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(Circulation Research. 1999;84:43-52.)
© 1999 American Heart Association, Inc.

Original Contribution

Coupling of ß₂-Adrenoceptor to G_i Proteins and Its Physiological Relevance in Murine Cardiac Myocytes

Rui-Ping Xiao, Pavel Avdonin, Ying-Ying Zhou, Heping Cheng, Shahab A. Akhter, Thomas Eschenhagen, Robert J. Lefkowitz, Walter J. Koch, Edward G. Lakatta

From the Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging (R.-P.X., Y.-Y.Z., H.C., E.G.L.), Baltimore, Md; Institute of Developmental Biology (P.A.), Russian Academy of Sciences, Moscow, Russia; Department of Surgery (S.A.A., W.J.K.), Department of Medicine and Howard Hughes Medical Institute (R.J.L.), Duke University Medical Center, Durham, NC; and Abteilung Allgemeine Pharmakologie, Universitats-Krankenhaus Eppendorf (T.E.), Hamburg, Germany.

Correspondence to Rui-Ping Xiao, MD, PhD, Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, 5600 Nathan Shock Dr, Baltimore, MD 21224. E-mail Xiaor{at}GRC.NIA.NIH.Gov

	Abstract

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Abstract—Transgenic mouse models have been developed to manipulate ß-adrenergic receptor (ßAR) signal transduction. Although several of these models have altered ßAR subtypes, the specific functional sequelae of ßAR stimulation in murine heart, particularly those of ß₂-adrenergic receptor (ß₂AR) stimulation, have not been characterized. In the present study, we investigated effects of ß₂AR stimulation on contraction, [Ca²⁺]_i transient, and L-type Ca²⁺ currents (I_Ca) in single ventricular myocytes isolated from transgenic mice overexpressing human ß₂AR (TG4 mice) and wild-type (WT) littermates. Baseline contractility of TG4 heart cells was increased by 3-fold relative to WT controls as a result of the presence of spontaneous ß₂AR activation. In contrast, ß₂AR stimulation by zinterol or isoproterenol plus a selective ß₁-adrenergic receptor (ß₁AR) antagonist CGP 20712A failed to enhance the contractility in TG4 myocytes, and more surprisingly, ß₂AR stimulation was also ineffective in increasing contractility in WT myocytes. Pertussis toxin (PTX) treatment fully rescued the I_Ca, [Ca²⁺]_i, and contractile responses to ß₂AR agonists in both WT and TG4 cells. The PTX-rescued murine cardiac ß₂AR response is mediated by cAMP-dependent mechanisms, because it was totally blocked by the inhibitory cAMP analog Rp-cAMPS. These results suggest that PTX-sensitive G proteins are responsible for the unresponsiveness of mouse heart to agonist-induced ß₂AR stimulation. This was further corroborated by an increased incorporation of the photoreactive GTP analog [-³²P]GTP azidoanilide into subunits of G_i2 and G_i3 after ß₂AR stimulation by zinterol or isoproterenol plus the ß₁AR blocker CGP 20712A. This effect to activate G_i proteins was abolished by a selective ß₂AR blocker ICI 118,551 or by PTX treatment. Thus, we conclude that (1) ß₂ARs in murine cardiac myocytes couple to concurrent G_s and G_i signaling, resulting in null inotropic response, unless the G_i signaling is inhibited; (2) as a special case, the lack of cardiac contractile response to ß₂AR agonists in TG4 mice is not due to a saturation of cell contractility or of the cAMP signaling cascade but rather to an activation of ß₂AR-coupled G_i proteins; and (3) spontaneous ß₂AR activation may differ from agonist-stimulated ß₂AR signaling.

Key Words: ß₂-adrenergic receptor • inhibitory G protein • cardiac contractility • L-type Ca²⁺ current • mice, transgenic

	Introduction

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Current opinion suggests that gene therapy may hold great promise for treatment of cardiovascular diseases that lead to chronic heart failure. The assimilation of rapid advances in mouse genetics into the realm of cardiovascular research has provided pharmacologists and physiologists a tremendous range of new opportunities to unravel the molecular secrets that govern cardiovascular structure and function in health and disease. Several lines of transgenic mice have been generated to target key proteins that govern transmembrane signal transduction or modulate the contractile properties of myocardial cell.^{1 2 3 4 5 6 7 8 9} One such model that has drawn substantial attention is a transgenic mouse model overexpressing the human ß₂-adrenergic receptor (ß₂AR) in a cardiac specific manner (TG4). In this model, while baseline myocardial contractility is successfully enhanced relative to wild-type (WT) littermates,¹ cardiac responsiveness to an acute administration of the ß-adrenergic receptor (ßAR) agonist isoproterenol (ISO) is totally lacking both in vivo and in isolated atria.^{1 7} These observations have led to a conclusion that ß-adrenergic modulation in the TG4 mouse heart is saturated in the basal state because of a greater number of ß₂ARs in the spontaneous active state (R* state) in the absence of agonists.^{1 7}

Because recent studies in other mammalian species have shown that physiological responses and signal transduction mechanisms of ß₂AR subtype stimulation are distinctly different from those of ß₁-adrenergic receptor (ß₁AR) stimulation,^{10 11 12 13 14 15} it is essential to characterize the individual ßAR subtypes in murine heart for optimal genetic manipulation of their signaling pathways. However, a close inspection of the studies in murine models to date^{1 2 3 6 7 8} surprisingly reveals that although the contractile effects of mixed ßAR or ß₁AR stimulation have been characterized, the functionality of the ß₂AR subtype at the cellular level has been ignored. In fact, whether ß₂AR agonists can elicit a contractile response in murine myocardium has been a matter of debate. Specifically, in WT mice, the mixed ßAR agonist ISO in the presence of the ß₁AR antagonist CGP 20712A (CGP) has virtually no positive inotropic effect.⁷ In addition, in ß₁AR knockout mice, the mixed ßAR agonist ISO fails to increase cardiac contractility.⁹ Thus, it is possible that an inability of ß₂AR stimulation by agonists to increase contractility in mouse heart per se masquerades as the observed "saturation" of ß₂-adrenergic signaling in the TG4 mouse.

Our previous studies have shown that in native rat cardiac myocytes, pertussis toxin (PTX) pretreatment selectively potentiates the positive inotropic effect of ß₂AR but not ß₁AR stimulation, suggesting that ß₂AR dually couples to G_s and to PTX-sensitive inhibitory G proteins.¹⁰ If the coupling of ß₂AR-G_i protein in murine heart were highly efficient, it might be expected to completely negate the G_s-mediated positive inotropic effect. Thus, a strong coupling of ß₂AR to G_i proteins in murine heart may explain the apparent and mysterious loss of ß₂AR contractile response in murine myocardium.

The present study was undertaken to characterize the effects of agonist-induced ß₂AR subtype stimulation on contraction, [Ca²⁺]_i transient, L-type Ca²⁺ currents (I_Ca), and activation of G proteins in both TG4 and WT ventricular myocytes. Surprisingly, ß₂AR stimulation by ISO plus the ß₁AR antagonist CGP or by zinterol was not able to enhance the contraction amplitude in either WT or TG4 myocytes. An analysis of the G protein activation profile indicated that ß₂AR stimulation in both WT and TG4 mice activated PTX-sensitive G proteins (G_i2 and G_i3) in addition to G_s. Pretreatment of myocytes with PTX rescued potent contractile, [Ca²⁺]_i, and I_Ca responses to ß₂AR agonists in WT as well as TG4 heart cells. These results indicate that in mouse ventricular myocytes, at normal or overexpressed receptor density, an activation of ß₂AR-coupled G_i proteins prevents the positive inotropic effect of agonist-induced ß₂AR stimulation.

	Materials and Methods

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Measurements of Cell Contraction and [Ca²⁺]_i Transient
Mouse ventricular myocytes were isolated from hearts of 2- to 3-month-old male transgenic mice overexpressing human ß₂AR (TG4) and WT littermates via a modified enzymatic technique.⁸ Cells were then perfused with HEPES buffer solution consisting of (in mmol/L) CaCl₂ 1.0, NaCl 137, KCl 5, dextrose 15, MgSO₄ 1.3, NaH₂PO₄ 1.2, and HEPES 20 (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 after electrical stimulation. In some experiments, cells were loaded with the fluorescent Ca²⁺ indicator Fluo-3 by incubation in 10 µmol/L Fluo-3 AM (Molecular Probes) for 10 minutes followed by a 20-minute wash.¹⁶ A laser scanning confocal microscope (Zeiss LSM410) was used to acquire fluorescence images every 2.09 ms along a line focused 5 to 10 µm into the cell. Both the Ca²⁺ signal and cell contraction were directly measured from the line-scan images using IDL (Research System) software. Criteria for viable mouse myocytes have been described in our previous study⁸ and include (1) a rod shape, (2) clearly defined sarcomeric striations, (3) a clear negative staircase after a rest period of 1.0 minute, and (4) a stable steady-state contraction amplitude for at least 5 minutes before drug administration.

I_Ca Measurements
I_Ca was measured via the whole-cell patch-clamp technique with the use of an Axopatch 1D amplifier (Axon Instruments Ltd). The resistance of electrode pipettes fabricated from the glass capillary tube (World Precision Instruments, Inc) ranged between 1 and 2 M. To selectively examine I_Ca, cells were voltage-clamped at -40 mV to inactivate the Na⁺ current and T-type Ca²⁺ current. The K⁺ currents were inhibited by the appropriate blockers in the pipette solution containing (in mmol/L) CsCl 100, TEACl 20, NaCl 10, HEPES 10, MgATP 5, and EGTA 5; the pH was adjusted to 7.2 with CsOH. In some experiments, Rp-cAMPS (100 µmol/L), an inhibitory cAMP analog, was included in the patch pipette solution and dialyzed into the cell for more than 10 minutes, as previously described.¹¹ The superfusion solution was the same as that used for cell length and [Ca²⁺]_i transient measurements. I_Ca was elicited from a depolarization from -40 to 0 mV and measured as the difference between the peak inward current and the current at the end of a 300-ms pulse.

