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• W2154620619 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 β and γ, but not αENaC. Temporal regulation of β and γENaC by PKC differed; γENaC decreased with a time constant of 3.7 ± 1.0 h, whereas βENaC 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 β and γENaC 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 β and γ but not αENaC protein. These results imply a PKC-dependent, long term decrease in Na+ reabsorption. 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 β and γ, but not αENaC. Temporal regulation of β and γENaC by PKC differed; γENaC decreased with a time constant of 3.7 ± 1.0 h, whereas βENaC 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 β and γENaC 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 β and γ but not αENaC protein. These results imply a PKC-dependent, long term decrease in Na+ reabsorption. epithelial Na+ channel(s) protein kinase C phorbol 12-myristate 13-acetate 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+ channels (ENaC)1 is rate-limiting for trans-epithelial Na+ reabsorption across the renal collecting duct and other Na+ 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: α, β, and γ (1Canessa C.M. Schild L. Buell G. Thorens B. Gautschi I. Horisberger J.D. Rossier B.C. Nature. 1994; 367: 463-467Crossref PubMed Scopus (1758) Google Scholar). Numerous results show that expression of ENaC subunit message and protein are differentially regulated in various tissues and species (reviewed by Refs. 2Verrey F. Am. J. Physiol. 1999; 277: F319-F327Crossref PubMed Google Scholar, 3Verrey F. Exp. Nephrol. 1998; 6: 294-301Crossref PubMed Scopus (28) Google Scholar, 4Verrey F. J. Membr. Biol. 1995; 144: 93-110Crossref PubMed Scopus (161) Google Scholar). For example, Masilamani and colleagues (5Masilamani S. Kim G.h Mitchell C. Wade J.B. Knepper M.A. J. Clin. Invest. 1999; 104: R19-R23Crossref PubMed Scopus (621) Google Scholar) recently showed in rat collecting duct principal cells that αENaC protein levels increase in response to aldosterone; however, we found in the amphibian A6 cell model of the collecting duct principal cell that αENaC is not significantly influenced by aldosterone, but βENaC protein levels are increased in response to steroid (Ref. 6Middleton, P., Al-Khalili, O. K., Yue, G., Zuckerman, J. B., Kleyman, T. R., and Eaton, D. C. (2000) Am. J. Physiol., in pressGoogle Scholar; also refer to Fig. 1 of the present study). Besides aldosterone, Zentner et al. (7Zentner M.D. Lin H.h Wen X. Kim K.J. Ann D.K. J. Biol. Chem. 1998; 273: 30770-30776Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) recently showed in the rat parotid epithelial cell line, Pa-4, that expression of αENaC mRNA and possibly protein was decreased within 6 h by protein kinase C activation.Activation of PKC decreases Na+ reabsorption across renal epithelium by affecting ENaC (8Garty H. Palmer L.G. Physiol. Rev. 1997; 77: 359-396Crossref PubMed Scopus (1033) Google Scholar, 9Duchatelle P. Ohara A. Ling B.N. Kemendy A.E. Kokko K.E. Matsumoto P.S. Eaton D.C. Mol. Cell. Biochem. 1992; 114: 27-34Crossref PubMed Scopus (36) Google Scholar, 10Ling B.N. Kemendy A.E. Kokko K.E. Hinton C.F. Marunaka Y. Eaton D.C. Mol. Cell. Biochem. 1990; 99: 141-150Crossref PubMed Scopus (32) Google Scholar, 11Rokaw M.D. West M. Johnson J.P. J. Biol. Chem. 1996; 271: 32468-32473Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). 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 (12Awayda M.S. Ismailov I.I. Berdiev B.K. Fuller C.M. Benos D.J. J. Gen. Physiol. 1996; 108: 49-65Crossref PubMed Scopus (81) Google Scholar, 13Frindt G. Palmer L.G. Windhager E.E. Am. J. Physiol. 1996; 270: F371-F376PubMed Google Scholar, 14Ling B.N. Eaton D.C. Am. J. Physiol. 1989; 256: F1094-F1103PubMed Google Scholar, 15Ishikawa T. Marunaka Y. Rotin D. J. Gen. Physiol. 1998; 111: 825-846Crossref PubMed Scopus (118) Google Scholar). 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 (14Ling B.N. Eaton D.C. Am. J. Physiol. 