Role of PKCalpha in feedback regulation of Na+ transport in an electrically tight epithelium

Mouhamed S. Awayda, Justin D. Platzer, Roxanne L. Reger, and Abderrahmane Bengrine

Department of Physiology, Tulane University Health Sciences Center, New Orleans, Louisiana 70112


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

It has long been known that Na+ channels in electrically tight epithelia are regulated by homeostatic mechanisms that maintain a steady state and allow new levels of transport to be sustained in hormonally challenged cells. Little is known about the potential pathways involved in these processes. In addition to short-term effect, recent evidence also indicates the involvement of PKC in the long-term regulation of the epithelial Na+ channel (ENaC) at the protein level (40). To determine whether stimulation of ENaC involves feedback regulation of PKC levels, we utilized Western blot analysis to determine the distribution of PKC isoforms in polarized A6 epithelia. We found the presence of PKC isoforms in the conventional (alpha  and gamma ), novel (delta , eta , and epsilon ), and atypical (iota , lambda , and zeta ) groups. Steady-state stimulation of Na+ transport with aldosterone was accompanied by a specific decrease of PKCalpha protein levels in both the cytoplasmic and membrane fractions. Similarly, overnight treatment with an uncharged amiloride analog (CDPC), a procedure that through feedback regulation causes a stimulation of Na+ transport, also decreased PKCalpha levels. These effects were additive, indicating separate mechanisms that converge at the level of PKCalpha . These effects were not accompanied by changes of PKCalpha mRNA levels as determined by Northern blot analysis. We propose that this may represent a novel regulatory feedback mechanism necessary for sustaining an increase of Na+ transport.

Western blotting; aldosterone; protein kinase Calpha ; epithelial Na+ channel


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IT IS WELL ACCEPTED that apical membrane Na+ permeability is the rate-limiting step for Na+ entry and, consequently, transepithelial Na+ transport in renal, electrically tight epithelia. By altering apical Na+ permeability, epithelia in the distal tubule can exhibit a wide range of salt and water transport. It is therefore not surprising that the activity of the Na+ channel in the apical membrane of these renal epithelia is highly regulated. Besides regulation by a variety of circulating hormones such as aldosterone and vasopressin, it is also well known that Na+ channels are regulated by feedback mechanisms that respond to changes of transport by attempting to restore a steady state (18, 41). Thus it follows that these feedback mechanisms are modified in hormonally stimulated cells; otherwise, they would tend to counteract the effects of hormones and attempt to restore baseline rates of Na+ transport. Despite a wealth of information to the existence of these self- or autoregulatory processes, little is known about their molecular mechanisms.

The mechanisms of short-term (minutes) feedback regulation of Na+ transport may be directly related to changes of intracellular Na+ concentration ([Na+]i) (4). However, the mechanisms of long-term processes are poorly understood but are likely to involve the actions of second-messenger protein kinases such as PKC. PKC is a ubiquitous enzyme with multiple isoforms that fall into three general categories classified according to their modes of activation (19). The conventional group (cPKC), which is composed of isoforms alpha , beta , and gamma , is activated by diacylglycerol (DAG), Ca2+, and phosphatidylserine (PS). Members of the second group (delta , epsilon , eta , and theta ) are stimulated by DAG and PS but do not require Ca2+ for activation and are referred to as novel PKC isoforms. Members of the third group (zeta , iota , and lambda ) are stimulated by PS alone and are considered atypical PKC isoforms.

Ample evidence exists supporting isoform-specific roles for PKC in nonpolarized and polarized low-resistance leaky epithelia (2, 6, 9, 10, 22, 23, 26, 36-38). However, the distribution and potential functions of these isoforms in electrically tight, Na+-absorbing epithelia are poorly understood. Preliminary evidence indicates specific functions for these isoforms. For example, DeCoy et al. (12) have reported that the rabbit cortical collecting duct (CCD) contains both PKCepsilon and PKCzeta and that inhibition of PKCepsilon enhances vasopressin-stimulated Na+ absorption, implicating this isoform in the tissue-specific transient stimulation of Na+ transport by arginine vasopressin. Wilborn and Schafer (43) found additional evidence for the presence of PKCalpha , -eta , and -theta in the CCD. Interestingly, these authors also found differences in the expression levels of PKCeta and PKCtheta between freshly isolated and cultured rabbit CCD cells.

Although the above findings provide strong indirect evidence in support of the hypothesis of distinct roles for PKC isoforms in the steady-state or hormonal regulation of the epithelial Na+ channel (ENaC) or of Na+ transport in high-resistance epithelia, little is know about these specific roles. Indeed, although a large number of studies have examined ENaC's regulation by PKC in various preparations (3, 7, 11, 15, 24, 27, 30, 32, 33, 39, 42), only a minor fraction have focused on the potential regulation by distinct isoforms or a group of isoforms. Moreover, these studies utilized pharmacological agents believed to be selective for certain isoforms. For example, Rokaw et al. (33, 34) utilized rapamycin, which has a higher specificity for cPKC isoforms, to determine that inhibition of PKCalpha transiently stimulates ENaC in A6 epithelia.

Recently, an additional important function for PKC has been elucidated. Stockand et al. (40) found that long-term stimulation of PKC results in downregulation of beta  and gamma  ENaC protein levels, indicating the presence of a chronic interaction between ENaC and PKC. The PKC isoforms involved in this process are undetermined. It is also unknown whether a reciprocal or feedback interaction exists between ENaC or Na+ transport and PKC.