Photolabeling of Membrane Proteins
Cardiac membranes were prepared by homogenizing WT and ß₂AR overexpressing transgenic mouse (TG4) ventricles in ice-cold lysis buffer (20 mmol/L Tris-HCl [pH 7.4], 250 mmol/L sucrose, 1 mmol/L EDTA, 10 µg/mL aprotinin, 10 µg/mL leupeptin, and 0.1 mmol/L PMSF). The samples were centrifuged at 10 000g for 10 minutes at 4°C. The supernatant was centrifuged at 100 000g for 2 hours at 4°C. The pellet was resuspended up to a final protein concentration of 4.5 to 5 mg/mL in a buffer containing 20 mmol/L Tris-HCl (pH 7.4) and 1 mmol/L EDTA. Membranes were aliquoted and stored at -80°C.

[-³²P]GTP-azidoanilide ([-³²P]GTP-AzA) was synthesized and purified according to the procedure described previously¹⁷ with some modifications. Briefly, 100 µL of 30 mg/mL 1-ethyl-3-(3-dimethylaminopropyl)carbodiide (N-DEC) (Fluka, Buchs, Switzerland) solution in 0.15 mol/L MES (pH 5.5) and 2 to 3 mCi of lyophilized [-³²P]-GTP were mixed for 10 minutes at room temperature. Then 50 µL of 4-azidoanilide (40 mg/mL in 1,4-dioxane) was added to this mixture and kept at 25°C to 28°C for 3 hours with constant mixing. The synthesized [-³²P]GTP-AzA was purified on a C-18 Sep-Pak Cartridge (Waters) and dried on a Speed-Vac Centrifuge. The purity of the final product was >90%, checked by thin-layer chromatography on PEI cellulose with 1 mol/L LiCl. The dried [-³²P]GTP-AzA was stored at -25°C. All synthesis procedures were performed in a dark room with red light illumination.

Membrane proteins (40 to 50 µg) were preincubated at 25°C for 10 minutes in 20 mmol/L Tris-HCl (pH 7.5), 50 mmol/L NaCH₃CO₂, 0.2 mmol/L EGTA, 1.0 mmol/L benzamidine, 2 mmol/L Mg Cl₂, 1.0 mmol/L EDTA, and 50 µmol/L GDP to load G protein subunits. ßAR agonists or antagonists and 5 to 10 µCi of [-³²P]GTP-Aza were then added to the samples and incubated for 4 minutes. The reaction was terminated by putting the samples on ice. All the subsequent procedures were performed at 4°C. After centrifugation (14 000g for 10 minutes), membrane pellets were carefully resuspended in 50 µL of ice-cold buffer (20 mmol/L Tris-HCl [pH 7.4], 1 mmol/L EDTA, and 1 mmol/L dithiothreitol), transferred to individual dimples in aluminum foil, and irradiated with a UV lamp (254 nm, 100 W) for 10 minutes at a distance of 10 cm. The irradiated samples were centrifuged at 14 000g for 30 minutes.

Immunoprecipitation of G Protein Subunits
Immunoprecipitation of G protein subunits was performed as previously described.¹⁸ Pellets of photolabeled membranes were solubilized in 40 µL of 2% SDS (wt/vol) at room temperature. Precipitation buffer (103 µL) containing 1% (wt/vol) Triton X-100, 1% (wt/vol) deoxycholate, 0.5% (wt/vol) SDS, 150 mmol/L NaCl, 1 mmol/L dithiothreitol, 1 mmol/L EDTA, 0.2 mmol/L PMSF, 10 µg/mL aprotinin, and 10 mmol/L Tris-Cl (pH 7.4) was added, and the solubilized membranes were centrifuged at 14 000g for 5 minutes at 4°C. Antisera (5 to 20 µL) was added to the supernatant. The samples were incubated overnight at 4°C under constant rotation. After adding washed protein A Sepharose beads, the samples were centrifuged at 14 000g for 5 minutes and washed with buffer A (1% [wt/vol] Igepal, 0.5% [wt/vol] SDS, 600 mmol/L NaCl, and 50 mmol/L Tris-HCl [pH 7.4]) and buffer B (300 mmol/L NaCl, 10 mmol/L EDTA, and 100 mmol/L Tris-HCl [pH 7.4]). The pellets of protein A Sepharose were dried with a Speed-Vac centrifuge. After a 15-minute incubation at room temperature, the samples were boiled for 10 minutes and centrifuged at 14 000g for 5 minutes. Thereafter, 20 µL of supernatants was subjected to SDS-PAGE electrophoresis according to Laemmli.¹⁹ The separating gel contained 9% acrylamide and 6 mol/L urea. Gels were stained with Coomassie blue. Photolabeled proteins were visualized by autoradiography.

PTX Treatment
For contraction, [Ca²⁺]_i transient, and I_Ca measurements, aliquots of cells were incubated with PTX (1.5 µg/mL at 37°C for at least 3 hours), as previously described.¹⁰ PTX-treated cells were compared with nontreated control myocytes from the same heart that had been kept at 37°C in the absence of PTX for an equal time. After PTX treatment, both PTX-treated and nontreated cells were kept at room temperature for the rest of the experimental day (6 to 8 hours). For biochemical measurements, mice were injected with PTX (150 µg/kg IP) 24 hours before the isolation of the hearts.²⁰

Materials
CGP was kindly supplied by Ciba-Geigy Corp, Basel, Switzerland; ICI 118,551 (ICI) was kindly supplied by Imperial Chemical Industries, London, UK; and zinterol was kindly supplied by Bristol-Myers, Evansville, Ind. Antibodies recognizing the subunits of G_s and G_i2 were obtained from Du Pont New England Nuclear (Wilmington, Del). The antibody recognizing the subunits of G_i3 was obtained from Santa Cruz Biotechnology, Calif. In our experiments, the antibodies against G_i3 (from Santa Cruz) dominantly react with G_i3, because in most experiments, the molecular weight (MW) of the G_i3 antibody-precipitated proteins is slightly greater than that precipitated by the G_i2 antibodies, as expected. However, these antibodies may also slightly cross-react with G_i2. In some experiments, double bands are visible, but the lower MW band, which has the same MW as that of the proteins precipitated by G_i2 antibodies (Figure 8C and 8D), is always much lighter. In addition, the G_i2 antibody-precipitated proteins are mainly G_i2, even though this antibody may cross-react weakly with G_i1. The reason for this is that in our preliminary experiments, we have found that the abundance of G_i1 in murine myocardium is much lower than that of G_i2 and G_i3 and is difficult to detect by Western analysis (data not shown). Control peptides of the G_i3 antibody were obtained from Santa Cruz. PTX, forskolin, ISO, and norepinephrine (NE) were purchased from Sigma, St. Louis, Mo. Rp-cAMPS was purchased from Biolog Life Science Institute, La Jolla, Calif.

View larger version (36K): [in this window] [in a new window]   Figure 8. Effects of ß2AR stimulation by zinterol or isoproterenol plus the ß1AR blocker CGP 20712A on the incorporation of the photoreactive GTP analog [-32P]GTP-AzA into subunits of Gi proteins. Membranes prepared from WT (A and B) or TG4 (C through F) mouse ventricles were photolabeled with [-32P]GTP-AzA. Photolabeled subunits were subsequently incubated with Gi3 or Gi2 polyclonal antibodies (anti-rabbit) and immunoprecipitated. Precipitated proteins were subjected to SDS-PAGE. Activated G protein subunits were visualized by autoradiography. A, Stimulatory effect of zinterol (Z) at 10-5 mol/L and the acetylcholine receptor agonist carbachol (cch, 10-5 mol/L) on the incorporation of [32P]GTP-AzA into subunits of Gi3. B, Photolabeling of subunits of Gi3 in the absence and presence of zinterol (Z). The right 2 lanes show that control peptides of Gi3 antibody at a concentration of 5-fold greater than that of Gi3 antibody completely abolished the Gi3 antibody-induced protein precipitation. C and D, Photolabeling of subunits of Gi3 and Gi2 in response to zinterol (Z) in the absence or presence of the ß2AR blocker ICI 118,551 (I) at 10-6 mol/L. E, Stimulatory effect of isoproterenol (IS) at 10-6 mol/L in the presence or absence of a selective ß2AR antagonist ICI (10-6 mol/L) or ß1AR blocker CGP (10-6 mol/L) on the photolabeling of subunits of Gi2. F, PTX treatment prevents the stimulatory effect of zinterol (Z) on Gi2 activation. In addition, in all experiments, preimmune antisera did not precipitate any photolabeled proteins, indicating that precipitation with the antisera used in the present study was specific (data not shown). Molecular masses (kDa) of standard proteins are shown on the left.

Statistics
Data reported are mean±SEM. Statistical comparisons were made by Student t test or paired t test when appropriate. Two-factor ANOVA was used to analyze the overall drug dose response. The significance between groups is analyzed by Bonferroni. A P value of <0.05 was considered to be statistically significant.

	Results

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Enhancement of Baseline Contractility of TG4 Ventricular Myocytes
In single ventricular myocytes isolated from TG4 or WT mice superfused with normal HEPES buffer solution with 1.0 mmol/L Ca²⁺, basal contractility was measured in the absence of any ßAR agonists. Figure 1 shows that baseline contraction amplitude of TG4 cells was enhanced by 3.2-fold relative to myocytes isolated from WT mice. The enhanced baseline contractility was markedly reduced by ICI (5x10^-7 mol/L), a ß₂AR inverse agonist (a class of receptor ligands that preferentially bind to inactive receptor, therefore driving the equilibrium between R and R* to the inactive conformation R), whereas ICI alone had no significant effect on WT cell basal contraction (Figure 1). However, there was no significant difference in the resting cell length between these 2 groups (144.5±5.6 µm, n=28 for TG4 versus 141.0±4.18 µm, n=22 for WT), consistent with the previous observations that the heart size is not changed in this transgenic model.¹

View larger version (12K): [in this window] [in a new window]   Figure 1. Baseline contraction amplitude of isolated, single WT and TG4 mouse ventricular myocytes in the absence and presence of the ß2AR inverse agonist ICI 118,551 (ICI, 5x10-7 mol/L). The baseline contraction of TG4 myocytes (n=22 cells from 8 hearts) was significantly greater than that of WT myocytes (n=28 from 8 hearts). *P<0.01. However, in the presence of the inverse ß2AR agonist ICI (5x10-7 mol/L), there was no significant difference between these 2 groups (n=9 cells for both groups).

