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Originally published In Press as doi:10.1074/jbc.M611160200 on May 3, 2007

J. Biol. Chem., Vol. 282, Issue 25, 18339-18347, June 22, 2007
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Epithelial Na+ Channel Stimulation by n-3 Fatty Acids Requires Proximity to a Membrane-bound A-kinase-anchoring Protein Complexed with Protein Kinase A and Phosphodiesterase*

Frédérique Mies{ddagger}, Corentin Spriet§1, Laurent Héliot§, and Sarah Sariban-Sohraby{ddagger}2

From the {ddagger}Physiology Department, Université Libre de Bruxelles, 808 Route de Lennik, CP604, 1070 Belgium and the §Biophotonique Cellulaire Fonctionnelle, Interdisciplinary Research Institute, 1 rue du Prof. Calmette, BP447, 59021 Lille Cedex, France

Received for publication, December 5, 2006 , and in revised form, March 29, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Essential polyunsatured fatty acids have been shown to modulate enzymes, channels and transporters, to interact with lipid bilayers and to affect metabolic pathways. We have previously shown that eicosapentanoic acid (EPA, C20:5, n-3) activates epithelial sodium channels (ENaCs) in a cAMP-dependent manner involving stimulation of cAMP-dependent protein kinase (PKA). In the present study, we explored further the mechanism of EPA stimulation of ENaC in A6 cells. Fluorescence resonance energy transfer experiments confirmed activation of PKA by EPA. Consistent with our previous studies, EPA had no further stimulatory effect on amiloride-sensitive transepithelial current (INa) in the presence of CPT-cAMP. Thus, we investigated the effect of EPA on cellular pathways which produce cAMP. EPA did not stimulate adenylate cyclase activity or total cellular cAMP accumulation. However, membrane-bound phosphodiesterase activity was inhibited by EPA from 2.46 pmol/mg of protein/min to 1.3 pmol/mg of protein/min. To investigate the potential role of an A-kinase-anchoring protein (AKAP), we used HT31, an inhibitor of the binding between PKA and AKAPs as well as cerulenin, an inhibitor of myristoylation and palmitoylation. Both agents prevented the stimulatory effect of EPA and CPT-cAMP on INa and drastically decreased the amount of PKA in the apical membrane. Colocalization experiments in A6 cells cotransfected with fluorescently labeled ENaC beta subunit and PKA regulatory subunit confirmed the close proximity of the two proteins and the membrane anchorage of PKA. Last, in A6 cells transfected with a dead mutant of Sgk, an enzyme which up-regulates ENaCs, EPA did not stimulate Na+ current. Our results suggest that stimulation of ENaCs by EPA occurs via SGK in membrane-bound compartments containing an AKAP, activated PKA, and a phosphodiesterase.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Omega-3 polyunsatured fatty acids (n-3 PUFAs)3 have been implicated in several physiological processes. They modulate enzyme activities, as well as ion transport function, through several transporters such as proton, chloride, ROMK, and calcium channels, nonselective cation channels, and the cardiac sodium/calcium exchanger (1-8). PUFAs also inhibit voltage-dependant sodium and calcium channels in cardiac myocytes, retinal glial cells, and sympathic neurons (3, 9-12). The mechanisms by which PUFAs modulate ion channels vary among the different cell types and channels. PUFAs may bind directly to and modulate the activity of some channels, whereas in other cases, they regulate channel activities indirectly through metabolites and protein kinases (13).

In a previous study, we investigated the effects of a PUFA, eicosapentaenoic acid (EPA, C20:5, n-3) on epithelial sodium channels (ENaCs). The regulation of this channel is essential for maintenance of renal sodium balance and, hence, of arterial blood pressure (14). In vivo, a number of hormones and others endogenous and exogenous factors including PUFAs regulate ENaCs.

In the A6 distal nephron cell line, which expresses highly selective sodium channels, we have shown that EPA activates these channels via stimulation of the cAMP-dependent protein kinase (PKA) (15). Numerous cellular processes are initiated, modulated, or terminated by cAMP-dependent pathways, mainly the PKA pathway. Although PKA broadly affects multiple intracellular pathways, its specificity and efficiency may be enhanced by its close proximity to specific molecular targets. In this regard, a family of anchoring proteins (A-kinase-anchoring proteins, AKAPs), which compartmentalize PKA to a specific subcellular domain has been shown to modulate cAMP-mediated signaling (16-23).

