Benzodiazepine diazepam regulates cell surface β₁-adrenergic receptor density in human monocytes

Downregulation of cell surface β-adrenergic receptors (β-AR) is an important adaptive response that prevents deleterious effects of receptor overstimulation. Various factors including reactive oxygen species cause β-AR downregulation. In this study, we evaluated the effects of ligands of the peripheral benzodiazepine receptor (PBR), a key protein in regulating oxidative stress, on surface density of endogenous β₁-and β₂-ARs in highly differentiated cells such as human monocytes, which express both β-AR subtypes. β-AR expression in human monocytes was evaluated by flow cytometry, qPCR and western blotting. Monocyte treatment with β-AR agonist isoproterenol did not change surface β₁-AR density while downregulating surface β₂-AR density. This effect was antagonized by the β-blocker propranolol. An opposite response was observed with benzodiazepine diazepam that led to a time-dependent reduction in β₁-AR density. In particular, while no significant downregulation was observed after 3 h of treatment, only 63% of β₁-ARs were still present on the cell surface after 48 h of treatment with diazepam at 1 μM. Treatment with the PBR antagonist PK11195, but not with propranolol, antagonized the effects of diazepam. No change in β₁-AR-mRNA or protein levels was observed at any time after diazepam treatment. We also found that diazepam did not affect Gs-protein or β-arrestin-2 recruitment for both β-ARs in engineered fibroblasts, further suggesting that diazepam activity on β₁-AR density is mediated by PBR. Finally, no sex-related differences were found. Collectively, these results indicate that monocyte β₁-ARs are resistant to catecholamine-mediated downregulation and suggest that PBR plays an important role in regulating β₁-AR density.