ß₂AR Agonists Fail to Increase Contractility of Either TG4 or WT Ventricular Myocytes
Despite the overwhelming expression (200-fold of WT) of ß₂ARs in TG4 myocytes,¹ the selective ß₂AR agonist zinterol, even at a maximal concentration (10^-5 mol/L), was unable to further augment contraction amplitude (Figure 2A) or the [Ca²⁺]_i transient (not shown). The inability of ß₂AR stimulation to further increase contraction amplitude in TG4 myocytes might suggest that the ß₂AR signaling to augment cell contractility in TG4 cells is already at the maximal level in the absence of agonist so that ß₂AR agonists would not be expected to further increase the contraction amplitude in these cells, as proposed previously.^{1 7} Alternatively, the unresponsiveness of TG4 cells might be due to some compensatory alterations, eg, a reduction in G_s-adenylyl cyclase signaling, or to a defect in excitation-contraction coupling machinery in these transgenic mice. The adenylyl cyclase activator forskolin was used to test these possibilities. If the contractility in TG4 heart cells were saturated at baseline, no positive inotropic effect would be observed after forskolin treatment. To the contrary, Figure 2B illustrates that forskolin (10^-6 mol/L) markedly and reversibly enhanced contraction amplitude in a representative TG4 ventricular myocyte. On average, forskolin increased TG4 cellular contraction by 2.4-fold (from 6.1±1.0% to 14.4±1.0% of resting cell length, n=5 cells from 3 hearts; P<0.01). This result indicates that both the excitation-contraction machinery and the ßAR signaling cascade downstream of the cyclase remain intact and are not saturated at baseline in TG4 mice. Therefore, we hypothesized that the unresponsiveness of TG4 ventricular myocytes to ß₂AR stimulation likely results from an impairment within the proximal ß₂AR signaling cascade.

View larger version (20K): [in this window] [in a new window]   Figure 2. Representative examples of contractile response to the selective ß2AR agonist zinterol (ZINT) (A) or to the adenylyl cyclase stimulator forskolin (B) in TG4 ventricular myocytes. Each panel shows a typical continuous chart recording of cell contraction (top, upward deflection) and traces on an expanded time scale (bottom; contraction is plotted as downward deflection) obtained at time points as indicated. Note that zinterol at 10-5 mol/L failed to elicit a positive inotropic effect, whereas forskolin (10-6 mol/L) markedly enhanced the contraction amplitude in TG4 cardiomyocytes.

To identify possible alterations of cardiac ß₂AR signaling in TG4 mice, we next examined the effects of ß₂AR stimulation in WT heart cells. Surprisingly, ß₂AR stimulation by the mixed ßAR agonist ISO (10^-6 mol/L) plus the ß₁AR blocker CGP was also unable to augment contraction amplitude in WT mouse ventricular myocytes (Figure 3). In contrast, the mixed ßAR stimulation by ISO alone or ß₁AR stimulation by ISO plus the ß₂AR blocker ICI markedly enhanced contraction amplitude in these WT myocytes (Figure 3), consistent with previous in vivo observations that ISO in WT mice enhances cardiac performance.^{1 7 21} Thus, the positive inotropic effect of ISO in WT cardiac myocytes or in vivo^{1 7 21} is largely, if not exclusively, mediated by ß₁AR subtype stimulation. Furthermore, as previously shown,⁸ the ß₁AR agonist NE plus the ₁-adrenergic blocker prazosin (10^-6 mol/L) produces a marked increase in contraction amplitude in these cells (Figure 4D). Again, the ß₂AR selective agonist zinterol at any concentration tested up to 10^-5 mol/L failed to enhance contraction amplitude in WT mouse cardiomyocytes (Figure 5A), despite the fact that ß₂ARs constitute about 24% of the total ßARs in WT mouse heart.¹ Taken together, the results so far indicate that ß₂AR stimulation induced by either zinterol or by ISO plus ß₁AR blockade was unable to augment contraction in either WT or TG4 mouse cardiomyocytes, whereas ß₁AR agonists or adenylyl cyclase activators potently augmented the contraction amplitude in these cells. The next question, then, is why ß₂AR stimulation cannot increase mouse cardiac contractility.

View larger version (16K): [in this window] [in a new window]   Figure 3. Isoproterenol (ISO)-induced contractile response in the absence and presence of the selective ß1AR antagonist CGP 20712A (CGP, 10-8 mol/L) or the selective ß2AR antagonist ICI 118,551 (ICI, 10-7 mol/L) in WT mouse ventricular myocytes. Note that even in WT myocytes, ß2AR stimulation by ISO+CGP has no significant effect on the contraction amplitude (n=5 to 8). *P<0.01 vs control and ISO+CGP groups.

View larger version (17K): [in this window] [in a new window]   Figure 4. Representative examples of continuous recording of cell contraction (upward deflections). A and B, In PTX-treated TG4 (A) and WT (B) mouse ventricular myocytes, zinterol (ZINT, 10-6 mol/L) markedly increased contraction amplitude, which was reversed by the selective ß2AR antagonist ICI 118,551 (ICI, 10-7 mol/L). C and D, In representative WT myocytes, the ß1AR antagonist CGP 20712A (CGP, 10-8 mol/L) could not reverse the PTX-rescued positive inotropic effect of zinterol (C), but it completely abolished the contractile response to the ß1AR agonist norepinephrine (NE, 10-7 mol/L) plus the 1-adrenergic blocker prazosin (10-6 mol/L) (D).

View larger version (16K): [in this window] [in a new window]   Figure 5. Dose response of contraction amplitude to the ß2AR agonist zinterol in WT (A) and TG4 (B) mouse ventricular myocytes with and without PTX treatment. Each cell was superfused with a single concentration of the ß2AR agonist zinterol. All measurements were obtained under steady-state conditions after 10 minutes of exposure to zinterol and are presented as mean±SEM (at each concentration, n=5 to 6). The overall drug effect (in both PTX-treated WT and TG4 cells) is significant at P<0.001 (by 2-way ANOVA). There is a significant difference between +PTX and -PTX groups (by Bonferroni, P<0.001 in both WT and TG4 groups).

Rescue of Contractile and [Ca²⁺]_i Responses to ß₂AR Stimulation by PTX Treatment
Because reconstituted ßARs can couple to both G_s and G_i in artificial systems^{22 23} and in rat ventricular myocytes, PTX treatment selectively potentiates the ß₂AR-mediated contractile response,^{10 11 24} we hypothesized that a dual coupling of ß₂AR to an inhibitory G protein in addition to a G_s protein might also exist in intact mouse ventricular myocytes and negate the contractile response mediated by the coupling to a G_s protein. To test this hypothesis, cells were incubated with PTX to abrogate G_i/G_o function via ADP ribosylation. Indeed, PTX pretreatment unmasked a potent positive inotropic effect after ß₂AR stimulation in both TG4 and WT heart cells, as shown in Figure 4A and 4B, in a representative PTX-treated TG4 and WT ventricular myocyte, respectively. The zinterol-induced (10^-6 mol/L) increase in contraction amplitude was completely abolished by the specific ß₂AR antagonist ICI (10^-7 mol/L). In contrast, the ß₁AR antagonist CGP (10^-8 mol/L) could not reverse the positive inotropic effect of zinterol (Figure 4C) but completely blocked the increase in contraction induced by the ß₁AR agonist NE (10^-7 mol/L) (Figure 4D). These results indicate that the PTX-rescued contractile response to zinterol is mediated by ß₂AR stimulation. The average dose response of contraction amplitude to the ß₂AR agonist zinterol is shown in Figure 5A and 5B for WT and TG4 cells, respectively. It is noteworthy that similar maximal contraction amplitude (15% of resting cell length) is obtained after zinterol in both PTX-treated WT and TG4 cells. Also note that the dose-response curve for PTX-treated TG4 cells is shifted leftward relative to that for WT cells (EC₅₀ is 1.5x10^-8 and 10^-7 mol/L for TG4 and WT groups, respectively), consistent with the greater ß₂AR density in TG4 cells. In addition, Figure 6 shows that the positive inotropic effect of zinterol in both PTX-treated TG4 and WT cells was accompanied by an increase in the [Ca²⁺]_i transient as indexed by the increase in the fluorescence signal of the Ca²⁺-sensitive probe Fluo-3. Thus, the full efficacy of ß₂AR stimulation is revealed in TG4 as well as in WT mouse cardiac cells only if cells were pretreated with PTX to eliminate the G_i-mediated inhibitory signaling.

View larger version (21K): [in this window] [in a new window]   Figure 6. Effect of the selective ß2AR agonist zinterol (ZINT, 10-6 mol/L) on the simultaneously recorded contraction and [Ca2+]i transient in PTX-treated TG4 and WT mouse ventricular myocytes. A and B, Typical examples of the effects of zinterol on [Ca2+]i transient (top, indexed by fold increase of Fluo-3 fluorescence [F/Fo]) and contraction amplitude (bottom) in PTX-treated WT and TG4 heart cells, respectively. Traces obtained before application of zinterol are indicated as control. C and D, Mean data for Ca2+ transient and contraction amplitude, respectively, before and after zinterol (n=8, *P<0.01, zinterol vs control; P<0.01, TG4 vs WT).

