Peroxynitrite inhibits amiloride-sensitive Na+ currents in Xenopus oocytes expressing alpha beta gamma -rENaC

M. D. DuVall1, S. Zhu1, C. M. Fuller2, and S. Matalon1,2,3

1 Departments of Anesthesiology, 2 Physiology and Biophysics, and 3 Pediatrics, University of Alabama at Birmingham, Birmingham, Alabama 35233

    ABSTRACT
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Abstract
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Materials & Methods
Results
Discussion
References

We examined the effect of peroxynitrite (ONOO-) on the cloned rat epithelial Na+ channel (alpha beta gamma -rENaC) expressed in Xenopus oocytes. 3-Morpholinosydnonimine (SIN-1) was used to concurrently generate nitric oxide (· NO) and superoxide (O-2 ·), which react to form ONOO-, a species known to promote protein nitration and oxidation. Under control conditions, oocytes displayed an amiloride-sensitive whole cell conductance of 7.4 ± 2.8 (SE) µS. When incubated at 18°C with SIN-1 (1 mM) for 2 h (final ONOO- concentration = 10 µM), the amiloride-sensitive conductance was reduced to 0.8 ± 0.5 µS. To evaluate whether the observed inhibition was due to ONOO-, as opposed to · NO, we also exposed oocytes to SIN-1 in the presence of urate (500 µM), a scavenger of ONOO- and superoxide dismutase, which scavenges O-2 ·, converting SIN-1 from an ONOO- to an · NO donor. Under these conditions, conductance values remained at control levels following SIN-1 treatment. Tetranitromethane, an agent that oxidizes sulfhydryl groups at pH 6, also inhibited the amiloride-sensitive conductance. These data suggest that oxidation of critical sulfhydryl groups within rENaC by ONOO- directly inhibits channel activity.

nitric oxide; reactive species; sodium conductance; tetranitromethane; 3-morpholinosydnonimine; oxidation; nitration

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

AMILORIDE-SENSITIVE SODIUM CHANNELS have been identified in the apical membranes of a variety of epithelial tissues and are in an important class of proteins that regulate Na+ transport and fluid homeostasis. These channels are classified into various categories based on their biophysical properties and the extent of their inhibition by amiloride and its structural analogs. One group of these channels [referred to as type Na(5)] have a low single-channel conductance (4-5 pS), high selectivity for Na+ to K+ (PNa/PK > 10), and long mean open and closed times (0.5-5 s) (32). Additionally, these channels have a high affinity for amiloride (Ki < 0.5 µM at high external Na+ concentrations) and display the following structure/inhibition pattern relationship: phenamil, benzamil > amiloride >>> N-ethyl-N-isopropyl amiloride (35). This group includes the so-called classic Na+ channels present in the apical membranes of epithelia with high transepithelial electrical resistance, such as frog skin, toad urinary bladder, mammalian colon, bovine renal papilla, and amphibian A6 cells (32, 35).

A cDNA encoding an amiloride-sensitive Na+ channel, known as alpha -rENaC (for the alpha -subunit of the rat epithelial Na+ channel), was cloned from the colon of salt-deprived rats using functional RNA expression (8, 10). When expressed in Xenopus oocytes, alpha -rENaC displayed the characteristics of Na(5) channels. Subsequently, Canessa et al. (10) identified and cloned two additional subunits of this channel, named beta -rENaC and gamma -rENaC. Coexpression of all three subunits in oocytes generated 100-fold higher amiloride-sensitive currents than alpha -rENaC alone (10).

Because of their location, epithelial tissues are often exposed to reactive oxygen and nitrogen species generated by a variety of intracellular and extracellular sources. In addition, most mammalian cells have the capacity to produce nitric oxide (· NO) from the oxidative deamination of L-arginine by either the Ca2+-sensitive (type I or III) or the Ca2+-insensitive (type II) forms of · NO synthase (14). · NO is an important signal transduction molecule with diverse physiological functions, including vasoregulation, neurotransmission, and immune host defense (29). For example, · NO may modulate the function of various proteins by bonding to transition metal centers. In the case of guanylate cyclase, perhaps the best understood example, this leads to synthesis of cGMP. However, the biological effects of · NO depend on its concentration, the biochemical composition of target molecules, and the presence of other free radicals. Under some circumstances, · NO is known to produce significant tissue injury. This is thought to be due, at least in part, to the rate-limited reaction of · NO with superoxide (O-2 ·) to form the highly reactive product peroxynitrite (ONOO-) (4). This species nitrates phenolics and oxidizes methionine and cysteine residues by one- or two-electron transfer. Both processes have been shown to result in altered protein function (15, 28, 30, 38).

There is thus considerable interest in identifying the contribution of · NO and its reactive intermediates to the initiation and propagation of injury to epithelial cells, including the effects on ion transport. Our previous results indicate that bolus addition of ONOO- decreases amiloride-sensitive 22Na+ uptake across membrane vesicles of colonic epithelial cells and freshly isolated alveolar type II cells (3, 21). However, the mechanisms by which reactive nitrogen intermediates alter Na+ transport have not been elucidated.

