Nitric oxide regulation of superoxide-dependent lung injury: Oxidant-protective actions of endogenously produced and exogenously administered nitric oxide

Free RadicalBiology& Medicine,Vol. 21, No. 1, pp. 43-52, 1996 Copyright© 1996ElsevierScienceInc. Printed in the USA.All rights reserved 0891-5849/96 $15.00 + .00

N:I I,:I ELSEVIER

SSDI 0891-5849(95)02226-0

Original Contribution NITRIC

OXIDE

INJURY:

REGULATION

OF

SUPEROXIDE-DEPENDENT

OXIDANT-PROTECTIVE

PRODUCED

AND

ACTIONS

EXOGENOUSLY

OF

LUNG

ENDOGENOUSLY

ADMINISTERED

NITRIC

OXIDE

HECTOR H. GUTIERREZ,* BEDFORD NIEVES, * PHILLIP CHUMLEY,t ARNOLD RIVERA,t and BRUCE A. FREEMAN* t* Departments of *Pediatrics, *Anesthesiology,and *Biochemistryand Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, USA (Received 7 June 1995; Revised 13 September 1995; Accepted 14 November 1995)

Abstract--The influence of endogenous cell "NO production and "NO derived from exogenous sources on oxidant injury to cultured fetal rat lung alveolar epithelium and an animal model of pulmonary oxidant injury was examined. Confluent fetal rat alveolar epithelial cell monolayers were stimulated to produce "NO after treatment with a combination of cytokines (IL-1/~, TNF-a, IFN-7), LPS and zymosan-activated serum (CZ). Cell injury, assessed by ~4C-adenine release, was significantly increased compared to basal and CZ-induced cells after inhibition of "NO synthesis by L-NMMA. Cell monolayer macromolecule barrier function was determined by the rate of diffusion of 125I-albumin from the apical to basolateral side of monolayers. Following exposure to CZ and/or 02"- generated by xanthine oxidase + lumazine (XO), endogenous cell "NO production and exogenously administered "NO (from "NO donors S-nitrosyl-glutathione and S-nitroso-N-acetylpenicillamine) significantly inhibited the increased monolayer permeability induced by exposure to reactive oxygen species• Furthermore, inhalation of 5-10 ppm of "NO significantly reduced the toxicity of > 95% oxygen to adult rats. We conclude that when cultured pulmonary epithelial cells and lung tissue in vivo are subjected to inflammatory mediators or acute oxidative stress, "NO can play a protective role by inhibiting O2"--dependent toxicity. Keywords--Nitric oxide, Superoxide, Peroxynitrite, Hydrogen peroxide, Free radical, Antioxidant, Lung injury, Alveolar epithelium

tion with iron-sulfur-containing respiratory enzymes, 8 and metal centers of cytochrome c oxidase. 9 Cellular replicative processes are inhibited by "NO reaction with ribonucleotide reductase. 1° Reactions with specific respiratory and biosynthetic enzymes ultimately lead to a general inhibition of D N A 10 and protein synthesisJ 1'1a Physiologically, "NO can exert its deleterious effects by affecting blood flow and permeability at the microcirculatory level. 13 Since tissue "NO concentrations are often low ( < 1 /zM), significant direct toxicity towards the afforementioned heme, iron-sulfur and thiol-containing proteins often require supraphysiologic concentrations of "NO or "NO-generating agents. 14'15 Since "NO has a relatively low reactivity for a free radical species, 16 it is becoming apparent that its toxicity is often the consequence of reaction with superoxide anion (O2"-) to yield secondary products that are more reactive and potentially cytotoxic. Nitric oxide reacts avidly with 02 "-17 to

INTRODUCTION Nitric oxide ( ' N O ) , a free radical species produced by a wide variety of mammalian cell types, has gained recognition as a key mediator of metabolic homeostatic processes, host defense mechanisms, and oxidant tissue injury.l Nitric oxide exerts a toxic role during such diverse pathologic events as neuroexcitotoxin-induced brain injury, 2,3 allograft transplant rejection, ischemiareperfusion phenomena, 4'5 and immune complex-stimulated pulmonary edema. 6'7 Nitric oxide mediates toxic reactions by impairing mitochondrial energy production via either direct or an indirect peroxynitrite ( O N O O - ) - m e d i a t e d reac-

Address correspondence to Bruce A. Freeman, Department of Anesthesiology, 946 Tinsley HarrisonTower, 619 South 19th Street, University of Alabama at Birmingham, Birmingham, AL 352336810. 43

44

H.H. GUTIERREZet al.

give peroxynitrite ( O N O O - ) and its conjugate acid, peroxynitrous acid ( O N O O H ) (k = 6.7 × 1 0 9 M - l " s - l ) , each of which have unique and potent reactivities towards diverse target molecules. 18-2° Peroxynitrite is a powerful one- and two-electron oxidant towards lipids, multiple amino acids, and can nitrate aromatic amino acids such as tyrosine. 19'21'22In these reactions, O N O O - can serve as a precursor for other potent reactive species, including "NO2, NO2 + and an intermediate with "OH-like reactivity. 23"24 Thus, it is often assumed that the toxicity of O2"- can be potentiated by reaction with "NO by leading to the generation of O N O O - . In support of these concepts, it has been demonstrated in humans that "NO-mediated production of O N O O - occurs in oxidative processes associated with atherogenesis 25.26and acute lung injury. 27'28 Recently, a protective role for "NO has also been observed in pathologic events associated with excess production of reactive oxygen species. Numerous reports in cerebrovascular, 29-31 hepatic, 32 splachnic, 33'34 renal, 35 pulmonary, 36.37 myocardial, 38.39inflammatory, and ischemia-reperfusion injury models have shown that stimulation of endogenous "NO production or exogenous administration of "NO-donating compounds often blunts the ultimate expression of tissue injury at both molecular and functional levels. Interestingly, this "NO-dependent protective effect is observed in processes having increased rates of 02" production and for which oxidant injury is proposed to play an etiological r o l e . 3°'32'4° There is now accumulating evidence that in the lung, "NO is produced by mammalian pulmonary airway epithelium, smooth muscle cells, vascular endothelium, and alveolar epithelial cells. 41-43 Basal pulmonary production of "NO mediates bronchodilation, 44 vasodilation, 45 and may also act as a neurotransmitter of nonadrenergic, noncholinergic pathways. 46 A significant increase in pulmonary "NO production occurs and may contribute to the pulmonary pathology secondary to deposition of immune complexes, 6'7 exposure to pertussis-derived tracheal cytotoxin, 47 and the toxicity induced by xenobiotic and air pollutant exposure. 48'49 These reports support the view that "NO would be a critical intermediary in the production of oxidant tissue damage. 49 With the increasing use of inhaled "NO in clinical situations where enhanced rates of production of reactive oxygen intermediates in the lung parenchyma occurs (i.e., during inflammation and ventilation with hyperoxic gas mixtures), it is imperative to have a clear understanding of both the deleterious and salutary properties of nitric oxide. To clarify the interdependence of the reactive species 02"- and "NO in cell and tissue injury, we examined the influence of endogenous cell "NO production and the effects of "NO derived

from exogenous sources on fetal rat lung alveolar epithelial type II cell injury, and extended in vitro observations to an animal model. We show herein that when cultured pulmonary epithelial cells and lung tissue in vivo are subjected to inflammatory mediators or acute oxidative stress, "NO can play a protective role by mediating the inhibition of 02"- and H202-dependent toxicity. EXPERIMENTAL METHODS