Compartmentalization of signaling molecules through association with anchoring proteins ensures specificity in signal transduction by placing enzymes close to their appropriate effectors and substrates. AKAPs bind to the regulatory subunit of PKA which directs the kinase to discrete intracellular locations. Functional studies aimed at disrupting AKAP-PKA complexes have demonstrated a role for anchored PKA in various cellular processes, including gene transcription, hormone-mediated insulin secretion, and ion-channel modulation (11). The A-kinase-anchoring proteins consists of two binding sites, a targeting motif to direct AKAPs to its subcellular localization and a conserved anchoring motif that binds the NH2 termini of the dimer of the RII subunit of PKA. Numerous AKAPs have been identified and are found in association with a variety of cellular compartments including centrosomes, dendrites, endoplasmic reticulum, mitochondria, nuclear membrane, cell membrane, and vesicles. Several studies have shown that AKAPs are required for the stimulatory effect of PKA on AQP-2 water channels (9), cystic fibrosis transmembrane conductance regulator chloride channels (5), L-type Ca2+ channels (9), ROMK channels (2), or voltage-sensitive sodium channels (17).


Figure 1
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  		FIGURE 1.
cAMP production/degradation balance. A, measurement of adenylyl cyclase activity in membrane preparation of A6 cells. Control represents basal adenylate cyclase activity. Forskolin (1 µM) served as positive control. **, p < 0.002 versus control, unpaired t test, n = 3. B, PDE activity. cAMP-3H hydrolysis was measured in membrane preparations of A6 cells without treatment (open circles) or with EPA 33 µM for 5 min (closed circles). Hydrolysis decreased with increased concentrations of non-labeled cAMP indicating specificity of action. *, p = 0.04 versus control, unpaired t test, n = 4. C, measurement of cAMP accumulation. Control represents basal cAMP accumulation. Forskolin (1 µM) served as positive control. Cells were treated for 20 min with 33 µM EPA before the assays. *, p = 0.04 versus control, unpaired t test, n = 3.

 
Although AKAPs have been defined on the basis of their interaction with PKA, an additional feature of many of these molecules is their ability to bind to other signaling enzymes. Of particular interest is the simultaneous binding of AKAPs to enzymes with opposing actions such as kinases and phosphatases or phosphodiesterases (24). AKAP-mediated localization of phosphodiesterase (PDE) near PKA is thought to control access of cAMP to the regulatory subunit of the kinase (24).

The data presented in this study suggest a model in which EPA inhibits membrane-bound phosphodiesterase activity. Local cAMP accumulation, in turn, activates AKAP-anchored PKA in the apical membrane in proximity to the Na+ channels.4


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Rolipram, dipyridamole, IBMX, and cerulenin were obtained from Calbiochem. st-HT31 was obtained from Promega. EPA, aldosterone, CPT-cAMP, and forskolin were obtained from Sigma-Aldrich.

Cell Culture—A6 cells (American Type Culture Collection derived originally from Xenopus laevis, passages 74-82) were maintained in culture on plastic flasks in Dulbecco's modified Eagle's medium:F-12 growth medium (Invitrogen), adapted for amphibian tissue culture osmolarity by a 20% dilution with distilled water, and supplemented with 5% fetal bovine serum (HyClone), 25 units/ml penicillin and 25 µg/ml streptomycin. Cells were grown at 28 °C in 1% CO2. For electrical measurements, cells were grown on permeable supports for 10 days (12-mm diameter inserts, Transwell, Costar). Maximum and stable values of transepithelial Na+ transport and electrical parameters are observed at this time indicating that apical Na+ channels are functional. All experiments were performed after 10 days of culture. Serum was omitted in the last 24 h.

Open-circuit transepithelial voltage and resistance were measured using an EVOM volt-ohmmeter (World Precision Instruments). The corresponding amiloride-inhibitable sodium current, INa, was calculated from these values. The INa is used as an estimate of the net transepithelial sodium current under the conditions of this study. All measurements were performed in parallel on control and experimental tissues.

Adenylate Cyclase Activity—Membrane were prepared from scraped cells which were pelleted and then lysed in 1 mM NaHCO3 and frozen in liquid nitrogen. After thawing, the lysate was first centrifuged at 4 °C for 10 min at 400 x g, and the supernatant was further centrifuged at 20,000 x g for 15 min. The pellet was resuspended in 1 mM NaHCO3 and used immediately as crude membrane fraction. Adenylate cyclase activity was determined as described previously by Salomon et al. (25, 26). Membrane protein (3-15 µg) was incubated in a total volume of 60 µl containing 0.5 mM ATP, 106 cpm of [{alpha}-32P]ATP/assay, 5 mM MgCl2, 0.5 mM EGTA, 1 mM cAMP, 1 mM theophylline, 10 mM phospho(enol)pyruvate, 30 µg/ml pyruvate kinase, and 30 mM Tris-HCl, pH 7.8, with or without 20 µM GTP. The reaction was initiated by membrane addition and was terminated after a 15-min incubation at 28 °C by adding 0.5 ml of a 0.5% sodium dodecyl sulfate solution containing 0.5 mM ATP, 0.5 mM cAMP, and 20,000 cpm of [3H]cAMP. cAMP was separated from ATP by two successive chromatographies on Dowex 50W-X8 and neutral alumina.

cAMP Accumulation Assay—The tests were run according to the manufacturer's notice (cAMP Biotrak Enzymeimmunoassay system, Amersham Biosciences). Briefly, cellular cAMP content was measured by enzyme-linked immunosorbent assay, in the presence of inhibitors of phosphodiesterase 4 and revealed colorimetrically.

