Although ligand-free, constitutive β2-adrenergic receptor (AR) signaling has been demonstrated in naive cell lines and in transgenic mice overexpressing cardiac β2-AR, it is unclear whether the dominant cardiac β-AR subtype, β1-AR, shares the ability of spontaneous activation. In the present study, we expressed human β1- or β2-AR via recombinant adenoviral infection in ventricular myocytes isolated from β1β2-AR double knockout mice, creating pure β1-AR and β2-AR systems with variable receptor densities. A contractile response to a nonselective β-AR agonist, isoproterenol, was absent in double knockout mouse myocytes but was fully restored after adenoviral β1-AR or adenoviral β2-AR infection. Increasing the titer of adenoviral vectors (multiplicity of infection 10-1000) led to a dose-dependent expression of β1- or β2-AR with a maximal density of 1207 ± 173 (36-fold over the wild-type control value) and 821 ± 38 fmol/mg protein (69-fold), respectively. Using confocal immunohistochemistry, we directly visualized the cellular distribution of β1-AR and β2-AR and found that both subtypes were distributed on the cell surface membrane and transverse tubules, resulting in a striated pattern. In the absence of ligand, β2-AR expression resulted in graded increases in baseline cAMP and contractility up to 428% and 233% of control, respectively, at the maximal β2-AR density. These effects were specifically reversed by a β2-AR inverse agonist, ICI 118,551 (10-7 M). In contrast, overexpression of β1-AR, even at a greater density, failed to enhance either basal cAMP or contractility; the alleged β1-AR inverse agonist, CGP 20712A (10-6 M), had no significant effect on basal contraction in these cells. Thus, we conclude that acute β2-AR overexpression in cardiac myocytes elicits significant physiological responses due to spontaneous receptor activation; however, this property is β-AR subtype specific because β1-AR does not exhibit agonist-independent spontaneous activation.
ASJC Scopus subject areas
- Molecular Medicine