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. 2003 Apr 11;138(7):1358–1366. doi: 10.1038/sj.bjp.0705191

Cardiac-specific overexpression of human β2 adrenoceptors in mice exposes coupling to both Gs and Gi proteins

A R G Hasseldine 1,2,*, E A Harper 2, J W Black 1,2
PMCID: PMC1573787  PMID: 12711637

Abstract

  1. Left atrial strips from transgenic (TG4) mice with cardiac-specific overexpression (∼200-fold) of the β2 adrenoceptor (β2AR) were isolated, and their isometric force of contraction (Fc) in response to electrical stimulation was measured.

  2. The βAR agonist isoprenaline elicited negative inotropic responses in all left atrial strips; in 6/11 preparations, it also had a small positive inotropic effect. This ‘up-phase' was observed from 0.1 to 10 nM, with the ‘down-phase' occurring at higher concentrations. Both phases were mediated by β2AR, as shown by their sensitivity to the β2AR antagonist ICI-118,551 (100 nM; pA2 8.60±0.07, 8.45±0.19, for ‘up-phase' and ‘down-phase,' respectively), but not the β1AR antagonist CGP-20712A (100 nM). Conversely, nontransgenic littermate preparations responded to isoprenaline treatment solely by an increase in Fc, which was β1AR-mediated.

  3. Pretreatment of left atrial strips with either 10 nM isoprenaline or 1 mM 8-bromo-cAMP significantly attenuated the TG4 ‘up-phase', while having no effect on either the TG4 ‘down-phase' or the littermate controls' responses. B. pertussis toxin treatment of the animals prevented isoprenaline's negative inotropic effects in TG4 preparations, but had no effect in littermate controls.

  4. The findings imply that the responses of TG4 left atrium to isoprenaline are because of β2AR coupling to Gs and Gi proteins, consistent with the model of Daaka et al., in which protein kinase A phosphorylation of the β2AR causes a switch from Gs to Gi protein coupling.

Keywords: β-Adrenoceptor, transgenic mice, receptor overexpression, protein kinase A, receptor coupling

Introduction

TG4 mice were first described by Milano et al. (1994), who reported that cardiac-selective high levels of β2AR overexpression could be attained by using an α-myosin heavy-chain promoter in front of the β2AR gene. The resulting phenotype was one of elevated right atrial rate and left atrial force of contraction, because of elevated basal cAMP generation. In a subsequent paper, evidence for β2AR constitutive activity was presented using the TG4 isolated left atrium (Bond et al., 1995). In this preparation, up to 50 nM isoprenaline (ISO) failed to evoke a response. Following the establishment of a TG4 colony by our laboratory in 1997, based on the kind donation of some breeding pairs by R. Lefkowitz, Prendergast et al. (2000) reported that the phenotype of the TG4 left atrium differed from the original reports. While ISO evoked increases in force of contraction (Fc) in nontransgenic littermate controls (LMC), it elicited mixed positive and negative inotropy in TG4s. Prendergast et al. (2000) hypothesised that the negative inotropic response was because of β2AR–Gi protein coupling.

G protein-coupled receptors (GPCR) are capable of coupling to more than a single class of G-protein (for review, see Kenakin, 1995), a phenomenon that has been observed for several disparate receptor classes, such as amine receptors (Berg et al., 1998; Brink et al., 2000), CB1 cannabinoid receptors (Bonhaus et al., 1998), and δ-opioid receptors (Prather et al., 1994), at both high receptor densities, and physiological expression levels. β2AR have typically been regarded as Gs protein-coupled receptors. β2AR coupling to Gi was first described by Asano et al. (1984), in reconstituted membranes, where β2AR, colocalised with Gi protein, were able to stimulate GTPγS binding and GTPase activity. Xiao et al. (1995) observed that this was not isolated to artificial systems, and could occur in intact rat cardiomyocytes. Later work (Xiao et al., 1999) uncovered the phenomenon in isolated cells from both TG4 and wild-type mice. The mechanism by which this occurs is reported to depend on the switching of β2AR signalling from Gs to Gi by protein kinase A (PKA) phosphorylation of the receptor (Daaka et al., 1997).

In this study, we have further investigated the behaviour of ISO in TG4 left atrium, and have endeavoured to elucidate the mechanism by which its positive and negative inotropic effects are mediated. A preliminary report, covering parts of the work, was made at the meeting of the British Pharmacological Society, Dublin, July 2001 (Hasseldine et al., 2001).

Methods

Genotyping of the mice

All procedures involving animals were carried out in accordance with the Animals (Scientific Procedures) Act, 1986. Mouse tail tips were removed under halothane anaesthesia, and used as a source of genomic DNA, which was extracted by phenol/chloroform method (Gross-Bellard et al., 1973). The DNA was UV-crosslinked to a nylon membrane and tested for the Q1presence of the transgene using a 32P-labelled (Stratagene Prime-it II Random Primer Kit) oligonucleotide probe, specific for the SV40 intron it contains. The blot was visualised using a phosphorimager.

