Effect of hydrogen peroxide on ROMK channels in the cortical collecting duct

Abstract

We used the patch-clamp technique to study the effect of H₂O₂ on the apical ROMK-like small-conductance K (SK) channel in the cortical collecting duct (CCD). The addition of H₂O₂ decreased the activity of the SK channels and the inhibitory effect of H₂O₂ was larger in the CCD from rats on a K-deficient diet than that from rats on a normal-K or a high-K diet. However, application of H₂O₂ did not inhibit the SK channels in inside-out patches. This suggests that the H₂O₂-mediated inhibition of SK channels was not due to direct oxidation of the SK channel protein. Because a previous study showed that H₂O₂ stimulated the expression of Src family protein tyrosine kinase (PTK) which inhibited SK channels (3), we explored the role of PTK in mediating the effect of H₂O₂ on SK channels. The application of H₂O₂ stimulated the activity of endogenous PTK in M-1 cells and increased tyrosine phosphorylation of ROMK in HEK293 cells transfected with GFP-ROMK1 and c-Src. However, blockade of PTK only attenuated but did not completely abolish the inhibitory effect of H₂O₂ on SK channels. Since H₂O₂ has also been demonstrated to activate mitogen-activated protein kinase, P38, and ERK (3), we examined the role of P38 and ERK in mediating the effect of H₂O₂ on SK channels. Similar to blockade of PTK, suppression of P38 and ERK did not completely abolish the H₂O₂-induced inhibition of SK channels. However, combined use of ERK, P38, and PTK inhibitors completely abolished the effect of H₂O₂ on SK channels. Also, treatment of the CCDs with concanavalin A, an agent which has been shown to inhibit endocytosis (19), abolished the inhibitory effect of H₂O₂. We conclude that addition of H₂O₂ inhibited SK channels by stimulating PTK activity, P38, and ERK in the CCD and that H₂O₂ enhances the internalization of the SK channels.

The cortical collecting duct (CCD) plays an important role in the final regulation of K secretion in the kidney (12, 26). K secretion is regulated by hormones such as vasopressin (1, 2, 8) and aldosterone and dietary K intake (11, 12, 26, 34). A large body of evidence has demonstrated that a low-K intake decreases, whereas a high-K intake increases renal K secretion (3, 4, 13, 24). The effect of K intake on renal K secretion is partially achieved by regulation of the apical K conductance: a low-K intake decreases (3), whereas a high-K intake increases the number of the apical small-conductance K (SK) channels (27, 34). The effect of a low-K intake on SK channels is mediated by increasing superoxide anions and related products such as H₂O₂ because depletion of superoxide anions significantly attenuates the inhibitory effect of low-K intake on SK activity and renal K secretion (4). We previously demonstrated that K restriction for 24 h significantly increased superoxide anion levels in renal cortex and outer medulla (3). The increase in superoxide levels may be important for preventing excessive K loss during K depletion.

Increase in superoxide levels during K restriction stimulates the expression of Src family protein tyrosine kinase (PTK) and activates MAPKs such as P38 and ERK (3, 4). Moreover, it is possible that activation of MAPKs is responsible for the early suppression effect of low-K intake on ROMK channels while increasing PTK activity is responsible for enhancing tyrosine phosphorylation of ROMK channels and endocytosis in the late stage of K restriction. Although the role of superoxide anions in mediating the effect of low-K intake on SK channels is strongly suggested, the direct evidence demonstrating the inhibitory effect of superoxide anions or related products on SK channels is absent. Thus the major goal of the present study is to demonstrate that H₂O₂ can acutely inhibit SK channels and to explore the mechanism by which H₂O₂ inhibits SK channels in the CCD.

METHODS

Preparation of CCDs

Pathogen-free Sprague-Dawley rats of either sex (5 wk) were used in experiments and were purchased from Taconic Farms (Germantown, NY). The animals were put on a high-K (HK; wt/wt, 10%), a normal-K (1.1%), or a K-deficient (KD) diet (<0.0001%; Harlan Teklad, Madison, WI) for 7 days before use. The rats were killed by cervical dislocation and the kidneys were removed immediately and cut into several thin slices (<1 mm) which were placed on an ice-cold Ringer solution until dissection. The dissection was carried out at room temperature and the single CCD was isolated and immobilized by placing the tubule on a 5 × 5-mm coverglass coated with polylysin. The glass-containing CCD was then transferred to a chamber (1,000 µl) mounted on an inverted Nikon microscope. The CCDs were superfused with HEPES-buffered NaCl solution and the temperature of the chamber was maintained at 37 ± 1°C by circulating warm water around the chamber. The CCD was cut open with a sharpened micropipette to expose the apical membrane.

