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Oxygen-induced lung toxicity: Effect on serotonin disposition and metabolism
64, 221-229(1982)
TOXICOLOGYANDAPPLIEDPHARMACOLOGY
Oxygen-induced
DALE Pharmacology
Lung Toxicity: Effect on Serotonin and Metabolism
E. MAIS, Section,
PATRICIA
Medical
Received
D. LAHR, AND TALMAGE
Sciences Program, Indiana Bloomington, Indiana 47405
January
25, 1981;
accepted
University
March
Disposition
R. BOSIN School
of Medicine,
9, 1982
Oxygen-Induced Lung Toxicity: Effect on Serotonin Disposition and Metabolism. MAIS, E., LAHR, P. D., AND BOSIN, T. R. (1982). Toxicol. Appl. Pharmacol. 64, 221-229. The effect of oxygen-induced lung injury on serotonin (5-hydroxytryptamine, 5-HT) disposition and metabolism was studied in the mouse. Animals were exposed to 100% oxygen under normobaric conditions, and the levels of endogenous 5-HT and exogenous 13H]S-HT were assessedafter 24 to 96 hr of exposure. By determining both 5-HT and [)H]S-HT, information concerning changes occurring over each 24-hr interval and at specific time points was obtained. After only 48 hr of oxygen exposure, lung contents of 5-HT and [3H]5-HT were significantly elevated (164 and 147%, respectively, of air-exposed controls) and continued to increase as a function of oxygen exposure time (96 hr, 225 and 2066, respectively). The opposite trend was observed in the plasma levels of 5-HT and [‘HIS-HT. The observed changes in 5-HT/ [)H]S-HT occurred prior to pulmonary edema formation and the earliest reported morphological changes. Lung slices obtained from animals exposed to 100% oxygen did not significantly differ from air-exposed controls in their ability to accumulate [14C]5-HT. Lung monoamine oxidase (MAO) activity was significantly decreased (24 to 37%) at 24 hr of oxygen exposure but returned to and occasionally exceeded control levels with continued exposure. It was shown that lung levels of 5-HT and [‘H]5-HT correlated with the degree of oxygeninduced injury to the pulmonary vasculature and suggested that 5-HT may be a factor in the pathogenesis of the vasculature injury and/or pulmonary edema formation. D.
The pulmonary toxicity of high partial pressures of oxygen is well known and results in alterations of both structure and function (Clark and Lambertsen, 197 1). These alterations lead to progressive respiratory distress and hypoxemia which may culminate in the death of the exposed animal. The earliest structural lesions in lungs of mice exposed to oxygen involve damage to capillary endothelial cells followed by granulocyte migration and interstitial and perivascular edema (Bowden et al., 1968; Adamson et al., 1970). Furthermore, changes in circulatory hemodynamics have been frequently reported as manifestations of oxygen toxicity (Wood et al., 1967, 1972). Since it has been
shown that the pulmonary endothelium plays a primary role in the regulation of the arterial concentration of several vasoactive agents (Fishman and Pietra, 1974; Gillis and Roth, 1976), a relationship may exist between oxygen-induced lung damage and the pathophysiology of altered hemodynamics. For example, circulating angiotensin I is rapidly converted to angiotensin II (Ng and Vane, 1967) by angiotensin converting enzyme which is associated with the luminal membrane of capillary endothelial cells (Ryan et al., 1976). The pulmonary endothelium is also involved in regulating the circulating levels of several biogenic amines, including serotonin (5hydroxytryptamine. 221
0041-008X/82/080221-09$02.00/0 Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
222
MAIS,
LAHR,