Materials

Minimal Essential Medium (MEM), Hank's balanced salt solution (HBSS), and antibiotic/antimycotic solution were from GIBCO Laboratories (Grand Island, NY). Heat-inactivated fetal calf serum was from Hyclone Laboratories (Logan, UT). Recombinant murine IL-1/3, IFN-y, and TNF-a were from R&D Systems (Minneapolis, MN). E. coli O11 l:B4 LPS, NC-mono methyl-L-arginine, zymosan, cysteine and lumazine were from Sigma Chemical Co. (St. Louis, MO). Timed-pregnant female Sprague-Dawley rats were from Charles River Laboratories (Wilmington, MA). Xanthine oxidase and CHAPS were from Calbiochem (La Jolla, CA). t4C-adenine and 125I was from NEN Products (Boston, MA). Albumin (Miles Scientific (Napersville, IL) was iodinated according to the method of Bolton and Hunter (50) with minor modifications. S-nitroso-N-acetylpenicillamine (SNAP) was from Biomol (Plymouth Meeting, PA). Dihydrorhodamine 123 was from Molecular Probes (Eugene, OR). Preparation and measurement of "NO

S-Nitroso-glutathione (GSNO) was synthesized at 25°C by reacting equimolar (200 mM) concentrations of reduced glutathione with sodium nitrite in 0.5 M HC1. 51 Stock solutions of SNAP were prepared in 100% ethyl alcohol. For analysis of "NO and O N O O production from SNAP, cysteine (100 # M ) and SNAP ( 5 0 / z M ) were added to HBSS in the presence or absence of 10 mU/ml xanthine oxidase and lumazine (100 # M ) , pH 7.4 at 37°C. Lumazine was chosen as a substrate because of its increased univalent flux of electrons to yield 02"- and the consequent absence of the "OH scavenger uric acid as a product. For production of "NO from GSNO, different concentrations were added to 100 mM potassium phosphate, pH 7.4, at 20°C under standard flourescent laboratory lightning conditions. Rates of "NO production were measured electrochemically using an "NO sensor (Iso-NO®, World Precision Instruments, Inc., Sarasota, FL). The "NO sensor was calibrated using a standard solution of "NO prepared by equilibrating "NO gas (Matheson, Madison, WI) for 30 min with argon-saturated deion-

Nitric oxide modulation of oxidant lung injury ized water. Any "NO2 present was eliminated by first bubbling "NO through 5 M NaOH. Aliquots of media from cell incubations were assayed for the stable oxidation products of "NO, NO2-, and NO3-. Samples were first incubated with E. coli nitrate reductase (No. 25922, ATCC) to reduce NO3- to NO 2- for quantitation of NO2- by the Greiss reaction. 52 Solutions of NaNO3 were prepared in media used for cell incubations to generate standard curves and to calculate the extent of NO3- reduction to NO2-.

In vivo exposure to "NO gas and/or hyperoxia Adult Sprague-Dawley rats weighing between 2 5 0 - 3 0 0 g were exposed to hyperoxia alone ( > 95% 02, control group) or hyperoxia plus "NO ( 5 - 1 0 ppm, "NO group) and survival assessed every 6 h during the first 60 h and then every 3 h until death or up to 144 h, when exposures were terminated. The "NO exposure group was exposed to "NO for the initial 48 h of > 95% 02 but remained under hyperoxia until completion of the experiment ( 144 h). All the experiments were performed in cylindrical plexigas chambers (900 RCS, Plas Labs, Lansing, MI) with a 7.5 1 volume and no dead space. Rats had access to food and water ad lib and were exposed to 1 0 - 1 2 h of light every 24-h cycle at 21.5°C to 27.0°C. Oxygen and "NO were mixed in the gas circuit immediately before reaching cages. Gas concentration were recorded continuously and delivered at a minimum flow of 15 LPM to avoid oxidation of "NO to "NO2. Nitric oxide and "NO2 were analyzed continuously and recorded by computer every 30 min by chemiluminescence (CLD 700 AL analyzer, Eco Physics, Multianalytical Inc., Mountain View, CA)

Detection of O N O O - production Peroxynitrite induces a concentration-dependent oxidation of dihydrorhodamine 123. 53 Xanthine oxidase (10 mU/ml) plus lumazine (100 # M ) and SNAP (50 # M ) plus cysteine ( 100 # M ) were combined with dihydrorhodamine 123 (50 # M ) in HBSS plus 1 mM DTPA. Absorbance was measured at 500 nm in reactions maintained at 37°C. Concentrations were calibrated against dihydrorhodarnine 123 oxidation induced by known concentrations of O N O O - , synthesized as previously described. TM

Cell culture Fetal rat lung type II epithelial cells were isolated and cultured as previously described. 54 Briefly, 19- or 20-d gestation fetuses (term = 22 d) were aseptically removed from dams, the lungs dissected, minced, and resuspended in cold calcium and phosphate-free

45

HBSS. The minced tissue was trypsinized for 20 min, filtered, and centrifuged. After several differential adherence steps, a 9 5 - 9 8 % pure suspension of epithelial cells was obtained as determined by cytokeratin and vimentin staining. 54 Cell numbers were determined using a Coulter particle counter (Coulter Electronics, Hialeah, FL) and then plated in 12-well culture dishes at 5 × 10 5 cells per well.