Measurement of receptor overexpression

Radioligand binding saturation analysis was used to determine the level of β2AR overexpression. Cell membranes were prepared from whole hearts using two sequential steps of tissue/pellet polytron homogenisation (Kinematica AG; PT-DA 3020/2TS; setting 12, ∼8 s), and low-speed centrifugation (100 × g for 7 min), followed by high-speed centrifugation (39,800 × g for 15 min) of the resulting pooled supernatant. The membranes were suspended in modified Krebs–Henseleit solution (118 mM NaCl, 2.5 mM CaCl2, 1.2 mM K2HPO4, 4.7 mM KCl, 1.2 mM MgSO4, 25 mM NaHCO3, 11.1 mM glucose). Selective β2AR binding was achieved with 0.01–3nM of the β1/β2AR antagonist radioligand [3H]-CGP 12177A, by including 5 μM β1AR-selective antagonist (CGP-20712A; β1AR pKI 9.6, β2AR pKI 5.7; Dooley et al., 1986). 1 μM propranolol was used to determine nonspecific binding. The mixture was incubated for 2.5 h at 32°C, before rapid filtration with a Brandell Cell Harvester. The filters were washed (3 × 3 ml) with ice-cold 50 mM Tris-HCl (pH 6.9 at 21±3 °C) before bound radioactivity was determined by liquid scintillation counting.

Bioassay of 1/2 left atrium

Methods were as described by Prendergast et al. (2000). Briefly, male and female TG4 mice and their nontransgenic LMC (24–31 g; 2–8 months old) were killed by cervical dislocation. Their hearts were removed, and the left atria resected. The remainder of the heart was immediately frozen on dry ice, and stored at −86°C until required for radioligand binding. The left atria were bisected, and each strip was tied with a loop of cotton at both ends, and suspended within a 20 ml organ bath, in constantly oxygenated (5% CO2 in O2) modified Krebs–Henseleit solution (as above) at 32°C. A force of 0.8 g, previously determined to be optimal for this preparation, was then applied. After the application of tension, the muscle strips relaxed to a resting tension of approximately half that applied. Strips were then stimulated electrically via punctate electrodes (1 ms square wave pulses at 1 Hz, 130% threshold V), and their force of contraction measured using an amplitude meter (source: noncommercial), which records dynamic force as the difference between resting and peak force. Experiments were performed in the presence of 100 nM desipramine, 10 μM corticosterone and 3 μM phentolamine, to block catecholamine uptake and αAR. βAR antagonists, where applied, were allowed to equilibrate for 90 min before agonist exposure.

B. pertussis toxin (PTX) treatment

Mice were treated by intraperitoneal injection of PTX in an aqueous suspension (100 μg kg−1; ∼3 μg in 0.15 ml), 48 h before assay. This dose was selected on the basis that it inhibited the negative inotropic effects of the adenosine receptor agonist 5′-(N-ethylcarboxamido)adenosine (NECA), indicating a loss of Gi protein function.

Analysis

Dose–response data were analysed by nonlinear regression, using Graphpad Prism 3.02, on a plot of effect (force of contraction: Fc) vs agonist concentration (log M), referred to as an E/[A] curve. A logistic function could be applied to some E/[A] curves (‘monophasic': Equation (1)). Others were classed as biphasic, as the agonist caused an increase followed by a decrease in Fc, and these were fitted by Equation (2)

graphic file with name 138-0705191e1.gif

where Fc is the measured force of contraction, Basal is the Fc immediately prior to agonist exposure, Max is the maximum Fc elicited by the agonist, p[A]50 is the midpoint curve location (negative logarithm of the molar concentration of A, which elicits half the maximum effect) and nH is the midpoint slope (Hill slope). The range of the curve was defined as (Max−Basal)

graphic file with name 138-0705191e2.gif

where, in addition to the above definitions, Min is the minimum Fc elicited by the agonist, and p[A]50u and p[A]50d are the midpoint curve locations of the ‘up' and ‘down' phases, respectively. The ‘up-range' of the curve was defined as (Max−Basal), with the ‘down-range' as (Max−Min). Both of these are therefore positive numbers. It was observed that a degree of overlap between the ‘up-phase' and the ‘down-phase' meant that the fitting programme had trouble discerning the true Max. Therefore, Max was constrained to the highest observed Fc when fitting biphasic curves.

The Hill slope (nH) of biphasic curves was not estimated. The inclusion of the slope factor would have introduced two extra parameters into the fitting equation, leading to an unacceptably high degree of redundancy (and interdependency) among the seven parameters.

Receptor saturation by radioligand binding produced data that were well described by a rectangular hyperbola (Equation (3)). These were also analysed using nonlinear regression, with Graphpad Prism3.02.

graphic file with name 138-0705191e3.gif

where [R]0 is the receptor density, pKD is the affinity (−log M concentration causing 50% occupancy) of the radioligand and [L] is its concentration.

