Aldosterone-induced increases in superoxide production counters nitric oxide inhibition of epithelial Na channel activity in A6 distal nephron cells.
Oxygen radicals play an important role in signal transduction and have been shown to influence epithelial sodium channel (ENaC) activity. We show that aldosterone, the principal hormone regulating renal ENaC activity, increases superoxide (O2*) production in A6 distal nephron cells. Aldosterone (50 nM to 1.5 microM) induced increases in dihydroethidium fluorescence in a dose-dependent manner in confluent A6 epithelial cells. Using single-channel measurements, we showed that sequestering endogenous O2* (with the O2* scavenger 2,2,6,6-tetramethylpiperidine 1-oxyl) significantly decreased ENaC open probability from 0.10 +/- 0.03 to 0.03 +/- 0.01. We also found that increasing endogenous O2* in A6 cells, by applying a superoxide dismutase inhibitor, prevented nitric oxide (NO) inhibition of ENaC activity. ENaC open probability values did not significantly change from control values (0.23 +/- 0.05) after superoxide dismutase and 1.5 microM NO coincubation (0.21 +/- 0.04). We report that xanthine oxidase and hypoxanthine compounds increase local concentrations of O2* by approximately 30%; with this mix, an increase in ENaC number of channels times the open probability (from 0.1 to 0.3) can be achieved in a cell-attached patch. Our data also suggest that O2* alters NO activity in a cGMP-independent mechanism, since pretreating A6 cells with ODQ compound (a selective inhibitor of NO-sensitive guanylyl cyclase) failed to block 2,2,6,6-tetramethylpiperidine 1-oxyl inhibition of ENaC activity.
Yu
Ling
L
The Center for Cell and Molecular Signaling, Department of Physiology, Emory University School of Medicine, Whitehead Biomedical Research Bldg., 615 Michael St., Atlanta, GA 30322, USA.
Bao
Hui-Fang
HF
Self
Julie L
JL
Eaton
Douglas C
DC
Helms
My N
MN
D000450:Aldosterone / Q000008:administration & dosage / Q000494:pharmacology*; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002460:Cell Line; D006152:Cyclic GMP / Q000378:metabolism; D003497:Cyclic N-Oxides / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D004791:Enzyme Inhibitors / Q000494:pharmacology; D062686:Epithelial Sodium Channel Blockers; D053503:Epithelial Sodium Channels / Q000378:metabolism*; D004996:Ethidium / Q000031:analogs & derivatives; D005456:Fluorescent Dyes; D019271:Hypoxanthine / Q000494:pharmacology; D009399:Nephrons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D026361:Reactive Nitrogen Species / Q000378:metabolism; D013482:Superoxide Dismutase / Q000037:antagonists & inhibitors / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism*; D014969:Xanthine Oxidase / Q000494:pharmacology; D014982:Xenopus laevis