Mitogen-Activated Protein Kinases Inhibit the ROMK (Kir 1.1)-Like Small Conductance K Channels in the Cortical Collecting Duct
Elisa Babilonia,
Dimin Li,
Zhijian Wang,
Peng Sun,
Dao-Hong Lin,
Yan Jin and
Wen-Hui Wang
Department of Pharmacology, New York Medical College, Valhalla, New York
Address correspondence to: Dr. Wen-Hui Wang, Department of Pharmacology, New York Medical College, Valhalla, NY 10595. Phone: 914-594-4120; Fax: 914-347-4956; E-mail: wenhui_wang{at}nymc.edu
Received for publication May 2, 2006.
Accepted for publication August 1, 2006.
It was demonstrated previously that low dietary potassium (K)intake stimulates Src family protein tyrosine kinase (PTK) expressionvia a superoxide-dependent signaling. This study explored therole of mitogen-activated protein kinase (MAPK) in mediatingthe effect of superoxide anions on PTK expression and ROMK (Kir1.1) channel activity. Western blot analysis demonstrated thatlow K intake significantly increased the phosphorylation ofP38 MAPK (P38) and extracellular signal–regulated kinase(ERK) but had no effect on phosphorylation of c-JUN N-terminuskinase in renal cortex and outer medulla. The stimulatory effectof low K intake on P38 and ERK was abolished by treatment ofrats with tempol. The possibility that increases in superoxideand related products that are induced by low K intake were responsiblefor stimulating phosphorylation of P38 and ERK also was supportedby the finding that application of H2O2 increased the phosphorylationof ERK and P38 in the cultured mouse collecting duct cells.Simultaneous blocking of ERK and P38 completely abolished theeffect of H2O2 on c-Src expression in mouse collecting ductcells. For determination of the role of P38 and ERK in the regulationof ROMK-like small-conductance K (SK) channels, the patch-clamptechnique was used to study the effect of inhibiting P38 andERK on SK channels in the cortical collecting duct from ratsthat were on a control K diet (1.1%) and on a K-deficient dietfor 1 d. Inhibition of ERK, c-JUN N-terminus kinase, or P38alone had no effect on SK channels. In contrast, simultaneousinhibition of P38 and ERK significantly increased channel activity.The effect of inhibiting MAPK on SK channels was not affectedin the presence of herbimycin A, a PTK inhibitor, and was largerin rats that were on a K-deficient diet than in rats that wereon a normal-K diet. However, the stimulatory effect of inhibitingERK and P38 on SK was absent in the cortical collecting ductthat was treated with colchicine. It is concluded that low Kintake–induced increases in superoxide levels are responsiblefor stimulation of P38 and ERK and that MAPK inhibit the SKchannels by stimulating PTK expression and via a PTK-independentmechanism.
The kidney plays a key role in maintaining potassium (K) homeostasis,which is essential for the function of a variety of cells, includingneurons, cardiac myocytes, and skeletal muscles (1). It is wellestablished that increases in K intake stimulate whereas decreasesin K intake suppress renal K excretion (1). Low K intake–inducedsuppression of K excretion is achieved by stimulation of K absorptionin intercalated cells (2,3) and inhibition of K secretion inprincipal cells (4) in the connecting tubule and the corticalcollecting duct (CCD). Inhibition of K secretion in principalcells is partially achieved by decreasing apical K channel expression(4,5). We previously demonstrated that low K intake decreasesthe apical small-conductance K (SK) channel activity (6). Theeffect of low K intake on the SK channels is mediated by a proteintyrosine kinase (PTK)-dependent pathway (7,8) because inhibitionof PTK increases the SK channel activity in the CCD (9,10).Moreover, we have shown that low K intake increases superoxidelevels which mediate the effect of low K intake on PTK expression(6) and that suppression of superoxide production with tempoldiminishes the effect of low K intake on c-Src expression (6).