Because of our interest in A6 cells as a model of Na+ transport by electrically tight epithelia, we examined their PKC isoform content. Three PKC isoforms have been cloned from Xenopus oocytes: PKI, PKCII (8), and PKCzeta (Ref. 13 and J. Moscat, M. M. Munico, and M. T. Diaz-Meco, unpublished observations; NCBI accession no. U12588). The first two, PKCI and PKCII, share the highest homology with mammalian PKCalpha and PKCbeta (13) and are likely to correspond to these isoforms. Xenopus PKCzeta shares homology with atypical PKCs and is likely to correspond to mammalian PKCzeta (J. Moscat et al., unpublished observations). However, it is uncertain which of these and other unidentified isoforms exist in A6 epithelia. Only PKCalpha has been identified at the protein level in A6 cells (1, 33). This isoform is stimulated by increasing [Ca2+]i, a procedure known to inhibit Na+ channels in tight epithelia (18). Given this effect, the data of Rokaw et al. (33, 34), and our own unpublished observations, it is likely that PKCalpha mediates channel inhibition. Thus this isoform would a priori make a good candidate for its involvement in feedback regulation of ENaC and Na+ transport.

To determine whether a reciprocal interaction exists between Na+ transport and these isoforms, and specifically PKCalpha , we examined the effects of 1) transport stimulation with the hormone aldosterone and 2) transport inhibition leading to feedback regulation, with the uncharged amiloride derivative CDPC. We report that polarized A6 epithelia contain PKC isoforms in all three groups. We also found a specific downregulation of PKCalpha by chronic aldosterone and CDPC treatment. Furthermore, the extent of Na+ transport activation with aldosterone or prolonged CDPC treatment could be enhanced with a specific cell-permeable inhibitor of PKCalpha , providing further functional evidence for the involvement of this isoform. We propose that by downregulating an inhibitory kinase, A6 cells are able to sustain a stimulation of Na+ transport, thereby resetting homeostatic mechanisms to a higher level.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A6 epithelia. Cells were obtained from American Type Culture Collection (ATCC, Manassas, VA) and were cultured as previously described by Awayda et al. (3). Cells were either plated on flasks or on permeable polyester membrane inserts (Transwell Clear; Corning-Costar, Acton, MA). Cell polarity was determined from their ability to develop a transepithelial open-circuit voltage (Voc). Monolayers which exhibited a Voc > 35 mV (basolateral with respect to apical) were used for subsequent experiments. These monolayers exhibited a transepithelial resistance (RT) in the range of 10 KOmega · cm2 and an equivalent short-circuit current (Isc) > 5 µA/cm2. Membrane voltage and resistance were determined by using a four-electrode system (EVOM; WPI, Sarasota, FL) designed for measurements of these parameters in tissue culture inserts without disrupting the cells. The equivalent Isc was calculated according to Ohm's law.

Confluent filters were treated with CDPC (6-chloro-3,5-diamino-2-pyrazinecarboxamide) aldosterone (Sigma, St. Louis, MO), PMA (CalBiochem, San Diego, CA), or a myristoylated PKCalpha C2 peptide inhibitor (N-Myr-Ser-Leu-Asn-Pro-Glu-Trp-Asn-Glu-Thr; Biomol, Plymouth Meeting, PA) as described in RESULTS. All reagents were of the highest grade available and were dissolved directly into medium or in stock solution of ethanol (EtOH), DMSO, or medium, respectively.

Cell fractionation and Western blotting. Confluent monolayers of A6 epithelia were harvested and immunoblotted as suggested by the manufacturer of the monoclonal antibodies against each of the PKC isoforms (Transduction Laboratories, San Diego, CA), with a few modifications as outlined below. Briefly, cells were lysed in boiling lysis buffer (10 mM Tris, pH 7.5, and 1 mM Na vanadate), scraped into 15-ml conicals, boiled for 5 min, and passed several times through a 27-gauge needle. Unbroken cells were pelleted, and an aliquot of the lysate was removed. This aliquot was the total protein fraction and was acetone (~6 volumes) precipitated and resuspended in 10 mM Tris, pH 7.5. The remaining lysate was centrifuged at 100,000 g for 1 h at 4°C. The supernatant contained the cytoplasmic fraction and was acetone precipitated as above. The pellet was resuspended in lysis buffer containing 1.0% Triton X-100 and incubated on ice for 2 h with occasional vortexing. The suspension was then centrifuged at 14,000 g for 20 min at 4°C, and the supernatant containing the solubilized membrane fraction was acetone precipitated as described above. Protein concentrations were determined with a Bradford-based assay (Bio-Rad, Chicago, IL) by using BSA as a standard.