1989; 256: F1094-F1103PubMed Google Scholar, 15Ishikawa T. Marunaka Y. Rotin D. J. Gen. Physiol. 1998; 111: 825-846Crossref PubMed Scopus (118) Google Scholar). Although most studies are consistent with PKC decreasing ENaC open probability initially and then subsequently reducing channel number, Els et al. (16Els W.J. Liu X. Helman S.I. Am. J. Physiol. 1998; 275: C120-C129Crossref PubMed Google Scholar) showed with blocker-induced noise analysis in A6 cells that PKC activation, besides producing the initial decrease in open probability and a longer term decrease in channel number, might also lead to a small initial compensatory increase in channel number presumably in response to decreased open probability. Nevertheless, actions on channel open probability likely precede effects on number and both reduce overall sodium transport. In a provocative study, Ishikawa and colleagues (15Ishikawa T. Marunaka Y. Rotin D. J. Gen. Physiol. 1998; 111: 825-846Crossref PubMed Scopus (118) Google Scholar) report biphasic actions of Ca2+-dependent processes (e.g. activation of PKC) on repression of ENaC activity with time constants ranging between 1–2 and 100–160 min. The current results are consistent with the latter time constant representing a decrease in ENaC number.Recently, Shimkets et al. (17Shimkets R.A. Lifton R. Canessa C.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3301-3305Crossref PubMed Scopus (162) Google Scholar) showed for the first time that PKC directly phosphorylates ENaC when this ion channel is overexpressed and PKC is substantially activated. It is unclear how PKC-mediated phosphorylation relates to ENaC kinetics and number and, thus, Na+ reabsorption. Moreover, the long term actions of PKC on transport and ENaC protein levels have not been studied.Because ENaC subunit levels can be differentially regulated in response to various factors and PKC is known to affect ENaC activity, perhaps, in part, through regulation of channel number, we tested the hypothesis that PKC differentially regulates subunit protein levels. This is the first report directly showing that ENaC subunit protein levels are differentially affected by PKC with both γ and β, but not αENaC, decreasing in response to kinase activation. The decrease in subunit levels is consistent with the long term actions of PKC on ENaC resulting in decreased Na+ channel number and, thus, transport.MATERIALS AND METHODSAll experiments were performed on A6 cells (American Tissue Type Culture; passages 75–80). A6 cells were cultured, as described previously (14Ling B.N. Eaton D.C. Am. J. Physiol. 1989; 256: F1094-F1103PubMed Google Scholar, 18Stockand J.D. Spier B.J. Worrell R.T. Yue G. Al-Baldawi N. Eaton D.C. J. Biol. Chem. 1999; 274: 35449-35454Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), in medium (3 parts Coon's F-12 and 7 parts Leibovitz's L-15) modified for amphibian cells (104 mmNaCl, 25 mm NaHCO3, pH 7.4) and supplemented with fetal bovine serum (10%) and aldosterone (1.5 μm). High resistance (>2 KΩ) A6 cell monolayers plated on permeable 3.8-cm2 inserts (0.02 μm Anopore membrane; Nalge NUNC International, Naperville, IL) avidly reabsorbing Na+ were used for all experiments.Trans-epithelial Na+ current was calculated as the ratio of trans-epithelial voltage to trans-epithelial resistance under open circuit conditions using a Millicel Electrical Resistance System with dual Ag/AgCl pellet electrodes (Millipore Corp.) to measure trans-epithelial potentials and resistances. If the current-voltage relationship of the monolayer is close to linear, this measurement is exactly equivalent to short circuit current. However, trans-epithelial currents calculated in this fashion do not require long term short circuiting of the tissue, which alters intracellular ion concentrations and, therefore, may alter single channel properties. The open circuit measurements, in addition, ensured that larger numbers of monolayers could be routinely sampled, thus ensuring that adequate cellular