Patch-clamp technique

An Axon200A patch-clamp amplifier was used to record channel current. The current was low-pass filtered at 1 KHz by an eight-pole Bessel filter (902LPF, Frequency Devices, Haverhill, MA) and digitized by an Axon interface (Digitada1200). Data were acquired by an IBM-compatible Pentium computer (Gateway 2000) at a rate of 4 KHz and analyzed using the pClamp software system 6.04 (Axon Instruments, Burlingame, CA). Channel activity was defined as NP_o, which was calculated from data samples of 60-s duration in the steady state as follows

$${\mathit{\text{NP}}}_{\mathrm{o}}={\displaystyle \sum (1t_1+2t_2+\dots {\mathrm{i}t}_{\mathrm{i}})}$$ (1)

where t_i is the fractional open time spent at each of the observed current levels. The effect of H₂O₂ on SK channels occured within 10–15 min at which time we selected a representative 60-s long recording to calculate channel activity.

Transfection of HEK293 cells

HEK293 cells were plated in 35-mm dishes and transfected with 1 µg of GFP-ROMK1 and 1 µg c-Src using 7 µl LT1 reagent (PanVera, Madison, WI) according to the manufacturer’s instructions. The experiments were carried out 2 days after the transfection. The successful rate for the cell transfection was between 60 and 70%. To determine the effect of H₂O₂ on the tyrosine phosphorylation level of ROMK1, PY20 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), which reacts with the tyrosine phosphorylated proteins, was used to detect the phosphorylated ROMK1. Changes in the tyrosine phosphorylated ROMK1 levels were normalized with the corresponding total ROMK1 protein, which was determined with ROMK antibody (Alomone Laboratories, Jerusalem, Israel). The density of the band was determined using Alpha DigiDoc 1000 (Alpha Innotech, San Leandro, CA).

Immunoprecipitation and Western blotting

The GFP antibody was added to the protein samples (500 µg) harvested from cell culture at a ratio of 5 µl/ml of solution. The mixture was gently rotated at 4°C overnight, followed by incubation with 25 µl protein A/G agarose (Santa Cruz Biotechnology) for an additional 2 h at 4°C. The tube containing the mixture was centrifuged at 3,000 rpm and washed twice with PBS containing 10 µl/ml PMSF and 10 µl of protease inhibitor cocktail per ml. The agarose pellet was resuspended in 25 µl 2× SDS sample buffer containing 4% SDS, 100 mM Tris • HCl (pH 6.8), 20% glycerol, 200 mM dithiothreitol, 0.2% bromophenol blue. After the sample was boiled for 5 min, proteins were separated by electrophoresis on 10% SDS-polyacrylamide gels and transferred to Immuno-Blot PVDF membrane (Bio-Rad, Hercules, CA). The membrane was blocked with 5% nonfat dry milk in Tris-buffered saline (TBS) and incubated overnight with the primary antibody at 4°C. The membrane was washed 3 × 15 min with TBS containing 0.05% Tween 20 followed by incubation for 30 min with respective second antibody horseradish peroxidase conjugate.

Cell cultures and measurement of PTK

Cultured M-1 cells were used to measure the activity of PTK and incubated in the presence or absence of H₂O₂ (200 µM). Cells were lysed in 100 µl of RIPA buffer [150 mM NaCl, 50 mM Tris • HCl (pH 7.4), 50 mM β-glycerophosphate, 50 mM NaF, 1 mM sodium orthovanadate, 2.5 mM EDTA, 5 mM EGTA, 0.5 mM dithiothreitol, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, 5 µg/ml aprotinin, 5 µg/ml leupeptin, and 2 µg/ml pepstatin]. For activity assay of PTK, 10 µl of the sample were incubated in a total volume of 30 µl containing 200 µM [³²P]ATP (1 cpm/fmol), 12 mM magnesium acetate, 2 mM MnCl₂, 0.3 mM dithiothreitol, 0.5 mM sodium orthovanadate, 0.5 mM ammonium molybdate, and 2 mM R-R-Src peptide (Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Gly). Reactions were quenched by adding 200 ml of 75 mM phosphoric acid, and 15 µl of cocktail were spotted on phosphocellulose paper. Following several washes, the amount of ³²P incorporated into R-R-Src peptide was assessed using a liquid scintillation counter. The increase in PTK activity was determined by comparing the radioactivity of the peptide mixed with the lysate from H₂O₂-treated cells to those from untreated cells at each time point.

