Cardiac-specific overexpression of human beta2 adrenoceptors in mice exposes coupling to both Gs and Gi proteins

Abstract

1. Left atrial strips from transgenic (TG4) mice with cardiac-specific overexpression ( approximately 200-fold) of the beta(2) adrenoceptor (beta(2)AR) were isolated, and their isometric force of contraction (F(c)) in response to electrical stimulation was measured. 2. The betaAR 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 beta(2)AR, as shown by their sensitivity to the beta(2)AR antagonist ICI-118,551 (100 nM; pA(2) 8.60+/-0.07, 8.45+/-0.19, for 'up-phase' and 'down-phase,' respectively), but not the beta(1)AR antagonist CGP-20712A (100 nM). Conversely, nontransgenic littermate preparations responded to isoprenaline treatment solely by an increase in F(c), which was beta(1)AR-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 beta(2)AR coupling to G(s) and G(i) proteins, consistent with the model of Daaka et al., in which protein kinase A phosphorylation of the beta(2)AR causes a switch from G(s) to G(i) protein coupling.

Figure 1

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.

Figure 2

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 F_c at the time of antagonist administration (90 min prior to isoprenaline).

Figure 3

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 F_c just prior to isoprenaline administration.

Figure 4

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 F_c just prior to isoprenaline administration.

Figure 5

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 F_c 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.

Figure 6

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-G_s signalling; two-state model). In b, application of an agonist reduces stimulus by activating receptors (e.g. G_i-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 K_APP, 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 K_APP, whereas that of an agonist is to the left of its K_APP. 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.