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Am J Physiol Heart Circ Physiol 277: H1505-H1512, 1999;
0363-6135/99 $5.00
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Vol. 277, Issue 4, H1505-H1512, October 1999

Loss of GSH and thiol enzymes in endothelial cells exposed to sublethal concentrations of hypochlorous acid

Juliet M. Pullar, Christine C. Winterbourn, and Margret C. M. Vissers

Free Radical Research Group, Department of Pathology, Christchurch School of Medicine, Christchurch, New Zealand


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We investigated the effect of sublethal concentrations of hypochlorous acid (HOCl) on intracellular thiol groups. Exposure of human umbilical vein endothelial cells to HOCl caused a decrease in cell viability, with concentrations of <= 25 µM HOCl being sublethal. At these concentrations, we saw a loss of glutathione and total protein thiol groups. Of the thiol enzymes we investigated, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was particularly susceptible to inactivation, creatine kinase was moderately susceptible, and lactate dehydrogenase was unaffected by HOCl at the concentrations used. Similar results were obtained with HOCl generated over 30 min by myeloperoxidase. GAPDH activity could be regenerated on reincubation of cells in Hanks' balanced salt solution or reduction with dithiothreitol. In contrast, glutathione loss was not reversible, and further decreased with time. Cellular ATP levels decreased with sublethal HOCl concentrations and this appeared to be unrelated to the inactivation of GAPDH. Our results demonstrate that intracellular thiol groups differ in their reactivity with HOCl and suggest that HOCl may be able to regulate specific cellular functions.

glyceraldehyde-3-phosphate dehydrogenase; creatine kinase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

HYPOCHLOROUS ACID (HOCl) is a powerful oxidant generated by the neutrophil enzyme myeloperoxidase (MPO) from H2O2 and chloride, and HOCl is thought to play a central role in the antimicrobial action of neutrophils (22). On stimulation and phagocytosis, HOCl production by the neutrophil accounts for up to 70% of the oxygen consumed, making it the major oxidative product of neutrophils (29). Although most of the HOCl generated in the inflammatory locus is directed at the target microorganism, the surrounding tissues are also vulnerable. HOCl has been implicated in the injury associated with neutrophil accumulation in myocardial reperfusion injury (59), inflammatory bowel disease (37), glomerulonephritis (30), rheumatoid arthritis (40), and respiratory distress syndrome (21).

HOCl is highly reactive with a range of biological substrates. It reacts readily with thiols and thioethers, Fe-S centers, and nucleotides (14, 61), with amines to form reactive chloramines (50), and with unsaturated fatty acids to form chlorohydrins (62). A body of work has been published focusing on the lethal, toxic nature of the oxidant, using bacteria (1, 24), red blood cells (56), tumor cells (9, 42), and mammalian cells (18) as targets. Most of this work has concentrated on the membrane as the target for HOCl reacting with cells (1, 4). However, previous work in this laboratory has shown that HOCl is able to pass through cell membranes (7, 58), and in red blood cells intracellular reduced GSH was a primary target (58). These studies have raised the possibility that lower, sublethal levels of HOCl may react with intracellular components and thereby affect cellular functions.

We investigated the reactivity of HOCl with intracellular constituents of cultured human umbilical vein endothelial cells (HUVECs). We investigated the effect of exposure to low levels of HOCl on cellular thiol status because HOCl reacts rapidly with thiol groups (14, 61), and thiol proteins play a crucial role in a number of cell processes, including ion transport, energy metabolism, and transcriptional regulation. The effects of HOCl on GSH, total cellular protein thiols and on the activities of the thiol-containing enzymes GAPDH, lactate dehydrogenase (LDH), and creatine kinase (CK) were investigated. We investigated the ability of the cell to recover from some of the HOCl-mediated redox changes and whether low, sublethal concentrations of HOCl caused depletion of cellular ATP.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Cell culture materials including medium 199, fetal bovine serum and penicillin/streptomycin were from GIBCO-Life Technologies (Auckland, New Zealand). CLS type I collagenase was from Worthington Biochemical (Freehold, NJ) and heparin was from Fisons (Sydney, Australia). Fibronectin was purified from human plasma by affinity chromatography on gelatin Sepharose as previously described (57). Purified human MPO was a gift from Dr. Tony Kettle (27). [51Cr]chromate was supplied by Amersham Australia. Sodium hypochlorite was from Reckitt and Coleman (Auckland, New Zealand). Monobromobimane was from Calbiochem (La Jolla, CA), and pyruvate kinase and lactate dehydrogenase were from Boehringer Mannheim (Mannheim, Germany). The ATP assay kit was supplied by Molecular Probes (Eugene, OR). The apoptosis test kit (Apoptest FITC kit) used to test cell viability was supplied by NeXins Research BV (The Netherlands). Protein was assayed using a kit supplied by Bio-Rad (Hercules, CA). Sigma Chemical (St. Louis, MO) supplied all other chemicals.