5-HT). Using isolated perfused lungs from various species it has been shown that 5-HT is removed from the pulmonary circulation by an active process and is metabolized by monoamine oxidase to 5-hydroxyindole-3acetic acid (5HIAA) (Alabaster and Bakhle, 1970; Junod, 1972; Iwasawa et al., 1973; Iwasawa and Gillis, 1974; Steinberg et al., 1975). The pulmonary endothelium has been identified as the site of 5-HT uptake by histofluorescence and autoradiography (Strum and Junod, 1972; Iwasawa et al., 1973; Cross et al., 1974). Since 5-HT is a potent vasoconstrictor (Gilbert et al., 1958; Brody and Stemmler, 1968), interference with its removal as a result of damage to the pulmonary endothelium could seriously affect the pulmonary and systemic circulation. Indeed, toxic agents known to injure pulmonary endothelial cells have been shown to decrease the clearance of 5-HT in isolated perfused lungs (Block and Fisher, 1977; Gillis et al., 1978; Roth et al., 1979; Fisher et al., 1980; Block and Schoen, 1981). For example, exposure of rats on 100% oxygen for 18 to 48 hr resulted in a 20 to 35% decrease in 5-HT clearance by isolated perfused lungs (Block and Fisher, 1977). Since previous work (Buckpitt et al., 1978; Bosin and Lahr, 198 1) had shown significant differences in 5-HT disposition in vivo versus the isolated lung (differences which may be due in part to the different techniques), the present work was done in the intact animal and involved the study of the effects of oxygen-induced lung injury on 5-HT and [ 3H]5-HT disposition and metabolism. METHODS Materials. Male, Swiss-Webster mice weighing 20 to 25 g were purchased from Laboratory Supply Company (Indianapolis, Ind.). All animals were fed Purina Rodent Chow, 5012 (Ralston Purina Co., St. Louis, MO.) and water ad libitum, maintained on a 12/12-hr light/dark cycle, and rested for 4 days prior to use. The following radiochemicals were purchased from New England Nuclear Corporation (Boston, Mass.): 5-[ 1,2-
AND
BOSIN
‘H(N)]hydroxytryptamine binoxalate (specific activity, 27.0 Ci/mmol); 5-[2-‘VZlhydroxytryptamine (spe51.5 mCi/mmol); @-[ethyl-lcific activity, ‘“Clphenylethylamine hydrochloride (specific activity, 50.0 mCi/mmol); [ I-i4C]tyramine (specific activity, 51.8 mCi/mmol). The purity of all labeled compounds was obtained by thin-layer chromatography on 250-Frn silica gel G plates purchased from Analtech, Inc. (Newart, Del.), in a solvent system composed of acetone2-propanol-water-ammonium hydroxide (50:40:7:3). The thin-layer plates were scanned on a Packard Model 7200 radiochromatogram scanner. Radiochemical purity was >98%. The following chemicals were purchased from Sigma Chemical Company (St. Louis, MO.): 5hydroxytryptamine binoxalate, phenylethylamine hydrochloride, and tyramine hydrochloride. Liquid scintillation supplies and hyamine hydroxide were purchased from Research Products International (Downers Grove, Ill.). oxygen exposure. Groups (8 to 10) of mice were exposed in 45-liter Plexiglas chambers to 100% oxygen or compressed air (3 to 4 liters/min) for varying periods of time. The chambers contained a carbon dioxide absorbent, and food and water were provided ad libitum. The concentrations of oxygen and carbon dioxide were monitored with an oxygen analyzer (Applied Technology Products, Denver, Colo.) and a carbon dioxide analyzer (Beckman Instruments Co., Fullerton, Calif.). During oxygen exposure the chamber concentration of oxygen was greater than 97% and the carbon dioxide less than 0.4%. The chamber temperature range was 22 to 25°C and the relative humidity varied from 50 to 70%. Lung indole analyses. After specific exposure times, a solution containing 0.2 &i [‘HIS-HT and 75 pmol of 5-HT binoxlate/jd was injected (1 PI/g) into the tail vein of mice. The mice were loosely restrained under an inverted loo-ml blackened beaker with the tail protruding from the lip of the beaker. Previous work has shown [‘HIS-HT accumulation by lung to be independent of 5-HT binoxalate concentration over the range 0.1 to 100 pmol/rl; the uptake of 5-HT was dose dependent whereas the percentage of the dose taken up by the lung was constant (Bosin and Lahr, 1981). Fifteen min after the administration of [‘HIS-HT, the animals were killed, and the lungs prepared (Mais et al., 1981). Blood was collected from the decapitation site in glass tubes previously rinsed with heparin dissolved in 0.9% saline (1800 units/ml) and dried. Hematocrits were routinely determined and were comparable to values previously reported (Friedman, 1959). Plasma samples were prepared by centrifugation for 0.5 hr at 750g in a Beckman J-6B centrifuge cooled to 4°C which produced essentially platelet-poor plasma. A 100~~1 aliquot of each plasma sample was transferred to a 125 X 15mm glass tube and stored at -80°C for analysis. The analysis of lung and plasma indole compounds was done