Drugs and chemicals

(−)Isoprenaline, CGP-20712A, ICI-118,551, 8-Br-cAMP, (−)propranolol, phentolamine, corticosterone and desipramine were purchased from Sigma-Aldrich (Dorset, UK). [3H]-CGP 12177A was purchased from NEN Life Science (Cambridge, UK), and B. pertussis toxin from CN Biosciences (Nottingham, UK). The Prime-it II Random Primer Kit was purchased from Stratagene (Amsterdam, Netherlands).

Statistical analysis

Best-fit parameters from nonlinear regression of E/[A] curves and other measured parameters are given as mean±s.e.mean, and were compared by ANOVA or t-test, as stated in the text, using Graphpad Prism 3.02. The P threshold for determination of statistical significance was 0.05, although exact P-values are given where possible.

Results

Radioligand binding

β2AR density in TG4 cardiac membranes was 55.7±7.8 fmol mg−1 (original wet weight; n=10/10 determinations/animals), compared with 0.25±0.02 fmol mg−1 in LMC (n=6/28), representing 219±10 fold overexpression of the β2AR (see Figure 1).

Figure 1.

Figure 1

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Panel a shows β2AR density data from TG4 and littermate control (LMC) cardiac cell membrane saturation binding with [3H]-CGP-12177A. Data are expressed as femtomoles specific binding per milligram of tissue wet weight; means are plotted with s.e.mean indicated by the error bars. For TG4 mice, n=10 determinations from 10 animals (10/10), and for LMC, n=6 determinations from 28 animals (6/28). Note that there is a dual ordinate scale, such that the LMC scale is stretched 200-fold relative to that of the TG4. In panel b, a typical β2AR saturation curve in TG4 heart membranes is shown.

Basal left atrial force of contraction (Fc) and antagonist effects on baseline

There was no statistically significant difference between the baseline Fc of half left atria from TG4 and LMC mice (183±14 mg, n=49 and 172±13 mg, n=47, respectively; P=0.6 by unpaired t-test).

In the absence of antagonists, baseline force of contraction (Fc) in LMC preparations faded by 28±5% (n=11) over the 90 min incubation period. In the presence of 100 nM CGP-20712A, fade in Fc was 41±5% (n=8), with 100 nM ICI-118,551 it was 43±6% (n=11), and with a combination of both of these, it was 49±8% (n=9). There was no statistically significant effect of either antagonist (two-way ANOVA, P=0.06, 0.1 and 0.6 for, CGP-20712A, ICI-118,551 and interaction, respectively). The Fc of untreated TG4 preparations faded by 13±3% (n=11), which was significantly less than that seen in LMC preparations (unpaired t-test, P=0.02). Fade in Fc was 30±3% with ICI-118,551 (n=7), 10±3% with CGP-20712A (n=7), and 26±8% with both antagonists (n=6). Two-way ANOVA revealed a significant effect of ICI-118,551 (P=0.0005), but not of CGP-20712A (P=0.5). There was no significant interaction (P=0.9).

Effect of ISO on left atrial Fc

In LMC preparations (n=10), 0.1–30 nM ISO elicited concentration-dependent increases in Fc with no further effects at higher concentrations (see Figure 2). The mean best-fit values for p[A]50 and range are shown in Table 1. The β1AR antagonist CGP-20712A (100 nM; n=8), shifted the ISO E/[A] curve, in parallel, to the right (see Figure 2), yielding a pA2 of 9.35±0.10 (βIAR pKI ∼9.6, β2AR pKI ∼5.7; Dooley et al., 1986). The β2AR selective antagonist ICI-118,551 (100 nM; n=10) also shifted the ISO E/[A] curve to the right, although to a much lesser extent (0.25±0.08 log units), yielding a pA2 of 6.89±0.08 (β1AR pKB ∼7.2, Henry & Goldie, 1990). Q2Each of the antagonists had a significant effect on p[A]50 (two-way ANOVA, P<0.0001 and 0.03, for CGP-20712A and ICI-118,551, respectively), but did not affect either the range or Hill slope of the curves (two-way ANOVA P>0.05 for both antagonists in both tests). In none of the three tests was there a significant interaction (P>0.05 for each). The relation between baseline fade in Fc and E/[A] curve range was examined: in ICI-118,551-treated preparations, range correlated with fade (Pearson, P=0.008), but in the absence of antagonist and in CGP-20712A-treated tissues, there was no significant correlation (Pearson, P=0.6, 0.1, respectively).

Figure 2.

Figure 2

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The effect of isoprenaline on littermate control (LMC. in panel a) and TG4 (panel b) left atrial strip contractility, in the presence and absence of antagonists selective for the β1AR (CGP-20712A) and β2AR (ICI-118,551). Mean data points are shown with curves that have been simulated using mean best-fit values (s.e.mean indicated by error bars) from nonlinear regression of individual data sets, using a 4–5 parameter logistic (see text for equations, Table 1 for mean best-fit data). The units of the ordinate axis are change in force of contraction, expressed in milligram; zero was set as the Fc at the time of antagonist administration (90 min prior to isoprenaline).

