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

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Abstract
We assessed regulation of volume-sensitive Cl− current (ICl,swell) by endothelin-1 (ET-1) and characterized the signalling pathway responsible for its activation in rabbit atrial and ventricular myocytes. ET-1 elicited ICl,swell under isosmotic conditions. Outwardly rectified Cl− current was blocked by the ICl,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 ICl,swell by osmotic swelling or angiotensin II (AngII) was suppressed by ETA blockade, whereas AngII AT1 receptor blockade failed to alter ET-1-induced current. Reactive oxygen species (ROS) produced by NADPH oxidase (NOX) stimulate ICl,swell. As expected, blockade of NOX suppressed ET-1-induced ICl,swell, but blockade of mitochondrial ROS production with rotenone also suppressed ICl,swell. ICl,swell was activated by augmenting complex III ROS production with antimycin A or diazoxide; in this case, ICl,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 ICl,swell via EGFR kinase, PI-3K, and NOX ROS production, which triggered mitochondrial ROS production. ETA receptors were downstream effectors when ICl,swell was elicited by osmotic swelling or AngII. These data suggest that ET-1-induced ROS-dependent ICl,swell is likely to participate in multiple physiological and pathophysiological processes.