Herein we isolated oocytes from Xenopus laevis, injected them with equivalent amounts (8.3 ng) of alpha -, beta -, and gamma -rENaC cRNA, and recorded whole cell currents 36-48 h later, in the presence and absence of amiloride. To quantify the effects of · NO and its by-products on the amiloride-sensitive currents, an index of Na+ channel activity, we incubated oocytes with ONOO- and · NO donors. In addition, these effects were compared with measurements obtained after exposing oocytes to tetranitromethane (TNM), which, like ONOO-, acts as a nitrogen dioxide (· NO2) donor and oxidizes sulfhydryl groups and, depending on the pH value, may also nitrate phenolics (36, 37). Our results indicate that ONOO-, but not · NO, decreased the amiloride-sensitive Na+ current in oocytes expressing alpha beta gamma -rENaC. Moreover, ONOO- most likely does this through oxidation of critical amino acid residues in the rENaC protein.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

RNA synthesis. The pSport plasmid (GIBCO BRL, Gaithersburg, MD), which contained either alpha -, beta -, or gamma -rENaC (a generous gift from Dr. B. Rossier, University of Lausanne, Switzerland), was linearized by overnight incubation with Not I (Promega, Madison, WI). Sense RNA was in vitro transcribed from purified plasmid DNA using T7 polymerase according to the manufacturer's instructions (Ambion, Austin, TX). The integrity of the cRNA was verified by denaturing gel electrophoresis through 1% agarose-formaldehyde gels.

Oocyte expression. Female X. laevis toads (Xenopus Express, Beverly Hills, FL) were maintained in dechlorinated tap water at 18°C and fed beef liver twice weekly. A partial ovarectomy was performed under anesthesia (immersion in ice-cold solution of 0.1% ethyl-m-aminobenzoate methane sulfonate; Sigma, St. Louis, MO) through a small (~5 mm) ventral abdominal incision. The ovarian tissue was then placed in half-strength Lebowitz-15 (1/2L15; GIBCO BRL) medium buffered with 15 mM HEPES, pH 7.6. Oocytes at maturation stages V and VI were manually defolliculated and incubated overnight in fresh oocyte culture media [1/2L15 medium supplemented with penicillin (100 U/ml) and streptomycin (100 µg/ml) at 18°C]. A Nanoject (Drummond, Broomall, PA) microinjector was used to inject 25 ng of total cRNA (8.3 ng of each rENaC subunit) in a 50-nl volume into each oocyte. Control oocytes were injected with 50 nl of KCl (100 mM). Injected oocytes were incubated in oocyte culture media at 18°C until use.

Electrophysiological measurements. Membrane currents were evaluated 36-48 h postinjection using a two-electrode voltage clamp. Oocytes were bathed in modified ND96 solution at 18°C. This solution contained (in mM) 96 NaCl, 2 KCl, 1 MgCl2, 0.2 CaCl2, and 15 HEPES at pH 7.6. Cells were impaled with two 3 M KCl-filled electrodes having resistances of 0.5-1.5 MOmega . The electrodes were connected to a Geneclamp 500 (Axon Instruments, Foster City, CA) current-voltage (I-V) clamp amplifier via Ag-AgCl pellet electrodes and referenced to an Ag-AgCl pellet connected to the bath via a 3 M KCl-agar bridge. After impalement, membrane potentials were allowed to stabilize before current measurements (5-10 min). The voltage clamp was controlled by a Macintosh IIci computer running Axodata (Axon Instruments) acquisition software.

Oocytes were clamped at a holding potential of 0 mV. Currents were recorded every 20 s by stepping from the holding potential to -100 mV for 400 ms, back to the holding potential for 50 ms, then to +100 mV for 400 ms. I-V relationships were determined before drug administration and at timed intervals thereafter by stepping from the holding potential to -100 mV through +60 mV in 20-mV increments for 500 ms. Current values sampled over the last 50 ms of each voltage step were averaged and used to construct I-V curves. Whole cell conductance values were calculated as the slope of the I-V curve over the range from -100 to -60 mV. Amiloride-sensitive I-V relationships were calculated by subtracting the I-V relationship after amiloride application (10 µM) from that obtained before.

Generation of ONOO- and · NO. Stock solutions of 100 mM 3-morpholinosydnonimine (SIN-1; Calbiochem, La Jolla, CA) in 10 mM phosphate buffer (pH 5.5), 10 mM urate (Sigma, St. Louis, MO) in 1/2L15 media, 78 mg/ml (5,200 U/mg) human Cu/Zn superoxide dismutase (SOD; provided to us by Dr. J. Beckman, Dept. of Anesthesiology, University of Alabama at Birmingham), 8.4 M TNM (Aldrich, Milwakee, WI) in ethanol, and 100 mM PAPA-NONOate (Cayman Chemical, Ann Arbor, MI) in 10 mM degassed phosphate buffer (pH 8.5) were stored at -20°C. Working solutions were prepared from stock solutions at the time of each experiment.

