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Epinephrine Activates Both G_s and G_i Pathways, but Norepinephrine Activates Only the G_s Pathway through Human ₂-Adrenoceptors Overexpressed in Mouse Heart

Institute of Pharmacology and Toxicology, Medical Faculty Carl Gustav Carus, Dresden University of Technology, Dresden, Germany (J.F.H., U.R.); and Department of Physiology, University of Cambridge, Cambridge, United Kingdom (A.J.K.)

Received August 13, 2003; accepted February 9, 2004

	Abstract

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Isoproterenol increases and decreases contractile force at low and high concentrations, respectively, through ₂-adrenoceptors overexpressed in transgenic mouse heart (TG4), consistent with activation of both G_s and G_i proteins. Using TG4 hearts, we demonstrated that epinephrine behaves like isoproterenol, but norepinephrine does not. Epinephrine both increased (-log EC₅₀M = 9.4) and decreased (-log EC₅₀M = 6.5) left atrial force. Pertussis toxin (PTX) abolished the negative inotropic effects of epinephrine, consistent with mediation through G_i protein. Norepinephrine only increased contractile force (-log EC₅₀M = 7.5). Norepinephrine (10-100 µM) prevented the positive inotropic effects but hardly affected the negative inotropic effects of epinephrine. Cardiodepressive epinephrine concentrations (1-10 µM) antagonized the positive inotropic effects of norepinephrine. In the free wall of TG4 right ventricle, norepinephrine and low epinephrine concentrations caused positive inotropic effects, and high epinephrine concentrations caused PTX-sensitive negative inotropic effects, as observed in the left atrium. Epinephrine (10 nM), a concentration causing maximum increase in contractile force, and norepinephrine (1 and 100 µM) increased cAMP-dependent protein kinase activity in TG4 left ventricle. Cardiodepressive concentrations of epinephrine (1 and 100 µM) did not increase cAMP-dependent protein kinase activity. The inotropic results were simulated with a model of two ₂-adrenoceptor sites. For one site involved in receptor coupling to G_s, both epinephrine and norepinephrine compete. The other site, recognized by epinephrine but not by norepinephrine, leads to receptor G_i coupling.

In addition to coupling to G_s protein, ₂-adrenoceptors, activated by isoproterenol, can also couple to G_i protein (recombinant receptors: Daaka et al., 1997; murine heart: Xiao et al., 1999; human heart: Kilts et al., 2000), but the relevance to human heart function is not clear. In a mouse phenotype descendent from the original TG4 mouse described by Milano et al. (1994), we (Heubach et al., 2003) and others (Hasseldine et al., 2003) have shown that isoproterenol both increases and decreases contractility through overexpressed ₂-adrenoceptors in the left atrium. The cardiodepressant effects of (-)-isoproterenol were abolished by pretreatment with pertussis toxin, consistent with mediation through ₂-adrenoceptors coupled to G_i protein (Hasseldine et al., 2003; Heubach et al., 2003).

We compared in this TG4 phenotype (Heubach et al., 2003) the effects of the physiological catecholamines norepinephrine and epinephrine on atrial and ventricular contractility. Daaka et al. (1997) proposed that isoproterenol-evoked coupling to G_s protein of the ₂-adrenoceptor induces PKA-catalyzed phosphorylation of the receptor, which in turn couples to G_i. This switch of G_s to G_i coupling of the ₂-adrenoceptor (Daaka et al., 1997) provides one plausible biochemical explanation for the positive (G_s coupling) and negative (G_i coupling) inotropic effects of isoproterenol (Hasseldine et al., 2003; Heubach et al., 2003). To further test the hypothesis of the PKA-induced switch of G protein, we compared the ventricular PKA activity before and during the administration of epinephrine and norepinephrine.

Both catecholamines caused cardiostimulation, but only epinephrine also elicited cardiodepression. The positive inotropic effects of epinephrine and norepinephrine were accompanied by an increase of ventricular PKA activity. The decline of force at high epinephrine concentrations was associated with a decrease of PKA activity. The inotropic results were simulated with a model for two ₂-adrenoceptor sites. For the site that couples to G_s, norepinephrine and epinephrine compete. The other site is only recognized by epinephrine, but not by norepinephrine, and leads to coupling to G_i.