EFFECT
OF HYPEROXIA
as previously described (Mais et al., 1981). No difference in lung S-HT/[‘H]S-HT levels was observed between heparin-in-saline perfused and nonperfused lungs. Lung biochemical analyses. After specific exposure times, animals were killed by decapitation, and the lungs prepared (Mais et al., 198 1). The lungs were homogenized (1:20) in 0.25 M sucrose, the homogenate was centrifuged for 10 min/4”C at 3OOOg, and the supernatant fraction was removed and stored at -80°C for analyses. Lung protein (Lowry et al, 1951) and DNA (Richards, 1974) contents were determined on the homogenate. Pulmonary monomine oxidase activity was determined for three enzyme substrates: 5-hydroxytryptamine (5HT), tyramine (TYR), and phenethylamine (PEA). The reaction mixture consisted of 50 ~1 of 0.2 M phosphate buffer (pH 7.2) 50 pl H20, and 100 ~1 of lung homogenate and was preincubated for 5 min at 37°C prior to the addition of 100 ~1 of the specific substrate (60 pM and 100 nCi/lOO ~1). The final substrate concentration was 20 /IM. The incubation was continued for 20 min at 37°C and the reaction was terminated by the addition of 200 pl of 2 N HCl. The reaction for each substrate was linear with both time and protein concentration. Blank samples were prepared by adding 200 &cl of 2 N HCI prior to the addition of substrate. The S-HT and TYR samples were extracted with 6 ml of a I:1 mixture of ethyl acetate/benzene and PEA samples were extracted with 6 ml of toluene. The extraction was done by vigorously mixing for 0.5 min; extraction efficiencies were >90%. After centrifugation, a 3-ml aliquot of the organic layer was added to 10 ml of a liquid scintillation mixture [5 g PPO/POPOP (98/2), and 100 g naphthalene per liter p-dioxane] and counted for 10 min. Blank values for 5-HT, TYR, and PEA were 0.6, 1.0, and 1.3 mmol product/lung/20 min, respectively. Counts were corrected for quench by internal standardization with [‘4C]toluene. [“C]S-HT uptake and acrumulation into lung slices. After specific exposure times, animals were killed by decapitation, and the lungs prepared (Maiset al., 1981). A Mcllwain tissue slicer (H. Mickel, Surrey, England) was employed to obtain 0.75mm slices, and only slices with two cut surfaces were used. Approximately 30 mg of tissue was placed in 2.45 ml of incubation medium (pH 7.4) consisting of NaCl (129 mM), KC1 (5 mM), CaCI, (1 .O mM), MgSO, (1.3 mM), Na,HPO, (10 mM), glucose (11 mM), and pargyline hydrochloride (0.4 mM). Pargyline effectively inhibited the metabolism of [ “‘C]5-HT by monoamine oxidase. Analysis of lung slice samples and medium by high-performance liquid chromatography (Mais et al.. 198 1) indicated a complete absence of metabolism. The slices were preincubated for 5 min at 37°C in a 95% 02/5% CO2 atmosphere prior to the addition of 50 ~1 (100 nCi) of [14C]5-HT (final 5-HT concentration, I FM). The slices were incubated for 15 min, washed
ON
LUNG
60 ’
SEROTONIN
24
48
72
96
TIME iHOUF6l
FIG. 1. Changes in lung parameters as a function of oxygen exposure. Data are expressed as percentage of control (air exposed) group and are X + SEM; asterisks indicate significant differences (p < 0.05) from control group. S f SEM (N) control group values were: lung wet weight, 0.202 f 0.007 g (64); percentage lung wet weight/body weight, 0.85 f 0.05 (64); DNA, CO.75 +0.06 mg/lung (16). twice with cold medium, blotted dry, weighed, and placed in liquid scintillation vials containing 0.5 ml of hyamine hydroxide. Solubilization was achieved by heating the samples (50°C) overnight. The hyamine hydroxide was neutralized with 32 pl of glacial acetic acid prior to the addition