The role of superoxide in the regulation of SK channels is demonstratedbest by findings that the SK channel activity in the CCD fromthe tempol-treated rats was higher than that without tempol.We hypothesized that low K intake stimulates superoxide levelsin the kidney and increases the expression of Src family PTK,which enhances the tyrosine phosphorylation of ROMK (Kir 1.1)channels in the CCD (8). As a consequence of tyrosine phosphorylation,SK channels were internalized. However, the mechanism by whichsuperoxide stimulates PTK expression is not understood. Also,the finding that increases in PTK expression were not observeduntil 2 to 3 d after K restriction whereas decreases in urinaryK excretion took place several hours after K restriction suggeststhat signal molecules other than PTK regulate the SK channelactivity in the early stage of K restriction. Increases in superoxidelevels have been shown to activate extracellular signal–regulatedkinase (ERK), P38, and c-JUN N-terminus kinase (JNK) (11–13).Moreover, stimulation of mitogen-activated protein kinase (MAPK)is known to increase the phosphorylation of transcription factorssuch as c-JUN (14). Therefore, it is conceivable that MAPK maybe a member of signaling molecules that are required for theeffect of low K intake on the Src family PTK and SK channelactivity. Also, MAPK has been demonstrated to regulate a varietyof ion channels (15–17). The goal of our study was todetermine whether low K intake also stimulates MAPK and to examinethe role of MAPK in the regulation of SK channel activity inthe CCD.
Tissue Preparation
Sprague-Dawley rats (6 to 8 wk, either sex) were purchased fromTaconic Farms (Germantown, NY). Rats were divided into threegroups: (1) Control group, in which animals were kept on a normalK (1.1%) diet; (2) low K group, in which rats were maintainedon a K-deficient (KD) diet (Harlan Teklad, Madison, WI) for7 d and had a daily injection of vehicle; and (3) tempol-treatedgroup, in which rats also were fed a KD diet and had a dailyintraperitoneal injection of tempol (15 mg/kg) for 1 wk. Ratswere killed by cervical dislocation, and kidneys were removedimmediately. The renal cortex and the outer medulla were separatedunder a dissecting microscope and suspended in RIPA buffer solution(1:8 ratio, wt/vol) that contained 1x PBS, 1% Nonidet P-40,0.5% sodium deoxycholate, and 0.1% SDS. A total of 10 µlof PMSF (10 mg/ml stock solution in isopropanol) and 10 µlof a cocktail of protease inhibitors (Sigma, St. Louis, MO)were added per milliliter of buffer at the time of lysis. Thesamples were homogenized on ice for 15 min with a mortar andpestle. The suspension was incubated at 4°C for 1 h in thepresence of DNAse (5 µg/ml) followed by centrifugationat 1800 rpm for 10 min, and the resultant supernatant was collected.Protein concentrations were measured in duplicate using a Bio-RadDc protein assay kit (Hercules, CA).
Metabolic Cage
Rats were housed in metabolic cages for 1 d to study the urinaryK excretion. After 3 d of training in the cage, rats were dividedinto a control group, in which rats were kept on a normal Kdiet, and the low K group, in which rats were maintained ona KD diet. Data regarding the 24-h food intake, body weight,and urine output were recorded. Urinary K concentrations weremeasured by a flame photometer, and daily K excretion were calculatedas mEq/24 h.
Measurement of Superoxide Anion
Renal cortex and outer medulla were isolated from rats thatwere on a normal or KD diet for 1 d. The tissue (100 mg) wascut into a small piece with a sharp blade and suspended in air-equilibratedMOPS-sucrose buffer (pH 7.4) that contained 5 µM lucigenin.The chemiluminescence that was elicited in the presence of lucigeninwas measured in a liquid scintillation counter with a singleactive photomultiplier tube positioned in out-of-coincidencemode. Blanks were subtracted from the average level of chemiluminescencesignal.
Preparation of Mouse Collecting Duct Cells
Mouse collecting duct (M-1) cells, a mouse CCD line, were purchasedfrom the American Type Culture Collection (Manassas, VA) andmaintained in RPMI 1640 medium supplemented with 10% FBS. BeforeH2O2 treatment, the cells were cultured in medium that contained1% FBS for 16 h, followed by incubation for an additional 30min in a solution that contained 22 mM HEPES (pH 7.4), 124 mMNaCl, 5 mM KCl, 1 mM MgCl2, 1.5 mM CaCl2, 0.16 mM HPO4, 0.4mM H2PO4, 5 mM NaHCO3, and 5.6 mM glucose. H2O2 (200 µM)was added directly to the cells in HEPES buffer for 60 to 120min. The viability of M-1 cells that were treated with H2O2as determined by Trypan Blue dye exclusion method was approximately90% of the corresponding control cells. To study the phosphorylationof MAPK, we washed the cells with ice-cold PBS twice after treatmentof H2O2 and incubated them for 30 min in RIPA lysis buffer.To study the expression of c-Src, we incubated cells for anadditional 2 h in the control medium after removal of H2O2.