Table 1.

Best−fit parameters from nonlinear regression of isoprenaline E/[A] curves in TG4 and LMC left atrial strips

    p[A]50u Up range (mg) p[A]50d Down range (mg) n
TG4: Biphasic controls 9.52±0.08 31±9 6.61±0.16 70±12 6
  Monophasic controls N/A 0±0 6.54±0.14 46±10 5
  All controls 9.52±0.08 17±7 6.57±0.10 59±8 11
  All+ICI−118,551 7.91±0.05 61±22 5.14±0.22 67±23 7
  Biphasic +CGP−20712A 9.43±0.28 19±8 6.70±0.16 63±1 4
  Monophasic +CGP−20712A N/A 0±0 6.74±0.12 89±9 3
  All+CGP−20712A 9.14±0.35 11±5 6.78±0.08 74±6 7
  All +both antagonists 7.60±0.06 42±9 5.36±0.25 61±13 6
LMC: controls 8.60±0.09 156±18 N/A N/A 11
  +ICI−118,551 8.35±0.06 155±25 N/A N/A 11
  +CGP−20712A 6.24±0.09 195±31 N/A N/A 8
  +both antagonists 6.14±0.06 116±18 N/A N/A 9
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ISO was added to half left atrial preparations in a cumulative fashion, in the presence or absence of 100 nM ICI−118,551, 100 nM CGP−20712A or a combination of the two. The data were fitted to a 4–5 parameter equation (see text for details). Best−fit values are expressed as mean±s.e.mean. Italicised p[A]50u values indicate that the figure is comprised only of those preparations whose responses permit the calculation of a p[A]50u value (i.e. biphasic preparations); the n for that row does therefore not apply to the italicised value.

In all TG4 preparations, ISO caused a decrease in Fc at concentrations in the range 0.1–10 μM. It was also observed that, in 6/11 preparations, an increase in Fc was elicited at lower concentrations (0.1–10 nM). Therefore, in order to employ the most accurate analytical technique of curve-fitting individual data sets rather than fitting the mean data, they had to be fitted by different equations (see methods, analysis section). Consequently, they are described as biphasic (6/11) or monophasic (5/11). The best-fit parameters from fitting of the individual E/[A] curves are shown in Table 1. There was no significant difference between biphasic and monophasic preparations in baseline before ISO, or midpoint location of the ‘down-phase' (p[A]50d; unpaired t-test, P>0.05 for each). There was no correlation observed between either initial basal Fc or baseline fade, and the magnitude of the ‘up-phase' (Spearman rank test, P=0.2, 0.8, respectively). As there was no a priori justification for the subgroup separation, statistical comparisons between control and antagonist-treated groups utilised the pooled best-fit parameters (n=11; see Table 1), where possible.

In the presence of the β2AR antagonist ICI-118,551 (100 nM), all ISO E/[A] curves in TG4s were biphasic, with ICI-118,551 causing a significant increase in the range of the ‘up-phase' (two-way ANOVA, P=0.004; see Table 1). ICI-118,551 had no effect on the range of the ‘down-phase' (two-way ANOVA, P=0.8). It was also observed that, in ICI-118,551-treated preparations, there was a significant positive correlation between the amount of baseline fade and the range of the ‘up-phase' (Pearson, P=0.0008). Both the ‘up' and ‘down-phases' of the ISO E/[A] curve were significantly shifted to the right by ICI-118,551 (two-way ANOVA, P<0.0001 for each), from which pA2 values of 8.60±0.07 and 8.45±0.19 respectively, were calculated (β2AR pKI ∼8.7, Bristow et al., 1989). The β1AR antagonist CGP-20712A (100 nM; β1AR pKI ∼9.6, β2AR pKI ∼5.7; Dooley et al., 1986) had no significant effect on any of the parameters measured or fitted, as tested by two-way ANOVA (see Table 1 and Figure 2; P>0.05 in every test). In none of the tests was there a significant interaction between the two antagonists (P>0.05 in every test).

Desensitisation assay

To study the mechanism of the biphasic response, the assay protocol was modified slightly. The main kinase enzymes that phosphorylate the β2AR are PKA and G protein-coupled receptor kinase (GRK). PKA, but not GRK, has been implicated in the switch from β2AR-Gs to -Gi coupling (Daaka et al., 1997). Selective PKA phosphorylation of β2AR can be achieved by elevating cAMP levels under conditions of low β2AR occupancy, because GRK, but not PKA, preferentially targets occupied receptors (Lohse et al., 1990). To this end, preparations were treated with 10 nM ISO until the positive inotropic response reached a plateau (∼10 min), after which it was washed out (three washes in ∼15 min). They were then exposed to cumulative concentrations of ISO. A pretreatment concentration of 10 nM was chosen because it was enough to elicit the maximum increase in Fc in both LMC and TG4, but is predicted to produce only ∼2% β2AR occupancy (Hasseldine et al., unpublished data, 1998, pKI 6.29±0.11). The second approach to selective activation of PKA was to pretreat with the cAMP analogue 8-bromo-cAMP rather than 10 nM ISO.