Experimental solutions and statistics

The pipette solution contained (in mM) 140 KCl, 1.8 MgCl₂, and 10 HEPES (pH 7.4). The bath solution was composed (in mM) of 140 NaCl, 5 KCl, 1.8 CaCl₂, 1.8 MgCl₂, 5 glucose, and 10 HEPES (pH 7.4). For inside-out patches, 100 µM MgATP and 2 mM DTT were added to the bath solution described above. Herbimycin A, SB202190, and PD098059 were purchased from Biomol (Plymouth Meeting, PA) and dissolved in the DMSO solution. The final concentration of DMSO was less than 0.1% and had no effect on channel activity. Data are shown as means ± SE, and on paired or unpaired Student’s t-test was used to determine the significance between the two groups. Statistical significance was taken as P < 0.05.

RESULTS

Figure 1A is a typical recording demonstrating the effect of 200 µM H₂O₂ on the activity of the SK channel in a cell-attached patch in the CCD from rats on a normal-K diet. It is apparent that addition of H₂O₂ inhibits the SK channels and decreases NP_o from 3.6 to 0. Data summarized in Fig. 1B demonstrate that addition of 200 µM H₂O₂ decreased channel activity from 3.6 ± 0.4 to 1.1 ± 0.3 (n = 9) in the CCD from rats on a normal-K diet. The channel activity in the present study was higher than that we reported previously (37) because we only calculated NP_o from those patches with channel activity and did not include many patches without channel activity. The effect of H₂O₂ on the SK channel is also regulated by dietary K intake: a low-K intake enhances, whereas a high-K intake reduces the effect H₂O₂. Figure 1B is a dose-response curve of H₂O₂ effect on the SK channels obtained from rats on different K diet. The application of 100 µM H₂O₂ decreased NP_o by 80% from 2.8 ± 0.3 to 0.6 ± 0.1 (n = 7) in the CCD from rats on a KD diet. In contrast, addition of 100 µM H₂O₂ inhibited channel activity only by 45% from 3.6 ± 0.3 to 2.0 ± 0.3 in the CCD from rats on a control K diet (n = 9) and 12% from 4.8 ± 0.3 to 4.2 ± 0.3 on a HK diet (n = 4). Again, the channel activity in the CCD from rats on KD diet was higher than the value reported previously (4) because these patches were selected to have a comparable channel activity as that in control animals. The effect of H₂O₂ on SK channels was not due to oxidation of SK channels because addition of 2 mM DTT, a reducing agent, did not abolish the H₂O₂-induced inhibition (Fig. 1C). Also, the inhibitory effect of H₂O₂ on the SK channels was observed only in cell-attached patches but not in inside-out patches.

Fig. 1.

A: channel recording showing the effect of 200 µM H₂O₂ on the small-conductance K (SK) channel activity in the cortical collecting duct (CCD). The experiment was performed in a cell-attached patch and the holding potential was 0 mV. Top trace: time course of the experiment and 2 parts of the trace indicated by numbers were displayed with fast time resolution. The channel closed level is indicated by “C.” B: dose-response curve of the effect of H₂O₂ on the SK channel activity in the CCD from rats on a K-deficient (KD; triangle), a normal-K (1%; ○), and a high-K diet (●). The experiments were performed in cell-attached patches and the experimental number ranges from 4 to 9. C: effect of H₂O₂ on SK channels in the presence (n = 4) or absence of 2 mM DTT (n = 9). *Significant difference (Student’s t-test) between control and experimental groups (**P < 0.01; *P < 0.05).

Figure 2 is a recording showing the effect of H₂O₂ on the SK channels in an inside-out patch in the presence of 2 mM DTT and 100 µM MgATP which is important to prevent channel run-down by a mechanism involving either PKA or PIP2 (16, 36). Clearly, addition of 200 µM H₂O₂ did not inhibit the channel activity and channel activity was 95 ± 4% of the control value (control 3.4 ± 0.7; H₂O₂ 3.2 ± 0.7; n = 5). This suggests that the effect of H₂O₂ on channel activity is not the result of oxidation of the SK channel protein.

Fig. 2.