	Materials and Methods

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Materials. [-³²P]ATP was obtained from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK). ICI 118,551 was from Tocris Cookson Inc. (Bristol, UK). CGP20712A was from Novartis (Basel, Switzerland). Pertussis toxin, prazosin, yohimbine, forskolin, (-)-epinephrine hydrochloride, and (-)-norepinephrine hydrochloride were from Sigma Chemie (Deisenhofen, Germany). Phenoxybenzamine was from Röhm Pharma (Darmstadt, Germany).

Transgenic Mice. The experiments and the use of pertussis toxin were approved by the German Home Office (Az 75-9168.11-1-2000-10). Transgenic mice were descendents from the TG4 mice at Duke University (Durham, NC) that overexpress human ₂-adrenoceptors in a heart-specific manner (Milano et al., 1994). Originally, -adrenoceptor density was increased 200-fold, and the overexpression was verified more recently for our TG4 colony (260- to 435-fold overexpression) (Heubach et al., 1999, 2001; Graf et al., 2001). TG4 mice were propagated in Dresden by breeding TG4 female mice with wild-type C57BL6 male mice until generation F₇. Genotypes were determined as described previously (Heubach et al., 2001).

Isolated Cardiac Tissues. Mice of either gender were killed by dislocation of the neck, and the hearts were dissected and placed in oxygenated, modified Tyrode's solution at room temperature containing 126.7 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 1.05 mM MgCl₂, 22.0 mM NaHCO₃, 0.42 mM NaH₂PO₄, 0.04 mM EDTA, 0.2 mM ascorbic acid, and 5.0 mM glucose. The solution was maintained at pH 7.4 by bubbling a mixture of 5% CO₂ and 95% O₂. Left atrium and the free wall of the right ventricle were rapidly dissected and mounted in pairs, attached to Swema 4-45 strain gauge transducers in an apparatus (Blinks, 1965) containing the above solution at 37°C, paced at 2 Hz, and stretched as described previously (Oostendorp and Kaumann, 2000; Heubach et al., 2002). Usually four thin left ventricular strips from each heart were also dissected for PKA assays. Contractile force was recorded through PowerLab amplifiers on a Chart for Windows, Version 4.0, recording program (ADInstruments Pty Ltd., Castle Hill, Australia).

All tissues, including left ventricular strips floating freely in the organ baths, were exposed to phenoxybenzamine (6 µM) for 90 min followed by washout to irreversibly block -adrenergic receptors and both neuronal and extraneuronal uptake of catecholamines (Gille et al., 1985). The experiments were carried out in the presence of CGP20712A (300 nM) to selectively block ₁-adrenoceptors (Heubach et al., 2002, 2003). We have previously shown in TG4 left atrium that the positive inotropic effects of norepinephrine are resistant to blockade by 300 nM CGP20712A and therefore are exclusively mediated through ₂-adrenoceptors (Heubach et al., 2003).

In Vivo PTX Treatment. Mice were injected with PTX (600 µg/kg i.p.) or 0.9% NaCl. Twenty-four hours later, left atrial and right ventricular free walls from PTX-treated mice were set up into the same organ bath. In wild-type C57BL6 mice, this procedure inhibited by 82% in vitro ADP-ribosylation of ventricles as measured by [³²P]ADP-ribose incorporation (Heubach et al., 2002).

