The reactive nitrogen species peroxynitrite is a potent inhibitor of renal Na-K-ATPase activity

Abstract

Peroxynitrite is a reactive nitrogen species produced when nitric oxide and superoxide react. In vivo studies suggest that reactive oxygen species and, perhaps, peroxynitrite can influence Na-K-ATPase function. However, the direct effects of peroxynitrite on Na-K-ATPase function remain unknown. We show that a single bolus addition of peroxynitrite inhibited purified renal Na-K-ATPase activity, with IC₅₀ of 107 ± 9 μM. To mimic cellular/physiological production of peroxynitrite, a syringe pump was used to slowly release (∼0.85 μM/s) peroxynitrite. The inhibition of Na-K-ATPase activity induced by this treatment was similar to that induced by a single bolus addition of equal cumulative concentration. Peroxynitrite produced 3-nitrotyrosine residues on the α, β, and FXYD subunits of the Na pump. Interestingly, the flavonoid epicatechin, which prevented tyrosine nitration, was unable to blunt peroxynitrite-induced ATPase inhibition, suggesting that tyrosine nitration is not required for inhibition. Peroxynitrite led to a decrease in iodoacetamidofluorescein labeling, implying that cysteine modifications were induced. Glutathione was unable to reverse ATPase inhibition. The presence of Na⁺ and low MgATP during peroxynitrite treatment increased the IC₅₀ to 145 ± 10 μM, while the presence of K⁺ and low MgATP increased the IC₅₀ to 255 ± 13 μM. This result suggests that the EPNa conformation of the pump is slightly more sensitive to peroxynitrite than the E(K) conformation. Taken together, these results show that peroxynitrite is a potent inhibitor of Na-K-ATPase activity and that peroxynitrite can induce amino acid modifications to the pump.

the contribution of elevated production of reactive oxygen and nitrogen species (ROS and RNS, respectively) to cell dysfunction in a variety of disease states via induction of oxidative damage to cell macromolecules, such as lipids, DNA, and proteins, is increasingly recognized (1, 17). Peroxynitrite is a damaging RNS that is produced in vivo when nitric oxide (NO) and superoxide react (for review see Refs. 17 and 21). The reaction rate between NO and superoxide is nearly diffusion limited; it is three- to fourfold higher than the reaction rate between superoxide and SOD (17, 18) when superoxide is less than its K_m. Thus, even if the NO concentration is as low as the SOD concentration, peroxynitrite is likely to form, even in the presence of SOD.

Peroxynitrite has a short (<1-s) half-life. It can react with tyrosine residues, generating 3-nitrotyrosine, which is stable. 3-Nitrotyrosine levels are elevated in a number of conditions, including cardiovascular diseases, hyperglycemia, and renal ischemia-reperfusion injuries (17, 25).

The effect of peroxynitrite on protein structure and function is of interest because of its potential to impact cell behavior and contribute to disease processes. Several lines of evidence in different tissue types, including the kidney, suggest that ROS and, perhaps, peroxynitrite play a role in regulating Na-K-ATPase activity and function (3, 26, 29). It is likely that peroxynitrite is produced in kidney cells, since they have been reported to produce NO and superoxide (10, 16, 22, 25, 29). Zhang et al. (29) showed that nanomolar concentrations of angiotensin II in the proximal renal tubule inhibit Na-K-ATPase activity compared with picomolar concentrations and that this inhibition was abolished in the presence of superoxide and peroxynitrite scavengers, implying that superoxide and/or peroxynitrite was responsible for the inhibition. Furthermore, they showed that angiotensin II increased total levels of 3-nitrotyrosine, suggesting that peroxynitrite levels were elevated. However, the direct effect of peroxynitrite on Na pump activity and function was not tested.

The extent to which peroxynitrite reacts with the Na pump in vivo is unknown. Deductively, one can see that different types of experiments can be carried out to investigate how a potential new modulator might alter protein activity/function. The first approach is to determine the effect of the modulator on intact animal tissue and/or cells. Such an investigation leads to physiologically relevant data but requires further experiments, in a simpler system, to determine whether the modulator directly or indirectly alters protein function. The second approach is to determine whether the modulator/inhibitor alters protein activity in a very simple system and then expand to more complicated physiologically relevant systems. We have chosen the second approach.

