expression was assessed by quantitative realtime RT-PCR. Moreover, to test whether the in vivo effect of propranolol onEgr1 induction depends on its ability to antagonize cardiac β-adrenoceptor-mediated effects of catecholamines, the β-blocker was
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administered in β1- and β2-adrenoceptor double knockout mice, in which the predominant β-adrenoceptor subtypes are lacking. Additionally, the capability of propranolol to induce Egr1 gene expression was also evaluated in cultured rat neonatal cardiomyocytes, an experimental setting in which catecholamines are not present. Using such experimental approaches, we found that propranolol increases Egr1 mRNA and protein expression in left ventricular myocardium as well as in cultured cardiomyocytes. We also demonstrate that this activity on Egr1 gene expression is mediated via β-adrenoceptors and is independent of its ability to antagonize the effects of catecholamines.
sure increment (+dP/dt) or fall (−dP/dt) and end-systolic elastance (Ees, a load-independent parameter of cardiac contractility) were computed.
2. Methods
2.7. RNA isolation and quantification
2.1. Animals
Total RNA was extracted from left ventricles of individual mice and from cultured neonatal rat cardiomyocytes by using SV Total RNA Isolation System (Promega, Madison, USA). cDNA out of total ventricular RNA was synthesized by using the High Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA, USA). RNA expression levels for Egr1 in the left ventricle was quantified with Real-Time TaqMan RT-PCR using 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). Taqman reactions were carried out in 96 well plates using cDNA, Taqman universal PCR MasterMix, predesigned and pre-optimized specific primers and fluorescent probes (Egr1, Mm00656724_m1; GAPDH, Mm99999915_g1 for mouse, and Egr1, Rn00561138_m1; GAPDH, Rn99999916_s1 for rat), and water to a final volume of 50 µl according to manufacturer's instructions. GAPDH mRNA was used as an endogenous control. The codes for primers and probes were obtained from the Applied Biosystems catalogue for quantitative gene expression analysis. No reverse transcriptase and no template controls were used to monitor for any contaminating amplification. The ΔCt was used for statistical analysis, and 2− ΔΔCt for data presentation (Livak and Schmittgen, 2001).
Male C57BL/6 12-week-old mice (Harlan, S. Pietro al Natisone, Italy) were used in most experiments and maintained in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). In some experiments, male β1- and β2-adrenoceptor double knockout mice were used (Jackson Laboratories, Ann Harbor, USA). Because knockout (KO) mice have a mixed background, we mated KO mice with C57BL/6 mice to produce F1 mice heterozygous for both KOs. Thereafter, F1 heterozygous mice were mated to produce F2 wild-type (WT) and double KO mice. Genotyping was performed on tail DNA using standard protocols. 2.2. Primary cardiomyocytes cultures Cardiomyocytes were obtained by 1- to 3-day-old neonatal Wistar rat ventricles as described (Sadoshima et al., 1992) and plated at a density of ≈5.0 × 105 cells per well in 6-well tissue culture plates. To induce Egr1 gene expression, cardiomyocytes were exposed to propranolol (1, 10 and 25 μM) for 24 h or (25 μM) 72 h. 2.3. Chronic administration of drugs Propranolol, metoprolol or isoproterenol were administered in a dose of 10 mg/kg/day, 10 mg/kg/day, and 15 mg/kg/day, respectively, for 14 consecutive days. The dosage for drugs was chosen on the basis of literature data (Boluyt et al., 1995; Keys et al., 2002; Zhang et al., 2007). Remarkably, at this dosage, isoproterenol does not induce arterial hypertension. The drugs were administered through osmotic minipumps (Alzet model 2002, Cupertino, CA, USA) implanted subcutaneously on the right side of the back. 2.4. Echocardiography In mice intubated and anesthetized with isoflurane (1% in 100% of oxygen), echocardiographic examination was performed with a SONOLINE G50 (Siemens AG, Erlangen, Germany) equipped with a 13-MHz imaging transducer as described (Marano et al., 2004).
2.6. Histological analysis Histological analysis was performed as reported previously (Marano et al., 2004). Briefly, left ventricular sections were cut and stained with hematoxylin and eosin for measurement of myocyte cross-sectional area by quantitative morphometry (Metamorph 6.1, Universal Imaging Corporation, PA, USA).
