Kiel, Alexander

Smoking contributes to or exacerbates diseases such as asthma and COPD, where airway hyperreactivity is a key feature. Enhanced Ca 2+ in airway smooth muscle contributes to airway hyperreactivity. We previously determined that the cytokine TNFα upregulates expression of Ca 2+ regulatory molecules such as membrane bound bifunctional enzyme CD38 and Ca 2+ influx regulatory TRPC3 in human airway smooth muscle cells (HASM). We hypothesized that cigarette smoke (CS) is an inflammatory stimulus that upregulates Ca 2+ regulatory mechanisms in HASM. Western analysis showed increased expression of CD38, TRPC3 and TRPC6 in lungs from mice following 4 week in vivo CS exposure. These proteins were also upregulated in HASM cells incubated in 1 or 2% CS extract (CSE), as well as in HASM samples from patients with COPD (compared to controls). In fura‐2 loaded HASM cells, exposure to CSE enhanced histamine‐mediated Ca 2+ mobilization as well as store‐operated calcium entry: effects substantially blunted by TRPC3 siRNAs. Finally, in living lung slices of mice exposed for 4 weeks to CS, ACh‐induced airway contractility was substantially greater, compared to controls. These results indicate that CS upregulates expression and function of Ca 2+ regulatory proteins, thus contributing to airway hyperreactivity in smoking related diseases. Supported by the Annenberg Foundation (MEW), HL090595 (CMP), FAMRI (RV, YSP).

The balance between mitochondrial fission and fusion is crucial for mitochondria to perform its normal cellular functions. We hypothesized that cigarette smoke (CS) disrupts this balance and enhances mitochondrial dysfunction in the airway. In nonasthmatic human airway smooth muscle (ASM) cells, CS extract (CSE) induced mitochondrial fragmentation and damages their networked morphology in a concentration-dependent fashion, via increased expression of mitochondrial fission protein dynamin-related protein 1 (Drp1) and decreased fusion protein mitofusin (Mfn) 2. CSE effects on Drp1 vs. Mfn2 and mitochondrial network morphology involved reactive oxygen species (ROS), activation of extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), protein kinase C (PKC) and proteasome pathways, as well as transcriptional regulation via factors such as NF-κB and nuclear erythroid 2-related factor 2. Inhibiting Drp1 prevented CSE effects on mitochondrial networks and ROS generation, whereas blocking Mfn2 had the opposite, detrimental effect. In ASM from asmatic patients, mitochondria exhibited substantial morphological defects at baseline and showed increased Drp1 but decreased Mfn2 expression, with exacerbating effects of CSE. Overall, these results highlight the importance of mitochondrial networks and their regulation in the context of cellular changes induced by insults such as inflammation (as in asthma) or CS. Altered mitochondrial fission/fusion proteins have a further potential to influence parameters such as ROS and cell proliferation and apoptosis relevant to airway diseases.