Phosphodiesterase Assay—PDE activity was measured as described by Wells and Hardman (27). A6 cells were grown on permeable supports for 10 days before experiments were performed. Cells were suspended in homogenization buffer (30 mM mannitol, 10 mM Tris, 10 mM MgCl2, pH 7.5, and protease inhibitor mixture; Roche Applied Science) and disrupted using a potter. Nuclei and cytosolic proteins were removed by centrifugation at 2164 x g for 10 min, and cell membranes were separated by centrifugation of the supernatant at 41,000 x g for 25 min. The pellet was resuspended in homogenization buffer and immediately used. Five µl of total protein were incubated in 100 µl of assay buffer (40 mM Tris-HCl, pH 7.4, 5 mM MgSO4, 5 mM 2-mercaptoethanol, cAMP between 10-7 and 10-4 M, and 15,000 cpm of [3H]cAMP) for 5 min at 30 °C. The reaction was stopped by boiling for 1 min and 0.1 unit of 5'-nucleotidase (from Crotalus atrox venom, Sigma) was added for 25 min. The reaction was terminated by the addition of 800 µl of stop buffer (20 mM ammonium formate, 15 mM EDTA, and 0.1 mM adenosine). [3H]Adenosine was separated by filtration on QAE-Sephadex A-25, and the filtrate was counted.


Figure 2
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  		FIGURE 2.
Effect of PDE inhibitors on transepithelial sodium current in A6 monolayers. Data are expressed as percent increase over the time 0 value set at 100%. A, rolipram does not prevent the effect of EPA. Cells were incubated with 3 µM of rolipram for 10 min prior the addition of 33 µM EPA. Effect of EPA alone (33 µM) is shown at 40 min and rolipram alone (3 µM) at 50 min. **, p < 0.001 versus rolipram, unpaired t test, n = 3. B, high dose of dipyridamole prevents the effect of EPA. Cells were incubated with 300 µM dipyridamole for 10 min prior the addition of 33 µM EPA. Effect of EPA alone (33 µM) is shown at 40 min and dipyridamole alone (300 µM) at 50 min. p > 0.05 versus dipyridamole, unpaired t test, n = 6.

 
Membrane Protein Biotinylation—Biotinylation and recovery of apical membrane proteins were performed using the cell surface protein isolation kit (Pierce) according to the manufacturer's notice. Briefly, A6 monolayers grown on permeable support were exposed to st-HT31 (20 µM) for 5 min or to cerulenin (30 µg/ml) for 3 h, prior to adding sulfo-NHS-SS-biotin to the apical compartment. The basolateral compartment received culture media supplemented with 10% fetal bovine serum to prevent biotinylation of basolateral membranes. Biotinylated proteins were eluted in SDS-PAGE sample buffer and analyzed by Western blotting. Actin was used as a control for the specificity of surface biotinylation. Protein was measured with BCA kit (Pierce).

Western Blotting—Biotinylated apical proteins were analyzed by Western blot using an anti PKARII{alpha} antibody (BD Biosciences). Detection was performed by addition of an horseradish peroxidase-conjugated secondary antibody and chemiluminescent substrate (SuperSignal West Pico chemiluminescent substrate, Pierce).

Colocalization PKA-ENaC—A6 cells were transfected with the regulatory and/or the catalytic subunit of PKA (fused with CFP or YFP, respectively) in pcDNA3 plasmids (kindly provided by Anna Terrin, University of Padua, Padua, Italy) and the beta or {gamma} subunit of ENaC tagged with CFP or YFP using Exgen 500 (Euromedex). Cells were fixed with paraformaldehyde 4% 48 h after transfection. Expression vectors for EYFP-betahENaC and ECFP-{gamma}hENaC{gamma} were generated by cloning PCR products into XhoI/BamHI sites of pEYFP-C1 and into HindIII/BamHI sites of pECFP-C1. Acquisitions were performed using a Leica SP2 confocal microscope with a 100x, NA 1.4, oil immersion objective. To ensure minimal cross-talk, we used a sequential acquisition procedure with wavelengths of 458 nm (excitation) and 465-495 nm (observation) for the CFP channel, and 514 nm (excitation) and 535-560 nm (observation) for the YFP channel. Three-dimensional acquisitions were reconstructed using the Imaris software (Bitplane Inc.) allowing selection of the plane with maximum membrane contribution. The ImarisColoc module (Bitplane Inc.) was used to generate a 2D histogram from this plane by plotting pixels according to intensity in each channel and to generate Pearson correlation coefficient. The Mander's correlation coefficient was calculated for each channel using the Intensity Correlation Analysis plug-in for ImageJ (Rasband, W. S., ImageJ, National Institutes of Health, Bethesda, MD (rsb.info.nih.gov/ij/)). Coefficient values of 100% represent a perfect correlation while 0% corresponds to a noncorrelated signal and a value of -100% to an inverse correlation. Correlation's means were calculated from 30 cells.


