Enhanced production of nitric oxide in A549 cells through activation of TRPA1 ion channel by cold stress

• Cold stress induced the enhanced production of NO in A549 cell.
• Cold-induced NO elevation was dependent on the extracellular Ca2+ inflow.
• The increased production of NO was through activation of cold-sensing TRPA1 channel induced by cold stress.

The respiratory epithelium is exposed to the external environment, and inhalation of cold air is common during the season of winter. In addition, the lung is a major source of nitric oxide (NO). However, the effect of cold stress on the production of NO is still unclear. In the present work, We measured the change of NO in single cell with DACF-DA and the change in cytosolic Ca2+ concentration ([Ca2+]c) in A549 cell. We observed that cold stress (from 20 °C to 5 °C) induced an increase of NO in A549 cell, which was completely abolished by applying an extracellular Ca2+ free medium. Further experiments showed that cold-sensing transient receptor potential subfamily member 1 (TRPA1) channel agonist (allyl isothiocyanate, AITC) increased the production of NO and the level of [Ca2+]c in A549 cell. Additionally, TRPA1 inhibitor, Ruthenium red (RR) and camphor, significantly blocked the enhanced production of NO and the rise of [Ca2+]c induced by AITC or cold stimulation, respectively. Taken together, these data indicated that cold-induced TRPA1 activation was responsible for the enhanced production of NO in A549 cell.