Approximately 7.5 µg of beta -mercaptoethanol (beta ME)-reduced protein were separated on 3.9%/7.5% Laemmli gels under denaturing conditions (SDS). Proteins were electrotransferred overnight at 4°C to nitrocellulose membranes. Blots were blocked in Blotto (10 mM Tris, pH 7.5, 100 mM NaCl, 0.05% Tween 20, and 5% nonfat dry milk) for 1 h and then incubated for 2 h in primary antiserum diluted in Blotto at the concentrations recommended by the manufacturer. All incubations were carried out at room temperature. For monoclonal antibodies, horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antiserum was diluted at 1:100,000 in Blotto, and the blots were incubated for 45-60 min. Rat anti-rabbit secondary antiserum was used at 1:160,000 for the polyclonal antibodies. Blots were washed in 10 mM Tris, pH 7.5, 100 mM NaCl, and 0.05% Tween 20 between antiserum incubations and in 10 mM Tris, pH 7.5, before being processed for chemiluminescence (SuperSignal West Dura Extended Duration Substrate; Pierce, Rockford, IL) according to the manufacturer's instructions. The blots were then exposed to film and developed in an automated developer. Films were scanned by using a flatbed scanner, followed by quantitation of signal intensity using SigmaScan (Jandel Scientific, San Rafael, CA). A similar procedure was carried out by using a positive control (rat brain extract) probed with anti-PKCalpha and anti-GAPDH antibodies to serve as calibration standards.

Equal loading was assessed by using three procedures: 1) protein concentration determination, 2) Ponceau S staining of the membranes after electrotransfer, and 3) Western blotting with a GAPDH-specific antibody (BioDesign International, Saco, ME). In this case, the bottom half of the blots (<50 kDa) was separated and probed with the GAPDH antibody.

The antibodies utilized in the present study were raised against highly conserved protein sequences and allow a high degree of isoform specificity. This specificity was also verified by testing the antibodies against known positive controls. These antibodies are also tested by the manufacturer and are known in many instances to recognize the same isoform from various species, including human, canine, rat, mouse, chicken, and frog. Moreover, isoform-specific anti-mammalian PKC antibodies have been tested by others and are known to recognize specific Xenopus PKC isoforms (1, 13, 31, 33).

Northern blotting. Total RNA was isolated from polarized A6 cells by utilizing the Trizol reagent (Life Technologies, Rockville, MD) following the manufacturer's instructions modified for a growth area of 4.5 cm2. Briefly, cells in each well were lysed in 500 µl (initial volume) of Trizol, scraped, and mixed with 0.2× initial volume chloroform, and the phases were separated by centrifugation at 12,000 g for 15 min at 4°C. The RNA was precipitated from the aqueous phase with 0.5× initial volume of isopropanol and, after 10 min at room temperature, pelleted by centrifugation at 12,000 g for 10 min at 4°C. The pellet was washed with 2.5× initial volume of 75% EtOH, vortexed, and centrifuged at 7,000 g for 5 min at 4°C. Total RNA was treated with RQ1 DNase (Promega, Madison, WI), phenol extracted, EtOH precipitated, and resuspended in RNase-free water. RNA concentrations were determined spectrophotometrically, and RNA integrity was verified on a 1% agarose gel by using formaldehyde loading buffer.

To construct the Northern probes, the isolated RNA served as the template for RT-PCR for the amplification of Xenopus PKCI (also known as PKCalpha ) and GAPDH DNA. Xenopus PKCI primers were designed to amplify a 303-bp fragment encompassing the V3 region and the adjacent C2 and C3 regions, bases 807-1110, as determined by Chen et al. (8). The PKCI forward primer was 5'-gctctttatcttcgggggtgtcagagctgatgaaaatg-3' and the reverse primer was 5'-ccagtgaattgtaatacgactcactatagggctgccaaccatgac-3'. Primers for Xenopus GAPDH were designed to encompass bases 911-1154 (accession no. GI1136598). The forward primer was 5'-ctcctccatctttgatgctgatgctggaattg-3' and the reverse primer was 5'-ccagtgaattgtaatacgactcactatagggaatgtttcatcatg-3'. The T7 RNA polymerase promoter was added to the 5' end of each reverse primer (underlined in sequences).

Briefly, 5 µg of control RNA were reverse transcribed (Thermoscript, Life Technologies) with the gene-specific primer for PKCI or oligo(dT)20 for GAPDH according to the manufacturer's protocol. The cDNAs (1/20 of each reaction) were then used as the template for PCR, incorporating the primers described above. Pfu DNA polymerase (Stratagene, La Jolla, CA) was used for touchdown PCR with the following parameters: initial denaturation at 95°C for 5 min; 15 cycles of 95°C for 30 s; 70°C for 30 s with a 1°C decrease in annealing temperature per cycle, and 74°C for 1 min, followed by 25 cycles of 95°C for 30 s, 54°C for 30 s, and 74°C for 1 min; and a final extension of 74°C for 10 min. The resulting products were purified on a 3.5% low-melt agarose gel, phenol extracted, EtOH precipitated, and resuspended in RNase-free water. Antisense RNA were generated from these products by in vitro transcription with T7 RNA polymerase (Promega). The RNA was labeled by using the BrightStar Psoralen-Biotin nonisotopic labeling kit (Ambion, Austin, TX) according to the manufacturer's protocol.

Total RNA (15 µg) from each condition for each of the probes was separated on a 1% agarose formaldehyde gel. The transfer to the nylon membrane (MSI) was done in the vacuum blotter according to the manufacturer's protocols (Bio-Rad Labs, Hercules, CA). After UV cross-linking, prehybridization (30 min) and hybridization (overnight) were performed at 55°C in ultrasensitive hybridization buffer (Ambion). The blots were washed twice at 55°C by using high-stringency wash solution (Ambion). Detection was accomplished by using the BrightStar BioDetect kit (Ambion) according to the manufacturer's protocol. Hybridization products were detected on Kodak X-ray film for 2 h.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isoform content of polarized A6 cells. A6 cells were grown on permeable supports as described in MATERIALS AND METHODS. Under these conditions, confluent cells exhibited a spontaneous Voc that stabilized at 49.5 ± 0.73 mV and an RT of 10.63 ± 0.16 KOmega · cm2 (n = 116). This resulted in an Isc of 4.71 ± 0.06 µA/cm2. Under these conditions, and consistent with data previously reported (3), the majority of the Isc is sensitive to amiloride and to removal of apical Na+.