material was available for subsequent biochemical analysis, as well as enabling the pairing of electrical and biochemical measurements. Movement of cations from the lumen to serosal fluids is represented as positive current. With this preparation, the majority of trans-epithelial current was amiloride-sensitive and carried by Na+ via ENaC from lumen to serosal fluid.A6 cells were extracted with RIPA buffer: 10 mmNaPO4, 150 mm NaCl, 1% deoxycholate, 1% Triton X-100, 0.1% sodium dodecyl sulfate, supplemented with 1 μm phenylmethylsulfonyl fluoride (pH 7.2). After clearing of cellular debris, standardization of total protein, and addition of sample buffer (containing 10% glycerol, 1% SDS, and 20 mmdithiothreotol), lysates were boiled at 85 °C for 10 min. Proteins were separated by standard SDS-polyacrylamide gel electrophoresis (7.5% gels) and subsequently electrophoretically transferred to nitrocellulose. Western blot analysis was performed using standard techniques and appropriate antibodies (see below). 0.1% Tween-20 and 5% dried milk (Carnation) were used as blocking agents. All secondary horseradish peroxidase conjugates were purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD). Western blots were developed with the ECL detection system (Amersham Pharmacia Biotech) and quantified with densitometric scanning using Sigmagel (Jandel Scientific). When possible, the flood configuration was used to measure band density.Protein kinase C was detected with the commercially available antibody PKC (MC5) purchased from Santa Cruz Biotech. (Santa Cruz, CA). This antibody identifies α, β, and γ isoforms of PKC. The anti-γ-xENaC antibody used in this study was a kind gift from John P. Johnson (Department of Medicine, University of Pittsburgh School of Medicine). This affinity purified, polyclonal antibody, developed against Xenopus laevis γENaC (residues 608–660) in chicken, has been described previously (19Rokaw M.D. Wang J.-M. Edinger R.S. Weisz O.A. Hui D. Middleton P. Shyonsky V. Berdiev B.K. Ismailov I. Eaton D.C. Benos D.J. Johnson J.P. J. Biol. Chem. 1998; 273: 28746-28751Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) and specifically identifies an intrinsic membrane protein of 85–95 kDa. The polyclonal anti-αENaC antibody used in this study was raised in rabbit (by Lofstrand Labs, Gaithersburg, MD) against residues 137–161 of X. laevis αENaC linked to keyhole limpet hemocyanin through an amino-terminal cysteine. Antiserum was subsequently affinity purified against inoculating immunogen using standard protocols to produce the final anti-αENaC antibody, AB586. Development and use of this antibody has been described previously (6Middleton, P., Al-Khalili, O. K., Yue, G., Zuckerman, J. B., Kleyman, T. R., and Eaton, D. C. (2000) Am. J. Physiol., in pressGoogle Scholar). This antibody specifically recognizes a membrane protein of appropriate size (85–90 kDa), as well as in vitro translated αENaC. The polyclonal anti-βENaC antibody used in this study was developed with a similar procedure. The anti-βENaC antibody (AB592) was against residues 624–647 of β-xENaC. Affinity purified AB592 specifically recognizes an intrinsic membrane protein of appropriate size (90–95 kDa) and in vitro translated βENaC. These three anti-ENaC subunit-specific antibodies have no detectable, inappropriate cross-reactivity with other subunits.All chemicals were purchased from Sigma, BIOMOL (Plymouth Meeting, PA), or Bio-Rad unless indicated otherwise. Phorbol 12-myristate 13-acetate (PMA) and 4α-PMA were prepared fresh in Me2SO prior to each experiment. Final concentrations of Me2SO never exceeded 0.05% and were without effect when applied alone. For time course experiments, PMA was replenished for the longer time points every 24 h.Data are reported as the means ± S.E. The t test and a one-way analysis of variance plus the Student-Newman-Keuls test were used to compare data where appropriate. p ≤ 0.05 was considered significant. Changes in relative protein levels and electrical parameters