Hydrogen Peroxide Decreases pH_i in Human Aortic Endothelial Cells by Inhibiting Na⁺/H⁺ Exchange

From the Department of Medicine (Q.H., Y.X., J.L.Z., R.C.Z.), Division of Cardiology, Johns Hopkins Bayview Medical Center, Johns Hopkins University School of Medicine, Baltimore, Md, and the Département d'Anesthésie-Réanimation Hôpital Lariboisière (S.C.), Paris, France.

Correspondence to Roy C. Ziegelstein, MD, Department of Medicine, Division of Cardiology, Johns Hopkins Bayview Medical Center, 4940 Eastern Ave, Baltimore, MD 21224-2780. E-mail rziegels{at}welchlink.welch.jhu.edu

	Abstract

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Abstract—Postischemic endothelial dysfunction may occur as a result of the effects of endogenous oxidants like hydrogen peroxide. Since endothelium-dependent vasodilator function may be affected by pH_i, the effect of hydrogen peroxide on endothelial pH_i was examined. Hydrogen peroxide (100 µmol/L for 10 minutes) decreased pH_i from 7.24±0.01 to 7.02±0.02 and inhibited recovery from an ammonium chloride–induced intracellular acid load in carboxy SNARF 1 (c-SNARF 1)–loaded human aortic endothelial cells in bicarbonate-free solution. Prior inhibition of Na⁺/H⁺ exchange with 5-(N-ethyl-N-isopropyl)amiloride (10 µmol/L), by removal of extracellular Na⁺, or by glycolytic inhibition with iodoacetic acid blocked the subsequent effect of hydrogen peroxide on pH_i. A 2-minute exposure to 100 µmol/L H₂O₂ decreased intracellular ATP levels by 40%; this was prevented by 3-aminobenzamide and nicotinamide (1 mmol/L each), inhibitors of the DNA repair enzyme poly(ADP-ribose) polymerase. Both 3-aminobenzamide and nicotinamide significantly inhibited the hydrogen peroxide–induced intracellular acidification and the effect of hydrogen peroxide on recovery from an intracellular acid load. Hydrogen peroxide decreases pH_i in human endothelial cells by inhibiting Na⁺/H⁺ exchange. This appears to be mediated by activation of the DNA repair enzyme poly(ADP-ribose) polymerase and subsequent depletion of intracellular ATP. Since a decrease in pH_i in this range may alter the activity of NO synthase or affect the synthesis of vasodilator prostaglandins, the effect of hydrogen peroxide on the endothelial Na⁺/H⁺ exchanger may be important in the pathogenesis of postischemic endothelial dysfunction.

Key Words: pH_i • endothelium • Na⁺/H⁺ exchange • free radical

	Introduction

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An abnormal endothelium-dependent vasodilator response occurs as a consequence of ischemia and reperfusion.^{1 2} The endothelial dysfunction of ischemia/reperfusion (I/R) injury is at least in part mediated by oxidants and oxygen-derived free radicals,³ such as hydrogen peroxide (H₂O₂) and the superoxide anion (O₂^- · ). The endothelium in vivo may be exposed to H₂O₂ as a consequence of the dismutation of O₂^- · or from the products of polymorphonuclear leukocytes or monocytes that accumulate in the blood vessel wall as a consequence of I/R.^{4 5} Previous studies have shown that exogenous H₂O₂ attenuates the endothelium-dependent vasodilator response of blood vessels in situ^{6 7} and of isolated coronary arteries in vitro,⁸ but the intracellular signaling pathways responsible for this effect are unknown.

H₂O₂ decreases pH_i in several cell types, including isolated rat cardiac myocytes,⁹ cultured rat cardiac myoblasts,⁹ and renal epithelial cells.¹⁰ In renal epithelial cells, this effect appears to be due, at least in part, to inhibition of Na⁺/H⁺ exchanger activity.¹⁰ The related compound tert-butyl hydroperoxide has been shown to decrease Na⁺/H⁺ antiport activity in bovine pulmonary artery endothelial cells.¹¹ Oxidants like H₂O₂ decrease cellular energy stores^{12 13} and induce DNA strand breaks^{13 14} in vascular endothelial cells. Within several minutes of exposure to micromolar concentrations of H₂O₂, activation of the nuclear DNA repair enzyme poly(ADP-ribose) polymerase (PARS, EC 2.4.2.30) occurs, which catalyzes the transfer of the ADP-ribosyl moiety of NAD⁺ to DNA.^{13 15} PARS activation rapidly decreases cellular NAD⁺ and, subsequently, ATP levels.¹⁵ The rapid intracellular ATP depletion induced by oxidants like H₂O₂^{16 17 18} may result from PARS activation, since it can be prevented by PARS inhibitors in type II pneumocytes.¹⁹ We hypothesized that H₂O₂ decreases endothelial pH_i by inhibiting the ATP-dependent^{20 21 22 23 24 25 26 27 28 29} Na⁺/H⁺ exchanger through a pathway involving PARS activation. The present study reports that H₂O₂ stimulates intracellular acidification and ATP depletion in vascular endothelial cells and that this may be prevented by PARS inhibition.