PKA Assay. The PKA activity ratio was assayed in frozen strips of left ventricle as described previously (Kaumann et al., 1989; Murray et al., 1990). Thin left ventricular strips, weighing 15 mg, were placed into modified Tyrode's solution. The tissues were quickly frozen in liquid nitrogen for PKA assays in control tissues and tissues 10 min after cumulative exposure to the last catecholamine concentration or to forskolin. The tissues were processed with a Polytron homogenizer (Kinematica, Basel, Switzerland) (7-mm probe at speed setting 8 for 10 s) in 40 volumes of ice-cold buffer, pH 6.8, containing 10 mM sodium phosphate, 10 mM EDTA, and 0.5 mM 3-isobutyl-L-methylxanthine and centrifuged at 4°C for 5 min at 6000g. PKA was determined by incubating 10 µl of resultant supernatant for 2 min at 30°C with 10 µl of [-³²P]ATP and 50 µl of assay buffer with final concentrations of 20 µM malantide, 0.3 mM [-³²P]ATP, 50 mM Na₂HPO₄, 10 mM MgCl₂, 1.0 mM EGTA, and 0.010% Tween 20 (w/w) and in the absence or presence of 2 µM cAMP. The reaction was terminated with 10 µl of 1 M HCl, after which 35 µl of sample was spotted onto phosphocellulose (P81) papers. These papers were washed six times for 2 min with 0.05% (w/v) tetraphosphoric acid/38 mM H₃PO₄, and then they were dried and counted in water by Cerenkov radiation. The activity ratio was calculated by dividing the radioactivity (counts per minute) obtained in the absence of cAMP by that obtained in the presence of cAMP after subtracting blank values (HCl added before [-³²P]ATP). Samples in the presence and absence of 2 µM cAMP were assayed in duplicate and assays replicated in 3 to 10 tissues.

Simulations. It is assumed that the ₂-adrenoceptor has one binding site for norepinephrine (NE) and two independent binding sites for epinephrine (E). The affinity of the binding sites for epinephrine differs by three orders of magnitude so that the highaffinity sites will be fully saturated when the low-affinity sites begin to form complexes with epinephrine. Binding to the high-affinity site is assumed to trigger coupling to G_s protein, whereas binding to the low-affinity site stimulates coupling to G_i protein. Because G_s coupling stimulates whereas G_i coupling inhibits force of contraction, this model can account for the inhibition of the receptor configuration that stimulates G_s protein. Scheme 1 represents this model by sequential binding of epinephrine because binding to the low-affinity site can only occur to a significant extent once the high-affinity site is fully saturated. As shown in Scheme 1, the R·NE and R·E complexes couple to G_s and the R·E·E complex couples to G_i, with K_NE, K_E, and K_EE as the corresponding equilibrium dissociation constants and K_EE >> K_E. NE and E are concentrations of norepinephrine and epinephrine. The total receptor population R_t = R + R·NE + R·E + R·E·E. The relative responses of norepinephrine (r_NE) and epinephrine (r_E) are

(1)

View larger version (12K): [in this window] [in a new window]   Scheme 1. Model for reactions between norepinephrine and epinephrine with the 2-adrenoceptor.

and

(2)

in which e_NE and e_E are relative maximum effects: e_NE = 1 in both left atrium and right ventricle, whereas e_E = 1.4 in left atrium and e_E = 1.25 in right ventricle. The relative effects of combinations of norepinephrine and epinephrine are given by

(3)

Data Analysis. Concentration-effect curves for the catecholamines were cumulative. -Log EC₅₀M and -log IC₅₀M values for catecholamines were estimated from fitting a Hill function with variable slopes to concentration-effect curves from individual experiments. The data are expressed as mean ± S.E.M. of n = number of mice. Significance of differences between means were assessed with the use of either Student's t test or analysis of variance followed by Bonferroni or Dunnett's post hoc test at P < 0.05 using Instat software (GraphPad Software Inc., San Diego, CA). Simulations were calculated with Sigma Plot software version 4 (SPSS Inc., Chicago, IL).

	Results

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Left Atrium. Epinephrine increased left atrial contractile force at low concentrations. (-log EC₅₀M = 9.40 ± 0.06 for maximal effects, 9.56 ± 0.06 for steady-state effects, n = 9, N.S.) (Fig. 1, A and B). At high concentrations, epinephrine decreased force (-log IC₅₀M = 6.45 ± 0.04 for maximal relaxations, n = 5), an effect prevented by PTX (Fig. 1C). The maximum negative inotropic effect was observed at 10 µM epinephrine and was followed by a slow increase of force. Norepinephrine only increased contractile force (-log EC₅₀M = 7.53 ± 0.09 for maximal effects, 7.44 ± 0.08 for steady-state effects, n = 6, N.S.) (Fig. 1B). The effects of norepinephrine and epinephrine were simulated on Fig. 1D. The effects of PTX on the epinephrine responses were simulated in Fig. 1E.