were fit using least squares nonlinear minimization (SigmaPlot 5.0, San Rafael, CA). Decreases were fit to a single exponential of the form f(t) =e−t/τ; increases were fit to a function of the form f(t) = (1 −e−t/τ)n wheret is time, τ is the time constant, and n is a factor that is a measure of the delay before an increase occurs.RESULTS AND DISCUSSIONA6 cells are models of the principal cell frequently used for the study of regulated Na+ reabsorption. These cells have been specifically used for investigating the contribution and modulation of ENaC during transport. A6 cell signal transduction and electrophysiological properties are believed to be similar to those of mammalian collecting duct principal cells. A6 cells express the epithelial Na+ channel in the luminal membrane with the this channel's gating kinetics and number influenced by cortico-steroids (2Verrey F. Am. J. Physiol. 1999; 277: F319-F327Crossref PubMed Google Scholar, 3Verrey F. Exp. Nephrol. 1998; 6: 294-301Crossref PubMed Scopus (28) Google Scholar, 10Ling B.N. Kemendy A.E. Kokko K.E. Hinton C.F. Marunaka Y. Eaton D.C. Mol. Cell. Biochem. 1990; 99: 141-150Crossref PubMed Scopus (32) Google Scholar). In the current study, we used the A6 cell line to investigate the regulation of ENaC subunit levels by protein kinase C. We show directly for the first time with ENaC subunit-specific antibodies that activation of PKC with PMA results in a decrease in γ and β but not αENaC protein levels; γENaC decreased prior to βENaC. PKC also decreased Na+reabsorption. Decreases in subunit levels and transport in response to PMA were reduced or eliminated with PKC inhibitors. Our results are consistent with activation of PKC resulting in long term (>4 h) depression of β and γENaC levels leading, in part, to a sustained (up to 48 h) decrease in Na+ reabsorption.The Western blot analysis in Fig. 1 is consistent with A6 cells expressing all three ENaC subunits: α, β, and γ. It is well documented both biochemically and electrophysiologically that A6 cells contain typical, fully functional (heteromultimeric) ENaC (2Verrey F. Am. J. Physiol. 1999; 277: F319-F327Crossref PubMed Google Scholar, 3Verrey F. Exp. Nephrol. 1998; 6: 294-301Crossref PubMed Scopus (28) Google Scholar, 10Ling B.N. Kemendy A.E. Kokko K.E. Hinton C.F. Marunaka Y. Eaton D.C. Mol. Cell. Biochem. 1990; 99: 141-150Crossref PubMed Scopus (32) Google Scholar). In fact, the cDNAs encoding these subunits have been cloned from A6 cells (20Puoti A. May A. Canessa C.M. Horisberger J.D. Schild L. Rossier B.C. Am. J. Physiol. 1995; 269: C188-C197Crossref PubMed Google Scholar). More importantly for the current study, results in Fig. 1 show that we have antibodies that specifically recognize each of the ENaC subunits.For Western blot analysis (representative blots shown in Fig. 1), each lane within a gel contained lysate with the same amount of total protein (∼80 μg). The blots of A and C in Fig. 1 were probed with anti-αENaC antibody AB586 and anti-βENaC antibody AB592, respectively. The first andthird lanes in these blots contained lysate harvested form A6 cells serum and aldosterone starved for >72 h (−). Thesecond and fourth lanes (+) had lysate from cells treated with aldosterone for >72 h. The right two lanes (+pep) of A and C were probed with antibody preabsorbed with 0.1 mg/ml of the respective immunogens. The top blot of B was probed with AB586. This blot was stripped and reprobed with anti-γ-xENaC (lower blot of B, where − and + have the same meaning). The effects of aldosterone on α and γENaC in A6 cells were inconsistent with steroid clearly increasing αENaC in two of four experiments (an increase is shown in A, and no effect is shown in the top blot of B) and increasing γENaC in one of four experiments (refer to bottom blot of B). In contrast, long term exposure to aldosterone (1.5 μm; ≥ 72 h), as shown by the Western blot of C and summarized in D, consistently and significantly increased βENaC 20.8 ± 5.5-fold (seven of seven experiments). For all experiments, aldosterone-treated cells had significantly more Na+ transport. These blots suggest interesting characteristics for ENaC subunits in A6 cells: 1) the relative molecular weight of subunits are α < γ < β and 2) β and possibly α are often observed as doublets perhaps indicating glycosylation. Both subunits contain putative glycosylation sites and previously have been reported to be glycosylated (5Masilamani S. Kim G.h Mitchell C. Wade J.B. Knepper M.A. J. Clin. Invest. 