2.8. Western blot analysis Mouse left ventricles were homogenized in RIPA-buffer supplemented with 10 mM NaF, 100 µM Na3VO4, 1 mM EDTA, 1 mM EGTA, 0,2 mM PMSF and 100 µM leupeptin. Insoluble material was removed by centrifugation at 1000 ×g at 4 °C for 10 min, and protein concentration measured. For each condition, 25 µg of protein were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were then blocked with 5% nonfat milk and incubated with rabbit polyclonal antibody anti-Egr1 (1:1000 dilution) (Santa Cruz Biotechnology, CA, USA) for 1 h at 25 °C. After several washes, the membranes were incubated with secondary antibodies (anti-rabbit IgG conjugated with horseradish peroxidase, 1:5000) for 1 h at 25 °C and visualized by the Amersham ECL system. The optical density of the bands (arbitrary units, AU) was measured with a GS-700 Imaging Densitometer (Bio-Rad) and referred to the corresponding control samples (taken as 1) run in the same gel. 2.9. Statistical analysis
2.5. Hemodynamic analysis Left ventricular hemodynamic measurements were obtained by direct left ventricular catheterization as described (Marano et al., 2004). Briefly, a 1.4-F, four-electrode pressure-volume catheter (model SPR839, Millar Instruments, Houston, TX, USA) was inserted into the left ventricle through the apex in the open chest anesthetized animal (isoflurane 1.5–2% in 100% of oxygen) and advanced along the long axis with proximal electrode just within the wall of the left ventricle. All pressure-volume loop data were analysed with cardiac pressurevolume analysis program (IOX 1.7; EMKA Technologies, Paris, France), and the heart rate, maximal left ventricular systolic pressure, left ventricular end-diastolic pressure, maximal slope of left ventricular pres-
Group means (±S.E.M.) were calculated for all relevant variables. Statistical analysis was performed by Student t-test or ANOVA with Bonferroni's multiple comparison test for post hoc analyses. A value of P b 0.05 was considered to be statistically significant. 3. Results 3.1. Propranolol upregulates Egr1 gene expression in left ventricular myocardium To evaluate whether propranolol promotes nuclear translocation of phospho-ERK and, in turn, gene expression in the myocardium, we
M. Patrizio et al. / European Journal of Pharmacology 587 (2008) 85–89 Table 1 Echocardiography and hemodynamic measurements 2 weeks after propranolol administration
N Body weight, g Heart rate, beats/min LVSP, mmHg LVEDP, mmHg −dP/dt, mmHg/s LVEDD, mm FS, % +dP/dt, mmHg/s Ees, mmHg/μl
Vehicle
Propranolol
5 25.3 ± 0.2 431 ± 11 67 ± 3 4.0 ± 0.5 4055 ± 32 3.6 ± 0.2 34 ± 2 4772 ± 67 6.1 ± 0.5
6 26.0 ± 0.3 398 ± 10 64 ± 3 4.1 ± 0.3 4331 ± 42 3.7 ± 0.2 35 ± 2 4902 ± 59 5.8 ± 0.4
LVSP, maximal left ventricular systolic pressure; LVEDP, left ventricular end-diastolic pressure; LVEDD, left ventricular end-diastolic diameter; +dP/dt and −dP/dt, maximum rate of left ventricular pressure rise or fall; FS, fractional shortening was calculated as ((LVEDD − LVESD) / LVEDD) × 100; LVESD, left ventricular end-systolic diameter; Ees, end-systolic elastance.
assessed the expression of Egr1 gene by real-time RT-PCR. Propranolol, administered by subcutaneous infusion at the dose of 10 mg/kg/day for 14 consecutive days in C57BL/6 mice, had no significant effect on heart rate, ventricular function evaluated as fractional shortening, +dP/dt and Ees, and chamber size in anesthetized animals (Table 1). In contrast, it caused a marked upregulation of Egr1 mRNA in left ventricular myocardium (≈19-fold, Fig. 1A). To evaluate whether the effects on Egr1 gene expression was also observed with a more selective β-adrenoceptor antagonist, a β1adrenoceptor antagonist, metoprolol, was also tested. Metoprolol, enhanced Egr1 gene expression, but the effects were significantly less than in propranolol-treated mice (Fig. 1A). Isoproterenol, a β-adrenoceptor agonist, which is known to increase Egr1 gene expression (Saadane et al., 1999), was used as a positive control. As expected, chronic infusion of isoproterenol caused a significant increase of Egr1 gene expression in left ventricular myocardium
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(Fig. 1A) associated with a 27% increase of the left ventricular weightto-body weight ratio (4.2 ± 0.2 mg/g). As a next step, induction of Egr1 gene was investigated at the protein level. Chronic administration of propranolol, metoprolol or isoproterenol caused a 2.1-fold, 2.0-fold and 1.7-fold increase of Egr1 protein levels in left ventricular myocardium, respectively (Fig. 1B). Taken together, these results indicate that the induction of Egr1 mRNA in myocardium by propranolol is followed by enhanced Egr1 protein expression. As Egr1 has been involved in the development of pathological cardiac growth (Buitrago et al., 2005), a detailed analysis of cardiomyocyte size was performed in both propranolol- and metoprolol-treated mice by transthoracic echocardiography and histopathological analysis. Compared to vehicle-treated mice, propranolol-treated mice showed a completely normal cardiac morphology (Fig. 1C–F). Similar results were observed in metoprolol-treated mice (Fig. 1D–F). Collectively, these results suggest that the increase in Egr1 gene expression alone is not sufficient to cause cardiac hypertrophy. 3.2. Propranolol induces Egr1 gene expression via a direct effect on β-adrenoceptors Propranolol is a competitive β-adrenoceptor antagonist and interacts with β1- and β2-adrenoceptors with equal affinity. Thus, the effect of propranolol on Egr1 induction could depend on its ability to antagonize cardiac β-adrenoceptor-mediated effects of catecholamines. To address this issue, quantitative analysis of Egr1 mRNA was performed in β1- and β2-adrenoceptor double knockout (KO) mice, in which the predominant cardiac β-adrenoceptor subtypes are lacking. We found a rather mild increase in Egr1 gene expression in left ventricles from KO mice (Fig. 2A). Remarkably, propranolol administration failed to increase Egr1 mRNA levels in KO mice (Fig. 2A). To further confirm that propranolol causes Egr1 gene induction independently of its property to antagonize the effects of catecholamines, we investigated the effects of propranolol in cultured neonatal cardiomyocytes at 24 h. As shown in Fig. 2B, propranolol induced a
Fig. 1. Propranolol upregulates Egr1 gene expression in the heart without affecting cardiac growth. (A) Expression of Egr1 in left ventricular myocardium. Data are presented as the fold change relative to the expression levels of vehicle-treated mice (2− ΔΔCt). Summary data from 4–5 separate experiments performed in triplicate are shown. ⁎ P b 0.05 when compared to vehicle group. (B) Western blot analysis of Egr1 protein expression in left ventricular myocardium. Propranolol, metoprolol or isoproterenol were administered in C57BL/ 6 mice for 14 consecutive days. (Top) Representative experiment. (Bottom) A graph representing a summary of repeated experiments is also shown (n = 3–4 animals per group). (C) Representative coronal sections of hearts from vehicle- and propranolol-treated mice. No abnormalities with normal atria and absence of cardiac hypertrophy of the ventricles were observed. (D) Left ventricular weight-to-body weight (LVW/BW) ratio (n = 5–6 animals per group). (E) Left ventricular posterior wall thickness (PWT). (n = 5–6 animals per group). (F) Morphometric analysis of cardiomyocytes. Data were from about 160 cardiomyocytes per group derived from 4 animals per group. Veh, vehicle; Pro, propranolol; Met, metoprolol; Iso, isoproterenol.