Because previous studies at the cellular level have shown that ROS and, perhaps, peroxynitrite might be involved in regulating the Na pump and studies on purified Na pump have shown that the pump is sensitive to superoxide, hydroxyl radical, and hydrogen peroxide, we investigated the functional effects of peroxynitrite, an RNS, on purified renal Na pump activity. Our results show that peroxynitrite is a potent inhibitor of Na-K-ATPase activity and that Na-K-ATPase activity is inhibited to a similar extent by a single bolus of concentrated peroxynitrite and constant release of a low concentration of peroxynitrite. We show that Na pump conformation has an influence, although small, on peroxynitrite-induced inhibition. Furthermore, we provide evidence that peroxynitrite modifies tyrosine and cysteine residues of the Na pump. We present evidence that nitrotyrosine formation is not the primary mode of inhibition of the pump, inasmuch as epicatechin, a flavonoid that prevents nitration, did not blunt peroxynitrite-induced ATPase inhibition. The endogenous redox regulator glutathione was unable to reverse peroxynitrite-induced ATPase inhibition. These results lay the groundwork for understanding how peroxynitrite affects Na pump activity and will prompt future studies that explore the extent to which peroxynitrite influences Na pump activity vivo.

METHODS

Peroxynitrite reactivity and concentration.

Peroxynitrite (catalog no. 516620, Calbiochem) was aliquoted under nitrogen gas and stored at −70°C. Peroxynitrite is stable in alkaline conditions; therefore, the stock peroxynitrite was supplied in 1.1 M NaOH. For determination of the exact concentration on the day of the experiment, an aliquot of stock peroxynitrite was thawed, 2.5 μl of peroxynitrite were diluted into 1 ml of 5 M NaOH, and the absorbance was measured at 302 nm. The stock peroxynitrite concentration for that day was calculated using the molar extinction coefficient of peroxynitrite (1,670 cm⁻¹·M⁻¹) and the dilution factor. The calculated stock concentration of peroxynitrite varied between 125 and 150 mM. Determining the concentration of stock peroxynitrite allowed us to add the appropriate volume of stock peroxynitrite (in 1.1 M NaOH) to the samples at the indicated concentrations. The final NaOH concentration was the same in the decomposed sample and the fresh sample and was between 0.7 and 7.5 mM, depending on the peroxynitrite concentration (well below the buffering capacity of the respective assay solutions).

To verify further that peroxynitrite exhibited the appropriate ROS activity, we incubated peroxynitrite with aminophenylfluorescein (APF), an ROS fluorescent probe. APF is nonfluorescent until it reacts with an ROS, particularly peroxynitrite or hydroxyl radical. As expected, incubation of APF with increasing concentrations of fresh peroxynitrite caused a dose-dependent increase in APF fluorescence (data not shown). However, very little, if any, fluorescence was observed when APF was incubated with increasing concentrations of decomposed peroxynitrite (data not shown). Peroxynitrite was decomposed by storage at room temperature and exposure to light and the atmosphere for ≥48 h.

Peroxynitrite treatment.

Purified renal Na pump was prepared by following the Jorgenson technique with slight modifications (7). Ten to 40 μg of purified Na pump in 100 mM Tris·HCl (pH 7.4) were treated with fresh or decomposed peroxynitrite administered as a single bolus or via a syringe pump that slowly released peroxynitrite. For single bolus additions, a pipette was used to dispense peroxynitrite (from the 1.1 M NaOH stock) into the Na pump-containing samples. After these samples were incubated with peroxynitrite for 5 min at 37°C, ATPase activity at 37°C was measured (see below). Given the reactivity and short half-life of peroxynitrite at pH 7.4, it is likely that, after 5 min, very little, if any, peroxynitrite remained. For the constant, slow release of peroxynitrite, a syringe pump (Pico Plus, Harvard Apparatus) was set to release peroxynitrite at a rate of 0.85 μM/min for 18 min into the Na pump-containing tubes at 37°C. Syringe pump parameters of 0.85 μM/s for 18 min were chosen, because if peroxynitrite could accumulate, it would be equivalent to the single bolus concentration used in the paired condition. In a separate experiment, the release rate of the syringe pump was validated by measurement of the total volume of colored water released over a given period of time. When indicated, purified renal Na pump was placed into the EPNa or E(K) conformation by incubation in the presence of 130 mM Na⁺-50 μM MgATP or 30 mM K⁺-50 μM MgATP, respectively, during peroxynitrite treatment. Also, when indicated, reduced glutathione (0.5, 2.5, or 5.0 mM; Acros Organics) was present during the peroxynitrite treatment at 37°C or was introduced for 30 min at 37°C only after peroxynitrite treatment. Stock concentrations of 100 and 25 μM epicatechin (catalog no. 157925, MP Biomedicals) dissolved in DMSO were used to selectively prevent tyrosine nitration.

Na-K-ATPase activity.