Confluent and polarized Na+-transporting A6 cells were fractionated and probed with isoform-specific PKC antibodies. As mentioned above, three PKC isoforms have been previously cloned from Xenopus oocytes. Given their homology with their mammalian counterparts, it is believed that Xenopus PKCI and PKCII correspond to PKCalpha and PKCbeta , respectively (8). Therefore, for the purposes of clarity and consistency with other reports in the literature, we will refer to these Xenopus isoforms as PKCalpha and PKCbeta .

Shown in Fig. 1 is a composite Western blot of the various immunoreactive species identified in these cells. Lanes probed with the secondary antibody alone, at the same concentration as that used with the primary, were blank (not shown). Specific PKC immunoreactivity was observed in lanes probed with monoclonal antibodies to PKCalpha , -gamma , -delta , -epsilon , -iota , and -lambda (Fig. 1A). PKC immunoreactivity was also observed with polyclonal antibodies to PKCeta and -zeta (Fig. 1B).


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Fig. 1.   PKC isoforms present in polarized A6 epithelia. Composite Western blot of A6 homogenate was probed with isoform-specific PKC antibodies. A: A6 proteins were probed with monoclonal antibodies which recognize unique PKC isoforms. Of the complement of monoclonal antibodies used, only those directed against PKCbeta and PKCtheta sequences did not cross-react with A6 protein (data not shown). B: A6 proteins were probed with polyclonal antibodies against PKCeta and PKCzeta . Lanes were separated and individually probed with a single isoform-specific antibody as described in MATERIALS AND METHODS.

These commercial isoform-specific antibodies are known to cross-react with various species, including frog. This is because antigenic regions chosen to construct these antibodies are conserved among various species. For example, the PKCalpha antibody was constructed against a large 156-amino acid region (amino acids 270-427) of human PKCalpha , which is 93% homologous to frog (Xenopus). Thus it is expected and indeed previously was demonstrated that this antibody will cross-react with amphibian PKCalpha . On the other hand, antibodies obtained from other commercial resources made against short regions with little or no homology between Xenopus and human failed to detect PKC immunoreactivity in A6 cells (data not shown).

In all cases, the Xenopus PKC isoforms recognized by these antibodies corresponded well with their mammalian counterpart observed in rat brain homogenate (data not shown; also see manufacturer's data). Our finding of PKCalpha in A6 cells is consistent with previous reports (1, 33), although the identification of other isoforms represents, to our knowledge, the first published report of such an attempt.

Summarized in Table 1 is the calculated mass for the various PKC isoforms in A6 cells. Some isoforms, e.g., delta  and eta  in the novel group and iota  in the atypical group, migrated as doublets. In these cases, the masses for each of the immunoreactive species are summarized in Table 1. With the exception of PKCalpha , there were no significant differences in the molecular weight of the identified isoform between the cytoplasmic and membrane fractions. On average there was a small shift in the calculated molecular weight for PKCalpha between the two cellular fractions, such that this isoform was ~6 kDa larger in the membrane fraction. It is possible that other isoforms exhibited a small shift in molecular weight between these two fractions; however, the differences in PKCalpha were highlighted once we focused our attention on this isoform (Fig. 4). The reason for this small shift in PKCalpha is unknown but may be attributed to posttranslational modification, e.g., phosphorylation of the membrane-associated PKCalpha (20).

                              
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Table 1.   Relative mobilities of Xenopus PKC isoforms in polarized A6 epithelia

As previously mentioned, conventional PKC isoforms, i.e., Ca2+-activated isoforms such as alpha  and gamma , are thought to mediate channel inhibition (18, 33, 44). Given these effects, our long-standing interest in understanding ENaC's inhibition by PKC (5), and our own unpublished observations with PKCalpha , we focused our attention on understanding the roles of these isoforms in feedback regulation of Na+ channels. Initial experimentation did not reveal consistent changes of PKCgamma , and we therefore focused our attention to examining PKCalpha protein and mRNA levels.

Role of PKCalpha in the response to aldosterone. To examine the effects of sustained stimulation of ENaC, we utilized the hormone aldosterone. Treatment with 1 µM aldosterone (24 h) caused a near doubling of Voc from 49.5 ± 0.73 to 96.5 ± 0.66 (n = 36), accompanied by a nearly fourfold increase of Isc (4.71 ± 0.06 to 15.75 ± 0.27), consistent with the previously reported effects of this hormone.

The accompanying changes of PKCalpha protein levels were determined by using Western blots of membrane and cytoplasmic fractions. As shown in Fig. 2, PKCalpha levels were reduced in both the cytoplasmic and membrane fractions. This decrease of PKCalpha protein levels was also confirmed by examining the levels of this isoform in the total cell homogenate. As shown in Table 2, a similar decrease of PKCalpha was observed among the cytoplasmic membrane and total fractions, indicating that this decrease was likely the result of a general downregulation of protein levels of this isoform.


