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(Circulation Research. 2000;87:705.)
© 2000 American Heart Association, Inc.

Molecular Medicine

ß₂-Adrenergic and Several Other G Protein–Coupled Receptors in Human Atrial Membranes Activate Both G_s and G_i

Presented in part at the 1998 Annual Meeting of the American Society of Anesthesiologists, Orlando, Fla, October 17–21, 1998.

Jason D. Kilts, Mark A. Gerhardt, Mark D. Richardson, Gautam Sreeram, G. Burkhard Mackensen, Hilary P. Grocott, William D. White, R. Duane Davis, Mark F. Newman, Joseph G. Reves, Debra A. Schwinn, Madan M. Kwatra

From the Departments of Anesthesiology (J.D.K., M.D.R., G.S., G.B.M., H.P.G., W.D.W., M.F.N., J.G.R., D.A.S., M.M.K.), Pharmacology and Cancer Biology (D.A.S., M.M.K.), and Surgery (R.D.D., D.A.S.), Duke University Medical Center, Durham, NC; and Department of Anesthesiology (M.A.G.), Ohio State University, Columbus, Ohio.

Correspondence to Madan M. Kwatra, PhD, Department of Anesthesiology, 146 Sands Bldg, Box 3094, Duke University Medical Center, Durham, NC 27710. E-mail kwatr001{at}mc.duke.edu

	Abstract

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Abstract—Cardiac G protein–coupled receptors that couple to G_s and stimulate cAMP formation (eg, ß-adrenergic, histamine, serotonin, and glucagon receptors) play a key role in cardiac inotropy. Recent studies in rodent cardiac myocytes and transfected cells have revealed that one of these receptors, the ß₂-adrenergic receptor (AR), also couples to the inhibitory G protein G_i (activation of which inhibits cAMP formation). If ß₂ARs could be shown to couple to G_i in the human heart, it would have important ramifications, because levels of G_i increase with age and in failing human heart. Therefore, we investigated whether ß₂ARs in the human heart activate G_i. By photoaffinity labeling human atrial membranes with [³²P]azidoanilido-GTP, followed by immunoprecipitation with antibodies specific for G_i, we found that G_i is activated by stimulation of ß₂ARs but not of ß₁ARs. In addition, we found that other G_s-coupled receptors also couple to G_i, including histamine, serotonin, and glucagon. When coupling of these receptors to G_i is disrupted by pertussis toxin, their ability to stimulate adenylyl cyclase is enhanced. These data provide the first evidence that ß₂AR and many other G_s-coupled receptors in human atrium also couple to G_i and that abolishing the coupling of these receptors to G_i increases the receptor-mediated adenylyl cyclase activity.

Key Words: human atrial G_s and G_i • ß₂-adrenergic receptor • cardiac G_s–coupled receptors

	Introduction

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Beta-adrenergic receptors (ßARs), which couple to G_s and stimulate adenylyl cyclase (AC) activity, play a key role in cardiac function.¹ An important recent finding in ßAR signaling is that the rodent cardiac ß₂AR couples to both stimulatory (G_s) and inhibitory (G_i) G proteins,^{2 3 4} as does the recombinant ß₂AR transiently expressed in HEK293 cells.⁵ The activation of G_i by ß₂ARs may have important clinical implications because cardiac G_i levels increase in failing heart^{6 7 8} and in aging.⁹ This increase in cardiac G_i could increase the number of ß₂ARs that are coupled to G_i, resulting in a decrease in cardiac ßAR function seen in older people and in patients with congestive heart failure. However, signaling through cardiac ß₂ARs exhibits considerable diversity among mammalian species (for review, see Xiao et al¹⁰ ). Therefore, before we implicate the coupling of ß₂AR to G_i as a factor in this observed reduction in cardiac ßAR function, ß₂ARs must first be shown to couple to G_i in the human heart as they do in rodents.

The present study sought to determine whether human cardiac ß₂ARs activate G_i. Using photoaffinity labeling with [³²P]azidoanilido-GTP ([³²P]AAGTP) followed by immunoprecipitation with antibodies specific for G_s and G_i, we show that stimulation of ßARs in human atrial membranes with isoproterenol leads to the activation of both G_s and G_i. Whereas both ß₁ARs and ß₂ARs activate G_s, only ß₂ARs activate G_i. Interestingly, cardiac G_i is also activated by stimulation of several other G_s-coupled receptors including histamine, serotonin, and glucagon. When the coupling of these receptors to G_i is disrupted by pertussis toxin (PTX), their ability to stimulate AC is enhanced. These data provide the first evidence that ß₂ARs and several other G_s-coupled cardiac G protein–coupled receptors (GPCRs) in human atrium couple to both G_s and G_i.

