Nitric oxide and lung surfactant

Nitric Oxide and Lung Surfactant Mikko Hallman and Kristina Bry

Inhalation of nitric oxide (NO) is an experimental treatment for severe pulmonary hypertension. Being rapidly metabolized by hemoglobin, inhaled NO causes selective vasodllation in the pulmonary vascular bed. In addition to the vascular smooth muscle, other pulmonary structures are exposed to inhaled NO, resulting in suppression of NO synthesis in a variety of pulmonary cells and in potential toxicity. NO is a free radical that interacts with a number of proteins, particularly metalloproteins. Together with superoxide radical, it rapidly forms highly toxic peroxynitrite. Peroxynitrite is involved in the killing of microbes by activated phagocytosing macrophages. In severe inflammation, peroxynitrite may be responsible for damaging proteins, lipids, and DNA. Peroxynitrite added to surfactant in vitro is capable of decreasing the surface activity, inducing lipid peroxidation, decreasing the function of surfactant proteins, SP-A and SP-B, and inducing protein-associated nitro-tyrosine. Exposure of animals for prolonged periods (48 to 72 hours) to inhaled NO (80 to 120 ppm) has been associated with a decrease in surface activity. This is caused by binding of surfactant to iron-proteins that are modified by NO (particularly methemoglobin), or by peroxynitrite induced damage of surfactant. In contrast, exposure of isolated surfactant complex to NO during surface cycling strikingly decreases the inactivation of surfactant, preventing the conversion of surfactant to small vesicles that are no longer surface-active, and preventing lipid peroxidation. This finding is consistent with the function of NO as a lipid-soluble chain-braking antioxidant. It is possible that this lipophilic gas has as yet undefined roles in regulation of surfactant metabolism and maintenance of surface activity. Deficiency in pulmonary NO may be present during the early neonatal period in respiratory distress syndrome and in persistent fetal circulation. The premature lung is likely to be sensitive to NO toxicity that may include lung damage, abnormal alveolarization, and mutagenicity. Defining of the indications, the dosage, and the toxicity of inhaled NO therapy remains the challenge for experimental and clinical research. Copyright 9 1996 by W.B.

Saunders Company

itric oxide (NO) is an i m p o r t a n t intercellular and intracellular messenger regulating many functions, including vascular tone, platelet adhesiveness, immunologic responses, and neurotransmission. 1-5According to c u r r e n t evidence, NO is a physiological vasodilator that is synthesized in endothelial cells and diffuses into vascular smooth muscle cells. NO activates guanylyl cyclase, catalyzing the formation of cyclic GMP that relaxes smooth muscle. NO is instantaneously b o u n d and inactivated by blood hemoglobin. E n h a n c e d endothelial NO synthesis contributes to the normal decline in pulmonary vascular resistance at birth. 6 T h e NO synthase, present in p u l m o n a r y endothelial cells, is suppressed a m o n g others by hypoxia and by competitive inhibitors of NO synthase. 7'8 According to r e c e n t reports, inhaled NO (I-NO) is an effective therapy in persistent p u l m o n a r y hypertension. 9'1~ It diffuses from the airways to p u l m o n a r y vascular adventitia, decreasing the vascular resistance, and possibly ameliorating

N

ventilation-perfusion mismatch. T h e efficacy of I-NO remains to be tested in large r a n d o m i z e d trials. During I-NO, many structures of the lung are exposed to exogenous NO that is an environmental pollutant (fossil fuels, tobacco smoke). 11 High endogenous NO may also cause serious organ damage (CNS, liver, cardiovascular system, islets of Langerhans).12 NO, being a free oxygen radical, reacts rapidly with a n u m b e r of biomolecules. Many of the reaction products are essential for normal homeostasis, whereas others are toxic. Inhaled NO expectedly increases its reaction products in the epithelial lining fluid of the airFrom the Neonatal Research Program, Department of Pediatrics, University of California, I*vine, CA. Address reprint requests to Mikko Hallman, MD, Division of Neonatology, University of California Irvine Medical Center, 101 City Drive, Bldg 29A, Rte 81, Orange, CA 92668. Copy*.ight 9 1996 by W.B. Saunders Company O146-0005/96/2003-0003505. 00/0

Seminars in Pelinatology, Vol 20, No 3 (June), 1996: pp 173-185

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Hallman and Bry

ways and airspaces. In addition, the epithelial lining of the airways and airspaces is exposed to variable, generally low endogenous concentrations of NO. However, during severe inflammatory conditions, activation of alveolar macrophages and other inflammatory cells result in bursts of high NO concentrations that favor the formation of peroxinitrite ( O N O O - ) and its toxic reaction products. ~3-a5 This article on metabolism of NO deals with its effects on the epithelial lining, with particular reference of the surfactant system during I-NO.

The Reactivity of Nitric Oxide NO' is a free radical that is oxidized, reduced, or c o m p l e x e d with other biomolecules depending on the microenvironment. 16 NO is one of several nitrogen oxides (NOx; ie, N20, N2Os, NO2, N204, N20~). In aqueous oxygenated envir o n m e n t interconversion among NOx species occurs with the formation of end-products, nitrite (NO2) and nitrate (NO~).17 The rate constant of NO oxidation is two orders of magnitude faster in aqueous solution than in the air (k 6 • 106 M -2 s-mvs ~ 1 • 104 M -2 s-l). In aqueous solution, the calculated half-life of NO at estimated concentrations required for signal transduction (10 to 100 n m o l / L ) would be more than 40 minutes. This is clearly longer than the biological half-life of NO, estimated to be only 1 to 10 seconds. Transition metals both oxidize and reduce NO, and oxidize NO2 to NO~. In addition, NO rapidly reacts with both non-heme and h e m e metalloproteins. The main trap for NO is oxyhemoglobin (Hb-Fe2+-O2). NO binds to oxyhemoglobin about two orders of magnitude faster than carbon m o n o x i d e and five to six orders of magnitude faster than oxygen. ~6 The reaction with HbFe2+-O2 produces nitrate and methemoglobin (Hb-FeS+). Hb-Fe2+-O2 + NO = Hb-Fe2+-OONO Hb-Fe ~+ + NO2 Besides oxyhemoglobin, NO reacts with a variety of other h e m e and non-heine metalloproreins, such as guanylyl cyclase. The basis of many biological actions of NO is the activation of guanylyl cyclase through binding of NO to the h e m e prosthetic group of the enzyme and formation

