Abstract Rationale Airway smooth muscle (ASM) contraction is a hallmark of asthma. Our group previously showed that PI320, a novel imidazobenzodiazepine, can relax lower airways in mice by inhibiting intracellular Ca2+ oscillations in ASM. We proposed a mechanism in which PI320 inhibits inositol triphosphate receptor (IP3R)-dependent Ca2+ mobilization from the sarcoplasmic reticulum, as evidenced by caged IP3 studies. However, the exact pathway by which PI320 inhibits the IP3R pathway remains to be further elucidated. Here we study the effect of diazepam, a clinically relevant benzodiazepine, on its ability to relax mouse peripheral airways and further explore its mechanism in the cAMP/protein kinase A pathway. Methods All studies were IACUC approved. Precision-cut lung slices (PCLS) were mounted on a perfusion chamber on an inverted microscope and suprafused with drugs. Ca2+ oscillation experiments were conducted in PCLS prepared from transgenic mice globally and constitutively expressing the fluorescent intracellular Ca2+ indicator GCaMP6f. For caged-IP3 studies, PCLS from C57BL/6J mice were loaded with caged IP3-PM and the IP3 was released with 1sec UV light flash. In separate experiments, cAMP concentrations were measured in cultured immortalized human ASM (hASM). Results Diazepam significantly reduced peripheral airway constriction with methacholine (MCh) at 10µM, an effect not reversed by GABAAR antagonists, flumazenil or picrotoxin, nor mimicked by a GABAAR agonist, muscimol. Diazepam also reversibly inhibited MCh-induced Ca2+ oscillations in mouse ASM concurrent with airway relaxation. Moreover, diazepam inhibited airway constriction induced by IP3-uncaging, suggesting that diazepam directly or indirectly inhibits IP3R. Importantly, diazepam prolonged ASM relaxation induced by β2-adrenoreceptor agonist terbutaline similarly to rolipram, a potent phosphodiesterase-4 (PDE4) inhibitor (Figure 1). Corresponding cAMP assays show diazepam potentiating cAMP concentrations in cultured hASM cells. This effect was not reversed by flumazenil and not replicated by muscimol, reiterating a GABAAR-independent pathway for diazepam’s relaxing effects on ASM and suggesting PDE4 inhibition as the underlying mechanism. Conclusion We show that benzodiazepines relax peripheral mouse airways in a non-GABAAR-mediated manner. Our data supports the hypothesis that PDE4 inhibition with subsequent increase in cAMP levels and IP3R inhibition is the underlying mechanism for diazepam’s effect on mouse ASM. Notably, literature exists in other organ systems such as cardiac tissue that diazepam may directly inhibit PDE4 activity. Future studies should include purified PDE4 enzyme activity assays, exploring other benzodiazepines as potential targets for reactive airway disease therapy, and considering the effects of benzodiazepines on inflammation, the other pathophysiology behind allergic lung disease. This abstract is funded by: NIH T32
Hwang et al. (Fri,) studied this question.