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J. Biol. Chem., Vol. 275, Issue 28, 21773-21779, July 14, 2000

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Inhibition of Spontaneous ₂-Adrenergic Activation Rescues ₁-Adrenergic Contractile Response in Cardiomyocytes Overexpressing ₂-Adrenoceptor^*

Sheng-Jun Zhang, Heping Cheng, Ying-Ying Zhou, Ding-Ji Wang, Weizhong Zhu, Bruce Ziman, Harold Spurgoen, Robert J. Lefkowitz, Edward G. Lakatta, Walter J. Koch^§, and Rui-Ping Xiao^¶

From the Laboratory of Cardiovascular Sciences, Gerontology Research Center, NIA, National Institutes of Health, Baltimore, Maryland 21224 and ^§ Department of Surgery and  Department of Medicine and Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710

Received for publication, November 24, 1999, and in revised form, April 6, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cardiac-specific overexpression of the human ₂-adrenergic receptor (AR) in transgenic mice (TG4) enhances basal cardiac function due to ligand-independent spontaneous ₂-AR activation. However, agonist-mediated stimulation of either ₁-AR or ₂-AR fails to further enhance contractility in TG4 ventricular myocytes. Although the lack of ₂-AR response has been ascribed to an efficient coupling of the receptor to pertussis toxin-sensitive G_i proteins in addition to G_s, the contractile response to ₁-AR stimulation by norepinephrine and an ₁-adrenergic antagonist prazosin is not restored by pertussis toxin treatment despite a G_i protein elevation of 1.7-fold in TG4 hearts. Since -adrenergic receptor kinase, ARK1, activity remains unaltered, the unresponsiveness of ₁-AR is not caused by ARK1-mediated receptor desensitization. In contrast, pre-incubation of cells with anti-adrenergic reagents such as muscarinic receptor agonist, carbachol (10⁵ M), or a ₂-AR inverse agonist, ICI 118,551 (5 × 10⁷ M), to abolish spontaneous ₂-AR signaling, both reduce the base-line cAMP and contractility and, surprisingly, restore the ₁-AR contractile response. The "rescued" contractile response is completely reversed by a ₁-AR antagonist, CGP 20712A. Furthermore, these results from the transgenic animals are corroborated by in vitro acute gene manipulation in cultured wild type adult mouse ventricular myocytes. Adenovirus-directed overexpression of the human ₂-AR results in elevated base-line cAMP and contraction associated with a marked attenuation of ₁-AR response; carbachol pretreatment fully revives the diminished ₁-AR contractile response. Thus, we conclude that constitutive ₂-AR activation induces a heterologous desensitization of ₁-ARs independent of ARK1 and G_i proteins; suppression of the constitutive ₂-AR signaling by either a ₂-AR inverse agonist or stimulation of the muscarinic receptor rescues the ₁-ARs from desensitization, permitting agonist-induced contractile response.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In addition to traditional pharmacological approaches, mouse genomic manipulation has opened new avenues of investigations of transmembrane signal transduction, particularly those signals mediated by G protein-coupled receptors. Owing to the critical role of -adrenergic receptor (-AR)¹ in cardiac functional regulation, several transgenic and gene-targeted mouse models have been recently developed to alter -AR signaling components (1). One such model that has drawn substantial attention is the transgenic mouse overexpressing the human ₂-AR in a cardiac-specific manner (TG4 mice) (2). In this model, due to the presence of spontaneously activated ₂-ARs (designated as R*s), multiple components of base-line cardiac function, including heart rate, the intracellular Ca²⁺ ([Ca]_i) transient, and contractility, are substantially enhanced relative to those of wild type (WT) littermates (2-5). It has been suggested that this constitutive ₂-AR stimulation may provide a novel mechanism for the inotropic support of the end stage, dilated, chronically failing heart (2), in which agonist-mediated -AR contractile response is markedly diminished. Surprisingly, in the context of substantially enhanced constitutive ₂-AR stimulation, acute administration of either ₁-AR or ₂-AR agonists fails to elicit any contractile or chronotropic response in the intact animals (4), isolated hearts, (2, 3) or single cardiac myocytes (5-8).

The loss of -AR responsiveness had initially been interpreted to indicate a saturation in contractility caused by the constitutive ₂-AR signaling (2, 3). Our more recent studies, however, have demonstrated that this is not the case, because TG4 ventricular myocytes still respond to a direct activation of adenylyl cyclase by forskolin (5). Furthermore, ₂-AR agonist-mediated contractile response is rescued by pertussis toxin (PTX)-induced G_i ribosylation, indicating that an efficient coupling of ₂-AR to G_i proteins (G_i2 and G_i3) negates the contractile response to ₂-AR agonists (5-8).

The loss of ₁-AR responses in the context of ₂-AR overexpression in TG4 mice, however, remained unexplained. It has been reported that acutely overexpression of ₂-AR in rat C6 glioma cells also results in null ₁-AR response (9). This observation and those in the TG4 mouse heart point to a complex interplay among the closely related members of -AR family, although the underlying mechanism was unknown.