Figure 3
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  		FIGURE 3.
A, time course of stimulation of transepithelial sodium current (INa) in A6 monolayers. Open circles, control; closed circles, 100 µM CPT-cAMP. The arrow indicates addition of EPA at 30 min. Control wells received an equivalent amount of ethanol. Control values were stable for the duration of experiments (data not shown). Data are expressed as percent increase compared with time 0 values of experimental tissues. EPA was added to the apical bath at a final concentration of 33 µM (1:1000 dilution). Measurements were carried out in triplicate (n = 15). B, dose response of activation of INa by cAMP. Open circles, CPT-cAMP alone at concentrations ranging between 5 and 100 µM. Closed circles, cells were incubated with 20 µM EPA (inactive concentration) and low concentrations of CPT-cAMP (10, 25, and 50 µM). INa was measured at 40 min. Data are expressed as percent increase compared with time 0 values of experimental tissues. Measurements were carried out in triplicate (*, p = 0.002; **, p < 0.001, unpaired t test n = 9).

 
Fluorescence Lifetime Imaging Microscopy and Analysis—Experiments were performed on fixed A6 cells 48 h after transfection (Exgen 500, Euromedex) with the regulatory and/or the catalytic subunit of PKA fused with CFP or YFP, respectively. We used a two-photon pulsed excitation at a wavelength of 830 nm for CFP excitation. Photon detection was performed using the Hamamatsu MCP R3809U-52 PMT. The omega XF3075 band pass filter allowed specific detection of CFP (465-500 nm). Images were acquired using a 100x, NA 1.4, oil immersion objective. The Becker & Hickl TCSPC 730 card determined the time between fluorophores excitation and photon emission. This card was driven by the Single Photon Counting software. Fluorescence decay curve fits, allowing lifetime determination, were obtained using the SPCImage software (28, 29) using the following equation,

Formula(Eq.1)

where b is background level, IRF is instrumental response function of our system, a is a proportion of each component, and {tau} is the corresponding lifetime. The selection between a mono-exponential and a bi-exponential decay was performed according to the reduced {chi}2 parameter (29).

Creation of Stably Transfected Clones—A6 cells were transfected with pCI-neo expression vectors containing the D222A sgk1 mutant with a myc tag on the NH2 terminus (kindly provided by N. Perrotti, Università Magna Graecia, Cantazaro, Italy). The cDNA was introduced to subconfluent cells growing in 24 wells plates using FuGENE (Roche Applied Science) in serum-free medium. Cells were maintained in a toxic concentration (2 mg/ml) of Geneticin (Sigma) for 10 days after which clones were subcultured in 96-well plates and maintained under the selective pressure of 0.5 mg/ml Geneticin. The various clones were tested for their response to aldosterone and to EPA.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of EPA Suggests Local Changes in cAMP—Consistent with our previous study (15), EPA (C20:5, n-3) transiently activated amiloride-sensitive open-circuit current (INa) from a basal value of 4.15 ± 0.24 µA/cm2 to 7.45 ± 0.48 µA/cm2 within 30 min of exposure (n = 15, p < 0.001). Since cAMP is necessary for the effect of EPA (15), we sought to measure the activity of the enzymes involved in the production/degradation balance of cAMP, namely adenylyl cyclase and cyclic nucleotide PDE. EPA did not activate adenylyl cyclase in membrane preparations (Fig. 1A) but inhibited cAMP-PDE activity by 30% (Fig. 1B). However, this inhibition was not accompanied by an increase in total cellular cAMP (Fig. 1C). These results suggest that EPA may generate a local increase in cAMP level, i.e. undetectable in total cellular cAMP, possibly by simply inhibiting a membrane-bound PDE. To get more insight in the type of PDE involved, we tested inhibitors of cAMP and cGMP PDEs. Rolipram, a selective inhibitor of cAMP phosphodiesterase, increased INa by 62.86 ± 6.77% (Fig. 2A, first column) as expected but did not prevent the effect of EPA (second column). The arithmetic sum of the effect of rolipram alone and of EPA alone was not statistically different from the combined effect of rolipram and EPA (second column). Similar results were obtained with IBMX, a nonselective inhibitor of cAMP PDE (data not shown). These data suggest that EPA does not act on a rolipram- or IBMX-sensitive PDE. Furthermore, pretreatment of A6 cells with a high concentration of dipyridamole (300 µM), a selective inhibitor of cGMP phosphodiesterase, which at high dose inhibits cAMP-PDE8, which is insensitive to all other inhibitors, prevented the effect of EPA (Fig. 2B). The combined results obtained with rolipram, IBMX, and dipyridamole strongly suggest the involvement of PDE8.

