Diazepam Relaxes Murine Peripheral Airway Smooth Muscle Through Inhibition of Phosphodiesterase 4 and Intracellular Calcium Oscillations

Benzodiazepines are widely used for sedation and anxiolysis but may also exert unrecognized beneficial effects on airway smooth muscle (ASM) tone in clinical settings where they are routinely administered. While their primary mechanism involves GABAA receptor (GABAAR) modulation in the central nervous system, their direct effects on peripheral airways remain poorly understood. Using mouse precision-cut lung slices (PCLS) and video phase-contrast microscopy, we show that diazepam, lorazepam, and midazolam induce robust, reversible, and dose-dependent relaxation of methacholine (MCh)-constricted peripheral airways with IC50 of 12, 22, and 23 µM, respectively. We selected diazepam to further investigate the cellular and molecular mechanisms underlying airway relaxation. GABAAR antagonists picrotoxin and flumazenil failed to block relaxation, and diazepam inhibited MCh-induced airway constriction even in the absence of extracellular Ca2+, consistent with a GABAAR-independent mechanism. Diazepam-induced relaxation correlated with strong inhibition of concurrent intracellular Ca2+ oscillations in ASM cells. Diazepam inhibited airway constriction and Ca2+ transients elicited by intracellular IP3 uncaging but not by caffeine, indicating specific modulation of IP3-receptor mediated Ca2+ signaling. Furthermore, low concentrations of diazepam (1 µM) significantly prolonged terbutaline-induced airway relaxation, mirroring the effects of selective phosphodiesterase 4 (PDE4) inhibitor rolipram. Diazepam inhibited purified PDE4D2 activity and potentiated forskolin-induced cAMP accumulation in human ASM cells. These findings indicate that diazepam produces bronchodilation at least in part through direct PDE4 inhibition and modulation of IP3 receptor-mediated Ca2+ oscillations in ASM. The synergistic interaction between benzodiazepines and β2-adrenoreceptor agonists at therapeutic concentrations has important clinical implications for bronchodilation in high-risk patients.