	Materials and Methods

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Culture of Human Aortic Endothelial Cells
Human aortic endothelial cells (HAECs) were obtained as proliferating quaternary cultures (Clonetics) and were grown to confluence at passages 5 to 9 in endothelial cell growth medium supplemented with 2% FBS, 10 µg/L human recombinant epidermal growth factor, 1 mg/L hydrocortisone, 50 µg/mL gentamicin, 50 ng/mL amphotericin B, and 12 µg/mL bovine brain extract (Clonetics) in a 37°C humidified atmosphere of 95% air/5% CO₂. To examine the effects of H₂O₂ on HAEC pH_i, HAECs were plated at an approximate concentration of 1x10⁵/mL and grown to 70% confluence on 25-mm-diameter circular glass coverslips (VWR Scientific) precoated with 2% gelatin solution (Sigma Chemical Co). The glass coverslips were washed 3 times with PBS (Quality Biological, Inc) before cell seeding.

Measurement of pH_i
HAEC pH_i was measured by using the fluorescent pH indicator carboxy SNARF 1 (c-SNARF 1) as previously reported.³⁰ HAEC monolayers on glass coverslips were incubated with culture medium containing 5 µmol/L of the acetoxymethyl ester form of c-SNARF 1 (Molecular Probes) and maintained at room temperature in a 95% air/5% CO₂ incubator for 30 minutes. Monolayers were washed for 30 minutes with indicator-free HEPES-buffered saline (HBS) of the following composition (in mmol/L): NaCl 137, KCl 4.9, CaCl₂ 1.5, MgSO₄ 1.2, NaH₂PO₄ 1.2, D-glucose 15, and HEPES 20 (pH adjusted to 7.40 at room temperature with NaOH). The glass coverslips were then transferred to a perfusion chamber and mounted on the stage of a modified Nikon Diaphot inverted epifluorescence microscope. c-SNARF 1 fluorescence was excited at 530±5 nm with the use of a Xenon short-arc lamp (UXL-75 XE, Ushio Inc), and bandpass interference filters (Omega Optical) selected fluorescence wavelength bands of 590±5 and 640±5 nm, corresponding to the H⁺-bound and H⁺-free forms of c-SNARF, respectively. The ratio of the emission fluorescence was monitored by using a spectrofluorometer (PTI, Deltascan). Autofluorescence from unloaded HAECs was <2% of c-SNARF–loaded HAECs and was subtracted automatically from c-SNARF fluorescence recordings.

A pH_i calibration was performed as previously described³⁰ from c-SNARF 1–loaded endothelial monolayers exposed to solutions of varying pH values containing 140 mmol/L KCl, 20 µmol/L nigericin (Sigma), 1 µmol/L valinomycin (Calbiochem), and 1 µmol/L carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (Sigma) at room temperature.

Measurement of H⁺ Production
The amount of H⁺ production in response to H₂O₂ was determined as previously described.^{10 31} HAEC monolayers were trypsinized, harvested, and centrifuged at 1000 rpm for 8 to 10 minutes at room temperature. The pellet from three 100-mm tissue culture dishes was then washed with 0.5 mmol/L HEPES-buffered saline (pH 7.40). The cell suspension (final volume, 4 mL) was then stirred by a magnetic driver during continuous pH measurement at room temperature using a pH meter (Beckman Instruments, Inc) and covered to prevent equilibration with air. The rate of H⁺ production after the addition of H₂O₂ (final concentration, 100 µmol/L) was calculated from the amount of OH^- required per unit time to maintain the pH of the cell suspension at a constant value of 7.40±0.01 using 0.1 mmol/L NaOH.

Measurement of Intracellular Buffer Capacity
The intrinsic intracellular buffer capacity (ß, mmol/L per pH unit) was calculated as previously described¹⁰ using the NH₄Cl acid-loading method. Briefly, the acid load was estimated as [NH₄]_i after exposure to 20 mmol/L NH₄Cl, assuming that all NH₄⁺ exits the cell as NH₃ after washout, yielding H⁺ in the process. The last measured pH_i value in the presence of NH₄Cl (pH_pre) was used to calculate [NH₄]_i, with pH_post considered the nadir pH_i value after NH₄Cl washout, assuming a pK of 9.0 and that [NH₃]_o=[NH₃]_i at a pH_o of 7.40. The change in pH_i (pH_i) was determined from the difference of the pH_pre and the pH_post and ß was calculated as follows: ß=[NH₄⁺]_i/pH_i=([NH₃]_ox10^pK-pH_pre)/pH_i =0.49x10^9-pH_pre/(pH_pre-pH_post)

Experimental Protocol
HAEC monolayers were perfused (flow, 1.8 mL/min) with H₂O₂ (1 µmol/L to 1 mmol/L) made from a 3% stock solution (Sigma) in HBS. In some experiments, HAECs were acid-loaded by the NH₄Cl prepulse method³² by replacing 20 mmol/L NaCl of the standard HBS with 20 mmol/L NH₄Cl (J.T. Baker Chemical Co). To examine the role of the Na⁺/H⁺ exchanger, some monolayers were exposed to 5-(N-ethyl-N-isopropyl)amiloride (EIPA, Molecular Probes) or to a Na⁺-free HBS in which NaCl was replaced by 138.2 mmol/L choline chloride (Sigma) and NaH₂PO₄ was replaced by 1.2 mmol/L KH₂PO₄ (Sigma); the pH was adjusted to 7.40 at room temperature with KOH. An EIPA concentration of 10 µmol/L was used since this concentration has been shown to inhibit endothelial Na⁺/H⁺ exchange activity.³³ Cells were exposed to EIPA for 30 to 60 minutes or to Na⁺-free HBS for 20 to 30 minutes before the remainder of the experimental protocol to allow pH_i to achieve a stable baseline after inhibition of Na⁺/H⁺ exchange.