	Materials and Methods

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Human Atrial Samples
Human atrial samples were obtained under an Institutional Review Board–approved protocol from 25 patients age 51 to 67 years (mean 57.8±4.9 years) undergoing cardiac surgery involving cardiopulmonary bypass at Duke University Medical Center. A piece of right atrial appendage weighing 60 to 150 mg was obtained at the time of atrial cannulation, frozen immediately in liquid nitrogen, and stored at -80°C. Eleven atrial samples were used solely for photoaffinity labeling experiments, 12 were used solely for AC experiments, and 2 were used for both types of experiments. Ten of the 13 samples used for photoaffinity labeling, and 11 of the 14 samples used for AC assays were taken from patients taking ßAR antagonists. There was no discernible difference in results between patients who received ßAR antagonists and those who did not.

Atrial Membrane Preparation
Atrial samples were homogenized and centrifuged. The membrane pellets were resuspended at 2 to 4 mg protein/mL in either buffer A (containing [in mmol/L] HEPES [pH 7.4] 50, EDTA 1, NaCl 50, and benzamidine 2) for photoaffinity labeling assays or buffer B (containing [in mmol/L] Tris [pH 7.4] 75, MgCl₂ 12.5, EDTA 2, and benzamidine 1, and [in mg/L] soybean trypsin inhibitor 10, leupeptin 10, and aprotinin 5) for AC assays.

Photoaffinity Labeling With [³²P]AAGTP and Immunoprecipitation
[³²P]AAGTP was synthesized according to published procedures and purified by thin-layer chromatography on polyethylenimine cellulose (J.T. Baker).^{11 12} Photoaffinity labeling of human atrial membranes (85 µg protein) was performed using 2 µCi of [³²P]AAGTP in a total volume of 60 µL in the presence of various drugs indicated in the figure legends. Immunoprecipitation of photolabeled G subunits and subsequent analysis by SDS-PAGE/autoradiography was performed as described previously.^{11 12}

PTX Treatment of Atrial Membranes and AC Assays
PTX (50 ng/µL) was activated by incubation with 100 mmol/L DTT and 0.25% SDS for 30 minutes at 30°C as described previously,¹³ with minor modifications, and mixed with atrial membranes. A second tube containing the same volumes of all constituents, with the exception of H₂O in place of PTX, was treated in the same fashion and used as a control. AC activity was measured according to the method of Salomon et al¹⁴ and Johnson and Salomon¹⁵ as described previously,¹⁶ with the addition of 0.4 mmol/L 3-isobutylmethylxanthine to the reaction mixture.

Statistical Analysis
Results are presented as mean±SD. Because of the large variations in basal activities between patients, the data are reported as percentage change. The statistical significance of agonist stimulation on the incorporation of [³²P]AAGTP into G proteins and agonist stimulation of AC were determined by paired t tests on the percentage change from basal. A paired t test was also used to compare AC activities in the control membranes and PTX-treated membranes. To compare the PTX-induced increase in basal, forskolin-stimulated, and ß₁AR-stimulated AC activities with the PTX-induced increase in AC stimulation of receptors that couple to both G_s and G_i, a repeated-measures mixed-effects general linear regression model was used.^{17 18} P<0.05 was considered significant for all comparisons.

An expanded Materials and Methods section can be found in an online data supplement available at http://www.circresaha.org.

	Results

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ß₂ARs, but Not ß₁ARs, Stimulate Both G_s and G_i in Human Atrial Membranes
As shown in Figure 1, stimulation of human atrial ßARs with isoproterenol activates G_s (both the long and short isoforms)¹⁹ and G_i; this is revealed by increased photoaffinity labeling of these G subunits with [³²P]AAGTP. On average, isoproterenol significantly increased the incorporation of [³²P]AAGTP, to 209±48% of control in G_s and to 212±33% of control in G_i. This increase in photoaffinity labeling of both G_s and G_i is blocked in the presence of the ß₂AR antagonist ICI 118,551, indicating that activation of G_s and G_i is mediated through ß₂ARs. Importantly, the extent of G_i activation by isoproterenol in these membranes is similar to that seen after stimulation of prototypical G_i-coupled muscarinic receptors with carbachol (data not shown).

