Aldosterone-induced increases in superoxide production counters nitric oxide inhibition of epithelial Na channel activity in A6 distal nephron cells
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.
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ABSTRACT: We used a PKC-α knockout model to investigate the regulation of alveolar ENaC by PKC. Primary alveolar type II (ATII) cells were subjected to cell-attached patch clamp. In the absence of PKC-α, the open probability (Po) of ENaC was decreased by half compared to wildtype mice. The channel density (N) was also reduced in the knockout mice. Using in-vivo biotinylation, membrane localization of all three ENaC subunits (α, β, and γ) was decreased in the PKC-α knockout lung, compared to the wildtype. Confocal microscopy of lung slices, showed elevated levels of reactive oxygen species (ROS) in the lungs of the PKC-α knockout mice, versus the wildtype. High levels of ROS in the knockout lung can be explained by a decrease in both cytosolic and mitochondrial superoxide dismutase (SOD) activity. Elevated levels of ROS in the knockout lung activates PKC-δ, as well as leading to reduced dephosphorylation of ERK 1/2 by MAP-kinase phosphatase, which in turn causes increased internalization of ENaC via ubiquitination by the ubiquitin-ligase Nedd4-2. In addition, in the knockout lung, PKC-δ activates ERK causing a decrease in ENaC density at the apical alveolar membrane. PKC-δ also phosphorylates MARCKS, leading to a decrease in ENaC Po. The effects of ROS and PKC-δ were confirmed with patch-clamp experiments on isolated ATII cells in which the ROS scavenger, Tempol, or a PKC-δ specific inhibitor added to patches reversed the observed decrease in ENaC apical channel density, and Po. These results explain the decrease in ENaC activity in PKC-α knockout lung.AJP Lung Cellular and Molecular Physiology 07/2014; 307(5). DOI:10.1152/ajplung.00040.2014 · 4.04 Impact Factor
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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. DOI:10.1016/j.cardfail.2013.10.001 · 3.07 Impact Factor
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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; 306(3). DOI:10.1152/ajprenal.00519.2013 · 4.42 Impact Factor