In LMC left atrial strips, 10 nM ISO elicited an increase in Fc that was not different from the response to 10 nM ISO as part of a cumulative E/[A] curve (104±25 mg acute, vs 136±29 mg cumulative in pre-exposed preparations, paired t-test, P=0.07; or vs 129±11 mg in those not pre-exposed, unpaired t-test, P=0.8; n=5 each). Pre-exposure to ISO had no effect on the slope, p[A]50 and range of the cumulative ISO E/[A] curve (see Figure 3). The best-fit values for pre-exposed and control preparations, respectively, were p[A]50 8.69±0.07 and 8.82±0.05, nH 1.54±0.12 and 1.31±0.08 and range 146±29 and 146±8 mg.

Figure 3.

Figure 3

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The effect of pretreatment with ISO (10 nM, 10 min) on littermate control (LMC; a) and TG4 (b) left atrial strips to subsequent, cumulative, isoprenaline. Columns indicate the mean effect (with s.e.mean) of acute exposure to 10 nM isoprenaline. Mean data points are shown with curves that have been simulated using mean best-fit values (s.e.mean indicated by error bars) from nonlinear regression of individual data sets, using a 4–5 parameter logistic (see text for equations). The units of the ordinate axis are change in force of contraction, expressed in milligram; zero was set as the Fc just prior to isoprenaline administration.

In TG4 preparations, 10 nM ISO elicited a small increase in Fc (in 6/7 preparations, a positive inotropic response was seen; 16±5 mg, n=7), which was not different from the effect of 10 nM ISO, applied as part of a cumulative curve, in time controls (30±7 mg, n=10; unpaired t-test, P=0.2). However, in no TG4 preparation pre-exposed to 10 nM ISO was there a quantifiable increase in Fc upon reapplication of ISO (Figure 3), so that E/[A] curves were all monophasic. By contrast, all time controls (which were also washed) were, in this experiment, biphasic. The p[A]50 of the monophasic curves was 6.69±0.04, with range 59±8 mg. These estimates were not different (unpaired t-tests, P>0.05) from the p[A]50d and range of the ‘down-phase' of the control curves (6.77±0.05, 63±7 mg). The p[A]50u and ‘up-phase' range, in controls, were 9.49±0.07 and 31±7 mg.

1 mM 8-Br-cAMP pretreatment (refer Figure 4) for 10 min caused an increase in TG4 left atrial Fc of 48±17 mg(n=5), which was not significantly different from its effect on LMC preparations (39±12 mg, n=4; unpaired t-test, P=0.7). Its effects mimicked those of ISO pretreatment: in LMC preparations, exposure to 8-Br-cAMP had no effect on later responses to cumulative ISO, whereas in TG4 preparations, the range of the ISO E/[A] curve's ‘up-phase' was significantly decreased (73±27 mg in time control, 9±5 mg in pre-exposed preparations; Mann–Whitney U-test, P=0.03).

Figure 4.

Figure 4

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The effect of pretreatment with adenosine cyclic monophosphate analogue 8-Br-cAMP (1 mM, 15 min) on littermate control (LMC; a) and TG4 (b) left atrial responses to subsequent, cumulative, isoprenaline. Columns indicate the mean effect (with s.e.mean) of acute exposure to 1 mM 8-Br-cAMP. Mean data points are shown with curves that have been simulated using mean best-fit values (s.e.mean indicated by error bars) from nonlinear regression of individual data sets, using a 4–5 parameter logistic (see text for equations). The units of the ordinate axis are change in force of contraction, expressed in milligram; zero was set as the Fc just prior to isoprenaline administration.

Effect of B. pertussis toxin (PTX) treatment

Baseline TG4 left atrial Fc was unchanged by PTX treatment (387±73 mg, n=5 and 370±86 mg, n=4; whole left atria from PTX and vehicle-treated animals, respectively). PTX treatment prevented the negative inotropic effects of ISO (n=5; see Figure 5), whereas vehicle treatment (n=4) had no effect on the curve shape or location compared with half left atria from untreated TG4. PTX significantly increased the magnitude of the ISO E/[A] curve's ‘up-phase' (126±32 mg with PTX, 21±10 mg with vehicle; unpaired t-test P=0.03), but had no significant effect on its location (p[A]50u 9.21±0.29, n=5 vs 9.37±0.16, n=3; unpaired t-test P=0.7). As well as the initial ‘up-phase', there was a second increase in Fc in some preparations (see Figure 5), at ISO concentrations greater than 10−6 M. The reason for this was not clear, and only the first phase was analysed. Other vehicle-treated TG4 best-fit parameters are as follows (cf. Table 1): p[A]50d=6.32±0.07, ‘up-range'=21±10 mg, ‘down-range'=123±28 mg – of the four curves, three were biphasic and one monophasic. Following ISO administration, in vehicle-treated TG4 preparations, 10 μM of the adenosine receptor agonist NECA reduced Fc below the minimum elicited by ISO (−226±98 mg from baseline before ISO). In PTX-treated preparations, NECA was unable even to reverse the positive inotropic effects of ISO, having only a very small negative inotropic effect (104±18 mg from before ISO; see Figure 5). PTX had no measurable effects on any parameters measured in LMC preparations (data not shown).