Since adenylyl cyclase-cAMP-PKA signaling and the contractile machinery are intact in TG4 cardiomyocytes (5-7), the loss of the ₁-AR contractile response could be attributed to either a defect of the receptor per se, e.g. desensitization or down-regulation, or to interference of receptor signaling at a more distal level. As is true for most G protein-coupled receptors, prolonged exposure of -ARs to an agonist leads to a decrease in receptor responsiveness, i.e. desensitization. When this occurs in the context of diminished responsiveness to a diverse array of other agonists (heterologous desensitization), it generally results from a negative feedback regulation by second messenger-stimulated kinases. For instance, activation of PKA downstream from various G_s-coupled receptors may lead to phosphorylation of -AR (at PKA consensus sequences in the carboxyl terminus and/or the third intracellular loop) which, in turn, initiates receptor desensitization (10, 11).

Exposure to an agonist also can result in a diminished response to only that particular agonist, i.e. homologous (or agonist-specific) desensitization, which is generally mediated by the second messenger-independent mechanism, G protein-coupled receptor kinases (12, 13). -Adrenergic receptor kinase 1 (ARK1), the prototypic G protein-coupled receptor kinase, has been shown to phosphorylate activated -ARs (both ₁ and ₂ subtypes) in vitro (13, 14). Increased cardiac expression (3-5-fold) of ARK1 in transgenic mice leads to diminished -AR agonist-stimulated cardiac contractility in vivo (15) and in isolated ventricular myocytes (16). Phosphorylated receptors become binding substrates for a class of inhibitor proteins, -arrestins, which inhibit further G protein coupling (17). The chronic spontaneous ₂-AR activation in TG4 cardiomyocytes could result in an increased G protein-coupled receptor kinase activity, as is the case in transgenic mice overexpressing G_s (18). Additionally, in both humans (19) and animals (20), chronic heart failure is often associated with elevated plasma catecholamine levels (21-23) and an increase in ARK1 abundance and enzymatic activity, which at least in part contribute to the reduced efficacy of -AR stimulation under these pathophysiological conditions.

In this study, we investigate potential mechanisms that nullify ₁-AR in the context of constitutive ₂-AR activation. Specifically, we have determined 1) whether ₁-AR in TG4 hearts is desensitized via a PKA- or ARK1-dependent mechanism, 2) whether inhibitory mechanisms, particularly G_i proteins, cross-talk with ₁-AR-coupled G_s signaling and counteract its contractile response, and 3) whether inhibition of the spontaneous ₂-AR activation by either a ₂-AR inverse agonist or stimulation of G_i-coupled muscarinic receptor restores the desensitized ₁-AR signaling. Furthermore, using a modified adenovirus-mediated gene transfer technique recently developed in this laboratory (24), we have reproduced these experiments in cultured WT mouse cardiomyocytes infected with adenovirus carrying the human ₂-AR gene (adeno-₂-AR). Our results indicate that the defect of ₁-AR stimulation in TG4 cardiomyocytes is conferred to the enhanced constitutive ₂-AR activation and is likely caused by PKA-dependent heterologous receptor desensitization, independent of ARK1 and G_i activation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Measurements of Cell Contraction-- Mouse ventricular myocytes were isolated by a modified enzymatic technique (16, 24). Cells were then perfused with Hepes buffer solution consisting of 1.0 mM CaCl₂, 137 mM NaCl, 5 mM KCl, 15 mM dextrose, 1.3 mM MgSO₄, 1.2 mM NaH₂PO₄, 20 mM Hepes, pH 7.4, and were electrically stimulated at 0.5 Hz at 23 °C. Cell length was monitored from the brightfield image of the cell by an optical edge-tracking method using a photodiode array (Reticon Model 1024 SAQ) with a 3-ms time resolution. Cell contraction was indexed by the percent reduction of cell length following electrical stimulation (25).

Adult Mouse Cardiomyocyte Culture and Adenoviral Infection-- The methods for adult mouse cardiomyocyte culture and adenoviral infection have been described previously (24). Briefly, freshly isolated cardiac myocytes were washed 3 times with minimal essential medium (MEM, Sigma) containing 1.2 mM Ca²⁺, 2.5% pre-selected fetal bovine serum (FBS), and 1% penicillin-streptomycin. Then myocytes were plated at 0.5~1 × 10⁴/cm² in culture dishes pre-coated with 10 µg/ml mouse laminin and containing MEM, 2.5% FBS, and 1% penicillin-streptomycin. After a 1-h culture, the culture media was changed to FBS-free MEM.

Adenovirus-directed gene transfer was implemented after cells were cultured for 1 h to achieve attachment. After the culture media was aspirated along with unattached cells, one-half of the volume (e.g. 1 ml for 35-mm Petri dish) of the FBS-free MEM containing adenovirus carrying the human ₂-AR gene (adeno-₂-AR) at multiplicity of infection (m.o.i.) of 1,000 was added into the dish. The other half-volume of the FBS-free MEM was supplied after culture for another 1-2 h. Biochemical and physiological measurements were performed after 24 h culture.