of guanylyl cyclase-heme-Fe2+-NO. 1 The activated enzyme catalyzes the formation of cyclic GMP. O t h e r NO-sensitive metalloproteins include NO synthases, ferritin, 18 ceruloplasmin, myoglobin, cyclo-oxygenase, catalase, ribonucleotide diphosphate reductase, 19'2~ and several components of the mitochondrial respiratory chain. 21-2~ These reactions have wide implications on the physiological and toxic effects of NO. Nitric oxide radical rapidly reacts with sup e r o x i d e radical (Og), with the rate constant (k) o f a b o u t 10 -9 M -1 s -1, forming peroxynitrite anion (ONOO-).13'14 In neutral pH, peroxynitrite rapidly forms peroxynitrous acid ( O N O O H ) . In the absence o f target molecules, the primary d e c o m p o s i t i o n p r o d u c t of p e r o x y n i t r o u s acid is nitrate. NO" + O~ = O N O O - + H + = O N O O H HNO3 = NO~- + H + Peroxynitrite oxidizes a wide spectrum of biological molecules, such as lipids, proteins, and DNA constituents. 13a5 According to one proposal, reaction of peroxynitrite with target molecules results in products characteristic of both nitrogen dioxide and hydroxyl radical (OH') as reactive intermediates. 24'25 O N O O H ~ NO2 + OH" A superoxide-rich environment and the neutral p H favors generation of O O N O - by the activated alveolar macrophages. Peroxynitrite has an important microbicidal and tumoricidal functions26'27; it may also activate guanylyl cyclase, z8 However, generation of excess of O N O O - leads to lung damage. Nitrogen dioxide is a major pollutant, known to have adverse effects on the airways, 11,zg'a~whereas the hydroxyl radicals possess extreme reactivity with a potential for destroying lipids, proteins, and DNA. 31 Peroxynitrite nitrates tyrosine residues of proteins in a reaction catalyzed by transition metals and by Zn,Cu-superoxide dismutase; 15 it also oxidizes m e t h i o n i n e . 32 A n u m b e r o f biological electrophilic molecules are nitrosated in the p r e s e n c e o f N O or peroxynitrite. Ironnitrosyls, nitrosamines (RNH-NO), and S-nitrosothiols (RS-NO) are a m o n g these products. S-Nitrosothiols may act as a scavenger for peroxynitrite in a h e m o g l o b i n - f r e e microenvi-

175

Nitric Oxide and Lung Surfactant

r o n m e n t , such as the epithelial lining fluid. SNitrosothiols slowly release NO, activating NOm e d i a t e d functions (vasorelaxation, inhibition o f p l a t e l e t aggregation).16 T h e f o r m a t i o n o f n i trosamines requires supraphysiological concentrations of NO, possibly present d u r i n g smoking (NO 200 to 1,000 p p m of cigarette smoke), 11 infection, or d u r i n g I-NO therapy. Nitrosamines and aryldiazonium derivatives o f d e o x y n u c l e o t i d e s have b e e n studied with respect o f their carcinogenicity. 33

Synthesis a n d F a t e of Nitric O x i d e in the Lung Most cells have a potential of generating NO. The cells capable of producing NO in the lung include macrophages, granulocytes, endothelial cells, fibroblasts, vascular smooth muscle cells, mast cells, and epithelial cells, including the type II alveolar cells. Nitric oxide is formed by NO synthase (NOS) that catalyzes a five-electron oxidation of guanidine group of L-arginine to generate citrulline and NO. 7

Arginine

NOS is bound to plasma membrane, generating extracellularly released NO. 7 Proinflammatory cytokines decrease the activity of endothelial NOS. s5 Under a nonstimulated state, macrophages possess little if any inducible NOS mRNA or protein. Lipopolysaccharide or cytokine (interferon% TNF, IL-1 and others) elicit new NOS protein synthesis over 2 to 4 hours. Glucocorticoids, epidermal growth factor, ferric iron, transforming growth factor-/5, IL-10, IL-4, and serine protease inhibitors attenuate this endotoxin/cytokine-induced increase in N O S . 27'36-39 In addition to macrophages and granulocytes, inducible NOS activity, elicited by inflammatory stimuli, is present among others in hepatocytes, vascular smooth muscle cells, fibroblasts, and epithelial cells. Two distinct NOS mRNA species, similar to Ca2+-dependent brain and inducible hepatic NOS, are present in h u m a n bronchial and alveolar type II cells, a~ T h e synthesis of inducible, CaZ+-independent NOS in these epithelial cells is increased by cytokines, particularly by interferon-y but not by lipopolysaccharide alone. Dexamethasone, epidermal growth factor, and transforming growth factor-/3 diminish the cyto-

