Functional ?-adrenergic receptor signalling on nuclear membranes in adult rat and mouse ventricular cardiomyocytes

Abstract

Objective We sought to determine if different β-adrenergic receptor (βAR) subtypes, and their associated signalling machinery, are functionally localized to nuclear membranes. Methods Employing enriched nuclear preparations, we assayed the specific presence of βAR by measuring ¹²⁵I-cyanopindolol (CYP) binding, Western blotting, confocal microscopy and functional assays. Results Western blots of rat heart nuclear fractions and confocal immunofluorescent analysis of adult rat and mouse ventricular cardiomyocytes displayed the presence of β₁AR and β₃AR but, surprisingly, not the β₂AR on nuclear membranes. Nuclear localization of downstream signalling partners Gs, Gi and adenylyl cyclases II and V/VI was also demonstrated. The functional relevance of nuclear βAR was shown by receptor-mediated stimulation of adenylyl cyclase activity by isoproterenol but not the β₃AR-selective agonist CL 316243 in enriched nuclear preparations. We also examined the effect of subtype-selective ligands on the initiation of RNA synthesis in isolated nuclei. Both isoproterenol and another β₃AR-selective agonist, BRL 37344, increased RNA synthesis which was inhibited by pertussis toxin (PTX). Neither a β₁AR-selective agonist, xamoterol, nor a β₂AR-selective agonist, procaterol, was able to stimulate transcription. However, both CGP 20712A and ICI 118,551 blocked isoproterenol-mediated effects to varying extents. PTX treatment also revealed that nuclear βAR may be coupled to other signalling pathways in addition to Gi, as stimulation under these conditions reduced initiation of transcription below basal levels. Conclusion These results highlight differential subcellular localization for βAR subtypes and indicate that βAR may have specific roles in regulating nuclear function in cardiomyocytes.