Receptor Radioligand Binding-- To prepare crude myocardial membranes, WT or TG4 ventricular myocytes were homogenized with 15 strokes on ice in a lysis buffer (5 mM Tris·HCl, pH 7.4, 5 mM EGTA). -AR radioligand binding studies were performed in membranes (25 µg/tube) from either WT or TG4 ventricular myocytes using the non-selective -AR antagonist ligand ¹²⁵I-cyanopindolol (1-300 pM) as described previously (26). Binding was allowed to occur for 1 h at 37 °C. Nonspecific binding was determined in the presence of 10 µM propranolol.

For competition isotherms, membranes (5-25 µg total protein) were incubated with 50 pM ¹²⁵I-cyanopindolol and increasing dilutions of ICI 118,551 (ICI), a selective ₂-AR antagonist, as described previously (26). The percentage of ₂-AR from the high affinity binding sub-population was calculated using GraphPad Prism.

cAMP Measurement-- In some experiments cells were incubated with carbachol (CCh, 10⁵ M) or ICI 118,551 (ICI, 5 × 10⁷ M) for 1 h at 37 °C. cAMP levels were assayed using standard protocols described previously (27). Briefly, 10 µl of membrane vesicles (20 µg total protein) was added to a 40-µl reaction mix to make a final concentration of 4 mM Tris-EDTA and 10 µM Ro 20-1724 (an inhibitor of phosphodiesterase IV). The reaction was performed for 15 min at 37 °C, and 25 µl of supernatant was assayed using a cAMP ³H assay kit obtained from Amersham Pharmacia Biotech.

Western Analysis of G_i Proteins-- Membranes from mouse left ventricles were used for G_i Western analysis. Equal protein amounts (50-70 µg) were electrophoresed under denaturing conditions on 12% polyacrylamide/Tris-glycine gels and transfected to nitrocellulose membranes, as described (26). Protein immunoblotting was done with the antibody recognizing the -subunits of G_i 1-3 (Santa-Cruz) and secondary antibodies using standard chemiluminescence protocol (ECL^®; Amersham Pharmacia Biotech).

ARK1 Activity in Rhodopsin Phosphorylation Assays-- Left ventricles from either WT or TG4 mice were homogenized in ice-cold lysis buffer (5 mM Tris-HCl, pH 7.5, 5 mM EDTA, 5 mM EGTA, 10 µg of leupeptin/ml, 20 µg of aprotinin/ml, and 1 mM phenylmethylsulfonyl fluoride). The samples were centrifugated at 800 × g for 14 min at 4 °C to clear the homogenate of cellular debris and nuclei. Subsequent supernatants were filtered through cheesecloth and centrifuged at 25,000 × g for 30 min at 4 °C. The final supernatant was used for G protein-coupled receptor kinase assays in which the primary G protein-coupled receptor kinase activity is ARK1 (15). The supernatants were concentrated using a Centricon 30 (Amicon) microconcentrator. Concentrated cytosolic extracts (300 µg of protein) were incubated with rhodopsin-enriched rod outer segments in 75 µl of lysis buffer with 10 mM MgCl₂ and 0.1 mM ATP containing [-³²P]ATP. The reactions were incubated in white light for 15 min at room temperature and quenched with 300 µl of ice-cold lysis buffer and then centrifuged for 15 min at 13,000 × g. Sedimented proteins were resuspended in 25 µl of protein-gel loading dye and electrophoresed through SDS, 12% polyacrylamide gels. Phosphorylated rhodopsin was visualized by autoradiography of dried polyacrylamide gels and quantified using a Phosphorlmager (Molecular Dynamics).

Incubation of Cells with Carbachol or ICI 118,551-- To antagonize possible PKA-dependent receptor desensitization induced by the spontaneous ₂-AR activation, aliquots of ventricular myocytes from TG4 hearts or adeno-₂-AR (m.o.i. 1000)-infected WT myocytes cultured for 24 h were incubated with a muscarinic receptor agonist, CCh (10⁵ M), for 1 h at 37 °C. Then the cells were transferred to perfusion chamber containing 10⁷ M CCh. CCh at this subthreshold concentration (in uninfected WT myocytes) was used to permit detection of a contractile response to ₁-AR agonist stimulation. Ligands of -AR subtypes were administered in the continuous presence of CCh (10⁷ M) unless indicated otherwise. In some experiments, cells were first treated with PTX (see below) before incubation with CCh. In another subset of experiments, cells were incubated with the ₂-AR inverse agonist, ICI 118,551 (ICI, 5 × 10⁷ M), for 1 h at 37 °C to inhibit spontaneous ₂-AR activation, and ₁-AR stimulation by norepinephrine (NE) was subsequently elicited in the continuous presence of ICI (10⁷ M).

PTX Treatment-- To inhibit G_i protein function, aliquots of cells were incubated with PTX (1.5 µg/ml, at 37 °C for at least 3 h), as described previously (28). Successful functional inactivation of PTX-sensitive G proteins was routinely verified by a loss of the ability of adenosine or CCh to reverse the positive inotropic effect of ₁AR stimulation by NE in WT myocytes (data not shown). PTX-treated cells were compared with myocytes from the same heart, which had been kept at 37 °C in the absence of PTX for an equal time.