Immunoprecipitation and Western Blot
The corresponding antibody was added to the protein samples(500 µg) that were harvested from cell cultures at a ratioof 5 µl/ml solution. The mixture was rotated gently at4°C overnight, followed by incubation with 25 µl ofprotein A/G agarose (Santa Cruz, CA) for an additional 2 h at4°C. The tube that contained the mixture was centrifugedat 3000 rpm and washed twice with PBS that contained 10 µl/mlPMSF and 10 µl/ml protease inhibitor cocktail. The agarosepellet was resuspended in 25 µl of 2x SDS sample bufferthat contained 4% SDS, 100 mM Tris-HCl (pH 6.8), 20% glycerol,200 mM dithiothreitol, and 0.2% bromophenol blue. After thesample was boiled for 5 min, proteins were separated by electrophoresison 10% SDS–polyacrylamide gels and transferred to Immuno-BlotPVDF membrane (Bio-Rad). The membrane was blocked with 5% nonfatdry milk in Tris-buffered saline and incubated overnight withthe primary antibody at 4°C. The membrane was washed threetimes for 15 min with Tris-buffered saline that contained 0.05%Tween 20 followed by incubation for 30 min with respective secondantibody horseradish peroxidase conjugate. ECL plus (AmershamPharmacia Biotech, Piscataway, NJ) was used to detect the proteinbands, and the intensity of the bands of interest was determinedusing Alpha DigiDoc 1000 (Alpha Innotech, San Leandro, CA).
Preparation of CCD for Patch-Clamping
We used rats that were on either a normal K diet or a KD dietfor 1 d for the patch-clamp experiments. Single CCD were isolated,placed on a 5 x 5-mm coverglass coated with polylysine, andtransferred to a chamber (1000 µl) mounted on an invertedNikon microscope. The CCD were superfused with HEPES bufferedNaCl solution that contained (in mM) 140 NaCl, 5 KCl, 1.8 CaCl2,1.8 MgCl2, and 5 HEPES (pH 7.4). The pipette solution was composedof (in mM) 140 KCl, 1.8 MgCl2, and 5 HEPES (pH 7.4). The temperatureof the chamber was maintained at 37 ± 1°C by circulationof warm water around the chamber. The CCD was cut open witha sharpened micropipette to expose the apical membrane. An Axon200A patch-clamp amplifier was used to record channel current.The current was low-pass filtered at 1 KHz by an eight-poleBessel filter (902LPF; Frequency Devices, Haverhill, MA) anddigitized with Axon interface (Digidata 1200). Data were analyzedusing the pClamp software system 6.04 (Axon Instruments, Burlingame,CA). Channel activity was defined as NPo, which was calculatedfrom data samples of 60-s duration in the steady state as follows:
where ti is the fractionalopen time spent at each of the observed current levels.
Experimental Materials
Antibodies to phospho-P38, P38, phospho-ERK, ERK, phospho-JNK,JNK, and c-Src were purchased from Santa Cruz Biotechnology(Santa Cruz, CA). Herbimycin A, SB202190, SP600125, and PD098059were purchased from Biomol (Plymouth Meeting, PA).
Statistical Analyses
The data are presented as mean ± SEM. We used pairedt test to determine the statistical significance. P < 0.05was considered to be significant.
To determine the role of MAPK in mediating the effect of lowK intake on the SK channels, we first examined the effect oflow K intake on the expression and phosphorylation of P38, ERK,and JNK in the renal cortex and outer medulla. Figure 1A isa representative Western blot demonstrating the effect of lowK intake on the expression of total P38 (middle) and the phosphorylationof P38 (top). From inspection of Figure 1A, it is apparent thatK restriction for 7 d stimulates the phosphorylation of P38by 155 ± 20% (P < 0.001; n = 5 rats). We also examinedthe effect of low K intake on the expression and phosphorylationof ERK in the renal cortex and outer medulla. From inspectionof Figure 1B, it is apparent that low K intake also stimulatedthe phosphorylation of ERK by 150 ± 20% (P < 0.001;n = 5; top) but had no effect on the expression of total ERK(middle). However, low K intake did not significantly altereither phosphorylation or expression of JNK (Figure 1C). Therefore,our data demonstrate that low K intake increases the activityof ERK and P38 but not JNK in renal cortex and outer medulla.