Rp-cAMPS Reversed the PTX-Rescued I_Ca Response to ß₂AR Stimulation
Because I_Ca is the key factor of the ß₂AR-mediated positive inotropic effect in rat and canine myocytes,^{10 11 12 14} we next measured the I_Ca response of mouse cardiomyocytes to ß₂AR stimulation. Similar to the contractile response, in the absence of PTX treatment, the ß₂AR agonist zinterol (10^-5 mol/L) could not increase I_Ca in WT or TG4 myocytes (data not shown). However, in PTX-treated cells, zinterol significantly enhanced the current amplitude in both WT and TG4 mice (Figure 7, left panels). The PTX-restored stimulatory effect of zinterol on I_Ca was completely abolished by a specific cAMP-dependent protein kinase A (PKA) inhibitor, an inhibitory cAMP analog Rp-cAMPS (Figure 7, right panels), consistent with previous observations in other species.¹¹ Similar results were obtained from the other 4 WT and TG myocytes. These results suggest that the PTX-rescued murine cardiac ß₂AR function is mediated by a cAMP-dependent signaling pathway.

View larger version (14K): [in this window] [in a new window]   Figure 7. Inhibition of PTX-rescued ICa response to ß2AR stimulation by the specific PKA inhibitor Rp-cAMPS in both TG4 and WT myocytes. Left panels, Superimposed traces of ICa in the absence (con, thin line) and presence (thick line) of the ß2AR agonist zinterol (zint, 10-6 mol/L). Right panels, Results obtained in the presence of the PKA inhibitor Rp-cAMPS (100 µmol/L included in pipette solution).

ß₂AR Stimulation Selectively Increases G_i Activation
The PTX sensitivity of the ß₂AR effect in murine (Figure 5) and rat hearts^{10 11 24} suggest that cardiac ß₂AR couples to G_i proteins. However, these results neither prove a direct interaction of ß₂AR and G_i proteins nor identify which specific G proteins couple to ß₂AR. Theoretically, the effect of PTX could be the consequence of disruption of tonic inhibitory actions of G_i proteins. To determine the interaction of ß₂AR and G_i proteins directly and to identify which specific PTX-sensitive G proteins are involved, we measured the G protein activation by photoaffinity labeling subunits of G proteins with the photoreactive GTP analog [-³²P]GTP-AzA.¹⁷ As the binding of an agonist to G protein-coupled receptors increases the rate of exchange of GTP for GDP on G protein subunits (see Reference 25²⁵ for a review), the magnitude to which [-³²P]GTP-AzA incorporates into subunits of G proteins affords a direct assessment of G protein activation in response to receptor stimulation. Subsequent precipitation with specific antisera was carried out to determine which specific G protein(s) was activated during ß₂AR stimulation.

As expected, the incorporation of [-³²P]GTP-AzA into subunits of G_s was clearly increased in WT mouse cardiac membranes after ß₂AR stimulation by zinterol. On average, the signal was enhanced by 1.5-fold (157.66±21.15% of control, n=7; P<0.05) in response to zinterol. Meanwhile, the ß₂AR agonist zinterol increased the incorporation of [-³²P]GTP-AzA into the subunits of G_i3 and G_i2 (Figure 8A through 8D), without affecting the incorporation into G_o or G_i1 proteins (data not shown). On average, zinterol (10^-5 mol/L) increased the incorporation of [-³²P]GTP-AzA into subunits of G_i2 and G_i3 to 146.3±8.8% of control (P<0.01, n=12) and 148.9±5.6% of control (P<0.01, n =13), respectively, in TG4 cardiomyocytes (Figure 9A). Similar results were obtained from WT mouse myocardium (Figure 9B). The magnitude of the ß₂AR-induced increases in the subunits of G_i3 and G_i2 photolabeling is similar to that induced by the muscarinic acetylcholine receptor agonist carbachol (10^-5 mol/L) (Figures 8A and 9B). The stimulatory effect of the ß₂AR agonist was specifically and significantly abolished by the ß₂AR antagonist ICI (Figures 8C, 8D, and 9A). Furthermore, the nonselective ßAR agonist ISO also clearly enhanced the incorporation of [-³²P]GTP-AzA into subunits of both G_i2 and G_i3 (Figures 8E and 9A), and this activation was specifically abolished by the ß₂AR antagonist ICI but not by the selective ß₁AR antagonist CGP (Figures 8E and 9A). Similarly, the ß₁AR agonist NE (10^-6 mol/L) had no significant effects on G_i activation (Figure 9B). Thus, the G_i coupling is specific for ß₂AR in both WT and TG4 myocardium. Finally, the ß₂AR-stimulated G_i activation was prevented by PTX treatment in both TG4 and WT mice (Figures 8F and 9B). Taken together, the present biochemical data, in conjunction with the physiological data described above, provide direct and compelling evidence that ß₂ARs but not ß₁ARs in native myocardium couple to PTX-sensitive G proteins G_i2 and G_i3.

View larger version (34K): [in this window] [in a new window]   Figure 9. Mean data of Gi protein photolabeling. Data are presented as percent of control (mean±SEM). A, Data obtained from TG4 mice. *P<0.01, P<0.05 vs control; n=12 and 13 for Gi2 (ZINT) and Gi3 (ZINT), respectively; n=3 to 5 for all other groups. B, Data from WT mice. *P<0.01 vs control; n=8 and 16 for Gi2 (ZINT) and Gi3 (ZINT), respectively; n=3 to 5 for all other groups.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Concurrent Coupling of ß₂AR to G_i Proteins Negates the ß₂AR-G_s–Mediated Contractile Response
Using a photoaffinity labeling technique in conjunction with specific antibodies of different G proteins, we found that ß₂AR stimulation increases activation of G_i proteins G_i2 and G_i3 (Figures 8 and 9), in a PTX- and ICI-sensitive manner, in addition to activation of G_s. These data provide the first direct biochemical evidence that ß₂ARs in the native cellular environments can interact with PTX-sensitive G proteins, specifically, G_i2 and G_i3. Physiological data showed that the concurrent coupling to G_i proteins completely negates the ß₂AR-G_s–mediated contractile, [Ca²⁺]_i, and I_Ca responses. As a result, ß₂ARs in murine hearts appear to be at an apparently dormant state in terms of these cardiac responses. More importantly, the cardiac ß₂AR function was fully restored after inhibiting G_i by PTX, as manifested by the robust effects of the ß₂AR agonist zinterol to augment [Ca²⁺]_i, I_Ca, and contraction amplitudes in both PTX-treated TG4 and WT cells. An examination of the dose responses of contraction with and without PTX treatment in both TG4 and WT myocytes further reveals that the ß₂AR-G_i coupling completely negates the G_s-mediated contractile response over a wide range of receptor densities and agonist concentrations. Thus, the concurrent coupling of ß₂ARs to G_i proteins provides an explanation for the "mysterious" loss of agonist-induced ß₂AR contractile response in TG4 and WT murine hearts.^{1 7 21} In addition, similar reasoning may also be applicable to explain the unresponsiveness of cardiac contractility to ß₂AR stimulation in the ß₁AR "knockout" mouse model,⁹ as well as in myocardium of other mammalian species (eg, guinea pig), in which ß₂ARs are present but nonfunctional in terms of cardiac contractile modulation.^{26 27}

PTX-Rescued Murine Cardiac ß₂AR Function Requires cAMP-Dependent PKA Activation
There is plenty of evidence indicating a coupling of ß₂AR to adenylyl cyclase to increase cAMP.^{13 28 29} In the present study and previous studies, the specific cAMP inhibitory analog Rp-cAMPS prevented the ß₂AR-stimulated increase in I_Ca in PTX-treated TG4 and WT myocytes (Figure 7) and in rat heart cells (with or without PTX),¹¹ indicating that cAMP-dependent PKA activation is obligatory for mammalian ß₂AR cardiac responses. Thus, the positive inotropic effects induced by both ß₁AR and ß₂AR subtypes are mediated by cAMP-PKA signaling pathways.^{11 30} However, ß₁AR does not behave the same way as ß₂AR with respect to coupling to G_i proteins under our experimental conditions (Figures 8 and 9). Similarly, our previous studies have shown that in rat ventricular myocytes, PTX pretreatment selectively enhances the positive inotropic effect of ß₂AR stimulation,^{10 11 24} suggesting that PTX-sensitive G_i-proteins specifically interact with ß₂AR but not ß₁AR. Therefore, the essential difference between ß₁AR- and ß₂AR-mediated cardiac responses is largely due to the additional coupling of ß₂AR to G_i proteins, which provides a negative feedback to the ß₂AR-stimulated cAMP-dependent signaling.

Dual Coupling of ß₂AR to G_i Proteins Mediates the Difference Between ßAR Subtypes and the Species-Dependent Diversity in Cardiac ß₂AR Responses
The coupling of ß₂AR to G_i proteins is not unique to native murine ß₂AR or to human ß₂AR surrogated in mouse cardiac myocytes. Our previous studies have shown that although stimulation of both ß₁AR and ß₂AR increases the contraction amplitude in rat and canine ventricular myocytes, numerous differences have been noted. Specifically, the ß₂AR- but not the ß₁AR-stimulated positive inotropic effect and increase in cytosolic Ca²⁺ transient are dissociated from cAMP production and occur without increasing phosphorylation of cytoplasmic proteins, eg, the sarcoplasmic reticulum membrane protein phospholamban.^{13 14} Interestingly, in rat myocytes, PTX treatment not only potentiates the positive inotropic effect of ß₂AR stimulation,¹⁰ it also largely reverses the differences between ß₁AR and ß₂AR,¹¹ indicating ß₂AR-activated G_i proteins play a key role in the differential cardiac response to ß₂AR versus ß₁AR subtype stimulation. A similar potentiating effect of PTX on ß₂AR contractile response has also been observed in normal canine ventricular myocytes (Zhou et al, unpublished data, 1998). These results reinforce the idea that the concurrent coupling of ß₂AR to functionally opposing G proteins is a universal phenomenon in mammalian hearts.