NADPH 1/2 NADPH , ~ Hydroxy-arginine

~

02

H20

The above reaction uses 02 and NADPH as co-substrates, and tetrahydropteridin, flavin adenine dinucle0tide, flavin mononucleotide, thiol, and h e m e as cofactors. The availability of arginine may also be rate-limiting for NO synthesis. In addition, nitric oxide suppresses its own synthesis by binding to the h e m e moiety of NOS. a4 Functionally NOS isoforms are classified either as Ca2+-dependent, constitutive, or Ca2+-indepen dent, inducible. The calcium requirement for NOS activity is typical for a calmodulin-activated enzyme, whereas Ca 2+ independence is a result of exceptionally tight binding of NOS to calmodulin. The endothelial CaU+-dependent NOS activity is stimulated by acetylcholine, bradykinin, and other agents that increase intracellular Ca 2+. Vascular endothelium contains a major membrane-bound Ca2+-dependent NOS and a soluble Cag+-depen dent NOS. Phosphorylation of endothelial NOS regulates both its activity and its subcellular distribution: the nonphosphorylated, catalytically active

02

Citrulline + NO

H20

kine-induced activity of inducible NOS in the epithelial cells. 40,4a At resting state, there may not be e n o u g h intracellular Ca u+ to activate the Ca2+-dependent NOS in type II alveolar epithelial cells. 42 The maximal cytokine-induced NO production by isolated type II cells is one order of magnitude lower than the cytokine-induced NO production by isolated alveolar macrophages. Short exposure to ozone in situ increases the expression of inducible NOS and production of NO by isolated type II cellsY Ozone is known to react with NO to form NO2. The possibility that NO regulates the metabolism of surfactant (synthesis, intracellular transport, exocytosis, recycling, or catabolism) remains to be studied.

Methods Detecting the Effects of I n h a l e d Nitric Oxide The expired air of healthy adults contains small quantities of NO (ie, 6 to 90 ppb). In reactive

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Hallman and B,y

airway diseases and infectious/inflammatory diseases, the concentration of NO in expired air is increased. 44 Intravenous acetylcholine and cardiovascular stress 45 increase the concentrations of NO in expired air, suggesting that the pulmonary vascular endothelium contributes to expired NO. Nitrosylated peptides (250 n m o l / L ) , mainly nitrosylated glutathione, and N O 2- (15 #mol L) are present in epithelial lining fluid of n o r m a l subjects: 46 In humans, the n u m b e r of alveolar macrophages correlates with NO2 concentration in BAL, whereas there is no correlation between neutrophils in BAL return and NO)- concentrations. 46 Data on nitrosylated peptide and nitrite/nitrate concentrations in epithelial lining fluid in various lung diseases are not available presently. NO is sparingly soluble in water (36 m L / L at 37~ 760 m m Hg). Exposure of the aqueous phase to 100 p p m of NO-containing gas phase would result in 170 n m o l / L of NO in the liquid phase. This is higher than required for signal transduction in vivo (10 to 100 n m o l / L ) , but lower than apparent NO concentrations seen in ischemic brain or in the vicinity of activated macrophages (1 to 10 #mol/L).47 However, the solubility of NO in the body is higher than in the water, because NO as a lipid-soluble gas concentrates within the membranes: The metabolism rate of NO is a function of NO concentration and of the reactivity of NO with other molecules in the microenvironment. During I-NO, monitoring of inhaled NO and exhaled N O 2 concentration, blood methemoglobin, and bleeding time is r e c o m m e n d e d . With the exception of accidental overdosage of NO, resulting in severe methemoglobinemia, shock and death, exhaled N O 2 and blood methemoglobin levels generally remain normal or modestly elevated during I-NO (--<20 ppm) therapy, 1~ and there have not b e e n reports on increased incidence of serious hemorrhages. However, the studies are small and nonrandomized. ~176T h e metabolic end-products of NO, nitrite and nitrate, are low in urine and serum of newborns with persistent pulmonary hypertension; 49 to date no reports on the effects of I-NO on nitrate/nitrite concentrations are available. T h e variable dietary intake of nitrate/nitrite complicates the estimate of total body NO production. The action of peroxynitrite on tyrosine residues of various proteins produces protein-nitro-

tyrosine that may perturb functional characteristics o f proteins. In severe respiratory failure in adults, nitro-tyrosine is detectable in the lung by immunohistochemistry, suggesting NO-dependent lung injury. 25 Similar evidence of NO toxicity may be f o u n d in some cases of severe respiratory failure in newborn infants as well. Airway specimens for nitro-tyrosine could be useful for detection of pulmonary toxicity caused by endogenous or exogenous NO. T h e r e is a need to develop noninvasive methods for detection of nitro-tyrosine. 5~ In isolated, perfused hypoxic or angiotensin II-treated lung, the pulmonary vascular resistance bears an inverse relationship with the concentration of inhaled NO ranging from 1 to at least 1,000 ppm, and the effect of NO is reversible. 51 However, the dose relationship between I-NO and the degree of respiratory failure in persistent pulmonary hypertension shows a more limited dose range. According to one study in newborn infants, the maximal response was obtained with 10 ppm; further increase in the concentration of I-NO did not improve the gas exchange. 52 A high dose of I-NO may increase the ventilation-perfusion mismatch, 9 or cause other adverse effects. 44'53 The impact of NO in experimental setting is assessed by administering of I-NO, NO-donors (S-nitroso-N-acetylpenicillamine and others), or Larginine analogues (N%nitro-L-arginine methyl ester, I~NAME, and others) that are competitive inhibitors of NOS; the latter are not lung-specific)