Measurement of Intracellular ATP Concentrations
Intracellular ATP concentrations in HAECs were measured by HPLC as previously described.³⁴ After HAECs in culture dishes were exposed to H₂O₂ (or other reagents described below), the reaction was stopped by the addition of 3 mol/L ice-cold HClO₄. The cells were then harvested, centrifuged at 16 000g, and frozen at -20°C. For measurements of intracellular ATP concentrations, cells were thawed and vortex-mixed. After ultrasonication (Dower 10-15v, Heat Systems, Inc) 3 times on ice, HAECs were centrifuged at 16 000g for 3 minutes at 4°C, and the supernatant was neutralized by 1:3 (vol:vol) mixing with Freon/trioctylamine (4:1). The mixture was centrifuged at 16 000g for 3 minutes at 4°C, and the upper aqueous layer was recovered for HPLC analysis. Reverse-phase HPLC was performed using a Waters Bondapak C18 column and a Waters HPLC system (Waters Associates) with a model 484 UV detector, 2 model 510 reciprocating pumps, and Maxima software. The intracellular ATP concentration was expressed as nanomoles per 10⁶ cells.

Data Analysis and Statistics
Data are reported as mean±SE. Statistical comparisons were made using the Student t test for paired and unpaired groups. ANOVAs were performed when multiple comparisons were involved. A difference was considered significant at P<0.05.

	Results

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Effects of H₂O₂ on HAEC pH_i
When HAEC monolayers were exposed to H₂O₂ for 10 minutes, a concentration-dependent intracellular acidification was observed. The threshold of this effect was 10 µmol/L (pH_i decrease of 0.15±0.02 at a concentration of 10 µmol/L, n=7, P<0.05 versus control, Figure 1A). The decrease in HAEC pH_i was typically observed within 2 minutes of exposure (Figure 1B) and was partially reversible on washout of H₂O₂ at concentrations of 1 to 100 µmol/L. At a concentration of 100 µmol/L H₂O₂, pH_i decreased from 7.24±0.01 to 7.02±0.02 (n=12) and then was partially reversible (pH_i=7.16±0.01 at 15 minutes) in 5 of the 6 monolayers in which pH_i was examined during H₂O₂ washout. At H₂O₂ concentrations of 250 µmol/L to 1 mmol/L, the intracellular acidification was irreversible on washout of H₂O₂ (n=7 for each concentration). Since 100 µmol/L H₂O₂ was the maximal concentration for which a reversible effect on pH_i was observed, this concentration was used in subsequent experiments.

View larger version (14K): [in this window] [in a new window]   Figure 1. Effects of H2O2 on HAEC pHi. A, Averaged data showing the relationship between H2O2 concentration (1 µmol/L to 1 mmol/L) and the decrease in pHi from baseline (pHi). H2O2 initiated a concentration-dependent decrease in pHi, which became significant at a concentration of 10 µmol/L (P<0.05 vs control, n=12 monolayers for 100 µmol/L concentration and n=7 for all others). B, Representative c-SNARF 1 fluorescence from an HAEC monolayer exposed to 100 µmol/L H2O2 in HEPES buffer with 1.5 mmol/L Ca2+. A partially reversible decrease in pHi that occurred within 2 minutes of exposure was observed (n=12).

Effect of Na⁺/H⁺ Exchange Inhibition on the H₂O₂-Induced Intracellular Acidification
To determine whether the effect of H₂O₂ on pH_i was related to Na⁺/H⁺ exchange inhibition, experiments were performed in which the Na⁺/H⁺ exchanger was inhibited before H₂O₂ exposure either pharmacologically (by pretreatment with EIPA), by removal of extracellular Na⁺ and replacement with equimolar choline,^{35 36} or by metabolic inhibition.^{21 26 27} Prior inhibition of the Na⁺/H⁺ exchanger by any of these 3 methods blocked the subsequent effect of H₂O₂ on pH_i. When HAEC monolayers were pretreated with 10 µmol/L EIPA, the effect of 100 µmol/L H₂O₂ on pH_i was significantly inhibited (pH_i=0.04±0.01 pH unit at 10 minutes, n=5, P<0.05 versus 100 µmol/L H₂O₂ without EIPA and P=NS versus time control). When HAEC monolayers were exposed to Na⁺-free buffer (Figure 2A), pH_i decreased over an 20-minute period from 7.23±0.01 to 6.98±0.02 (n=6). After this decrease in pH_i, the subsequent exposure to 100 µmol/L H₂O₂ in Na⁺-free buffer (n=4) did not significantly affect pH_i (pH_i=0.03±0.01 pH unit, P=NS versus control and P<0.05 versus 100 µmol/L H₂O₂ in Na⁺-containing HBS).