Assessment of cell injury Release of preincorporated ~4C-adenine was used as a marker of cell injury. Upon reaching confluence ( 2 4 36 h), cells were incubated with 0.2 #Ci/ml 14C-adenine in complete medium for 4 h. The 14C-adenine containing medium was removed, and cells washed three times with HBSS at 37°C. Previous studies revealed that - 8 0 % of incorporated radiolabel is acid soluble. At specific time points ( 12 and 24 h) medium was removed for measurement of ~4C-adenine prelabel release. Cells treated with 0.5% CHAPS in 10 mM potassium phosphate, pH 7.4, indicated 100% monolayer lysis. The percent of cell lysis was calculated as: % cell lysis = cpm in medium/ (cpm in media of 100% lysed cells). (100%) Cells were sometimes preincubated with various stimuli or inhibitors, including CZ (a combination of IFNy (100 U/ml), T N F a (500 U/ml), IL-I/3 (300pM), zymosan-activated rat serum ( 1% v / v ) , and E. coli LPS (10 /zg/ml), L-NMMA (1.0 mM), XO (10 mU/ml) + lumazine (50 # M ) , SNAP (50 # M ) , GSNO (25 # M ) , and combinations thereof. The cytokine-exposed cell monolayers (in the presence or absence of L-NMMA) were incubated for 24 h prior to other additions. Cell monolayer integrity is an overall expression of intact cell metabolism; thus, increased permeability to a macromolecule like albumin is a sensitive indicator of cell damage. 55 Transwell® plates (Costar, Cambridge, MA), consisting of an upper well separated from a lower well by a collagen micropore filter (2.4cm diameter, 4.5-cm 2 surface area, 0.4-/zm pore size), were utilized for permeability studies. Filters were pretreated for 1 h with 5 # g / c m 2 poly-D-lysine (Boehringer-Mannheim) in HBSS, the poly-D-lysine solution removed, 5 × l0 s cells per well were plated in the upper well and grown to confluency ( 3 6 - 4 8 h), in MEM + 5% FCS. Upon reaching confluency ( 3 6 48 h), monolayers were washed twice with HBSS and exposed to various experimental conditions in MEM plus 5% FCS. The lower well contained 2.65 ml medium and the upper well 1.5 ml total medium volume,

H.H. GtrrlERREZet al.

46

which included added cytokines or S-nitrosothiols compounds when appropriate. As for 14C-adenine release studies, the cytokine-exposed cell monolayers (in the presence or absence of L-NMMA) were incubated for 24-h prior to other additions. Then, ~25I-albumin ( 1 x 106 cpm) and XO (10 mU/ml) + lumazine (50 mM), were added to the lower well. The total volumes in both upper and lower wells resulted in an equivalent fluid volume level and no hydrostatic pressure difference. With the addition of 125I-albumin, XO and lumazine being t = 0, sequential 10 #1 samples were taken from the upper well at 15-min intervals for the first hour and at 30-min intervals the next 3 h. During the sampling period, the plates were placed on an shaker at 15 rpm to minimize unstirred layer effects and maintained at 37°C. Radioactivity was assessed by gamma counting (LKB-Wallac 1275 Minigama, Finland).

when comparing different treatments to a common control. Survival analysis was performed using the log rank and Wilcoxon tests. RESULTS

Detection of 02"-, "NO, and O N O O - production Addition of 50 # M S-nitroso-N-acetylpenicillamine (SNAP) to buffer systems employed for cell studies resulted in "NO production for approximately 20 min, achieving a peak "NO concentration of 8.3 #M in the absence of a source of O2"- (Fig. 1,A). The maximal initial rate of "NO production was 1.8 #M" min-1. The addition of 100 # M lumazine + 10 mU/ml XO resulted in almost complete suppression of "NO detectability as before, 56 inferring the reaction of "NO + 02"- with subsequent production of O N O O - (Fig. 1,B). Addition of 1 mM GSNO resulted in "NO production for more than 90 min, with a initial rate of 0.80 # M . min -1, achieving a maximum concentration of 3.7 #M in the absence of 02"- (not shown). Peroxynitrite oxidizes dihydrorhodamine 123 to the chromophore rhodamine. 53 In HBSS, dihydrorhodamine 123 was readily oxidized when 02"- (XO plus lumazine) and "NO generating systems (either SNAP or GSNO) are combined, but not by independent exposure to 02"- or "NO separately (Fig. 2). This supports that XO plus lumazine-derived 02"- yields O N O O - in

Statistical analysis All values represent mean ___ SEM. For all experiments assessing extents of cell "NO production, 5 × 105 cells per well were plated into 12-well (2.2 cm 2) or Transwell® plates, with six separate wells dedicate to each test condition. Each experiment was repeated at least three times. Statistical significance (p < .05) was determined by Student's two-tailed paired t-test. Duncan's multiple comparison analysis was performed

A

m

,q,.

--

u -t

e,i

--

e~

.z

B

I 0

I

I 12

I

I 24

I

I 36

I

I 48

I

I 60

Time (rain)

Fig. 1. Measurementof steady state "NO concentrationin the presence or absence of 02"-. Using SNAP, a peak concentration of 8.3/zM of "NO was achievedwith a mean initial rate of rate of 1.8 #M "min-~. Data is a representativeexperimentrepeated three times, m represents HBSS; 1 represents HBSS + SNAP; n represents HBSS + SNAP + XO + lumazine; o represents HBSS + XO + lumazine.

Nitric oxide modulation of oxidant lung injury the presence of "NO. 53'56 Calibration of dihydrorhodamine 123 oxidation by known concentrations of pure O N O O - quantitatively yielded O N O O - from Oz'- and "NO in cell and protein-free buffer systems (not shown). For these experimental conditions, control studies showed that there was no significant inhibition of XO due to substrate depletion or from 02"-, "NO, O N O O - , and/or secondary products generated by reaction systems.

Endogenous production of "NO by rat fetal alveolar epithelial type H cells A wide range of cell types express an inducible form of nitric oxide synthase (iNOS) following exposure to cytokines and other agents, often in synergy with an IFNT or an IFNy-inducing agent like bacterial lipopolysaccharide (LPS). 1,57We have recently shown that fetal rat alveolar epithelial type II cells express iNOS are capable of producing high yields of "NO 43 and can be stimulated to produce "NO after treatment with a combination of cytokines plus endotoxin. Basal NO production was noted in unstimulated cells, indicated by a 48 h accumulation of 7.1 ___ 0.8 #M NO2- + N O 3 - ' m g protein -~ (accumulated in 1 ml medium), while there was a sevenfold increase in "NO production in the CZ-treated cells (Table 1 ).

The influence of endogenous and exogenously-added "NO on pulmonary type H alveolar epithelial cell response to oxidant stress 14C-adenine-labeled cell monolayers were either stimulated to produce endogenous "NO by cytokines plus endotoxin or incubated with the nitrosothiols

1.75 1.50 "

1.25 LO0

~

o.Ts

~I

0.50

47

Table 1. Endogenous "NO Production by Fetal Rat PulmonaryCeils Condition Control cells + L-NMMA + XO + Cytokinemix (CZ) + CZ + L-NMMA + CZ + XO + CZ + XO + L-NMMA

~M NO~ + NO~-mg protein7.1 _ 0.8 2.1 _+ 0.2* 0.6 ___1.0* 46.7 _ 3.6* 5.4 + 0.7 38.2 _+ 5.1" 4.1 _+ 1.3

Confluent cell monolayers were incubated on 12-well plates on 1 ml media with a combination of IFNT (100 U/ml), TNFc~(500 U/ml), IL-1/3(300 pM), ZAS (1% v/v) and E. coli LPS (10/~g/ml) in the presence and absence of L-NMMA(1 mM) and XO (10 mU/ ml + 50 #M lumazine). * Represents p < .05 compared with control and CZ + LNMMA. *Represnts p < .05 compared with control cells.