Channel recording showing that 200 µM H₂O₂ failed to block the SK channel activity in excised patches. Top trace: time course of the experiment and 2 parts of the trace indicated by numbers were displayed with fast time resolution. The channel closed level is indicated by “C” and the holding potential was −40 mV (hyperpolarization).

After demonstrating that addition of H₂O₂ inhibited SK channels, we explored the mechanism by which H₂O₂ blocks SK channels. Although a previous study showed that H₂O₂ stimulated the expression of c-Src (3) which inhibits the SK channel activity (37), it is unlikely that this mechanism plays a role in mediating the effect of H₂O₂ on SK channels because the effect of H₂O₂ occurred within 15 min. However, it has been reported that H₂O₂ stimulates the activity of PTK (6, 25). Therefore, we tested whether H₂O₂ stimulates the activity of PTK which, in turn, increases the tyrosine phosphorylation of ROMK. We used specific peptide as PTK substrate and measured the radiolabeled ATP incorporation as an index of PTK activity. Results summarized in Fig. 3 demonstrate that treatment of M-1 cells with 200 µM H₂O₂ significantly increased the ³²P incorporation into peptide in 60 and 120 min. This suggests that H₂O₂ treatment stimulates the PTK activity or suppresses the activity of protein tyrosine phosphatase. We next examined the effect of H₂O₂ on tyrosine phosphorylation of ROMK channels using HEK293 cells transfected with GFP-ROMK1 and c-Src. Two days after transfection, cells were treated with 200 µM H₂O₂-containing media for 2 h, harvested, and homogenated. ROMK1 proteins were collected with GFP antibody and the phosphorylated ROMK1 were detected with PY20, an antibody which recognizes the tyrosine phosphorylated protein. Figure 4A is a Western blot showing the effect of H₂O₂ on tyrosine phosphorylation of ROMK1. The addition of H₂O₂ significantly increases the level of tyrosine phosphorylation of ROMK1 by 120 ± 15% (n = 3; Fig. 4B) but did not affect the expression of ROMK1. Thus the present results strongly suggest that treatment of the CCD with H₂O₂ may stimulate the PTK activity and increase the phosphorylation of SK channels.

Fig. 3.

Time course of the effect of H₂O₂ on the ³²P incorporation to the protein tyrosine kinase (PTK) substrates. The increase in PTK activity was determined by comparing the radioactivity of the peptide mixed with the lysate from H₂O₂-treated cells to those from untreated cells (control) at each time point (n = 4). *Significant difference between the corresponding control and experimental groups (P < 0.05, Student’s t-test).

Fig. 4.

A: effect of H₂O₂ on the tyrosine phosphorylation of ROMK channels. ROMK channels were harvested through immunoprecipitation and tyrosine phosphorylated ROMK was detected with tyrosine phosphorylation antibody. The GFP-ROMK and c-Src were expressed in HEK 293 cells which were treated with 200 µM H₂O₂. B: bar graph summarizes the effect of H₂O₂ on ROMK1 tyrosine phosphorylation (n = 3). The changes in the phosphorylation levels were normalized by total ROMK expression. *Significant difference determined by Student’s t-test.

To determine the role of PTK in mediating the effect of H₂O₂ on SK channels, we examined the effect of H₂O₂ on SK channels in the presence of herbimycin A, an inhibitor of PTK. The CCD of rats on a KD diet was incubated in herbimycin A-containing bath for 30 min followed by addition of 100 µM H₂O₂ in the continuous presence of PTK inhibitor. Figure 5A shows the data calculated from 10 experiments demonstrating that inhibition of PTK significantly attenuated the effect of H₂O₂ on SK channels and that addition of 100 µM H₂O₂ decreased channel activity by 53 ± 3% from 3.4 ± 0.4 to 1.6 ± 0.3 (P < 0.05), whereas the same concentration of H₂O₂ decreased channel activity by almost 80% (see Fig. 1B). However, inhibition of PTK failed to completely block the H₂O₂-induced inhibition of channel activity. This suggests that signalings other than PTK are also involved in mediating the effect of H₂O₂ on SK channels.

Fig. 5.