View larger version (32K): [in this window] [in a new window]   Fig. 1. Effects of norepinephrine and epinephrine on TG4 left atrium and influence of PTX. A, representative experiments. B, comparison of the effects of epinephrine and norepinephrine. Maximum positive inotropic and negative inotropic effects at each agonist concentration were assessed for the construction of the concentration-effect curves. C, PTX-treatment of the mice abolished the negative inotropic effects of epinephrine. D, simulation of the effects of epinephrine and norepinephrine using, eqs. 1 and 2. E, simulation of the effects of epinephrine with and without PTX treatment, using eq. 2.

Norepinephrine (10-100 µM) prevented the increases in left atrial contractile force by epinephrine and slightly reduced the cardiodepressant potency of epinephrine, as shown in Fig. 2, A to C, and simulated in Fig. 2D. The -log IC₅₀M values for the relaxant effects of epinephrine were 6.45 ± 0.04 (n = 5), 6.28 ± 0.25 (n = 3, P = 0.4) and 5.96 ± 0.04 (n = 3, P = 0.05) in the absence and presence of 10 and 100 µM norepinephrine, respectively.

View larger version (28K): [in this window] [in a new window]   Fig. 2. Antagonism by norepinephrine of the positive inotropic effects of epinephrine but hardly of the negative inotropic effects of epinephrine on TG4 left atrium. A and B are representative experiments. A concentration-effect curve to norepinephrine was carried out up to 10 µM (A) or 100 µM (B), followed by a curve for epinephrine. C, curves for steady-state positive inotropic effects and maximal negative inotropic effects at each epinephrine concentration in the absence and presence of indicated concentrations of norepinephrine. The positive inotropic effects of norepinephrine are steady-state effects. D, simulation of the experiments shown in A, B, and C using eqs. 2 and 3.

Force-reducing concentrations of epinephrine (1 and 10 µM) antagonized the positive inotropic effects of norepinephrine in surmountable manner (Fig. 3, A-C). The -log EC₅₀M values for the steady-state effects of norepinephrine were 7.49 ± 0.08 (n = 6), 4.37 ± 0.10 (n = 3, P < 0.001), and 3.46 ± 0.25 (n = 3, P < 0.001) in the absence and presence of 1 and 10 µM epinephrine, respectively. The blockade of the norepinephrine effects by epinephrine was simulated in Fig. 3D.

View larger version (26K): [in this window] [in a new window]   Fig. 3. Antagonism by epinephrine of the positive inotropic effects of norepinephrine on TG4 left atria. A and B are representative experiments. A concentration effect curve to epinephrine up to 1 µM (A) and 10 µM (B) was carried out, followed by a curve for norepinephrine. C, curves for steady-state positive inotropic effects of norepinephrine in the absence and presence of the indicated epinephrine concentrations. D, simulation of the experiments shown in A, B, and C using eqs. 1 and 3.

The noncumulative administration of 10 µM epinephrine elicited positive inotropic responses (Fig. 4, B and C) that were smaller than the response to norepinephrine (100 µM, Fig. 4A). The responses tended to be biphasic (Fig. 4, B and C), with an initial fast component followed by a brief plateau and a slow component leading to a steady-state increase of 34 ± 4% of forskolin (n = 3). This biphasic pattern was not modified by additional treatment with the combination of ₁-selective prazosin (1 µM) plus ₂-selective yohimbine (1 µM) (Fig. 4D), leading to a steady-state increase of 36 ± 14% of forskolin (n = 3). The steady-state positive inotropic effect observed with the noncumulative administration did not differ from the steady-state positive inotropic effect of 10 µM epinephrine added cumulatively (30 ± 4% of forskolin). Thus, the same conclusions can be drawn from cumulative and noncumulative effects of 10 µM epinephrine on the left atrium.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Kinetics after noncumulative administration of a single high epinephrine concentration (B-D). Comparison of norepinephrine (A) and epinephrine (B). Shown are representative left atria. The effects of 10 µM epinephrine on force of contraction were similar in the absence (C) and presence (D) of the additional -adrenoceptor blockers prazosin (1 µM) and yohimbine (1 µM).