Figure 5.

Figure 5

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The negative inotropic effects of isoprenaline in TG4 left atrial strips are sensitive to B. pertussis toxin treatment. Controls were treated with vehicle (water). Mean data points are shown (s.e.mean indicated by error bars) with curves generated by nonlinear regression, using a five parameter logistic (see text for equations, Table 1 for mean best-fit data). The units of the ordinate axis are change in force of contraction, expressed in milligram; zero was set as the Fc immediately prior to isoprenaline administration. The point labelled ‘NECA' is the mean response of each group to 10 μM NECA, administered after the highest dose of isoprenaline.

Discussion

Radioligand binding has confirmed that TG4 mice still have ∼200-fold overexpression of cardiac β2AR, as previously reported (Milano et al., 1994; Bond et al., 1995). This therefore excludes the possibility that discrepancies between the respective bioassay results are because of a change in overexpression.

In isolated TG4 left atrial strips, ISO elicited both increases and decreases in force of contraction, contrasting with its solely positive inotropic effect in LMCs. The effects of ISO in LMC preparations are mediated by the β1AR, as shown by the high potency of CGP-20712A (pA2 9.35±0.10; β1AR pKI ∼9.6, β2AR pKI ∼5.7, Dooley et al., 1986), and the low potency of ICI-118,551 (pA2 6.94±0.08; β1AR pKB ∼7.2, Henry & Goldie, 1990; β2AR pKI ∼8.7, Bristow et al., 1989). This is consistent with the behaviour of left atria from other nontransgenic strains, such as Charles River CD1 (data not shown) or C57BL/10ScSn mice (Lu & Hoey, 2000), in Q3which ISO also elicits β1AR-mediated increases in Fc.

The positive and negative inotropic effects of ISO in TG4 left atria appeared to be solely β2AR-mediated, as shown by the shift that 100 nM ICI-118,551, but not 100 nM CGP-20712A, produced. It was therefore concluded that β2AR were coupling to both stimulatory and inhibitory pathways. Although β2AR are usually considered to be Gs protein-coupled receptors, it has been shown that β2AR–Gi coupling may occur in several systems (Asano et al., 1984; Xiao et al., 1995, 1999; Luo et al., 1999), and its patho-/physiological importance has been reviewed by Xiao (2000).

From previous work in our laboratory, it had been concluded that β2AR-Gi coupling occurred in TG4 isolated left atria, but it was thought that the β1AR also played a part in the positive inotropic effects of ISO (Prendergast et al., 2000). This was intuitively attractive, as there is no obvious reason for the disappearance of the functional β1AR population seen in LMC left atria. However, the shift produced by CGP-20712A was far smaller than expected for a β1AR-mediated effect (pA2 7.1 vs β1AR pK1 ∼9.6, β2AR pK1 ∼5.7, Dooley et al., 1986)–an enigma that was not resolved. The present work has demonstrated that β2AR mediate both phases of the TG4 response. To explain the apparent disappearance of the β1AR, it was considered that the excess β2AR may mask their signal. However, this was discounted on the basis that the β2AR-mediated positive inotropy can be desensitised by pretreatment with 10 nM ISO or 1 mM 8-Br-cAMP, whereas the β1AR (in LMC) cannot. Therefore, if β1AR played a part in the TG4 response, one would expect the positive inotropic effect of ISO to be maintained in TG4 preparations after pretreatment, mediated by the β1AR. The absence of this effect does not preclude the existence of a β1AR population in the tissue, but it suggests that β1AR do not play a part in the TG4 left atrial contractile response to ISO.

β2AR coupling to both Gs and Gi proteins was first shown by Asano et al. (1984). Later, a biochemical mechanism for the switch from Gs to Gi was proposed by Daaka et al. (1997): PKA phosphorylation of the β2AR. This mechanism, described in HEK293 cells, was confirmed in mouse submandibular gland cells (Luo et al., 1999). Consistent with this, we found that ISO pretreatment of TG4 left atrial strips was able to repress the ‘up-phase' of a subsequent ISO E/[A] curve. Moreover, exposure of the tissue to the cAMP analogue 8-Br-cAMP mimicked this effect, demonstrating that this phenomenon is cAMP-mediated, rather than dependent on a prior interaction between ISO and the β2AR. The efficacy of the wash out procedure and the β2AR-specificity of the Gs signalling inhibition were demonstrated by the absence of desensitisation phenomena in LMC preparations treated with ISO or 8-Br-cAMP. The involvement of PKA could not be tested directly in the TG4, as PKA is necessary not only for the switch in coupling to Gi, but also for downstream effects of β2AR-GS protein signalling (Zhou et al., 1997; Xiao et al., 1999).