Figure 1. Effect of potassium (K) depletion on the phosphorylation of P38 mitogen-activated protein kinase (P38 MAPK; A), extracellular signal–regulated kinase (ERK; B), and c-JUN N-terminus kinase (JNK; C) in rats that received tempol treatment or vehicle. (Top) Phosphorylated P38, ERK, and JNK, respectively. (Middle) Corresponding total amount of P38, ERK, and JNK. Data are summarized as a bar graph at the bottom of each panel. Changes of the band intensity are determined by calculation of the ratio between phosphorylation band intensity (Exp)/control value times the ratio between total protein intensity (normal)/Exp. *P < 0.01, control versus experimental group.
We then explored the possibility that superoxide anions or relatedproducts may mediate the effect of low K intake on MAPK. Thisnotion was based on the previous finding that low K intake increasesthe superoxide production and that superoxide anions and relatedproducts mediate the effect of low K intake on PTK expression(6). We examined the effect of H2O2 on MAPK phosphorylationin M-1 cells, a mouse CCD line. Previously, we demonstratedthat treatment of M-1 cells with H2O2 mimicked the effect oflow K on c-Src expression (6). Figure 2A is a typical Westernblot showing the effect of H2O2 on the phosphorylation and expressionof P38 in M-1 cells that were treated with 200 µM H2O2for 60, 90, and 120 min. Treatment of M-1 cells with H2O2 for60, 90, and 120 min increased phosphorylation of P38 by 30 ±10, 90 ± 30 (P < 0.01; n = 5), and 130 ± 40%(P < 0.001), respectively. We also examined the effect ofH2O2 on the phosphorylation and expression of ERK in M-1 cells.Figure 2B is a Western blot showing that treatment of M-1 cellswith 200 µM H2O2 for 60, 90, and 120 min increased phosphorylationof ERK by 30 ± 15, 70 ± 20 (P < 0.01; n = 5),and 110 ± 30% (P < 0.001), respectively. Also, treatmentof M-1 cells with H2O2 did not significantly stimulate the phosphorylationof JNK in M-1 cells. Therefore, the effect of H2O2 on P38 andERK in M-1 cells was similar to those observed in tissue, suggestingthat superoxide or related products may be responsible for mediatingthe effect of low K intake on P38 and ERK in the kidney.
Figure 2. Effect of H2O2 on the phosphorylation of P38 MAPK (A), ERK (B), and JNK (C) in mouse collecting duct (M-1) cells. (Top and middle) Phosphorylated MAPK and the total MAPK, respectively. Data are summarized as a bar graph at the bottom of each panel. *P < 0.01, control versus experimental group.
The notion that superoxide anions and related products may beresponsible for the effect of low K on MAPK phosphorylationis established further by experiments in which the effect oflow K intake for 7 d on MAPK phosphorylation was examined inrats that were treated with tempol for 7 d. We previously showedthat tempol treatment reduced the level of superoxide anionsthat are induced by low K intake (6). From inspection of Figure 1,it is apparent that tempol treatment abolished the effect oflow K intake on the phosphorylation of P38 and ERK. The effectof tempol must be related specifically to the reduction of superoxidebecause tempol treatment did not significantly change the phosphorylationof P38 and ERK in rats that were on a normal K diet, which hasa low superoxide level. Also, tempol treatment did not significantlyaffect the phosphorylation level of JNK. This suggests thatlow K intake–induced superoxide did not regulate the phosphorylationof JNK in renal cortex and outer medulla in our experimentalsettings. Therefore, our study strongly suggests that low Kintake–induced increase in superoxide anions is responsiblefor the stimulation of phosphorylation of P38 and ERK duringK restriction.
