Article

Aldosterone-induced increases in superoxide production counters nitric oxide inhibition of epithelial Na channel activity in A6 distal nephron cells.

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.
American journal of physiology. Renal physiology (Impact Factor: 3.3). 12/2007; 293(5):F1666-77. DOI: 10.1152/ajprenal.00444.2006
Source: PubMed

ABSTRACT 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.

0 Bookmarks
 · 
61 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: ENaC is negatively regulated by protein kinase C (PKC) as shown using PKC activators in a cell culture model. To determine whether PKCα influences ENaC activity in vivo, we examined the regulation of ENaC in renal tubules from PKCα(-/-) mice. Cortical collecting ducts were dissected and split open and the exposed principal cells were subjected to cell-attached patch clamp. In the absence of PKCα, open probability (Po) of ENaC was increased three-fold vs wild-type SV129 mice (0.52 ± 0.04 vs 0.17 ± 0.02). The number of channels per patch was also increased. Using confocal microscopy, we observed an increase in membrane localization of α, β, and γ subunits of ENaC in principal cells in the cortical collecting ducts of PKCα(-/-) mice compared to wild-type mice. To confirm this increase, one kidney from each animal was perfused with biotin and membrane protein was pulled down with streptavidin. The nonbiotinylated kidney was used to assess total protein. While total ENaC protein did not change in PKCα(-/-) mice, membrane localization of all the ENaC subunits was increased. The increase in membrane ENaC could be explained by the observation that ERK1/2 phosphorylation was decreased in the knockout mice. These results imply a reduction in ENaC membrane accumulation and Po by PKCα in vivo. The PKC-mediated increase in ENaC activity was associated with an increase in blood pressure in knockout mice fed a high-salt diet.
    AJP Renal Physiology 12/2013; · 4.42 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The significance of basal renal nitric oxide (NO) availability in the regulation of renal perfusion and sodium excretion in human congestive heart failure (CHF) has not been described previously. We studied the effects of acute systemic NO synthesis inhibition with N(G)-monomethyl-L-arginine (L-NMMA) in 12 patients with CHF and 10 healthy control subjects (CON) in a randomized placebo-controlled study. Effect parameters were renal plasma flow (RPF), renal vascular resistance (RVR), glomerular filtration rate (GFR), urine sodium excretion and plasma levels of vasoactive hormones. L-NMMA was associated with a significant decrease in RPF (CON-LNMMA: -13 ± 3% [P = .014]; CHF-LNMMA: -17 ± 7% [P = .017]) and a profound increase in RVR in both CHF and CON (CON-LNMMA: +26 ± 6% [P = .009]; CHF-LNMMA: +37 ± 70% [P = .005]). Significant decreases in sodium excretion were found in both CHF-LNMMA and CON-LNMMA. Relative changes from baseline were not statistically different between CHF-LNMMA and CON-LNMMA. After L-NMMA, RPF values correlated inversely with plasma aldosterone in CHF-LNMMA (P = .01). L-NMMA induced an increase in A-type natriuretic peptide (ANP) only in CHF-LNMMA (+18 ± 8%; P = .035), which correlated significantly with basal ANP levels (P = .034). There was no difference in the renal response to L-NMMA in CHF vs CON, suggesting that the impact of NO on renal perfusion and sodium excretion is maintained in stable CHF. We suggest that NO influences the release of ANP during high levels of atrial stretch in CHF.
    Journal of cardiac failure 11/2013; 19(11):776-785. · 3.07 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Nadph oxidase 4 is an important cellular source of reactive oxygen species (ROS) generation in the kidney. Novel anti-oxidant drugs, such as Nox4-inhibitor compounds, are being developed. There is however, very little experimental evidence for the biological role and regulation of Nadph oxidase isoforms in the kidney. Herein, we show that Fulvene-5 is an effective inhibitor of Nox-generated ROS and report the role of Nox isoforms in activating epithelial sodium channels (ENaC) in A6 distal nephron cells via oxidant signaling and cell stretch activation. Using single channel patch clamp analysis, we report that Fulvene-5 blocked the increase in ENaC activity that is typically observed with H2O2 treatment of A6 cells: average ENaC NPo values decreased from a baseline level of 1.04±0.18 (mean±SE) to 0.25±0.08 following Fulvene-5 treatment. H2O2 treatment failed to increase ENaC activity in the presence of Fulvene-5. Moreover, Fulvene-5 treatment of A6 cells blocked the osmotic-cell stretch response of A6 cells; indicating that stretch activation of Nox-derived ROS plays an important role in ENaC regulation. Together, these findings indicate that Fulvene-5, and perhaps other classes of antioxidant inhibitors, may represent a novel class of compounds useful for the treatment of pathological disorders stemming from inappropriate ion channel activity, such as hypertention.
    AJP Renal Physiology 07/2013; · 4.42 Impact Factor