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Fig. 2.   Downregulation of PKCalpha by aldosterone. Representative Western blot of the effect of aldosterone on the distribution of PKCalpha in A6 cell fractions. T, M, and C refer to total, membrane, and cytoplasmic fractions, respectively. + and - refer to 24-h treatment with 1 µM aldosterone or ethanol vehicle, respectively. Data are representative of 7 experiments. See MATERIALS AND METHODS for more details.


                              
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Table 2.   Relative changes of PKCalpha distribution in aldosterone-treated A6 epithelia

To determine whether this effect is attributed to a decrease of PKCalpha protein synthesis, a question especially important given that aldosterone is a steroid hormone with multiple genomic and nongenomic effects, we utilized Northern blotting with riboprobes constructed from Xenopus PKCalpha as described in MATERIALS AND METHODS. A similar signal was observed in RNA from control and aldosterone-treated cells (Fig. 3), indicating that the decrease of PKCalpha protein levels was not the result of a direct genomic downregulation of the PKCalpha gene by aldosterone. However, we were uncertain whether these effects were specifically attributed to the actions of aldosterone or generally related to prolonged stimulation of Na+ transport, leading to potential secondary changes of [Na+]i. To address these issues, we carried out experiments in which Na+ channels were stimulated by aldosterone but in which the effects on transport were restored to near baseline by overnight (24 h) incubation with a specific ENaC blocker.


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Fig. 3.   Lack of direct genomic effects of aldosterone on PKCalpha . A: Northern blotting with PKCalpha -specific probes. B: parallel Northern blot with a GAPDH-specific probe was carried out to assure equality of loading. The intensity of ethidium-stained ribosomal RNA was also examined (data not shown). Conditions were as described in MATERIALS AND METHODS, except that 10 µg of the total RNA were used and washes were carried out at 65°C. This may have resulted in decreased signal intensity (see Fig. 6 for comparison). The intensities of control cells (lanes 1 and 3) were similar to those observed from aldosterone-treated cells (lanes 2 and 4). Note the presence of 2 products in both control and aldosterone-treated cells. These may represent different forms of PKCalpha and correspond to sizes of ~3.6 and 6.5 kB, respectively. Data are representative of 3 experiments.

Effects of CDPC: feedback regulation. We utilized the electroneutral amiloride analog CDPC to reduce Na+ transport in aldosterone-treated tissues to control prestimulated levels. We specifically chose this blocker because it is electroneutral and is therefore not susceptible to changes of intracellular voltage leading to changes of its blocking efficacy. We initially chose a concentration of 50-100 µM CDPC, which is in the range of the half-maximal block for ENaC by this compound (3). However, because of the distribution of channels from the closed to open and blocked states, this concentration was not sufficient in providing an appreciable inhibition of the Isc, even under short-term conditions. We increased this concentration and found that 300-400 µM CDPC acutely decreased the Isc in aldosterone-treated cells to values similar to those in the untreated ones.

The effects of separate and combined CDPC and aldosterone treatment are shown in Fig. 4. It is clear that the effects of aldosterone on PKCalpha were not mediated by a simple increase of Na+ transport, because the protein levels of this isoform also decreased in epithelia treated with aldosterone and CDPC. However, and surprisingly, tissues treated with both CDPC and aldosterone exhibited the largest decrease of PKCalpha . Again, this was observed in the cytoplasmic and membrane fractions and was a reflection of decreased PKCalpha protein levels. Consistent with this observation was the finding that CDPC alone was also capable of decreasing PKCalpha levels. Although these findings with aldosterone and CDPC may appear puzzling given the opposite effects of these agents on Na+ transport, these effects may be reconciled by the ability of both CDPC and aldosterone to cause prolonged stimulation of transport (see below).


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Fig. 4.   Additive downregulation of PKCalpha protein levels by aldosterone and uncharged amiloride analog (CDPC). A: representative Western blot demonstrating that PKCalpha is downregulated by the separate and combined treatments with aldosterone and CDPC. Note that the largest decrease was observed in cells treated with both aldosterone and CDPC. 0, A, C, and C + A refer to control (vehicle), 1 µM aldosterone, 400 µM CDPC, and CDPC and aldosterone, respectively. B: corresponding signal intensities relative to those observed in the control ethanol-treated cells (lanes 1 and 2). Data are representative of 4 experiments with 400 µM CDPC and 5 additional experiments with 200-300 µM CDPC. Aldosterone and CDPC were added for 24 and 21 h, respectively.

Shown in Fig. 5 are the effects of aldosterone and CDPC on the Na+ transport properties of A6 cells. As seen above, aldosterone caused a threefold increase of the Isc. Short-term treatment with CDPC (10 min) caused a large decrease of the Isc in both control and aldosterone-treated cells. However, at 21 h, the Isc in cells treated with CDPC and aldosterone exhibited a complete recovery to values even higher than those found in tissues treated with aldosterone alone. A similar recovery to values above control was also observed in tissues treated with CDPC alone. These processes are likely a continuation of the short-term feedback or autoregulation described in many Na+-transporting epithelia, where the Isc gradually recovers toward control values after inhibition of Na+ transport, manifested by the ability of cells to stimulate the apical Na+ channels (16, 18, 41). Thus, in both the CDPC- and aldosterone-treated cells, a downregulation of PKCalpha was associated with conditions that require a sustained increase of Na+ transport. As with the effects of aldosterone alone, the decrease of PKCalpha protein levels was not accompanied by a decrease of mRNA levels (Fig. 6). These findings indicate that these effects were not the result of direct genomic modulation of the PKCalpha gene.