In A6 cells pretreated with 100 µM CPT-cAMP, a permeant analog of cAMP, INa increased as expected and no additive effect of EPA was observed indicating a common pathway (Fig. 3A, closed circles). Alone, lower concentrations of CPT-cAMP, ranging between 5 and 50 µM, had no effect on INa (Fig. 3B, open circles). Similarly, a low concentration of EPA (20 µM) had no effect on INa. However, in combination, 20 µM EPA with CPT-cAMP concentrations ranging from 10 to 50 µM yielded significant increases of INa averaging between 30 and 50% (Fig. 3B, closed circles). These results indicate that in the presence of EPA, small increases in cAMP become sufficient to activate PKA. Since no increase in total cell cAMP was detected in cells exposed to EPA, we hypothesized that local increases in cAMP near sodium channels may be attributed to PDE inhibition specifically at the apical membrane.


Figure 4
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  		FIGURE 4.
Colocalization analysis of A6 cells coexpressing the regulatory subunit of PKA fused to CFP and the beta subunit of ENaC fused to YFP. The image in A depicts a high colocalization level (white), particularly in the cell membrane. A plane corresponding only to cell membrane was extracted from this image (B). The Pearson correlation level was calculated from all contributing pixels (C).

 
Local Changes in cAMP Activate Membrane-associated PKA—To test for membrane localization of PKA, A6 cells were transiently cotransfected with the regulatory subunit of PKA (RII) fused to CFP and with the beta subunit of ENaC fused toYFP. Fig. 4A shows the colocalization of the two proteins in whole cells (white). Expression level of betaENaC-YFP was heterogeneous but seemed colocalized with RII-CFP, preferentially at the membrane level (Fig. 4B) where CFP versus YFP fluorescence emission was highly correlated (Fig. 4C) as indicated by a Pearson's correlation of 93.1 ± 1.7% and by Mander's correlation coefficients of 98.7 ± 0.5% and 94.2 ± 1.6% for CFP and YFP channels, respectively.

Since PKA and ENaC are colocalized, it was of interest to examine the influence of EPA on a membrane-specific activity of PKA. An important step in PKA activation is the dissociation of the catalytic from the regulatory subunit of the enzyme. This was examined using fluorescence lifetime imaging where the fluorescent energy transfer between catalytic and regulatory subunits of PKA was determined in response to EPA. As a reference for no interaction between subunits, we used A6 cells expressing the recombinant PKA RII alone tagged with cyan fluorescent protein (RII-CFP; mean lifetime of 1956 ± 46 ps, Fig. 5A). Fig. 5B shows A6 cells coexpressing RII-CFP simultaneously with the PKA catalytic subunit tagged with yellow fluorescent protein (C-YFP). Fluorescence resonance energy transfer (FRET) was detected as indicated by a decrease in the measured lifetime (1724 ± 49 ps). Treatment with EPA resulted in a loss of FRET as evidenced by the mean lifetime increasing back toward control levels (1866 ± 53 ps, Fig. 5C) consistent with dissociation of the regulatory and catalytic subunits of PKA. These data are summarized in Fig. 5D. In the absence of EPA, a detectable energy transfer between associated RII-CFP and C-YFP subunits was observed that disappeared in EPA stimulated cells. These data indicate activation of membrane associated PKA by EPA.

Effect of EPA Involves an AKAP—As PKA and many of its substrates are present throughout the cell, one means of having a localized action would result from compartmentalization of PKA. AKAP 18, a low molecular weight anchoring protein, targets PKA to the cell membrane (23), which, if near Na+ channels, could facilitate activation by cAMP-dependant phosphorylation.

To test the role of an AKAP in our model, we used st-HT31, a peptide that disrupts the binding between AKAP and PKA. Fig. 6A shows that pretreatment of cells with st-HT31 (20 µM) for 5 min prevented the EPA stimulation of sodium current. Similarly, pretreatment of cells with st-HT31 also prevented stimulation of INa by 100 µM CPT-cAMP. Inactive st-HT31 control peptide did not prevent EPA's effect (Fig. 6B).