Materials-- CGP 20712A (CGP) was kindly supplied by Ciba-Geigy Corp. ICI 118,551 (ICI) was kindly supplied by Imperial Chemical Industry, and zinterol was kindly supplied by Bristol-Myers Squibb Co. The antibody recognizing the -subunits of G_i 1-3 was obtained from Santa-Cruz. MEM, pertussis toxin, isoproterenol, norepinephrine, prazosin, and carbachol were purchased from Sigma. FBS, penicillin-streptomycin, and mouse laminin were purchased from Life Technologies, Inc.

Statistics-- Data reported are the mean ± S.E. Statistical comparisons were made by student's t test, paired t test, or analysis of variance when appropriate. A P value of < 0.05 was considered to be statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Loss of Contractile Response to ₁-AR Stimulation in TG4 Ventricular Myocytes-- Fig. 1A shows that a non-selective -AR agonist, isoproterenol (ISO, 10⁷ M) robustly increases contraction amplitude in a representative ventricular myocyte from a WT littermate. This effect is reversed by the ₁-AR antagonist, CGP (3 × 10⁷ M), but not by the ₂AR blocker, ICI (10⁷ M), indicating that stimulation of ₁-AR, but not ₂-AR, in WT mouse myocytes enhances contraction amplitude. Similarly, a selective ₁-AR agonist, NE, in the presence of an -adrenergic antagonist, prazosin (10⁶ M), augments the contraction amplitude of WT cardiomyocytes in a dose-dependent manner (Fig. 1B). However, ₂-AR stimulation by zinterol at any concentration tested is unable to augment contraction (Fig. 1B) due to a strong coupling of ₂-AR to G_i proteins in mouse cardiomyocytes (5).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   1-AR stimulation augments contraction amplitude in WT mouse ventricular myocytes. Panel A shows a continuous chart recording of cell contraction in response to a non-selective -AR agonist, isoproterenol (ISO, 107 M), in the presence and absence of the 1-AR selective antagonist CGP 20712A (3 × 107 M) or the 2-AR antagonist ICI 118,551 (107 M). Panel B shows the average dose response of contraction amplitude to the 1-AR agonist NE in the presence of an 1-AR antagonist, prazosin (106 M), or to the 2-AR agonist zinterol (ZINT) in WT cardiomyocytes. Each cell was superfused with a single concentration of NE or zinterol. All measurements were obtained under steady-state conditions after a 10-min exposure to the agonists. The results are presented as mean ± S.E. (n = 6~8 cells from 5~6 hearts).

The base-line contraction amplitude of TG4 myocytes is markedly elevated relative to WT cells (Refs. 5-7, also see below), which is in agreement with the previous notion that overexpression of cardiac ₂-AR exacerbates the physiological effects of ligand-free spontaneous receptor activation (2-7) and that spontaneously activated ₂-AR differs from agonist-stimulated ₂-AR in terms of its ability to augment cardiac contractility (6, 7). However, agonist-mediated ₁-AR stimulation by NE at 10⁷ M or higher concentrations in the presence of an ₁-AR antagonist, prazosin (10⁶ M), fails to augment contractility in these cells (Fig. 2A). The contraction amplitudes in the absence and presence of NE are 4.13 ± 0.76 and 4.31 ± 0.69% of cell rest length (n = 9), respectively. In cells from the same TG4 hearts, forskolin, an adenylyl cyclase activator, increases contraction amplitude by 2.3-fold (236.1 ± 16.5% of base-line, n = 7, p < 0.01). These results suggest that ₁-ARs in the TG4 heart are either completely lost or desensitized as a consequence of the transgenic overexpression of ₂-AR. Alternatively, ₁-AR could be intact, but its functionality might be antagonized by some inhibitory mechanisms.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   PTX treatment rescues 2-AR but not 1-AR mediated contractile response in TG4 ventricular myocytes. The contractile response to 1-AR stimulation by NE (107 M) plus the 1-AR antagonist, prazosin (PRAZ, 106 M), in a representative untreated (panel A) or PTX-treated (panel B) TG4 ventricular myocyte. NE fails to increase the contraction amplitude in either PTX-treated (n = 7) or untreated (n = 9) TG4 cells (114.1 ± 8.7% and 110.3 ± 8.5% of base-line contraction amplitude, respectively). Panel C shows the 2-AR agonist, zinterol (ZINT, 106 M), induced contractile response and its blockade by the 2-AR antagonist, ICI 118,551 (107 M), in a typical PTX-treated TG4 myocyte.

Because no absolutely selective -AR subtype ligand is presently available, a direct appraisal by the classic radioligand binding assay for ₁-AR density in TG4 myocardium, in which ₂-AR predominates by ~100-fold (2), is precluded. However, rescuing ₁-AR function would not only verify the physical existence of ₁-AR but also illuminate specific mechanisms for the loss of its function. We next searched for possible experimental interventions to restore ₁-AR contractile response in TG4 cardiomyocytes.