We then explored the physiologic role of P38 and ERK in mediatingthe effect of low K on the expression of c-Src, which servesas a representative member of Src family PTK. We previouslyshowed that low K intake stimulates the expression of c-Src(6,9) and that the effect of low K intake on c-Src expressionis mediated by superoxide anions. Therefore, we used M-1 cellsto examine whether inhibiting MAPK could abolish the effectof H2O2 on c-Src expression. Figure 3 is a Western blot demonstratingthe effect of H2O2 on c-Src expression in the absence (control)and presence of SB202190 (5 µM), PD098059 (50 µM),SP600125 (10 µM), SB202190+PD098059, or three inhibitors.We confirmed the previous report that H2O2 increased the expressionof c-Src by 200 ± 20% (P < 0.001; n = 5). However,inhibition of ERK with PD098059 (18) or JNK with SP600125 (19)or P38 with SB202190 (20) only attenuated the effect of H2O2on c-Src expression and still increased the expression of c-Srcby 80 ± 25 (P < 0.01; n = 5), 90 ± 20 (P <0.01; n = 5), and 60 ± 10% (P < 0.01; n = 5), respectively.When P38 and ERK MAPK were inhibited simultaneously, the effectof H2O2 on the expression of c-Src was completely abolished.Also, application of triple inhibitors did not cause furtherinhibition of the effect of H2O2 on c-Src expression. In consideringthat low K intake did not significantly increase the JNK phosphorylationin the rat kidney and that H2O2 did not affect the phosphorylationof JNK in M-1 cells, it is possible that the suppressing effectof SP600125 may be the result of inhibiting MAPK other thanJNK, such as P38 or ERK. This view is also supported by theWestern blot analysis that is shown in Figure 3B, in which applicationof SP+PD had a similar effect of SB+PD, whereas SP+SB did notcompletely abolish the effect of H2O2 on c-Src expression inM-1 cells (n = 3). Thus, P38 and ERK MAPK are involved mainlyin mediating the effect of low K or superoxide on the expressionof c-Src. However, considering the possible cross-action ofPD098059 or SB202190, we could not completely exclude the roleof JNK in mediating the effect of H2O2 on c-Src expression.
Figure 3. (A) Effect of H2O2 on the expression of c-Src in M-1 cells in the absence of MAPK inhibitors and in the presence of PD098059 (50 µM), SP600125 (10 µM), SB202190 (5 µM), PD+SB, or three inhibitors. Data are summarized and presented as bar graph at the bottom of the figure. **P < 0.001 and *P < 0.01, control versus experimental group. IP, immunoprecipitation; IB, immunoblot. (B) A Western blot showing the effect of H2O2 on the expression of c-Src in M-1 cells in the presence of SP600125+SB202190, SP600125+PD098059, and SB202190+PD098059.
Because PTK has been shown to phosphorylate ROMK channels andfacilitate the internalization (6,21), activation of P38 andERK was expected to inhibit the ROMK-like SK channels via aPTK-dependent pathway. Therefore, it was conceivable that P38and ERK are two molecules of the PTK-dependent pathway, whichregulates the SK channels in the CCD. Moreover, MAPK have beenshown to regulate ion channels by a PTK-independent pathway(17,22,23). Therefore, we carried out the patch-clamp experimentsto determine whether inhibition of P38, ERK, or JNK could increasethe SK channel activity in the CCD. Inhibition of ERK (n = 12)or blocking JNK (n = 8) had no significant effect on the SKchannels (control 1.5 ± 0.2; 50 µM PD098059 1.5± 0.2; 10 µM SP600125 1.55 ± 0.2). Also,suppression of P38 with 5 µM SB202190 slightly but insignificantlyincreased SK channel activity from 1.4 ± 0.2 to 1.6 ±0.2 (n = 12), but when both ERK and P38 were inhibited simultaneouslyby PD098059 and SB202190, the SK channel activity increasedsignificantly. Figure 4 is a channel recording showing thatapplication of SB202190+PD098059 stimulates the activity ofSK channels and increased NPo from 0 to close to 2.8 in thisparticular patch of the CCD from rats that were on a controlK diet. Data that are summarized in Figure 5 show that applicationof PD098059+SB202190 increased SK channel activity from 1.3± 0.3 to 2.4 ± 0.3 (n = 10; P < 0.01) in theCCD from rats that were on a control K diet. From inspectionof Figure 4, it also is apparent that the stimulatory effectof SB202190+PD98059 on the SK channels occurs within 5 min.In 10 experiments, the onset time of the effect of MAPK inhibitorson SK channels took place in <10 min. In contrast, the stimulatoryeffect of inhibiting PTK on the SK channels was observed within20 to 30 min (7). Therefore, it is unlikely that the effectof inhibiting MAPK on the SK channels was