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Fig. 2. Propranolol induces Egr1 gene expression via β-adrenoceptors. (A) Administration of propranolol in β-adrenoceptor double knockout (KO) mice. After 14 days, the β-blocker failed to increase the expression of Egr1 mRNA in KO mice (n = 4–5 animals per group). (B) Effects of propranolol on Egr1 mRNA levels in cultured neonatal cardiomyocytes 24 h and 72 h after drug exposure. Propranolol induced a significant increase of Egr1 gene expression in a concentration-dependent manner. (C) Western blot analysis of Egr1 protein expression in cultured cardiomyocytes. Cells were treated for 72 h with or without propranolol (25 µM). (Top) Representative experiment. (Bottom) Summary data from 3 separate experiments are also shown. Ctr, control; WT, wild-type; Pro, propranolol. ⁎P b 0.05, vs corresponding control group.
significant increase in Egr1 gene expression in a concentrationdependent manner. Additionally, to test whether the effects of propranolol on Egr1 mRNA levels were maintained over time, cultured cardiomyocytes were treated with propranolol for 72 h. Treatment with propranolol caused a significant increase in Egr1 mRNA levels similar to that observed at 24 h (Fig. 2B). Again, the induction of Egr1 mRNA was followed by enhanced Egr1 protein expression (Fig. 2C). To determine whether the propranolol-induced upregulation of Egr1 mRNA was a consequence of changes in the rate of gene transcription, cultured cardiomyocytes were treated with the transcription inhibitor actinomycin D (2.5 µg/ml) plus propranolol (25 μM) for 24 h. We found a 5.1 ± 0.1-fold increase of Egr1 mRNA in propranololtreated cells. This response was prevented when cells were treated with actinomycin D, indicating that transcriptional activation is the more likely mechanism involved. 4. Discussion The product of the early growth response gene Egr1, which is also known as NGFI-A, zif268, krox24, or Tis8, is a transcriptional factor that is rapidly induced in many different cell types by a variety of growth factors, cytokines, and harmful stimuli. In the present study, we demonstrate for the first time that propranolol, a β-adrenoceptor antagonist, upregulates mRNA levels of Egr1 in the mouse heart under basal conditions. These data extend observations reported recently by Azzi et al. (2003) of the propranolol-induced activation of transcriptional factors in Cos1 cell line, into cardiomyocytes. An additional major finding of this study is that propranolol causes Egr1 gene induction via both β-adrenoceptor subtypes, but independently of its ability to antagonize the effects of catecholamines. This is supported by 3 independent lines of evidence. First, propranolol effects on Egr1 are abrogated in β1- and β2-adrenoceptor double knockout mice, in which the predominant cardiac β-adrenoceptor subtypes are lacking. Second, propranolol is able to induce Egr1 gene expression in cultured neonatal cardiomyocytes in a concentrationdependent manner. Third, the selective β1-AR antagonist, metoprolol, also increased Egr1 gene expression but to a less extent than in propranolol-treated mice. Egr1 gene expression is increased in the heart after adrenoceptor stimulation (Saadane et al., 1999). Here, we show that both propranolol, a β-adrenoceptor antagonist, and isoproterenol, a βadrenoceptor agonist, upregulate Egr1 gene expression in the mouse heart. However, this is an apparent discrepancy because both compounds have been reported to show agonistic properties towards the ERK signaling pathway. Indeed, Galandrin and Bouvier (2006) have reported that propranolol induces partial agonist responses for
the ERK1/2 pathway via both β1- and β2-adrenoceptors. Additionally, Azzi et al. (2003) have observed that propranolol activates ERK1/2 and promotes its translocation from the cytoplasm into the nucleus, where it phosphorylates Elk-1 which plays a primary role in regulating Egr1 gene expression. With few exceptions, at the transcriptional level, cardiac hypertrophy is characterized by a specific gene expression pattern including upregulation of immediate–early response genes such as Egr1 as well as of fetal genes such as atrial natriuretic peptide and the β-isoform of myosin heavy chain. At this time, the significance of these changes remains to be fully understood. Antifibrotic effects or growth promoting activities have been attributed to Egr1, depending on the experimental context. For example, Egr1 knockout mice show a greater susceptibility to develop fibrotic lesions than wild-type mice under basal conditions (Saadane et al., 2000). On the contrary, mouse hearts with specific overexpression of Nab1, a transcriptional repressor of Egr1, or Egr1-deficient mice show a marked reduction in ventricular hypertrophic response when subjected to pressure overload which is consistent with a growth promoting activity of Egr1 under conditions of enhanced cardiac stress (Buitrago et al., 2005). In the present study we demonstrate that propranolol-induced upregulation of Egr1 gene expression seems to be functionally silent being associated with a completely normal ventricular morphology. In summary, our data indicate that propranolol is able to promote Egr1 gene expression in cardiomyocytes via β-adrenoceptors with a mechanism which is independent of its ability to antagonize the effects of catecholamines. It is also suggested that cardiomyocyte growth and Egr1 gene overexpression are not obligate processes. Acknowledgements We are grateful to Mr. Valerio Vago for technical support. This work was supported in part by grants from the Italian Minister of Health to G.M (2004/N01). K.F. was recipient of a fellowship from FIRB the Italian Ministry for University and Research (Grant RBNE03FMCJ). References Azzi, M., Piñeyro, G., Pontier, S., Parent, S., Ansanay, H., Bouvier, M., 2001. Allosteric effects of G protein overexpression on the binding of β-adrenergic ligands with distinct inverse efficacies. Mol. Pharmacol. 60, 999–1007. Azzi, M., Charest, P.G., Angers, S., Rousseau, G., Kohout, T., Bouvier, M., Piñeyro, G., 2003. β-adrenoceptorrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for G protein-coupled receptors. Proc. Natl. Acad. Sci. U S A. 100, 11406–11411. Baker, J.G., Hall, I.P., Hill, S.J., 2003. Agonist and inverse agonist actions of β-blockers at the human β2-adrenoceptor provide evidence for agonist-directed signaling. Mol. Pharmacol. 64, 1357–1369.