PTX Treatment Cannot Restore ₁-AR Contractile Response-- The failure of ₂-AR stimulation to increase cardiac contractility in both WT and TG4 mice has been explained by a "cross-talk" between the G_s and G_i signaling pathways concurrently coupled to ₂-ARs (5). Analogous to the situation of chronic in vivo infusion of catecholamines (29, 30), chronic ₂-AR stimulation in TG4 heart could adaptively elevate G_i abundance or activity, offsetting G_s-cyclase-stimulated inotropic response. We therefore sought to determine the potential role of G_i proteins in the unresponsiveness of ₁-ARs by incubating TG4 cardiomyocytes with PTX to disrupt G_i function. In the presence of PTX treatment, the ₁-AR agonist, NE, at a saturating concentration (10⁷ M) in the presence of prazosin (10⁶ M) is still unable to increase contraction amplitude (Fig. 2B), whereas in these PTX-treated cells contractile response to ₂-AR stimulation by zinterol (10⁶ M) is fully restored (Fig. 2C; also see Ref. 5). Similar results are obtained with ISO (10⁷ M) in the presence of -AR subtype-selective antagonists (data not shown). Nevertheless, Western blotting reveals that the amount of G_i in TG4 is 1.7-fold more abundant than that in WT myocardium (Fig. 3A), similar to the situation of in vivo chronic infusion of catecholamines (29, 30). Thus, despite the elevated G_i protein level in TG4 heart, the lack of ₁-AR contractile response is not caused by a G_i-mediated inhibitory signaling. This conclusion is consistent with the idea that ₁-AR, unlike ₂-AR, does not couple to G_i proteins in the heart (5, 26, 28) and implicates distinctly different mechanisms for the loss of ₁-AR versus ₂-AR subtype function in TG4 mice.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Gi protein abundance and ARK1 activity in TG4 and WT myocardium. Panel A shows the average data of Gi abundance assayed by Western blot in WT and TG4 cardiac membranes (* p < 0.05 versus WT, n = 7). The inset shows a typical example of the Western blot (Con refers to WT). Panel B illustrates the average soluble myocardial G protein-coupled receptor kinase (ARK1) activity in TG4 (n = 8) and WT (n = 7) mice. There is no significant difference in ARK1 activity between WT and TG4 myocardium. Phosphorylated rhodopsin was counted using a Molecular Dynamics Phosphorlmager.

Basal ARK1 Activity and cAMP Accumulation in TG4 Heart-- A prolonged exposure of -ARs to agonists leads to receptor desensitization via either PKA or ARK1-mediated receptor phosphorylation, both interfering with -AR-G_s coupling (10-16). Thus, we tested the hypothesis that chronic spontaneous ₂-AR activation in TG4 cardiomyocytes may induce ₁-AR desensitization via a super-activation of these kinases. Using the rhodopsin phosphorylation assay, we have found no evidence for an increase in the basal ARK1 enzymatic activity in TG4 heart (Fig. 3B), suggesting that ARK1 is unlikely involved in the loss of ₁-AR responsiveness. However, baseline adenylyl cyclase activity (2, 3) and cAMP level (Fig. 4A) are markedly enhanced in TG4 hearts relative to WT controls. We have therefore assumed that the constitutive ₂-AR signaling per se could increase cAMP-dependent PKA activation and enhance the basal phosphorylation of -ARs, causing -AR, particularly, ₁-AR desensitization. If this were the case, an abolition of the constitutive ₂-AR activation would be expected to resensitize ₁-ARs in TG4 cardiomyocytes by reducing the basal cAMP levels.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Effects of carbachol or ICI 118,551 incubation on basal cAMP levels and contractility. Panel A, incubation cardiac membranes with CCh (105 M) or ICI 118,551 (5 × 107 M) at 37 °C for 1 h markedly reduces the basal cAMP accumulation in TG4 but not WT (*, p < 0.01 versus WT or TG4 in the presence of CCh or ICI, n = 7 for each group). Panel B, incubation of ventricular myocytes with either CCh (105 M) or ICI (5 × 107 M) at 37 °C for 1 h significantly decreases the base-line contractility in TG4 but not WT myocytes (, p < 0.05 versus WT and TG4 in the presence of CCh or ICI, n = 12~38 cells).