the result of suppressionof PTK. This speculation also was supported by experiments inwhich the effect of SB202190+PD098059 on SK channels was examinedin the presence of herbimycin A to block PTK. From inspectionof Figure 6, it is apparent that inhibition of MAPK still canstimulate the SK channel activity after inhibition of PTK becauseNPo increased from 0 to 1.9 in this patch. Figure 5 summarizeddata showing that inhibiting MAPK increased channel activityfrom 1.5 ± 0.6 to 2.9 ± 0.3 (n = 5; P < 0.05).This suggests that the inhibitory effect of P38 and ERK on SKchannels did not require the involvement of PTK. To determinethe mechanism by which inhibiting MAPK stimulates the SK channelactivity, we examined the effect of PD098059+SB202190 on SKchannels in the CCD that were treated with colchicine. We previouslyshowed that colchicine treatment blocked the effect of inhibitingPTK on SK channels (24), suggesting the role of microtubulein mediating the insertion of SK channels induced by inhibitionof PTK. In 10 experiments, inhibition of microtubule completelyabolished the effect of PD098059 and SB202190 on ROMK channelactivity (control, NPo = 1.1 ± 0.2; PD+SB, NPo = 1.2± 0.25). This suggests that microtubule is required forthe effect of MAPK on SK channels in the CCD.
Figure 4. A channel recording showing that simultaneous inhibition of ERK with PD098059 (PD) and P38 MAPK with SB202190 (SB) activates the small-conductance K (SK) channels in the cortical collecting duct (CCD) from a rat that was on a normal K diet. The experiments were performed in cell-attached patches, and the holding potential was 30 mV. The top trace is the time course of the experiment, whereas three parts of trace, indicated by numbers, are extended to show the fast time resolution. The channel close level is indicated by "C." The arrow indicates the addition of MAPK inhibitors that were present throughout the experiments. Also, the electric noise after addition of PD+SB was artifact.
Figure 5. Effect of PD098059+SB202190 on the SK channels in the presence and absence of herbimycin A in the CCD from rats that were on a normal K diet. The experiments were performed in cell-attached patches. Herbimycin A (1 µM) was used to treated the CCD for 15 min and present throughout the experiment. *P < 0.05 and **P < 0.01 versus control.
Figure 6. A channel recording showing that simultaneous inhibition of ERK and P38 MAPK activates the SK channels in the presence of herbimycin A. The experiments were performed in cell-attached patches, and the holding potential was 30 mV. The top trace is the time course of the experiment, whereas three parts of trace, indicated by numbers, are extended to show the fast time resolution. The channel close level is indicated by "C," and the gap of the top trace is 180 s. The arrow indicates the addition of MAPK inhibitors, which were present throughout the experiments.
Because low K intake–induced increase in the expressionof Src family PTK was not observed until 2 to 3 d after K restriction(9), we speculated that activation of MAPK may serve as an earlysignal molecule during K depletion and that it suppresses apicalSK channels and renal K excretion. This notion is supportedby experiments in which superoxide levels and phosphorylationof ERK and P38 were examined in renal cortex and outer medullafrom rats that were on a KD diet for 24 h. Results that aresummarized in Figure 7A demonstrate that superoxide levels weresignificantly higher (190 ± 30% of the control valuein the kidney from rats that were on a KD diet for 1 d thanthose that were on a control K diet). Moreover, K restrictionfor 1 d increased the phosphorylation of P38 and ERK by 100± 10% (n = 3) but had no significant effect on the expressionof c-Src (Figure 7B). We also examined the effect of P38 andERK on the SK channel activity (Figure 7C) in the CCD and usedthe metabolic cage to examine the urinary K excretion in ratsthat were on a KD diet for 1 d. Low K intake for 1 d reducedchannel activity to 0.7 ± 0.1 (n = 18), and inhibitionof ERK and P38 with PD098059 and SB202190 significantly increasedchannel activity to 2.1 ± 0.3 (n = 11). Therefore, theeffect of inhibiting ERK and P38 on the SK channels was larger(200% increase) in the CCD from rats that were on a KD dietfor 1 d than that from rats that were on control K diet (changesin NPo from 1.3 to 2.4, an 85% increase). Also, metabolic cagestudy shows that urinary K secretion decreased from 3.5 ±0.3 to 0.41 ± 0.02 mEq/L (n = 6) when rats were maintainedon a KD diet for 24 h (data not shown).