Ouabain-sensitive Na-K-ATPase activity was measured via a previously described ascorbic acid-molybdate colorimetric assay capable of detecting inorganic phosphate (8). Briefly, 1–4 μg of untreated or peroxynitrite-treated Na pump were diluted into ∼600–700 μl of assay medium and incubated for 10 min at 37°C. The assay medium contained 2.5 mM ATP, 100 mM NaCl, 10 mM KCl, and 5.0 mM MgCl₂. These concentrations of NaCl, KCl, MgCl₂, and ATP are typical of this assay and allow the pump to be saturated with Na⁺ and K⁺. Under physiological conditions, K⁺ is saturated, and the Na⁺ saturation depends on the cell Na⁺ concentration. After the 10-min ATPase incubation period, the ascorbic acid-molybdate colorimetric assay was performed. For each experiment, an ouabain condition, in which the Na pump ATPase reaction was carried out in the presence of 1 mM ouabain, was included. The resulting background value was subtracted from all other experimental conditions that yielded ouabain-sensitive Na-K-ATPase activity. Furthermore, for all ATPase experiments, each condition was performed in quadruplicate, and data were normalized to the activity in the absence of peroxynitrite.

Iodoacetamidofluorescein labeling of free thiol groups.

Forty micrograms of purified renal Na pump in 300 μl of 200 mM Tris·HCl (pH 7.4) were incubated for 5 min at 37°C in the presence or absence of fresh or decomposed peroxynitrite. Samples were incubated with 0.5% SDS for 15 min at room temperature and then with 500 μM iodoacetamidofluorescein (IAF) for 30 min in the dark at room temperature. Cysteine (5 mM) was added to quench any nonreacted IAF.

SDS-PAGE/Western blotting.

Na pump samples were incubated with lane marker reducing sample buffer (catalog no. 3900, Pierce) at 1 volume sample buffer to 4 volumes protein sample. Equal protein concentrations from each sample were then loaded into 4–20% Tris·HCl Ready Gels (catalog no. 161-1159, Bio-Rad) and run for ∼40 min at 200 V. A 12-μl volume of Precision Plus protein dual-color standards (catalog no. 161-0374, Bio-Rad) was used for molecular weight markers. Gel running buffer consisted of 1× Tris-glycine-SDS buffer (catalog no. 161-0732, Bio-Rad).

Protein was then transferred from the gel to an Immobilon polyvinylidene difluoride (0.45-μm pore size) transfer membrane (catalog no. IPVH10100, Millipore) in 100 mM 3-cyclohexylamino-1-propane sulfonic acid-10% MeOH (pH 11.0). The protein transfer took place over 1 h 15 min at 150 mA. The membrane was then blocked for 1 h at room temperature with Superblock Dry Blend [Tris-buffered saline (TBS)] blocking buffer (catalog no. 37545, Pierce).

3-Nitrotyrosine residues were detected using 1 mg/ml rabbit anti-nitrotyrosine (catalog no. A21285, Molecular Probes) diluted 1:10,000 into 1× TBS with 0.05% Tween 20 (Fisher). IAF-labeled thiol groups were detected using 1 mg/ml mouse anti-fluorescein-Oregon Green (catalog no. A6421, Molecular Probes) diluted 1:5,000 into 1× TBS with 0.05% Tween 20. Membranes were incubated with the appropriate primary antibody for 1 h at room temperature and then washed five times in 1× TBS with 0.05% Tween 20. Membranes were incubated again for 1 h with goat anti-rabbit or goat anti-mouse horseradish peroxidase-conjugated secondary antibody (catalog nos. 31460 and 31430, Pierce) diluted 1:50,000 into 1× TBS with 0.05% Tween 20 and washed five times, for 5 min each, in fresh 1× TBS with 0.05% Tween 20. Membranes were then incubated for 5 min with SuperSignal West Pico chemiluminescent substrate: 5 ml each of stable peroxide solution (Super Signal West Pico) and luminol enhancer solution (product no. 34080, Pierce, Thermo Scientific). The membrane was placed on a plastic sheet, and excess substrate was wiped away. Classic Blue autoradiography film (Midsci) was exposed to the membrane and developed with a mini-medical X-ray film processor (AFP Imaging).

Statistical analysis.

Values are means ± SD. Kaleidagraph version 4.0 was used for curve fitting. Curves were fit to all individual data points from each experiment to provide Kaleidagraph with as much information as possible. The IC₅₀ values obtained from these curve fits are reported in results; however, means ± SD are plotted on Figs. 1–3, 5, and 7. When appropriate, Student's t-test was used to determine significance between groups. P < 0.05 was considered significant.

Fig. 1.