View larger version (16K): [in this window] [in a new window]   Figure 2. Effect of Na+/H+ exchange inhibition on the H2O2-induced change in pHi. A, Representative c-SNARF 1 fluorescence from an HAEC monolayer exposed to 100 µmol/L H2O2 in Na+-free HEPES buffer. After exposure to HEPES buffer in which Na+ was replaced by equimolar choline (0-Na+), pHi decreased over 20 minutes (n=6). After reaching a stable plateau at a new baseline pHi, exposure to 100 µmol/L H2O2 did not significantly affect pHi (P=NS vs control and P<0.05 vs 100 µmol/L H2O2 in Na+-containing buffer, n=4). B, Representative c-SNARF 1 fluorescence from an HAEC monolayer exposed to 100 µmol/L H2O2 in HEPES buffer with 100 µmol/L IAA. After exposure to IAA, pHi decreased over 15 to 20 minutes. After reaching a stable plateau at a new baseline pHi, exposure to 100 µmol/L H2O2 did not significantly affect pHi (P=NS vs control, n=5). The subsequent exposure to 4.7 mmol/L PA decreased pHi, indicating that the effect of IAA on H2O2-induced acidification was not due to a decrease in pHi alone.

In some experiments, the effect of Na⁺/H⁺ exchange inhibition by ATP depletion on the H₂O₂-induced acidification was examined by exposing cells for 20 minutes to the glycolytic inhibitor iodoacetic acid (IAA), a nonspecific alkylating agent that irreversibly and noncompetitively inhibits GAPDH.³⁷ This concentration of IAA was used because a previous study³⁸ showed that 10 minutes after exposure to 100 µmol/L IAA, endothelial pH_i decreased to a similar extent as did pH_i after exposure to 100 µmol/L H₂O₂ in the present study. As shown in Figure 2B, 100 µmol/L IAA decreased pH_i from 7.24±0.01 to 6.96±0.03 and inhibited the effect of a subsequent H₂O₂ exposure on pH_i (pH_i=0.02±0.01, n=5, P=NS versus control). This was not a nonspecific effect of IAA, since the subsequent exposure to 4.7 mmol/L propionic acid (PA) initiated a further decrease in pH_i of 0.11±0.02 pH unit, which was no different from that observed when HAEC monolayers were exposed to 4.7 mmol/L PA without IAA pretreatment (pH_i=0.13±0.00, n=4, P=NS versus pH_i change induced by PA after IAA).

Effect of H₂O₂ on Recovery From an NH₄Cl Prepulse-Induced Intracellular Acid Load
The NH₄Cl prepulse method of intracellular acid loading was used to examine the effects of H₂O₂ on Na⁺/H⁺ exchanger activity, since pH_i recovery from an acid load in bicarbonate-free buffer is due to H⁺ extrusion via the Na⁺/H⁺ exchanger and is blocked by pharmacological inhibition of the exchanger.³⁹ As shown in Figure 3, when HAECs were exposed to NH₄Cl in HBS, pH_i increased from 7.23±0.01 to 7.83±0.02 (n=9) because of the rapid diffusion of NH₃ into cells and its combination with H⁺.³² During exposure to NH₄Cl, pH_i gradually began to recover because of the slower diffusion of NH₄⁺ into cells, which subsequently dissociates into NH₃ and H⁺. When HAEC monolayers were then returned to NH₄Cl-free HBS, pH_i decreased to 7.03±0.02 because of the rapid diffusion of NH₃ from cells, leaving an excess of intracellular H⁺.³² Recovery then proceeded slowly over 15 to 20 minutes, with an increase in pH_i of 0.15±0.02 pH unit at 10 minutes. After pH_i recovered to baseline, a second NH₄Cl prepulse stimulated a similar effect on pH_i (Figure 3A) with an increase in pH_i of 0.12±0.01 pH unit at 10 minutes (n=9).

View larger version (14K): [in this window] [in a new window]   Figure 3. Effect of H2O2 on recovery from an intracellular acid load. A, Representative c-SNARF 1 fluorescence from an HAEC monolayer exposed sequentially to 20 mmol/L ammonium chloride (NH4Cl) in HEPES buffer with 1.5 mmol/L Ca2+. During exposure to NH4Cl, an increase in pHi was observed, with some pHi recovery during continued exposure. On washout of NH4Cl, pHi decreased below baseline before recovering over 15 to 20 minutes (n=9). B, Representative c-SNARF 1 fluorescence from an HAEC monolayer exposed to 20 mmol/L NH4Cl under control conditions (first exposure) and in the presence of the Na+/H+ exchange inhibitor EIPA (10 µmol/L, second exposure) during washout. EIPA inhibited pHi recovery (n=6, P<0.05 vs no EIPA). C, Representative c-SNARF 1 fluorescence from an HAEC monolayer exposed to 20 mmol/L NH4Cl under control conditions (first exposure) and in the presence of 100 µmol/L H2O2 (second exposure) during the washout. H2O2 inhibited pHi recovery (n=6, P<0.05 vs no H2O2).