In murine cardiomyocytes, PTX permits a de novo contractile response (Figure 5). In contrast to mice, PTX pretreatment only augments an already extant positive ß₂AR contractile response in other species examined.^{10 11 24} This diversity in cardiac ß₂AR stimulation among species or within species under different circumstances may be largely accounted for on the basis of quantitative differences in the extent of ß₂AR-G_i coupling. For example, the ß₂AR-G_i coupling would be expected to be extremely robust in mouse heart, as manifested by the absence of a ß₂AR-mediated positive inotropic effect without PTX pretreatment. In rat, an augmentation of the extent of ß₂AR coupling to G_i proteins during development could explain the greater sensitivity of neonatal than that of adult heart cells to ß₂AR activation in the absence of PTX.¹⁵ The difference between ß₁AR and ß₂AR in their G protein coupling profiles may also provide new insight for understanding the role of ßAR subtypes in health and diseased mammalian heart (References 12 through 15^{12 13 14 15} ; also see subsequent sections).

Peculiar Features of TG4 Myocytes
The results of the present study show that TG4 mouse ventricular myocytes overexpressing human ß₂AR exhibit a markedly enhanced baseline contractility, which can be reversed by the inverse ß₂AR agonist ICI (Figure 1). Because our experiments were conducted in superfused single, isolated ventricular myocytes, possible endogenous catecholamine contamination, which might complicate the interpretation of observations of previous studies in vivo and in isolated atria,^{1 7 21} can be completely ruled out. Thus, the results of the present study confirm and extend previous studies and provide evidence at the single cell level for the functional existence of spontaneous active ß₂ARs in TG4 mice. Conceptually, a small fraction of receptors undergoes spontaneous transition to an active state (R*) at any time, even in the absence of agonist.⁷ The 200-fold overexpression of the ß₂AR in TG4 hearts results in more receptors in the R* state, which constitutively increase basal adenylyl cyclase activity^{1 7} and baseline cellular contractility (Figure 1).

The results of the present study also show that the inability of ß₂AR agonists to augment the contractility of TG4 cardiomyocytes cannot be explained by a saturation of contractility at baseline. The reason for this is that the adenylyl cyclase activator forskolin can further increase contraction amplitude over the enhanced basal contraction, indicating that the cAMP-PKA signaling is still capable of modulating the contractility of TG4 heart cells. More importantly, the lack of ß₂AR positive inotropic effect was also observed in WT mouse myocytes, indicating that the null contractile response to ß₂AR agonists has nothing to do with the receptor overexpression or chronic spontaneous ß₂AR activation in the transgenic model, but it is a fundamental property of ß₂AR signaling in murine heart. In addition, the results of the present study indicate that the ß₂AR-G_i coupling is retained over a wide range of ß₂AR densities and agonist concentrations.

Another peculiar feature of TG4 hearts is an absence of contractile response to ß₁AR stimulation, as manifested by the inability of ß₁AR stimulation by NE or ISO in vivo^{7 21} or the inability of ISO plus the ß₂AR blocker ICI to increase cardiac contractility in single isolated myocytes (data not shown). Although PTX treatment fully rescued the ß₂AR responsiveness in TG4 cardiomyocytes, it was not able to rescue the lost cardiac response to ß₁AR stimulation in these transgenic mice (data not shown). This is consistent with the observation that ß₁AR does not couple to G_i proteins (Figures 8 and 9). These results suggest that a different mechanism might be involved in the subsensitivity of TG4 hearts to ß₁AR stimulation (eg, desensitization of the receptor via an enhanced basal PKA-dependent receptor phosphorylation or by the ß-adrenergic receptor kinase ßARK^{2 31 32} ).

Do Spontaneous Active ß₂ARs Differ From Ligand-Stimulated ß₂ARs?
According to the current "two-state" model of receptor theory,⁷ receptors exist in equilibrium of an inactive state (R) and an active state (R*) in terms of the ability to interact with G proteins. This model predicts that spontaneous active receptors should be identical to ligand-stimulated active receptor species (LR*), given that there is a sole active state. The results of the present study, however, provide several lines of evidence to suggest that spontaneously activated ß₂AR may differ from the ligand-stimulated ß₂AR. First, whereas spontaneous active ß₂ARs in TG4 heart, presumably only a small fraction of total receptor population,¹ increased the cell contractility by about 3-fold, ß₂AR agonists, at maximal concentrations that would be expected to occupy a large quantity of the excessive ß₂ARs in TG4 cells, were unable to further increase contraction amplitude, even though the cell contractility and ßAR-cAMP signaling are not saturated. Second, PTX treatment only slightly potentiated the basal contractility (in TG4 cells only) but had a disproportionally large potentiating effect on the agonist-stimulated contractile response in both TG4 and WT heart cells (Figure 5), suggesting that the spontaneously activated ß₂AR, unlike the agonist activated ß₂AR, only weakly couples or does not couple to G_i proteins. In this respect, recent studies in transgenic mice with high or medium overexpression of cardiac ß₂AR have demonstrated that spontaneously activated ß₂ARs coprecipitate with G_s but not G_i/G_o proteins in the absence of agonist.³³ Our preliminary data have also consistently shown that the ß₂AR inverse agonist ICI (5x10^-7 mol/L) reduced the basal incorporation of [-³²P]GTP-Aza into subunits of G_s but not G_i proteins in TG4 mice (Avdonin et al, unpublished data, 1998). Taken together, we suggest that spontaneous active ß₂ARs are predominantly coupled to G_s with little or no coupling to G_i proteins, whereas the ligand-activated ß₂ARs couple to both G_s and G_i proteins. The distinct difference between the spontaneous and ligand-induced active ß₂ARs demonstrated in the present study and in previous studies of murine myocardium requires a reformulation of the current model⁷ to describe receptor–G protein coupling in the physiological context.

Implications of ß₂AR-G_i Coupling in the Heart
In addition to modulating the ß₂AR-G_s–mediated enhancement in cardiac contractility, the ß₂AR-stimulated G_i activation might have chronic effects, eg, cellular metabolism or excitability or cell growth, which requires additional investigations. In this regard, it is intriguing that ß₂AR overexpression in TG4 mice is not associated with a cardiac or cellular hypertrophy¹ and exhibits no change in the size of single isolated cardiomyocytes (as shown in the present study), whereas a genetic manipulation of G_s-cAMP signaling system³ or chronic ßAR stimulation by agonists^{34 35} is often associated with cardiac hypertrophy or heart failure. Thus, we speculate that ßAR subtypes may differentially regulate cell growth as a result of the additional ß₂AR-G_i coupling. In addition, it has been shown that inhibition of G_i function by PTX treatment increases the occurrence of spontaneous cell contractions in rat ventricular myocytes¹⁰ and arrhythmia in intact rats (Eschenhagen et al, unpublished data, 1998) during ßAR agonist stimulation. Thus, an activation of the ß₂AR-coupled G_i proteins may have some cardiac protective functions.

The demonstration that ß₂AR couples to G_s and G_i also provides new insights for the pathogenesis of heart failure. It is generally acknowledged that heart failure in human and animal models is characterized by a deterioration in cardiac contractility and a reduced catecholamine responsiveness, which are associated with an increase in G_i mRNA levels,³⁶ G_i activity as indicated by PTX-induced ribosylation³⁷ or G_i protein amount in human³⁸ or in animal models³⁹ and an increase in the ratio of ß₂AR to ß₁AR as a result of a selective downregulation of ß₁ARs.^{40 41 42} It has been proposed that the upregulation of G_i proteins may contribute to the suppressed ßAR, particularly ß₁AR, contractile response in the failing hearts.^{36 37 38 39} However, this hypothesis has not been directly examined, because most previous studies failed to determine whether the increased G_i activity differentially affected ßAR subtype signaling. On the basis of biochemical and physiological evidence for a coupling of ß₂AR to G_i proteins (References 10 and 11^{10 11} and the present study), it is possible that, on one hand, the upregulation of G_i proteins could protect the diseased heart from Ca²⁺ overloading and arrhythmia; and on the other hand, the upregulated G_i signaling in failing hearts could offset or mask the ß₂AR-stimulated positive inotropic effect, resulting in an attenuation or loss of the overall ßAR-mediated inotropic response. Additional studies are required to test these provocative hypotheses.

In summary, we demonstrate that ß₂AR stimulation cannot augment contractile function in isolated single WT or receptor overexpression transgenic (TG4) cardiac myocytes, although spontaneous ß₂AR activation enhances the baseline contractility of TG4 myocytes. We also provide the first biochemical evidence that in murine cardiac myocytes, ß₂AR is dually coupled to inhibitory G proteins G_i2 and G_i3 in addition to G_s. PTX treatment permits ß₂AR stimulation to induce a robust augmentation in contraction, associated with an increase in I_Ca and [Ca²⁺]_i transient. The PTX-restored cardiac ß₂AR response can be reversed by the inhibitory cAMP analog Rp-cAMPS. Thus, the ß₂AR-coupled G_i pathway exerts a strong negative feedback to the ß₂AR-mediated, cAMP-dependent cardiac contractile and [Ca²⁺]_i transient and I_Ca responses. These findings may have important implications not only for understanding signaling mechanisms and functionality of cardiac ßAR subtypes but also for devising future strategies for the treatment of human heart failure via genetic therapy.

	Acknowledgments

 
P. Avdonin was partially supported by the Russian Foundation for Basic Research (grant No. 96-04-49921).

Received June 24, 1998; accepted October 6, 1998.