Peroxynitrite is a potent inhibitor of purified renal Na-K-ATPase activity in 100 mM Tris·HCl (pH 7.4). Purified renal Na pump was treated with 0–1,000 μM fresh peroxynitrite or an equivalent volume of decomposed peroxynitrite at 37°C and then incubated for 5 min. Then ouabain-sensitive Na-K-ATPase activity was measured in the presence of 3.0 mM ATP, 4.0 mM MgCl₂, 100 mM NaCl, and 12 mM KCl. Data were fit to the following equation: V = V₀ × IC₅₀/(IC₅₀ + P), where P is peroxynitrite concentration and V₀ is maximum velocity in the absence of peroxynitrite (i.e., P = 0). Treatment with fresh peroxynitrite yielded IC₅₀ = 107 ± 9 μM (fit 1, all data points) or 125 ± 9 μM (fit 2). Treatment with decomposed peroxynitrite yielded IC₅₀ = 4,480 ± 603 μM. Values are means ± SD of data from 3 paired experiments.

Fig. 3.

Sensitivity to peroxynitrite is greater for EPNa than for E(K) conformation. Purified renal Na pump was placed in EPNa or E(K) conformation by incubation in the presence of 130 mM Na⁺-50 μM MgATP or 30 mM K⁺-50 μM MgATP, respectively, during peroxynitrite treatment. Solutions for ATPase were identical. A: data were fit to equation in Fig. 1 legend. IC₅₀ = 145 ± 10 μM for the EPNa conformation and 255 ± 13 μM for the E(K) conformation. Values are means ± SD of data from 5 paired experiments. P = 0.007 (Student's t-test), IC₅₀ = 145 ± 10 μM vs. 255 ± 13 μM. B: IC₅₀ values determined for each individual paired experiment and mean IC₅₀ values for all 5 experiments. *P < 0.001.

Fig. 5.

Epicatechin enhances peroxynitrite-induced Na-K-ATPase inhibition. Purified renal Na pump was treated with 0, 275, or 490 μM peroxynitrite in the absence or presence of 100 μM epicatechin or DMSO. After peroxynitrite treatment, ouabain-sensitive Na-K-ATPase activity was measured in the presence of 2.5 mM ATP, 7.0 mM MgCl₂, 95 mM NaCl, and 15 mM KCl. Values are means ± SD of 3 experiments. *P < 0.001.

Fig. 7.

Presence of glutathione (GSH) during peroxynitrite treatment confers protection, whereas presence of glutathione only after peroxynitrite treatment does not reverse ATPase inhibition. Purified renal Na pump was not treated or treated with 100 μM peroxynitrite in the presence of 0–5.0 mM glutathione, or glutathione was introduced only after peroxynitrite treatment. After treatment, ouabain-sensitive Na-K-ATPase activity was measured in the presence of 3 mM ATP, 4 mM MgCl₂, 100 mM NaCl, and 12 mM KCl. Values are means ± SD of 2 experiments. *P < 0.001 vs. control (0.0 peroxynitrite and 0.0 glutathione).

RESULTS

Figure 1 shows that single bolus additions of fresh peroxynitrite decreased Na pump ATPase activity, with an IC₅₀ of 107 ± 9 μM. Single bolus additions of decomposed peroxynitrite required much higher concentrations (IC₅₀ > 4 mM). The IC₅₀ fit for fresh peroxynitrite is not very good. At the two highest concentrations of peroxynitrite, there is very little activity, and these points are difficult to obtain with great confidence. Omitting the data points obtained at the two highest concentrations, of course, improved the fit and only slightly changed IC₅₀ to 126 ± 9 μM (fit 2).

We are unaware of reports examining whether peroxynitrite can be produced at hundreds of micromolar within cells. However, at least under some pathological conditions, a constant, low concentration of peroxynitrite may be produced intracellularly. To mimic this type of condition, a syringe pump was used to inject peroxynitrite into samples containing purified Na pump at <1.0 μM/s for 18 min. As shown in Fig. 2, Na pump ATPase activity was inhibited to a similar extent by a constant exposure to low concentrations of peroxynitrite and a single bolus addition of peroxynitrite at much higher concentrations. As shown in Fig. 1, decomposed peroxynitrite administered as a single bolus or with the syringe pump caused ∼10–15% inhibition.

Fig. 2.

Na-K-ATPase activity is inhibited to a similar extent by administration of a single bolus of concentrated peroxynitrite (ONOO) and release of a constant low concentration of peroxynitrite. Purified renal Na pump was treated with fresh (FR) or decomposed (DC) peroxynitrite administered as a single, bolus addition or via a syringe pump to generate a constant, low concentration of peroxynitrite as might be produced intracellulary. Syringe pump parameters of 0.85 μM/s for 18 min were chosen, because if peroxynitrite could accumulate at pH 7.4, it would yield a cumulative concentration equal to that used in the single bolus conditions. After peroxynitrite treatment, ouabain-sensitive Na-K-ATPase activity was measured in the presence of 2.5 mM ATP, 7.0 mM MgCl₂, 95 mM NaCl, and 15 mM KCl. Values are means ± SD of 4 paired experiments.