1999; 104: R19-R23Crossref PubMed Scopus (621) Google Scholar, 6Middleton, P., Al-Khalili, O. K., Yue, G., Zuckerman, J. B., Kleyman, T. R., and Eaton, D. C. (2000) Am. J. Physiol., in pressGoogle Scholar, 21May A. Puoti A. Gaeggeler H.P. Horisberger J.D. Rossier B.C. J. Am. Soc. Nephrol. 1997; 8: 1813-1822Crossref PubMed Google Scholar). Moreover, β but neither γ nor αENaC subunit levels correlated well with the actions of aldosterone to increase transport suggesting that βENaC may be limiting for long term steroid-regulated reabsorption across A6 cells. However, these results are preliminary in this regard, and thus, this notion needs to be investigated further prior to making more definitive statements on this topic.It is well documented in A6 cells that ENaC transcript and protein subunit levels are differentially regulated by aldosterone and other factors (reviewed in Refs. 2Verrey F. Am. J. Physiol. 1999; 277: F319-F327Crossref PubMed Google Scholar, 3Verrey F. Exp. Nephrol. 1998; 6: 294-301Crossref PubMed Scopus (28) Google Scholar, and 8Garty H. Palmer L.G. Physiol. Rev. 1997; 77: 359-396Crossref PubMed Scopus (1033) Google Scholar). The results of Fig. 1 are consistent with findings we have published previously (6Middleton, P., Al-Khalili, O. K., Yue, G., Zuckerman, J. B., Kleyman, T. R., and Eaton, D. C. (2000) Am. J. Physiol., in pressGoogle Scholar) showing that β (detected with an antibody distinct from the one used in the current study) but not α and γENaC protein levels are reproducibly increased in response to long term administration of aldosterone. Similarly, J. P. Johnson and colleagues 2J. P. Johnson, M. D. Rokaw, and R. S. Edinger, personal communication. also find in A6 cells that β but not α and γENaC protein is increased by aldosterone. It is unclear why these results differ from those of Mayet al. (21May A. Puoti A. Gaeggeler H.P. Horisberger J.D. Rossier B.C. J. Am. Soc. Nephrol. 1997; 8: 1813-1822Crossref PubMed Google Scholar) in A6 cells and Masilamani et al. (5Masilamani S. Kim G.h Mitchell C. Wade J.B. Knepper M.A. J. Clin. Invest. 1999; 104: R19-R23Crossref PubMed Scopus (621) Google Scholar) in rat collecting duct principal cells showing that only αENaC is consistently increased in response to aldosterone. Nonetheless, our antibodies, as well as those of others, are useful tools for studying the regulation of ENaC subunit protein levels.Although PKC is known to decrease ENaC activity (reviewed in Refs. 9Duchatelle P. Ohara A. Ling B.N. Kemendy A.E. Kokko K.E. Matsumoto P.S. Eaton D.C. Mol. Cell. Biochem. 1992; 114: 27-34Crossref PubMed Scopus (36) Google Scholar,10Ling B.N. Kemendy A.E. Kokko K.E. Hinton C.F. Marunaka Y. Eaton D.C. Mol. Cell. Biochem. 1990; 99: 141-150Crossref PubMed Scopus (32) Google Scholar, and 22Eaton D.C. Becchetti A. Ma h Ling B.N. Kid. Int. 1995; 48: 941-949Abstract Full Text PDF PubMed Scopus (66) Google Scholar), the direct actions of PKC on ENaC subunit levels has not been investigated. In addition, a temporal correlation between PKC effects on ENaC subunit levels and Na+ reabsorption has not been established. Thus, we tested the hypothesis that PKC differentially regulates ENaC subunit levels and also temporally correlated effects on subunit levels with changes in transport.The representative Western blots of Fig.2 A show that γ (bottom blot) and β (middle blot) but not αENaC (top blot) levels are decreased within 24 h in response to PMA (100 ng/ml) activation of PKC. This effect was not observed with inactive phorbol ester (4α-PMA; 100 ng/ml). The Western blots and bar graph of Fig. 2 (B and C) summarize the actions of PMA at differing concentrations on β and γENaC subunit levels after 24 h (n = 3). Neither PMA (100 ng/ml) nor 4α-PMA affected αENaC with relative (to time 0) levels being 1.0 ± 0.1 and 1.0 ± 0.1 before treatment and 0.9 ± 0.1 (n = 5) and 0.9 ± 0.1 (n = 2) after 24 h of treatment, respectively. In contrast, PMA (100 ng/ml) decreased βENaC levels from 1.0 ± 0.03 to 0.4 ± 0.04 (n = 10) and γENaC levels from 1.0 ± 0.02 to 0.2 ± 0.02 (n = 8). 