SIN-1 was added directly to 1/2L15 media and the oocytes incubated for 2 h before electrophysiological recording. At physiological pH, SIN-1 undergoes a base-catalyzed reaction, leading to the simultaneous release of · NO and O-2 ·, which react rapidly to form ONOO- (13, 22). Because SIN-1 consumes oxygen during its decomposition, the oocyte medium was aerated every 15 min during the incubation period. Exposure of oocytes to SIN-1 was repeated in the presence of urate (500 µM), a nonspecific scavenger of ONOO-, or SOD (3,000 U/ml), which, by enhancing the dismutation of O-2 · to hydrogen peroxide, converts SIN-1 from a ONOO- to a · NO donor. Current measurements were also performed in the presence of PAPA-NONOate, which decomposes by first-order kinetics to produce · NO.

To assess whether intracellular Ca2+ was responsible for the observed effects of SIN-1 on amiloride-sensitive Na+ currents, oocytes were incubated with 50 µM 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM; Calbiochem, La Jolla, CA) for 3 h before treatment with SIN-1 and BAPTA-AM for an additional 2 h. Currents were then measured and compared with timed controls (no BAPTA-AM or SIN-1) and SIN-1-treated (no BAPTA-AM) oocytes. To evaluate whether oxidative or nitrating reactions were involved in the observed SIN-1 effects, oocytes were also treated with TNM (50 µM). For these experiments, oocytes were perfused with solutions at pH 6.0 (ND96 in which MES was substituted for HEPES) or pH 7.6. Depending on the pH, TNM acts as either a nitrating or an oxidizing agent. Little or no nitration is observed at pH 6 (36, 37). TNM was added directly to the bath following equilibration of the basal currents.

Quantitation of ONOO- production. ONOO- generation by SIN-1 (1 mM) was quantified by measuring the rate of rhodamine formation from the oxidation of dihydrorhodamine 123 (DHR) as previously described (16). All measurements were conducted in 1/2L15 media at 25 or 18°C. Rates were also measured at 18°C in the presence of SOD and urate. Samples were taken at either 10- or 15-min (SOD and urate) intervals. Rhodamine formation was monitored in a spectrophotometer at 500 nm for 2 min (epsilon 500nm = 78,000 M-1 · cm-1) in cuvettes containing DHR (50 µM). Because ONOO- oxidizes DHR with an efficiency of 45%, the rate of ONOO- formation was calculated by dividing the rate of rhodamine formation by 0.45.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

SIN-1 forms ONOO- in the oocyte recording solution. The time course of ONOO- formation resulting from the addition of SIN-1 (1 mM) to the 1/2L15 media (pH 7.6) is shown in Fig. 1. At 25°C, ONOO- levels rose gradually to >30 µM by 2 h. In contrast, only ~10 µM ONOO- was produced over the same time course at 18°C, the temperature at which oocytes were exposed to SIN-1 and at which electrophysiological recordings were performed.


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Fig. 1.   Generation of ONOO- after incubation of 3-morpholinosydnonimine (SIN-1; 1 mM) in oocyte 1/2L15 culture media. Data are shown at 25 and 18°C and at 18°C in the presence of urate (500 µM) and superoxide dismutase (SOD; 3,000 U/ml) during a 2-h period. At 18°C, 1 mM SIN-1 released ~10 µM ONOO- over 2 h. Urate and SOD blocked the oxidative effects of ONOO-. Results are means for n = 3.

SIN-1 inhibits amiloride-sensitive whole cell currents in oocytes expressing alpha beta gamma -rENaC. All measurements of I-V relationships across oocytes were obtained after 2-h exposure to SIN-1 at 18°C. A representative experiment illustrating the whole cell currents present in oocytes expressing alpha beta gamma -rENaC in the absence (control) and presence of SIN-1 (1 mM; 2 h) is shown in Fig. 2. The basal whole cell current for control oocytes at -100 mV was -1,257 ± 418 nA (means ± SE; n = 6) and decreased significantly to -437 ± 109 nA after the addition of amiloride (10 µM) to the bathing solution. KCl-injected oocytes did not express amiloride-sensitive currents (data not shown). Oocytes treated with SIN-1 (1 mM) for 2 h had a current at -100 mV of -580 ± 88 nA under basal conditions. Importantly, these currents were not significantly changed following amiloride treatment (-519 ± 98 nA).


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Fig. 2.   Representative current responses at clamping voltages from -100 to 60 mV recorded from Xenopus oocytes expressing alpha -, beta -, and gamma -subunits of rat epithelial Na+ channels (alpha beta gamma -rENaC). Top: responses from control alpha beta gamma -rENaC-expressing oocytes. Bottom: responses from an oocyte treated with SIN-1 (1 mM) for 2 h. Basal responses are shown on left and responses 5 min after amiloride (10 µM) treatment are shown on right. Amiloride-sensitive currents were inhibited by SIN-1 treatment sufficient to release ~10 µM ONOO-.