The Na pump undergoes several conformational changes during its cycle, and it is likely that changes in cell Na⁺ concentration change the predominant conformation. Furthermore, Na pump conformation has been shown to alter aspects of its regulation. For example, conformation has been shown to alter regulatory phosphorylation of the Na pump (6). Therefore, we examined whether Na pump conformation would alter the ability of peroxynitrite to inhibit the pump. In the presence of Na⁺ and low concentrations of MgATP, the Na pump is catalytically phosphorylated. We wanted to compare this conformation with conformation in the presence of K⁺ but in the absence of Na⁺. Because there is the potential for MgATP to scavenge peroxynitrite, we chose to have the same MgATP concentrations in both conditions, so that any scavenging would be equivalent. The effect of Na pump conformation on peroxynitrite-induced inhibition was determined by incubation of the purified pump with 50 μM MgATP and either Na⁺ or K⁺ in paired experiments. The IC₅₀ for peroxynitrite-induced inhibition of ATPase activity was 145 ± 10 μM in the presence of Na⁺; in the presence of K⁺, it was 255 ± 13 μM (Fig. 3A). There was some day-to-day variation in IC₅₀ values. The results of the five individual paired experiments are shown in Fig. 3B. Each individual experiment did show a lower IC₅₀ value in the EPNa conformation than in the E(K) conformation.

To test whether peroxynitrite promotes tyrosine nitration of the Na pump, purified Na pump was exposed to increasing concentrations (0, 50, 250, 500, and 750 μM) of peroxynitrite. Figure 4 is a representative Western blot demonstrating that peroxynitrite induces tyrosine nitration of the 100-kDa α-subunit, 50-kDa β-subunit, and 15-kDa FXYD subunit (lanes 1–5). It has been reported that the flavonoid epicatechin selectively prevents nitration but not other peroxynitrite-induced oxidative reactions (19). We confirmed that 100 or 25 μM epicatechin prevents peroxynitrite-induced tyrosine nitration, even at the highest concentration (750 μM) of peroxynitrite (Fig. 4, lanes 6 and 7). We also showed that DMSO alone, used as the epicatechin vehicle control, does not prevent nitration (Fig. 4, lanes 8 and 9).

Fig. 4.

Western blot showing an increase in peroxynitrite-induced tyrosine nitration of α, β, and FXYD subunits and prevention of nitration by epicatechin. Equal amounts of purified renal Na pump were treated with 0, 50, 250, 500, or 750 μM peroxynitrite and probed with an anti-nitrotyrosine antibody. Prominent bands at 100, 50, and 15 kDa represent α, β, and FXYD subunits, respectively (lanes 1–5). Epicatechin at 100 or 25 μM prevents tyrosine nitration at the highest concentration of peroxynitrite (lanes 6 and 7). DMSO, a vehicle control for epicatechin, did not prevent tyrosine nitration (lanes 8 and 9).

Having verified that epicatechin prevents tyrosine nitration, we next determined whether tyrosine nitration accounts for loss of ATPase activity. We treated purified Na pump with 0, 275, or 490 μM peroxynitrite in the presence and absence of 100 μM epicatechin or DMSO (vehicle control). In the absence of peroxynitrite, 100 μM epicatechin alone had no effect on Na pump ATPase activity (Fig. 5). However, at 275 and 490 μM peroxynitrite, 100 μM epicatechin enhanced ATPase inhibition. At 275 and 490 μM peroxynitrite, DMSO, used as a vehicle control, had a slight protective effect.

Thiol groups on cysteine residues are particularly sensitive to oxidative modifications. To determine whether peroxynitrite modified cysteines, we labeled nontreated and peroxynitrite- or decomposed peroxynitrite-treated Na pump free thiol groups with IAF. Figure 6 shows a decrease in IAF labeling in peroxynitrite-treated pump compared with untreated and decomposed peroxynitrite-treated pump. This result demonstrates that peroxynitrite modifies Na pump cysteine groups. N-ethylmaleimide, a known thiol-blocking agent, was used as a control to verify that blockade of thiol groups decreased IAF labeling (data not shown).

Fig. 6.