M. Patrizio et al. / European Journal of Pharmacology 587 (2008) 85–89 Boluyt, M., Long, X., Eschenhagen, T., Mende, U., Schmitz, W., Crow, M.T., Lakatta, E.G., 1995. Isoproterenol infusion induces alterations in expression of hypertrophyassociated genes in rat heart. Am. J. Physiol. 269, H638–H647. Buitrago, M., Lorenz, K., Maass, A.H., Oberdorf-Maass, S., Keller, U., Schmitteckert, E.M., Ivashchenko, Y., Lohse, M.J., Engelhardt, S., 2005. The transcriptional repressor Nab1 is a specific regulator of pathological cardiac hypertrophy. Nat. Med. 11, 837–844. Galandrin, S., Bouvier, M., 2006. Distinct signaling profiles of β1 and β2 adrenergic receptor ligands toward adenylyl cyclase and mitogen-activated protein kinase reveals the pluridimensionality of efficacy. Mol. Pharmacol. 70, 1575–1584. Guillemot, L., Levy, A., Raymondjean, M., Rothhut, B., 2001. Angiotensin II-induced transcriptional activation of the cyclin D1 gene is mediated by Egr-1 in CHO-AT1A cells J. Biol. Chem. 276, 39394–39403. Keys, J.R., Greene, E.A., Koch, W.J., Eckhart, A.D., 2002. Gq-coupled receptor agonists mediate cardiac hypertrophy via the vasculature. Hypertension 40, 660–666. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using realtime quantitative PCR and the 2− ΔΔCT method. Methods 25, 402–408. Marano, G., Vergari, A., Catalano, L., Gaudi, S., Palazzesi, S., Musumeci, M., Stati, T., Ferrari, A.U., 2004. Na+/H+ exchange inhibition attenuates left ventricular remodeling and preserves systolic function in pressure-overloaded hearts. Br. J. Pharmacol. 141, 526–532.
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Saadane, N., Alpert, L., Chalifour, L.E., 1999. Expression of immediate early genes, GATA-4, and Nkx-2.5 in adrenergic-induced cardiac hypertrophy and during regression in adult mice. Br. J. Pharmacol. 127, 1165–1176. Saadane, N., Alpert, L., Chalifour, L.E., 2000. Altered molecular response to adrenoreceptor-induced cardiac hypertrophy in Egr-1-deficient mice. Am. J. Physiol. 278, H796–H805. Sadoshima, J., Jahn, L., Takahashi, T., Kulik, T.J., Izumo, S., 1992. Molecular characterization of the stretch-induced adaptation of cultured cardiac cells. An in vitro model of load-induced cardiac hypertrophy. J. Biol. Chem. 267, 10551–10560. Wei, H., Ahn, S., Barnes, W.G., Lefkowitz, R.J., 2004. Stable interaction between β-adrenoceptorrestin 2 and angiotensin type 1A receptor is required for β-adrenoceptorrestin 2-mediated activation of extracellular signal-regulated kinases 1 and 2. J. Biol. Chem. 279, 48255–48261. Zhang, G.X., Ohmori, K., Nagai, Y., Fujisawa, Y., Nishiyama, A., Abe, Y., Kimura, S., 2007. Role of AT1 receptor in isoproterenol-induced cardiac hypertrophy and oxidative stress in mice. J. Mol. Cell. Cardiol. 42, 804–811.