View larger version (31K): [in this window] [in a new window]   Figure 1. Activation of both Gs and Gi by ßARs in human heart. Human atrial membranes were photoaffinity labeled with [32P]AAGTP in the presence of no drug (Basal), isoproterenol (Iso), or isoproterenol and ICI 118,551 (ICI), each drug at 100 µmol/L, as described in Materials and Methods. The photolabeled membranes were immunoprecipitated with either RM/1 antibody (for Gs) or AS/7 antibody (for Gi), and immunoprecipitates were subjected to SDS-PAGE followed by autoradiography. Gs-L and Gs-S indicate the long and short isoforms of Gs, respectively. A representative autoradiogram is shown; the experiment was repeated 4 times with similar results.

In rodent hearts, ß₂ARs, but not ß₁ARs, activate G_i.^{2 3 4} To determine whether this is also the case in human heart, we used the following 2 approaches: (1) we stimulated ßARs with agonists selective for either ß₁ARs or ß₂ARs and (2) we stimulated ßARs with the nonselective agonist isoproterenol in the presence of a ß₁AR- or ß₂AR-selective antagonist (Figures 2 and 3, respectively). As shown in Figure 2, whereas both zinterol (agonist for ß₂ARs) and dobutamine (agonist for ß₁ARs) stimulate G_s (to 195±38% and 165±26% of control, respectively), only zinterol stimulates G_i (to 204±19% of control). Similar results are obtained using ß₁/ß₂-selective antagonists; Figure 3 shows that stimulation of G_i by isoproterenol is blocked in the presence of ß₂AR-selective antagonist ICI 118,551 but not in the presence of ß₁AR-selective antagonist CGP 20712A. Taken together, these data establish that ß₂ARs, but not ß₁ARs, in human atrial membranes activate G_i.

View larger version (23K): [in this window] [in a new window]   Figure 2. ß2AR, but not ß1AR, couples to Gi. Human atrial membranes were photoaffinity labeled with [32P]AAGTP in the presence of no drug (Basal), 100 µmol/L dobutamine (Dobut, ß1AR agonist), or 100 µmol/L zinterol (Zint, ß2AR agonist) as described in Materials and Methods. Photolabeled membranes were immunoprecipitated with either RM/1 antibody (for Gs) or AS/7 antibody (for Gi), and immunoprecipitates were subjected to SDS-PAGE followed by autoradiography. Gs-L and Gs-S indicate the long and short isoforms of Gs, respectively. A representative autoradiogram is shown; the experiment was repeated 3 times with similar results.

View larger version (61K): [in this window] [in a new window]   Figure 3. Blockade of ß2AR, but not of ß1AR, abolishes isoproterenol-induced stimulation of Gi. Human atrial membranes were photoaffinity labeled with [32P]AAGTP in the presence of no drug (Basal), isoproterenol (Iso), or Iso and either ICI 118,551 (ICI, ß2AR antagonist) or CGP 20712A (CGP, ß1AR antagonist), each drug at 100 µmol/L, as described in Materials and Methods. The photolabeled membranes were immunoprecipitated with AS/7 antibody (for Gi), and immunoprecipitates were subjected to SDS-PAGE followed by autoradiography. A representative autoradiogram is shown; the experiment was repeated 3 times with similar results.

Stimulation of Histamine, Serotonin, and Glucagon Receptors in Human Atrial Membranes Also Activates G_i
In addition to ßARs, cardiac inotropy is also mediated through histamine, serotonin, and glucagon receptors.²⁰ Like ßARs, these receptors stimulate AC through G_s. Therefore, it was of interest to determine whether these receptors can also activate G_i. As shown in Figure 4, G_i is strongly activated in the presence of histamine and glucagon and moderately activated in the presence of serotonin. Histamine, glucagon, and serotonin all significantly increase the incorporation of [³²P]AAGTP into G_i, to 189±16%, to 210±58%, and to 121±11% of control, respectively. A selective antagonist for each receptor can block the stimulation of G_i by that receptor, indicating that activation of G_i is a consequence of receptor stimulation (Figure 4).