The maximum contractile force, observed under forskolin (3 µM), administered to terminate the experiments was not significantly different in the experimental groups shown in Figs. 1, 2, 3, 4.

Right Ventricle. The catecholamines tended to produce arrhythmias on the free wall of the right ventricle. Representative recordings from nonarrhythmic tissues are illustrated in Fig. 5. As observed in left atrium, both norepinephrine (Fig. 5A) and epinephrine (Fig. 5, B-D) produced positive inotropic effects, but only epinephrine elicited negative inotropic effects (Fig. 5, C and D). PTX prevented the negative inotropic effect of epinephrine (Fig. 5B). Also as observed in the left atrium, epinephrine antagonized the positive inotropic effects of noradrenaline (Fig. 5, C and D). Quantitative data and simulations from a limited number of arrhythmiafree ventricles are shown in Fig. 6, A and B. The -log EC₅₀M values for the positive inotropic effects of epinephrine were 9.04 ± 0.07 (n = 4) and 9.36 ± 0.25 (n = 3, P = 0.2) in ventricular preparations from PTX-untreated and PTX-treated mice. The -log IC₅₀M for the relaxant effects of epinephrine was 6.88 ± 0.09. The -log EC₅₀M values for the positive inotropic effects of norepinephrine in two ventricular preparations were 6.77 and 6.89.

View larger version (23K): [in this window] [in a new window]   Fig. 5. Representative experiments of the effects of norepinephrine and epinephrine, isolated or in combination, on TG4 right ventricle. A, effects of norepinephrine. B, effects of epinephrine in a ventricle obtained from a PTX-treated TG4 mouse. Carb, carbachol. C, effects of epinephrine (up to 1 µM) and norepinephrine. D, effects of epinephrine (up to 10 µM) and norepinephrine.

View larger version (28K): [in this window] [in a new window]   Fig. 6. A, inotropic effects of epinephrine and norepinephrine on TG4 right ventricle, and effects of PTX. B, simulations of the ventricular effects of epinephrine and of PTX treatment using eqs. 1 and 2.

PKA Activation. A low concentration of epinephrine (10 nM) and micromolar concentrations of norepinephrine (1 and 100 µM) increased the left ventricular PKA activity ratio (Table 1). However, micromolar concentrations of epinephrine (1 and 100 µM) failed to increase the PKA activity ratio (Table 1). The increases of PKA activity ratio by epinephrine (10 nM) and norepinephrine (1-100 µM) amounted to 28% and 38-30%, respectively, of the increase in PKA activity ratio caused by forskolin (3 µM) (Table 1).

	Discussion

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Our experiments demonstrate that the physiological agonists epinephrine and norepinephrine act differently through human ₂-adrenoceptors overexpressed in mouse heart. The results and simulations are consistent with a model of interaction of norepinephrine and epinephrine with and competition for the ₂-adrenoceptor coupled to G_s protein. In addition, high concentrations of epinephrine but not of norepinephrine induce coupling of the ₂-adrenoceptors to G_i protein. The mode of action of epinephrine resembles that of isoproterenol, which also has cardiostimulant and depressant effects in the TG4 phenotype used in this work (Heubach et al., 2003).