of 10 ml of liquid scintillation fluid. Samples were allowed to stand for 2 hr in the dark to diminish the chemiluminescence before being counted in Beckman LS-8000 liquid scintillation counter (Beckman Instrument Co.). Statistics. Problems associated with expressing data derived from damaged lung have been reviewed, and it has been suggested that the most appropriate means of expressing such data is per total lung (Witschi, 1975). In the present study, the lung data have been expressed as units per whole lung and the plasma data as units per milliliter. Student’s t test (Steel and Torrie, 1960) was used for statistical analysis of air-exposed versus oxygen-exposed groups, and a significance level of p < 0.05 (two-tailed) was selected for rejection of the null hypothesis.
RESULTS The time course of select pulmonary changes resulting from oxygen-induced lung injury is shown in Fig. 1. During the first 72 hr of oxygen exposure the animals appeared normal, while at later exposure times the toxic effects of oxygen became rapidly apparent (LT50 - 108 hr). At 48 hr of oxygen exposure no change in lung wet weights or
224
600
MAIS, LAHR, AND BOSIN
TIME biO”RS;2
FIG. 2. Changes in lung indole compounds as a function of oxygen exposure. Data are expressed as percentage of control (air exposed) group and are X f SEM; asterisks indicate significant differences (p < 0.05) from control group. X f SEM (N) control group values were: TRP, 1.85 f 0.24 pg/lung (32); 5-HT, 0.58 + 0.04 fig/ lung (64); r3H]5-HT, 277 + 16 nCi/Iung (64).
percentage lung wet weight/body weight was observed; however, significant increases in these values, indicative of pulmonary edema formation, were observed at 72 and 96 hr. Lung deoxyribonucleic acid (DNA) content, which has been suggested as an appropriate basis for expressing lung data (Cronin and Giri, 1974), was markedly elevated 48 hr after initiation of oxygen exposure. In order to determine if these oxygen-induced lung changes alter the pulmonary disposition of endogenous 5-HT as well as exogenously administered [ 3H]5-HT, lung and plasma levels of these compounds were determined after select periods of oxygen exposure (Figs. 2 and 3). By determining both endogenous 5-HT and exogenous [ 3H]5-HT levels it is possible to obtain information concerning the effect of exposure on 5-HT disposition over an entire 24-hr time period as well as its effect on [3H]5-HT disposition over a discrete time period. The levels of tryptophan (TRP), a serotonin precursor, were also measured. Although the analytical procedure (Mais et al., 198 1) was capable
of measuring the immediate 5-HT precursor, 5-hydroxytryptophan (5-HTP), and primary metabolite, 5-hydroxyindole-5-acetic acid (5-HIAA), these compounds were not routinely detected. The levels of 5-HIAA-0 conjugate were determined however, and did not significantly differ from control values. During the oxygen exposure period there was a significant increase in lung levels of both 5-HT and [3H]5-HT as early as 48 hr after initiation of exposure (Fig. 2) and these levels continued to increase with increasing exposure time. Similarly, the lung levels of tryptophan were elevated as a function of exposure time, being significant at the 72- and 96-hr time points. In contrast, the plasma levels of these compounds showed a different time course. After 72 hr of oxygen exposure, the plasma levels of TRP, 5-HT, and [3H]5-HT were decreased by 34,76, and 78%, respectively. Thus, oxygen-induced changes in lung TRP, 5-HT, and [3H]5-HT levels are reflected by opposite changes in the plasma levels of these compounds. The parallel changes observed in lung and plasma levels of 5-HT and [3H]5-HT in oxygen and
FIG. 3. Changes in plasma indole compounds as a function of oxygen exposure. Data are expressed as percentage of control (air exposed) group and are 2 k SEM; asterisks indicate significant differences (p < 0.05). i + SEM (N) control group values were: TRP, 14.8 + 2.5 pg/ml(32); 5-HT, 1.56 f 0.22 rg/ml(32); [‘HIS-HT, 595 k 26 nCi/ml(32).