Interactions of Nitric Oxide With Lung Surfactant The following evidence indicates that the alveolar epithelial lining and the surfactant system in the normal lung are at least intermittently exposed to NO: (1) the alveolar macrophages and alveolar type II cells possess the capacity of NO synthesis; (2) NO diffuses across the epithelium from pulmonary vascular endothelial cells45; (3) NO is detectable in expired air; and (4) the epithelial lining fluid contains S-nitrosothiols (RS-NO) that form when NO (or peroxynitrite) reacts with SH-groups. T h e r e is no reliable m e t h o d to analyze the " N O load" on the alveolar epithelial lining. Surfactant affects lung mechanics, gas exchange, 54 and microbicidal defense functions. 55 Perturbations in the surfactant

Nitric Oxide and Lung Surfactant

system as a result of I-NO are difficult to detect in patients, because the underlying lung disease is associated with a range o f abnormalities, including surfactant dysfunction, altered composition of surfactant, and altered surfactant concentration in the epithelial lining fluid. 56 To our knowledge, there are no published studies on p u l m o n a r y surfactant recovered f r o m lung effluent during I-NO t r e a t m e n t in patients with severe respiratory failure. Indirect Effects o f Nitric O x i d e on Lung Surfactant

T h e p u l m o n a r y defense functions are interdep e n d e n t . N O influences c a r d i o p u l m o n a r y functions that have an effect on the surfactant system. 17 Inhibition of e n d o g e n o u s N O formation increases platelet-activating factor-evoked protein extravasation. 57 In contrast, I-NO (50 p p m ) prevents endothelial d a m a g e and vascular leak in perfused lung 58 and in i s c h e m i a / r e p e r f u sion, 5~ potentially decreasing the blood-derived inhibitors of surface activity in epithelial lining fluid. N O (either e n d o g e n o u s or exogenous) improves the survival in traumatic s h o c k , 6~ in endotoxin- or microbe-iduced s h o c k , 61'62 and in hyperoxia. 63-66N O maintains perfusion by decreasing the high vascular resistance and preventing the endothelial adhesion of trombocytes and granulocytes. In addition, by acting as a chainbreaking antioxidant, 67 N O may protect against oxidant injury. However, in the presence of high local concentrations (and of low concentrations of antioxidants), N O b e c o m e s toxic, decreasing the cardiac output and causing oxidant injury. High quantities of N O a n d superoxide are g e n e r a t e d in inflammatory diseases and during reperfusion, lz'68 Superoxide and N O f o r m peroxynitrite, a major toxic intermediate of N O metabolism causing o x i d a n t injury, 47'69 and potentially disturbing any function in the lung. N O toxicity threshold is lowered as a result of immaturity or acquired defect in antioxidants. 7~ Lack of thiol groups (proteins, r e d u c e d glutathione), and decrease in unsaturated iron-binding capaci@ 8 are i m p o r t a n t factors potentiating the toxicity of NO. A n o t h e r concern is that as a result o f inhibitory effect of I-NO on NOS, the endogenous synthesis of N O is suppressed, causing local p e r t u r b a t i o n in N O - m e d i a t e d functions (microbicidal effects, vasodilatation). Mercer et a171 r e p o r t e d that the exposure o f

177

adult rat to N O for 9 weeks at 0.5 to 1.5 p p m twice daily (mimicking exposure to polluted outd o o r environment) caused d e g e n e r a t i o n of interstitial cells, interstitial matrix, and connective tissue. An identical dosage of N O 2 had similar, although smaller effects on lung interstitium. T h e pathogenetic mechanisms of these findings are unknown; N O may activate metalloproteinases, or inhibit protein and DNA synthesis. 26'7a Exposure of rats for 6 weeks to N O (2 p p m ) has led to emphysematous changes with large airspaces and destruction o f alveolar septa. 72 Connective tissue and interstitial fibroblasts have a major influence on the differentiation of the alveolar type II cells 73 and on the alveolarization of the airspaces. 74 It is essential to confirm and extend the findings of Mercer et al, 71 a n d investigate the effects of I-NO in lung immaturity and in chronic lung disease. Alveolar inflammatory cells influence the surfactant function. The m a c r o p h a g e s catabolize surfactant, 75 and activated neutrophils deteriorate the surfactant f u n c t i o n . 76 I-NO given to healthy y o u n g rats (100 p p m for 24 hours in r o o m air or in 95% 02) neither decreases Fc receptors n o r alters the respiratory burst activity of alveolar macrophages. I-NO does n o t affect the total or the differential cell counts of the bronchoalveolar lavage (BAL) return either. 77 IN O (15 p p m for 10 hours) does not affect the H2O 2 p r o d u c t i o n or the expression of/32 integrin by neutrophils recovered by BAL f r o m rats treated with intratracheal endotoxin and 85% 02 .78 Further studies are required to assess the influences of I-NO on the p u l m o n a r y inflammatory ceils in various disease states and in different age groups. Studies o n Pulmonary Surfactant After Inhaled N O

To date only few studies have addressed the direct effects of I-NO on the surfactant system. In some studies the exposure c h a m b e r is flushed with N O 79 whereas in others N O is given to the nose area of spontaneously breathing animals 77 or via the intubation tube. 8~ No adverse effects on surfactant are detected in piglets during the 48-hour exposure to 100 p p m N O in 90% oxygen or in air. 79 However, a similar exposure of ventilated piglets decreases the surface activity of the lavageable surfactant a m o n g the N O and 90% O2-exposed animals,