Because lipid modification through myristoylation and palmitoylation is necessary to promote the specific association of AKAP 18 with the cell membrane (23), we tested the effect of cerulenin, an acylation reaction and fatty acid biosynthesis inhibitor, on the action of EPA. As shown in Fig. 6C, pretreatment of A6 cells with 30 µg/ml cerulenin for 30 min prevented the stimulatory effect of EPA on INa. st-HT-31 peptide and cerulenin also caused a significant decrease of PKA levels in apical membranes of A6 cells as shown in Fig. 7. Densitometry scanning revealed a 84.5 ± 3.9% decrease in PKA with HT31 and a 85.8 ± 2.7% decrease with cerulenin. These results confirm that PKA is present at the apical membrane and point to the role of an AKAP. Results obtained with cerulenin further suggest that AKAP 18 is involved.


Figure 5
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  		FIGURE 5.
Fluorescence lifetime imaging microscopy analysis of the CFP fluorescence in fixed A6 cells expressing CFP-RII regulatory subunit of PKA alone (A) or coexpressing CFP-RII regulatory subunit of PKA and YFP-C catalytic subunit of PKA without treatment (B) and treated with 33 µM EPA (C). Only the CFP fluorescence is monitored by use of a cyan fluorescent protein-specific emission filter. The CFP fluorescence lifetime image of individual cells is calculated on a pixel-by-pixel basis and pseudocolored. Red-orange colors in the lifetime image depict low fluorescence lifetime (FRET). The blue colors in the lifetime image depict higher fluorescence lifetimes (no FRET). Focus and depth of field were consistent with localization of fluorescent signal to the apical membrane. D, histogram of the mean fluorescence lifetime for each condition (*, p = 0.0018; **, p < 0.001, unpaired t test, n = 30).

 
EPA Leads to Activation of Serum- and Glucocorticoid-induced Protein Kinase (Sgk)—The EPA-induced PKA stimulation of INa could be due to direct phosphorylation of the Na+ channels, phosphorylation of intermediate pathways, or both. In this regard, ENaC activity is also stimulated by cAMP-dependant PKA phosphorylation of Sgk1 (30). To better understand the mechanism of the action of EPA, we examined whether the PKA-induced stimulation of transport of EPA was retained after inhibiting Sgk1. Previous studies demonstrated that A6 cells transfected with a D222A Sgk1 kinase-dead mutant producing a dominant negative effect exhibit a basal sodium current lower than the parental line and an attenuated response to aldosterone, reflecting the lack of Sgk1 activity (31). Similarly, we generated A6 cells stably transfected with a D222A sgk1 kinase dead mutant to examine the functional response of the clonal cell line to EPA. The presence of Sgk1 in the cell membranes of transfected A6 cells was confirmed by Western blotting using an anti-myc antibody (data not shown). As expected, the Sgk1-transfected cells exhibited a basal INa significantly lower than that observed in the parent cell line (0.5 versus 3.9 µA/cm2; compare the fifth and first columns in Fig. 8). Similarly, the stimulation of INa by aldosterone was reduced from 111.2 ± 8.8% in the parental line to 36.0 ± 5.1% in the transfected cells (Fig. 8). The response of EPA was also reduced in the transfected cell line, with an increase of INa of 24.8 ± 2.3% as compared with 77.6 ± 10.4% in the parental line (Fig. 8).


Figure 6
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  		FIGURE 6.
Transepithelial sodium current in A6 monolayers. Data are expressed as percent of the time 0 value. Inhibition of binding of PKA to AKAPs by st-HT31 peptide blunts the effects of EPA and cAMP. Cells were incubated with st-HT31 peptide (20 µM) for 5 min prior addition of 33 µM EPA or 100 µM CPT-cAMP. Effect of EPA alone (33 µM) and CPT-cAMP alone (100 µM) is shown at 40 min. *, p < 0.001 versus HT31 5 min; #, p < 0.03 versus HT31 40 min; §§, p < 0.0001 versus HT31+EPA 40 min; §, p = 0.002 versus HT31 + cAMP, unpaired t test. B, inactive mutant of st-HT31 peptide does not modify the effects of EPA and cAMP. Cells were treated in the same manner as indicated in A.*, p < 0.0001 versus HT31 alone at 40 min, nonsignificant versus EPA at 40 min, unpaired t test. C, inhibition of acylation reactions with cerulenin blunts the effect of EPA. Cells were incubated with cerulenin (30 µg/ml) for 30 min prior addition of 33 µM EPA. *, p < 0.001 versus cerulenin 30 min; #, p < 0.001 versus cerulenin 30 + 40 min; §, p < 0.0001 versus cerulenin (30 min) + EPA (40 min), unpaired t test.