The hypothesis that β2AR couple to Gi in TG4 left atrium is further supported by the effect of PTX, which, when administered 48 h prior to sacrifice, prevented the negative inotropic effects of ISO, and enhanced the magnitude of its positive effects. The latter suggests that β2AR-Gs signalling may be regulated (functionally antagonised) by Gi, consistent with literature reports, in which it has been shown that β2AR–Gi interactions limit the spread of β2AR–Gs signalling through the cytosol (‘compartmentalisation' of Gs signalling, Kuschel et al., 1999). Compartmentalisation, or functional antagonism, of β2AR-Gs signalling, could explain β2AR-mediated positive inotropy (ISO in TG4 without PTX) is lesser in magnitude than ISO's effect in LMC (β1AR mediated), or forskolin in LMC/TG4 (βAR independent; data not shown). Clearly, the small size of the β2AR-mediated positive inotropic effect is not because of low receptor density, as we have confirmed that β2AR are overexpressed in this system, and functionally, ISO's p[A]50u in TG4 is approximately 3 log units to the left of its binding affinity. The compartmentalisation hypothesis is also attractive because no left-shift in the ‘up-phase' was seen in response to PTX, implying that the functional antagonism of Gs by Gi affects the magnitude of stimulus by altering its range rather than its coupling efficiency (e.g. in Appendix, EMAX, rather than β). The small negative inotropic effect of NECA after PTX treatment indicates that the ADP-ribosylation of Gi was probably incomplete, but this does not alter the conclusion from the experiment, that Gi mediates ISO's negative inotropic action.

The observation that PTX did not affect baseline Fc suggests that there is no tonic Gi protein activation. Hence there is no evidence for β2AR constitutive activity – had this been present, but curtailed by Gi, PTX treatment would have increased basal left atrial Fc. The absence of β2AR constitutive activity may be because of enhanced levels of β2AR phosphorylation, which would also account for the lack of shift in the ISO E/[A] curve ‘down-phase' after ‘up-phase' desensitisation. In this situation, the phosphorylated β2AR is present in large numbers, meaning that overexpression of unphosphorylated receptor, in effect, occurs at a considerably lower level than 200-fold. When the tissue is pretreated, the loss of unphosphorylated receptors represents significant receptor depletion (and consequent loss of signal). By contrast, the concomitant increase in phosphorylated β2AR levels is slight, relative to the high numbers already present, and hence the p[A]50d does not change appreciably.

There are certain similarities between the present data and those from the original descriptions of TG4 mice (Milano et al., 1994; Bond et al., 1995), in which the two-state model of receptor activation (for model description, see Leff, 1995) was proposed to account for the observations. That is, some of the previous authors' data are also consistent with β2AR Gs/Gi coupling. The lack of response to ⩽50 nM ISO seen in TG4 atria may have been because the ‘up-phase' responses are small, when present, and difficult to characterise. Furthermore, it has been suggested that the negative inotropic effect of ICI-118,551 involves β2AR-Gi-directed agonism (Gong et al., 2002). However, despite the similarities, the data of Bond et al. (1995) cannot be reconciled with β2AR–Gi coupling, principally for two reasons. Firstly, force of contraction in the absence of agonist was reportedly three-fold higher in TG4 than LMC mice, implying that constitutive Gs-coupled β2AR activity existed. Secondly, by depletion of the β2AR population with pindobind, Bond et al. (1995) were able to cause the negative inotropic E/[A] curve of ICI-118,551 (its ‘inverse agonist' curve) to collapse to the left, as predicted by the two-state model. By contrast, removal of receptors should cause Gi-directed agonist E/[A] curves to collapse to the right (see Figure 6). This criterion is, in general, useful for distinguishing inverse agonism from ‘traditional' agonism phenomena, which may otherwise be difficult to separate.

Figure 6.

Figure 6

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Simulation showing the effect of reducing receptor density in two models of agonism. The large arrow in each graph refers to a progressive reduction in receptor number, which may be, for example, the result of receptor alkylation, or differential expression levels. In a, application of an inverse agonist reduces stimulus by reversing the constitutive receptor activity (‘deactivating' receptors, e.g. β2AR-Gs signalling; two-state model). In b, application of an agonist reduces stimulus by activating receptors (e.g. Gi-coupled receptors, signalling in the presence of elevated basal cAMP; traditional model). Stimulus units are arbitrary, and should not be used for comparison between the models. The E/[A] curve always collapses towards KAPP, which is, in both cases, the compound's ‘affinity constant,' when receptor density is reduced. An inverse agonist curve may be seen to lie to the right of its KAPP, whereas that of an agonist is to the left of its KAPP. The effect on curve location of reducing receptor number is thus a qualitative diagnostic, permitting the determination of the appropriate model. The equations and parameters used in the simulations are detailed in the Appendix.