Cell culture. After we obtained informed consent from patients, HUVECs were isolated from umbilical cords by collagenase digestion (26). The cells were cultured in medium 199 supplemented with 20% FBS, 100 µg/ml heparin, 30 µg/ml endothelial cell growth factor, 25 U/ml penicillin, and 25 µg/ml streptomycin. Flasks and plates were precoated with 20 µg/ml fibronectin, and cultures were maintained in a 37°C incubator with 5% CO2. Cells were used after the second or third passage. HUVECs were identified by their typical morphology, including the presence of Wiebel-Palade bodies and cobblestone pattern on confluency.

Cell treatment. HOCl concentration was established by reaction with 5-thio-2-nitrobenzoic acid and measurement of A412 (epsilon  = 28,200 M-1 · cm-1) (55) or alternatively using the extinction coefficient of hypochlorite epsilon 292 = 350 M-1 · cm-1 at pH 10-12 (16). At pH 7.4 solutions contained about equimolar concentrations of HOCl and OCl- (pKa = 7.6) but are referred to as HOCl. HOCl was diluted in Hanks' balanced salt solution (HBSS): PBS, pH 7.4, containing 5.5 mM glucose, 0.5 mM magnesium, and 1.0 mM calcium to final concentrations ranging from 0 to 50 µM. Confluent cells were washed in HBSS three times, 1 ml of the relevant HOCl solution was quickly added to each well, and the cells were incubated at 37°C for the times indicated. In some cases, cells were reincubated after the 10-min HOCl treatment by removal of the supernatant and incubation of the cells in fresh HBSS for an additional hour. All assays were performed in 24-well plates with confluent cultures (~1.2 × 105 cells/well). Therefore, a 50 µM dose of HOCl is equivalent to ~50 nmol of HOCl per 1.2 × 105 cells. Each well contained ~40 µg of protein.

Alternatively, HUVECs were exposed to HOCl generated over 30 min by an MPO-H2O2-Cl- system, with continuous generation of H2O2 by glucose oxidase. The H2O2 generation rate was measured using a YSI model 25 oxidase meter fitted with a YSI 2510 probe (Yellow Springs Instruments, Yellow Springs, OH), as described previously (28). HUVECs were exposed to a flux of HOCl by incubating cells in 1 ml of HBSS containing 100 nM MPO and sufficient glucose oxidase to give 0, 15, 25, 50, and 100 µM H2O2 over 30 min at 37°C.

Cytotoxicity assays. Confluent cells were preincubated with [51Cr]chromate in complete medium for 3-4 h, washed in HBSS and exposed to HOCl. Aliquots of the supernatant were removed and spun, and the radioactivity was counted. Released 51Cr is expressed as a percentage of the total cellular radioactivity estimated after lysis with Nonidet P-40 (NP-40).

Alternatively, the number of viable cells was determined using a flow cytometry assay to distinguish between viable, apoptotic, and necrotic cells (52). This assay uses annexin V to detect the phosphatidylserine that transfers to the outer leaflet of the membrane during apoptosis and propidium iodide uptake to detect necrotic cells. Confluent cells were washed and the medium was retained. Cells were then treated with HOCl (0-50 µM) for 1 h at 37°C. The HBSS was removed, centrifuged to pellet any detached cells, and both the detached cells and those remaining on the plate were reincubated in the conditioned medium at 37°C for 5 h. After this procedure, adherent cells were removed from the plate with trypsin, pooled with the previous detached cells, and washed. Annexin V was added according to manufacturer's instructions, along with 1 µl of 2 mg/ml propidium iodide, and the cells were left for 10 min in the dark. The cell fluorescence was measured using a bivariate flow cytometer. Results were analyzed as the percentage of viable cells and expressed as percentage of control (see Fig. 3A).