4α-PMA did not affect either γ (relative density at 24 h was 0.9 ± 0.03;n = 3) or βENaC levels (relative density at 24 h was 1.2 ± 0.1; n = 5). Note that 10 but not 0.1 ng/ml PMA decreased β and γENaC levels. The dose response of PMA on Na+ transport (measured after 24 h.) is summarized in Fig. 2 D. Relative transport in the absence of PMA and in the presence of PMA at 100, 10, 1, 0.1, and 0.001 ng/ml were 0.95 ± 0.04, 0.05 ± 0.01, 0.70 ± 0.06, 0.79 ± 0.06, 1.1 ± 0.06, and 1.1 ± 0.05 (n = 6), respectively. These results show that PKC differentially regulates ENaC subunits levels. The mechanism by which PKC decreases γ and βENaC protein levels remains to be determined.Figure 2PMA decreases the levels of β and γ but not α ENaC. A, top,middle, and bottom blots probed with AB586, AB592, and anti-γ-xENaC, respectively. Whole A6 cell lysate was extracted from cells treated with vehicle (CON, < 0.05% Me2SO), 4α-PMA (4α, 100 ng/ml; 162 nm), and PMA (100 ng/ml; 162 nm) for 24 h.B, typical blots showing the PMA dose response on β (top) and gENaC (bottom) levels 24 h after treatment (100 ng/ml = 162 nm, 10 ng/ml = 16.2 nm, 1 ng/ml = 1.6 nm, 0.1 ng/ml = 0.16 nm, and 0.01 ng/ml = 0.02 nm).C, summary graph of the effects of PMA at various doses on ENaC subunit levels. *, significance difference from levels in the absence of PMA; **, significance difference from αENaC levels at the same dose. D, dose response of PMA on relative Na+ current measured at 24 h. *, significance from levels in the absence of PMA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Our findings that γ and β but not αENaC subunit levels decrease in response to PKC activation are different than the results of Zentneret al. (7Zentner M.D. Lin H.h Wen X. Kim K.J. Ann D.K. J. Biol. Chem. 1998; 273: 30770-30776Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) in which PKC was found only to decrease αENaC transcript levels in this latter study. Also in this latter study, preliminary evidence was provided that PKC effects on αENaC transcript levels translated into decreased subunit protein expression. Thus, even though they have not directly examined subunit protein levels, it seems likely that there is a difference in the response to PMA of salivary gland cells and A6 cells. There are many possible explanations of the difference. The simplest would be that the complement of transcription factors present in salivary cells and A6 cells is different and that, therefore, regulation of gene expression is different. A second simple possibility is that the signaling pathways activated by PMA are different in the two cell types so that the relative activation of different transcription factors would be different. Interestingly, the time course of PKC action on ENaC subunits agrees with the results of Zentner et al. (7Zentner M.D. Lin H.h Wen X. Kim K.J. Ann D.K. J. Biol. Chem. 1998; 273: 30770-30776Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Nonetheless, more research is necessary to understand the regulation of ENaC subunits in different epithelia.Fig. 3 shows the time course of PMA actions on ENaC subunit and PKC levels. The representative blots of Fig. 3 (A–C) show that although αENaC levels (A) are not effected by PMA (100 ng/ml) for up to 96 h, βENaC (B) and γENaC (C) levels decrease prior to 24 h after activation of PKC. Both β and γENaC increase toward pretreatment levels 72 h after PMA addition. PMA also decreased PKC levels beginning 2–3 h after addition. PKC levels remained