M₂-Muscarinic Stimulation Reduces Basal cAMP Levels and Rescues ₁-AR Contractile Response-- It is widely recognized that stimulation of PTX-sensitive G_i proteins antagonizes -AR stimulation via, at least in part, inhibiting adenylyl cyclase activity (31). We have employed a M₂-muscarinic receptor agonist, CCh, in an attempt to activate G_i proteins and subsequently reduce basal cAMP production in TG4 hearts. Fig. 4A shows that incubation of cardiac myocytes with CCh (10⁵ M) at 37 °C for 1 h decreases the basal cellular cAMP to a level similar to that of WT control. Concomitantly, CCh incubation significantly decreases the base-line contraction amplitude of TG4 ventricular myocytes (Fig. 4B), whereas it has no effect on the base-line contractility or cAMP levels in WT mouse hearts (Fig. 4). These observations indicate that muscarinic stimulation can effectively interact with and negate the ligand-independent constitutive ₂-AR signaling. More importantly, in 10⁵ M CCh-pretreated TG4 ventricular myocytes in the continuous presence of 10⁷ M CCh, the ₁-AR agonist, NE (10^{- 7} M), plus prazosin (10⁶ M) markedly augments the contraction amplitude, as shown by the typical example in Fig. 5A and the average data in Fig. 5D. The inclusion of a low concentration of CCh is devised to tonically suppress spontaneous ₂-AR signaling with a minimal effect on ligand-elicited ₁-AR stimulation. It is noteworthy that in the very same cell, contractile response to ₂-AR stimulation by zinterol is not rescued by CCh treatment (Fig. 5A), reinforcing the idea that different mechanisms underlie the loss of -AR-subtype function in TG4 hearts. Fig. 5B shows a typical example that NE-induced positive inotropic effect is specifically abolished by the ₁-AR antagonist, CGP (3 × 10⁷ M) (Fig. 5B). In myocytes pre-treated with both CCh and PTX, the ability of CCh to restore the ₁-AR contractile response is abolished (Fig. 5C), indicating that the effect of CCh is mediated by a PTX-sensitive G protein-coupled signaling pathway. On average, the CCh-rescued maximal ₁-AR contractile response is lower than that induced by forskolin (10⁶ M) in un-treated myocytes (Fig. 5D).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Carbachol or ICI 118,551 incubation rescues the 1-AR-mediated contractile response in TG4 myocytes. Results in panels A-C were obtained in CCh-treated myocytes, in the continuous presence of 107 M CCh. Panel A shows that 1-AR stimulation with NE (107 M) plus prazosin (PRAZ, 106 M), but not 2-AR stimulation with zinterol (ZINT, 106 M), increases contraction amplitude. Panel B shows the antagonistic effect of the 1-AR blocker, CGP 20712A (3 × 107 M), on the CCh-rescued 1-AR contractile response. Panel C shows that in a representative myocyte pretreated with both CCh and PTX pretreatment, 1-AR contractile response is not restored. Panel D illustrates the average data on the contractile response induced by forskolin (FORS) or 1-AR stimulation in the presence and absence of PTX, CCh, or ICI incubation (*, p < 0.01 versus NE and NE (PTX); , p < 0.01 versus NE, NE (PTX), NE (CCh), and NE (ICI), n = 7~12 cells).

Inhibition of Spontaneous ₂-AR Activation Restores ₁-AR-mediated Contractile Response-- The aforementioned results suggest that ₁-AR stimulation in TG4 cells can be rescued by antagonizing constitutive ₂-AR signaling. To further test this idea, we incubate ventricular myocytes with the ₂-AR inverse agonist, ICI (5 × 10⁷ M), since an inverse agonist stabilizes the receptor in its inactive state and thereby abrogates the constitutive receptor signaling (2, 3). Similar to CCh, ICI also reduces the basal cAMP accumulation in TG4 heart (Fig. 4A), consistent with previous observations that ICI decreases the base-line adenylyl cyclase activity (2, 3). In ICI-treated TG4 myocytes, NE (10⁷ M) plus prazosin (10⁶ M) in the presence of ICI (10⁷ M) significantly enhances contraction amplitude (Fig. 5D). Thus, we conclude that ligand-free ₂-AR activation induces a "heterologous" receptor desensitization of ₁-AR and that the desensitized ₁-AR can be revived by either M₂-muscarinic stimulation or the ₂-AR inverse agonist ICI, both suppressing the constitutive ₂-AR activation and reducing basal cAMP production and cAMP/PKA-dependent signaling.

Rescuing Diminished ₁-AR Contractile Response in WT Myocytes Overexpressing ₂-AR-- As is true for most transgenic mouse models, a specific or even a global remodeling process accompanies the up-regulation or down-regulation of a gene or set of genes, particularly during early development (32). To determine whether potential chronic compensatory changes may complicate the interpretation of our experimental findings, we next acutely overexpress the human ₂-AR in cultured ventricular myocytes using an adenoviral gene transfer technique (24). In mouse cardiomyocytes infected by adeno-₂-AR at m.o.i. of 1000 and cultured for 24 h, radioligand binding assays revealed that ₂-AR is overexpressed by 69-fold greater relative to that of WT myocytes (Fig. 6A). The acute overexpression of ₂-AR significantly enhances basal cellular cAMP level and contraction amplitude by 4.8- and 2.5-fold, respectively (Figs. 6, B and C), indicating a robust ligand-independent constitutive ₂-AR activation. Similar to the observation in TG4 myocytes, the ₁-AR-mediated contractile effect in these adeno-₂-AR infected WT myocytes is markedly attenuated but is fully restored when the cells are pre-incubated with CCh (Fig. 6D). Thus, the results of in vitro single-cell gene manipulation corroborate the observations in TG4 myocytes, reinforcing our conclusion that spontaneous ₂-AR activation results in heterologous desensitization of ₁-AR and that suppression of the spontaneous ₂-AR signaling can restore the diminished ₁-AR signaling.