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / e j p h a r
Short communication
Propranolol promotes Egr1 gene expression in cardiomyocytes via β-adrenoceptors Mario Patrizio a,1, Marco Musumeci a,1, Tonino Stati a, Katia Fecchi a, Elisabetta Mattei c, Liviana Catalano b, Giuseppe Marano a,⁎ a b c
Dipartimento del Farmaco, Istituto Superiore di Sanità, Rome, Italy Centro Nazionale Sangue, Rome, Italy Istituto di Neurobiologia e Medicina Molecolare, Consiglio Nazionale delle Ricerche, Rome, Italy
A R T I C L E
I N F O
Article history: Received 14 September 2007 Received in revised form 17 March 2008 Accepted 2 April 2008 Available online 10 April 2008 Keywords: Adrenoceptors Propranolol Gene expression Egr1 Cardiomyocytes
A B S T R A C T Recent research has revealed that propranolol, a β-adrenoceptor antagonist, causes extracellular signalregulated kinase (ERK) cascade activation, nuclear translocation of phospho-ERK and increased transcriptional activity in cultured cell lines. Given the importance of β-adrenoceptor antagonists in the treatment of heart failure, we evaluated the capability of propranolol of promoting the ERK-dependent gene expression at the cardiomyocyte level. To this end, the gene expression of the early growth response factor 1 (Egr1), a wellrecognized indicator of nuclear extracellular signal-regulated kinase 1/2 (ERK1/2) activation, was assessed by quantitative real-time RT-PCR in vivo as well as in vitro experiments. Propranolol, administered at the dose of 10 mg/kg/day in C57BL/6 mice, caused a ≈19-fold increase of Egr1 mRNA expression in left ventricular myocardium along with a ≈2.1-fold increase of Egr1 protein expression. Isoproterenol, a nonselective β-adrenoceptor agonist, also increased Egr1 mRNA and protein expression but to a lesser degree. Remarkably, isoproterenol administration was associated with the development of cardiac hypertrophy, whereas propranolol-treated mice showed a completely normal cardiac morphology. The effect of propranolol on Egr1 mRNA expression was abrogated in mice lacking β1- and β2-adrenoceptors indicating that propranolol increases Egr1 mRNA expression in a β-adrenoceptor-dependent manner. The role of β-adrenoceptors was further confirmed by showing that propranolol was able to increase Egr1 mRNA and protein levels in cultured neonatal cardiomyocytes. Collectively, these results indicate that propranolol promotes Egr1 gene expression in cardiomyocytes via β-adrenoceptors with a mechanism which is independent of its ability to antagonize the effects of catecholamines. It is also suggested that cardiomyocyte growth and Egr1 gene overexpression are not obligate processes. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Over the past several years, much attention has been focused on β-adrenoceptor antagonists because of their efficacy in the treatment of common cardiovascular pathologies. Propranolol, a nonselective β-adrenoceptor antagonist, remains the prototype of this class of drugs and that with which all others are compared. Propranolol has been generally reported to be a pure antagonist, unable to activate β-adrenoceptors, or to be an inverse agonist (Azzi et al., 2001), that is, it reduces the basal accumulation of cAMP. However, recent research has revealed that propranolol can also act as an agonist for the extracellular signal-regulated kinase 1/2 (ERK1/2). Indeed, propranolol has been found to cause ERK cascade activation (Baker et al., 2003; Galandrin and Bouvier, 2006), nuclear translocation of phospho-ERK and increased gene transcription in a Gs/i-independent
⁎ Corresponding author. Dipartimento del Farmaco, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. Tel.: +39 0649903412; fax: +39 0649387104. E-mail address: [email protected] (G. Marano). 1 These authors contributed equally to the work. 0014-2999/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.04.017
manner in cultured cell lines (Azzi et al., 2003). However, whether propranolol is able to promote ERK-induced gene expression in cardiomyocytes remains to be ascertained. Given the importance of β-adrenoceptor antagonists in the treatment of heart failure, elucidation of molecular mechanisms underlying pharmacological action of propranolol could provide important clues for the identification of additional therapeutic targets. ERKs are primarily cytoplasmic in most quiescent cells, but activation of ERK1/2 promotes their translocation from the cytoplasm into the nucleus, where they phosphorylate and activate several transcription factors, including Egr1, a prototypical member of zincfinger family of transcription factors, which is a well-recognized indicator of nuclear ERK1/2 activation in a variety of cells (Guillemot et al., 2001; Wei et al., 2004). In the present study, propranolol was chronically administered to mice by osmotic minipumps for 2 weeks and the resulting effect on cardiac Egr1 gene expression was assessed by quantitative realtime RT-PCR. Moreover, to test whether the in vivo effect of propranolol onEgr1 induction depends on its ability to antagonize cardiac β-adrenoceptor-mediated effects of catecholamines, the β-blocker was
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administered in β1- and β2-adrenoceptor double knockout mice, in which the predominant β-adrenoceptor subtypes are lacking. Additionally, the capability of propranolol to induce Egr1 gene expression was also evaluated in cultured rat neonatal cardiomyocytes, an experimental setting in which catecholamines are not present. Using such experimental approaches, we found that propranolol increases Egr1 mRNA and protein expression in left ventricular myocardium as well as in cultured cardiomyocytes. We also demonstrate that this activity on Egr1 gene expression is mediated via β-adrenoceptors and is independent of its ability to antagonize the effects of catecholamines.
sure increment (+dP/dt) or fall (−dP/dt) and end-systolic elastance (Ees, a load-independent parameter of cardiac contractility) were computed.
2. Methods
2.7. RNA isolation and quantification
2.1. Animals
Total RNA was extracted from left ventricles of individual mice and from cultured neonatal rat cardiomyocytes by using SV Total RNA Isolation System (Promega, Madison, USA). cDNA out of total ventricular RNA was synthesized by using the High Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA, USA). RNA expression levels for Egr1 in the left ventricle was quantified with Real-Time TaqMan RT-PCR using 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). Taqman reactions were carried out in 96 well plates using cDNA, Taqman universal PCR MasterMix, predesigned and pre-optimized specific primers and fluorescent probes (Egr1, Mm00656724_m1; GAPDH, Mm99999915_g1 for mouse, and Egr1, Rn00561138_m1; GAPDH, Rn99999916_s1 for rat), and water to a final volume of 50 µl according to manufacturer's instructions. GAPDH mRNA was used as an endogenous control. The codes for primers and probes were obtained from the Applied Biosystems catalogue for quantitative gene expression analysis. No reverse transcriptase and no template controls were used to monitor for any contaminating amplification. The ΔCt was used for statistical analysis, and 2− ΔΔCt for data presentation (Livak and Schmittgen, 2001).