Recovery from the intracellular acid load was almost completely inhibited when NH₄Cl washout occurred in the presence of 10 µmol/L EIPA (Figure 3B), with an increase in pH_i of 0.02±0.00 pH unit at 10 minutes (n=6, P<0.05 versus no EIPA). Recovery from the NH₄Cl prepulse-induced acid load was also inhibited when washout occurred in the presence 100 µmol/L H₂O₂ (Figure 3C, increase in pH_i of 0.02±0.00 pH unit at 10 minutes, n=6, P<0.05 versus no H₂O₂). To determine whether H₂O₂ had a similar effect under conditions of greater acid loading, which would be expected to result in more significant activation of the Na⁺/H⁺ exchanger, HAECs were exposed sequentially to 2 separate 50 mmol/L NH₄Cl prepulses, the first in HBS and the second in HBS with 100 µmol/L H₂O₂. Under these conditions, pH_i decreased to a nadir of 6.78±0.01 after washout of the first prepulse and 6.79±0.08 after washout of the second prepulse. Recovery from the 50 mmol/L NH₄Cl prepulse-induced acid load was inhibited by 100 µmol/L H₂O₂ (0.46±0.07 versus 0.18±0.06 pH unit, both at 10 minutes, n=3, P<0.02). Thus, the effects of this concentration of H₂O₂ on pH_i and on recovery from an intracellular acid load may be explained by Na⁺/H⁺ exchange inhibition.

Effect of H₂O₂ on H⁺ Production and on Intracellular Buffer Capacity
The rate of H⁺ production was assessed as net cellular H⁺ efflux from HAEC suspensions at baseline and after addition of H₂O₂. Under control conditions, HAECs produce H⁺ at a rate of 0.24±0.03 µmol/h per 10⁶ cells (n=6). There was a nonsignificant trend for the rate of acid production to decrease after the addition of 100 µmol/L H₂O₂ (0.21±0.02 µmol/h per 10⁶ cells, n=6, P=0.06 versus control). There was no significant change in the rate of acid production in time control experiments in which H₂O₂ was not added.

The effect of H₂O₂ on intracellular buffer capacity was examined using the NH₄Cl acid-loading method, since this represents an important mechanism to maintain pH_i homeostasis. In HBS, the calculation of buffer capacity ß from the first NH₄Cl exposure was 26.66±1.71 mmol/L per pH unit (n=21), and that from the second NH₄Cl exposure was 28.07±2.70 mmol/L per pH unit (n=9, P=NS). Addition of 100 µmol/L H₂O₂ during washout of a second NH₄Cl prepulse did not affect the ß value (31.50±3.19 mmol/L per pH unit, n=6, P=NS versus second control NH₄Cl exposure).

Effect of H₂O₂ on Intracellular ATP Concentration With and Without PARS Inhibition
Since ATP depletion reduces the activity of the Na⁺/H⁺ exchanger^{21 27} and H₂O₂ decreases cellular ATP in several cell types,^{16 18} the effect of H₂O₂ on intracellular ATP concentration in HAECs was examined. In these experiments, the effect of a 2-minute exposure to H₂O₂ was studied, since the effect of H₂O₂ on pH_i begins within this time period (see Figure 1B). As shown in Figure 4, 100 µmol/L H₂O₂ decreased intracellular ATP levels by 40% (9.95±0.76 versus 16.61±1.15 nmol/10⁶ cells, n=4 for each, P<0.05). This effect was partially reversible at this concentration of H₂O₂ after a 15-minute washout (13.10±0.85 nmol/10⁶ cells, n=4, P=NS versus control). A higher concentration of H₂O₂ (1 mmol/L) decreased ATP levels by 54% (7.65±0.21 nmol/10⁶ cells, n=4, P=NS versus 100 µmol/L H₂O₂), but the effect on intracellular ATP levels at this concentration was irreversible (P=NS versus no washout). Cell viability, as measured by Trypan blue exclusion, was not significantly affected by either concentration of H₂O₂.

View larger version (20K): [in this window] [in a new window]   Figure 4. Effect of H2O2 on intracellular ATP. Averaged data show the effect of a 2-minute exposure to H2O2 on intracellular ATP levels in HAECs. H2O2 produced a significant (*P<0.05 vs control, n=4) decrease in intracellular ATP levels, which was partially reversible on washout and was prevented by the PARS inhibitors 3-AB (1 mmol/L, n=5) and NIC (1 mmol/L, n=4) (+P<0.05 vs 100 µmol/L H2O2). At a higher concentration of H2O2 (1 mmol/L), ATP depletion was irreversible (n=4).