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suggesting exacerbation of 02 toxicity by NO. s~ Others have r e p o r t e d that periodic exposure of adult rats to 100 p p m NO improves the survival during hyperoxia, 64 whereas administration of NOS inhibitor (L-NAME) decreases the survival in hyperoxia. 6~'66 The acute effects on the gas exchange and on the lung mechanics of I-NO used in combination with exogenous surfactant appear conflicting, too: one study finds an attenuated surfactant response on the gas exchange in NO-treated (undisclosed dose) pigs in BALinduced respiratory failure, sl whereas others find that I-NO potentiates the beneficial effects of exogenous surfactant in BAL-induced respiratory failure in rabbits (I-NO 20 ppm),S2's3 or increases gas exchange in surfactant-treated immature lambs with RDS (gestation 115 days, I-NO 20 p p m for 3 hours).S4 The differences between the results may be a result of differences in age and species o f animals, dose and quality of INO, and of differences in the ancillary treatment practices. In one study, adult rats breathed in nose-only, n o n r e b r e a t h i n g exposure chambers s~ for 24 hours in one of the following four humidified environments: (1) air; (2) 95% O2; (3) NO (final concentration 100 ppm) in air; or (4) NO in 95% O2. T h e animals survived the exposures without a significant respiratory distress, and histology of the lung showed no atelectasis or increase in inflammatory cells. In animals exposed to NO and hyperoxia, the mean concentration of N O 2 in breathing zone was 3.1 ppm. The two groups of animals on I-NO had a lower rectal temperature than the animals exposed to air or O2 (31 to 32~ vs 35 to 36~ indicating impaired thermoregulation during the high dose of I-NO. T h e recoveries of phosphatidylcholine (PC) and saturated PC in BAL were similar in all four groups exposed to NO a n d / o r 02. However, the m i n i m u m surface tension ()~min), at low surfactant concentration (1.5 #mol P C / m L ) from animals exposed to 100 ppm NO (in air or O2) was higher and the surface adsorption rate lower than the Xmin or the surface adsorption in the control groups. When studied at high surfactant concentration (4 #mol P C / m L ) , the differences of the surface activity between the four treatment groups tended to disappear. The surfactant PC concentration in the epithelial lining fluid of the normal lung was higher than 1.5 # m o l / m l . s6 The inactivation of surfactant found

after I-NO may thus be clinically significant only in surfactant deficiency syndromes (particularly in RDS). T h e non-sedimentable BAL protein fraction from animals exposed to I-NO contains a higher activity of surfactant inhibitor than the corresponding fraction from the controls. Exposure to NO (_+ hyperoxia) has no effect on the quantity or on the molecular weight distribution of the BAL protein. The non-sedimentable BAL from the control animals was exposed to NO in vitro, s5 As a result of the NO exposure, the BAL protein fraction becomes an active inhibitor of the surface activity. Mechanism o f I - N O - I n d u c e d Increase in Surfactant Inhibition by Proteins

BAL proteins from NO-exposed animals contain small quantifies of Hb that is quantitatively oxidized to methemoglobin (Hb-Fe~+). Similar quantities of H b are recovered in the BALs from the controls, but this Hb is mostly oxyhemoglobin (Hb-Fe2+-O2). The blood hemoglobin from the NO-exposed animals is reduced (methemoglobin content 5% to 7% of total Hb), as methemoglobin reductase in red blood cells rapidly converts methemoglobin to Hb-Fe2+-O2 (Fig 1). Methemoglobin and H b exposed to NO inhibit the surface activity at a concentration as low as 30 # g / m L , ie, low e n o u g h to be f o u n d in the epithelial lining fluid in mild lung injury. Methemoglobin (unlike Hb-Fe2+-O2) binds to surfactant, decreasing surface activity after the u n b o u n d methemoglobin is removed by sedimentation. T h e acidic surfactant lipid, phosphatidylglycerol, increases the binding of methemoglobin to surfactant. Both surfactant protein B (SP-B) and the acidic lipid are necessary in eliciting the Hb-Fe~+-induced inhibition of surface activity (Table 1). Methemoglobin decreases the surface activity and disturbs the electrostatic interactions s7 between the acidic surfactant lipid and the cationic SP-B. ss The surface activity-inhibiting characteristics of methemoglobin are shown by applying surfactant together with methemoglobin (or oxyhemoglobin, or placebo as controls) to the airways of newborn rabbits at premature birth on day 27 (term, 31 days). T h e surfactant-induced ventilatory response and the post-ventilatory lung volumes are decreased by methemoglobin. Increasing the dose of surfactant decreases the

Nitric Oxide and Lung Surfactant

Shock

Peroxynitrous acid (ONOOH)

Organ damage - lipid peroxidation - protein inactivation

NO 2

Peroxynitdte (ONOO')

Superoxide (O2- )

,91_•

179

N itrosothiols (RS - NO)

Pulmonary vasodilation Improved ventilation/pe rfusion

Hydroxyl (OH,)

]

/ ~ release

Bronchodilatation

Nitro-tyrosine Damage to surfactant

Improved surfactant function

Fe 3+

Increased resistance against - oxidative damage

, Nitrosoamines (RNH - NO)

Growth inhibition

- trombosis - reperfusion injury

Deaminated DNA

Mutagenicity

- microbes, tumor cells

(ONOO-)

I

surfactant dysfunction

Methemoglobin (Hb - Fe3+ ) ~

I Oxyhemoglobin I (Hb - Fe2+- 02)

Methemoglobin , ~

NAD DAMAGE

~

NADH2 ~

~

PROTECTION

Figure 1. Possible effects of inhaled (I) and endogenous (E) nitric oxide in the lung. Depending on the concentrations of NO in different lung compartments, NO has beneficial or adverse effects. Interactions of NO with a n u m b e r of metal-proteins cause many of NO's physiological actions and side effects.

i n h i b i t o r y effect o f m e t h e m o g l o b i n . O x y h e m o g l o b i n has n o effect o n the efficacy of e x o g e n o u s surfactant, ss Besides c o n v e r t i n g o x y h e m o g l o b i n to m e t h e m o g l o b i n , s9 N O f o r m s S-nitrosothiols with proteins, 16 reacts with i r o n - c o n t a i n i n g p r o t e i n s , re-

leasing n o n - h e m e i r o n f r o m ferritin, is a n d m a y b e c o n v e r t e d to p e r o x y n i t r i t e that catalyzes nitration of proteins. N o n e o f these r e a c t i o n s appreciably c h a n g e t h e m o l e c u l a r weight, b u t c o u l d c h a n g e the capacity of p r o t e i n s to decrease the surface activity. U n d e r the c o n d i t i o n s used, S-

Table 1. Methemoglobin Binding to Surfactant and Effect of the Methemoglobin on the Minimum Surface Tension (Xmi.)

