β2‐ but not β1‐adrenoceptor activation modulates intracellular oxygen availability

β‐Adrenoceptors (β‐ARs) play a critical role in the regulation of cardiovascular function. Intracellular oxygen homeostasis is crucial for the survival of cardiomyocytes. However, it is still unclear whether β‐AR activation can modulate intracellular oxygen. Here we used mitochondrial and cytosolic target Renilla luciferase to detect intracellular oxygen concentration. Pharmacological experiments revealed that β2‐AR activation specifically regulates intracellular oxygen in cardiomyocytes and COS7 cells. This effect was abrogated by inhibitory G protein (Gi) inhibition, endothelial nitric oxide synthase (eNOS) blockade, and NO scavenging, implicating that the β2‐AR–Gi–eNOS pathway is involved in this regulation. β2‐AR activation increased the AMP/ATP ratio, AMPK activity, ROS production and prolyl hydroxylase activity. These effects also contribute to the regulation of β2‐AR signalling, thus providing an additional layer of complexity to enforce the specificity of β1‐AR and β2‐AR signalling. Collectively, the study provides novel insight into the modulation of oxygen homeostasis, broadens the scope of β2‐AR function, and may have crucial implications for β2‐AR signalling regulation. β‐Adrenoceptors (β‐ARs) are prototypical G protein‐coupled receptors that play a critical role in the regulation of cardiovascular function. Precise and rapid redistribution of intracellular oxygen is crucial for cardiomyocyte survival in response to various stimuli. We found that β2‐AR but not β1‐AR activation specifically modulates intracellular oxygen availability through the Beta2‐AR coupled inhibitory G protein and endothelial nitric oxide synthase signalling pathway. This in turn affects AMP‐activated kinase activity, reactive oxygen species generation, and prolyl hydroxylase activity. The study provides novel insight into the modulation of oxygen homeostasis, broadens the scope of β2‐AR function, and may have crucial implications for β2‐AR signalling regulation.