Imidazobenzodiazepine PI320 Relaxes Mouse Peripheral Airways by Inhibiting Calcium Mobilization

Asthma is a common respiratory disease characterized, in part, by excessive airway smooth muscle (ASM) contraction (airway hyperresponsiveness). Various GABAR (γ-aminobutyric acid type A receptor) activators, including benzodiazepines, relax ASM. The GABAR is a ligand-operated Cl channel best known for its role in inhibitory neurotransmission in the central nervous system. Although ASM cells express GABARs, affording a seemingly logical site of action, the mechanism(s) by which GABAR ligands relax ASM remains unclear. PI320, a novel imidazobenzodiazepine designed for tissue selectivity, is a promising asthma drug candidate. Here, we show that PI320 alleviates methacholine (MCh)-induced bronchoconstriction and relaxes peripheral airways preconstricted with MCh using the forced oscillation technique and precision-cut lung slice experiments, respectively. Surprisingly, the peripheral airway relaxation demonstrated in precision-cut lung slices does not appear to be GABAR-dependent, as it is not inhibited by the GABAR antagonist picrotoxin or the benzodiazepine antagonist flumazenil. Furthermore, we demonstrate here that PI320 inhibits MCh-induced airway constriction in the absence of external Ca, suggesting that PI320-mediated relaxation is not mediated by inhibition of Ca influx in ASM. However, PI320 does inhibit MCh-induced intracellular Ca oscillations in peripheral ASM, a key mediator of contraction that is dependent on sarcoplasmic reticulum Ca mobilization. Furthermore, PI320 inhibits peripheral airway constriction induced by experimentally increasing the intracellular concentration of inositol triphosphate (IP). These novel data suggest that PI320 relaxes murine peripheral airways by inhibiting intracellular Ca mobilization in ASM, likely by inhibiting Ca release through IPRs (IP receptors).