Since H₂O₂-induced ATP depletion may occur as a result of DNA strand breaks,^{12 13} with depletion of cellular energy stores occurring as a result of the subsequent activation of the repair enzyme PARS,¹⁵ the effect of the PARS inhibitors 3-aminobenzamide (3-AB) and nicotinamide (NIC) on H₂O₂-induced ATP depletion was examined. When HAEC monolayers were pretreated for 15 minutes with either 3-AB or NIC (each 1 mmol/L) before exposure to 100 µmol/L H₂O₂, ATP depletion was largely prevented. This concentration of 3-AB and NIC was used on the basis of previous studies showing that these PARS inhibitors prevented oxidant stress–induced DNA strand breaks and ATP depletion in several cell types, including endothelial cells.^{13 19 40 41 42} In HAEC monolayers pretreated with 3-AB, ATP levels were 93% of control after exposure to 100 µmol/L H₂O₂ (n=5, P=NS versus control). ATP levels were similarly preserved by NIC pretreatment (94% of control after 100 µmol/L H₂O₂, n=4, P=NS versus control). Neither 3-AB nor NIC alone affected ATP levels (93% and 95%, respectively; P=NS versus control, n=4 for each). In contrast to the effect of PARS inhibition before exposure to 100 µmol/L H₂O₂, 3-AB pretreatment did not prevent the effect of 1 mmol/L H₂O₂ on ATP levels (47% depletion, n=4, P<0.05 versus control, P=NS versus 1 mmol/L H₂O₂ without 3-AB pretreatment).

To determine whether prevention of ATP depletion by PARS inhibitors occurred by a nonspecific effect rather than one related to damage and repair of DNA, the effect of 3-AB pretreatment was examined before ATP depletion by a mechanism that is not known to involve PARS activation. HAECs were exposed to the glycolytic inhibitor IAA for 2 minutes. Exposure to 30, 40, and 100 µmol/L IAA decreased ATP levels by 36%, 45%, and 60%, respectively (n=4, P<0.05 versus control, P=NS versus 100 µmol/L H₂O₂ for each), but this was not affected by 3-AB pretreatment for either IAA concentration examined (44% depletion for 40 µmol/L IAA and 54% depletion for 100 µmol/L IAA, n=4, P=NS versus no 3-AB, not shown). IAA did not affect cell viability at these concentrations, as assessed by Trypan blue exclusion.

Effect of PARS Inhibitors on H₂O₂-Induced Intracellular Acidification and Inhibition of Recovery From an Intracellular Acid Load
Neither 3-AB nor NIC pretreatment (1 mmol/L for 15 minutes) affected HAEC pH_i (pH_i=0.02±0.01 pH unit for 3-AB and 0.02±0.02 pH unit for NIC, n=6 for both, P=NS versus control). When HAEC monolayers were exposed to 100 µmol/L H₂O₂ after 3-AB or NIC pretreatment, the effect of H₂O₂ on pH_i was significantly inhibited (pH_i=0.05±0.01 and 0.03±0.01 pH unit for 3-AB and NIC, respectively; n=6 for both, P<0.05 versus 100 µmol/L H₂O₂ without pretreatment, Figure 5). The ability of PARS inhibitors to attenuate the H₂O₂-induced acidification was not observed when HAEC monolayers were acidified by exposure to IAA. After 3-AB pretreatment, the subsequent exposure to 100 µmol/L IAA decreased pH_i by 0.24±0.01 pH unit (P=NS versus IAA without 3-AB pretreatment).

View larger version (13K): [in this window] [in a new window]   Figure 5. Effect of inhibition of PARS on H2O2-induced pHi. A, Representative c-SNARF 1 fluorescence from an HAEC monolayer exposed to 100 µmol/L H2O2 for 15 minutes after pretreatment with the PARS inhibitor 3-AB (1 mmol/L present throughout). 3-AB inhibited the subsequent effect of H2O2 on pHi (n=6, P<0.05 vs 100 µmol/L H2O2 without 3-AB). B, Averaged data showing the effect of 100 µmol/L H2O2 on pHi with and without pretreatment with 3-AB or NIC. Both 3-AB and NIC (each 1 mmol/L) inhibited the subsequent effect of H2O2 on pHi (n=6, *P<0.05 vs H2O2 alone for each).

The effect of PARS inhibition on the recovery from an NH₄Cl prepulse-induced acid load was examined with and without H₂O₂ present in the washout period (Figure 6). When HAEC monolayers were exposed to NH₄Cl in the presence of either 3-AB (n=7) or NIC (n=6), pH_i recovery on washout (first exposure in Figure 6A and 6B) was no different from that in the absence of these inhibitors (0.13±0.01 pH unit at 10 minutes for both 3-AB and NIC, P=NS versus control). As summarized in Figure 6C, both 3-AB and NIC blocked the inhibition of pH_i recovery induced by 100 µmol/L H₂O₂ during the second NH₄Cl washout period (0.09±0.01 pH unit 10 minutes after washout of NH₄Cl for 3-AB pretreatment and 0.09±0.01 pH unit for NIC pretreatment, P<0.05 versus 100 µmol/L H₂O₂ and P=NS versus control for both). Neither 3-AB nor NIC pretreatment affected the calculated ß value (34.15±4.24 mmol/L per pH unit for 3-AB and 33.08±2.73 mmol/L per pH unit for NIC, n=6 and P=NS versus control for each).