In this study, we did not use pindobind, as the negative inotropic effects of ICI-118,551 appeared to be independent of β2AR, for the following reasons. They were not antagonised by the neutral βAR antagonist alprenolol (no significant shift with 100 nM. pKB 8.62, Hopkinson et al., 2000); secondly, the range of concentrations over which they were seen was 0.1–10 μM (p[A]50 6.39±0.23, n=7) – concentrations considerably higher than required for β2AR occupancy. Hence, the negative inotropic effects of ICI-118,551 may have been because of another action, perhaps direct inhibition of the G protein-gated inward rectifying potassium channel, which has been reported to occur at concentrations ⩾0.1 μM (Wellner-Kienitz et al., 2001). This channel is regulated by the βγsubunit of Gi protein, which has also been implicated in β2AR–Gi signalling (Daaka et al., 1997). Gong et al. (2002) proposed that the negative inotropic effect of ICI-118,551 is not only Gi- but also β2AR-dependent, but, as stated, evidence to the contrary was found by the present authors. In addition to the findings outlined above, separate experiments showed that the degree of baseline Fc fade in preparations from PTX-treated TG4 mice, in the presence of ICI-118,551 (31±4%, n=4), was comparable with that from non-PTX-treated half left atria, implying a Gi-independent mechanism of action. In the results presented here, the natural tendency of the left atrial contractions to ‘fade' with time, presumably because of fatigue or biochemical run-down of some sort, generally complicated the examination of the negative inotropic actions of ICI-118,551. The experiments undertaken with ICI-118,551 and alprenolol (alluded to above) were conducted over a shorter time course than the 90 min incubation period, after a long equilibration time, to overcome this problem.

Clearly, the phenotype of the TG4 is now different from that which was first described. Although the transgene is still present and functional – β2AR density has not changed – adaptations may have occurred to compensate for it. The TG4 colony has not been outbred since they were obtained from the USA, but it has been noted that, in our colony, perinatal mortality was initially higher in TG4 litters than in those of the descendants of the original LMCs (C. E. Prendergast, 2001, PhD thesis, University of London). It is possible that natural selection has influenced the TG4 ‘genetic background', which is an important determinant of phenotype (Wolfer & Lipp, 2000). Alternatively, the change in environment, with different pathogens, food and storage conditions, may have in itself had an effect. Whatever the reason, the phenotype now appears to be stable, with recent data closely resembling observations made 4–5 years ago. The data are also similar to those reported by other groups using the same strain of transgenic mouse (Xiao et al., 1999; Gong et al., 2000), suggesting that either the phenotypic change occurred fairly soon after the initial work was published, before the mice were distributed to other investigators, or that the adaptation is intrinsic to the long-term effect of β2AR overexpression.

In conclusion, this study has shown, by using selective antagonists, that β2AR in TG4 left atria mediate both positive and negative inotropic effects of ISO. These responses – as shown by the effects of Gi protein inactivation with PTX, or cAMP preactivation with 10 nM ISO or 1 mM 8-Br-cAMP – are consistent with β2AR coupling to Gs and Gi proteins, respectively, modulated by a PKA switch, as proposed by Daaka et al. (1997).

Acknowledgments

We acknowledge Drs C. E. Prendergast and N. P. Shankley, who set up the mouse colonies used in the current work and helped to develop the half left atrium assay, and Dr J. Morris, who assisted with mouse genotyping. Financial support for this work was kindly provided by the James Black Foundation.

Abbreviations

Fc

force of contraction

GRK

G protein-coupled receptor kinase

E/[A] curve

plot of effect, E, against agonist concentration [A]

ISO

isoprenaline

LMC

littermate control

NECA

5′-(N-ethylcarboxamido)adenosine

PKA

protein kinase A

PTX

B. pertussis toxin

Appendix : Simulation for Figure 6

The simulations were performed using Microsoft Excel 97. For comparison of ‘traditional' and inverse agonism, the same equations were used (two-state model) but with constitutive activity set at zero for the former

graphic file with name 138-0705191e4.gif
graphic file with name 138-0705191e5.gif
graphic file with name 138-0705191e6.gif

where [R]0 is the receptor density, [A] is the concentration of the agonist or inverse agonist, KA is the affinity of A for the receptor in its inactive state, α is the factor by which A is selective for the inactive over the active state and L is the equilibrium constant governing the isomerisation of unliganded receptor. The second equation is a simple sigmoid function, where the conversion of stimulus to effect is governed by the maximum effect parameter, EMAX, and the equilibrium constant, β.

For both simulations, [R]0 was set between 0.1 and 1000, at log unit intervals; KA was 10−7 M; EMAXwas 100; and β was 0.2.

For modelling ‘traditional' agonism, α was set at 4 × 10−6 and L at 106. When modelling inverse agonism, these were both set at 100. The pKAPP values are therefore 7.1 and 7.0, respectively.

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