EFFECT
OF HYPEROXIA
air-exposed animals produced no change in [ 3H]5-HT specific activity. During the entire oxygen exposure the lung/plasma ratios for 5-HT and [3H]5-HT ranged from 1.8 to 23, reflecting its avid pulmonary accumulation, while the corresponding ratios for TRP were always less than one. These results suggest that the elevated lung levels of 5-HT and [ 3H]5-HT produced by oxygen exposure could result from stimulation of the transcellular uptake and accumulation of 5-HT and/or the depression of its intracellular metabolism by monoamine oxidase (MAO). When the uptake and accumulation of [14C]5-HT into lung slices obtained from air and oxygen-exposed animals were examined, no significant difference was observed. Since increased 5-HT uptake and accumulation were not responsible for the observed changes, altered metabolism was examined. The activity of pulmonary MAO was measured in the lungs of oxygen and air-exposed animals (Table 1). Three substrates, which have known specificities for the two forms of the enzyme, types A and B (Kung and Wilson, 1979), were evaluated: 5-HT (type A), tyramine (TYR, types A and B), and phenylethylamine (PEA, type B). The effect of oxygen exposure on pulmonary MAO occurred very early in the course of exposure. At 24 hr of oxygen exposure the activity of both forms of the enzyme was reduced by 27 and 37% respectively. With continued oxygen exposure the activity of the two forms of MAO returned to and occasionally exceeded air control levels. DISCUSSION The pathophysiology of oxygen-induced lung damage has been well characterized (Clark and Lambertsen, 1971) and can be divided into two phases; an immediate exudative phase and a delayed proliferative phase. The exudative phase is characterized by destruction of capillary endothelial and
ON
LUNG
‘25
SEROTONIN TABLE
EFFECT
Treatment
1
OF OXYGEN EXPOSURE ON MOUSE LUNG MONOAMINE OXIDASE ACTIVITY’ Time (hr)
Substrate
MAO activityb (nmol product/ lung/20 min)
Air
24
S-HT TYR PEA
41.5 f 91.2i 129.2 +
3.8 76 5.7
Oxygen
24
S-HT TYR PEA
30.9 + 59.8 + 94.6 +
I 9’ 3.8’ 3.8’
Air
48
5-HT TYR PEA
59.4 -fr 91.8 k 104.4 C
I 8 7.2 2.6
Oxygen
48
5-HT TYR PEA
67.0 -+ 3.0 99.5 F 3.0 134.0 -+ 7 1’
Air
72
5-HT TYR PEA
78.8 f 9.0 128.3 + 12.4 160.0 -+ 13.5
Oxygen
12
5-HT TYR PEA
83.2 It 107.6 + 136.0 +-
4.0 7.1 2.0
Air
96
5-HT TYR PEA
61.7 f 93.5 +123.4 rt
4.7 6.6 9.8
Oxygen
96
5-HT TYR PEA
76.7 c 102.2 f 125.7 f
1.5‘ 1.5 1.5
a Mice (20 to 25 g) were exposed to air or 100%’ oxygen. b X f SEM; four animals per time point. c Significantly different from air control group (p < 0.05).