Figure 7. (A) A bar graph shows the effect of K restriction for 1 d on superoxide levels (n = 3). *Significant difference. (B) Western blots demonstrate the effect of 24-h K restriction on the phosphorylation of P38 and ERK and the expression of c-Src. (C) Effect of K restriction for 24 h on the SK channel activity in the presence or absence of inhibitors of MAPK (PD098059+ SB202190). *Significant different between control and K-deficient (KD) group; #significant difference between KD and KD+MAPK inhibitor groups. The experimental number is indicated under each bar.
The main findings of our study are that low K intake activatesthe ERK and P38 and that the MAPK regulate ROMK channel activityin the CCD. Our data suggest that the SK channel activity issuppressed by P38 and ERK under physiologic conditions. Therefore,inhibition of MAPK relieves the inhibition of SK channels thatis induced by P38 and ERK. Three lines of evidence indicatethat superoxide is a mediator for the effect of low K intakeon the expression of ERK and P38: (1) Low K intake increasedthe superoxide production in the renal cortex and outer medulla,(2) treatment of M-1 cells with H2O2 mimicked the effect oflow K and increased the expression of ERK and P38, and (3) suppressionof superoxide production with tempol abolished the effect oflow K on the phosphorylation of MAPK. We previously showed thatlow K intake stimulates the expression of Src-family PTK (6,9),which in turn increases the tyrosine phosphorylation of ROMKchannels and facilitates the internalization (6,8,25,26). Wealso showed that superoxide anion and the related products mediatethe effect of low K intake on PTK activity, because decreasingsuperoxide levels abolished the effect of low K intake on PTKexpression (6). Now we have demonstrated that superoxide anionsand related products also mediate the effect of low K intakeon ERK and P38.
Superoxide and the related products initially were identifiedto be involved in the regulation of immunoresponse and celldeath. However, a large body of evidence supports the notionthat superoxide anions play an important role in mediating avariety of cell functions (27). Superoxide anions have beendemonstrated to modulate the activity of various protein kinasesand phosphatases (12,28–32). H2O2 has been shown to inhibitprotein tyrosine phosphatase (30,33) and activate several membersof Src family PTK, such as Lck and Fyn (31,32). Superoxide anionshave been shown to mediate the effect of nerve growth factorin neuronal cells (34) and EGF in human epidermoid carcinomacells (35). Moreover, elimination of H2O2 by catalase has beendemonstrated to inhibit EGF and nerve growth factor receptors.Stimulation of insulin receptors has been shown to augment theformation of superoxide (36), and low concentrations of H2O2can potentiate the insulin effect in insulin-responsive tissues(37). Furthermore, high concentrations of H2O2 can induce insulin-likeeffects in the absence of insulin via stimulation of the insulin-independenttyrosine phosphorylation of the insulin receptor (38). H2O2mediates the stimulatory effect of angiotensin II on nitricoxide production in endothelial cells (39). Also, H2O2 stimulatescGMP generation and causes the transient relaxation of calfcoronary arteries (40).
Numerous experiments have demonstrated that superoxide and therelated products play an important role in the regulation ofMAPK (11,12,14,28). The activity of MAPK is regulated by phosphorylationor dephosphorylation on serine or tyrosine residues (13,14).H2O2 has been shown to increase the serine phosphorylation ofJNK, P38, and ERK1/2 (13). In our study, low K intake stimulatessignificantly the phosphorylation of P38 and ERK but does notsignificantly alter the phosphorylation of JNK in the renalcortex and outer medulla. Although we cannot completely excludethe role of JNK in mediating the effect of low K intake on PTKactivity and SK channel, the observation that inhibition ofP38 and ERK abolished the effect of H2O2 on c-Src expressionin M-1 cells suggests that P38 and ERK are responsible mainlyfor mediating the effect of low K intake. Although inhibitionof JNK attenuated the H2O2-induced increases in c-Src expression,the effect may be the results of inhibition of ERK or P38. Thisnotion is supported by two lines of evidence: (1) H2O2 did notsignificantly alter the phosphorylation of JNK in M-1 cells,and (2) inhibition of JNK did not have additive effect in comparisonwith that with inhibitors of P38 and ERK. Therefore, it is likelythat ERK and P38 are two molecules that are involved in mediatingthe effect of low K on the expression of c-Src. We propose thatlow K intake increases superoxide anions and the related productsthat stimulate the phosphorylation of ERK and P38. Activationof MAPK increased the activity of some transcription factorsand, accordingly, the expression of Src family PTK. Indeed,it has been reported that H2O2-induced activation of MAPK playsa key role in the stimulation of c-Jun activity (41).