A: effect of H₂O₂ on the SK channel activity in the presence of herbimycin A (1 µM) alone or PD98059 (PD; 50 µM) and SB202190 (SB; 5 µM) or triple inhibitors. The experiments were performed in cell-attached patches and CCDs were treated with herbimycin A (n = 10), SB + PD (n = 4), or SB + PD + herbimycin A (n = 6) before adding H₂O₂. *Significant difference between experimental and corresponding controls (Student’s t-test). B: channel recording showing the effect of 200 µM H₂O₂ on the SK channel activity in the CCD. The experiment was carried out in a cell-attached patch. Top trace: time course and 2 parts of the trace indicated by numbers were displayed with fast time resolution. The channel closed level is indicated by “C” and the holding potential was 0 mV.

In addition to stimulation of PTK, H₂O₂ has also been shown to activate P38 and ERK (3), which inhibit ROMK channel activity. Thus we examined whether the effect of H₂O₂ on SK channels is mediated also by stimulation of P38 and ERK. Similar to inhibition of PTK, blocking P38 and ERK significantly diminished but did not completely abolish the inhibitory effect of H₂O₂ (Fig. 5A). In the presence of 5 µM SB202190 (P38 inhibitor) and 50 µM PD098059 (ERK inhibitor), H₂O₂ decreased NP_o by 45 ± 4% from 1.7 ± 0.2 to 0.93 ± 0.1 (n = 4, P < 0.05). We then examined the effect of H₂O₂ on SK in the presence of triple inhibitors to block ERK, P38, and PTK. Figure 5B is a representative channel recording from six such experiments in which the effect of H₂O₂ on SK channels was tested in the presence of 5 µM SB202190, 50 µM PD098059, and 1 µM herbimycin A. It is apparent that inhibition of P38, ERK, and PTK completely abolished the effect of 100 µM H₂O₂ on SK (control 2.7 ± 0.4; H₂O₂ 2.6 ± 0.4; n = 6; Fig. 5A). This indicates that the effect of H₂O₂ on SK channels is the result of activation of PTK and MAPKs.

Since stimulation of PTK has been shown to enhance internalization (32) and activation of MAPKs may also facilitate the endocytosis (3), we tested whether the H₂O₂-induced inhibition of SK channels was also the result of enhancing internalization. Thus we examined the effect of H₂O₂ on channel activity in the presence of concanavalin A, an agent which has been shown to block the internalization of membrane receptor and channels (23). After the tubules were treated with 250 µg/ml concanavalin A for 30 min, the effect of H₂O₂ on channel activity was tested in the presence of concanavalin A. Figure 6 summarizes results demonstrating the effect of H₂O₂ on the SK channels in the CCD treated with concanavalin A. The addition of 200 µM H₂O₂ did not inhibit the SK channels in the presence of concanavalin A. Thus it is likely that the effect of H₂O₂ on the SK channel is the result of stimulation of endocytosis.

Fig. 6.

Effect of H₂O₂ on SK channels in the CCD treated with concanavalin A (250 µg/ml). The experiments were performed in cell-attached patches and the CCDs were treated with concanavalin A before adding H₂O₂ (n = 5). *Significant difference between experimental group and the corresponding control (Student’s t-test).

DISCUSSION

The main findings of the present study are that addition of H₂O₂ inhibited SK channels and that the inhibitory effect of H₂O₂ was abolished by blocking PTK, P38, and ERK. We previously demonstrated that superoxide anions and related products are involved in mediating the effect of low-K intake on SK channels because suppression of superoxide anion levels attenuated the inhibitory effect of low-K intake on SK channels and increased channel activity (4). We further showed that treatment of M-1 cells with H₂O₂ mimics the effect of low-K intake on c-Src expression and the phosphorylation of ERK and P38 (3). Now, we demonstrated that acute application of H₂O₂ inhibits ROMK-like SK channels in the CCD. Thus these results strongly suggest that superoxide anions are involved in mediating the effect of low-K intake on ROMK channels. Although concentrations of H₂O₂ used in the present experiments were in the range reported in the literature (20, 22), they may be higher than those under physiological conditions. However, the permeability of the cell membrane to H₂O₂ is presumably low. Thus the real concentration of intracellular H₂O₂ should be significantly lower than that in media and may be in a similar range as occurred in living renal tubule cells.