Control oocytes had mean whole cell conductances of 14.2 ± 4.5 and 5.5 ± 1.3 µS under basal and amiloride-treated conditions, respectively. Additionally, the reversal potential (ERev) in alpha beta gamma -rENaC-expressing oocytes, calculated from the I-V relationship, was significantly depolarized at -7 ± 3 mV (n = 6), consistent with the presence of a constitutively active Na+ conductance. Amiloride treatment shifted the ERev to -22 ± 4 mV, further supporting the presence of a constitutively active Na+-selective conductive pathway. These findings are in agreement with previous studies (10, 25). In contrast, SIN-1-treated oocytes displayed whole cell conductances under basal and amiloride-treated conditions of 6.9 ± 1.3 and 5.2 ± 0.9 µS, respectively. These values were significantly lower than those of control oocytes under basal conditions but were not different from values in control oocytes after amiloride treatment. The ERev of the SIN-1-treated oocytes was -22 ± 1 mV (n = 7), did not change after amiloride treatment (-22 ± 1 mV), and was not different from control values after amiloride treatment (-22 ± 4 mV; see above). The effects of SIN-1 (1 mM) on the amiloride-sensitive I-V relationships in rENaC-expressing oocytes are summarized in Fig. 3. Additionally, SIN-1 (1 mM) did not alter the I-V relationships of oocytes injected with KCl (100 mM; Fig. 4). In those oocytes, the ERev under control conditions and after SIN-1 treatment were -24 ± 2 and -26 ± 1 mV, respectively, and were not different from those of amiloride- or SIN-1-treated oocytes expressing rENaC. These data indicate that oocytes injected with alpha beta gamma -rENaC cRNA express an amiloride-sensitive conductance that was significantly inhibited after treatment with SIN-1 and that SIN-1 did not alter other conductive pathways in the oocytes.


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Fig. 3.   Amiloride-sensitive current-voltage (I-V) relationships from Xenopus oocytes expressing alpha beta gamma -rENaC. bullet , Values from oocytes expressing rENaC but not exposed to SIN-1 (means ± SE; n = 6). open circle , Values from oocytes expressing rENaC and exposed to SIN-1 (1 mM) for 2 h (means ± SE; n = 7). Conductance (slope of I-V relationship) was significantly different from zero in control oocytes (P < 0.01) but not in oocytes exposed to SIN-1.


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Fig. 4.   I-V relationships from either KCl (100 mM)-injected control oocytes (means ± SE; n = 4) or those exposed to SIN-1 (1 mM; means ± SE; n = 4) for 2 h.

The inhibitory effect of SIN-1 is mediated by ONOO-. When SIN-1 was added to 1/2L15 culture media in the presence of urate (500 µM) or SOD (3,000 U/ml), there was a >90% drop in detectable DHR oxidation, indicating a significant decrease in ONOO- production (Fig. 1). As shown in Table 1, similar values for the amiloride-sensitive conductance of alpha beta gamma -rENaC-expressing oocytes were seen in controls (7.4 ± 2.8 µS; n = 6), those treated with SIN-1 in the presence of urate (7.7 ± 2.2 µS; n = 7), and those treated with SIN-1 in the presence of SOD (6.0 ± 1.6 µS; n = 7). However, SIN-1 alone profoundly inhibited the amiloride-sensitive conductance (0.8 ± 0.5 µS; n = 7). We further examined the effect of · NO on the alpha beta gamma -rENaC-expressing oocytes by treating them with 400 µM PAPA-NONOate, which generates >3 µM · NO (18). However, even at this supraphysiological concentration, · NO failed to decrease either the total or the amiloride-sensitive currents (data not shown). These data indicate that the inhibitory effect of SIN-1 on the amiloride-sensitive conductance was due to production of ONOO- and not · NO or hydrogen peroxide.

                              
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Table 1.   Amiloride-sensitive conductances of alpha beta gamma -rENaC-injected oocytes

The inhibitory effects of SIN-1 are not mediated through a Ca2+-dependent pathway. Oocytes expressing alpha beta gamma -rENaC and pretreated with BAPTA-AM (50 µM) for 3 h followed by exposure to SIN-1 (1 mM) and BAPTA-AM for 2 h had amiloride-sensitive conductance values that were not different from those of oocytes exposed to SIN-1 alone [17.7 ± 6.4 µS (n = 4) vs. 21.4 ± 4.1 µS (n = 6)]. In this set of experiments, these values were ~55% lower than the amiloride-sensitive conductance of control oocytes expressing rENaC (47.4 ± 3.0 µS; n = 3; P < 0.05). These data suggest that SIN-1 did not inhibit the amiloride-sensitive conductance through a Ca2+-dependent pathway. This is further supported by the fact that SIN-1 did not activate the endogenous Ca2+-activated Cl- currents present in KCl-injected oocytes (Fig. 4).

TNM inhibits amiloride-sensitive conductances. Because ONOO- may act as an · NO2 donor to modify proteins, we chose to examine the effects of another · NO2 donor, TNM, on amiloride-sensitive whole cell currents in alpha beta gamma -rENaC-expressing oocytes. When alpha beta gamma -rENaC-expressing oocytes were exposed to TNM (50 µM) at pH 6.0, the mean current at -100 mV was inhibited by 54 ± 7% (n = 6; Fig. 5A). This was significantly greater than the 12 ± 4% (n = 4) current inactivation observed in control oocytes over a comparable period. When the oocytes were subsequently treated with amiloride following TNM, the residual current was further inhibited to ~30% of the total basal current. In contrast, when oocytes were first treated with amiloride, the current decreased ~70%, and subsequent application of TNM (50 µM) had no further effect (Fig. 5B).