SNAP (100 # M ) or GSNO (25 # M ) as exogenous sources of "NO. No significant "NO-dependent increase 14C-adenine release was observed in cell monolayers treated with CZ or following exposure to "NO donors alone (Fig. 3). Notably, both basal and cytokine-activated cell monolayer 14C-adenine release were significantly increased after inhibition of endogenous cell "NO synthesis with N~-monomethyl-t:arginine (LN M M A ) . Oxidant stress induced by XO-derived O2"-, H2Oz and "OH increased ~4C-adenine release, which was significantly attenuated by stimulation of endogenous cell "NO production and by the concomitant addition of the nitrosothiols SNAP (100 # M ) and GSNO (25 #M) (Fig. 4). The rate of transmonolayer diffusion of 125I-albumin after exposure of cells to CZ and/ or O2"-, H202, and "OH generated by xanthine oxidase plus lumazine was then assessed. Cell monolayers exposed to XO-derived reactive species showed progressive and significant loss of barrier function to macromolecules, which was attenuated by "NO derived from GSNO or SNAP decomposition (Fig. 5). Cytokinemediated induction of iNOS significantly blunted transmonolayer permeability increases induced by XO plus lumazine (Fig. 5), which was reversed by inhibition of cell "NO synthesis by L-NMMA.

The influence of inhaled "NO on rat survival to hyperoxic oxidant stress

O.N 12

24

36

48

60

72

Time (rain)

Fig. 2. Dihydrorhodamine 123 detection of ONOO- production. Dihydrorhodamine 123 (50 #M) was added to a combination of 10 mU/ml XO, 50/zM lumazine, 50/zM SNAP, and 100/~M cysteine. Absorbance was read periodically for 1 h. The approximate rate of ONOO- production was 500 nm" min -1 and for Oz'-, 1 #M " min -~. Data is a representative experiment repeated three times.

The mean of "NO concentration during exposure of rats to hyperoxia _+ "NO (n = 8 for each group) was 7.8 __ 0.2 ppm, and a mean "NO2 concentration of 0.1 + 0.0 ppm. At 63 h 50% of the control group had died, and only one survived during the 145 h period (12.5% survival). By contrast, the group exposed to "NO + hyperoxia had no mortality at 63 h and 50% mortality at 120 h, with no additional deaths thereafter

48

H . H . GUT1ERREZet al.

.$

L-NMMA

L-NMMA

Fig. 3. Effect of endogenously produced and exogenous "NO generation on cultured rat fetal lung cells. Cytokine incubation conditions were as for Table 1. Cells were then labeled with ~4C-adenine for 4 h prior to exposure to the noted experimental conditions, t4C-adenine release is expressed as % of lysis following 24 h of incubation. *Represents p < .05 compared with all the other groups. #Represents p < .05 compared with control.

(50% survival at 145 h), (Fig. 6). This difference in survival was statistically significant between both groups (p < .001 by log rank and Wilcoxon tests).

DISCUSSION

Nitric oxide and 0 2 ° a r e important mediators of both physiologic and pathologic events. Because of the transient nature of free radical species and their often broad range of reactivities, it becomes challenging to define the mechanisms of tissue injury in processes of oxidant stress when a diverse spectrum of reactive species are produced. The data reported herein reveals the relationship between the cytotoxic properties of O2" and "NO when generated alone or together. Our results show that basal rates of cytolysis and O2" -dependent oxidative damage in a fetal rat lung epithelial cell model is attenuated by either (a) enhanced rates of endogenous cellular "NO production upon cytokine-mediated induction of iNOS expression (Figs, 4 and 5 ), or (b) exogenous "NO generated by the decomposition of nitrosothiols GSNO and SNAP (Figs. 4 and 5). Moreover, in a 24-h period the fetal lung epithelial cells were not adversely affected (i.e., no increase in nucleotide release through intracellular degradation and/or membrane leak or loss of macromolecular barrier function) when exposed to high concentrations of endogenously produced or exogenously added "NO. Finally, when control and cytokine-stimulated cells were treated with L-NMMA, a competitive inhibitor of multiple NO synthase isoforms, cell injury assessed by 14C-adenine release increased significantly, suggesting a physiologic protective role for endogenously produced "NO (Table 1, Figs. 3, 4, and 5). This demonstrates that "NO or its secondary products were not appreciably cytotoxic under the con-

ditions employed (Fig. 3). This is further supported by the observation that enhanced cellular production of "NO in iNOS-transfected human bronchial epithelial cells did not alter clonal cell growth. 58 Inducible NOS is expressed in morphologically normal respiratory epithelium of cartilaginous human airways, 59 and epithelial cells lines, 4j thus reflecting a "constitutive" expression in normal tissues, where "NO presumably serves homeostatic roles. 41'57 While our results support this hypothesis, it is also possible that the stress of isolation and primary culture of cells stimulated iNOS induction. The alveolar epithelium plays a critical role in maintenance of the integrity of the air-blood barrier and the synthesis of pulmonary surfactant. When these properties are compromised in acute lung injury, pulmonary edema and loss of respiratory function occurs. Acute lung injury results from a complex interplay between the damaging effects of reactive species generated by alveolar, vascular, and inflammatory cells and humoral mediators such as cell growth factors, which are subsequently directed towards the cells of the pulmonary air-blood barrier. 6° A number of pulmonary diseases are caused or made worse by the necessary use of therapeutic hyperoxia, including ARDS, bronchopulmonary dysplasia, pulmonary inflammation, drug-induced pulmonary disease, and ischemiareperfusion injuryY Previous studies have shown that in lung tissue, the rate of partial reduction of oxygen to 02" , H202, and secondary species (i.e., "OH, ONOO- ) is directly proportional to tissue oxygen tension. Nonrespiratory tissue oxygen consumption is normally 9% of total and increases four- to five-fold in > 95% oxygen. 61'62 Similarly, a large percentage of this noncytochrome c oxidase-dependent oxygen con-

100

80 ¸

60

20 ¸

L-NMMA Endozenouscell .NO prOduCtion

+ XO

+ XO

Exogenous-NO addition

Fig. 4. Influence of endogenously produced "NO or exogenous "NO generation, on 02" -dependent injury of rat fetal lung cell monolayers. Cytokine incubation conditions were as for Table 1. Cells were labeled with ~4C-adenine for 4 h prior to the noted experimental conditions. ~4C-Adenine release is expressed as % of lysis following 24 h of incubation. *Represents p < .05 compared with XO + lumazine group. #Represents p < .05 compared with the CZ + XO group.