Type I alveolar epithelial cells, interstitial and alveolar edema, and hyaline membrane formation. During the proliferative phase there is a proliferation of Type II alveolar and interstitial cells, infiltration of blood cells and fibroblasts, and interstitial fibrosis. In the mouse lung, the capillary endothelium has been identified as the distinctive cellular target of oxygen toxicity and similar phases have been described (Bowden et al., 1968; Adamson et al., 1970). Since the pulmonary
226
MAIS,
LAHR,
endothelium is a prominent site of oxygen damage and is intimately involved in the regulation of circulating vasoactive substances (Gillis and Roth, 1976), we undertook to study the effect of oxygen-induced endothelial cell damage on the disposition and metabolism of one such vasoactive substance, 5-HT. Earlier work has shown that pretreatment of animals with toxic compounds which damage the pulmonary capillary endothelium such as the pyrrolizidine alkaloid, monocrotaline (Gillis et al., 1978), the rat poison, a-naphthylthiourea (Block and Schoen, 198 1 ), or the herbicide, paraquat (Roth et al., 1979), resulted in a decreased clearance of 5-HT by the isolated perfused lung. Furthermore, Block and Fisher (1977) have shown that exposure of rats to 1 atm of 100% oxygen for as little as 18 hr resulted in a 20% decrease in 5-HT clearance when the lungs were isolated and perfused. The depressed 5-HT clearance was directly related to oxygen exposure time (35% at 48 hr) and provided an early indication of pulmonary endothelium change. In the present study, the effect of oxygeninduced lung injury on 5-HT disposition and metabolism was studied in the intact animal. Previous work (Buckpitt et al., 1978; Bosin and Lahr, 198 1) had indicated some significant differences between the results obtained in vivo and those obtained using isolated perfused lungs, differences which may in part reflect the differences in the techniques. Thus, the marked increases in both endogenous 5-HT and exogenous [ 3H]5-HT levels (Fig. 2) reported herein, contrast sharply with the reported decrease in 5-HT clearance using the isolated perfused lung (Block and Fisher, 1977). In an attempt to understand the mechanisms responsible for these changes in lung 5-HT content, oxygen-induced alterations in 5-HT uptake, accumulation, and metabolism were evaluated. The uptake of 5-HT is an active process and represents the rate-
AND
BOSIN
limiting step in the clearance of 5-HT by the isolated perfused lung (Junod, 1972). Uptake and accumulation of 5-HT by lung slices which allow 5-HT access to cells other than endothelium cells closely resemble the isolated perfused lung (Smith et al., 1976; Junod and Ody, 1977; Wekberg and Hede, 198 1). Since the endothelium accounts for approximately 40% of the lung cells (Fisher, 1978) and oxygen toxicity can reduce their number by 47% (Report of Workshop on the Pulmonary Endothelial Cell, 1979), significant changes therein should be reflected in changes in 5-HT uptake and accumulation. Indeed, monocrotaline, which damages the endothelium and depresses 5-HT clearance in the perfused lung (Gillis et al., 1978), also significantly decreases 5-HT uptake and accumulation into lung slices (Hilliker and Roth, 1980). When 5-HT uptake and accumulation were examined in lung slices obtained from oxygen-exposed animals, no significant difference from controls was observed. The lung monoamine oxidase (MAO) system is affected by pulmonary toxicants (Mustafa et al,, 1977; Roth et al., 1979). Acute exposure to high concentrations of ozone, which primarily affects the pulmonary epithelium, resulted in decreased MAO activity in lung homogenates, mitochondria, and microsomes, while chronic exposure to low ozone concentrations was associated with increased MAO activity. In addition, paraquat decreased pulmonary MAO activity (Roth et al., 1979). In this study, we found that exposure of mice to 100% oxygen caused an initial 25 to 35% decrease in lung MAO activity followed by recovery and occasional stimulation with continued exposure (Table 1). The time course of these oxygen-induced changes in pulmonary MAO activity correlated with the described (Mustafa and Tierney, 1978) morphological and metabolic changes in lung mitochondria, initial depression followed by stimulation. Although the lung homogenate data do not to-
