Differential Effects of Protein Kinase C on the Levels of Epithelial Na+ Channel Subunit Proteins

Abstract

Regulation of epithelial Na؉ channel (ENaC) subunit levels by protein kinase C (PKC) was investigated in A6 cells. PKC activation altered ENaC subunit levels, differentially decreasing the levels of both ␤ and ␥, but not ␣ENaC. Temporal regulation of ␤ and ␥ENaC by PKC differed; ␥ENaC decreased with a time constant of 3.7 ؎ 1.0 h, whereas ␤ENaC decreased in 13.9 ؎ 3.0 h. Activation of PKC also resulted in a decrease in trans-epithelial Na ؉ reabsorption for up to 48 h. PMA activation of PKC resulted in negative feedback inhibition of PKC protein levels beginning within 4 h. Both ␤ and ␥ENaC levels, as well as transport tended toward pretreatment values after 48 h of PMA treatment. PKC inhibitors attenuated the effects of PMA on ENaC subunit levels and Na ؉ transport. These results directly show for the first time that PKC differentially regulates ENaC subunit levels by decreasing the levels of ␤ and ␥ but not ␣ENaC protein. These results imply a PKC-dependent, long term decrease in Na ؉ reabsorption.Sodium homeostasis is essential to proper maintenance of total body water and electrolyte content, and thus, blood pressure control. The activity of luminal, epithelial Na ϩ channels (ENaC) 1 is rate-limiting for trans-epithelial Na ϩ reabsorption across the renal collecting duct and other Na ϩ reabsorbing epithelium. Thus, understanding regulation of ENaC activity is relevant to physiology as well as to treating disease with associated fluid imbalance.ENaC is a heterotetrameric channel complex composed of at least three homologous but distinct subunits: ␣, ␤, and ␥ (1). Numerous results show that expression of ENaC subunit message and protein are differentially regulated in various tissues and species (reviewed by Refs. 2-4). For example, Masilamani and colleagues (5) recently showed in rat collecting duct principal cells that ␣ENaC protein levels increase in response to aldosterone; however, we found in the amphibian A6 cell model of the collecting duct principal cell that ␣ENaC is not significantly influenced by aldosterone, but ␤ENaC protein levels are increased in response to steroid (Ref. 6; also refer to Fig. 1 of the present study). Besides aldosterone, Zentner et al. (7) recently showed in the rat parotid epithelial cell line, Pa-4, that expression of ␣ENaC mRNA and possibly protein was decreased within 6 h by protein kinase C activation.Activation of PKC decreases Na ϩ reabsorption across renal epithelium by affecting ENaC (8 -11). Studies of single channel properties show that in amphibian, rat, and rabbit distal tubule cells, ENaC activity is decreased within 5-10 min after activation of PKC (12-15). A rapid initial decrease in ENaC open probability is, in part, responsible for the early change in activity; however, it appears that PKC may also subsequently affect the number of functional channels (14, 15). Although most studies are consistent with PKC decreasing ENaC open probability initially and then subsequently reducing channel number, Els et al. (16) showed with blocker-induced noise analysis in A6...

Inhibition of Na+ Transport in Lung Epithelial Cells by Respiratory Syncytial Virus Infection

“…P2Y receptors (the main targets of UTP) are G protein-coupled and act via the inositol phosphate pathway to stimulate Ca 21 release from intracellular stores, but can also act via multiple secondary signal transduction pathways, including protein kinase C (PKC) (36). Activation of PKC has been shown to reduce ENaC activity and modify its subunit composition (37,38). Our previous data indicate that infection of mice with RSV results in activation of PKCz as indicated by its phosphorylation and translocation from the cytoplasm to the plasma membrane (27).…”

P2Y4-Mediated Regulation of Na+Absorption in the Reissner's Membrane of the Cochlea

“…It has been reported that activation of PLC signaling inhibits ENaC activity either by activating PKC or depletion of the inner membrane PI(4,5)P 2 content (Kunzelmann et al, 2005). The activation of PKC is known to evoke later response and to maintain long-term (up to 48 h) downregulation of ENaC by decreasing plasma membrane levels of the channel (Stockand et al, 2000;Booth and Stockand, 2003). The level of ␥-ENaC decreased by PKC with a time constant of 3.7 h, and the level of ␤-ENaC decreased in 13.9 h. The fact that treatment of tissues with 2-APB, a cell-permeable antagonist of inositol trisphosphate (IP 3 ) receptor and store-operated channels (Bootman et al, 2002), did not affect UTP effect on ENaC activity suggests that the involvement of the second branch of the PLC pathway (IP 3 production) in response to UTP is negligible.…”

New ideas about aldosterone signaling in epithelia

“…During this phase, there is a clear increase in the amount of ENaC protein within the cell, as well as at the apical membrane (100, 107, 189). However, controversy surrounds which subunits of the heteromultimeric ENaC are induced by aldosterone, with some studies showing increases in ␣-ENaC levels (100,107,110,114) and some increases in ␤-ENaC levels (158,189), and yet others reporting increases in both (42, 47). Moreover, controversy surrounds the idea that expression and insertion into the apical membrane of ENaC subunits are discordantly and independently regulated by aldosterone (67,100,107,139,189).…”