We assessed regulation of volume-sensitive Cl⁻ current (I_Cl,swell) by endothelin-1 (ET-1) and characterized the signalling pathway responsible for its activation in rabbit atrial and ventricular myocytes.

ET-1 elicited I_Cl,swell under isosmotic conditions. Outwardly rectified Cl⁻ current was blocked by the I_Cl,swell-selective inhibitor DCPIB or osmotic shrinkage and involved ET_A but not ET_B receptors. ET-1-induced current was abolished by inhibiting epidermal growth factor receptor (EGFR) kinase or phosphoinositide-3-kinase (PI-3K), indicating that these kinases were downstream. Regarding upstream events, activation of I_Cl,swell by osmotic swelling or angiotensin II (AngII) was suppressed by ET_A blockade, whereas AngII AT₁ receptor blockade failed to alter ET-1-induced current. Reactive oxygen species (ROS) produced by NADPH oxidase (NOX) stimulate I_Cl,swell. As expected, blockade of NOX suppressed ET-1-induced I_Cl,swell, but blockade of mitochondrial ROS production with rotenone also suppressed I_Cl,swell. I_Cl,swell was activated by augmenting complex III ROS production with antimycin A or diazoxide; in this case, I_Cl,swell was insensitive to NOX inhibitors, indicating that mitochondria were downstream from NOX. ROS generation in HL-1 cardiomyocytes measured by flow cytometry confirmed the electrophysiological findings. ET-1-induced ROS production was inhibited by blocking either NOX or mitochondrial complex I, whereas complex III-induced ROS production was insensitive to NOX blockade.

ET-1–ET_A signalling activated I_Cl,swell via EGFR kinase, PI-3K, and NOX ROS production, which triggered mitochondrial ROS production. ET_A receptors were downstream effectors when I_Cl,swell was elicited by osmotic swelling or AngII. These data suggest that ET-1-induced ROS-dependent I_Cl,swell is likely to participate in multiple physiological and pathophysiological processes.