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Fig. 5.   Feedback regulation of Na+ transport after prolonged inhibition with the electroneutral blocker CDPC. The initial effects of CDPC were measured 10 min after its addition to control or aldosterone-prestimulated cells (3-h treatment). Overnight changes were reported at 24 h after addition of aldosterone and 21 h after addition of CDPC. Lanes 1 and 2 refer to control and 3-h aldosterone-treated cells. Lanes 3 and 4 refer to the same tissues 10 min after the addition of CDPC. Lanes 5, 6, and 7 refer to filters chronically treated (21 or 24 h) with CDPC, aldosterone, and CDPC + aldosterone, respectively. As expected, CDPC caused a large initial inhibition of transport. However, at 24 h, cells were able to compensate and restore their transport rates to values slightly higher than those in the absence of blocker. Note the changes of transepithelial resistance (RT) observed with aldosterone and CDPC treatments. These changes indicate that, under our culture conditions, RT is predominantly a reflection of the cellular resistance and that the observation of an RT > 4 KOmega · cm2 under all experimental conditions indicates the absence of adverse cellular effects.



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Fig. 6.   Lack of direct genomic effects of CDPC and combined CDPC/aldosterone treatments on PKCalpha . Blots were conducted as described in MATERIALS AND METHODS. Lanes 1 through 4 correspond to RNA from control, aldosterone-, CDPC-, and CDPC + aldosterone-treated cells, respectively. The top panel was probed with a PKCalpha -specific probe, while the bottom panel was probed with a GAPDH-specific probe. Note that lanes 1 and 2 have similar intensities, whereas lanes 3 and 4 are slightly higher than 1 and 2. Thus the decrease of PKCalpha protein levels cannot be accounted for by its observed levels of RNA. As with Fig. 3, 2 bands are observed for PKCalpha . Data are representative of 3 additional experiments.

Given the importance of these findings, we further characterized Xenopus PKCalpha . Specifically, we tested whether this isoform exhibits cytoplasm-to-membrane translocation subsequent to its activation. As shown in Fig. 7A, 1-h treatment with 100 nM PMA was capable of translocating the majority of cytoplasmic PKCalpha to the plasma membrane. This indicates that the mechanism of activation of Xenopus PKCalpha is similar to that described in other systems. As expected, the addition of PMA caused a large decrease of Isc and Voc (Fig. 7B). This was accompanied by an increase of RT due to inhibition of the apically located ENaC.


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Fig. 7.   PMA causes translocation of PKCalpha to the membrane, subsequent to its acute activation, and inhibition of Na+ transport. A: Western blot of the effect of 1-h treatment with 100 nM PMA on the distribution of PKCalpha . This provides further evidence for the specificity of the PKCalpha antibody in recognizing Xenopus PKCI. B: transport properties of these cells were severely inhibited by PMA, consistent with a previous report by Els et al. (15). The increase of RT with PMA's inhibition of Isc provides further evidence for the integrity of the cells under our experimental conditions.

Role of PKCalpha in feedback stimulation. The above findings indicate that two different conditions that require a sustained stimulation of Na+ transport are also accompanied by a decrease of PKCalpha activity subsequent to a massive decrease of its protein levels. Thus it follows that specific inhibition of this kinase may result in sustaining higher rates of transport. This hypothesis was tested as shown in Fig. 8. In these sets of experiments, we examined the time course of the changes of Na+ transport and the effects of specific PKCalpha inhibition with a cell-permeable peptide inhibitor. The sequence of this inhibitor is derived from the C2 (Ca2+-binding) region of PKCs (35). Because beta  levels are not detectable, and because gamma  does not appear to participate in the response to aldosterone or CPDC, the effects of this peptide can be interpreted in terms of inhibition of PKCalpha membrane localization.


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Fig. 8.   Additional sustained stimulation of Na+ transport after prolonged inhibition of PKCalpha . Experiments for control, CDPC-, and aldosterone-treated cells were carried out as previously described. Experiments with the cell-permeable PKCalpha inhibitor (C2-4 peptide) were carried out by adding 10 µM peptide to the medium at time 0. The transient stimulation observed in untreated cells was purely due to the solution exchange. Data were normalized to the value of current immediately before the solution exchange. n = 9, 11, and 11 for A, B, and C, respectively. See text for additional details.

As shown in the Fig. 8A, addition of this peptide did not significantly affect Na+ transport over a 24-h period such that the equivalent current was indistinguishable from that observed in control untreated cells at 4, 18, and 24 h. On the other hand, the recovery of current after prolonged addition of CDPC was enhanced with the PKCalpha inhibitor such that the values at 2.5, 4, 18, and 24 h were significantly higher than those observed with CDPC alone (Fig. 8B). As shown in Fig. 8C, a similar trend was also observed with aldosterone treatment, except that the differences were only marginally significant at 18 and 24 h (P < 0.07).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Numerous studies have examined the regulation of epithelial Na+ channels. Although it is clear that many hormonal and humoral factors regulate this channel, it is also accepted that other partially defined processes act on these channels to maintain homeostasis and attempt to restore activity in response to various experimental stimuli. It is a priori expected that such mechanisms would also affect the response of the channel to hormones and circulating factors, and, thus, a better understanding of these feedback homeostatic processes is essential to our understanding of channel regulation in tight epithelia. We put forth the hypothesis that such processes are likely to involve modulation of channel regulation by kinases. We began by defining the PKC isoforms present in our model system. Second, we tested the above hypothesis by determining the effects of channel stimulation on an inhibitory PKC isoform, PKCalpha . We report that sustained stimulation of Na+ channels by hormonal and nonhormonal mechanisms is accompanied by specific, and potentially nongenomic, downregulation of this kinase. Moreover, both processes were additive in their downregulation of PKCalpha , indicating distinct signaling cascades that converge at the level of this kinase. We propose that this mechanism represents a novel regulatory pathway invoked when cells require sustained increase of Na+ transport.

