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


RESULTS
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 AT(1) 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.


CONCLUSIONS
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.
Deng
Wu
W
Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, VA 23298-0551, USA.
Baki
Lia
L
Baumgarten
Clive M
CM
D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D048429:Cell Size; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D019332:Endothelin-1 / Q000378:metabolism*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism; D005434:Flow Cytometry; D066298:In Vitro Techniques; D008564:Membrane Potentials; D049990:Membrane Transport Modulators / Q000494:pharmacology; D008929:Mitochondria, Heart / Q000187:drug effects / Q000201:enzymology*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000201:enzymology*; D009997:Osmotic Pressure; D018408:Patch-Clamp Techniques; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011817:Rabbits; D017382:Reactive Oxygen Species / Q000378:metabolism*; D044022:Receptor, Endothelin A / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014475:Uncoupling Agents / Q000494:pharmacology
D000804:Angiotensin II; D018118:Chloride Channels; D002712:Chlorides; D019332:Endothelin-1; D066246:ErbB Receptors; D049990:Membrane Transport Modulators; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D017382:Reactive Oxygen Species; D044022:Receptor, Endothelin A; D014475:Uncoupling Agents
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