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Fig. 5.   Representative current traces at -100 mV, depicting effects of tetranitromethane (TNM) on oocytes expressing alpha beta gamma -rENaC. A: TNM (50 µM) added to bathing solution induced a rapid decrease in current. Subsequent treatment with amiloride (Amil; 10 µM) resulted in further current inhibition, such that the basal current was inhibited ~70%. B: when amiloride was added to bath first, current decreased ~70%, and subsequent addition of TNM failed to further affect the current. C: summary of whole cell conductance in oocytes expressing alpha beta gamma -rENaC under basal conditions and after TNM (100 µM) at pH 6.0. Conductance measurements were made before and 5-10 min after addition of TNM to bathing solution. (* P < 0.05 compared with basal conductance values).

Figure 5C summarizes the effect of TNM (50 µM) on oocytes expressing alpha beta gamma -rENaC at pH 6.0. As illustrated, the whole cell conductance decreased significantly from 17.3 ± 5.2 to 4.6 ± 1.8 µS (n = 6) following TNM treatment. At higher pH values, the reactions of TNM with amino acids are more complex and include not only oxidation of sulfhydryl moieties but also nitration of tyrosines (37). Although we examined the effects of TNM at pH 7.6, the mean current responses were not different from untreated alpha beta gamma -rENaC-expressing oocytes (13 ± 22%; n = 4). The basal currents at -100 mV in pH 6.0 and 7.6 media were not different [-2,319 ± 477 nA (n = 6) and -2,598 ± 581 nA (n = 5), respectively], suggesting that the effects of TNM were not due to the effects of pH alone. However, these data do indicate that exposure of oocytes to TNM at a pH that produces sulfhydryl oxidation but not nitration resulted in an inhibition of the amiloride-sensitive conductance. Furthermore, these results support the conclusion that ONOO- acts through an oxidative pathway to inhibit rENaC activity.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In agreement with other studies (1, 9, 25), we show that coinjection of the alpha -, beta ,- and gamma -rENaC subunits in Xenopus oocytes resulted in the expression of a constitutively active amiloride-sensitive conductance. We further show that incubation of alpha beta gamma -rENaC-expressing oocytes with SIN-1 for >= 2 h resulted in a marked decrease in this amiloride-sensitive conductance. Importantly, the I-V relationships of rENaC-expressing oocytes treated with amiloride or exposed to SIN-1 were not different from those of KCl-injected oocytes. The changes in ERev, as well as the decrease in the whole cell conductance, support the conclusion that SIN-1 inhibited rENaC activity, rather than the amiloride sensitivity of the channel expressed in oocytes. This is an important distinction in light of recent work by Ismailov et al. (25), who demonstrated that deletion of amino acid residues 278-283 in the extracellular domain of the alpha -rENaC subunit reduced the amiloride sensitivity of the channel by two orders of magnitude without affecting the conductance of the channel. SIN-1-induced modifications of amino acid residues in this region of the protein could have affected amiloride binding and therefore the amiloride sensitivity. However, the whole cell conductance would be unchanged, and this was not the case in our study.

The decomposition of SIN-1 at physiological pH leads to the simultaneous generation of O-2 · and · NO, which react at diffusion-limited rates to form the highly reactive species ONOO- (20, 22). The following observations implicate ONOO- as the toxic agent involved in the decrease of the amiloride-sensitive Na+ current. First, amiloride-sensitive currents were decreased in the presence of SIN-1, which produces ONOO-, but not by SIN-1 plus SOD or PAPA-NONOate, which produce · NO. Second, the inhibitory effect of SIN-1 was ameliorated by urate, a nonspecific scavenger of ONOO-. Our data further confirm that, in the presence of SOD or urate, SIN-1 failed to oxidize DHR to rhodamine (Fig. 1), an index of ONOO- formation (16). The impermeable nature of SOD further suggests that ONOO- works within the extracellular medium to inhibit rENaC function. Although we have not specifically examined whether O-2 · is responsible for the effect of SIN-1, the fact that · NO reacts with O-2 · at a rate of 6.7 × 109 M-1 · s-1 (22) makes it very unlikely that any free O-2 · will be generated by the spontaneous decomposition of SIN-1.

At higher concentrations, ONOO- has been shown to induce the release of intracellular Ca2+ (31). This could result in an increase in protein kinase C activity, which may inhibit rENaC activity in Xenopus oocytes (2). However, we did not observe a decrease in the inhibitory effect of SIN-1 in the presence of BAPTA-AM. At concentrations of 1-10 µM, BAPTA-AM has been shown to block the effects of intracellular Ca2+ on expressed channel activity in a number of previous studies (34, 39, 40). It therefore seems unlikely that, under our experimental conditions, the effects of ONOO- on rENaC are mediated through an increase in intracellular Ca2+ concentration. As previously mentioned, this conclusion is also supported by the fact that SIN-1 did not activate the endogenous Ca2+-activated Cl- currents present in KCl-injected oocytes (Fig. 4).

