Coupling of β 2 -Adrenoceptor to G i Proteins and Its Physiological Relevance in Murine Cardiac Myocytes

Abstract

Abstract —Transgenic mouse models have been developed to manipulate β-adrenergic receptor (βAR) signal transduction. Although several of these models have altered βAR subtypes, the specific functional sequelae of βAR stimulation in murine heart, particularly those of β 2 -adrenergic receptor (β 2 AR) stimulation, have not been characterized. In the present study, we investigated effects of β 2 AR stimulation on contraction, [Ca 2+ ] i transient, and L-type Ca 2+ currents ( I Ca ) in single ventricular myocytes isolated from transgenic mice overexpressing human β 2 AR (TG4 mice) and wild-type (WT) littermates. Baseline contractility of TG4 heart cells was increased by 3-fold relative to WT controls as a result of the presence of spontaneous β 2 AR activation. In contrast, β 2 AR stimulation by zinterol or isoproterenol plus a selective β 1 -adrenergic receptor (β 1 AR) antagonist CGP 20712A failed to enhance the contractility in TG4 myocytes, and more surprisingly, β 2 AR stimulation was also ineffective in increasing contractility in WT myocytes. Pertussis toxin (PTX) treatment fully rescued the I Ca , [Ca 2+ ] i , and contractile responses to β 2 AR agonists in both WT and TG4 cells. The PTX-rescued murine cardiac β 2 AR response is mediated by cAMP-dependent mechanisms, because it was totally blocked by the inhibitory cAMP analog Rp-cAMPS. These results suggest that PTX-sensitive G proteins are responsible for the unresponsiveness of mouse heart to agonist-induced β 2 AR stimulation. This was further corroborated by an increased incorporation of the photoreactive GTP analog [γ- 32 P]GTP azidoanilide into α subunits of G i2 and G i3 after β 2 AR stimulation by zinterol or isoproterenol plus the β 1 AR blocker CGP 20712A. This effect to activate G i proteins was abolished by a selective β 2 AR blocker ICI 118,551 or by PTX treatment. Thus, we conclude that (1) β 2 ARs in murine cardiac myocytes couple to concurrent G s and G i signaling, resulting in null inotropic response, unless the G i signaling is inhibited; (2) as a special case, the lack of cardiac contractile response to β 2 AR agonists in TG4 mice is not due to a saturation of cell contractility or of the cAMP signaling cascade but rather to an activation of β 2 AR-coupled G i proteins; and (3) spontaneous β 2 AR activation may differ from agonist-stimulated β 2 AR signaling.