Xmi,, (mN/m)** Surfactant Natural surfactant (0.7 m g / m L ) Surfactant lipid extract (0.9 mg/mL) SP-B, dipalmitoyl PC and phosphatidylglycerol (0.9 mg/mL) Dipalmitoyl PC and phosphatidylglycerol (2.5 mg/mL) Dipalmitoyl PC (5 mg/mL)

Methemoglobin Binding* (#g/mg surfactant)

No Inhibitor

Methemoglobin 0.5 mg/mL

61 _+ 5 59 + 3

2 -+ 0 4 + 1

16 _+ I t 17 _+ 3t

44--+ 6

2-+ 0

14_+ 2t

44+3 12-+ 4 +

2+0 9-+ 1

2--+0 11 -+ 2

*Methemoglobin (0.5 mg/mL) was incubated with various surfactants (~0.5 mg PC/mL). Thereafter, surfactant was isolated by centrifugation and the binding of methemoglobin to surfactant quantitated. No detectable quantities of oxyhemoglobin bound to surfactant, ss ** Xm, in the absence or presence of methemoglobin was measured at surfactant concentrations indicated. Oxyhemoglobin increased XmintOO, but this is likely to be due to trace of methemoglobin contaminant. t P < .05 compared with Xmi,in the absence of inhibitor. $ P < .05 compared with methemoglobin binding to other surfactants.

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nitrosothiols have no effect of surfactant inhibition and f o r m a t i o n o f nitro-tyrosines is not detectable, s8 Animals exposed to N O have a higher iron saturation of transferrin in BAL than the controls, s5 suggesting that I-NO releases storage iron. Despite lowering the iron-binding capacity, I-NO did not decrease the non-sedimentable BAL proteins' capacity to prevent lipid oxidation. s5 Iron saturation of transferrin also increases the inhibition of surface activity by this protein (>0.5 m g / m L ) . 9~ However, the concentration of iron-saturated transferrin present in epithelial lining fluid is often too low to account for the decrease in surface activity. Direct E f f e c t s o f N O and its M e t a b o l i s m P r o d u c t s o n L u n g Surfactant

Exposure of surfactant to peroxynitrite in vitro decreases the surface activity. T h e surfactant d a m a g e by peroxynitrite is e n h a n c e d in the presence of Fe3+-EDTA. m'92 Peroxynitrite causes lipid peroxidation and decreases the ability of the maj o r hydrophilic surfactant protein (SP), SPA, to aggregate lipids and to act synergistically with o t h e r snrfactant proteins in lowering Xmin" T h e loss of this surface activity-promoting function of SP-A is associated with the generation o f nitrotyrosine residues, detectable by Western blots. 93 A mixture of the h y d r o p h o b i c SP-B and SP-C exposed to peroxynitrite does not attain low Xmin in the presence of snrfactant lipids. T h e surfactant damage, similar to that caused by peroxynitrite in vitro, may take place in vivo, when concentrations of N O are very high and superoxide anions are generated. 94 Sulfhydryl groups may protect against peroxynitrite-induced surfactant d a m a g e .91 Nitrogen dioxide and hydroxyl radical are metabolism products of NO. According to Mfiller et a l Y exposure of adult rats to inhaled NO2 for 1 to 3 days causes a dose-dependent increase in cellularity of BAL and a shift from m a c r o p h a g e s to granulocytes and lymphocytes. NO2 also increases the n u m b e r of type II alveolar cells and their activity o f PC synthesis. 96 T h e total alveolar protein a n d p h o s p h o l i p i d increase in quantity, with a decrease in portion of saturated PC. SP-A is unaltered as studied using two-dimensional gel electrophoresis. Exposure to NO2 (5 and 10 p p m ) increases the surface tensions as studied using the modified Wilhelmy balance, whereas low NO2 (0.8 p p m ) has no effect. 95 Hy-

70. 9 60"

~

Control Cycling in presence of 80 ppm NO in Air Cycling in Air

50' '~

40' 30'

"6 2o, 10' .

1.020

1.040

1.060

1.080

Density ( g / m l )

Figure 2. The density profile of surfactant particles after surface cycling in the presence or absence of NO (80 ppm) in the ambient air. The surface area was oscillated between 0.7 and 1.4 cm ~ at a rate of 0.4 Hz for 8 hours in 37~ NO delayed the conversion of the dense surfactant particles to less dense particles that are no longer surface-active (Yma*~> 14 mN/m).

droxyl radicals induce lipid peroxidation, damage of surfactant protein, and decrease the surface activity. 9~ Exposure of natural surfactant to N O (80 p p m for 4 to 12 hours) in air, while the surface area is cycled, protects the surfactant against the decrease in the surface activity that takes place during the surface cycling. T h e m e c h a n i s m of N O in protecting against the inactivation of surfactant is poorly understood. T h e interracial surfactant film is a small transient fraction of total surfactant. Cyclic compression of surface film, occurring during tidal ventilation, concentrates the fully saturated phospholipids on the interface, virtually eliminating the surface tension and the air-liquid contact, whereas during cyclic expansion of the surface, the surfactant particles f r o m the subphase rapidly adsorb to the interface, maintaining the surface tension close to equilibrium (ie, 22 to 26 m N / m ) . T h e surface cycling converts the heavy SP-rich aggregates to smaller, protein-poor lipid vesicles. As during the surface cycling, the large surfactant aggregates are converted to small, less dense vesicles, the surface activity decreases (ie, the Ymin increases and the surface adsorption decreases).98 N O decreases the rate of conversion of the large surfactant aggregates to smaller, less dense vesicles that no longer are surface-active (Fig 2). It has b e e n p r o p o s e d that surface cycling ten-