Our laboratory has previously demonstrated that ONOO- directly inhibits Na+ transport across alveolar type II cells and membrane vesicles isolated from colonic enterocytes (3, 21). In the former study, the decrease in amiloride-sensitive Na+ uptake coincided with a decrease in cellular metabolism. It was determined, however, that the inhibitory effect of ONOO- in alveolar type II cells was not due to these changes in metabolism, as cell viability and Na+-K+-ATPase activity were unaffected. In both of these former studies, the effects were observed following bolus application of ONOO- (0.1-1 mM) to the experimental media. In the present study, we elected to use SIN-1 to generate ONOO- over a period >= 2 h, which more closely resembles the profile of in vivo ONOO- generation. A novel and most interesting aspect of this study is that significant inhibition of the amiloride-sensitive current was observed at very small concentrations of ONOO-, likely to be encountered in vivo during inflammation. For example, Ischiropoulos et al. (23) reported that 106 rat alveolar macrophages stimulated with phorbol myristate acetate generated 0.1 nmol ONOO-/min. Direct evidence for the reactivity of ONOO- in vivo has been demonstrated by the presence of nitrotyrosine, the stable by-product of the interaction of ONOO- with tyrosine residues, under a number of inflammatory conditions (5, 17, 26).

Nitration, nitrosylation, or oxidation of key amino acid residues may account for the observed responses in the present study. For example, the external loop of alpha -rENaC contains 26 tyrosine, 6 tryptophan, and 14 cysteine residues. In addition, regions at the outer borders of the two transmembrane (TM) domains contain tyrosines (Y) in close proximity to one another (Y134 and Y137 in TM1 and Y482, Y484, and Y485 in TM2), which may form a portion of the outer opening of the channel. Oxidation or nitration of any or all of these amino acids by reactive oxygen-nitrogen species may affect the function of this channel.

We have attempted to determine the nature of ONOO--induced inhibition by comparing it with the effects of a second · NO2 donor, TNM. This compound has been shown to promote nitration at pH > 7.6 but not at pH <=  6.0 (19) and to oxidize sulfhydryl groups over a broad pH range (36, 37). In the present study, TNM significantly inhibited the amiloride-sensitive Na+ conductance, but only at pH 6.0. This finding is concordant with the conclusion that oxidative modification of specific sulfhydryl groups within the rENaC protein account for a decreased activity. All three subunits of the protein have highly conserved cysteine-rich regions within the putative extracellular region of the protein (10) that represent potential targets for oxidative modification. In previous work, ONOO- has been shown to oxidize methionine by a two-electron donation (33). Methionine oxidation by ONOO- was shown to account for the inactivation of alpha 1-antitrypsin by ONOO- (30). It is therefore highly possible that oxidative injury to the rENaC protein will account for the decreased activity observed in the present study. Oxidation of critical sulfhydryl groups in nonselective cation channels was found to alter their biophysical properties through a process that was reversed by dithiothreitol (27). However, the usefulness of dithiothreitol in ameliorating the oxidation of rENaC is questionable. It was shown that, at concentrations as low as 50 µM, dithiothreitol altered the gating of the rENaC channel, an effect attributed to the disruption of coordinated multichannel activity (24).

We believe our data strongly support the conclusion that ONOO-, acting on the extracellular side of the protein, directly inhibits alpha beta gamma -rENaC activity. However, it should be emphasized that we have no direct evidence for modification of rENaC by ONOO- at this time. It is possible that ONOO- modified other proteins, such as actin, which in turn may have altered the function of Na+ channels. Compeau et al. (12) have shown that endotoxin treatment of distal airway cells resulted in the reduction of Na+ channel density, corresponding to a reduction in F-actin. Moreover, the results were dependent on L-arginine, which suggested a role for · NO in the process. Other studies have established the role of the actin in normal Na+ channel activity (6, 7, 11, 12). Although we cannot exclude the potential effects of ONOO- on other proteins, the impermeable nature of SOD and the fact that this enzyme ameliorated the effect of SIN-1 strongly suggest that damage to intracellular regulators of rENaC function is not responsible for the ONOO--induced responses in this study.

In summary, our results indicate that, at concentrations as low as 10 µM, ONOO- decreases amiloride-sensitive currents across Xenopus oocytes expressing alpha beta gamma -rENaC. Moreover, this effect is likely due to the oxidation of specific amino acid residues within the extracellular domain of the channel proteins. Because ONOO- is produced in vivo during acute inflammation (5, 17), these findings point out that reactive nitrogen species released by inflammatory cells may interfere with Na+ and fluid homeostasis by damaging epithelial Na+ channels.

    ACKNOWLEDGEMENTS

We acknowledge the assistance of Dr. Lan Chen and Carpantato Myles.

    FOOTNOTES

This project was supported by National Heart, Lung, and Blood Institute Grants HL-31197 and HL-51173 and by a grant from the Office of Naval Research (N00014-97-1-0309).

M. D. DuVall is a Parker B. Francis Fellow.

Address for reprint requests: S. Matalon, Dept. of Anesthesiology, Univ. of Alabama at Birmingham, 619 South 19th St., Birmingham, AL 35233-6810.

Received 22 October 1997; accepted in final form 12 February 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Awayda, M. S., I. I. Ismailov, B. K. Berdiev, and D. J. Benos. A cloned renal epithelial Na+ channel protein displays stretch activation in planar lipid bilayers. Am. J. Physiol. 268 (Cell Physiol. 37): C1450-C1459, 1995[Abstract/Free Full Text].