View larger version (23K): [in this window] [in a new window]   Figure 4. Histamine, glucagon, and serotonin receptors couple to Gi. Human atrial membranes were photoaffinity labeled with [32P]AAGTP in the presence of no drug (Basal) or various agonists, or agonists plus corresponding antagonists, as described in Materials and Methods. Agonists included were isoproterenol (Iso, 100 µmol/L), histamine (Hist, 100 µmol/L), glucagon (Gluc, 50 µmol/L), and serotonin (5-HT, 100 µmol/L). Antagonists included were ICI 118,551 (ICI) for ß2ARs, cimetidine (Cimet) for histamine receptors, des-His1-(Glu9)-glucagon (des-Gluc) for glucagon receptors, and SDZ-205,557 (SDZ) for serotonin receptors (all antagonist concentrations were the same as for the respective agonists). Photolabeled membranes were immunoprecipitated with AS/7 antibody (for Gi), and immunoprecipitates were subjected to SDS-PAGE followed by autoradiography. A representative autoradiogram is shown; the experiment was repeated 3 times with similar results.

Effect of PTX Treatment on AC Activity
The data of Figures 1 through 4 indicate that ß₂AR and other G_s-coupled receptors couple to both G_s and G_i. Given that stimulation of G_i-coupled receptors inhibits AC, an obvious next question is whether the disruption of the coupling of these cardiac receptors to G_i removes this inhibitory component and increases receptor-mediated AC activation. Therefore, we treated human atrial membranes with PTX, which ADP-ribosylates G_i and disrupts its ability to interact with GPCRs.^{21 22} These data are shown in Figure 5.

View larger version (31K): [in this window] [in a new window]   Figure 5. PTX treatment of human atrial membranes increases AC activity. Control or PTX-treated human atrial membranes were assayed for AC activity, as described in Materials and Methods. Drugs included were the following (in µmol/L): isoproterenol (Iso, 100), ICI 118,551 (ICI, 100), CGP 20712A (CGP, 100), histamine (Hist, 100), glucagon (Gluc, 50), serotonin (5-HT, 100), and forskolin (Fsk, 50). Basal AC activity ranged from 11 to 254 pmol cAMP/mg protein per 15 minutes. Data are expressed as percentage of basal AC activity in control atrial membranes. The experiment was repeated 4 times with similar results. *P<0.05 compared with basal AC activity in control membranes; **P<0.05 compared with AC stimulation by same drug in control membranes.

In control membranes, AC activity increases with stimulation of ßARs by isoproterenol to 254±46% of control. Selective activation of ß₁ARs (by inclusion of both isoproterenol and the ß₂AR antagonist ICI 118,551) stimulates AC to 178±27% of control, whereas selective activation of ß₂ARs (by inclusion of both isoproterenol and the ß₁AR antagonist CGP 20712A) stimulates AC to 140±22% of control. In addition, histamine and glucagon receptors stimulate AC to 145±31% and 156±48% of control, respectively; the stimulation of AC by serotonin (105±16% of control) was not statistically significant. Forskolin, a direct activator of AC, stimulates AC to 1104±326% of control. These data on AC stimulation in human atrium are similar to that reported by other investigators.^{23 24}

In PTX-treated membranes, basal AC activity is significantly increased (to 442±266% of control), as is AC activity stimulated by agonists of various G_s-coupled receptors (ßARs, 1091±676% of control; ß₁ARs, 668±252%; ß₂ARs, 907±393%; histamine receptors, 787±486%; glucagon receptors, 895±505%; and serotonin receptors, 852±448%) and by forskolin (1896±543% of control) (Figure 5). Although PTX treatment increases AC activity under every condition tested, there is a greater increase in AC stimulation through receptors that also couple to G_i. The PTX-induced increase in basal, forskolin-stimulated, and ß₁AR-stimulated AC activities ranged from 1.7- to 4.4-fold, whereas the PTX-induced increase in AC activity after stimulation of receptors that couple to both G_s and G_i ranged from 5.4- to 8.1-fold, and the difference between the 2 groups is statistically significant (P<0.05). These results indicate that disruption of the coupling of G_s-coupled receptors to G_i enhances their ability to stimulate AC. Note that whereas stimulation of AC by serotonin in control membranes is not significant, it is highly significant in PTX-treated membranes.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The main findings of this study are that, in human atrial membranes, (1) ß₂AR activates both G_s and G_i; (2) both G_s and G_i are also activated by several other receptors including glucagon, histamine, and serotonin; and (3) the coupling of these G_s-coupled receptors to G_i is functional, given that AC stimulation by these receptors is increased by PTX treatment. These findings add a new level of complexity into the signaling of human cardiac inotropic receptors.

