Acute cigarette smoke or extract exposure rapidly activates TRPA1-mediated calcium influx in primary human airway smooth muscle cells

Jinheng Lin,Malcolm Brodlie,Michael A Gray,Robert Tarran,M Flori Sassano,Lee Borthwick,Michael Taggart,Andrew Fisher

doi:10.1038/s41598-021-89051-4

Abstract

Tobacco smoking is the largest risk factor for developing chronic obstructive pulmonary disease (COPD), and is associated with hyperresponsiveness of airway smooth muscle (ASM). Chronic exposure to cigarette smoke (CS) leads to airway inflammation and remodelling. However, the direct effect of gaseous CS or CS extract (CSE) on human airway smooth muscle cell (hASMC) function remains poorly understood. This study investigated the acute effect of CS/CSE on calcium homeostasis, a key regulator of ASM physiology and pathophysiology. Primary hASMC were isolated from non-smoking donor lungs, and subjected to Ca2+ imaging studies. We found that both CS, and CSE, rapidly elevated cytosolic Ca2+ in hASMC through stimulation of plasmalemmal Ca2+ influx, but excluded store-operated and L-type Ca2+ channels as mediators of this effect. Using a specific pharmacological inhibitor, or shRNA-driven knockdown, we established that both CS and CSE stimulated Ca2+ influx in hASMC through the neurogenic pain receptor channel, transient receptor potential ankyrin 1 (TRPA1). CS/CSE-dependent, TRPA1-mediated Ca2+ influx led to myosin light-chain phosphorylation, a key process regulating ASM contractility. We conclude that TRPA1 is likely an important link between CS/CSE exposure and airway hyperresponsiveness, and speculate that acute CS/CSE-induced Ca2+ influx could lead to exacerbated ASM contraction and potentially initiate further chronic pathological effects of tobacco smoke.

Highlights

Abbreviations chronic obstructive pulmonary disease (COPD) Chronic obstructive pulmonary diseases ROS Reactive oxygen species SOCE Store-operated calcium entry [Ca2+]i Cytosolic Ca2+ concentration CPA Cyclopiazonic acid MLC Myosin light-chain p-MLC Phospho-myosin light-chain HRP Horseradish peroxidase transient receptor potential ankyrin 1 (TRPA1) Transient receptor potential ankyrin 1 EGTA Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid DMEM/F-12 Dulbecco’s modified eagle’s medium/Ham’s nutrient mixture F-12 NaHEPES 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid sodium salt LB Lysogeny broth
Our results show that acute exposure to either gaseous cigarette smoke (CS) or to CS extract (CSE) rapidly stimulates C a2+ influx in primary human ASM cells (hASMC) through activation of TRPA1 channels, which induces downstream phosphorylation of myosin lightchain, presenting TRPA1 as an important link between smoking and airway hyperresponsiveness
Several groups have previously reported that CSE mobilises calcium in neuronal cells, lung epithelia and fibroblasts via T RPA123,26–28, observations which are strongly substantiated by our results

Summary

Introduction

Abbreviations COPD Chronic obstructive pulmonary diseases ROS Reactive oxygen species SOCE Store-operated calcium entry [Ca2+]i Cytosolic Ca2+ concentration CPA Cyclopiazonic acid MLC Myosin light-chain p-MLC Phospho-myosin light-chain HRP Horseradish peroxidase TRPA1 Transient receptor potential ankyrin 1 EGTA Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid DMEM/F-12 Dulbecco’s modified eagle’s medium/Ham’s nutrient mixture F-12 NaHEPES 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid sodium salt LB Lysogeny broth. Airway smooth muscle (ASM) is at the centre of AHR and is more sensitive to contractile stimuli in the diseased state This may be exacerbated by airway inflammation and remodelling, which are extensively linked to tobacco smoking[5,6,7,8,9]. Delivery of CS can be performed by direct aerosolisation into a closed chamber, commonly used for animal smoking studies, but have been applied in in vitro experiments. Another delivery model is known as CS extract (CSE), produced by bubbling CS into a buffer solution, which can be applied to in vitro or ex vivo samples, or collected for chemical analysis. This makes it difficult to extrapolate to what may occur in these circumstances in hASMCs

Methods

Results

Conclusion