Glutathione measurements. Reduced glutathione was analyzed using an adaptation of the method of Martin and White (36). Cells were treated with HOCl or the MPO system, and thiol groups were blocked with iodoacetic acid and subsequently derivatized with dansyl chloride. Cell extracts were then separated by HPLC, using a 5-µm NH2 silica LiChrospher column (E Merck, Alltech) with a fluorescent detection system (excitation 328 nm and emission 542 nm). The retention times of the cell extract peaks were compared with standard GSH.

Total protein thiols. After treatment with HOCl, cells were brought to pH 8.0 with KOH, and reacted with 12 mM monobromobimane for 20 min in the dark (8). Protein was precipitated with 5% ice-cold perchloric acid and washed three times with 5% trichloroacetic acid. Samples were resuspended in 1% sodium dodecyl sulfate and their fluorescence was measured (excitation 394 nm and emission 480 nm). Protein concentrations of samples were measured using a detergent-compatible Bio-Rad protein assay based on the Folin-Lowry method. Results are expressed as percent control after correcting for protein levels. To obtain molar values for control HUVEC protein thiols, perchloric acid extracts of cells were reacted with 100 µM 5,5'-dithiobis(2-nitrobenzoic acid), and absorbances were measured at 412 nm (epsilon  = 14,100 M-1 · cm-1) (5).

Enzyme assays. Ten minutes after exposure to HOCl or after 30 min with the MPO system, cells were washed and lysed by the addition of 0.025% NP-40 for 30 min at 37°C. Samples were removed from the well and spun to remove debris, giving a cytoplasmic extract. GAPDH (5), LDH (11), and CK (49) were assayed using standard spectrophotometric techniques adapted for use in a 96-well plate reader.

The ability of the cells to regenerate GAPDH activity was investigated. The enzyme activity was assayed either after reincubation with fresh HBSS for 1 h or in cytoplasmic extracts of the cells that were prepared after 10 min of HOCl treatment then incubated with 5 mM dithiothreitol (DTT) for 1 h at 37°C.

ATP levels. ATP concentrations in cell samples were measured with a Molecular Probes kit using luciferase and luciferin (12) and a Bio · Orbit 1253 luminometer. The kit was altered to make it more sensitive by decreasing the luciferin concentration to 20 µM and increasing the final tricine buffer concentration to 100 µM. After HOCl treatment, ATP was extracted from cells by adding ice-cold perchloric acid (3% vol/vol), 2 mM EDTA, and 0.5% Triton X-100 to wells and leaving the wells on ice for 5 min. Extracts were removed from the well, centrifuged (13,000 g, 1 min), and either snap frozen and stored at -80°C for later analysis or placed on ice for analysis that same day. Perchloric acid-extracted samples were neutralized with a potassium hydroxide solution (2 M KOH, 2 mM EDTA, 50 mM MOPS) and left on ice for 10 min. Samples were then spun and the supernatant assayed for ATP as per kit instructions.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Characterization of HOCl cytotoxicity. HOCl is rapidly consumed on addition to HUVECs. With 35 µM HOCl, half of the HOCl was consumed within 30 s of its addition, with the remainder disappearing during the next 15 min. In the subsequent experiments, most of the assays were performed 10 min after the addition of HOCl to the cells.

HOCl (0-50 µM) was added to the cells in a 1-ml volume; addition in a larger volume changes the ratio of oxidant to cells and alters the toxicity profile. Exposure of HUVECs to HOCl (0-50 µM) produced a concentration- and time-dependent release of 51Cr from the cells (Fig. 1). At the higher HOCl concentrations 51Cr leakage was apparent within 30 min of exposure. At these HOCl concentrations, maximum 51Cr release rarely exceeded 50%. However, up to 90% release was seen with higher HOCl concentrations (not shown). At <25 µM HOCl, minimal leakage of 51Cr was observed over time. This correlated well with the visual changes seen in the cells. Control cells looked very similar to cells exposed to 20 µM HOCl after 3 h of treatment (Fig. 2, A and B). Interestingly, 20 µM HOCl at 10 min caused contraction in cells with the formation of intercellular spikes. However, the original morphology was recovered within 1 h of treatment. At higher doses, effects were more extreme; within 10 min of exposure, 50 µM HOCl caused contraction and rounding of the cells (Fig. 2C). Cells were beginning to detach after 1 h, with a majority of cells detached after 3 h (Fig. 2D).


