The activation of M3 mAChR in airway epithelial cells promotes IL-8 and TGF-ß1 secretion and airway smooth muscle cell migration.

BACKGROUND: Muscarinic acetylcholine receptors (mAChRs) have been identified in airway epithelium, and epithelium-derived chemokines can initiate the migration of airway smooth muscle (ASM) cells. However, the mAChRs that are expressed in airway epithelium and the mechanism underlying the regulation of ASM cell migration are not clear. The aim of this study was to test whether the effects of the epithelium-derived chemokines on ASM cell migration could be modulated by mAChRs. METHOD: Human epithelial cells (A549 cells) were stimulated with cigarette smoke extract (CSE) or the mAChRs agonist carbachol. IL-8 and TGF-ß1 production were measured by ELISA, and human ASM cell migration was measured using the transwell migration assay and scratch assay. The mRNA levels of the mAChRs subtypes and the acetylcholine concentrations were measured using RT-PCR and LC-MS/MS, respectively. RESULTS: ASM cell migration toward CSE-stimulated A549 cells was markedly reduced by Ac-RRWWCR-NH2 (IL-8 inhibitor) and SB431542 (TGF-ß1 inhibitor). CSE-induced ASM cell migration was also suppressed by the mAChRs antagonist tiotropium. Interestingly, carbachol-stimulated A549 cells also induced ASM cell migration; this migration event was suppressed by tiotropium, Ac-RRWWCR-NH2 and SB431542. In addition, the effects of CSE on ASM cell migration were significantly and cooperatively enhanced by carbachol compared to CSE alone. Carbachol-induced ASM cell migration was reduced by selective inhibitors of PI3K/Akt (LY294002) and p38 (SB203580), suggesting that it occurred through p38 and Akt phosphorylation, which was inhibited by the M3 mAChR antagonist 4-DAMP. CONCLUSIONS: These findings indicate that M3 mAChR may be important therapeutic target for obstructive airway diseases, as it regulates the effects of the epithelial-derived chemokines on ASM cell migration, which results in lung remodeling.

Targeted inhibition of KCa3.1 channel attenuates airway inflammation and remodeling in allergic asthma.

KCa3.1 has been suggested to be involved in regulating cell activation, proliferation, and migration in multiple cell types, including airway inflammatory and structural cells. However, the contributions of KCa3.1 to airway inflammation and remodeling and subsequent airway hyperresponsiveness (AHR) in allergic asthma remain to be explored. The main purpose of this study was to elucidate the roles of KCa3.1 and the potential therapeutic value of KCa3.1 blockers in chronic allergic asthma. Using real-time PCR, Western blotting, or immunohistochemical analyses, we explored the precise role of KCa3.1 in the bronchi of allergic mice and asthmatic human bronchial smooth muscle cells (BSMCs). We found that KCa3.1 mRNA and protein expression were elevated in the bronchi of allergic mice, and double labeling revealed that up-regulation occurred primarily in airway smooth muscle cells. Triarylmethane (TRAM)-34, a KCa3.1 blocker, dose-dependently inhibited the generation and maintenance of the ovalbumin-induced airway inflammation associated with increased Th2-type cytokines and decreased Th1-type cytokine, as well as subepithelial extracellular matrix deposition, goblet-cell hyperplasia, and AHR in a murine model of asthma. Moreover, the pharmacological blockade and gene silencing of KCa3.1, which was evidently elevated after mitogen stimulation, suppressed asthmatic human BSMC proliferation and migration, and arrested the cell cycle at the G0/G1 phase. In addition, the KCa3.1 activator 1-ethylbenzimidazolinone-induced membrane hyperpolarization and intracellular calcium increase in asthmatic human BSMCs were attenuated by TRAM-34. We demonstrate for the first time an important role for KCa3.1 in the pathogenesis of airway inflammation and remodeling in allergic asthma, and we suggest that KCa3.1 blockers may represent a promising therapeutic strategy for asthma.

Role of M3 mAChR in in vivo and in vitro models of LPS-induced inflammatory response.

OBJECTIVE: We tested the potential role of the mAChR in lipopolysaccharide (LPS)-induced inflammatory response in in vivo and in vitro models and a possible signaling pathway involved in the inflammatory process. METHODS: Anesthetized mice were challenged with intratracheal LPS to induce acute lung injury. The cytology and histopathology changes, expression of cytokines and pulmonary vascular permeability were used to evaluate the effects of the cholinergic agent. Alveolar macrophage cell line NR8383 was also used to confirm the role of mAChRs and the molecular mechanisms underlying the LPS-induced events. RESULTS: LPS-induced acute lung injury (ALI) was significantly improved by atropine (a non-selective mAChR antagonist) and 4-DAMP (a M3 mAChR antagonist), as indicated by the diminution of neutrophil infiltration, pulmonary vascular permeability and IL-6 and TNF-α production. LPS-induced TNF-α production from the alveolar macrophage was significantly inhibited by atropine and 4-DAMP, but not pirenzepine (a M1 mAChR antagonist) and methoctramine (a M2 mAChR antagonist). Interestingly, LPS-induced TNF-α production was enhanced by the muscarinic receptor agonist pilocarpine, and treatment with pilocarpine alone was able to trigger TNF-α production from the alveolar macrophage, which was effectively attenuated by 4-DAMP. Western blot analysis showed that LPS-induced degradation of IκBα was strongly blocked by atropine/4-DAMP both in vivo and in vitro, indicating that M3 mAChR was involved in LPS-induced lung inflammation by mediating the NF-κB signaling pathway. CONCLUSION: Our findings bring the evidence that the blockage of mAChR exerts anti-inflammatory properties, in which the M3 mAChR plays an important role in the LPS-induced lung inflammation.

Protective effects of anisodamine on cigarette smoke extract-induced airway smooth muscle cell proliferation and tracheal contractility.

Anisodamine, an antagonist of muscarinic acetylcholine receptors (mAChRs), has been used therapeutically to improve smooth muscle function, including microvascular, intestinal and airway spasms. Our previous studies have revealed that airway hyper-reactivity could be prevented by anisodamine. However, whether anisodamine prevents smoking-induced airway smooth muscle (ASM) cell proliferation remained unclear. In this study, a primary culture of rat ASM cells was used to evaluate an ASM phenotype through the ability of the cells to proliferate and express contractile proteins in response to cigarette smoke extract (CSE) and intervention of anisodamine. Our results showed that CSE resulted in an increase in cyclin D1 expression concomitant with the G0/G1-to-S phase transition, and high expression of M2 and M3. Functional studies showed that tracheal hyper-contractility accompanied contractile marker α-SMA high-expression. These changes, which occur only after CSE stimulation, were prevented and reversed by anisodamine, and CSE-induced cyclin D1 expression was significantly inhibited by anisodamine and the specific inhibitor U0126, BAY11-7082 and LY294002. Thus, we concluded that the protective and reversal effects and mechanism of anisodamine on CSE-induced events might involve, at least partially, the ERK, Akt and NF-κB signaling pathways associated with cyclin D1 via mAChRs. Our study validated that anisodamine intervention on ASM cells may contribute to anti-remodeling properties other than bronchodilation.