It has been proposed that PKA-catalyzed phosphorylation of the ₂-adrenoceptor induces a switch to G_i coupling from G_s coupling (Daaka et al., 1997), and our results with epinephrine, as well as previous results with isoproterenol (Hasseldine et al., 2003; Heubach et al., 2003), are apparently in line with this hypothesis. However, our norepinephrine data are inconsistent with the hypothesis of the PKA-evoked switch because increases in PKA activity and contractile force were observed in a 100-fold concentration range (1-100 µM), but the high norepinephrine concentration did not decrease PKA activity and contractile force. The increases in PKA activity by 10 nM epinephrine and 1 µM norepinephrine, concentrations causing maximum increases in contractile force, were of similar magnitude. As expected from the G_s G_i switch hypothesis, increasing the concentration of epinephrine 100-fold to 1 µM and even 10,000-fold to 100 µM reduced the PKA activity from its higher level produced by low epinephrine concentrations, consistent with uncoupling from G_s protein and coupling to G_i protein. On the contrary, when the norepinephrine concentration was increased 100-fold to 100 µM, the PKA stimulation persisted, suggesting persistent coupling to G_s without a switch to G_i coupling. Our results therefore suggest that PKA activation through the ₂-adrenoceptor and the subsequent shift of coupling from G_s to G_i is agonist-dependent: Norepinephrine (1-100 µM) stabilizes a G_s-coupled receptor configuration, which is consistently observed with both contractile force and PKA stimulation. Unlike norepinephrine, however, high epinephrine concentrations (1-100 µM) reduced both previously elevated PKA activity and contractility, probably through coupling of the ₂-adrenoceptor to G_i protein. The negative inotropic effect of epinephrine was prevented by PTX pretreatment, consistent with mediation through the G_i-coupled ₂-adrenoceptor.

Agonist-dependent coupling of a receptor to more than one G protein has been observed previously with other receptor systems (Kenakin, 1995b). Interestingly, epinephrine exhibits a 200-fold greater potency for activating the G_i pathway than activating the G_s pathway at recombinant _2C(10)-adrenoceptors, whereas the imidazoline agonist oxymetazoline only stimulates the G_i pathway (Eason et al., 1994). Furthermore, during submission of this work, an article supporting the concept of agonist-dependent selectivity for coupling of rat cardiac ₂-adrenoceptors was published (Xiao et al., 2003). These authors showed that the positive inotropic effects of the ₂-selective agonists salbutamol, zinterol, and procaterol, but not of fenoterol, are enhanced by PTX. Thus, fenoterol only activates the G_s pathway in rat heart expressing native ₂-adrenoceptors, as found by us for the effects of norepinephrine mediated through ₂-adrenoceptors overexpressed in mouse heart.

The increases and decreases in left atrial and right ventricular contractility caused by epinephrine are mirrored by increased and decreased PKA activity in left ventricle at low and high epinephrine concentrations, respectively, consistent with the G_s G_i switch in the three cardiac regions. On the other hand, the inotropic and PKA data with norepinephrine are consistent with G_s coupling but not with G_i coupling in the three cardiac regions.

The maximum inotropic effects of norepinephrine tended to be smaller than those of epinephrine in both atrium and ventricle (i.e., e_NE e_E). As expected from competition for binding of epinephrine to the G_s-coupled ₂-adrenoceptor site, increasing concentrations of norepinephrine that partially activate the receptor through this site antagonized the cardiostimulant effects of epinephrine. This pattern resembles that of a classic partial agonist (norepinephrine) antagonizing the effects of a full agonist (epinephrine). As expected from a lack of interaction with the receptor site that would couple to G_i protein, norepinephrine hardly affected the cardiodepressive effects of epinephrine, which are mediated through this site. The small decrease in negative inotropic potency of epinephrine, observed under 100 µM norepinephrine, was anticipated by the model (simulation in Fig. 2D).

Under concentrations that depress contractile force, epinephrine becomes a competitive antagonist of the positive inotropic effects of norepinephrine. The experimental difference of -log EC₅₀ values of norepinephrine in the absence and presence of 1 µM epinephrine (7.49 - 4.37 = 3.12 log units) is similar to the 3.4 log units theoretically expected using a dissociation equilibrium constant K_E = 0.4 nM for epinephrine as antagonist at the ₂-adrenoceptor site coupled to G_s. The logCR = log(1 + [E]/K_E) = log(1 + 1000/0.4) = 3.4, where CR is the EC₅₀ ratio of norepinephrine in the presence and absence of 1 µM epinephrine ([E]) (Fig. 3). Similarly, the observed EC₅₀ ratio of norepinephrine under 10 µM epinephrine of 4 log units (7.49-3.46 = 4.03) was similar to the expected 4.4 log units. These quantitative agreements support the use of the atrial positive inotropic potency of epinephrine, EC₅₀ 0.4 nM, as K_E = 0.4 nM in the model.