View larger version (28K): [in this window] [in a new window]   Fig. 6.   2-AR overexpression desensitizes 1-AR contractile response in cultured WT myocytes infected with adeno-2-AR. Panel A, acute overexpression of 2-AR by infecting WT mouse myocytes with adeno-2-AR at m.o.i. 1000 for 24 h (see "Experimental Procedures"). Panels B and C, 2-AR overexpression elevates basal cAMP (B) and contraction amplitude (C) in the absence of any ligand (*, p < 0.01 versus uninfected myocytes, n = 4~12). Panel D, 2-AR overexpression markedly reduces the contractile response to 1-AR stimulation by NE (107 M) plus prazosin (106 M); incubation of the infected cells with CCh (105 M) for 1 h at 37 °C completely restores the diminished 1-AR responsiveness. Base-line contraction amplitude of infected cells is reduced by CCh pretreatment to 3.34 ± 0.26% of cell rest length, which is similar to that of uninfected myocytes. Data are presented as percent of control (*, p < 0.01 versus base-line contraction amplitude, n = 8~10 for each group).

Finally, to corroborate that muscarinic receptors rather than nicotinic receptors are involved in the CCh-induced inhibition of constitutive ₂-AR signaling, we examined antagonistic effects of atropine (10¹⁰~10⁷ M), a muscarinic receptor antagonist, and hexamethonium, a nicotinic receptor antagonist, on the CCh-induced reduction in base-line contraction amplitude in mouse myocytes overexpressing ₂-AR. Atropine (10⁷ M) rapidly and fully reverses the inhibitory effect of CCh, whereas the nicotinic receptor antagonist, hexamethonium (10⁷ M), virtually has no antagonistic effect in myocytes infected with adeno-₂-AR at m.o.i. 1000 (Figs. 7, A and B). Fig. 7C shows that atropine antagonizes the effect of CCh in a dose-dependent manner, with an EC₅₀ of 3.5 × 10⁹ M. These results indicate that muscarinic receptors, but not nicotinic receptors, are involved in the inhibitory effect of CCh on constitutive ₂-AR signaling.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Effects of muscarinic and nicotinic antagonists on carbachol-induced inhibition of base-line contractility in WT myocytes infected with adeno-2-AR at m.o.i. 1000. Panel A, the muscarinic receptor antagonist, atropine (107 M), rapidly reverses the CCh-induced reduction in base-line contraction amplitude, whereas a nicotinic receptor antagonist, hexamethonium (Hex × 107 M), has no effect in a typical infected WT myocyte. The top shows a continuous recording of the cell contraction (an upward deflection indicating cell shortening). The bottom displays individual contractile traces at time points as indicated (a downward deflection indicating cell shortening). Panel B, the average data of the experiments shown in panel A (* p < 0.01 versus control (Con) and CCh + hexamethonium + atropine; n = 8 cells from 5 hearts). Panel C, the average dose-response curve of atropine to antagonize the inhibitory effect of CCh on the base-line contractility in WT mouse myocytes overexpressing 2-AR. Data are presented as % of maximal antagonistic effect of atropine (n = 6~8 cells from 5 hearts for each data point).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Using the genetically manipulated TG4 mouse model, the present and previous studies have revealed several intriguing phenomena related to interactions among receptor types or subtypes. For instance, overexpression of the human ₂-AR induces a complete loss of ₁-AR modulation of cardiac contractility and heart rate in vivo (4) and in isolated atria (3), consistent with previous observations in rat C6 glioma cells that overexpression of ₂-AR results in a loss of ₁-AR response (9). The present study at the single cell level further demonstrates that this is likely caused by the spontaneous ₂-AR activation and its concomitant cAMP/PKA-dependent ₁-AR desensitization. More importantly, these observations in TG4 myocytes are entirely reproducible in vitro by acute, single-cell gene manipulation, excluding possible complications from chronic or developmental compensatory changes in the transgenic animal model. Thus, we conclude that heterologous receptor desensitization can result from ligand-directed receptor activation and from ligand-independent constitutive receptor activation as well. Although it is well established that G_i-coupled muscarinic receptor stimulation antagonizes -AR-G_s-mediated contractile response (33-35), the principal novel finding of the present study is that activation of muscarinic receptor can resensitize heterologously desensitized ₁-AR signaling in cardiomyocytes overexpressing ₂-AR (Figs. 5 and 6). In other words, both positive and negative interactions between the adrenergic and muscarinic receptors can be demonstrated when the signaling system is predisposed differently. This observation exemplifies a principle that stimulation of a given receptor may elicit qualitatively different physiological responses in the same cell, depending on the set point of overt and "covert" parameters of the signaling circuitry. Specifically, these dual effects of muscarinic receptor stimulation appear to be related to different phases of -AR and muscarinic receptor interaction. When -AR and muscarinic receptor are simultaneously activated, muscarinic stimulation (at high agonist concentrations) negates the -AR-mediated positive inotropic effect, as reported previously in many mammalian species (31, 33-35) including mouse (24). In contrast, in mouse cardiomyocytes in which a high level of constitutive ₂-AR signaling is present, pretreatment of the cells with CCh or acetylcholine (data not shown) sensitizes ₁-AR response, presumably via reducing the basal PKA activation, thereby decreasing the basal -AR phosphorylation. A similar cross-regulation between G_i activation and the G_s-coupled -AR signaling has been demonstrated in hamster smooth muscle DDT₁MF-2 cells, in which short term (60 min) pretreatment with an A₁-adenosine agonist enhances -AR-stimulated increases in adenylyl cyclase activity (36). Taken together, these in vivo and in vitro studies emphasize the subtlety and complexity of cross-talk between -AR subtypes and between G_s- and G_i-coupled receptors.