Peroxynitrite-induced decrease in iodoacetamidofluorescein labeling implies that peroxynitrite modifies cysteine thiol groups. Purified Na pump was not treated (lane 1) or treated with increasing concentrations of fresh (FR) peroxynitrite (lanes 3, 5, 7, and 9) or an equivalent volume of decomposed (DC) peroxynitrite (lanes 2, 4, 6, and 8).

Having verified that cysteine thiol groups are modified by peroxynitrite, we next asked whether glutathione, an intracellular tripeptide capable of reducing some ROS-induced thiol modifications, was able to reverse peroxynitrite-induced ATPase inhibition. Figure 7 shows that introduction of glutathione after peroxynitrite treatment did not reverse ATPase inhibition. In contrast, the presence of glutathione during peroxynitrite treatment had a protective effect on ATPase activity. That is, in the presence of increasing concentrations of glutathione, ATPase activity returned to normal. The presence of 2.5 mM glutathione during 840 μM peroxynitrite treatment of 50 μM l-tyrosine prevented the formation of nitrotyrosine, as determined by the absorbance near 420 nm (data not shown). Thus it appears that glutathione effectively “scavenges” available peroxynitrite and, thus, prevents Na pump modification.

DISCUSSION

In the present study, we investigated the effects of peroxynitrite on purified renal Na pump activity with the goal of characterizing the effects of peroxynitrite on Na-K-ATPase activity. Two different types of intracellular peroxynitrite production are considered. 1) Peroxynitrite concentrations could be elevated in localized areas (“hot spots”) likely centered around sites of superoxide production because of the difference between NO and superoxide reactivity and diffusion distances. 2) Peroxynitrite might also be produced at low concentrations over a long time course. We also discuss whether peroxynitrite modified tyrosine and cysteine residues and whether these account for the observed ATPase effects. Finally, we consider how these modifications might contribute to altered Na pump signaling, which could in turn lead to disturbed degradation and trafficking.

Our data provide direct evidence that peroxynitrite is a potent inhibitor of Na-K-ATPase activity. Decomposed peroxynitrite had a small effect on ATPase activity. The inhibition that was caused by decomposed peroxynitrite (∼15% at the highest concentration tested) could be due to one or more of the following mechanisms: 1) incomplete decomposition of the decomposed peroxynitrite, 2) inhibition induced by a breakdown product such as nitrite, or 3) NaOH influences on pH. Gutiérrez-Martín et al. (12) found the K_0.5 (IC₅₀) of peroxynitrite on sarco(endo)plasmic reticulum Ca-ATPase (SERCA) activity to be between 200 and 300 μM. Given the high degree of structural and sequence homology between SERCA and the Na pump α-subunit, our results suggest that peroxynitrite, in general, is a potent inhibitor of P-type ATPases and Na-K-ATPase activity. Although these results are not paired, they also suggest that the Na pump might have an even lower IC₅₀ than SERCA.

We also addressed how alterative models of peroxynitrite production might differentially influence Na-K-ATPase inhibition. Specifically, we modeled two extremes of intracellular peroxynitrite production by administration of peroxynitrite as a single concentrated bolus or a constant release of a low concentration via a syringe pump. We show that Na-K-ATPase is inhibited to a similar extent by addition of a single bolus of concentrated peroxynitrite and by constant release of lower concentrations of peroxynitrite. This result is significant, because it provides evidence that a constant release of a low concentration of peroxynitrite, as might be produced intracellularly, could, over time, induce significant inhibition of Na pumps. Certainly, red blood cell Na pumps are likely to be exposed to (fluctuating) low concentrations of peroxynitrite, inasmuch as the pumps and the red blood cell itself must last for 120 days. Moreover, red blood cells are faced with high oxygen concentrations and are also likely scavengers of excess “spillover” NO from vascular cells.

The precise rate and concentration of renal peroxynitrite production in vivo have not been determined. For example, peroxynitrite production very well might occur, such that it is constantly produced in a chronic manner at low concentrations (nanomolar to picomolar), but it is also possible that local hot spots, where subcellularly isolated concentrations of peroxynitrite are substantially higher (micromolar) than surrounding areas, might also exist. This seems feasible if we consider that NO is fairly stable and able to diffuse over relatively long distances, whereas superoxide is much more reactive and has a much shorter half-life. Therefore, peroxynitrite could be produced in discrete localized areas within the cell, most likely centered around sites of superoxide production, such as mitochondria and NAD(P)H oxidase, xanthine oxidase, and other superoxide production sites. In either case, peroxynitrite could lead to inhibition of cell Na pumps if our purified preparation behaves similarly to cell Na pumps.