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  		Fig. 1.   Time course of hypochlorous acid (HOCl)-mediated chromium-51 release. Confluent human umbilical vein endothelial cells (HUVEC) were exposed to HOCl (0-50 µM), aliquots were removed at intervals, and results are presented as percentages of total cellular counts (means ± SD of 3 experiments). Concentration of HOCl was 0 µM (), 10 µM (), 20 µM (black-triangle), 25 µM (black-down-triangle ), 35 µM (black-diamond ), and 50 µM (hexagon).



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  		Fig. 2.   Morphology of HUVECs after HOCl treatment. Confluent HUVECs were treated with 0, 20, and 50 µM HOCl for times indicated: control at 0 time (A), 20 µM HOCl at 3 h (B), 50 µM HOCl at 10 min (C), and 50 µM HOCl at 3 h (D). Magnification ×100.

More information on the concentrations of HOCl that were sublethal was obtained using a flow-cytometry method to distinguish viable cells from those that were necrotic or apoptotic. HOCl, depending on the concentration, causes endothelial cells to die by apoptosis or necrosis (unpublished observations). Flow-cytometry measurements, made 6 h after HOCl treatment, confirmed that concentrations of <= 25 µM HOCl were sublethal with a progressive decrease in viability above this (Fig. 3B).


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  		Fig. 3.   Cell viability after 6 h of HOCl treatment as determined by flow cytometry. A: fluorescence-activated cell sorter (FACS) analysis of HUVECs. Viable, apoptotic, and necrotic cells were distinguished on basis of apoptotic cells being positive for annexin V (R2) and necrotic cells being positive for both propidium iodide (PI) and annexin V (R3). Percentage of viable cells represents proportion within R1. B: dependence of viability on HOCl concentration ranging from 0-50 µM. Results are expressed as means ± SD of 3 separate experiments. Results of 1-way ANOVA indicate a significant decrease in viability with increasing HOCl (P < 0.005).

Cell thiol changes. With increasing concentrations of HOCl, there was a progressive loss of intracellular GSH at 10 min posttreatment (Fig. 4). This loss became apparent at concentrations higher than 10 µM HOCl, and at 50 µM HOCl most of the GSH was oxidized. At this point the ratio of HOCl added to GSH lost was ~40. With extended incubation there was an additional drop in GSH levels. Investigation of the reaction of GSH with HOCl in endothelial cells indicated that very little GSSG was formed, and that more unusual products were generated. These results are reported in detail elsewhere (unpublished observations).


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  		Fig. 4.   Loss of intracellular GSH after exposure of HUVECs to HOCl. GSH levels were assayed after 10 min of HOCl treatment and after an additional 1-h incubation in fresh Hanks' buffered saline solution (HBSS). Results are means ± SD of 2-4 experiments with duplicates in each experiment. Each well contained 1.66 ± 0.44 nmol of GSH. A 1-way ANOVA established a significant decrease in GSH with increasing HOCl at 10 min (P < 0.001). Two-way ANOVA established a significant difference between the 2 curves (P < 0.001).

Each well of HUVECs contained ~25 nmol of protein thiols. Consequently the concentration of protein thiols was 15 times that of the GSH. With increasing concentrations of HOCl there was a decrease in endothelial protein thiol groups (Fig. 5). At 50 µM HOCl at least 50% of protein thiols were modified, and at this concentration, at least one in every four HOCl molecules was reacting with a protein thiol group.


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  		Fig. 5.   Loss of HUVEC protein thiols on exposure to HOCl. Results were calculated as fluorescence U/µg protein and further expressed as percentage of control. Results are means ± SD of 3 experiments. Each well contained 25 ± 11 nmol of protein thiol groups. A 1-way ANOVA established a significant decrease in protein thiol groups with increasing HOCl (P < 0.005). Individual values for HOCl-treated samples were significantly different from control at >= 15 µM HOCl as determined by a paired Student's t-test, P < 0.05.

Thiol enzyme inactivation. GAPDH, LDH, and CK exhibited very different susceptibilities to HOCl (Fig. 6). GAPDH was particularly sensitive, with >= 10 µM HOCl causing a significant loss in activity. CK was moderately susceptible, whereas LDH underwent only a very small loss in activity at 50 µM HOCl.


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  		Fig. 6.   Relative susceptibilities of endothelial cell thiol-containing enzymes to HOCl. Top: Lactate dehydrogenase (LDH) activity; middle: creatine kinase (CK) activity; bottom: glyceraldehyde 3-phosphate dehydrogenase (GAPDH) activity. Results are percentage of control and are means ± SE of 4-10 separate experiments. LDH activity was not significantly different from control at any HOCl concentration tested as determined by Mann-Whitney's rank sum test. CK activity differed from control >= 15 µM (P < 0.02) and GAPDH from >= 10 µM (P < 0.05; Mann-Whitney's rank sum test).

There were also changes in thiol enzymes and GSH when HUVECs were exposed to a continuous flux of HOCl using the MPO-H2O2-Cl- system (Fig. 7). GAPDH was particularly susceptible to inactivation by a continuous generation of HOCl and creatine kinase was moderately susceptible. A loss in cellular GSH levels was apparent with a flux of HOCl. These changes were not a result of the H2O2 generated by glucose oxidase, as there was no effect in the absence of MPO (not shown). Thus the relative susceptibilities of these thiols were identical whether HOCl was generated continuously or added in a bolus.


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  		Fig. 7.   Cell thiol loss after treatment of HUVECs with myeloperoxidase (MPO)-H2O2-Cl- system. H2O2 was generated by glucose oxidase over 30 min in presence of MPO. Cells were then assayed for GAPDH, GSH, and CK. Results are expressed as percentage of control and are means ± SD for 2-3 experiments, each performed in duplicate. A 1-way ANOVA on each curve established a significant decrease in GSH levels and GAPDH activity with increasing HOCl (P < 0.05). A significant decrease was not established for CK activity.

The ability of the cells to recover GAPDH activity was investigated. After HOCl treatment, cells were reincubated for 1 h in HBSS. GAPDH activity was almost completely regenerated at the lower HOCl concentrations (Fig. 8A). At 50 µM HOCl, GAPDH activity appeared to be only partially restored, although there was some variability at this concentration. Similarly, CK activity lost after exposure to HOCl could be restored on reincubation in HBSS (not shown).


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  		Fig. 8.   Regeneration of GAPDH activity by reincubation in HBSS and by dithiothreitol (DTT). A: reincubation in HBSS. After a 10-min HOCl treatment, fresh HBSS was added for an additional hour. Reincubated samples were significantly different from respective 10-min controls at 10 and 25 µM HOCl (* P < 0.05). B: DTT treatment. After 10 min with HOCl, cells were lysed and extract was DTT treated. Results are means ± SD of 3 and 5 experiments for reincubation curve and DTT, respectively. DTT treatments at 25 and 50 µM HOCl were significantly different from their respective 10-min controls (* P < 0.05) as determined by a paired Student's t-test.

Extracts from HOCl-exposed cells were also treated with DTT, which at the concentrations used can reduce protein-disulfides and many sulfenic acid residues (2). DTT raised the basal GAPDH activity, suggesting that a proportion was present in disulfide form in controls (Fig. 8B). At 10 and 25 µM HOCl, GAPDH activity was restored by DTT treatment, whereas at 50 µM HOCl it was only partially regenerated (Fig. 8B). DTT regenerated CK activity in a similar fashion, although this was not always significant as a result of the relatively small HOCl-induced loss in CK activity (not shown).

ATP levels. The effect of HOCl exposure on ATP levels was measured to determine whether the loss in GAPDH activity affected cellular energy status. After the 10-min treatment, a progressive loss in ATP was observed with increasing HOCl concentrations above 15 µM (Fig. 9). After reincubation in fresh HBSS for 1 h there was an additional drop in cellular ATP levels, particularly at the higher HOCl doses. The decreased ATP levels seen at 1 h were maintained over the next 5 h (not shown).


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  		Fig. 9.   ATP levels in cells exposed to HOCl (0-50 µM). Results are expressed as percentage of control and are means ± SD of 4-6 separate experiments. ATP concentration was measured 10 min after treatment and after additional 1-h reincubation in fresh HBSS buffer. Controls contained 5.06 ± 2.05 nmol ATP/well (n = 7). A 1-way ANOVA established a significant decrease in ATP at 10 min (P < 0.005); this became significant only at 50 µM HOCl (P < 0.05). After reincubation for an additional hour there was a significant decrease in ATP levels compared with control levels (P < 0.0001); loss became significant at 35 and 50 µM HOCl (P < 0.05). Significant difference between the 2 curves was established using 2-way ANOVA (P < 0.05).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Exposure to reactive oxygen species is known to affect cells in a variety of ways. High levels of exposure are cytotoxic, and cell death can occur via an overwhelming onslaught to many vital cell components, including membranes, protein, and DNA (20). More recently it has been demonstrated that low levels of oxidants can modulate cell processes and affect signal transduction (46). In this way sublethal levels of H2O2 have been shown to affect the levels of transcription factors (3) and heat shock proteins (25), the activity of specific kinases (39, 60), and to induce apoptosis (15), cell proliferation (6), and growth arrest (10). Similar effects also occur with nitric oxide (13), organic peroxides (19), and peroxynitrite (31). We were interested in whether HOCl could act in a similar manner. Given the prevalence of neutrophils at inflammatory sites, HOCl and chloramines derived from it are relevant oxidants in vivo. Although neutrophils generally direct their oxidants at phagocytosed organisms, the surrounding tissues can also be a target and can be exposed to varying doses of HOCl or chloramines. In support of this, recent studies have demonstrated the presence of markers of HOCl at inflammation sites (23, 34).

A number of studies have determined the effects of HOCl on cell thiol status and have shown that HOCl can cause GSH loss in erythrocytes (58), neutrophils (7), and other mammalian cells (48). Although previous investigations (7, 48) have shown a loss in GSH and protein thiols, this is the first more detailed look at the relative susceptibilities of GSH and specific thiol proteins at sublethal levels of HOCl exposure. We have shown that low amounts of HOCl, either added as a reagent or generated by MPO cause significant loss of cellular thiols in cultured HUVECs. Both GSH and protein thiols were affected and some thiol-containing enzymes were preferential targets for HOCl. When HOCl was continuously generated by the MPO system over 30 min, the pattern of thiol loss was similar to that seen with a bolus addition of HOCl, indicating that the relative susceptibilities of the targets for HOCl was independent of the time of exposure to the oxidant.

To assess relative susceptibilities of different cell parameters, including viability, 50% inhibitory concentrations for HOCl were calculated (Fig. 10). Of the thiols measured, GAPDH was most susceptible to inactivation by HOCl. The IC50 value for GAPDH was much lower than that for the total protein thiols, reinforcing the comparative sensitivity of GAPDH to HOCl. GSH was more sensitive to oxidation than the total protein thiol groups, as indicated by its lower IC50. We have shown a range of susceptibilities for different thiol proteins and would expect GAPDH and other sensitive targets to account for the loss in total protein thiols at the low HOCl concentrations that did not affect GSH. Particularly at the lower concentrations, a major proportion of the HOCl could be accounted for, on a molar basis, as thiol loss. Protein thiols are present at a 15-fold excess over GSH, and accounted for most of this HOCl. Other targets of HOCl are also likely, and could be significant in HUVECs at higher, lethal doses of HOCl. We have previously detected chloramine formation and protein cross-linking in red blood cells, and this latter reaction, rather than thiol oxidation, correlated with red blood cell lysis (54).


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  		Fig. 10.   HOCl concentrations that gave 50% inhibition (IC50) for different cell parameters; GSH, PSH (protein thiol groups), ATP10 (ATP levels at 10 min), ATP70 (ATP levels after 1-h reincubation), VC (viable cells as measured by flow cytometry), CK activity, and GADPH activity.

Our findings that HOCl shows a high reactivity with apparent selectivity for particular thiols raises the possibility that HOCl could regulate cell function through specific thiol oxidation. Support for this idea comes from the finding that HOCl can raise the levels of the transcription factor p53 in human skin fibroblasts (53). A study by Schoonbroodt et al. (41) has shown that HOCl can cause the activation of the transcription factor NF-kappa B in a lymphocytic cell line. However, the HOCl was added in the presence of whole medium, and this effect is likely to be caused by the action of secondary chloramines. Whether either of these mechanisms involves the oxidation of specific thiols is unknown.

The difference in susceptibility of the protein thiols to HOCl may be related to a number of factors, including accessibility and reactivity. GAPDH is also sensitive to other oxidants (51), and oxidative regulation of its activity may be important more generally (35). Studies have shown that the GAPDH thiol in the active site is particularly reactive and that it is ionized at neutral pH (17). Our finding that treatment of the cells with DTT raises the basal levels of GAPDH suggests that a proportion of this enzyme is oxidized under control conditions. However, Lind and co-workers (32) have recently demonstrated that S-thiolation of GAPDH does not inactivate the enzyme, suggesting that S-thiolation is unlikely to be the mechanism for the inactivation of GAPDH by HOCl.

Although the products of the HOCl-induced oxidation of thiols were not determined in this study, we found very little GSSG or evidence for protein S-thiolation in HOCl-treated endothelial cells. GSH loss was irreversible and actually increased with time, which is consistent with limited GSSG or mixed-disulfide formation. The products are under investigation and will be described elsewhere (unpublished results). In contrast, reincubation of the cells in HBSS or with DTT was able to restore the enzyme activity, suggesting the formation of disulfides or a sulfenic acid, as is seen with other oxidative systems (33, 45). At higher levels of HOCl exposure, the activity of GAPDH and CK was not reliably restored, suggesting that the thiol group was further oxidized to a higher oxidation state, or that there was irreversible modification of another residue on the protein.

We were interested in whether the decreased activity of the glycolytic enzyme GAPDH transcribed to an effect on cellular energy status. ATP levels fell within 10 min of HOCl treatment and decreased further after a 1-h incubation. Although it is unclear whether the early drop in ATP levels is related to GAPDH, the later loss is unlikely to be caused by the inactivation of GAPDH because at this time point enzyme activity was mostly restored. The direct reaction of ATP with HOCl could cause the initial decrease (38) but not the further drop, and several mechanisms could explain this loss of cellular ATP. A decrease in ATP synthesis either by impairment of glycolysis (other than GAPDH inhibition) or mitochondrial oxidative phosphorylation is one possibility. Alternatively, HOCl could increase the activity of ATPase pumps in the membrane (47). Loss in ATP caused by the activation of poly(ADP-ribose) polymerase by DNA damage (43) is unlikely, because HOCl is not known to cause alterations in DNA at sublethal levels (44).

In summary, our results show that at sublethal levels HOCl causes a loss of cell thiols. The results suggest that this oxidant may be able to regulate cell function by altering the redox balance of the cell or by selectively reacting with thiol proteins that control critical cell processes. The endothelial cell is a particularly relevant model for this interaction because of its unique association with neutrophils in the inflammatory response. Further work is required to characterize these reactions and to determine their significance.


    ACKNOWLEDGEMENTS

We thank Dr. John Babson for help in setting up the glutathione assay.


    FOOTNOTES

This work was supported by the Health Research Council of New Zealand and the University of Otago in Dunedin, New Zealand.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. Pullar, Free Radical Research Group, Christchurch School of Medicine, PO Box 4345, Christchurch, New Zealand (E-mail: juliet.pullar{at}chmeds.ac.nz).

Received 19 November 1998; accepted in final form 20 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Albrich, J. M., J. H. Gilbaugh, III, K. B. Callahan, and J. K. Hurst. Effects of the putative neutrophil-generated toxin, hypochlorous acid, on membrane permeability and transport systems of Escherichia coli. J. Clin. Invest. 78: 177-184, 1986.

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Am J Physiol Heart Circ Physiol 277(4):H1505-H1512
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