Hydrogen Peroxide Stimulates the Epithelial Sodium Channel through a Phosphatidylinositide 3-Kinase-dependent Pathway

Abstract

ENaC 2 in the distal nephron plays an important role in maintaining Na ϩ homeostasis and consequently controls systemic blood pressure. Excess sodium reabsorption due to enhanced ENaC activity can lead to hypertension, as is seen in Liddle syndrome (1, 2) and other genetic diseases (3). Surprisingly, whether high salt intake induces hypertension by targeting ENaC has not been examined. High salt intake can elevate both superoxide (O 2 . ) (4, 5) and H 2 O 2 (6) in the kidney by stimulating NAD(P)H oxidase (7-9), and O 2 . can increase aldosterone-me- , stimulate the non-receptor tyrosine kinase, c-Src (15). c-Src is known to be directly associated with p85, the regulatory subunit of PI3K (16). Tyrosine phosphorylation of p85 reduces its inhibitory effect on PI3K (17). These studies indicate that ROS may stimulate PI3K and consequently promote the synthesis of PI(3,4,5)P 3 . We (18 -20) and others (21,22) have previously shown that PI(3,4,5)P 3 stimulates ENaC. Taken together, these studies suggest that H 2 O 2 may stimulate ENaC by elevating PI(3,4,5)P 3 through activation of PI3K. In the present study, using patch clamp techniques combined with confocal microscopy analysis of apical PI(3,4,5)P 3 levels, we show that H 2 O 2 stimulates ENaC in A6 distal nephron cells via PI3K-dependent elevation of PI(3,4,5)P 3 in the apical membrane. MATERIALS AND METHODSCell Culture-Xenopus A6 distal nephron cells were purchased from the American Type Culture Collection (Manassas, VA). Under previously used culture conditions, basal activity of ENaC in A6 cells is relatively high (23). It is difficult to see and analyze any stimulatory effect when basal ENaC activity is high. Therefore, in the present study, we modified the culture medium to reduce basal ENaC activity. The medium contained 2 parts of DMEM/F-12 (1:1) medium (Invitrogen), 1 part of H 2 O, 15 mM NaHCO 3 (total Na ϩ ϭ 101 mM, which is ideal for amphibian A6 cells), 2 mM L-glutamine, 10% fetal bovine serum (Invitrogen), 25 units/ml penicillin, 25 units/ml streptomycin. A6 cells were cultured in plastic flasks in the presence of 1 M aldosterone at 26°C and 4% CO 2 . After the cells became 70% * This work was supported, in whole or in part, by National Institutes of Health Grant 5R01-DK067110 (to H.-P. M.

Early bioelectric activities mediate redox-modulated regeneration

“…The sharp TEP increase until 6 hpa supports this and might suggest the ubiquitous Na + /K + -ATPase as a candidate (Dubé et al, 2010). Thus, it is possible that H 2 O 2 (or other ROS) modulates additional ion translocators (Ma, 2011;Matalon et al, 2003). Exogenous H 2 O 2 rescued and induced regeneration and induced ectopic tails, suggesting that H 2 O 2 provides indispensable morphogenetic information.…”

Angiotensin II Increases Activity of the Epithelial Na+ Channel (ENaC) in Distal Nephron Additively to Aldosterone

“…Furthermore, we directly demonstrated that Ang II increases ROS production in cultured mouse principal cells. A recent report suggests that ROS, specifically H 2 O 2 , possess a stimulatory effect on ENaC P o in A6 cells (49). This effect was not direct and involved activation of PI3K.…”

Lovastatin-Induced Phosphatidylinositol-4-Phosphate 5-Kinase Diffusion from Microvilli Stimulates ROMK Channels

“…43 Briefly, to detect the localization of ROMK1, prominin-1, and PI(4)P5K I g in mpkCCD c14 cells, the cells were fixed with 4% paraformaldehyde for 10 minutes, washed two times, permeabilized with 0.1% Triton X-100 in NaCl bath solution for 15 minutes, and then washed two times. The cells were incubated with rabbit polyclonal antibodies to ROMK1 (APC-001; Alomone Laboratories) or PI(4)P5K type I g (ABS190; EMD Millipore) for 1 hour, washed two times, incubated with either a secondary antibody (Alexa Fluor 594, goat antirabbit IgG, 5 mg/ml) or directly with Alexa Fluor 488-conjugated antibody to prominin-1 (53-1331-80, lot: E031633; eBioscience) or Alexa Fluor 488-conjugated CTX subunit B (C34775, lot: 1316686; Life Technologies) at room temperature for 1 hour.…”