 


Figure 7
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  		FIGURE7.
st-HT31 and cerulenin decrease apical membrane PKA localization in polarized A6 cell monolayers. The presence of PKA in the apical membrane was detected by Western blotting after cell surface biotinylation. Lane 1, negative control (treatment with st-HT31 inactive peptide, 20 µM). Lane 2, after treatment with st-HT31 (20 µM) for 5 min. Lane 3, after treatment with cerulenin (30 µg/ml) for 3 h. One representative experiment is shown; three additional experiments gave comparable results. Densitometry analysis results are the mean ± S.D. of three experiments; **, p < 0.01, unpaired t test.

 


Figure 8
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  		FIGURE 8.
EPA's effect in the A6 parental cell line and in A6 stably transfected with D222A Sgk1 kinase-dead mutant. Basal current and response to aldosterone (0.1 µM for 4 h) and to EPA (33 µM for 30 min) are shown. Values are represented as means ± S.E. ***, p < 0.0001 versus control; **, p < 0.001 versus vehicle; #, p < 0.05 versus Sgk1 kinase-dead mutant, unpaired t test, n = 9.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we sought to determine the signaling pathway for the stimulation of apical sodium transport by EPA through ENaCs in A6 epithelia. ENaCs, which can be activated by phosphorylation, are integral membrane proteins localized in discrete membrane compartments (32, 33). Total cellular levels of cAMP and PKA do not correlate with channel activity under all conditions. For example, decrease in the level of either or both of these molecules does not affect Na+ transport (15, 34). EPA was previously shown to increase PKA activity downstream of cAMP generation (15). Our data show that EPA increases PKA activity without increasing adenylyl cyclase activity or total intracellular cAMP levels. However, EPA inhibits a membrane-bound phosphodiesterase, which presumably increases levels of cAMP locally and also shifts a set point for the intracellular levels of cAMP causing activation of PKA. The activity of adenylyl cyclase provides basal levels of cAMP, while inhibition of its degradation by PDE inhibition allows increases of cAMP levels that are sufficient for PKA activation. The ability to detect low changes in cellular cAMP levels is an important tool for understanding spatial and temporal cAMP responses (24, 35). Fluorescence lifetime imaging microscopy allowed us to detect activation of the PKA pathway related to local changes in cAMP levels. Localized increases of cAMP would require compartmentalization of PKA near the apical Na+ channels to elicit a specific, timely, and spatially orchestrated stimulation of transport. AKAP-mediated tethering of PKA to subcellular compartments is thought to localize PKA to specific sites and to limit its access to a subset of substrates. PKA can also be associated with proteins involved in the modulation of effector proteins. To this end, we demonstrated that the regulatory subunit of the PKA (RII) is colocalized with the beta subunit of ENaC at the apical cell membrane. The detection of PKA in biotinylated A6 cells corroborate the presence of PKA at the apical membrane. A working model depicting this compartmentalization is shown in Fig. 9. The central feature of this model is the colocalization of ENaC and PKA activity. We also provided evidence that compartmentalization of PKA is mediated by an protein kinase A anchoring protein (AKAP). A direct interference with AKAP was shown using st-HT31, a peptide designed to fit into the putative binding site of the regulatory PKA subunit (RII) on the AKAP, thereby competing for the RII-AKAP interaction (21). We found that st-HT31 disrupted AKAP function, prevented the stimulatory effect of EPA, and significantly decreased the amount of PKA present in the apical membrane. A number of AKAPs have been described that compartmentalize PKA to various subcellular domains. In particular, AKAP 18, which we used in the present study, is targeted to the plasma membrane through lipid modification, i.e. myristoylation and palmitoylation of NH2-terminal residues (23). Consistent with these observations, we have demonstrated the lipid dependence by blocking fatty acid synthesis using cerulenin which led to the loss of EPA's stimulation of sodium transport. In renal collecting duct principal cells, AKAP 18 has been shown to be a key component in the fusion of vesicles containing aquaporin-2 water channels with the cell membrane, a process that requires activation of cAMP-dependant PKA (16, 20). In A6 amphibian-derived cells, the use of a rabbit antibody failed to detect AKAP 18. However, our data with st-HT31 and cerulenin confirm that PKA is anchored in the apical membrane by an AKAP and results with cerulenin further suggest that AKAP 18 is involved. Membrane localization of AKAP 18 has been demonstrated in Madin-Darby canine kidney cells (22) and recently in oocytes where it was shown to regulate ENaC feedback inhibition (36). Anchorage of PKA via an AKAP has not been described previously in A6 cells and could represent a new mechanism of regulation of sodium channels in epithelia.


Figure 9
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  		FIGURE 9.
Proposed model of the effect of EPA on ENaC. EPA crosses the cell membrane and inhibits membrane-bound PDE activity, leading to increased concentration in cAMP. The PKA is activated and phosphorylates Sgk1, which becomes in turn activated. Sgk1 either controls the retrieve of the channels or acts directly on ENaC. This action occurs in or near a compartment where PDE and PKA are in close proximity to ENaC by means of an AKAP. Whether Sgk1 is also tethered to the membrane by an AKAP remains to be determined.