Male C57BL/6 12-week-old mice (Harlan, S. Pietro al Natisone, Italy) were used in most experiments and maintained in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). In some experiments, male β1- and β2-adrenoceptor double knockout mice were used (Jackson Laboratories, Ann Harbor, USA). Because knockout (KO) mice have a mixed background, we mated KO mice with C57BL/6 mice to produce F1 mice heterozygous for both KOs. Thereafter, F1 heterozygous mice were mated to produce F2 wild-type (WT) and double KO mice. Genotyping was performed on tail DNA using standard protocols. 2.2. Primary cardiomyocytes cultures Cardiomyocytes were obtained by 1- to 3-day-old neonatal Wistar rat ventricles as described (Sadoshima et al., 1992) and plated at a density of ≈5.0 × 105 cells per well in 6-well tissue culture plates. To induce Egr1 gene expression, cardiomyocytes were exposed to propranolol (1, 10 and 25 μM) for 24 h or (25 μM) 72 h. 2.3. Chronic administration of drugs Propranolol, metoprolol or isoproterenol were administered in a dose of 10 mg/kg/day, 10 mg/kg/day, and 15 mg/kg/day, respectively, for 14 consecutive days. The dosage for drugs was chosen on the basis of literature data (Boluyt et al., 1995; Keys et al., 2002; Zhang et al., 2007). Remarkably, at this dosage, isoproterenol does not induce arterial hypertension. The drugs were administered through osmotic minipumps (Alzet model 2002, Cupertino, CA, USA) implanted subcutaneously on the right side of the back. 2.4. Echocardiography In mice intubated and anesthetized with isoflurane (1% in 100% of oxygen), echocardiographic examination was performed with a SONOLINE G50 (Siemens AG, Erlangen, Germany) equipped with a 13-MHz imaging transducer as described (Marano et al., 2004).
2.6. Histological analysis Histological analysis was performed as reported previously (Marano et al., 2004). Briefly, left ventricular sections were cut and stained with hematoxylin and eosin for measurement of myocyte cross-sectional area by quantitative morphometry (Metamorph 6.1, Universal Imaging Corporation, PA, USA).
2.8. Western blot analysis Mouse left ventricles were homogenized in RIPA-buffer supplemented with 10 mM NaF, 100 µM Na3VO4, 1 mM EDTA, 1 mM EGTA, 0,2 mM PMSF and 100 µM leupeptin. Insoluble material was removed by centrifugation at 1000 ×g at 4 °C for 10 min, and protein concentration measured. For each condition, 25 µg of protein were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were then blocked with 5% nonfat milk and incubated with rabbit polyclonal antibody anti-Egr1 (1:1000 dilution) (Santa Cruz Biotechnology, CA, USA) for 1 h at 25 °C. After several washes, the membranes were incubated with secondary antibodies (anti-rabbit IgG conjugated with horseradish peroxidase, 1:5000) for 1 h at 25 °C and visualized by the Amersham ECL system. The optical density of the bands (arbitrary units, AU) was measured with a GS-700 Imaging Densitometer (Bio-Rad) and referred to the corresponding control samples (taken as 1) run in the same gel. 2.9. Statistical analysis
2.5. Hemodynamic analysis Left ventricular hemodynamic measurements were obtained by direct left ventricular catheterization as described (Marano et al., 2004). Briefly, a 1.4-F, four-electrode pressure-volume catheter (model SPR839, Millar Instruments, Houston, TX, USA) was inserted into the left ventricle through the apex in the open chest anesthetized animal (isoflurane 1.5–2% in 100% of oxygen) and advanced along the long axis with proximal electrode just within the wall of the left ventricle. All pressure-volume loop data were analysed with cardiac pressurevolume analysis program (IOX 1.7; EMKA Technologies, Paris, France), and the heart rate, maximal left ventricular systolic pressure, left ventricular end-diastolic pressure, maximal slope of left ventricular pres-
Group means (±S.E.M.) were calculated for all relevant variables. Statistical analysis was performed by Student t-test or ANOVA with Bonferroni's multiple comparison test for post hoc analyses. A value of P b 0.05 was considered to be statistically significant. 3. Results 3.1. Propranolol upregulates Egr1 gene expression in left ventricular myocardium To evaluate whether propranolol promotes nuclear translocation of phospho-ERK and, in turn, gene expression in the myocardium, we
M. Patrizio et al. / European Journal of Pharmacology 587 (2008) 85–89 Table 1 Echocardiography and hemodynamic measurements 2 weeks after propranolol administration
N Body weight, g Heart rate, beats/min LVSP, mmHg LVEDP, mmHg −dP/dt, mmHg/s LVEDD, mm FS, % +dP/dt, mmHg/s Ees, mmHg/μl
Vehicle
Propranolol
5 25.3 ± 0.2 431 ± 11 67 ± 3 4.0 ± 0.5 4055 ± 32 3.6 ± 0.2 34 ± 2 4772 ± 67 6.1 ± 0.5
6 26.0 ± 0.3 398 ± 10 64 ± 3 4.1 ± 0.3 4331 ± 42 3.7 ± 0.2 35 ± 2 4902 ± 59 5.8 ± 0.4
LVSP, maximal left ventricular systolic pressure; LVEDP, left ventricular end-diastolic pressure; LVEDD, left ventricular end-diastolic diameter; +dP/dt and −dP/dt, maximum rate of left ventricular pressure rise or fall; FS, fractional shortening was calculated as ((LVEDD − LVESD) / LVEDD) × 100; LVESD, left ventricular end-systolic diameter; Ees, end-systolic elastance.