View larger version (16K): [in this window] [in a new window]   Figure 6. Effect of inhibition of PARS on H2O2-induced inhibition of recovery from an intracellular acid load. A, Representative c-SNARF 1 fluorescence from an HAEC monolayer exposed to 20 mmol/L NH4Cl under control conditions (first exposure) and in the presence of the 100 µmol/L H2O2 during washout (second exposure). The PARS inhibitor 3-AB (1 mmol/L) was present throughout. Inhibition of pHi recovery from an NH4Cl prepulse-induced acid load by H2O2 was prevented by 3-AB pretreatment (n=7, P=NS vs control). B, Representative c-SNARF 1 fluorescence from an HAEC monolayer exposed to 20 mmol/L NH4Cl under control conditions (first exposure) and in the presence of the 100 µmol/L H2O2 during washout (second exposure). The PARS inhibitor NIC (1 mmol/L) was present throughout. Inhibition of pHi recovery from an NH4Cl prepulse-induced acid load by H2O2 was prevented by NIC pretreatment (n=6, P=NS vs control). C, Averaged data showing the effect of PARS inhibitors on H2O2-induced inhibition of recovery from an intracellular acid load. The average pHi recovery 10 minutes after washout of 20 mmol/L NH4Cl is shown on the y-axis. H2O2 (100 µmol/L) significantly inhibited recovery from the intracellular acid load (*P<0.05 vs control, n=6). This effect was inhibited by pretreatment with either 3-AB (1 mmol/L, n=7) or NIC (1 mmol/L, n=6) (#P<0.05 vs 100 µmol/L H2O2 alone and P=NS vs control for each).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study demonstrates that H₂O₂ decreases pH_i, depletes intracellular ATP, and inhibits the Na⁺/H⁺ exchanger in human endothelial cells by a mechanism that is prevented by inhibition of the DNA repair enzyme PARS. At a concentration of 100 µmol/L, H₂O₂ decreases HAEC pH_i by 0.22 pH unit, inhibits pH_i recovery from an NH₄Cl prepulse-induced intracellular acid load, and decreases intracellular ATP levels by 40%. Although we cannot exclude the possibility that the effect of H₂O₂ on endothelial pH_i is in part due to an increase in acid production resulting from ATP hydrolysis, no effect on H⁺ efflux was observed when HAECs were exposed to 100 µmol/L H₂O₂ in the present study, and H₂O₂ did not increase H⁺ production in other cell types.¹⁰ The effects of H₂O₂ on pH_i and cellular ATP are prevented by pretreatment with the PARS inhibitors 3-AB and NIC, which themselves have no effect on pH_i, on pH_i recovery from an acid load, on intracellular buffer capacity, or on ATP levels. The H₂O₂-induced acidification is prevented after inhibition of the Na⁺/H⁺ exchanger by EIPA, by removal of extracellular Na⁺, or by glycolytic inhibition with IAA.

Plasmalemmal Na⁺/H⁺ exchangers are present in all mammalian cells and are involved in pH_i regulation in many cell types, including endothelial cells.^{35 36 43} The Na⁺/H⁺ exchanger extrudes H⁺ from the cell in electroneutral exchange for Na⁺ in a 1:1 stoichiometry.²⁹ Inhibition of the Na⁺/H⁺ exchanger by oxidant stress has been reported previously in several cell types,^{10 44} including vascular endothelial cells,¹¹ although the mechanism of this effect has not been determined.

The present study shows that H₂O₂ induces rapid inhibition of Na⁺/H⁺ exchange in human endothelial cells by a process associated with PARS activation and depletion of intracellular ATP. It is likely that this concentration of H₂O₂ does not result in complete inhibition of the exchanger, since pH_i recovery from an acid load was significantly, although not completely, inhibited by 100 µmol/L H₂O₂ when 50 mmol/L NH₄Cl was used to decrease HAEC pH_i to a degree that would be expected to maximally activate the Na⁺/H⁺ exchanger. H₂O₂ has been shown to rapidly induce time- and concentration-dependent intracellular ATP depletion in several cell types^{12 16 17 18} without affecting cell viability.^{12 17} In the present study, H₂O₂ decreased HAEC pH_i within minutes of oxidant exposure and produced a similarly rapid depletion of intracellular ATP. After only a 2-minute exposure, 100 µmol/L H₂O₂ decreased intracellular ATP concentration by 40%. This is similar to previously reported observations in alveolar type II cells, in which a 2-minute exposure to 500 µmol/L H₂O₂ reduced ATP levels by 60%.¹⁶

Changes in the concentration of intracellular ATP induced by H₂O₂ would be expected to affect the activity of the Na⁺/H⁺ exchanger. Intracellular ATP depletion inhibits the exchanger in several cell types, including vascular smooth muscle cells,²¹ rat ventricular cells,²² sheep Purkinje fibers,²⁶ A431 carcinoma cells,²⁰ alveolar type II cells,²⁴ renal epithelial cells,²³ and fibroblasts transfected with the growth factor–sensitive isoform of the exchanger, NHE-1.²⁷ Indeed, ATP depletion results in decreased activity of Na⁺/H⁺ exchange by all 3 exchanger isoforms.^{25 28} Although it has been suggested that ATP depletion affects basal Na⁺/H⁺ exchanger activity by decreasing phosphorylation of the antiporter,^{22 45 46} this concept has been challenged, since ATP depletion rapidly inhibits the Na⁺/H⁺ exchanger in NHE-1–transfected fibroblasts without affecting NHE-1 phosphorylation.²⁷ The mechanism of ATP depletion may be important in determining the effect on exchanger activity. In Purkinje fibers, glycolytic inhibition by 2-deoxyglucose inhibits the Na⁺/H⁺ exchanger, whereas the oxidative inhibitor cyanide does not, despite similar degrees of intracellular ATP depletion (70%) produced by each.²⁶ The Na⁺/H⁺ exchanger has a relatively low affinity for ATP, with inactivation reported at ATP concentrations in the millimolar range, consistent with a 10-fold lower affinity for ATP than the Na⁺-K⁺ pump.⁴⁷