 
In addition to anchoring PKA, AKAPs can also bind to other signaling enzymes such as protein kinase C, protein phosphatases, and phosphodiesterases (21). For example, phosphodiesterases have been found in complexes with AKAPs and PKA (24, 18). As transduction of cAMP signals involved in regulating ion channel currents occurs in milliseconds, the close association of AKAP·PKA·PDE complexes with ENaCs would allow for rapid cAMP-dependant phosphorylation and specific activation of these channels.

Another kinase that is an important regulator of ENaC is Sgk1, which is present in membrane preparations of A6 cells. When cells were transfected with a kinase-dead mutant of Sgk1, a large decrease of control INa was observed suggesting that constitutive Sgk1 strongly contributes to resting Na+ transport rate. Perrotti et al. (30) demonstrated that cAMP-dependant PKA phosphorylation of Sgk1 stimulates ENaC activity consistent with our observation that the kinase-dead mutant Sgk1 also drastically reduced the EPA stimulation of Na+ transport. Since this implies that Sgk1 mediates the effect of EPA, it must either reside in, or transiently enter, the AKAP/ENaC compartment depicted in Fig. 9. Preliminary experiments have shown localization of Sgk1 at the apical membrane. Whether an AKAP is required to anchor Sgk1 remains to be determined.

Previously, we found that the stimulation of Na+ transport by EPA is transient, and after 30 min of EPA, INa decreases back toward control values (15). Although the mechanism of this time course is unknown, it could be explained by PDE phosphorylation, which would lead to activation of the enzyme and to lowering of cAMP. Phosphorylation of isoforms of most PDE classes has been reported, and in some cases, phosphorylation was associated with changes in the hydrolytic activity (37). We have not directly identified the type of PDE involved in our study but several arguments point to PDE8. First, PDE8 is expressed in kidney tissue. Second, it uniquely possesses a PAS domain in its NH2 terminus (38). Such domains are known to mediate protein-protein interactions and to complex ligands (39). The PAS domain of PDE8 could therefore both allow interaction with an AKAP and bind EPA. Third, PDE8 is insensitive to rolipram, a selective inhibitor of cAMP-PDE, and to IBMX, a nonspecific inhibitor of PDEs. We have confirmed that both rolipram and IBMX stimulation of INa+ are indeed additive to the effect of EPA. Furthermore, PDE8 is sensitive to high concentrations of dipyridamole, which was also confirmed in this study.

In summary, we propose that EPA activation of ENaCs in which the fatty acid acts on membrane-bound compartments containing an protein kinase A-anchoring protein, which permits the simultaneous anchorage of a PDE, most likely PDE8, and PKA to the cell membrane, in close proximity of ENaCs. The inhibition of membrane-bound PDE by EPA would allow a local increase in cAMP sufficient to activate PKA. Activated PKA in turn phosphorylates both Sgk, leading to increased Na+ transport and PDE, which eventually provides the negative feedback to decrease transport.


   FOOTNOTES
 
* This work was supported by funds from Université Libre de Bruxelles and from the Fonds National pour la Recherche Scientifique (FNRS), the ANR-05-MIIM-042, and the Mission des Ressources et Compétences Technologiques. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Supported by Region-Nord-Pas-de-Calais, ANR-05-MIIM-042, and MRCT. Back

2 To whom correspondence should be addressed: National Institutes of Health, Bldg. 31, Rm. 6A22, Bethesda, MD 20892. Tel.: 301-451-6763; Fax: 301-451-6764; E-mail: sohrabys{at}nei.nih.gov.

3 The abbreviations used are: PUFA, polyunsatured fatty acid; EPA, eicosapentaenoic acid; ENaC, epithelial sodium channel; PKA, cAMP-dependent protein kinase; AKAP, A-kinase-anchoring protein; IBMX, isobutylmethylx-anthine; PDE, phosphodiesterase; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; FRET, fluorescence resonance energy transfer. Back

4 The present results were presented in part at the 2006 Experimental Biology Meeting (Mies, F., Spriet, C., Héliot, L., Shlyonsky, V., Goolaerts, A., Roch, A., and Sariban-Sohraby, S. (2006) FASEB J. 20, A347). Back


   ACKNOWLEDGMENTS
 
We thank Dr. M. Waelbroek for help with the adenylyl cyclase assay, Dr. S. Swillens for help in measuring PDE activity, Dr. V. Shlyonskiy for critical comments, Dr. A. Terrin for providing the PKA plasmids, Dr. N. Perrotti for providing the SGK plasmids, Dr. E. Klussmann for providing anti-AKAP18 antibody, and Dr. R. Naeije for support.



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TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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