2.   Awayda, M. S., I. I. Ismailov, B. K. Berdiev, C. M. Fuller, and D. J. Benos. Protein kinase regulation of a cloned epithelial Na+ channel. J. Gen. Physiol. 108: 49-65, 1996[Abstract].

3.   Bauer, M. L., J. S. Beckman, R. J. Bridges, C. M. Fuller, and S. Matalon. Peroxynitrite inhibits sodium uptake in rat colonic membrane vesicles. Biochim. Biophys. Acta 1104: 87-94, 1992[Medline].

4.   Beckman, J. S., T. W. Beckman, J. Chen, P. A. Marshall, and B. A. Freeman. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc. Natl. Acad. Sci. USA 87: 1620-1624, 1990[Abstract].

5.   Beckman, J. S., Y. Z. Ye, P. G. Anderson, J. Chen, M. A. Accavitti, M. M. Tarpey, and C. R. White. Extensive nitration of protein tyrosines in human atherosclerosis detected by immunohistochemistry. Biol. Chem. Hoppe-Seyler 375: 81-88, 1994[Medline].

6.   Berdiev, B. K., A. G. Prat, H. F. Cantiello, D. A. Ausiello, C. M. Fuller, B. Jovov, D. J. Benos, and I. I. Ismailov. Regulation of epithelial sodium channels by short actin filaments. J. Biol. Chem. 271: 17704-17710, 1996[Abstract/Free Full Text].

7.   Berdiev, B. K., V. G. Shlyonsky, O. Senyk, D. Keeton, Y. Guo, S. Matalon, H. F. Cantiello, A. G. Prat, D. A. Ausiello, I. I. Ismailov, and D. J. Benos. Protein kinase A phosphorylation and G protein regulation of type II pneumocyte Na+ channels in lipid bilayers. Am. J. Physiol. 272 (Cell Physiol. 41): C1262-C1270, 1997[Abstract/Free Full Text].

8.   Canessa, C. M., J. D. Horisberger, and B. C. Rossier. Epithelial sodium channel related to proteins involved in neurodegeneration. Nature 361: 467-470, 1993[Medline].

9.   Canessa, C. M., J. D. Horisberger, L. Schild, and B. C. Rossier. Expression cloning of the epithelial sodium channel. Kidney Int. 48: 950-955, 1995[Medline].

10.   Canessa, C. M., L. Schild, G. Buell, B. Thorens, I. Gautschi, J. D. Horisberger, and B. C. Rossier. Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature 367: 463-467, 1994[Medline].

11.   Cantiello, H. F. Role of the actin cytoskeleton on epithelial Na+ channel regulation. Kidney Int. 48: 970-984, 1995[Medline].

12.   Compeau, C. G., O. D. Rotstein, H. Tohda, Y. Marunaka, B. Rafii, A. S. Slutsky, and H. O'Brodovich. Endotoxin-stimulated alveolar macrophages impair lung epithelial Na+ transport by an L-Arg-dependent mechanism. Am. J. Physiol. 266 (Cell Physiol. 35): C1330-C41, 1994[Abstract/Free Full Text].

13.   Darley-Usmar, V. M., N. Hogg, V. J. O'Leary, M. T. Wilson, and S. Moncada. The simultaneous generation of superoxide and nitric oxide can initiate lipid peroxidation in human low density lipoprotein. Free Radic. Res. Commun. 17: 9-20, 1992[Medline].

14.   Forstermann, U., E. I. Closs, J. S. Pollock, M. Nakane, P. Schwarz, I. Gath, and H. Kleinert. Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions. Hypertension 23: 1121-1131, 1994[Abstract].

15.   Greis, K. D., S. Zhu, and S. Matalon. Identification of nitration sites on surfactant protein A by tandem electrospray mass spectrometry. Arch. Biochem. Biophys. 335: 396-402, 1996[Medline].

16.   Haddad, I. Y., J. P. Crow, P. Hu, Y. Ye, J. Beckman, and S. Matalon. Concurrent generation of nitric oxide and superoxide damages surfactant protein A. Am. J. Physiol. 267 (Lung Cell. Mol. Physiol. 11): L242-L249, 1994[Abstract/Free Full Text].

17.   Haddad, I. Y., G. Pataki, P. Hu, C. Galliani, J. S. Beckman, and S. Matalon. Quantitation of nitrotyrosine levels in lung sections of patients and animals with acute lung injury. J. Clin. Invest. 94: 2407-2413, 1994[Medline].

18.   Haddad, I. Y., S. Zhu, J. Crow, E. Barefield, T. Gadilhe, and S. Matalon. Inhibition of alveolar type II cell ATP and surfactant synthesis by nitric oxide. Am. J. Physiol. 270 (Lung Cell. Mol. Physiol. 14): L898-L906, 1996[Abstract/Free Full Text].

19.   Haddad, I. Y., S. Zhu, H. Ischiropoulos, and S. Matalon. Nitration of surfactant protein A results in decreased ability to aggregate lipids. Am. J. Physiol. 270 (Lung Cell. Mol. Physiol. 14): L281-L288, 1996[Abstract/Free Full Text].

