ENaC activity and expression is decreased in the lungs of protein kinase C-α knockout mice

Abstract

We used a PKC-α knockout model to investigate the regulation of alveolar epithelial Na(+) channels (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 with wild-type 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 with the wild-type. Confocal microscopy of lung slices showed elevated levels of reactive oxygen species (ROS) in the lungs of the PKC-α knockout mice vs. the wild-type. High levels of ROS in the knockout lung can be explained by a decrease in both cytosolic and mitochondrial superoxide dismutase activity. Elevated levels of ROS in the knockout lung activates PKC-δ and leads to reduced dephosphorylation of ERK1/2 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.

Novel mechanisms for crotonaldehyde-induced lung edema

“…It seems that there are different roles for ROS in the regulation of ENaC, both inhibitory and stimulatory. Elevated ROS could cause a decrease in ENaC activity, as seen in the PKC-α knockout lung [ 1 , 51 ], as well as previous literature had reported that ROS had a stimulatory effect on ENaC activity [ 49 , 52 ]. The discrete effects of extracellular and intracellular ROS on different cell and tissue models might regulate alveolar ENaC differently.…”

“…AFC appears to play an essential part in lung fluid balance in both physiological and pathological conditions, as studies have showed that patients suffering from acute lung injury or the more severe acute respiratory distress syndrome have dysfunctional alveolar fluid clearance [ 37 ]. Much evidence has confirmed that Na + reabsorption is a vital means of maintaining lung fluid balance and that ENaC plays a key role in Na + reabsorption [ 1 , 3 , 38 – 41 ].…”

“…In the lung, a thin layer of fluid is critical for efficient gas exchange on the apical surface of the alveolar epithelium [ 1 , 2 ], and lung edema is resolved by active salt and water transport [ 3 ]. Epithelial sodium (Na + ) channels (ENaC) are responsible for the majority of Na + transport across the apical membranes of alveolar epithelial cells [ 1 , 4 , 5 ], and increasing evidence has shown that ENaC, which is inhibited by the drug amiloride, appears to be essential in lung fluid clearance [ 6 , 7 ].…”

“…In the lung, a thin layer of fluid is critical for efficient gas exchange on the apical surface of the alveolar epithelium [ 1 , 2 ], and lung edema is resolved by active salt and water transport [ 3 ]. Epithelial sodium (Na + ) channels (ENaC) are responsible for the majority of Na + transport across the apical membranes of alveolar epithelial cells [ 1 , 4 , 5 ], and increasing evidence has shown that ENaC, which is inhibited by the drug amiloride, appears to be essential in lung fluid clearance [ 6 , 7 ]. Five ENaC subunits have been cloned to date, namely, α-, β-, γ-, δ- and ε-ENaC [ 8 , 9 ], and, when expressed in heterologous systems, the amiloride sensitivity and other properties of cloned αβγ-ENaC reflect of the characteristic of highly Na + selective of the channels found in native epithelial cells [ 8 ].…”

Inhibition of ENaC by Endothelin-1

“…The receptor for advanced glycation end-products (RAGE) regulated lung fluid balance via PKC-gp91 phox signaling to ENaC (Downs, Kreiner, Johnson, Brown, & Helms, 2014). Kinases SGK, PKC and PKA up-regulate ENaC activity in a variety of epithelial cell systems (Baines, 2013; Eaton, Yue, Eaton, & Bao, 2014). Some of these signaling molecules and pathways are well known components of signal transduction by ET-1.…”