To model our experiments, we assumed as a first approximation that our potency estimates (EC₅₀ values) were equivalent to the K values (K_NE and K_E) used in the model. Our affinity estimates for norepinephrine, deduced from inotropic EC₅₀ values in TG4 atrium (K_NE = 30 nM) and ventricle (K_NE = 150 nM) were similar to the dissociation equilibrium constant for norepinephrine (K_NE = 210 nM), estimated from inhibition of membrane binding of (-)-¹²⁵I-cyanopindolol to human ventricular ₂-adrenoceptors (Kaumann et al., 1995). However, for epinephrine, the inotropic EC₅₀ values in TG4 atrium (K_E = 0.4 nM) and TG4 ventricle (K_E = 0.9 nM) were lower than the corresponding dissociation equilibrium constant from binding inhibition (K_E = 15 nM) (Kaumann et al., 1995). The discrepancy between the binding K_E estimate from human ventricle and our EC₅₀ for the positive inotropic effects of epinephrine in TG4 myocardium may be caused by the oversimplification of equating EC₅₀ = K_E. However, for two reasons, our EC₅₀ values, estimated functionally, seem to reflect a high-affinity state. First, our EC₅₀ values for epinephrine and norepinephrine agree with the corresponding EC₅₀ values (1 and 100 nM, respectively) for cAMP accumulation induced by epinephrine and norepinephrine through recombinant ₂-adrenoceptors (Swaminath et al., 2004). Second, the stimulant potency (EC₅₀) and blocking potency of epinephrine against norepinephrine on the left atrium were consistently subnanomolar, suggesting that epinephrine causes half-maximal ₂-adrenoceptor occupancy at the estimated K_E of 0.4 nM, consistent with the mass law assumptions of the model.

Although the simple mass law model yielded satisfactory simulations for several experimental conditions, it predicted nearly complete reversal of the positive inotropic effects of epinephrine 10 µM and abolishment at 100 µM, which was not observed (Figs. 1D and 3D). In the left atrium, the experimental maximum negative inotropic effect occurred at 10 µM epinephrine with a residual force equivalent to 19 to 25% of the forskolin response. This negative inotropic response was followed by a slow increase in force (Fig. 1). Furthermore, 100 µM epinephrine tended to increase the force of contraction with slow kinetics. The slow increase in contractile force was also observed after application of 10 µM epinephrine as a single concentration, and the effect was resistant to -adrenoceptor blockade by the combined treatment with phenoxybenzamine, prazosin, and yohimbine. The discrepancy between the prediction of the model and the experimental results could be attributed, at least in part, to the slow positive inotropic effects of epinephrine, of unknown nature, which became apparent at 10 and 100 µM and would partially oppose the predicted cardiodepression. However, it is also plausible that the G_i activation failed to oppose completely the G_s activation produced by epinephrine in the left atrium. In contrast to the left atrium, in ventricle, high epinephrine concentrations completely reversed the positive inotropic effects of low concentrations, as predicted by the model (Figs. 5D and 6, A and B).

The kinetics of the positive inotropic effects of 10 µM epinephrine, administered noncumulatively to the left atrium, were biphasic. As expected from the blunting effect of G_i stimulation, the positive inotropic response to epinephrine was considerably smaller than that of a maximally effective concentration of norepinephrine (Fig. 4). We interpret the fast initial component as residual effects mediated through G_s not completely opposed by G_i, and the late slow component as the unknown effect resistant to -adrenoceptor blockade. Consistent with this interpretation is that PTX treatment abolished the negative inotropic effect of epinephrine (Fig. 1).

The inotropic results from the right ventricle were quantitatively similar but not identical with those of the left atrium. The negative inotropic effects of epinephrine seemed more pronounced in the ventricles (Figs. 5 and 6) than in the left atrium (Fig. 1). The negative inotropic effects of epinephrine occurred at lower concentrations in ventricle than in atrium. The ratio between K_EE and K_E in ventricle was only 147, but it was 1000 in the left atrium. G_i-mediated inhibition of contractile force by epinephrine would therefore be expected to oppose G_s-mediated increases in contractile force more in ventricle because the cardiodepression occurs at lower epinephrine concentrations than in atrium. Inactivation of G_i with PTX would consequently be predicted to enhance further the positive inotropic effect of epinephrine in ventricle than in atrium, as observed in the simulation (compare Figs. 1E and 6B). However, because of large errors in basal force, the maximum positive inotropic effects of epinephrine were not significantly different in atria and ventricles from PTX-untreated and PTX-treated mice.

