The Ca2+–NO–ROS Crosstalk Induced by Arachidonic Acid in Human Lung Fibroblasts: Implications for Pulmonary Fibrosis

Arachidonic acid (AA) is an emerging regulator of fibroblast activity in pulmonary fibrosis; however, the underlying intracellular mechanisms remain unclear. This study investigated the effects of AA on the free intracellular calcium concentration ([Ca2+]i), nitric oxide (NO), and reactive oxygen species (ROS) in human WI-38 lung fibroblasts. Using fluorescent imaging and pharmacological tools, we demonstrate that AA evokes a robust, concentration-dependent increase in [Ca2+]i. This response is initiated by G protein-coupled receptor 40 (GPR40), which leads to endoplasmic reticulum Ca2+ release through inositol 1,4,5-trisphosphate receptors (IP3Rs) and lysosomal Ca2+ mobilisation through two-pore channels (TPCs). Sustained Ca2+ elevation is primarily mediated by influx through transient receptor potential vanilloid 4 (TRPV4) channels, with a minor contribution from store-operated Ca2+ entry. The AA-induced Ca2+ signal stimulates endothelial NO synthase-dependent NO production, which in turn triggers ROS generation, revealing a tightly coupled Ca2+–NO–ROS signalling network. Our findings identify AA as a potent modulator of Ca2+ and redox signalling in lung fibroblasts, and highlight GPR40, TRPV4, IP3Rs and lysosomal TPCs as potential therapeutic targets for intervening in pulmonary fibrosis.