Nitric oxide modulation of oxidant lung injury 32 ¸

'~, 24'

8

L-NMMA Endogenous cell .NO production

XO

GSNO

Exogenous .NO addition

Fig. 5. Influence of endogenously produced "NO or exogenous "NO generation, on O~'--dependentinjury of rat fetal lung cell monolayer macromolecule barrier function. Effect assessed by apical ~25I-albumin flux. 5 × 105 cells/well were plated on Transwell plates as described in Experimental Methods. *Representsp < .05 compared with the XO + lumazine group. #Representsp < .05 compared with the CZ + XO group.

sumption represents the production of reactive oxygen species. 61'62 A threefold increase in lung 02"- production occurs in reperfusion injury, with protective actions lent by administration of superoxide dismutase and "OH scavengers. 63 Survival of adult rats in > 95% oxygen is significantly enhanced by inhalation of "NO (Fig. 6). In oxidant-induced lung injury (i.e., hyperoxia, ARDS, and ischemia-reperfusion phenomena) "NO could, thus, serve a dual role, as either a protective species or as a pro-oxidant by serving as a precursor for ONOO . Evidence for the production of ONOO - in rodent lung tissue following hyperoxia, as well as in human lung tissue obtained from patients with ARDS and sepsis, has recently been reported. 27'28 The formation of tissue nitrotyrosine derivatives, while providing supporting evidence for one of the reaction pathways of O N O O - , does not provide causal evidence for a toxic role of this species or its precursor "NO in tissue injury processes. It remains possible that in our more acute studies ranging from 24 h (cells) to 144 h (hyperoxic exposure of rats), the oxidant reaction pathways influenced by "NO would result in tissue protection. Thus, under more chronic or less oxidizing conditions (several days to weeks), it remains possible that enhanced rates of tissue "NO (hence, O N O O - ) production would exacerbate pathologic processes. The protection offered by both endogenous and exogenous "NO might occur in several ways. First, endogenous "NO production or addition of exogenous "NO may exert a protective effect by reacting with free coordination sites of iron, thus indirectly acting as an iron chelator 64 by forming iron-nitrosyl complexes, thus possibly limiting Fenton chemistry and iron-dependent electron transfer reactions. 65'66 This is not a particularly tenable

49

explanation, because the extensive reaction of "NO with heme, iron-sulfur, and other metalloproteins requires high concentrations of "NO or "NO-generating agents. Also, the presence or absence of redox active metal complexes does not influence the ability of "NO to inhibit XO-dependent lipid peroxidation. 22'67Finally, electron spin resonance analysis of oxidant reaction systems containing Fe-EDTA complexes or the nonheme ironcontaining protein soybean lipoxygenase did not reveal iron-nitrosyl complex formation when rates of "NO production were in a biological range ( 1 - 2 #M" min 1 o r l e s s ) . 22'67 It should also be noted that in "NO-metal interactions, "NO can exert prooxidant effects as well, by reducing ferric iron complexes. 68 Second, "NO may protect by activating guanylate cyclase with subsequent induction of cGMP-dependent effects. In animal models of reperfusion injury, "NO inhibits neutrophil and platelet aggregation and adherence to the vessel wall, the latter associated with increased cGMP levels, 69-7~ Two recent reports add strength to this possibility. A rapid fall in tissue "NO production was shown to occur upon lung reperfusion, with augmentation of the "NO pathways at the level of cGMP enhancing lung preservation and function following transplantation. 36 Also, there was a protective effect of the "NO donor SPM-5185 by apparent supression of neutrophil adherence to and diapedesis through vascular endothelium, thereby protecting subjacent myocytes from neutrophil-generated reactive oxygen species. 72 In the case of hyperoxic-exposed rats, inhalation of "NO may have induced salutory bronchorelaxation 73 and microvascular perfusion via induction of smooth muscle relaxation. Third, "NO serves as a potent inhibitor of oxidantinduced membrane and lipoprotein oxidation by annihilation of lipid radical species, thus terminating radical chain propagation reactions. By this mechanism,

100

75 ¸

50'

I

25 ¸

Hyperoxia

24

48

72

9'6

120

alone

144

Time (hr)

Fig. 6. Survival rates after in vivo exposure to "NO gas and/or hyperoxia. Adults rats were subjected to hyperoxia _+ "NO as noted in Experimental Methods. *Representsp < .01 compared with hyperoxia alone.

50

H.H. GUTIERREZet al.

"NO inhibits 0 2 " - , O N O O - , H202, "OH, and lipoxyg e n a s e - d e p e n d e n t lipid peroxidation, y i e l d i n g unstable nitrogen-containing derivatives o f o x i d i z e d lipids as products. 22'67 Nitric o x i d e reaction with 02"- to y i e l d the highly toxic peroxynitrite m o l e c u l e was e x p e c t e d to exacerbate O2"--induced toxicity, but the p r o o x i d a n t vs. antioxidant o u t c o m e o f these reactions, which are sensitive to "NO regulation, a p p e a r to be critically dep e n d e n t on the relative concentrations and rates o f production o f "NO and 02"-22,74,75 Fourth, "NO m a y divert O2"--mediated toxic reactions to other oxidative and less d a m a g i n g , pathways, thus protecting O2"--sensitive target molecules. Nitric o x i d e undergoes a facile r a d i c a l - r a d i c a l reaction with O2"-, J7 to yield O N O O - , a reaction that is three times faster than the S O D - c a t a l y z e d dismutation o f 02"-.76 This diversion o f 02"- through O N O O - oxidation and d e c o m p o s i t i o n p a t h w a y s w o u l d also limit H202 accum u l a t i o n and subsequent reactions o f H202, by decreasing the a m o u n t o f 02"- available for spontaneous or S O D - c a t a l y z e d dismutation. 75 Finally, is p o s s i b l e that "NO could lead to transcriptional activation o f antioxidant e n z y m e gene expression or transcriptional inhibition o f p r o i n f l a m m a t o r y mediators with the net result being e n h a n c e d survival in a m o d e l o f acute lung injury as o b s e r v e d from in vivo studies (Fig. 6 ) . F o r e x a m p l e , constitutive expression o f i N O S in h u m a n bronchial epithelial cells will induce c - f o s . 58 This is not a full explanation o f the presently reported in vitro p h e n o m e n a for two reasons. First, acute inhibition o f cell "NO production and shortterm e x p o s u r e to "NO donors significantly affected cell responses to oxidant stress within a time frame that excludes influences o f altered gene expression. A d d i tionally, xanthine o x i d a s e - m e d i a t e d injury to isolated perfused lungs was attenuated b y inhalation o f "NO under similar time constraints ( 1 - 2 h ) , w h i c h w o u l d exclude transcriptional events. 37 In spite o f these qualifying points, the "NO-mediated protection o f rats from h y p e r o x i c lung injury m a y include s o m e contributory role for altered regulation o f transcriptional expression in the m e c h a n i s m o f "NO action. In s u m m a r y , both e n d o g e n o u s "NO p r o d u c t i o n or e x o g e n o u s "NO addition offered protection to both in vitro and in vivo m o d e l s o f O2"--dependent oxidant lung injury. W e c o n c l u d e that differences in the local concentrations o f reactive species, antioxidants, and mediators o f tissue-free oxidant injury will critically influence the o b s e r v e d toxic p r o o x i d a n t or tissue-protective effects o f "NO. The relative rates o f production and steady-state concentrations o f 02"- and "NO, their sites o f production, and the d o m i n a n t m e c h a n i s m s o f o x i d a n t injury ocurring in tissues having simultaneous p r o d u c t i o n a n d / o r e x p o s u r e to 02" and "NO will in-

fluence whether "NO attenuates or exacerbates 02"-d e p e n d e n t cell and tissue injury. Acknowledgements - - We thank Drs. J. P. Crow, V. Darley-Usmar,