substantially depressed throughout the course of these experiments (n = 6). In contrast, 4α-PMA had no effect on PKC levels between 0–96 h (n = 4; not shown). These findings show that PMA addition to A6 cells does in fact activate PKC because phorbol activation of PKC is well known to cause self-inhibition via a cellular negative feedback loop resulting in decreased kinase expression.Figure 3Temporal effects of PMA on PKC and ENaC protein subunit levels. A, typical blots probed with AB586. Lysate was extracted from cells after the indicated PMA (100 ng/ml; 162 nm) treatment time. αENaC did not consistently decrease in response to PMA. B, typical blots probed with AB592. Numbers indicate in hours the time cells were treated with PMA. βENaC consistently decreased with the decrease reaching maximal by 24 h. Note that by 96 h βENaC levels tended toward pretreatment levels. C, typical blots probed with anti-γ-xENaC antibody. γENaC levels begin to decrease soon after PMA treatment with decrease reaching a maximum by 8 h. After 48 h of sustained PMA addition γENaC tended toward pretreatment levels. D, typical blots probed with anti-PKC antibody. PMA treatment leads to a decline in PKC levels within 2 h with maximal decline by 48 h. PKC levels remained suppressed in the presence of chronic PMA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The effects of PMA on ENaC subunit levels (A) and calculated current (B), and trans-monolayer resistances (C) and voltages (D) are summarized in Fig.4. The mean ± S.E. (n ≥ 3) describing the relative (compared with time 0) density of each subunit at the indicated times are shown with the short dashed (●), long-dashed (○), and solid (▿) regression lines fitting α, β, and γENaC, respectively. γENaC begins to decrease soon after PMA addition with a time constant of 3.7 ± 1.0 h. The decrease in βENaC levels is described by a time constant of 13.9 ± 3.0 h. The change in αENaC over 96 h was not significantly different from that at time 0 (p < 0.01). 4α-PMA had no effect on α, β, and γENaC levels at any time point (times detected = 2, 6, 8, 12, and 24 h; not shown). The decrease in both γ and βENaC were fit best by a single exponential, suggesting that PMA had only one major effect on ENaC protein levels. Currently, it is unclear whether the action of PMA results in a decreased subunit expression or increased subunit degradation. In the continued presence of PMA, presumably because of self-inhibition of PKC, β and γENaC subunits levels began to return to pretreatment levels after 24–48 h (with time constants of 32 ± 8.1 and 52 ± 10 h for β and γENaC, respectively, which are not statistically different from one another;p = 0.128). The levels of γ and βENaC at 96 h were significantly greater then the levels of these subunits at 24 h. Importantly, the increase toward pretreatment levels for γ and βENaC after 48 h demonstrate the reversibility of PKC actions on subunit levels. It is unclear why there is an extended lag time after PKC self-inhibition prior to β and γENaC levels returning toward pretreatment values. This observation needs to be more carefully examined but suggests some prolonged suppression of ENaC levels that is first triggered by PKC and then sustained after kinase down-regulation. Alternatively, the results imply that ENaC synthesis is quite slow.Figure 4Summary graphs showing the time course of PMA decreases on relative ENaC protein subunit levels, calculated Na+ current, and trans-monolayer voltages and resistances. A, temporal effects of PMA (100 ng/ml; 162 nm) on ENaC subunit levels (α = ●, β = ○, and γ = ▿). T" @default.
• W2154620619 created "2016-06-24" @default.
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• W2154620619 date "2000-08-01" @default.
• W2154620619 modified "2023-10-13" @default.
• W2154620619 title "Differential Effects of Protein Kinase C on the Levels of Epithelial Na+ Channel Subunit Proteins" @default.
• W2154620619 cites W1850052335 @default.
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