In the present work, the effects of 100 µmol/L H₂O₂ on pH_i and ATP levels were at least partially reversible after washout, but at a higher concentration (1 mmol/L), these effects were irreversible. At high concentrations (>100 µmol/L), H₂O₂ inhibits ATP generation by affecting the glycolytic enzyme GAPDH.^{40 48} This appears to be due to direct oxidation of sulfhydryl groups on the active site of GAPDH.⁴⁰ At lower concentrations (100 µmol/L), GAPDH may be partially inhibited,⁹ but intracellular ATP depletion also occurs as a result of H₂O₂-induced DNA damage, subsequent activation of PARS, and consumption of NAD⁺.⁴⁸ Of note, in the present study, pretreatment with 3-AB or NIC prevented the effects of 100 µmol/L H₂O₂ on pH_i and intracellular ATP levels, but PARS inhibition did not prevent these effects of H₂O₂ at a concentration of 1 mmol/L and did not alter the effects of the GAPDH inhibitor IAA.³⁷ These data are most consistent with the predominant effect of 100 µmol/L H₂O₂ resulting from PARS activation in HAECs. Our results differ from those of Wu et al,⁹ who reported that 100 µmol/L H₂O₂ decreased pH_i in freshly dissociated rat cardiac myocytes and rat cardiac myoblasts by inhibiting glycolysis. This may relate to differences in cell type and to cell type–specific differences in glycolytic activity and dependence on glycolysis for ATP production. The effects of 3-AB and NIC cannot be explained by free radical scavenging activity, since neither of these PARS inhibitors affected free radicals generated by use of a Fenton reaction system⁴⁹ containing H₂O₂ (100 µmol/L) and 10 µmol/L Fe³⁺-nitrilotriacetate and assessed by electron paramagnetic resonance spectroscopy (data not shown).

Previous studies show that H₂O₂ induces rapid and concentration-dependent DNA strand breaks in vascular endothelial cells.^{13 14} DNA strand breaks activate the nuclear enzyme PARS, resulting in depletion of intracellular NAD⁺ and ATP levels. Pharmacological inhibition of PARS has been shown to prevent this effect in many cell types, including vascular endothelial cells.¹³ In murine macrophages, Schraufstatter et al¹⁵ showed that DNA single-strand breaks occur within seconds of exposure to micromolar concentrations of H₂O₂. Only 26% of DNA remained double-stranded after 5 minutes of exposure to 100 µmol/L H₂O₂. Within 1 minute, decreases in NAD and ATP levels were observed, and within several minutes, activation of PARS was noted.¹⁵

The effect of H₂O₂ on endothelial pH_i may play a role in the endothelial dysfunction that occurs as a consequence of ischemia and reperfusion. On reperfusion after a period of ischemia, the response to endothelium-dependent vasodilators is diminished, and previous work shows that oxidants play a role in this process.³ The H₂O₂-induced decrease in pH_i and Na⁺/H⁺ exchange inhibition may affect agonist-stimulated vasodilation due to either NO or prostaglandins. Relatively small pH changes within a narrow physiological range affect the activity of the constitutive NO synthase in endothelial cells,⁵⁰ with a decrease in pH_i inhibiting the enzyme. Pharmacological inhibition of endothelial Na⁺/H⁺ exchange significantly blocked the production of NO >15 minutes after stimulation with bradykinin, suggesting a role for the exchanger or endothelial pH_i in determining the activity of agonist-induced endothelium-dependent vasodilator function. The decrease in pH_i from 7.24 to 7.02 (due to exposure to 100 µmol/L H₂O₂ reported in the present study) would be expected to decrease the activity of constitutive NO synthase by 35% (I. Fleming, unpublished data, 1997). Inhibition of Na⁺/H⁺ exchange activity may also reduce endothelial synthesis or release of vasodilatory prostaglandins, since the stimulation of the exchanger appears to mediate agonist-induced prostaglandin release.⁴³ It is interesting that a recent study reports that inhibitors of PARS activity reduce myocardial infarct size in a rabbit model of I/R injury.⁵¹ Since 100 µmol/L H₂O₂ has been shown to mimic some of the pathophysiological responses observed in I/R injury^{52 53} and oxidants are associated with impaired endothelium-dependent relaxation,³ the effects of micromolar concentrations of H₂O₂ on pH_i described in the present study may be important in the pathogenesis of postischemic endothelial dysfunction.

	Acknowledgments

 
This study was supported in part by National Heart, Lung, and Blood Institute grants HL-03102 and HL-52315.

Received March 10, 1998; accepted June 30, 1998.

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