title: ROS production as a common mechanism of ENaC regulation by EGF, insulin, and IGF-1.
abstract: The epithelial Na(+) channel (ENaC) is a key transporter participating in the fine tuning of Na(+) reabsorption in the nephron. ENaC activity is acutely upregulated by epidermal growth factor (EGF), insulin, and insulin-like growth factor-1 (IGF-1). It was also proposed that reactive oxygen species (ROS) have a stimulatory effect on ENaC. Here we studied whether effects of EGF, insulin, and IGF-1 correlate with ROS production in the mouse cortical collecting duct (mpkCCD(c14)) cells. Western blotting confirmed the expression of the NADPH oxidase complex subunits in these cells. Treatment of mpkCCD(c14) cells with EGF, insulin, or IGF-1 evoked an increase in ROS production as measured by CM-H(2)DCF-DA fluorescence. ROS production caused by a xanthine-xanthine oxidase reaction also resulted in a significant elevation in short-circuit current through the mpkCCD(c14) monolayer. Transepithelial current measurements showed an acute increase of amiloride-sensitive current through the mpkCCD(c14) monolayer in response to EGF, insulin, or IGF-1. Pretreatment with the nonselective NADPH oxidase activity inhibitor apocynin blunted both ROS production and increase in ENaC-mediated current in response to these drugs. To further test whether NADPH oxidase subunits are involved in the effect of EGF, we used a stable M-1 cell line with a knockdown of Rac1, which is one of the key subunits of the NADPH oxidase complex, and measured amiloride-sensitive currents in response to EGF. In contrast to control cells, EGF had no effect in Rac1 knockdown cells. We hypothesize that EGF, insulin, and IGF-1 have a common stimulatory effect on ENaC mediated by ROS production.
authors.affiliation: Medical College of Wisconsin, Dept. of Physiology, 8701 Watertown Plank Rd., Milwaukee, WI 53226, USA.
authors.forename: Daria V
authors.initials: DV
authors.lastname: Ilatovskaya
authors.forename: Tengis S
authors.initials: TS
authors.lastname: Pavlov
authors.forename: Vladislav
authors.initials: V
authors.lastname: Levchenko
authors.forename: Alexander
authors.initials: A
authors.lastname: Staruschenko
mesh_terms: D000818:Animals; D002461:Cell Line, Transformed; D004815:Epidermal Growth Factor / Q000502:physiology*; D053503:Epithelial Sodium Channels / Q000378:metabolism* / Q000502:physiology; D007004:Hypoglycemic Agents / Q000737:chemistry / Q000378:metabolism; D007328:Insulin / Q000502:physiology*; D007334:Insulin-Like Growth Factor I / Q000502:physiology*; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D051379:Mice; D009479:Neuropeptides / Q000172:deficiency / Q000235:genetics; D018384:Oxidative Stress / Q000502:physiology; D017382:Reactive Oxygen Species / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000172:deficiency / Q000235:genetics; D020830:rac1 GTP-Binding Protein
chemical_list: D053503:Epithelial Sodium Channels; D007004:Hypoglycemic Agents; D007328:Insulin; D009479:Neuropeptides; C484602:Rac1 protein, mouse; D017382:Reactive Oxygen Species; D004815:Epidermal Growth Factor; D007334:Insulin-Like Growth Factor I; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein