Novel synthesis of S-nitrosoglutathione and degradation by human neutrophils

ANALYTICAL

204,

BIOCHEMISTRY

365-371

(1992)

Novel Synthesis of S-Nitrosoglutathione and Degradation by Human Neutrophils Robert

M. Clancy

and Steven

B. Abramson

Department of Medicine, Division of Rheumatology, New York University Medical Center, 530 First Avenue, Neua York, New York 10016 and Department of Rheumatic Diseases at the Hospital for Joint Diseases, 301 East 17th Street, New York, New York 10003

Received

March

19, 1992

S-nitrosoglutathione (SNO-GSH), a stable derivative of nitric oxide, is an endothelium-derived relaxation factor, which provokes vasodilation, inhibits platelet aggregation, and inhibits neutrophil (PMN) superoxide anion (0:) generation. We have established a novel method for synthesis of S-nitrosoglutathione using a column containing S-nitrosothiol covalently attached to agarose. S-nitrosoglutathione was a product as assessed after separation using C-18 reverse-phase HPLC and absorption spectroscopy. We examined the stability of SNO-GSH in the presence or absence of PMN. The halflife (mercuric acid diazotization) of SNO-GSH in Hepes was >60 min. The addition of resting PMN did not affect the Ti of SNO-GSH. PMN exposed to N-fMet-LeuPhe (FMLP, lo-’ M) reduced measurable SNO-GSH (15 FM) at 5 min (48 + 5.6% control, P < 6.05). Incubation (5 min, 37°C) of PMN with 10 pM tenidap (an anti-inflammatory drug which inhibits PMN activation) before addition of FMLP blocked the PMN-dependent degradation of SNO-GSH (42 2 3 vs 78 + 1.3% control, P = 0.01). We confirmed the recovery of SNO-GSH through measurements by bioassay (platelet aggregation) and HPLC analysis. The degradation of S-nitrosothiols by activated neutrophils may reverse the inhibitory effect of S-nitrosothiols on PMN functions and contribute to tissue injury at sites of inflammation. Q is92 Academic Press,

trophil-endothelial cell interaction by inhibiting neutrophi1 functions which include LTB, synthesis, release of 0; and neutrophil adhesion to vascular endothelium (l-3). Since nitric oxide reacts with reduced thiol to form S-nitrosothiol, the focus on biological effects of nitric oxide has expanded to include S-nitrosothiols (4,5). These compounds (S-nitrosocysteine, S-nitrosoglutathione) also inhibit neutrophil functions (2). Thus, release of nitric oxide and S-nitrosothiols by endothelial cells may modulate neutrophil margination and diapedesis. Nitric oxide rapidly degrades to inactive inorganic salts, which limits the possible biological effects of nitric oxide (4-8). It is known that oxidants such as superoxide anion react to form nitrate and nitrites (9). However, the susceptibility of S-nitrosothiols to oxidants derived from activated neutrophils has yet to be investigated. The purpose of this study is to report a novel method of S-nitrosothiol synthesis as well as to examine the stability of S-nitrosothiols in aqueous solution and neutrophil cell suspensions. The results show that activated neutrophils play a pivotal role in the metabolism of S-nitrosothiol and thereby modulate the biological effects of EDRF. METHODS

Inc.

Neutrophils and endothelial cells undergo a dynamic interaction that is required for the migration of neutrophils from peripheral blood to sites of inflammation. Recent studies suggest that nitric oxide, an endothelialderived relaxation factor (EDRF)’ contributes to neu’ Abbreviations used: Ol, SNO-GSH, S-nitrosoglutathione; 0003.2697192

Copyright All rights

0 of

$5.00 1992

superoxide HgCl,,

by Academic Press, Inc. reproduction in any form reserved.

anion; PMN, neutrophils; mercuric chloride; FMLP,

Neutrophil studies. Human neutrophils were isolated from whole blood after centrifugation through Hypaque-Ficoll gradients, sedimentation through dextran (6% wt/vol), and hypotonic lysis of red blood cells

N-formyl-methionine-leucine-phenylalanine; GSH, glutathione; Hepes, N-2hydroxyethylpiperazine-N’-2-ethanesulfonic acid; Tris, tris(hydroxymethyl)aminomethane; ADP, adenosine diphosphate; NSAID, nonsteroidal anti-inflammatory drug; NaNO,, sodium nitrite; EDRF, endothelial-derived relaxation factor; DMSO, dimethyl sulfoxide. 365

366

CLANCY

AND

(4). The order of addition for the cell reaction mixture was neutrophils (5 X 106/ml), cysteine (30 PM), S-nitrosoglutathione (varied concentration), cytochalasin B (Sigma, 50 pg/ml), with or without N-fMet-Leu-Phe (FMLP; Sigma, 10e7 M). In some experiments, neutrophils were incubated (5 min, 37°C) with tenidap (varied concentration, Pfizer), piroxicam (IO0 pM, Sigma), salicylate (100 pM, Sigma), or superoxide dismutase (Boehringer-Mannheim, 50 pglml). Platelet aggregation studies. Platelets were obtained as a platelet-rich plasma, which was derived by centrifugation of whole blood (containing citrate as anticoagulant) at 200g for 20 min at room temperature. Aggregation (induced by 2.5 pM ADP) was measured by the change in light transmittance through the stirred suspension of cells (3 X 105/mm3) as monitored in a dual chamber aggregometer (Bio Data Corp, Model PAP4) (7). Quantitative determination of thiols and S-nitrosothiols. The concentration of cysteine was determined after reaction with 1 mM dithionitrobenzoic acid (Sigma) at pH 8.1 by using E = 12.3,, - 1 at 412 nm (Milton Roy 1201 spectrophotometer). S-Nitrosocysteine and S-nitrosoglutathione were quantitated using Greiss reaction as the difference (measured spectrophotometrically) between sample incubated with or without prior treatment with mercuric chloride. Briefly, treatment of S-nitrosocysteine with mercuric chloride (0.01%) hydrolyzed the S-nitrosbond and results in release of an equivalent of nitrite (10). The Greiss reaction employed diazotization of nitrite with sulfanilamide (Aldrich, 0.3% in 0.1 M phosphoric acid) and subsequent coupling with N-(l-naphthyl) ethylenediamine (Aldrich, 0.03% in water) (11). Nitrite concentration was determined using E = 45,, - 1 at 548 nm (Milton Roy 1201 spectrophotometer). Synthesis of red agurose. Dithiobis(ethylamine) diHC1 (ICN, 150 mg/l ml DMSO) was added to 25 ml Affi-Gel 10 (Bio-Rad) plus 0.4 ml triethylamine (Fisher) and was allowed to react for 30 min at room temperature. Resin was washed and equilibrated with 0.1 M Tris, pH 8.1. The disulfide bond was reduced with addition of 0.1 M fi-mercaptoethanol for 30 min at 40°C. Bio-Gel A-SH was obtained when HCl(O.2 M) was added and the resin was washed and equilibrated with 0.1 M acetate, pH 2.0. Bio-Gel A-SNO was obtained when 690 mg of sodium nitrite was added to 10 ml of Bio-Gel A-SH and allowed to react for 2 min at 37°C. Resin was washed and equilibrated with 0.1 M acetate, pH 2.0. Red agarose was prepared by combining 1 part Bio-Gel A-SH to 9 parts of Bio-Gel A-SNO. Red agarose was equilibrated with 0.9% saline, 25 mM Tris, pH 8.1. Synthesis of S-nitrosothiol. Red agarose (1 ml/l part resin: 1 part 25 mM Tris, pH 8.1, plus 0.9% NaCl) was incubated with different concentrations of cysteine

ABRAMSON

(Sigma), glutathione (Sigma), or N-acetylcysteine (Sigma) for 5 min (37°C). Reaction fluid was transferred to polypropylene Econo-column (Bio-Rad) and eluate containing S-nitrosothiol was obtained. Alternatively, S-nitrosoglutathione was synthesized by the acid-NaNO, method (11). Briefly, the reaction mixture includes glutathione (75 nmol/ml), NaNO, (750 nmol/ml), and 0.1 M acetic acid (5 min, 37°C). After reaction, the pH of the mixture was adjusted to pH 7.0 through addition of NaOH. Measurement of oxidant production by neutrophils exposed to FMLP. Superoxide anion generation was monitored by determination of reduction of cytochrome c in the presence or absence of superoxide dismutase (12). Neutrophils were exposed to FMLP ( 10d7 M, 5 min, 37°C) with or without 5 min prior treatment with tenidap (10 PM), salicylate (100 PM), or piroxicam (100 PM). HPLC analysis of S-nitrosoglutathione. HPLC separation of S-nitrosoglutathione was accomphshed using a C-18 reverse-phase column with a mobile phase consisting of 20% methanol, 80% 50 mM sodium phosphate, pH 2.2, and 1 mM octane sulfonic acid (13). Ultraviolet detection at 220 nm was used. The retention time of authentic S-nitrosoglutathione was assigned after analysis of eluate for S-nitrosothiol using the HgCl, diazotization method (described above) and using the glutathione recycling assay to detect glutathione (performed after the sample was reduced with NaBH,). RESULTS Synthesis of S-nitrosothiols. A novel method for synthesis of fluid phase S-nitrosothiols was developed which employed a transfer of the S-nitroso moiety from an S-nitrosothiol immobilized on resin to a fluid phase thiol. The former was prepared by coupling 2,2-dithiobis(ethylamine) to Bio-Gel A through amide linkage. Treatment with dithiothreitol as a reductant gave a reduced thiol immobilized on agarose (Bio-Gel A-SH). Red agarose was prepared by a reaction between BioGel A-SH and sodium nitrite at pH 2.0. Red agarose was washed and placed in 0.1 M Tris, pH 8.1. Bio-Gel A-SH and red agarose were chemically characterized and found to contain 10.4 2 0.8 ymol/ml resin thiol (detected using dithionitrobenzoic acid) and 8.5 ? 1.3 gmol/ml resin S-nitrosothiol (detected using the HgCl, Greiss reaction), respectively. In addition, resin (red agarose) was red, which is characteristic for absorption of S-nitrosothiols in 500-600 nm spectrum (not shown). When thiols such as cysteine or glutathione were added to red agarose, a transfer of the S-nitroso moiety from immobilized thiol to fluid phase thiol was observed as shown in Fig. 1. Formation of S-nitrosoglutathione from the reaction between reduced glutathione and red agarose was proportional over a wide range of concentrations (5-150 PM). Stoichiometric conversion of cys-

NEUTROPHIL

ACTIVATION

RESULTS

200

0

30

Glutathione

60

90

or Cysteine

120

150

(pM)

FIG. 1. Synthesis of S-nitrosothiols from glutathione (0) and cysteine (0) using the red agarose technique. Red agarose (1 ml/l part resin: 1 part 25 mM Tris, pH 8.1) was incubated with different concentrations of glutathione or cysteine (5 min, 37’C). S-nitrosothiols recovered in fluid phase were analyzed using the HgCI, Greiss reaction as described under Methods. Results are expressed as the mean of two separate determinations from different preparations of red agarose. Mean concentration (*SE, n = 3) of S-nitrosothiols was determined. Stoichiometric conversion of cysteine and glutathione to S-nitrosothiols was observed.

teine to S-nitrosocysteine was also observed. When 100 PM cysteine was added, 94 t 7.5 ~.LM S-nitrosocysteine was recovered after a 5-min reaction at 37°C with red agarose. This reaction is consistent with a previous study which demonstrated a transfer between S-nitroso-N,N-acetylpenicillamine and N-acetylcysteine (14). The pH varied for the reaction of red agarose with glutathione (5 min, 37°C) and the results are shown in Fig. 2. For the reaction at pH below 6.3 formation of S-nitrosoglutathione was negligible. There was significant formation of S-nitrosothiol at pH 7.4 as 340 nmoll ml of S-nitrosoglutathione was formed from the reaction between 1 mM glutathione and 1 ml red agarose. The optimal reaction was obtained at pH 8.1 as glutathione was stoichiometrically converted to S-nitrosoglutathione. S-nitrosoglutathione was analyzed using 220 nm uv absorption after separation by C-18 reverse-phase HPLC. The reaction between glutathione and red agarose resulted in a reaction product with the elution time of S-nitrosoglutathione (Fig. 3, bottom). Sodium nitrite or other 220-nm uv absorbing reaction products were not detected. In addition, the product was analyzed for absorption properties (not shown) and we observed apparent extinction coefficients (mM-‘) at 340 and 550 nm of 0.68 2 0.08 and 0.020 -t 0.005, respectively. For comparison we also analyzed S-nitrosoglutathione synthe-

WITH

S-NITROSOTHIOL

367

DEGRADATION

sized by the method of Saville (10). The reaction between glutathione (75 nmol) and sodium nitrite (750 nmol) at acid pH resulted in the formation of S-nitrosoglutathione (68 nmol, n = 2). In contrast to the red agarose method, the reaction mixture also contained SOdium nitrite (Fig. 3, top). Inactivation of S-nitrosoglutathione requires neutrophi1 actiuation. The stability of S-nitrosoglutathione was examined after incubation with neutrophils. S-nitrosoglutathione (3-15 PM) was incubated (5 min, 37°C) with neutrophils in the presence or absence of FMLP (10e7 M). Following the 5-min incubation, the reaction mixture was centrifuged and the supernatant assayed (HgCl, diazotization method) for S-nitrosoglutathione. As shown in Table 1, the addition of resting neutrophils did not effect S-nitrosoglutathione recovery at 5 min. In contrast, the exposure of neutrophils to FMLP resulted in the loss of S-nitrosothiol over the entire range of concentrations examined. For example, incubation of 15 /*M S-nitrosoglutathione with neutrophils in the absence and presence of FMLP resulted in recovery of 91 + 4 and 29 + 5% (P < 0.01; control vs FMLP), respectively. Thus, the recovery of S-nitrosoglutathione was reduced by activated but not by resting neutrophils. To confirm the above results (which were based on the HgC1, diazotization reaction), we employed two additional assays for S-nitrosoglutathione. First, supernatant fluids were analyzed for S-nitrosoglutathione after separation by C-18 reverse-phase HPLC as shown in Fig. 4. S-nitrosoglutathione was efficiently recovered from fluid obtained from resting neutrophils (102% applied, n = 2). Recovery of S-nitrosog-

1000

5 -a

,

800

0 ’ 0

3

6

9

PH FIG. 2. Effect of pH on recovery of S-nitrosoglutathione. Red agarose (1 ml) was incubated with 1.0 mM glutathione (5 min, 37°C) at varied pH. Buffers were 50 mM acetate (pH 2-5.5), 50 mM phosphate (pH 6-7.5), and 50 mM Tris (pH 8.1). Results are expressed as the mean of two experiments. S-nitrosoglutathione was analyzed using the HgCl, Griess reaction as described under Methods.

368

CLANCY .I2 .lO

i3 o_

.08-

3 E Ji *

.06-

AND

ABRAMSON

Anti-inflammatory drugs block the capacity of activated neutrophils to degrade S-nitrosoglutathione. Since anti-inflammatory agents are known to inhibit the neutrophil respiratory burst we examined their effects on the capacity of activated neutrophils to degrade S-nitrosoglutathione. Neutrophils plus S-nitrosoglutathione (15 PM) were exposed to FMLP in the presence or absence of three drugs. After 5 min the neutrophils were removed by centrifugation and the amount of S-nitrosoglutathione remaining in the supernatant was measured. As shown in Table 2, tenidap (10 PM) reduced FMLP-dependent superoxide anion release to 49% of control. This inhibition of superoxide anion release was associated with a significant increase of S-nitrosoglutathione recovery [42 * 3 in the absence of tenidap vs 78 + 1% in its presence (P < O.Ol)]. Tenidap alone does not interfere with the assay (not shown). Piroxicam, which effectively inhibits superoxide anion production (la), also reversed the capacity of activated neutrophils to degrade S-nitrosoglutathione. Salicylate had no effect on either superoxide anion production or on S-nitrosoglutathione recovery.

1 \

1 AM.02 O-

.12 .I0

DISCUSSION

We have established a novel method for synthesis of S-nitrosothiols using red agarose. That the product was a pure S-nitrosothiol and not a reduced thiol or nitrite was established by four criteria: first, the S-nitrosothiol bond released nitrite after reaction with mercuric chloride; second, the parent sulfhydryl moiety was no longer

.02 0 b

I 1

;

;

;

b

;1

Time (min) FIG. 3. Analysis of S-nitrosoglutathione synthesized from the acid-NaNO, method (top) and red agarose method (bottom) using C-18 reverse-phase HPLC and absorption spectroscopy. S-nitrosoglutathione was synthesized by the acid-NaNO, method by combining GSH (75 nmol) and NaNO, (750 nmol) as described under Methods and 25 ~1 was analyzed. Alternatively, 150 nmol was incubated with red agarose as described in the legend to Fig. 1. HPLC was performed as described under Methods. Retention times for reduced glutathione, oxidized glutathione, S-nitrosoglutathione, and NaNO, correspond to 4.1, 5.3, 7.0, and 8.4 min, respectively.

lutathione following neutrophil activation was reduced to 54% applied (n = 2). The recovery of S-nitrosoglutathione was also measured by bioassay: aggregation of human platelets after challenge with ADP. S-nitrosoglutathione prepared by the novel red agarose method inhibited platelet aggregation to 5% control. As shown in Fig. 5B S-nitrosoglutathione present in the supernatant of resting neutrophils effectively inhibited ADP-induced platelet aggregation. In contrast, supernatants derived from suspensions of FMLP-activated neutrophils failed to inhibit platelet aggregation (Fig. 5C).

TABLE Effect

1

of Neutrophil Activation of S-Nitrosoglutathione S-Nitrosothiol

S-Nitrosothiol Applied (PM) SNO-GSH 3 6 9 12 15

Resting neutrophils

1.7 4.0 7.1 8.5 13.6 f 0.6*

on Recovery

recovered

(kM)

Activated [FMLP

neutrophils (W7 M)]

1.0 1.0 1.5 2.4 4.3 +- 0.8*

Note. S-nitrosoglutathione (SNO-GSH, varied concentration) was incubated with neutrophils (5 X 10s) in the absence (control) or presence of 10m7 M FMLP (5 min, 37°C). S-nitrosoglutathione was prepared as described under Methods. Prior to analysis using the HgCl, diazotization assay, neutrophils were removed by centrifugation and SNO-GSH in supernatant was assayed. Data for 3-12 pM represent the mean of two determinations on cells from different donors; Data for 15 PM SNO-GSH represent the mean of five determinations on cells from different donors (*P < 0.01). Recovery at 5 min of SNOGSH (15 PM) in the absence of neutrophils was 14.1 k 0.2 pM.

NEUTROPHIL

ACTIVATION

RESULTS

WITH

S-NITROSOTHIOL

369

DEGRADATION

.30 1.12 .lO g

.08

5

.-J(j

f 2

64 .02

VW 0’ I

1

I

I

1

1

I

I

I

I

I

I

1

I

0

1

3

5

7

9

II

0

I

3

5

7

9

II

Time (mill) FIG. 4. Effect S-nitrosoglutathione analyzed. HPLC

lht

of neutrophils exposed to FMLP on recovery of S-nitrosoglutathione. in the absence (left) and presence (right) of 10v7 M FMLP was performed as described under Methods

detected by means of the dithionitrobenzoic acid assay; third, the product possessed an absorption profile characteristic of S-nitrosothiol (X,,, at 335 and 550 nm); and fourth, after HPLC separation, we observed a single uv absorbing product with the retention time of synthetic S-nitrosoglutathione. In addition to these four criteria, we have shown functional properties of the synthetic S-nitrosoglutathione [such as the capacity (a) to inhibit platelet aggregation (Fig. 5), (b) to promote ADP ribosylation of a 37-kDa neutrophil cytosolic protein (15), and (c) to raise intracellular cGMP production by cultured lymphocytes (X5)] which support its identity as an S-nitrosothiol. Reactions between various concentrations of thiols and red agarose resulted in stoichiometric formation of S-nitrosothiols (Fig. 1). The reaction correlated with the pK,, of the thiol, which suggests that S-nitrosothiol synthesis proceeds via a nucleophilic substitution reaction between fluid phase thiol and immobilized thiol to yield S-nitrosothiol as a pure product. Thiols (but not amines or amides, unpublished observation), reacted with red agarose presumably because thiols are better nucleophiles than amines or amides under the mild reaction conditions used in this study. Several factors favor the red agarose technique over currently available methods used to synthesize S-nitrosothiols. For example, while S-nitrosothiols can be synthesized by direct addition of nitric oxide to thiol(8,14), nitric oxide is unstable in aqueous solution (Ti < 30 s) and has limited solubility. Alternatively, S-nitrosothiols have been previously synthesized from a mixture of

Neutrophils (5 min, 37°C).

(mh)

(5 X 106) were incubated with 150 nmol Cells were removed and fluid (25 ~1) was

thiol and NaNO, at acidic pH. However, the product may also contain NaNO, and the reaction is nonspecific since NaNO, is also reactive with amines and amides (16). The facile synthesis of S-nitrosothiols by the red agarose technique permits the systematic study of the properties of these compounds in aqueous and neutrophi1 suspensions. Using the red agarose technique, which avoids the problems associated with the synthesis of pure S-nitrosothiols using nitric oxide and NaNO,, we show that S-nitrosoglutathione is stable (Ti > 60 min at 37”C, unpublished observation). This is consistent with previously published half-life values for S-nitrosoglutathione. There is increasing interest in the properties of S-nitrosothiols since the parent compound, nitric oxide, has been identified as an EDRF (17-20). Nitric oxide reacts with tissue sulfhydryls to form S-nitrosothiol compounds, which are more stable and potent (21,22). Both cysteine and glutathione are abundant sources of free sulfhydryl groups in mammalian tissue (23). S-nitrosothiols, synthesized by the reaction between nitric oxide and thiols, activates soluble guanylate cyclase of platelets and smooth muscle cells (48). The resultant rise of cytosolic cycle GMP mediates smooth muscle relaxation and inhibits platelet aggregation (4,6). Because it has been suggested that S-nitrosoglutathione may be taken up by cells and therein yield nitric oxide after a one-electron reduction, several investigators have suggested that the physiological role of nitric oxide may be mediated by this and other S-nitrosothiols (21,23).

370

CLANCY

AND

It is now evident that cells other than vascular endothelium synthesize nitric oxide, including fibroblasts, macrophages, neutrophils, and neurons (24-28). Key inflammatory mediators such as bradykinin, endotoxin, y-interferon, and interleukin-1 activate nitric oxide synthesis (20,29), suggesting that nitric oxide may exert effects at sites of inflammation. The evidence to date indicates anti-inflammatory properties: Nitric oxide produced by suppressor macrophages, for example, inhibits proliferation of rat lymphocytes (30). In addition, previous studies demonstrate that nitric oxide and/or S-nitrosothiol derivatives inhibit superoxide anion production, adhesion to vascular endothelium, and leukotriene B, production by activated neutrophils (l-3). Our current studies indicate that activated neutrophils degrade S-nitrosothiols, an effect which may attenuate their potential anti-inflammatory effects. The mechanism(s) by which neutrophils degrade S-nitrosothiols is not clear. S-nitrosoglutathione was extensively degraded after incubation with neutrophils exposed to FMLP. In addition, tenidap and piroxicam, agents which inhibit 0: blocked the capacity of activated neutrophils to degrade S-nitrosoglutathione. Salicylate, which had no effect on respiratory burst, also had no effect on S-nitrosothiol recovery. One possible explanation is that an oxidant produced by activated neutrophils is responsible for S-nitrosothiol degradation.

100 A

B

ABRAMSON TABLE

2

Effect of Treatment late

with Tenidap, of S-Nitrosoglutathione

on Recovery

duction for Neutrophils

S-Nitrosoglutathione recovered (percentage control)

Dw None Tenidap Piroxicam

Exposed to

Piroxicam and Salicyand on Oxidant ProFMLP Neutrophil 0~ release (percentage control)

42 f 3 (10 pM) (I00

pM)

(100

pM)

SaliCylate

78+

100 r 14 49 5 10* 68 k 3*,” lOl& 9

1*

56 + 8 46 f 5

Note. SNO-GSH (15 gM) was incubated with neutrophils (5 X 106) in the absence (control) or presence of 10m7 M FMLP (5 min, 37°C). SNO-GSH was prepared and analyzed as described in the footnote to Table 1. Oxidant production for neutrophils exposed to FMLP (5 min, 37°C) in the presence or absence of anti-inflammatory drug was determined as described under Methods. Control 0; release was 19.7 -+ 2.8 nmol/106 cells/5 min. Data represent the mean of three separate determinations on cells from different donors. Results (expressed as percentage control) indicate that the inhibition of neutrophil function by anti-inflammatory agents reverses the capacity to degrade S-nitrosoglutathione. a From (12). * P < 0.01 compared to minus drug addition (None).

Taken together, these data indicate that neutrophils have the potential to degrade S-nitrosothiols and to lower S-nitrosothiol tissue levels, which may limit their biological activity. The degradation of anti-phlogistic Snitrosothiols by activated neutrophils may contribute to tissue injury at sites of inflammation.

C

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2

4

6

TIME (mid FIG. 5. Effect of neutrophils on the capacity of S-nitrosoglutathione to inhibit platelet aggregation. S-nitrosoglutathione (15 MM) was incubated with neutrophils (5 X 106) in the absence or presence of 10m7 M FMLP (5 min, 37°C). Neutrophils were removed and fluid was analyzed for biological activity. Prior to exposure to ADP, platelets were incubated in buffer (A, control) or supernatants derived from resting (B) or activated neutrophils (C). Cellular supernatant (1:lO dilution) was analyzed using aggregation of human platelets after challenge with 2.5 pM ADP. Data are one representative experiment of two determinations on cells from different donors.

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RESULTS

G. (1990)

ZnfEammation

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DEGRADATION

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