It is noteworthy that G_i activation by CCh significantly reduces the base line of cAMP and contraction amplitude in TG4 myocytes and in cultured WT mouse myocytes overexpressing ₂-AR, whereas it has no effect in control WT myocytes (Fig. 4). These results suggest that the magnitude of the anti-adrenergic effect of muscarinic receptor stimulation is very much dependent on the existing level of the cyclase/cAMP/PKA signaling. This is consistent with previous observations that M₂ muscarinic receptor stimulation has no significant effect on base-line ventricular contraction but markedly inhibits -AR-induced positive inotropic response (33-35).

Several cellular mechanisms are involved in modulating the throughput of -AR signaling, e.g. PKA- or G protein-coupled receptor kinases-dependent receptor desensitization via receptor phosphorylation, receptor down-regulation, and other counterbalancing signaling. The failure of ₂-AR to increase contractility in TG4 heart has been explained by the coupling of the receptor to G_i proteins (5). The loss of ₁-AR contractile response in TG4 heart appears to be independent of either G_i or ARK1 activation (Figs. 2 and 3). In contrast, pre-activation of G_i proteins by CCh selectively revitalizes the ₁-AR contractile response in TG4 heart. Similarly, the desensitization of ₁-AR induced by the acute overexpression of ₂-AR in vitro is also fully rescued by CCh pretreatment. Since ₁-AR desensitization is accompanied by an enhanced cAMP accumulation, and conversely, its rescue by CCh is associated with a reduction in basal cAMP level, we conclude that the loss of ₁-AR contractile response in cells overexpressing ₂-AR is attributable largely to the cAMP/PKA-mediated heterologous receptor desensitization. Further studies are required to elucidate the exact mechanisms by which CCh and ICI rescue the desensitized ₁-AR, e.g. a reduction in basal ₁-AR phosphorylation, or a translocation of the receptors from cytoplasmic pools to sarcolemmal membrane.

Additionally, incubation cells with CCh or ICI only partially restores the ₁-AR-mediated positive inotropic effect in TG4 ventricular myocytes. The maximal contractile response induced by ₁-AR stimulation in CCh-treated cells is still lower than that induced by ₁-AR stimulation in WT myocytes (Fig. 1) or by the adenylyl cyclase activator, forskolin, in TG4 cells (Fig. 5D). In contrast, CCh fully rescues the ₁-AR signaling in WT myocytes overexpressing the human ₂-AR (Fig. 6D). These results suggest that additional unidentified mechanisms might be involved in the diminution of ₁-AR contractile response in TG4 cardiac myocytes, e.g. reduction in ₁-AR density (₁-AR down-regulation), or a dislocation between ₁-ARs and downstream signaling molecules, reducing the signal transmission efficiency. Unfortunately, a direct view of ₁-AR subcellular distribution or a measurement of the receptor density is technically impossible due to the vast overexpression of ₂-ARs and the absence of absolutely selective ₁-AR ligands.

Finally, the present findings may have important pathophysiological and therapeutic implications. In light of distinct mechanisms underlying ₁-AR and ₂-AR desensitization and their G protein coupling, the defects of ₁-AR versus ₂-AR subtype signaling in the failing heart may be mediated by different cellular pathways. Under certain circumstances, blunted -AR responsiveness is, at least in part, due to receptor desensitization. For example, the diminished ₁-AR contractile response in chronic heart failure is associated with enhanced circulating catecholamine levels (21-23). To resensitize these receptor functions, a new strategy might involve activation of G_i-coupled receptors to a critical level that is sufficient to rescue or prevent receptors from desensitization but not to directly antagonize the -AR-G_s-mediated positive inotropic effects, as demonstrated in the present study.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Laboratory of Cardiovascular Sciences, Gerontology Research Center, NIA, NIH, 5600 Nathan Shock Dr., Baltimore, Maryland 21224. E-mail: xiaor@grc.nia.nih.gov. Tel.: 410-558-8662; Fax: 410-558-8150.

Published, JBC Papers in Press, April 27, 2000, DOI 10.1074/jbc.M909484199

	ABBREVIATIONS

The abbreviations used are: ₂-AR, ₂-adrenoceptor; WT, wild type; PTX, pertussis toxin; PKA, protein kinase A; ARK1, -AR receptor kinase 1; MEM, minimal essential medium; FBS, fetal bovine serum; m.o.i., multiplicity of infection; CCh, carbachol; NE, norepinephrine.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES