Villalba Dominic R, Jannu Arun K, Javed Elham, Dandekar Isha, Wang Ruping, Deshpande Deepak A, An Steven S, Panettieri Reynold A, Tang Dale D, Penn Raymond B, Nayak Ajay P
Department of Medicine, Center for Translational Medicine & Division of Pulmonary and Critical Care Medicine; Jane and Leonard Korman Lung Center, Thomas Jefferson University, Philadelphia, PA, 19107, USA.
Rutgers University, New Brunswick, NJ, USA.
Respir Res. 2025 May 13;26(1):183. doi: 10.1186/s12931-025-03268-9.
BACKGROUND: We recently reported that the ovarian cancer G protein-coupled receptor-1 (OGR1) can be pharmacologically biased with specific benzodiazepines to couple with distinct heterotrimeric G proteins in human airway smooth muscle (ASM) cells. Lorazepam stimulated both G and G signaling via OGR1, whereas sulazepam only stimulated G signaling in ASM cells. The present study sought to determine the effects of sulazepam and lorazepam on contraction of human precision cut lung slices (hPCLS), and detail the biochemical mechanisms mediating these effects. METHODS: Models of histamine (His) -stimulated contraction included imaging of ex vivo human precision cut lung slices (hPCLS) and Magnetic Twisting Cytometry (MTC) analysis of human ASM cell stiffness. To explore mechanisms of regulation, we examined effects on myosin light chain (pMLC) phosphorylation and PKA activity in primary human ASM cultures, as well as actin cytoskeleton integrity as defined by changes in the ratio of F to G actin assessed by immunofluorescence. RESULTS: In a dose-dependent manner, sulazepam relaxed His-contracted hPCLS and reduced baseline cell stiffness. Lorazepam did not relax His-contracted hPCLS, and only at a maximal dose (100 μM) did lorazepam relax baseline cell stiffness. The G-biased ligand sulazepam stimulated PKA activity as evidenced by significant induction of VASP and HSP20 phosphorylation, which was associated with significant inhibition of His-induced pMLC phosphorylation. Conversely, the balanced ligand lorazepam did not significantly increase HSP20 phosphorylation or VASP phosphorylation and did not significantly inhibit His-induced MLC phosphorylation. Sulazepam was also able to inhibit histamine induced F-actin formation. CONCLUSIONS: The G-biased OGR1 ligand sulazepam relaxed contracted ASM in both tissue- and cell- based models, via inhibition of MLC phosphorylation in a PKA-dependent manner and through inhibition of actin stress fiber formation. The relative inability of the balanced ligand lorazepam to influence ASM contractile state was likely due to competitive actions of concomitant G and G signaling.
背景：我们最近报道，卵巢癌G蛋白偶联受体-1（OGR1）可被特定苯二氮䓬类药物进行药理学偏向，从而在人气道平滑肌（ASM）细胞中与不同的异源三聚体G蛋白偶联。劳拉西泮通过OGR1刺激G和G信号传导，而舒拉西泮仅在ASM细胞中刺激G信号传导。本研究旨在确定舒拉西泮和劳拉西泮对人精密切割肺切片（hPCLS）收缩的影响，并详细阐述介导这些影响的生化机制。 方法：组胺（His）刺激收缩模型包括体外人精密切割肺切片（hPCLS）成像和人ASM细胞硬度的磁扭转细胞术（MTC）分析。为了探索调节机制，我们研究了对原代人ASM培养物中肌球蛋白轻链（pMLC）磷酸化和PKA活性的影响，以及通过免疫荧光评估的F肌动蛋白与G肌动蛋白比率变化所定义的肌动蛋白细胞骨架完整性。 结果：舒拉西泮以剂量依赖性方式使His收缩的hPCLS舒张，并降低基线细胞硬度。劳拉西泮不能使His收缩的hPCLS舒张，仅在最大剂量（100 μM）时劳拉西泮才能使基线细胞硬度舒张。G偏向性配体舒拉西泮刺激PKA活性，VASP和HSP20磷酸化的显著诱导证明了这一点，这与His诱导的pMLC磷酸化的显著抑制相关。相反，平衡配体劳拉西泮没有显著增加HSP20磷酸化或VASP磷酸化，也没有显著抑制His诱导的MLC磷酸化。舒拉西泮还能够抑制组胺诱导的F肌动蛋白形成。 结论：G偏向性OGR1配体舒拉西泮在基于组织和细胞的模型中均通过以PKA依赖性方式抑制MLC磷酸化并通过抑制肌动蛋白应力纤维形成来舒张收缩的ASM。平衡配体劳拉西泮相对无法影响ASM收缩状态可能是由于伴随的G和G信号传导的竞争性作用。
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