Endothelin signalling regulates volume-sensitive Cl- current via NADPH oxidase and mitochondrial reactive oxygen species

We assessed regulation of volume-sensitive Cl- current (I-Cl,I-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,I-swell under isosmotic conditions. Outwardly rectified Cl- current was blocked by the I-Cl,I-swell-selective inhibitor DCPIB or osmotic shrinkage and involved ETA but not ETB 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,I-swell by osmotic swelling or angiotensin II (AngII) was suppressed by ETA blockade, whereas AngII AT(1) receptor blockade failed to alter ET-1-induced current. Reactive oxygen species (ROS) produced by NADPH oxidase (NOX) stimulate I-Cl,I-swell. As expected, blockade of NOX suppressed ET-1-induced I-Cl,I-swell, but blockade of mitochondrial ROS production with rotenone also suppressed I-Cl,I-swell. I-Cl,I-swell was activated by augmenting complex III ROS production with antimycin A or diazoxide; in this case, I-Cl,I-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-ETA signalling activated I-Cl,I-swell via EGFR kinase, PI-3K, and NOX ROS production, which triggered mitochondrial ROS production. ETA receptors were downstream effectors when I-Cl,I-swell was elicited by osmotic swelling or AngII. These data suggest that ET-1-induced ROS-dependent I-Cl,I-swell is likely to participate in multiple physiological and pathophysiological processes.