We hypothesized that peroxynitrite was nitrating Na pump tyrosine residues, which might account for loss of ATPase activity. We show here that all three subunits (α, β, and FXYD) of the Na pump contain 3-nitrotyrosine residues after treatment with peroxynitrite. The sheep α, β, and FXYD subunits contain 25, 20, and 3 tyrosine residues, respectively, and clearly one or more tyrosine residues on each subunit is being nitrated. Tyrosine nitration itself has been implicated in altering phosphotyrosine-dependent signaling, protein-protein interactions, and enzymatic activity (11, 13, 24, 28). To test whether tyrosine nitration was the sole reason for inhibition of ATPase activity, we used epicatechin. Epicatechin is a polyphenol flavonoid found in dark chocolate, green tea, and red wine and is a reported selective inhibitor of nitration but not other peroxynitrite-induced oxidation reactions (19). Schroeder et al. (19) hypothesized that the selectivity of epicatechin is a result of its ability to scavenge the tyrosyl radical, a peroxynitrite intermediate, rather than directly scavenge peroxynitrite. We verified that the presence of 100 μM epicatechin prevents nitration.

Having confirmed that epicatechin prevented nitration, we predicted that the presence of epicatechin would blunt peroxynitrite-induced inhibition. This would suggest that tyrosine nitration partially or fully accounts for the loss of Na-K-ATPase activity. Contrary to our prediction, the presence of epicatechin did not blunt inhibition, and, actually, the presence of epicatechin enhanced ATPase inhibition. That is, even when tyrosine nitration was prevented with epicatechin, ATPase inhibition was still present and actually enhanced. This result shows that tyrosine nitration is not required for inhibition. However, it does not rule out that tyrosine modification is required for inhibition. For example, it could be that the product of tyrosine, peroxynitrite, and epicatechin modifies tyrosine residues to something other than nitrotyrosine, which eludes our detection, and that this tyrosine conjugate causes the inhibition.

There are several explanations for enhancement of peroxynitrite-induced inhibition by epicatechin. Epicatechin itself could have altered the conformation of the pump, in turn exposing more peroxynitrite-modifiable residues, which enhanced the inhibition. However, it should be pointed out that the epicatechin by itself did not alter ATPase activity. Alternatively, it could be that the product of epicatechin and peroxynitrite itself damages the pump. To our knowledge, the product of the reaction between epicatechin and peroxynitrite has not been described in the literature.

In contrast to the Na pump, in SERCA, nitrotyrosine does account for some of the inhibition. Gutiérrez-Martín et al. (12) found that epicatechin reversed a sizable fraction of the inhibition caused by peroxynitrite, whereas we found that epicatechin actually increased the inhibition (25). A number of SERCA studies have mapped the oxidative modifications, including 3-nitrotyrosine, to tyrosine and cysteine residues (15, 20, 23, 24, 27). These studies have been very helpful in sorting out tyrosine vs. cysteine modification for the different SERCA isoforms, which show very different amino acid modification patterns. For example, for SERCA, a number of tyrosine (and cysteine) residues that are modified by peroxynitrite do not lead to enzyme inhibition (2), apart from the critical tyrosine residues in SERCA2a and critical cysteine residues in SERCA1 that are modified by peroxynitrite and lead to enzyme inhibition. Aging and cardiovascular disease are associated with increased nitration of SERCA residues Y294 and Y295 (27). Both of these residues are located in the M4 segment of SERCA which, as in the Na pump, serves an important role in the structural-coupling mechanism that links ATP hydrolysis, phosphorylation, and transport. Bigelow (2) hypothesized that nitrating SERCA residues Y294 and Y295 might significantly perturb helix-helix interactions at the M4-M5 interface and inhibit the coordinated movements of membrane helices required for active transport by the Ca²⁺-ATPase. Given the high degree of homology between SERCA and the Na pump, it would be tempting to think that a similar mechanism of inhibition might occur with the Na pump. However, our epicatechin data suggest otherwise. Interestingly, the Na pump has two adjacent tyrosine residues at 146 and 147, but these are in located in M2, not M4; indeed, the Na pump has no tyrosine residues in M4. Whether the Na pump also accumulates nitrated residues under similar conditions remains unknown, but because we have shown that peroxynitrite inhibits the Na pump, we are looking for nitrotyrosines in the Na pump in vivo. If the Na pump is modified by peroxynitrite in vivo, then the Na pump, SERCA, and, perhaps, P-type ATPases, in general, might act as RNS “sensors,” as has been suggested (2). A question would be raised if the Na pump is not modified by peroxynitrite in vivo in conditions under which other proteins (e.g., SERCA) are modified, since peroxynitrite can modify the pump in vitro. Two solutions to this potential puzzle are 1) local production of peroxynitrite that is spatially constrained [e.g., near the sarco(endo)plasmic reticulum and away from the plasma membrane] and 2) local interactions or scavengers that protect the Na pump from damage.