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				B. Yoo, A. Lemaire, S. Mangmool, M. J. Wolf, A. Curcio, L. Mao, and H. A. Rockman {beta}1-Adrenergic receptors stimulate cardiac contractility and CaMKII activation in vivo and enhance cardiac dysfunction following myocardial infarction Am J Physiol Heart Circ Physiol, October 1, 2009; 297(4): H1377 - H1386. [Abstract] [Full Text] [PDF]

				F. Vandeput, J. Krall, R. Ockaili, F. N. Salloum, V. Florio, J. D. Corbin, S. H. Francis, R. C. Kukreja, and M. A. Movsesian cGMP-Hydrolytic Activity and Its Inhibition by Sildenafil in Normal and Failing Human and Mouse Myocardium J. Pharmacol. Exp. Ther., September 1, 2009; 330(3): 884 - 891. [Abstract] [Full Text] [PDF]

				S. Dai, D. D. Hall, and J. W. Hell Supramolecular Assemblies and Localized Regulation of Voltage-Gated Ion Channels Physiol Rev, April 1, 2009; 89(2): 411 - 452. [Abstract] [Full Text] [PDF]

				A. Y.-H. Woo, T.-B. Wang, X. Zeng, W. Zhu, D. R. Abernethy, I. W. Wainer, and R.-P. Xiao Stereochemistry of an Agonist Determines Coupling Preference of {beta}2-Adrenoceptor to Different G Proteins in Cardiomyocytes Mol. Pharmacol., January 1, 2009; 75(1): 158 - 165. [Abstract] [Full Text] [PDF]

				J. Davis, M. V. Westfall, D. Townsend, M. Blankinship, T. J. Herron, G. Guerrero-Serna, W. Wang, E. Devaney, and J. M. Metzger Designing Heart Performance by Gene Transfer Physiol Rev, October 1, 2008; 88(4): 1567 - 1651. [Abstract] [Full Text] [PDF]

				R. Stones, A. Natali, R. Billeter, S. Harrison, and E. White Voluntary exercise-induced changes in {beta}2-adrenoceptor signalling in rat ventricular myocytes Exp Physiol, September 1, 2008; 93(9): 1065 - 1075. [Abstract] [Full Text] [PDF]

				J. G. Ryall, J. D. Schertzer, K. T. Murphy, A. M. Allen, and G. S. Lynch Chronic {beta}2-adrenoceptor stimulation impairs cardiac relaxation via reduced SR Ca2+-ATPase protein and activity Am J Physiol Heart Circ Physiol, June 1, 2008; 294(6): H2587 - H2595. [Abstract] [Full Text] [PDF]

				K. Janssens, M. Boussemaere, S. Wagner, K. Kopka, and C. Denef {beta}1-Adrenoceptors in Rat Anterior Pituitary May Be Constitutively Active. Inverse Agonism of CGP 20712A on Basal 3',5'-Cyclic Adenosine 5'-Monophosphate Levels Endocrinology, May 1, 2008; 149(5): 2391 - 2402. [Abstract] [Full Text] [PDF]

				G. S. Lynch and J. G. Ryall Role of {beta}-Adrenoceptor Signaling in Skeletal Muscle: Implications for Muscle Wasting and Disease Physiol Rev, April 1, 2008; 88(2): 729 - 767. [Abstract] [Full Text] [PDF]

				B. R. DeGeorge Jr, E. Gao, M. Boucher, L. E. Vinge, J. S. Martini, P. W. Raake, J. K. Chuprun, D. M. Harris, G. W. Kim, S. Soltys, et al. Targeted Inhibition of Cardiomyocyte Gi Signaling Enhances Susceptibility to Apoptotic Cell Death in Response to Ischemic Stress Circulation, March 18, 2008; 117(11): 1378 - 1387. [Abstract] [Full Text] [PDF]

				F. A. Faucher, F. E. Gannier, J. M. Lignon, P. Cosnay, and C. O. Malecot Roles of PKA, PI3K, and cPLA2 in the NO-mediated negative inotropic effect of {beta}2-adrenoceptor agonists in guinea pig right papillary muscles Am J Physiol Cell Physiol, January 1, 2008; 294(1): C106 - C117. [Abstract] [Full Text] [PDF]

				F. Vandeput, S. L. Wolda, J. Krall, R. Hambleton, L. Uher, K. N. McCaw, P. B. Radwanski, V. Florio, and M. A. Movsesian Cyclic Nucleotide Phosphodiesterase PDE1C1 in Human Cardiac Myocytes J. Biol. Chem., November 9, 2007; 282(45): 32749 - 32757. [Abstract] [Full Text] [PDF]

				L. P. Collis, S. Srivastava, W. A. Coetzee, and M. Artman beta2-Adrenergic receptor agonists stimulate L-type calcium current independent of PKA in newborn rabbit ventricular myocytes Am J Physiol Heart Circ Physiol, November 1, 2007; 293(5): H2826 - H2835. [Abstract] [Full Text] [PDF]

				H. Ruan, S. Mitchell, M. Vainoriene, Q. Lou, L.-H. Xie, S. Ren, J. I. Goldhaber, and Y. Wang Gi{alpha}1-Mediated Cardiac Electrophysiological Remodeling and Arrhythmia in Hypertrophic Cardiomyopathy Circulation, August 7, 2007; 116(6): 596 - 605. [Abstract] [Full Text] [PDF]

				D. J. Dupre, M. Robitaille, N. Ethier, L. R. Villeneuve, A. M. Mamarbachi, and T. E. Hebert Seven Transmembrane Receptor Core Signaling Complexes Are Assembled Prior to Plasma Membrane Trafficking J. Biol. Chem., November 10, 2006; 281(45): 34561 - 34573. [Abstract] [Full Text] [PDF]

				V. O. Nikolaev, M. Bunemann, E. Schmitteckert, M. J. Lohse, and S. Engelhardt Cyclic AMP Imaging in Adult Cardiac Myocytes Reveals Far-Reaching {beta}1-Adrenergic but Locally Confined {beta}2-Adrenergic Receptor-Mediated Signaling Circ. Res., November 10, 2006; 99(10): 1084 - 1091. [Abstract] [Full Text] [PDF]

				J. T. Hulme, R. E. Westenbroek, T. Scheuer, and W. A. Catterall Phosphorylation of serine 1928 in the distal C-terminal domain of cardiac CaV1.2 channels during beta1-adrenergic regulation PNAS, October 31, 2006; 103(44): 16574 - 16579. [Abstract] [Full Text] [PDF]

				L. R. Queen, Y. Ji, B. Xu, L. Young, K. Yao, A. W. Wyatt, D. J. Rowlands, R. C. M. Siow, G. E. Mann, and A. Ferro Mechanisms underlying {beta}2-adrenoceptor-mediated nitric oxide generation by human umbilical vein endothelial cells J. Physiol., October 15, 2006; 576(2): 585 - 594. [Abstract] [Full Text] [PDF]

				R. C. Balijepalli, J. D. Foell, D. D. Hall, J. W. Hell, and T. J. Kamp From the Cover: Localization of cardiac L-type Ca2+ channels to a caveolar macromolecular signaling complex is required for beta2-adrenergic regulation PNAS, May 9, 2006; 103(19): 7500 - 7505. [Abstract] [Full Text] [PDF]

				S. Calaghan and E. White Caveolae modulate excitation-contraction coupling and {beta}2-adrenergic signalling in adult rat ventricular myocytes Cardiovasc Res, March 1, 2006; 69(4): 816 - 824. [Abstract] [Full Text] [PDF]

				R. Germack and J. M. Dickenson Induction of {beta}3-Adrenergic Receptor Functional Expression following Chronic Stimulation with Noradrenaline in Neonatal Rat Cardiomyocytes J. Pharmacol. Exp. Ther., January 1, 2006; 316(1): 392 - 402. [Abstract] [Full Text] [PDF]

				H. Fujino and J. W. Regan EP4 Prostanoid Receptor Coupling to a Pertussis Toxin-Sensitive Inhibitory G Protein Mol. Pharmacol., January 1, 2006; 69(1): 5 - 10. [Abstract] [Full Text] [PDF]

				N. Wettschureck and S. Offermanns Mammalian G Proteins and Their Cell Type Specific Functions Physiol Rev, October 1, 2005; 85(4): 1159 - 1204. [Abstract] [Full Text] [PDF]

				P. Dorian Antiarrhythmic Action of{beta}-Blockers: Potential Mechanisms Journal of Cardiovascular Pharmacology and Therapeutics, October 1, 2005; 10(4_suppl): S15 - S22. [Abstract] [PDF]

				W. Zhu, X. Zeng, M. Zheng, and R.-P. Xiao The Enigma of {beta}2-Adrenergic Receptor Gi Signaling in the Heart: The Good, the Bad, and the Ugly Circ. Res., September 16, 2005; 97(6): 507 - 509. [Full Text] [PDF]

				W.-Z. Zhu, K. Chakir, S. Zhang, D. Yang, C. Lavoie, M. Bouvier, T. E. Hebert, E. G. Lakatta, H. Cheng, and R.-P. Xiao Heterodimerization of {beta}1- and {beta}2-Adrenergic Receptor Subtypes Optimizes {beta}-Adrenergic Modulation of Cardiac Contractility Circ. Res., August 5, 2005; 97(3): 244 - 251. [Abstract] [Full Text] [PDF]

				S. M. MacDonnell, H. Kubo, D. L. Crabbe, B. F. Renna, P. O. Reger, J. Mohara, L. A. Smithwick, W. J. Koch, S. R. Houser, and J. R. Libonati Improved Myocardial {beta}-Adrenergic Responsiveness and Signaling With Exercise Training in Hypertension Circulation, June 28, 2005; 111(25): 3420 - 3428. [Abstract] [Full Text] [PDF]

				D. D. Denson, J. Li, X. Wang, and D. C. Eaton Activation of BK Channels in GH3 Cells by a c-PLA2-Dependent G-Protein Signaling Pathway J Neurophysiol, June 1, 2005; 93(6): 3146 - 3156. [Abstract] [Full Text] [PDF]

				B. Ait-Mamar, M. Cailleret, C. Rucker-Martin, A. Bouabdallah, G. Candiani, C. Adamy, P. Duvaldestin, F. Pecker, N. Defer, and C. Pavoine The Cytosolic Phospholipase A2 Pathway, a Safeguard of {beta}2-Adrenergic Cardiac Effects in Rat J. Biol. Chem., May 13, 2005; 280(19): 18881 - 18890. [Abstract] [Full Text] [PDF]