In addition to stimulation of PTK expression by which MAPK areinvolved in regulating SK channels, MAPK regulate the SK channelsin the CCD by a PTK-independent mechanism. This view is stronglysupported by the finding that inhibition of MAPK stimulatedSK channels in the presence of the inhibitor of PTK. The MAPK-inducedinhibition of the SK channel activity should have a significanteffect in response to K restriction. We have observed that Ksecretion decreases by 90% 1 d after maintaining rats on a KDdiet, whereas PTK expression is not significantly altered. Therefore,it is possible that a PTK-independent mechanism may be responsiblefor inhibiting the SK channels and decreasing K secretion inresponse to low K intake before PTK is fully effective. MAPK-inducedinhibition could serve as a PTK-independent mechanism by whichthe SK channels are blocked in response to K restriction. Thisnotion is supported by two lines of evidence: (1) K restrictionfor 1 d significantly increased superoxide levels and MAPK phosphorylationin renal cortex and outer medulla, and (2) inhibition of P38and ERK significantly increased the SK channel activity in theCCD from rats that were on a KD diet for 1 d whereas the effectof inhibiting PTK on the SK channels was still modest (9). Figure 8is a cell model illustrating the role of MAPK in the regulationof ROMK channel activity. We speculate that low K intake increasessuperoxide and the related products after K restriction, whichin turn activates both ERK and P38. Activation of MAPK inhibitsSK channels in the CCD, and this mechanism may contribute partiallyto the suppression of renal K excretion at the initial stageof K restriction. In addition, MAPK are involved in mediatingthe stimulatory effect of low K intake on PTK expression, whichfurther decreases channel activity by the internalization.
Figure 8. A cell model illustrating the role of MAPK in mediating the effect of low K intake on ROMK (Kir 1.1) channel activity in the CCD.
The mechanism by which P38 and ERK inhibit the SK channels isnot clear. It is well established that ERK and P38 also canregulate membrane proteins, including receptors and ion channels,in addition to the traditional role of regulating signal moleculesin the nucleus and cytosol (15–17,22,23). It has beenshown that P38 inhibits epithelial Na channels in alveolar epithelialcells by changing membrane expression or trafficking (16). Theobservation that inhibition of microtubule blocked the stimulatoryeffect of PD098059 and SB202190 on SK channels in the CCD suggeststhat microtubule is required for the effect of MAPK on SK channels.We previously showed that colchicine treatment abolished theROMK channel insertion that was induced by inhibition of PTK(24). The finding that colchicine also abolished the effectof inhibition of MAPK on SK channels suggests that blockingP38- and ERK-induced increase in channel activity may be theresult of altering channel trafficking. We need further experimentsto explore the mechanism by which MAPK inhibits the SK channelsin the CCD.
Low K intake stimulates superoxide levels, which activate ERKand P38. Activation of ERK and P38 inhibits the SK channelsthrough two possibilities: (1) ERK and P38 inhibit the SK channelsby a PTK-independent mechanism; and (2) ERK and P38 are involvedin stimulating the expression of Src-family PTK, which in turnenhances in tyrosine phosphorylation and internalization ofthe SK channels in the CCD. Furthermore, ERK and P38 may playa key role in suppressing SK channels and renal K secretionin the early phase after K restriction.
Acknowledgments
The work was supported by National Institutes of Health grantsDK 47402 and DK 54983.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
E.B. and D.L. contributed equally to this work and should beconsidered as co–first authors.
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Abstract
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