Given our finding that tyrosine nitration does not account for loss of activity, we next checked to see if cysteine modifications accompany peroxynitrite treatment. Cysteine thiol groups are particularly sensitive to ROS/RNS-mediated modifications, and there are a number of RNS/ROS-induced thiol modifications, including inter/intradisulfide formation, nitrothiol, and sulfenic/sulfinic/sulfonic acid. The decrease in IAF labeling that accompanied peroxynitrite treatments indicates that peroxynitrite is indeed modifying cysteine residues.

Glutathione, an intracellular tripeptide, is present during the peroxynitrite treatment-conferred protection. It is tempting to conclude that cysteine modifications are a primary mechanism for peroxynitrite-induced inhibition, because 1) cysteine residues are modified and 2) the presence of glutathione protects. However, we believe that this interpretation requires caution. When glutathione was present with peroxynitrite, it probably scavenged the peroxynitrite and, thus, likely prevented modifications, including cysteine, tyrosine, and other residues, to the Na pump. Indeed, we found that glutathione, when present with peroxynitrite and l-tyrosine, prevented the formation of nitrotyrosine. Thus the reduction in ATPase inhibition when peroxynitrite was added in the presence of glutathione probably reflects this scavenging activity and cannot provide definitive information about the residue(s) that is modified to cause Na-K-ATPase inhibition.

In addition to scavenging, glutathione is also capable of reducing certain kinds of RNS/ROS-induced thiol modifications under some conditions. Glutathione was unable to reverse peroxynitrite-induced ATPase inhibition. This result is consistent with two interpretations. 1) Glutathione led to recovery of cysteines but not activity; thus the inhibition is the result of modifying an amino acid besides cysteine (and the epicatechin data suggest that it is not tyrosine). Other amino acids that react directly with peroxynitrite include methionine, tryptophan, phenylalanine, and histidine (1). 2) Cysteine modifications do account for the inhibition; however, glutathione is not chemically strong enough to reverse the peroxynitrite-dependent cysteine-modified product.

It is well known that the Na pump undergoes a very coordinated series of conformation changes between the E1 and E2 conformations during the pump enzymatic cycle (see Ref. 14 for review of Na pump ATPase cycle). Interestingly, there is evidence that conformation-dependent processes exist. Feschenko and Sweadner (6) reported that PKC and PKA regulatory phosphorylation occurs in rat Na pumps in a conformation-dependent manner. Furthermore, compounds such as 2-(4′-maleimidylanilino)naphthalene 6-sulfonic acid and FITC react with specific Na pump residues, Cys⁵⁷⁷ and Lys⁵⁰¹, respectively, in a conformation-dependent manner (5, 9). If peroxynitrite damage of the Na pump were to play a signaling/sensor function, the signaling might be conformation dependent and, thus, a way for the cell to respond differently to low and high cell Na⁺ concentrations. We investigated whether peroxynitrite inhibition was conformation dependent by treating the Na pump with peroxynitrite in two distinct conformations: EPNa and E(K). Na⁺-low MgATP was used to place the Na pump in the EPNa conformation, whereas K⁺-low MgATP was used to place the Na pump in the E(K) conformation. Our results show that conformation had a small, but reproducible, effect on peroxynitrite-induced inhibition, such that the EPNa conformation is slightly more sensitive to peroxynitrite than the E(K) conformation. This could be due to residues that are exposed in the EPNa, but not the E(K), conformation.

If we had found no effect of peroxynitrite on purified Na pump, then doing the experiments in a more physiological system would be irrelevant in terms of direct effects on the Na pump. Our positive effects make the more complicated experiments worth pursuing and also suggest some key information for those studies: for example, the pump conformation is unlikely to alter the interaction substantially, and peroxynitrite can inhibit the pump even if no nitrotyrosine is formed. Furthermore, if the future experiments under physiological conditions clearly show that peroxynitrite is produced but that the Na pump is not inhibited, then the combination of studies would allow us to deduce that peroxynitrite produced is localized away from the Na pump or that there is some method for local protection of the Na pump.

In summary, we have shown that peroxynitrite is a potent irreversible inhibitor of Na-K-ATPase activity. Peroxynitrite also leads to nitration of α, β, and FXYD subunits and modification of cysteine residues. Glutathione did not reverse the ATPase inhibition. Although our results show that tyrosine nitration is not required for ATPase inhibition, nitration could alter trafficking and degradation of the Na pump.

GRANTS

This work was supported by National Institutes of Health Grants DK-37512 (M. A. Milanick) and GM-061583 (C. Gatto).