Connexin-hemichannels-mediated ATP release causes lung injury following chlorine inhalation

Chlorine (Cl 2 ) is a highly reactive halogen gas that undergoes rapid hydrolysis in lung epithelial lining fluid (ELF) upon inhalation, forming hypochlorous acid (HOCl) and hydrochloric acid (HCl). These products subsequently, through chemical reactions, modify the structure and the function of membrane proteins. Herein, we investigated the effects of Cl 2 on connexin-hemichannels and the release of ATP in the ELF. Adult C57BL/6 mice were subjected to 400 ppm Cl 2 for 30 minutes. Subsequent analysis revealed a marked increase in ATP levels within the BAL, with concentrations reaching 43.952 ± 9.553 nM at 2 hours and 30.554 ± 7.383 nM at 24 hours post-exposure, relative to control. Additionally, at 24 hours post-exposure, the lung wet/dry (W/D) ratio significantly increased from 4.48 ± 0.142 to 5.067± 0.359, while alveolar fluid clearance (AFC) decreased from 0.249 ± 0.019 to 0.145 ± 0.018. Electrophysiological recordings in alveolar type 2 (AT2) cells revealed reduced open probabilities (P o ) of both ENaC (4 pS) and a cation channel (18 pS), declining from 0.323 ± 0.021 and 0.202 ± 0.022 to 0.151 ± 0.042 and 0.091 ± 0.019, respectively. Instillation of 50 μl of 100 μg/ml Gap27—a connexin mimetic peptide selectively inhibiting connexin-hemichannels—administered 30 minutes post-exposure, restored ATP to control, normalized the W/D ratio, improved AFC, and reestablished ENaC function. Moreover, Gap27 normalized airway resistance following methacholine challenge. In human airway smooth muscle cells (hASMCs), 100 μM ATP induced Ca i 2+ elevation and depolarized V m to -40 mV, with both effects partially reversed by P2X 7 R inhibitor, A804598.