Effects of aldosterone. It is well established that the actions of aldosterone are to cause prolonged and sustained increases of Na+ transport. This is in contrast to the effects of other renal hormones, e.g., antidiuretic hormone (ADH), where the response is transient in nature. This sustained increase is observed despite the presence of homeostatic mechanisms that tend to inactivate Na+ channels. For example, channel activation by aldosterone would tend to increase [Na+]i, which in turn could inactivate the apical membrane resident ENaC via various feedback and self-inhibitory mechanisms (4, 18, 41). However, the response to aldosterone is clearly not a transient one, and one such possibility is the subsequent downregulation of kinases responsible for maintaining feedback inhibition, such as PKCalpha .

PKCalpha has been previously implicated in the regulation of ENaC and the response to aldosterone utilizing a pharmacological approach. Rokaw et al. (33, 34) found that the immunosuppressive agent rapamycin causes a transient (peak 1 h) stimulation of the Isc in A6 cells. This was later confirmed by Yue and colleagues (44) to be an effect on ENaC activity. Short-term treatment with this agent was found to inhibit PKC activity, despite what appears to be an increase in the membrane localization of this isoform (see Fig. 4 in Ref. 33). Long-term treatment (>18 h) with rapamycin was effective in nearly abolishing the stimulation of Na+ transport observed with aldosterone. Thus it is possible, and indeed likely, that the long-term effects of rapamycin on aldosterone are related to the increased translocation of PKCalpha in the membrane which, consistent with our hypothesis, would tend to counteract the stimulatory effects of aldosterone.

The precise link between PKCalpha and aldosterone is unknown. Doolan and Harvey (14) previously attributed some of the nongenomic and rapid (minutes) effects of aldosterone to stimulation of PKC. Until recently, the isoforms involved in this response were undetermined. In a recent followup (21), it was concluded that aldosterone directly stimulates PKCalpha activity in vitro. This effect was specific to PKCalpha and was observed in a cell-free system containing purified enzyme. In these experiments, addition of 1 µM aldosterone caused an ~30% increase of PKCalpha activity. These experiments provide additional evidence in support of interaction between the aldosterone-signaling pathway and the alpha -isoform of PKC. However, it is important to recognize that the experiments of Doolan and Harvey represent a different time scale than those in the current study (minutes vs. 24 h) and that the effects on PKCalpha are opposite in direction. Nonetheless, it is possible that the early stimulatory effects of aldosterone on PKCalpha lead to prolonged inhibition. In this case, the long-term effects would be more physiologically relevant in understanding the sustained increase of Na+ transport, especially because PKCalpha is likely to inhibit ENaC (see below and Refs. 33, 34, and 44).

A second possibility that may explain the link between aldosterone and PKCalpha may involve changes of transport leading to potential changes of [Na+]i. This possibility is further explored with the addition of CDPC (see below). In either case, it is important to recognize that the findings from the Northern blot experiments are consistent with no direct effects of aldosterone on the PKCalpha gene. This does not rule out the possibility of indirect genomic effects on a PKCalpha protease or other upstream regulators of PKCalpha protein levels. Unfortunately, these issues could not be directly assessed, because experiments utilizing actinomycin D resulted in complete dissolution of polarized A6 cells at concentrations (5 µM) routinely used to inhibit de novo protein synthesis in other epithelial preparations (data not shown). Nonetheless, the similarity of these effects to those observed with CDPC argue against direct genomic effects.

Effects of CDPC: feedback regulation. As mentioned in RESULTS, CDPC was used to decrease Na+ transport in the presence of aldosterone to test the role of [Na+]i. These experiments were chosen in favor of those with reduced external apical [Na+], because custom formulation of Na+-free medium is prohibitively costly and, moreover, appreciable basolateral Na+ back-leak would drastically alter the [Na+] in the external vicinity of the channel. We found that CDPC also decreased PKCalpha protein levels and that this decrease was additive with aldosterone. These changes coincided with the ability of the cells to counteract the inhibitory effects of this passive blocker and restore Na+ transport to control levels either in the absence or presence of aldosterone. Because the Isc returned to pre-CDPC levels, it is likely that the steady-state [Na+]i was similar to that in the absence of CDPC. In this case, it is unlikely that prolonged changes in [Na+]i are the simple explanation that ties in the effects of aldosterone and CDPC. However, it remains possible that the time course of changes of [Na+]i is important in setting in motion a prolonged signaling cascade that may over the course of 24 h affect PKCalpha protein levels. This hypothesis awaits further experimental verification.