assessed the expression of Egr1 gene by real-time RT-PCR. Propranolol, administered by subcutaneous infusion at the dose of 10 mg/kg/day for 14 consecutive days in C57BL/6 mice, had no significant effect on heart rate, ventricular function evaluated as fractional shortening, +dP/dt and Ees, and chamber size in anesthetized animals (Table 1). In contrast, it caused a marked upregulation of Egr1 mRNA in left ventricular myocardium (≈19-fold, Fig. 1A). To evaluate whether the effects on Egr1 gene expression was also observed with a more selective β-adrenoceptor antagonist, a β1adrenoceptor antagonist, metoprolol, was also tested. Metoprolol, enhanced Egr1 gene expression, but the effects were significantly less than in propranolol-treated mice (Fig. 1A). Isoproterenol, a β-adrenoceptor agonist, which is known to increase Egr1 gene expression (Saadane et al., 1999), was used as a positive control. As expected, chronic infusion of isoproterenol caused a significant increase of Egr1 gene expression in left ventricular myocardium
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(Fig. 1A) associated with a 27% increase of the left ventricular weightto-body weight ratio (4.2 ± 0.2 mg/g). As a next step, induction of Egr1 gene was investigated at the protein level. Chronic administration of propranolol, metoprolol or isoproterenol caused a 2.1-fold, 2.0-fold and 1.7-fold increase of Egr1 protein levels in left ventricular myocardium, respectively (Fig. 1B). Taken together, these results indicate that the induction of Egr1 mRNA in myocardium by propranolol is followed by enhanced Egr1 protein expression. As Egr1 has been involved in the development of pathological cardiac growth (Buitrago et al., 2005), a detailed analysis of cardiomyocyte size was performed in both propranolol- and metoprolol-treated mice by transthoracic echocardiography and histopathological analysis. Compared to vehicle-treated mice, propranolol-treated mice showed a completely normal cardiac morphology (Fig. 1C–F). Similar results were observed in metoprolol-treated mice (Fig. 1D–F). Collectively, these results suggest that the increase in Egr1 gene expression alone is not sufficient to cause cardiac hypertrophy. 3.2. Propranolol induces Egr1 gene expression via a direct effect on β-adrenoceptors Propranolol is a competitive β-adrenoceptor antagonist and interacts with β1- and β2-adrenoceptors with equal affinity. Thus, the effect of propranolol on Egr1 induction could depend on its ability to antagonize cardiac β-adrenoceptor-mediated effects of catecholamines. To address this issue, quantitative analysis of Egr1 mRNA was performed in β1- and β2-adrenoceptor double knockout (KO) mice, in which the predominant cardiac β-adrenoceptor subtypes are lacking. We found a rather mild increase in Egr1 gene expression in left ventricles from KO mice (Fig. 2A). Remarkably, propranolol administration failed to increase Egr1 mRNA levels in KO mice (Fig. 2A). To further confirm that propranolol causes Egr1 gene induction independently of its property to antagonize the effects of catecholamines, we investigated the effects of propranolol in cultured neonatal cardiomyocytes at 24 h. As shown in Fig. 2B, propranolol induced a
Fig. 1. Propranolol upregulates Egr1 gene expression in the heart without affecting cardiac growth. (A) Expression of Egr1 in left ventricular myocardium. Data are presented as the fold change relative to the expression levels of vehicle-treated mice (2− ΔΔCt). Summary data from 4–5 separate experiments performed in triplicate are shown. ⁎ P b 0.05 when compared to vehicle group. (B) Western blot analysis of Egr1 protein expression in left ventricular myocardium. Propranolol, metoprolol or isoproterenol were administered in C57BL/ 6 mice for 14 consecutive days. (Top) Representative experiment. (Bottom) A graph representing a summary of repeated experiments is also shown (n = 3–4 animals per group). (C) Representative coronal sections of hearts from vehicle- and propranolol-treated mice. No abnormalities with normal atria and absence of cardiac hypertrophy of the ventricles were observed. (D) Left ventricular weight-to-body weight (LVW/BW) ratio (n = 5–6 animals per group). (E) Left ventricular posterior wall thickness (PWT). (n = 5–6 animals per group). (F) Morphometric analysis of cardiomyocytes. Data were from about 160 cardiomyocytes per group derived from 4 animals per group. Veh, vehicle; Pro, propranolol; Met, metoprolol; Iso, isoproterenol.