Nitric Oxide and Lung Surfactant

ders the surfactant protein(s) sensitive to an elastase-like enzyme activity that cleaves surfactant proteins and decreases the aggregate size of surfactant. 98 Increase in SP-A or SP-B decreases the rate of conversion from large aggregates to small vesicles. 99'1~176 NO-induced decrease in surfactant subtype conversion is also evident when the hydrophilic proteins (including SPA, proteases) are removed by lipid extraction. Therefore, the mechanism of NO-induced resistance to the spontaneous inactivation may not be enzymatic. Nitric oxide is a highly lipid-soluble gas that associates with surfactant. T h e effect of NO in protecting the surfactant aggregates during the surface cycling requires oxygen and presence of the hydrophobic surfactant proteins. When given in moderate quantities, this gas protects against oxidant injury. 63-67The proposed mechanisms are as follows: (1) NO serves as a chainbreaking antioxidant, destroying the toxic intermediates of lipid peroxidation (peroxyl radicals) that augment the generation of more free radicals: and (2) NO has shown to paradoxically decrease the generation of peroxynitrite in the presence of superoxide. This effect depends on the concentrations o f both NO and superoxide. 67 T h e hypothesis that NO solely deteriorates the surfactant system thus needs to be modified: besides surfactant inhibition by products of NO metabolism, NO additionally prevents the inhibition of surface activity during surface cycling. More studies are required to elucidate whether the NO-induced dynamic stability of the surface activity is expressed in situ, and whether the changes in surfactant metabolism influence the responsiveness of I-NO in lung diseases.

Summary and Conclusion Inhaled NO supplements and enhances the effects of endogenous NO in decreasing the pulm o n a r y vascular resistance in pulmonary hypertension 1'9 and, owing to its exceptional affinity to oxyhemoglobin, NO is likely to be inactivated within seconds after it is inhaled, resulting in fast onset and discontinuation of the vasodilatatory action. Besides being a selective pulmonary vasodilator, NO's capacity of decreasing vessel wall adhesion of trombocytes and granulocytes is likely to moderate the vascular permeability and the inflammatory response, and decrease the reperfusion injury.

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Being a free radical with both antioxidant and pro-oxidant properties, NO can be called a molecular chameleon. NO and superoxide may form peroxynitrite ( O N O O - ) that serves as an antimicrobial agent of macrophages. However, excess of O N O O damages vital structures by nitrating phenolic rings, oxidizing lipids, and by other mechanisms. Excess of NO ( O N O O - ) inhibits mitochondrial respiration, activates the energy-consuming DNA-repair enzyme, and may even be carcinogenic (Fig 1). Long-term exposure of adult rodents to a low dose of NO decreases lung interstitial cells, connective tissue, and alveolar septae by a mechanism that remains unknown. 71 As for other biological systems, NO may either activate or inhibit the pulmonary surfactant system. U n d e r conditions favoring generation of O N O O - , surfactant is degraded with formation of lipid peroxides, nitration of tyrosine residues of surfactant proteins, and with loss of the surface activity. Peroxynitrite additionally decreases the oxygen uptake and sodium transport in alveolar type II cells. 1~ The presumed degradation products of O N O O - , N O 2 and OH', both damage the surfactant complex. The presence of superoxide, transient metal, high concentrations of NO, oxygen, and the absence of thiol groups, urate, and ascorbate in the airways promote the destructive role o f NO, as the g e n e r a t i o n O N O O - is accelerated or the defense mechanisms against O N O O - toxicity are weakened. NO may increase free oxygen radicals by releasing ferritin-bound iron that may become a transient metal, required for the generation of hydroxyl radicals. Thiols and nitrosothiols (consisting of mostly reduced or nitrosylated glutathione) in the epithelial lining fluid are likely to control NO homeostasis. By becoming nitrosylated as a result of a burst of peroxynitrite (or NO), the thiols neutralize the toxic effects of peroxynitrite. In addition, by slowly releasing NO, S-nitrosothiols are an additional source of NO (Fig 1). During I-NO, oxyhemoglobin that may contaminate the epithelial lining fluid is converted to methemoglobin. Instead of becoming reduced by methemoglobin reductase, alveolar Hb-Fe ~+ binds to acidic surfactant lipids and decreases the surface activity in the presence of SP-B. This hydrophobic surfactant protein has intermittent positively charged amino acid resi-