There are two possibilities by which superoxide or its related products could regulate the SK channels: through direct oxidation or via modulation of cell signaling. Superoxide has been shown to interact with NO to form highly active oxidants such as peroxynitrite, which inhibits the basolateral K channels in the CCD (18). However, the first possibility was largely excluded by observation that H₂O₂ failed to inhibit SK channels in inside-out patches. Thus it is most likely that the inhibitory effect of H₂O₂ on SK channels is mediated by modulation of cell signaling. Three lines of evidence suggest that PTK is involved in mediating the effect of H₂O₂ on SK channels: 1) addition of H₂O₂ has been shown to stimulate the expression of Src family PTK (4); 2) H₂O₂ increased the activity of PTK and tyrosine phosphorylation of ROMK channels; and 3) inhibition of PTK partially attenuated the H₂O₂-induced inhibition of SK channels. However, the observation that inhibition of PTK did not completely abolish the effect of H₂O₂ on ROMK channels suggests that signaling other than PTK is also involved in mediating the inhibitory effect of H₂O₂ on SK channels.

Two lines of evidence suggest that activation of P38 and ERK is also responsible for mediating the effect of H₂O₂ on SK channels: 1) H₂O₂ increased the phosphorylation of ERK and P38 (3); and 2) the inhibitory effect of H₂O₂ on SK channels was partially blocked by suppressing P38 and ERK. Numerous experiments demonstrated that ${\mathrm{O}}_2^{-}$ and H₂O₂ play an important role in the regulation of MAPKs (9, 14, 17, 28). The MAPK family is mainly composed of three subfamilies with multiple members: ERK (28), JNK (17), and the P38 MAPK (29). The activity of MAPKs is regulated by phosphorylation or dephosphorylation on serine or tyrosine residues (9, 31). H₂O₂ has been shown to increase the serine phosphorylation of JNK, p38 MAPK, and ERK1/2 (31). However, our previous observation that low-K intake stimulated the phosphorylation of ERK and P38 but did not significantly affect the phosphorylation of JNK suggested that P38 and ERK were two major MAPKs responsible for mediating the effect of low-K intake on SK channels. The mechanism by which P38 and ERK inhibit SK channels is not clear. We previously showed that inhibition of P38 and ERK increased the SK channel activity and that the effect of inhibiting MAPKs was absent in the presence of colchicines, an agent which inhibits the microtubule assembling (3). Thus it is possible that stimulation of P38 and ERK may affect the SK channel expression by altering channel trafficking. This speculation is also supported by the finding that treatment of CCD with concanavalin A abolished the inhibitory effect of H₂O₂ on SK channels. Therefore, our data suggest that the effect of H₂O₂ on SK channels may be the result of stimulation of internalization and that activation of PTK, P38, and ERK is involved in mediating the effect of superoxide and related products on SK channels.

Although superoxide anions have been shown to be responsible for inducing oxidative stress in many cells, accumulated evidence has been merged to suggest that superoxide and its related product such as H₂O₂ are also involved in mediating physiological signaling. H₂O₂ has been shown to inhibit protein tyrosine phosphatase (10, 21) and activate several members of Src family PTK such as Lck and Fyn (6, 25). Superoxide has been shown to mediate the effect of nerve growth factor (NGF) in neuronal cells (33) and epidermal growth factor (EGF) in human epidermoid carcinoma cells (5). Stimulation of NGF and EGF receptors results in a transient increase in superoxide and H₂O₂ concentrations. Moreover, elimination of H₂O₂ by catalase has been demonstrated to inhibit EGF and NGF receptors. Stimulation of the insulin receptor has been reported to augment the formation of superoxide (20), and low concentrations of H₂O₂ can potentiate the insulin effect in insulin-responsive tissues (30). Moreover, high concentrations of H₂O₂ can induce insulin-like effects in the absence of insulin via stimulation of the insulin-independent tyrosine phosphorylation of the insulin receptor (15). H₂O₂ mediates the stimulatory effect of ANG II on NO production in endothelial cells (7). Also, H₂O₂ stimulates cGMP generation and causes the transient relaxation of calf coronary arteries (22).

In the present study, we observed that the inhibitory effect of H₂O₂ on SK channels depends on K intake such that SK channels have the highest sensitivity to H₂O₂ in the CCD from rats on KD diet. Although the mechanism by which K intake affects the effect of H₂O₂ on SK channels is not completely understood, we speculate that this may be related to the highest PTK and MAPK activity in the rats on a low-K diet. We previously showed that low-K intake stimulates the expression of Src family PTK and PKC and increases the phosphorylation of P38 and ERK (3, 4). Thus the SK channels are more sensitive to H₂O₂-induced inhibition in the CCD from rats on a low-K diet than those on a normal- or a high-K intake. We conclude that H₂O₂ inhibits SK channels by activation of PTK, ERK, and P38 and that their activation enhances the internalization of ROMK channels in the CCD.