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Fig. 2. Propranolol induces Egr1 gene expression via β-adrenoceptors. (A) Administration of propranolol in β-adrenoceptor double knockout (KO) mice. After 14 days, the β-blocker failed to increase the expression of Egr1 mRNA in KO mice (n = 4–5 animals per group). (B) Effects of propranolol on Egr1 mRNA levels in cultured neonatal cardiomyocytes 24 h and 72 h after drug exposure. Propranolol induced a significant increase of Egr1 gene expression in a concentration-dependent manner. (C) Western blot analysis of Egr1 protein expression in cultured cardiomyocytes. Cells were treated for 72 h with or without propranolol (25 µM). (Top) Representative experiment. (Bottom) Summary data from 3 separate experiments are also shown. Ctr, control; WT, wild-type; Pro, propranolol. ⁎P b 0.05, vs corresponding control group.
significant increase in Egr1 gene expression in a concentrationdependent manner. Additionally, to test whether the effects of propranolol on Egr1 mRNA levels were maintained over time, cultured cardiomyocytes were treated with propranolol for 72 h. Treatment with propranolol caused a significant increase in Egr1 mRNA levels similar to that observed at 24 h (Fig. 2B). Again, the induction of Egr1 mRNA was followed by enhanced Egr1 protein expression (Fig. 2C). To determine whether the propranolol-induced upregulation of Egr1 mRNA was a consequence of changes in the rate of gene transcription, cultured cardiomyocytes were treated with the transcription inhibitor actinomycin D (2.5 µg/ml) plus propranolol (25 μM) for 24 h. We found a 5.1 ± 0.1-fold increase of Egr1 mRNA in propranololtreated cells. This response was prevented when cells were treated with actinomycin D, indicating that transcriptional activation is the more likely mechanism involved. 4. Discussion The product of the early growth response gene Egr1, which is also known as NGFI-A, zif268, krox24, or Tis8, is a transcriptional factor that is rapidly induced in many different cell types by a variety of growth factors, cytokines, and harmful stimuli. In the present study, we demonstrate for the first time that propranolol, a β-adrenoceptor antagonist, upregulates mRNA levels of Egr1 in the mouse heart under basal conditions. These data extend observations reported recently by Azzi et al. (2003) of the propranolol-induced activation of transcriptional factors in Cos1 cell line, into cardiomyocytes. An additional major finding of this study is that propranolol causes Egr1 gene induction via both β-adrenoceptor subtypes, but independently of its ability to antagonize the effects of catecholamines. This is supported by 3 independent lines of evidence. First, propranolol effects on Egr1 are abrogated in β1- and β2-adrenoceptor double knockout mice, in which the predominant cardiac β-adrenoceptor subtypes are lacking. Second, propranolol is able to induce Egr1 gene expression in cultured neonatal cardiomyocytes in a concentrationdependent manner. Third, the selective β1-AR antagonist, metoprolol, also increased Egr1 gene expression but to a less extent than in propranolol-treated mice. Egr1 gene expression is increased in the heart after adrenoceptor stimulation (Saadane et al., 1999). Here, we show that both propranolol, a β-adrenoceptor antagonist, and isoproterenol, a βadrenoceptor agonist, upregulate Egr1 gene expression in the mouse heart. However, this is an apparent discrepancy because both compounds have been reported to show agonistic properties towards the ERK signaling pathway. Indeed, Galandrin and Bouvier (2006) have reported that propranolol induces partial agonist responses for
the ERK1/2 pathway via both β1- and β2-adrenoceptors. Additionally, Azzi et al. (2003) have observed that propranolol activates ERK1/2 and promotes its translocation from the cytoplasm into the nucleus, where it phosphorylates Elk-1 which plays a primary role in regulating Egr1 gene expression. With few exceptions, at the transcriptional level, cardiac hypertrophy is characterized by a specific gene expression pattern including upregulation of immediate–early response genes such as Egr1 as well as of fetal genes such as atrial natriuretic peptide and the β-isoform of myosin heavy chain. At this time, the significance of these changes remains to be fully understood. Antifibrotic effects or growth promoting activities have been attributed to Egr1, depending on the experimental context. For example, Egr1 knockout mice show a greater susceptibility to develop fibrotic lesions than wild-type mice under basal conditions (Saadane et al., 2000). On the contrary, mouse hearts with specific overexpression of Nab1, a transcriptional repressor of Egr1, or Egr1-deficient mice show a marked reduction in ventricular hypertrophic response when subjected to pressure overload which is consistent with a growth promoting activity of Egr1 under conditions of enhanced cardiac stress (Buitrago et al., 2005). In the present study we demonstrate that propranolol-induced upregulation of Egr1 gene expression seems to be functionally silent being associated with a completely normal ventricular morphology. In summary, our data indicate that propranolol is able to promote Egr1 gene expression in cardiomyocytes via β-adrenoceptors with a mechanism which is independent of its ability to antagonize the effects of catecholamines. It is also suggested that cardiomyocyte growth and Egr1 gene overexpression are not obligate processes. Acknowledgements We are grateful to Mr. Valerio Vago for technical support. This work was supported in part by grants from the Italian Minister of Health to G.M (2004/N01). K.F. was recipient of a fellowship from FIRB the Italian Ministry for University and Research (Grant RBNE03FMCJ). References Azzi, M., Piñeyro, G., Pontier, S., Parent, S., Ansanay, H., Bouvier, M., 2001. Allosteric effects of G protein overexpression on the binding of β-adrenergic ligands with distinct inverse efficacies. Mol. Pharmacol. 60, 999–1007. Azzi, M., Charest, P.G., Angers, S., Rousseau, G., Kohout, T., Bouvier, M., Piñeyro, G., 2003. β-adrenoceptorrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for G protein-coupled receptors. Proc. Natl. Acad. Sci. U S A. 100, 11406–11411. Baker, J.G., Hall, I.P., Hill, S.J., 2003. Agonist and inverse agonist actions of β-blockers at the human β2-adrenoceptor provide evidence for agonist-directed signaling. Mol. Pharmacol. 64, 1357–1369.
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