20.   Hogg, N., V. M. Darley-Usmar, M. T. Wilson, and S. Moncada. Production of hydroxyl radicals from the simultaneous generation of superoxide and nitric oxide. Biochem. J. 281: 419-424, 1992[Medline].

21.   Hu, P., H. Ischiropoulos, J. S. Beckman, and S. Matalon. Peroxynitrite inhibition of oxygen consumption and sodium transport in alveolar type II cells. Am. J. Physiol. 266 (Lung Cell. Mol. Physiol. 10): L628-L634, 1994[Abstract/Free Full Text].

22.   Huie, R. E., and S. Padmaja. The reaction of NO with superoxide. Free Radic. Res. Commun. 18: 195-199, 1993[Medline].

23.   Ischiropoulos, H., L. Zhu, and J. S. Beckman. Peroxynitrite formation from macrophage-derived nitric oxide. Arch. Biochem. Biophys. 298: 446-451, 1992[Medline].

24.   Ismailov, I. I., M. S. Awayda, B. K. Berdiev, J. K. Bubien, J. E. Lucas, C. M. Fuller, and D. J. Benos. Triple-barrel organization of ENaC, a cloned epithelial Na+ channel. J. Biol. Chem. 271: 807-816, 1996[Abstract/Free Full Text].

25.   Ismailov, I. I., T. Kieber-Emmons, C. Lin, B. K. Berdiev, V. G. Shlyonsky, H. K. Patton, C. M. Fuller, R. Worrell, J. B. Zuckerman, W. Sun, D. C. Eaton, D. J. Benos, and T. R. Kleyman. Identification of an amiloride binding domain within the alpha -subunit of the epithelial Na+ channel. J. Biol. Chem. 272: 21075-21083, 1997[Abstract/Free Full Text].

26.   Kaur, H., and B. Halliwell. Evidence for nitric oxide-mediated oxidative damage in chronic inflammation. Nitrotyrosine in serum and synovial fluid from rheumatoid patients. FEBS Lett. 350: 9-12, 1994[Medline].

27.   Koivisto, A., D. Siemen, and J. Nedergaard. Reversible blockade of the calcium-activated nonselective cation channel in brown fat cells by the sulfhydryl reagents mercury and thimerosal. Pflügers Arch. 425: 549-551, 1993[Medline].

28.   MacMillan-Crow, L. A., J. P. Crow, J. D. Kerby, J. S. Beckman, and J. A. Thompson. Nitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allografts. Proc. Natl. Acad. Sci. USA 93: 11853-11858, 1996[Abstract/Free Full Text].

29.   Moncada, S., and A. Higgs. The L-arginine-nitric oxide pathway. N. Engl. J. Med. 329: 2002-2012, 1993[Free Full Text].

30.   Moreno, J. J., and W. A. Pryor. Inactivation of alpha 1-proteinase inhibitor by peroxynitrite. Chem. Res. Toxicol. 5: 425-431, 1992[Medline].

31.   Packer, M. A., and M. P. Murphy. Peroxynitrite causes calcium efflux from mitochondria which is prevented by cyclosporin A. FEBS Lett. 345: 237-240, 1994[Medline].

32.   Palmer, L. G. Epithelial Na channels: function and diversity. Annu. Rev. Physiol. 54: 51-66, 1992[Medline].

33.   Pryor, W. A., X. Jin, and G. L. Squadrito. One- and two-electron oxidations of methionine by peroxynitrite. Proc. Natl. Acad. Sci. USA 91: 11173-11177, 1994[Abstract/Free Full Text].

34.   Saugstad, J. A., T. P. Segerson, and G. L. Westbrook. Metabotropic glutamate receptors activate G-protein-coupled inwardly rectifying potassium channels in Xenopus oocytes. J. Neurosci. 16: 5979-5985, 1996[Abstract/Free Full Text].

35.   Smith, P. R., and D. J. Benos. Epithelial Na+ channels. Annu. Rev. Physiol. 53: 509-530, 1991[Medline].

36.   Sokolovsky, M., D. Harell, and J. F. Riordan. Reaction of tetranitromethane with sulfhydryl groups in proteins. Biochemistry 8: 4740-4745, 1969[Medline].

37.   Sokolovsky, M., J. F. Riordan, and B. L. Vallee. Tetranitromethane. A reagent for the nitration of tyrosyl residues in proteins. Biochemistry 5: 3582-3589, 1966[Medline].

38.   Van der Vliet, A., D. Smith, C. A. O'Neill, H. Kaur, V. Darley-Usmar, C. E. Cross, and B. Halliwell. Interactions of peroxynitrite with human plasma and its constituents: oxidative damage and antioxidant depletion. Biochem. J. 303: 295-301, 1994[Medline].

39.   Yoshida, S., and S. Plant. A potassium current evoked by growth hormone-releasing hormone in follicular oocytes of Xenopus laevis. J. Physiol. (Lond.) 443: 651-667, 1991[Abstract].

40.   Yoshimura, M., S. Yoshida, and K. Taniyama. Property of receptor for vasoactive intestinal contractor (VIC) expressed in Xenopus oocytes injected with mRNA from rat intestine. Life Sci. 58: 1731-1736, 1996[Medline].


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