The finding that ß₂AR and other G_s-coupled receptors also couple to G_i is an important development. Initially, it was thought that G_s-coupled receptors and G_i-coupled receptors are activated by distinct hormones. For example, G_s-coupled ßARs are stimulated by the sympathetic hormones norepinephrine and epinephrine, and G_i-coupled muscarinic receptors are activated by the parasympathetic hormone acetylcholine. However, this apparently logical signaling scheme is complicated by the recent finding of Xiao et al² that cardiac ß₂ARs also activate G_i. Specifically, they showed that PTX pretreatment in rat cardiac myocytes increases ß₂AR-mediated responses, including increases in contraction amplitudes, calcium influx, and Ca_i transients. Their finding has been supplemented by additional data showing potentiation of ß₂AR functional responses by PTX as well as a direct labeling of G_i proteins due to ß₂AR stimulation in rat and mouse cardiac myocytes.^{3 5 25} In contrast, ß₁AR responses are not sensitive to PTX, and direct photoaffinity labeling of mouse heart membranes did not detect any ß₁AR-mediated G_i activation.³ In the present study, we show that a similar situation occurs in human atrial tissue. More importantly, we find that G_i is also activated by stimulation of glucagon, histamine, and serotonin receptors. Thus, coupling to G_i is not a unique property of cardiac ß₂ARs. In fact, with the exception of ß₁ARs, coupling to both G_s and G_i appears to be a general property of cardiac G_s-coupled receptors in human atrial tissue.

Having demonstrated that ß₂AR and several other G_s-coupled receptors also activate G_i, we sought to determine whether disruption of G_i increases AC stimulation through G_s-coupled receptors. The data of Figure 5 show that disruption of G_i causes an increase in AC activity under every condition tested. An increase in basal and forskolin-stimulated AC activity after PTX treatment implies that there exists a tonic inhibition of AC by G_i. This tonic inhibition of AC by G_i may also be responsible for the observed increase in ß₁AR-stimulated AC activity after PTX treatment. Compared with the effect of PTX on AC stimulation through ß₁ARs, which do not couple to G_i, the effect of PTX on AC stimulation through ß₂ARs (and other G_s-coupled receptors that also couple to G_i) is greater. These results indicate that coupling of G_s-coupled receptors to G_i has functional consequences at the level of AC. This conclusion, however, disagrees with similar studies in rat myocytes in which PTX treatment had no significant effect on ß₂AR-mediated cAMP formation²⁶ or activation of protein kinase A.²⁵ On the basis of these data, Xiao et al¹⁰ present a model depicting that the cross talk between the pathways stimulated by ß₂AR coupling to G_s and G_i occurs at a point distal to AC and protein kinase A. Clearly, this rat model is not supported by our data with human atrial membranes.

The physiological function of the coupling of G_s-coupled receptors to G_i also remains to be fully evaluated. The ability of G_s-coupled receptors to also activate G_i raises the possibility that this pathway may become exaggerated when G_i levels are increased in both failing and aged hearts. Indeed, a decrease in ßAR function in failing human heart and in the elderly has been observed.^{27 28} In addition to ßARs, histamine and serotonin receptors show decreased ability to stimulate AC in failing heart.²³ Whether this decrease in AC stimulation through G_s-coupled receptors in failing heart and aging is the result of an enhanced coupling to G_i remains to be determined. Interestingly, it was recently shown that activation of G_i by ß₂AR stimulation opposes the apoptotic action of ß₁AR stimulation in rat cardiac myocytes, and it was suggested that an increased activation of G_i may be of therapeutic value.²⁹

In summary, the present study provides the first evidence that ß₂ARs and several other GPCRs in human atrial tissue couple to both G_s and G_i; disruption of the coupling of these receptors to G_i enhances their ability to stimulate AC.

	Acknowledgments

 
This research was supported by NIH Grants AG15817 (to M.M.K.), HL57447 (to D.A.S. and M.M.K.), AG00745 (to D.A.S.), and 2-T32-AG00029 (to M.D.R.), and GCRC759, as well as a Foundation for Anesthesia Education and Research grant (to M.A.G.). We thank Dr David Kellogg of Duke University for editorial assistance. We thank Wayne Cohen, Kelli Colgan, Keinya Lee, and Scott Lee for their efforts in the collection of tissue samples.

Received April 14, 2000; revision received August 21, 2000; accepted August 21, 2000.

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