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dues, mostly arginine. These positively charged amino acids are essential for promoting tile critical surfactant properties, fast surface adsorption and low minimum surface tension. By binding to the acidic lipid (phosphatidylglycerol), HbFe 3+ disturbs the electrostatic interactions between SP-B and the acidic lipid. The adverse effect of Hb-Fe 3+ to the surfactant system is eliminated by increasing the concentration of surfactant. In respiratory distress syndrome, I-NO may thus promote the ventilatory response by exogenous surfactant. NO also has a potential of improving the surfactant function. When lung surfactant is cycled in vitro (ie, the air-liquid interface is cyclically compressed and expanded, mimicking breathing movements), addition of NO to the gas phase improves the surface activity. NO delays the conversion of the large surfactant aggregates to small vesicles that no longer are surface-active. The mechanism and physiological significance of this effect remains to be studied. Being highly soluble in lipid and sparingly soluble in water, NO (unlike the water-soluble superoxide) concentrates within the surfactant structures and the membranes in general. By acting as a chainbreaking antioxidant, NO serves as an antioxidant. 67 At the interfacial surfactant lining, NO could protect against oxidation of lipids and hydrophobic proteins. The patient's response to I-NO is determined by the degree of pulmonary vasodilatation, or by other pulmonary effects of NO. Inhaled NO potentially alters the pulmonary liquid balance, permeability, interstitial cells and connective tissue, immunological defense, responsiveness of the airway musculature, and the function of the surfactant system; the effects of NO on these systems may be positive or negative, depending on the dosage and the host. The challenge for the future is to better understand the pharmacodynamics of I-NO. Monitoring of the effects of both endogenous and inhaled NO need to be improved. Better understanding of the roles of NO during normal lung development and in severe lung diseases would eventually improve the treatment practices and the outcome. References 1. Moncada S, Higgs A: The L-arginine- nitric oxide pathway. N Engl J Med 329:2002-2012, 1992

2. Buga GM, Gold ME, Wood KS, et al: Endothelium derived nitric oxide relaxes nonvascular smooth muscle. EurJ Pharmacol 161:61-72, 1989 3. Culotta E, Koshland DE Jr: NO news is good news. Science 258:1862-1865, 1992 4. KerwinJF, Heller M: The arginine-nitric oxide pathway: A target for new drugs. Med Res Rev 14:23-74, 1994 5. Snyder SH: Nitric oxide: First in a new class of neurotransmitters? Science 257:494-496, 1992 6. Abman SH, Chatfield BA, Hall SL, et al: Role of endothelium-derived relaxing factor during transition of pulmonary circulation at birth. Am J Physiol 259: H1921-H1927, 1990 7. Bredt DS, Snyder SH: Nitric Oxide: A Physiologic Messenger Molecule. Aunu Rev Biochem 63:175-195, 1994 8. Kourembanas S: Hypoxic responses of the neonatal endothelium. Semin Perinatol 16:140-146, 1992 9. Kinsella JP, Abman SH: Recent developments in the pathophysiology and treatment of persistent puhnonary hypertension of the newborn. J Pediatr 126:853864, 1995 10. Mupanemunda RH, Edwards AD: Treatment of newborn infants with inhaled nitric oxide. Arch Dis Child 72:F131-F134, 1995 11. Eiserich JP, Vossen V, O'Neill CA, et al: Molecular mechanisms of damage by excess nitrogen oxides: Nitration of tyrosine by gas-phase cigarette smoke. FEBS Lett 353:53-56, 1994 12. Angg~rd E: Nitric oxide: Mediator, murderer, and medicine. Lancet 343:1199-1206, 1994 13. Beckman JS, Beckman TW, Chen J, et al: Apparent hydroxyl radical production by peroxynitrite: hnplications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA 87:1620-1624, 1990 14. Huie RE, Padmaja S: The reaction rate of nitric oxide with superoxide. Free Rad Res Commun 18:195-199, 1993 15. Ischiropoulos H, Zhu L, Beckman JS: Peroxynitrite formation from macrophage-derived nitric oxide. Arch Biochem Biophys 298:446-451, 1992 16. Stamler FH, Singel DJ, LoscalzoJ: Biochemistry of nitric oxide and its redox-activated forms. Science 258:18981902, 1992 17. Gaston B, DrazenJM, LoscalzoJ, Stamler JS: The biology of nitrogen oxides in the airways. Am J Crit Care Med 149:538-551, 1994 18. Reif DW, Simmons RD: Nitric oxide mediates iron release from ferritin. Arch Biochem Biophys 283:537-541, 1990 19. Kwon NS, Stuehr DJ, Nathan CF: Inhibition of tumor cell ribonucleotide reductase by macrophage-derived nitric oxide. J Exp Med 174:761-767, 1991 20. Henry Y, Ducrocq C, DrapierJ-C, et al: Nitric oxide: A biological effector: electron paramagnetic resonance detection of nitrosyl-iron-protein complexes in whole cells. Eur BiophysJ 1991:1-15, 1991 21. Granger DL, Lehninger AL: Sites of inhibition of mitochondrial electron transport in macrophage-injured neoplastic cells. J Cell Biol 95:52%535, 1982 22. Welsh N, Eizirik DL, Bendtzen K, Sandler S: Interleukin-1/3-induced nitric oxide production in isolated rat pancreatic islets requires gene transcription and may

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91. Haddad IY, Ischiropoulos H, Holm BA, et ah Mechanisms of peroxynitrite-induced injury to pulmonary surfactants. Am J Physiol 265:L555-L564, 1993 92. CifuentesJ, Ruiz-OronozJ, Myles C, et al: Interaction of surfactant mixtures with reactive oxygen and nitrogen specie. J Appl Physiol 78:1800-1805, 1995 93. Haddad IY, CrowJP, Hu P, et al: Concurrent generation of nitric oxide and superoxide damages surfactant protein A. Am J Physiol 267:L242-L249, 1994 94. Freeman BA, Crapo JD: Hyperoxia increases oxygen radical production in rat lungs and lung mitochondria. J Biol Chem 256: 10986-10992, 1981 95. M~ller B., SchSfer H, Barth P, yon Wichert P: Lung surfactant components in bronchoalveolar lavage after inhalation of N02 as markers of altered surfactant metabolism. Lung 172:61-72, 1994 96. Mfiller B, yon Wichert P: Effect of nitrogen dioxide inhalation on surfactant phosphatidylcholine synthesis in rat alveolar type II cells. Biochim Biophys Acta 1170:38-43, 1993

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