S. Matalon, and A. K. Tanswell for their comments and suggestions in the preparation of the manuscript. This work was supported in part by the National Heart, Lung and Blood Institute Grants PO1HL-48676, RO1-HL-40458 (B.A.F.), and by a Grant-in-Aid from the American Hearth Association, Alabama affiliate (H.H.G). H.H.G. is supported by a Parker B. Francis Fellowship in Pulmonary Research.

REFERENCES

1. Nathan, C.; Xie, Q.-W. Nitric oxide synthases: Roles, tolls, and controls. Cell 78:915-918; 1994. 2, Dawson, V. L.; Dawson, T. M.; London, E. D.; Bredt, D. S.; Snyder, S. H. Nitric oxide mediates neurotoxicity in primary cortical cultures. Proc. Natl. Acad. Sci. USA 88:6368-6371; 1991. 3. Snyder, S. H. Nitric oxide: First in a new class of neurotransmitters? Science 257:494-496; 1993. 4. Matheis, G.; Sherman, M. P.; Buckberg, G. D.; Haybron, D. M.; Young, H. H.; Ignarro, L. J. Role of L-arginine-nitric oxide pathway in myocardial reoxygenation injury. Am. J. Physiol. 262"H616-H620; 1992. 5. Lopez-Belmonte, J.; Whittle, B. J. R.; Moncada, S. The actions of nitric oxide donors in the prevention or induction of injury to the rat gastric mucosa. Br. J. Pharmacol. 108:73; 1993. 6. Mulligan, M. S.; Hevel, J. M.; Marietta, M. A.; Ward, P. A. Tissue injury caused by deposition of immune complexes is Larginine-dependent. Proc. Natl. Acad. Sci. USA 88:6338-6342; 1991. 7. Mulligan, M. S.; Warren, J. S.; Smith, C. W.; Anderson, D. C.; Yeh, C. G.; Rudolph, A. R.; Ward, P. A. Lung injury after deposition of IgA immune complexes: Requirement for CD18 and L-arginine. J. lmmunol. 148:3086-3092; 1992. 8. Hibbs, J. B., Jr.; Taintor, R. R.; Vavrin, Z.; Granger, D. L.; Drapier, J. C.; Amber, I. J.; Lancaster, J. R., Jr. Synthesis of nitric oxide from terminal guanidino nitrogen atom of L-arginine: A molecular mechanism regulating cellular proliferation that targets intracellular iron. In: S. Moncada, S.; Higgs, E. A., eds. Nitric oxide from L-arginine: A bioregulatory system. Amsterdam: Elsevier; 1990:189-223. 9. Cleeter, M. W. J.; Cooper, J. M.; Darley-Usmar, V. M.; Moncada, S.; Schapira, A. H. V. Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide. Implications for neurodegenerative diseases. FEBS Lett. 345:50-54; 1994. 10. Lepoivre, M.; Flaman, J-M.; Bobr, P.; Lemaire, G.; Henry, Y. Quenching of the tyrosyl free radical of ribonucleotide reductase by nitric oxide: Relationship to cytostasis induced in tumor cells by macrophages. J. Biol. Chem. 269:21891-21897; 1994. 11. Kwon, N. S.; Stuehr, D. H.; Nathan, C. F. Inhibition of tumor cell ribonucleotide reductase by macrophage-derived nitric oxide. J. Exp. Med. 174:761-767; 1991. 12. Curran, R. D.; Ferrari, F. K.; Kispert, P. H.; Stadler, J.; Stuehr, D. H.; Simmons, R. L.; Billiar, T. R. Nitric oxide and nitric oxide-generating compounds inhibit hepatocyte protein synthesis. FASEB J. 5:2085-2092; 1991. 13. Kubes, P.; Granger, D. N. Nitric oxide modulates microvascular permeability. Am. J. Physiol. 263:H611-615; 1992. 14. Wink, D. A.; Osawa, Y.; Darbyshire, J.; Jones, C. R.; Eshenaur, S.; Nims, R. W. Inhibition of cytochromes P450 by nitric oxide and a nitric oxide-releasing agent. Arch. Biochem. Biophys. 300:115-123; 1993. 15. Fukahori, M.; Ichimori, K.; Ishida, H.; Nakagawa, H.; Okino, H. Nitric oxide reversibly supresses xanthine oxidase activity. Free Radic. Res. 21:203-212; 1994.