				J. G. Burniston, L.-B. Tan, and D. F. Goldspink {beta}2-Adrenergic receptor stimulation in vivo induces apoptosis in the rat heart and soleus muscle J Appl Physiol, April 1, 2005; 98(4): 1379 - 1386. [Abstract] [Full Text] [PDF]

				A. Das, L. Xi, and R. C. Kukreja Phosphodiesterase-5 Inhibitor Sildenafil Preconditions Adult Cardiac Myocytes against Necrosis and Apoptosis: ESSENTIAL ROLE OF NITRIC OXIDE SIGNALING J. Biol. Chem., April 1, 2005; 280(13): 12944 - 12955. [Abstract] [Full Text] [PDF]

				M. Gallego, R. Setien, L. Puebla, M. d. C. Boyano-Adanez, E. Arilla, and O. Casis {alpha}1-Adrenoceptors stimulate a G{alpha}s protein and reduce the transient outward K+ current via a cAMP/PKA-mediated pathway in the rat heart Am J Physiol Cell Physiol, March 1, 2005; 288(3): C577 - C585. [Abstract] [Full Text] [PDF]

				V. Leblais, S.-H. Jo, K. Chakir, V. Maltsev, M. Zheng, M. T. Crow, W. Wang, E. G. Lakatta, and R.-P. Xiao Phosphatidylinositol 3-Kinase Offsets cAMP-Mediated Positive Inotropic Effect via Inhibiting Ca2+ Influx in Cardiomyocytes Circ. Res., December 10, 2004; 95(12): 1183 - 1190. [Abstract] [Full Text] [PDF]

				S. A. Grandy, E. M. Denovan-Wright, G. R. Ferrier, and S. E. Howlett Overexpression of human {beta}2-adrenergic receptors increases gain of excitation-contraction coupling in mouse ventricular myocytes Am J Physiol Heart Circ Physiol, September 1, 2004; 287(3): H1029 - H1038. [Abstract] [Full Text] [PDF]

				I. Ahmet, M. Krawczyk, P. Heller, C. Moon, E. G. Lakatta, and M. I. Talan Beneficial Effects of Chronic Pharmacological Manipulation of {beta}-Adrenoreceptor Subtype Signaling in Rodent Dilated Ischemic Cardiomyopathy Circulation, August 31, 2004; 110(9): 1083 - 1090. [Abstract] [Full Text] [PDF]

				L. Barki-Harrington, C. Perrino, and H. A Rockman Network integration of the adrenergic system in cardiac hypertrophy Cardiovasc Res, August 15, 2004; 63(3): 391 - 402. [Abstract] [Full Text] [PDF]

				S. Pepe, O. W.V van den Brink, E. G Lakatta, and R.-P. Xiao Cross-talk of opioid peptide receptor and {beta}-adrenergic receptor signalling in the heart Cardiovasc Res, August 15, 2004; 63(3): 414 - 422. [Abstract] [Full Text] [PDF]

				M. A Movsesian Altered cAMP-mediated signalling and its role in the pathogenesis of dilated cardiomyopathy Cardiovasc Res, June 1, 2004; 62(3): 450 - 459. [Abstract] [Full Text] [PDF]

				J. F. Heubach, U. Ravens, and A. J. Kaumann Epinephrine Activates Both Gs and Gi Pathways, but Norepinephrine Activates Only the Gs Pathway through Human {beta}2-Adrenoceptors Overexpressed in Mouse Heart Mol. Pharmacol., May 1, 2004; 65(5): 1313 - 1322. [Abstract] [Full Text]

				J.R. Keys and W.J. Koch The Adrenergic Pathway and Heart Failure Recent Prog. Horm. Res., January 1, 2004; 59(1): 13 - 30. [Abstract] [Full Text]

				A. El-Armouche, O. Zolk, T. Rau, and T. Eschenhagen Inhibitory G-proteins and their role in desensitization of the adenylyl cyclase pathway in heart failure Cardiovasc Res, December 1, 2003; 60(3): 478 - 487. [Abstract] [Full Text] [PDF]

				K. Foerster, F. Groner, J. Matthes, W. J. Koch, L. Birnbaumer, and S. Herzig Cardioprotection specific for the G protein Gi2 in chronic adrenergic signaling through {beta}2-adrenoceptors PNAS, November 25, 2003; 100(24): 14475 - 14480. [Abstract] [Full Text] [PDF]

				K. Chakir, Y. Xiang, D. Yang, S.-J. Zhang, H. Cheng, B. K. Kobilka, and R.-P. Xiao The Third Intracellular Loop and the Carboxyl Terminus of {beta}2-Adrenergic Receptor Confer Spontaneous Activity of the Receptor Mol. Pharmacol., November 1, 2003; 64(5): 1048 - 1058. [Abstract] [Full Text] [PDF]

				R.-P. Xiao, S.-J. Zhang, K. Chakir, P. Avdonin, W. Zhu, R. A. Bond, C. W. Balke, E. G. Lakatta, and H. Cheng Enhanced Gi Signaling Selectively Negates {beta}2-Adrenergic Receptor (AR)- but Not {beta}1-AR-Mediated Positive Inotropic Effect in Myocytes From Failing Rat Hearts Circulation, September 30, 2003; 108(13): 1633 - 1639. [Abstract] [Full Text] [PDF]

				L. A. Hu, W. Chen, N. P. Martin, E. J. Whalen, R. T. Premont, and R. J. Lefkowitz GIPC Interacts with the {beta}1-Adrenergic Receptor and Regulates {beta}1-Adrenergic Receptor-mediated ERK Activation J. Biol. Chem., July 3, 2003; 278(28): 26295 - 26301. [Abstract] [Full Text] [PDF]

				M. T. Borchers, T. Biechele, J. P. Justice, T. Ansay, S. Cormier, V. Mancino, T. M. Wilkie, M. I. Simon, N. A. Lee, and J. J. Lee Methacholine-induced airway hyperresponsiveness is dependent on G{alpha}q signaling Am J Physiol Lung Cell Mol Physiol, July 1, 2003; 285(1): L114 - L120. [Abstract] [Full Text] [PDF]

				L. Chu, R. Takahashi, I. Norota, T. Miyamoto, Y. Takeishi, K. Ishii, I. Kubota, and M. Endoh Signal Transduction and Ca2+ Signaling in Contractile Regulation Induced by Crosstalk Between Endothelin-1 and Norepinephrine in Dog Ventricular Myocardium Circ. Res., May 16, 2003; 92(9): 1024 - 1032. [Abstract] [Full Text] [PDF]

				N. R. Lenard, T. W. Gettys, and A. J. Dunn Activation of beta 2- and beta 3-Adrenergic Receptors Increases Brain Tryptophan J. Pharmacol. Exp. Ther., May 1, 2003; 305(2): 653 - 659. [Abstract] [Full Text] [PDF]

				B. Schwarz, E. Percy, X.-M. Gao, A. M. Dart, G. Richardt, and X.-J. Du Altered calcium transient and development of hypertrophy in {beta}2-adrenoceptor overexpressing mice with and without pressure overload Eur J Heart Fail, March 1, 2003; 5(2): 131 - 136. [Abstract] [Full Text] [PDF]

				S. Bartel, E.-G. Krause, G. Wallukat, and P. Karczewski New insights into {beta}2-adrenoceptor signaling in the adult rat heart Cardiovasc Res, March 1, 2003; 57(3): 694 - 703. [Abstract] [Full Text] [PDF]

				J. Kim, A. D. Eckhart, S. Eguchi, and W. J. Koch beta -Adrenergic Receptor-mediated DNA Synthesis in Cardiac Fibroblasts Is Dependent on Transactivation of the Epidermal Growth Factor Receptor and Subsequent Activation of Extracellular Signal-regulated Kinases J. Biol. Chem., August 23, 2002; 277(35): 32116 - 32123. [Abstract] [Full Text] [PDF]

				A. M. Zamah, M. Delahunty, L. M. Luttrell, and R. J. Lefkowitz Protein Kinase A-mediated Phosphorylation of the beta 2-Adrenergic Receptor Regulates Its Coupling to Gs and Gi. DEMONSTRATION IN A RECONSTITUTED SYSTEM J. Biol. Chem., August 16, 2002; 277(34): 31249 - 31256. [Abstract] [Full Text] [PDF]

				J. D. Kilts, T. Akazawa, M. D. Richardson, and M. M. Kwatra Age Increases Cardiac Galpha i2 Expression, Resulting in Enhanced Coupling to G Protein-coupled Receptors J. Biol. Chem., August 16, 2002; 277(34): 31257 - 31262. [Abstract] [Full Text] [PDF]

				S.-H. Jo, V. Leblais, P. H. Wang, M. T. Crow, and R.-P. Xiao Phosphatidylinositol 3-Kinase Functionally Compartmentalizes the Concurrent Gs Signaling During {beta}2-Adrenergic Stimulation Circ. Res., July 12, 2002; 91(1): 46 - 53. [Abstract] [Full Text] [PDF]

				H. Gong, H. Sun, W. J. Koch, T. Rau, T. Eschenhagen, U. Ravens, J. F. Heubach, D. L. Adamson, and S. E. Harding Specific {beta}2AR Blocker ICI 118,551 Actively Decreases Contraction Through a Gi-Coupled Form of the {beta}2AR in Myocytes From Failing Human Heart Circulation, May 28, 2002; 105(21): 2497 - 2503. [Abstract] [Full Text] [PDF]

				D. Motlagh, K. J Alden, B. Russell, and J. Garcia Sodium current modulation by a tubulin/GTP coupled process in rat neonatal cardiac myocytes J. Physiol., April 1, 2002; 540(1): 93 - 103. [Abstract] [Full Text] [PDF]

				M. Zaugg, M. C. Schaub, T. Pasch, and D. R. Spahn Modulation of {beta}-adrenergic receptor subtype activities in perioperative medicine: mechanisms and sites of action Br. J. Anaesth., January 1, 2002; 88(1): 101 - 123. [Abstract] [Full Text] [PDF]