The stimulation observed with CDPC is similar to that observed with a variety of other maneuvers that alter Na+ transport and has been historically termed feedback regulation. This is a homeostatic mechanism that represents the first line of cellular regulation of Na+ transport. This process is thought to involve potential direct effects of the Na+ ions, a process termed self-inhibition, in addition to delayed cell signaling-mediated effects termed feedback inhibition. Two previous reports from the Palmer and Eaton groups (17, 25) indicate that the latter phenomenon involves the activation of PKC leading to inhibition of apical ENaCs. Our findings extend those observations to specifically implicate PKCalpha and to provide additional evidence supporting the notion that downregulation of this isoform is accompanied by feedback stimulation of Na+ transport. Our findings from Western blotting are also functionally validated, as further stimulation of this process is observed by the addition of a specific PKCalpha inhibitor.

Isform-specific regulation by PKCalpha . Our findings of isoform-specific regulation of a physiological process in tight epithelia is consistent with the emerging hypothesis of distinct roles of PKC isoforms (see Introduction). In the present study, we find that specific inhibition of PKCalpha with a cell-permeable peptide inhibitor did not alter baseline rates of transport. After the initial transient stimulation observed with cell feeding and solution exchange, the Isc returned to values not different from control. This indicates little or no regulation of baseline rates of transport by PKCalpha (Fig. 8). These data are in contrast to the observations from other laboratories examining the baseline regulation by PKC. An important difference between the majority of previously published observations and our present findings is that we are dealing with inhibition of a specific PKC isoform. Because it is our overall hypothesis that the regulation of ENaC by PKC is isoform specific, it is nearly impossible to compare our present findings with those using pharmacological agents that affect multiple isoforms in all three PKC subtypes.

However, two previously published reports indicate that rapamycin inhibits Na+ transport at the macroscopic (33) and microscopic (44) levels. Because rapamycin has been elegantly demonstrated by Rokaw et al. (33) to affect purified PKCalpha in vitro, and because the effects of rapamycin could be mimicked by inhibiting conventional PKC with a myristoylated pseudosubstrate peptide, these data would appear to contradict our conclusion of no baseline regulation by this isoform. A major difference between those studies and ours is that our inhibitor binds to the C2 domain (Ca2+ binding) of PKCs and inhibits their translocation to the plasma membrane (35), whereas the peptide used by Rokaw et al. acts as a pseudosubstrate for cPKCs. Thus, in our case, we expect reduced membrane distribution of PKCalpha with little or no effects on the activity of cytoplasmic PKC, whereas in the experiments of Rokaw et al., we expect reduced activity throughout the cell. A second possibility is differences in the levels of PKCalpha or the presence of PKCbeta between cells grown under our conditions and those of Rokaw et al. (33) and Yue et al. (44). Because PKCbeta levels are not detectable under our conditions, effects of these inhibitors on this isoform can be ignored.

A third potential explanation is that the previously published experiments with rapamycin are short term, whereas ours extend to the time scale of many hours (24 h). Thus it is possible that the short-term stimulatory effects of rapamycin are masked in our experiments by the transient effects observed from the medium exchange that result in current stimulation by itself. On the other hand, the prolonged effects of PKCalpha inhibition, which are not complicated by the feeding transient, also indicate a lack of transport inhibition. However, these data may be consistent with the experiments with rapamycin and the cPKC pseudosubstrate, because prolonged incubation with these agents may eventually lead to the return of transport to baseline levels. This indeed is the trend observed after 60 min from the data of Rokaw et al. (see Fig. 3 in Ref. 33). Resolution of these issues awaits the development of additional isoform-specific agonists/antagonists.

Our conclusions are different with CDPC and aldosterone, where the Isc was sustained at 25 and 45% higher levels (24 h), respectively. Because PKCalpha levels are already downregulated by aldosterone and CDPC, it is not expected that further pharmacological inhibition will result in dramatic changes in the sustained level of stimulation. Thus the observed small additional stimulation is entirely consistent with this explanation. Moreover, in both cases (CDPC and aldosterone treatment), the changes of transport were apparent at longer time points, consistent with the hypothesis that PKCalpha is downregulated under conditions where long-term stimulation of Na+ transport is required.

In conclusion, we have described the PKC isoforms present in A6 epithelia. We have also identified a novel mechanism for the regulation of Na+ transport in these epithelia involving a specific isoform, PKCalpha . Two different conditions that require sustained Na+ transport resulted in the downregulation of this isoform. These effects are not mediated via steady-state changes of [Na+]i, because aldosterone and CDPC are expected to have opposite effects on this parameter. This can also be ruled out because the Isc and presumably [Na+]i are essentially the same at 24 h between untreated and CDPC-treated tissues. The decrease of PKCalpha protein levels is unlikely to be a direct genomic effect, because PKCalpha mRNA levels are unchanged and, moreover, it is unexpected that a passive channel blocker would exhibit genomic effects. The simplest explanation for our findings is that stimulation of ENaC is sustained by downregulation of PKCalpha through novel signaling mechanisms yet to be defined. It is noteworthy that the transient stimulation of Na+ transport observed in various epithelia in response to ADH has been linked to secondary activation of PKC and, more specifically, Ca2+-dependent cPKC isoforms (1). Thus it seems plausible that PKCalpha may be under precise cellular control by various feedback and feedforward mechanisms that impinge on long-term regulation of Na+ transport.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-55626.


    FOOTNOTES

Address for reprint requests and other correspondence: M. S. Awayda, Dept. of Physiology, SL 39, Tulane University Health Sciences Center, New Orleans, LA 70112 (E-Mail: mawayda{at}tulane.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

May 29, 2002;10.1152/ajpcell.00142.2002

Received 29 March 2002; accepted in final form 23 May 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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