Nitric oxide modulation of oxidant lung injury 16. Knowles, R. G.; Moncada, S. Nitric oxide as a signal in blood vessels. Trends Biochem. Sci. 17:399-402; 1993. 17. Huie, R. E.; Padmaja, S. Reaction of NO with 02-. Free Radic. Res. Commun. 18:195-199; 1993. 18. Beckman, J. S.; Beckman, T. W.; Chen, J.; Marshall, P. A.; Freeman, B. A. Apparent hydroxyl radical production by peroxynitrite: Implications for endothelial injury by nitric oxide and superoxide. Proc. Natl. Acad. Sci. USA 87:1620-1624; 1990. 19. Radi, R.; Beckman, J. S.; Bush, K. M.; Freeman, B. A. Peroxynitrite-induced membrane lipid peroxidation: The cytotoxic potential of superoxide and nitric oxide. Arch. Biochern. Biophys. 288:481-487; 1991. 20. Koppenol, W. H.; Moreno, J. J.; Pryor, W. A.; Ischiropoulos, H.; Beckman, J. S. Peroxynitrite, a cloaked oxidant formed by nitric oxide and superoxide. Chem. Res. Toxicol. 5:834-840; 1992. 21. Radi, R.; Beckman, J. S.; Freeman, B. A. Peroxynitrite oxidation of sulhydryls: The cytotoxic potential of endothelial-derived superoxide and nitric oxide. J. Biol. Chem. 266:4244-4250; 1991. 22. Rubbo, H.; Radi, R.; Trujillo, M.; Telleri, R.; Kalyanaraman, B.; Barnes, S.; Kirk, M.; Freeman, B. A. Nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation: Formation of novel nitrogen-containing oxidized lipid derivatives. J. Biol. Chem. 269:26066-26075; 1994. 23. Ischiropoulos, H.; Zhu, L.; Chen, J.; Tsai, M.; Martin, J.; Smith, C.; Beckman, J. S. Peroxynitrite-mediated nitration of tyrosine catalized by superoxide dismutase. Arch. Biochem. Biophys. 298:431-437; 1992. 24. Beckman, J. S.; Chen, J.; Ischiropoulos, H.; Crow, J. P. Oxidative chemistry of peroxynitrite. Methods Enzymol. 233:229240; 1994. 25. White, R.; Brock, T.; Chang, L.; Crapo, J.; Briscoe, P.; Ku, D.; Bradley, W.; Gianturco, S.; Gore, J.; Freeman, B. A.; Tarpey, M. M. Superoxide and peroxynitrite in atherosclerosis. Proc. Natl. Acad. Sci. USA 91:1044-1048; 1994. 26. Beckman, J. S.; Ye, Y. Z.; Anderson, P. G.; Chen, J.; Accavitti, M. A.; Tarpey, M. M.; White, R. Extensive nitration of protein tyrosines in human atherosclerosis detected by immunohistochemistry. Biol. Chem. Hoppe-Seyler 375:81-88; 1994. 27. Haddad, I.; Pataki, G.; Hu, P.; Galliani, C.; Beckman, J. S.; Matalon, S. Quantitation of nitrotyrosine levels in lung sections of patients and animals with acute lung injury. J. Clin. Invest. 94:2407-2413; 1994. 28. Kooy, N. W.; Royall, J. A.; Ye, Y. Z.; Beckman, J. S. Peroxynitrite is produced during human adult respiratory distress syndrome. Am. J. Respir. Crit. Care Med. 151:1250-1254; 1995. 29. Yamamoto, S.; Golanov, E. V.; Berger, S. B.; Reis, D. J. Inhibition of nitric oxide synthesis increases focal ischemic infarction in rat. J. Cereb. Blood Flow Metab. 12:717-726; 1992. 30. Wink, D. A.; Hanbauer, I.; Krishna, M. C.; DeGraff, W.; Gamson, J.; Mitchell, J. B. Nitric oxide protects against cellular damage and cytotoxicity from reactive oxygen species. Proc. Natl. Acad. Sci. USA 90:9813-9817; 1993. 31. Kuluz, J. W.; Prado, R. J.; Dietrich, D.; Schleien, C. L.; Watson, B. D. The effect of nitric oxide synthase inhibition on infarct volume after reversible focal cerebral ischemia in conscious rats. Stroke 24:2023-2029; 1993. 32. Bautista, A. P.; Spitzer, J. J. Inhibition of nitric oxide formation in vivo enhances superoxide release by the perfused liver. Am. J. Physiol. 266:G783-G788; 1994. 33. Payne, D.; Kubes, P. Nitric oxide donors reduce the rise in reperfusion-induced intestinal mucosa permeability. Am. J. Physiol. 265:GI89-G195; 1993. 34. Lfiszl6, F.; Whittle, B. J. R. Constitutive nitric oxide modulates the injurious actions of vasopressin on rat intestinal microcirculation in acute endotoxaemia. Eur. J. Pharmacol. 260:265-268; 1994. 35. Shultz, P. J.; Raij, L. Endogenously synthesized nitric oxide prevents endotoxin-induced glomemlar thrombosis. J. Clin. Invest. 90:1718-1725; 1992. 36. Pinsky, D. J.; Naka, Y.; Chowdhury, N. C.; Liao, H.; Oz, M. C.; Michler, R. E.; Kubaszewiski, E.; Malinski, T.; Stern, D. M. The nitric oxide/cyclic GMP pathway in organ trans-

51

plantation: Critical role in successful lung preservation. Proc. Natl. Acad. Sci. USA 91:12086-12090; 1994.

37. Kavanagh, B. P.; Mouchawar, A.; Goldsmith, J.; Pearl, R. G. Effects of inhaled nitric oxide and inhibition of endogenous nitric oxide synthesis in oxidant-induced acute lung injury. J. Appl. Physiol. 76:1324-1329; 1994. 38. Cooke, J. P.; Tsao, P. S. Cytoprotective effects of nitric oxide. Circulation 88:2451-2454; 1993. 39. Siegfried, M. R.; Carey, C.; Ma, X.; Lefer, A. M. Beneficial effects of SPM-5185, a cysteine-contalning nitric oxide donor in myocardial ischemia-reperfusion. Am. J. Physiol. 263:H771H777; 1992. 40. Clancy, R. M.; Leszcynska-Piziak, J.; Abramson, S. B. Nitric oxide, an endothelial cell relaxation factor, inhibits neutrophil superoxide anion production via a direct action on the NADPH oxidase. J. Clin. Invest. 90:1116-1121; 1992. 41. Asano, K.; Chee, C. B. E.; Gaston, B.; Lilly, C. M.; Gerard, C.; Drazen, J. M.; Stamler, J. S. Constitutive and inducible nitric oxide synthase gene expression, regulation, and activity in human lung epithelial cells. Proc. Natl. Acad. Sci. USA 91:1008910093; 1994. 42. Robbins, R. A.; Barnes, P. J.; Springall, D. R.; Warren, J. B.; Kwon, O. J.; Buttery, L. D. K.; Wilson, A. J.; Geller, D. A.; Polak, J. M. Expression of inducible nitric oxide in human lung epithelial cells. Biochem. Biophys. Res. Commun. 203:209-218; 1994. 43. Gutierrez, H. H.; Pitt, B. R.; Schwarz, M.; Watkins, S. C.; Lowenstein, C.; Caniggia, I.; Chumley, P.; Freeman, B. A. Pulmonary alveolar epithelial inducible nitric oxide synthase gene expression: Regulation by inflammatory mediators. Am. J. Physiol. 268:L501-L508; 1995. 44. Dupuy, P. M.; Shore, S. A.; Drazen, J. M.; Frostell, C.; Hill, W. A.; Zapol, W. M. Bronchodilator action of inhaled nitric oxide in guinea-pigs. J. Clin. Invest. 90:421-428; 1992. 45. Pepke-Zaha, J.; Higenhottan, T. W.; Dinh-Xuan, A. T.; Stone, D.; Wallwork, J. Inhaled nitric oxide as a cause of selective pulmonary vasodilatation in pulmonary hypertension. Lancet 338:1173-1174; 1991. 46. Belvisi, M. G.; Stretton, C. D.; Yacoub, M.; Barnes, P. J. Nitric oxide is the endogenous neurotranmitter of bronchodilator nerves in human airways in guinea-pig airways. Eur. J. Pharmacol. 210:221-222; 1992. 47. Heiss, L. N.; Lancaster, J. R.; Corbett, J. A.; Goldman, W. E. Epithelial autotoxicity of nitric oxide: Role in the respiratory cytopathology of pertussis. Proc. Natl. Acad. Sci. USA 91:267270; 1994. 48. Punjabi, C. J.; Laskin, J. D.; Pendino, K. J.; Goller, N. L.; Durham, S. K.; Laskin, D. L. Production of nitric oxide by rat type II penumocytes: Increased expression of inducible nitric oxide synthase following inhalation of a pulmonary irritant. Am. J. Respir. Cell Mol. Biol. 11:165-172; 1994. 49. Berisha, H. I.; Pakbaz, H.; Absood, A.; Said, S. I. Nitric oxide as a mediator of oxidant lung injury due to paraquat. Proc. Natl. Acad. Sci. USA 91:7445-7449; 1994. 50. Bolton, A. E.; Hunter, W. M. The labeling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agents. Biochem. J. 133:529-539; 1973. 51. Stamler, J. S.; Loscalzo, J. Capillary zone electrophoretic detection of biological thiols and their S-nitrosated derivatives. Anal. Chem. 64:779-785; 1992. 52. Green, L. C.; Wagner, D. A.; Glogowski, J.; Skipper, P. L.; Wishnok, J. S.; Tannenbaum, S. R. Analysis of nitrate, nitrite, and [~4N] nitrite in biological fluids. Anal. Biochem. 126:131138; 1982. 53. Kooy, N. W.; Royal1, J. A.; Ischiropoulos, H.; Beckman, J. S. Peroxynitrite-mediated oxidation of dihydrorhodamine 123. Free Radic. Biol. Med. 16:149-156; 1994. 54. Caniggia, I.; Tseu, I4 Han, R. N.; Smith, B. T.; Tanswell, A. K.; Post, M. Spatial and temporal differences in fibroblast behavior in fetal rat lung. Am. J. Physiol. 261:L424-L433; 1991. 55. Folz, R. J.; Crapo, J. D. Pulmonary oxygen toxicity. Curr. Pulmonol. 15:113-136; 1994.