				Y. G. Wang, E. N. Dedkova, S. F. Steinberg, L. A. Blatter, and S. L. Lipsius {beta}2-Adrenergic Receptor Signaling Acts via No Release to Mediate Ach-Induced Activation of Atp-Sensitive K+ Current in Cat Atrial Myocytes J. Gen. Physiol., January 1, 2002; 119(1): 69 - 82. [Abstract] [Full Text] [PDF]

				J. T. Auman, F. J. Seidler, C. A. Tate, and T. A. Slotkin beta -Adrenoceptor-mediated cell signaling in the neonatal heart and liver: responses to terbutaline Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2001; 281(6): R1895 - R1901. [Abstract] [Full Text] [PDF]

				R. S. Ostrom, C. Gregorian, R. M. Drenan, Y. Xiang, J. W. Regan, and P. A. Insel Receptor Number and Caveolar Co-localization Determine Receptor Coupling Efficiency to Adenylyl Cyclase J. Biol. Chem., November 2, 2001; 276(45): 42063 - 42069. [Abstract] [Full Text] [PDF]

				S. Magne, D. Couchie, F. Pecker, and C. Pavoine beta 2-Adrenergic Receptor Agonists Increase Intracellular Free Ca2+ Concentration Cycling in Ventricular Cardiomyocytes through p38 and p42/44 MAPK-mediated Cytosolic Phospholipase A2 Activation J. Biol. Chem., October 19, 2001; 276(43): 39539 - 39548. [Abstract] [Full Text] [PDF]

				R.-P. Xiao {beta}-Adrenergic Signaling in the Heart: Dual Coupling of the {beta}2-Adrenergic Receptor to Gs and Gi Proteins Sci. Signal., October 16, 2001; 2001(104): re15 - re15. [Abstract] [Full Text] [PDF]

				A. D. Eckhart and W. J. Koch Transgenic Studies of Cardiac Adrenergic Receptor Regulation J. Pharmacol. Exp. Ther., October 1, 2001; 299(1): 1 - 5. [Abstract] [Full Text] [PDF]

				E. Devic, Y. Xiang, D. Gould, and B. Kobilka beta -Adrenergic Receptor Subtype-Specific Signaling in Cardiac Myocytes from beta 1 and beta 2 Adrenoceptor Knockout Mice Mol. Pharmacol., September 1, 2001; 60(3): 577 - 583. [Abstract] [Full Text] [PDF]

				W.-Z. Zhu, M. Zheng, W. J. Koch, R. J. Lefkowitz, B. K. Kobilka, and R.-P. Xiao Dual modulation of cell survival and cell death by beta 2-adrenergic signaling in adult mouse cardiac myocytes PNAS, February 13, 2001; 98(4): 1607 - 1612. [Abstract] [Full Text] [PDF]

				M. Jain, C. C. Lim, K. Nagata, V. M. Davis, D. S. Milstone, R. Liao, and R. M. Mortensen Targeted inactivation of G{alpha}i does not alter cardiac function or {beta}-adrenergic sensitivity Am J Physiol Heart Circ Physiol, February 1, 2001; 280(2): H569 - H575. [Abstract] [Full Text] [PDF]

				M. Flesch, S. Ettelbruck, S. Rosenkranz, C. Maack, B. Cremers, K.-D. Schluter, O. Zolk, and M. Bohm Differential effects of carvedilol and metoprolol on isoprenaline-induced changes in {beta}-adrenoceptor density and systolic function in rat cardiac myocytes Cardiovasc Res, February 1, 2001; 49(2): 371 - 380. [Abstract] [Full Text] [PDF]

				A. Chesley, M. S. Lundberg, T. Asai, R.-P. Xiao, S. Ohtani, E. G. Lakatta, and M. T. Crow The {beta}2-Adrenergic Receptor Delivers an Antiapoptotic Signal to Cardiac Myocytes Through Gi-Dependent Coupling to Phosphatidylinositol 3'-Kinase Circ. Res., December 8, 2000; 87(12): 1172 - 1179. [Abstract] [Full Text] [PDF]

				Y.-Y. Zhou, D. Yang, W.-Z. Zhu, S.-J. Zhang, D.-J. Wang, D. K. Rohrer, E. Devic, B. K. Kobilka, E. G. Lakatta, H. Cheng, et al. Spontaneous Activation of beta 2- but Not beta 1-Adrenoceptors Expressed in Cardiac Myocytes from beta 1beta 2 Double Knockout Mice Mol. Pharmacol., November 1, 2000; 58(5): 887 - 894. [Abstract] [Full Text]

				K. Wenzel-Seifert and R. Seifert Molecular Analysis of beta 2-Adrenoceptor Coupling to Gs-, Gi-, and Gq-Proteins Mol. Pharmacol., November 1, 2000; 58(5): 954 - 966. [Abstract] [Full Text]

				X.-J. Du, X.-M. Gao, G. L. Jennings, A. M. Dart, and E. A. Woodcock Preserved ventricular contractility in infarcted mouse heart overexpressing beta 2-adrenergic receptors Am J Physiol Heart Circ Physiol, November 1, 2000; 279(5): H2456 - H2463. [Abstract] [Full Text] [PDF]

				R.-P. Xiao Cell Logic for Dual Coupling of a Single Class of Receptors to Gs and Gi Proteins Circ. Res., October 13, 2000; 87(8): 635 - 637. [Full Text] [PDF]

				J. D. Kilts, M. A. Gerhardt, M. D. Richardson, G. Sreeram, G. B. Mackensen, H. P. Grocott, W. D. White, R. D. Davis, M. F. Newman, J. G. Reves, et al. {beta}2-Adrenergic and Several Other G Protein-Coupled Receptors in Human Atrial Membranes Activate Both Gs and Gi Circ. Res., October 13, 2000; 87(8): 705 - 709. [Abstract] [Full Text] [PDF]

				I. H. Derweesh, M. A. Wheeler, and R. M. Weiss Alterations in G-Proteins and beta -Adrenergic Responsive Adenylyl Cyclase in Rat Urinary Bladder during Aging J. Pharmacol. Exp. Ther., September 1, 2000; 294(3): 969 - 974. [Abstract] [Full Text]

				Z. Nagykaldi, D. Kem, R. Lazzara, and B. Szabo Conditioning of beta 1-adrenoceptor effect via beta 2-subtype on L-type Ca2+ current in canine ventricular myocytes Am J Physiol Heart Circ Physiol, September 1, 2000; 279(3): H1329 - H1337. [Abstract] [Full Text] [PDF]

				Y. G. Wang, A. M Samarel, and S. L Lipsius Laminin binding to {beta}1-integrins selectively alters {beta}1- and {beta}2-adrenoceptor signalling in cat atrial myocytes J. Physiol., August 15, 2000; 527(1): 3 - 9. [Abstract] [Full Text] [PDF]

				M. Zaugg, W. Xu, E. Lucchinetti, S. A. Shafiq, N. Z. Jamali, and M. A. Q. Siddiqui {beta}-Adrenergic Receptor Subtypes Differentially Affect Apoptosis in Adult Rat Ventricular Myocytes Circulation, July 18, 2000; 102(3): 344 - 350. [Abstract] [Full Text] [PDF]

				Y.-Y. Zhou, S.-Q. Wang, W.-Z. Zhu, A. Chruscinski, B. K. Kobilka, B. Ziman, S. Wang, E. G. Lakatta, H. Cheng, and R.-P. Xiao Culture and adenoviral infection of adult mouse cardiac myocytes: methods for cellular genetic physiology Am J Physiol Heart Circ Physiol, July 1, 2000; 279(1): H429 - H436. [Abstract] [Full Text] [PDF]

				I. Kouchi, O. Zolk, F. Jockenhovel, G. Itter, W. Linz, B. Cremers, and M. Bohm Increase in Gi{alpha} Protein Accompanies Progression of Post-Infarction Remodeling in Hypertensive Cardiomyopathy Hypertension, July 1, 2000; 36(1): 42 - 47. [Abstract] [Full Text] [PDF]

				H. K. Ranu, J. C. W. Mak, P. J. Barnes, and S. E. Harding Gi-dependent suppression of beta 1-adrenoceptor effects in ventricular myocytes from NE-treated guinea pigs Am J Physiol Heart Circ Physiol, June 1, 2000; 278(6): H1807 - H1814. [Abstract] [Full Text] [PDF]

				A. Sabri, E. Pak, S. A. Alcott, B. A. Wilson, and S. F. Steinberg Coupling Function of Endogenous {alpha}1- and {beta}-Adrenergic Receptors in Mouse Cardiomyocytes Circ. Res., May 26, 2000; 86(10): 1047 - 1053. [Abstract] [Full Text] [PDF]

				R. J. Lefkowitz, H. A. Rockman, and W. J. Koch Catecholamines, Cardiac {beta}-Adrenergic Receptors, and Heart Failure Circulation, April 11, 2000; 101(14): 1634 - 1637. [Full Text] [PDF]

				K. Singh, C. Communal, D. B. Sawyer, and W. S. Colucci Adrenergic regulation of myocardial apoptosis Cardiovasc Res, February 1, 2000; 45(3): 713 - 719. [Abstract] [Full Text] [PDF]

				J. L. Leaney, G. Milligan, and A. Tinker The G Protein alpha Subunit Has a Key Role in Determining the Specificity of Coupling to, but Not the Activation of, G Protein-gated Inwardly Rectifying K+ Channels J. Biol. Chem., January 14, 2000; 275(2): 921 - 929. [Abstract] [Full Text] [PDF]

				X.-J. Du, D. J. Autelitano, R. J. Dilley, B. Wang, A. M. Dart, and E. A. Woodcock {beta}2-Adrenergic Receptor Overexpression Exacerbates Development of Heart Failure After Aortic Stenosis Circulation, January 4, 2000; 101(1): 71 - 77. [Abstract] [Full Text] [PDF]

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