The experimental errors of the effects of the catecholamines were large. However, using the experimental parameters, the model was able to simulate several effects of the catecholamines, separately and in combination, despite the large experimental errors of the effects. This was possible because of the relatively small errors of the catecholamine concentrations causing half-maximal effects under various conditions. The simple mass law relations of the model agreed reasonably well with some interactions of catecholamines with the ₂-adrenoceptor, as reflected through the inotropic effects.

The coupling of the ₂-adrenoceptor to G_i protein has been proposed to exert a cardioprotective role against G_s protein-mediated cardiac overstimulation, especially in patients with heart failure who have high noradrenaline levels (Xiao, 2000). Stimulation of ₁-but not ₂-adrenoceptors produces apoptosis in rat heart (Communal et al., 1999), and the ₂-adrenoceptor of murine heart seems to deliver antiapoptotic signals through G_i-dependent coupling to phosphatidylinositol 3'-kinase (Chesley et al., 2000; Zhu et al., 2001). Both norepinephrine and isoproterenol have been reported to prevent hypoxia-induced cellular nuclear fragmentation through G_i-coupled ₂-adrenoceptors in cultured myocytes from the hearts of neonatal rats (Chesley et al., 2000). Our results, demonstrating that norepinephrine does not induce coupling of human ₂-adrenoceptors to G_i protein in TG4 ventricle, suggest a difference with murine ₂-adrenoceptors. The suggestion of Xiao (2000) was derived from data of Kilts et al. (2000), who used isoproterenol to demonstrate coupling of human atrial ₂-adrenoceptors to G_i protein. However, from an extrapolation of our results, it would seem that sympathetic nerve stimulation is unlikely to cause G_i-mediated protection, because interaction of the physiological neurotransmitter norepinephrine with the human ₂-adrenoceptor would not result in coupling to G_i protein but would actually enhance cardiostimulation through coupling to G_s protein. Further work is necessary to understand the differences between murine and human ₂-adrenoceptors regarding G_i protein coupling.

Simultaneous coupling of a receptor to more than one G-protein often becomes more evident at a high density of recombinant receptors (Eason et al., 1992; Kenakin, 1995a). The relevance of our finding with overexpressed human ₂-adrenoceptors into murine heart needs to be tested at native human cardiac ₂-adrenoceptors expressed at physiological density.

We conclude that coupling of the human ₂-adrenoceptor to G_i protein is agonist-dependent. Epinephrine and isoproterenol, but not norepinephrine, interact at a binding site of the ₂-adrenoceptor that will lead to coupling to G_i protein and which is different from the site that induces coupling to G_s protein. The failure of norepinephrine to affect substantially the epinephrine-induced relaxation is consistent with a lack of recognition of the site which, when activated by epinephrine, leads to the G_i-coupled ₂-adrenoceptor.

	Acknowledgements

 
We are grateful to Dr. V. L. Lew for inspiring suggestions with the simulations and to Romy Kempe and J. A. Lynham for excellent technical assistance.

	Footnotes

 
This work was supported by grants from the Deutsche Forschungsgemeinschaft and the British Heart Foundation.

ABBREVIATIONS: TG4, transgenic mouse with cardiac overexpression of the human ₂-adrenoceptor; CGP20712A, 2-hydroxy-5(2-((2-hydroxy-3-(4-((methyl-4-trifluomethyl)-1H-imidazole-2-yl)-phenoxy)propyl)-amino)ethoxy)-benzamide monomethane sulfonate; PTX, pertussis toxin; PKA, cAMP-dependent protein kinase; ICI 118,551, (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol; NE, norepinephrine; E, epinephrine.

Address correspondence to: Dr. Alberto J. Kaumann, Department of Physiology, University of Cambridge, Downing Street, Cambridge CB2 3EG, United Kingdom. E-mail: ajk41{at}hermes.cam.ac.uk

	References

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