52

H.H. Gtrnmu~z et al.

56. Haddad, I. Y.; Crow, J. P.; Hu, P.; Ye, Y.; Beckman, J. S.; Matalon, S. Concurrent generation of nitric oxide and superoxide damages surfactant protein A. Am. J. Physiol. 267:L242L249; 1994. 57. Nathan, C.; Xie, Q.-W. Regulation of biosynthesis of nitric oxide. J. Biol. Chem. 269:13725-13728; 1994. 58. Felley-Bosco, E.; Ambs, S.; Lowenstein, C. J.; Keefer, L. K.; Harris, C. C. Constitutive expression of inducible nitric oxide synthase in human bronchial epithelial cells induces c-fos and stimulates the cGMP pathway. Am. J. Respir. Cell Mol. Biol. 11:159-164; 1994. 59. Kobzik, L.; Bredt, D. S.; Lowenstein, C. J.; Drazen, J.; Gaston, B.; Sugarbaker, D.; Starnler, J. S. Nitric oxide synthase in human and rat lung: Immunocytochemical and histochemical localization. Am. J. Respir. Cell Mol. Biol. 9:371-377; 1993. 60. Repine, J. E. Scientific perspectives on adult respiratory distress syndrome. Lancet 339:466-469; 1992. 61. Freeman, B. A.; Crapo, J. D. Hyperoxia increases oxygen radical production in rat lungs and lung mitochondria. J. Biol. Chem. 256:10986-10992; 1981. 62. Turrens, J. F.; Freeman, B. A.; Crapo, J. D. Hyperoxia increases H202 release by lung mitochondria and microsomes. Arch. Biochem. Biophys. 217:411-421; 1982. 63. Kennedy, T. P.; Ran, N. V.; Hopkins, C.; Pennington, L.; Tolley, E.; Hoidal, J. R. Role of reactive oxygen species in reperfusion injury of the rabbit lung. J. Clin. Invest. 83:1326-1335; 1989. 64. Ignarro, L. J. Biosynthesis and metabolism of endothelium-derived nitric oxide. Annu. Rev. Pharmacol. Toxicol. 30:335-360; 1990. 65. Reddy, D.; Lancaster, J.; Comforth, D. P. Nitrite inhibition of Clostridium botulinum: Electron spin resonance detection of iron-nitric complexes. Science 221:769-770; 1983.

66. Kanner, J.; Harel, S.; Granit, R. Nitric oxide as an antioxidant. Arch. Biochem. Biophys. 289:130-136; 1991. 67. Rubbo, H.; Parthasarathy, S.; Kalyanaraman, B.; Barnes, S.; Kirk, M.; Freeman, B. A. Nitric oxide inhibition of lipoxygenase-dependent liposome and low density lipoprotein oxidation: Formation of novel nitrogen-containing oxidized lipid derivatives. Arch. Biochem. Biophys. 3(24):15-25; 1995. 68. Reif, D. W.; Simmons, R. D. Nitric oxide mediates iron release from ferritin. Arch. Biochem. Biophys. 283:537-541; 1990. 69. Radomski, M. W.; Palmer, R. M. J.; Moncada, S. Endogenous nitric oxide inhibits human platelet adhesion to vascular endothelium. Lancet 2:1057-1058; 1987. 70. Kubes, P.; Suzuki, M.; Granger, D. M. Nitric oxide: An endogenous modulator of leukocyte adhesion. Proc. Natl. Acad. Sci. USA 88:4651-4655; 1991. 71. Kurose, I.; Wolf, R.; Grisham, M. B.; Granger, D. N. Modulation of ischemia-reperfusion-induced microvascular dysfunction by nitric oxide. Ore. Res. 74:376-382; 1994. 72. Lefer, D. J.; Nakanishi, K.; Johnston, W. E.; Vinten-Johansen, J. Anti-neutrophil and myocardial protecting actions of a novel nitric oxide donor following acute myocardial ischemia and reperfusion. Circulation 88:2337-2350; 1993. 73. Gaston, B.; Drazen, J. M.; Loscalzo, J.; Stamler, J. S. The biology of nitrogen oxides in the airways. Am. J. Respir. Crit. Care Med. 149:538-551; 1994. 74. Rubbo, H.; Denicola, A.; Radi, R. Peroxynitrite inactivates thiolcontaining enzymes of T. cruzi oxidative metabolism and inhibits cell respiration. Arch. Biocher~ Biophys. 308:96-102; 1994. 75. Chumley, P.; Rubbo, H.; Gutierrez, H. H.; Freeman, B. A. Nitric oxide regulation of superoxide, hydrogen peroxide and peroxynitrite-dependent injury to vascular endothelium. Free Radic. Biol. Med. (submitted). 76. Klug, D.; Rabani, J.; Fridovich, I. A direct demonstration of the catalytic action of superoxide dismutase through the use of pulse radiolysis. J. Biol. Chem. 247:4839-4842; 1972.