EFFECT
OF
HYPEROXIA
ON
LUNG
SEROTONIN
127
cell tally reflect the nature of MAO in lung alveoli is consistent with endothelial mitochondria, the mitochondrial fraction damage. Since PMN can release potent oxaccounts for greater than 77% of the total idants which can damage endothelial cells lung MAO activity (Kung and Wilson, (Yamada et al., 1979) and which can also 1979). Thus changes in the mitochondrial cause the release of platelet 5-HT (Handin enzyme would be expected to result in et al., 1977) they may significantly contribchanges in lung homogenate activity. Due ute to the pathophysiology of oxygen toxto differences in the time course and extent icity. Indeed, Shasby et al. ( 198 1) have of inhibition, these data indicate that altered shown that depletion of PMN protects ani5-HT metabolism by MAO appears not to mals from oxygen-induced lung injury. be a significant mechanism responsible for Platelets have been frequently implicated the elevated lung 5-HT levels observed dur- in lung microvascular injury. Not only are ing oxygen exposure. they components of microemboli, but they Earlier work has demonstrated a relationare capable of accumulating and releasing ship between pulmonary vasculature injury, a number of vasoactive substances, including edema, and increased lung 5-HT levels 5-HT. Recently, Yamada et al. (1979) dem(Kind et al., 1961; Skillen et al., 1961). onstrated that platelets and platelet products Acute exposure of animals to ozone pro- (5-HT) potentiate PMN-mediated endotheduced significant pulmonary edema and el- lial cell injury. They showed that platelets evated levels of 5-HT (Skillen et al., 1961). augment PMN adherence to endothelial Such increases in lung 5-HT content followcells and strikingly amplify PMN-induced ing oxygen- or ozone-induced injury may endothelial cell damage. Furthermore, they represent either a consequence of the injury found that 5-HT could completely mimic the or may be a factor in the response of pul- platelet effect, and they concluded that 5monary microvasculature to injury. It is easy HT functions as an effector of PMN-endothelial cell interactions. Since platelets to envision how damage to the pulmonary endothelium might allow 5-HT access to contain most of the circulating 5-HT, oxyother lung cells capable of accumulating the gen-induced alterations of platelet 5-HT upvasoactive amine, and how lung 5-HT con- take, storage, or release could result in intent might then parallel the extent of injury. creased levels of the free amine which could Alternatively, several lines of evidence potentiate endothelial cell damage by the suggest a possible role for 5-HT and other mechanisms described above. blood-borne substances in the response of the pulmonary microvasculature to injury. Teleologically, one might expect the pulmonary ACKNOWLEDGMENTS microvasculature to be insensitive to bloodborne mediators of inflammation, since all This work was supported by a grant-in-aid from the the systemic venous blood flows through the American Heart Association, Indiana Affiliate, and by lungs and concentrations of injurious sub- a Young Investigator Pulmonary Research Grant (HL 19573) from the National Institute of Heart, Lung. and stances would be expected to be greatest Blood. there. Recent data, however, indicate that this may not be the case. Fox et al. (198 1) REFERENCES have correlated the number of alveolar polymorphonuclear leukocytes (PMN) with the ADAMSON, 1. Y. R., BOWDEN, D. H., AND WYATT. degree of pulmonary edema in animals exJ. P. (1970). Oxygen poisoning in mice. Uhrastrucposed to 100% oxygen. This correlation of tural and surfactant studies during exposure and recovery. Arch. Pathol. 90, 463-472. pulmonary edema with influx of PMN into
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GILLIS, C. N., AND ROTH, J. A. (1976). Pulmonary disposition of circulating vasoactive hormones. Biothem. Pharmacol. 25,2547-2553. GILLIS, C. N., HUXTABLE, R. J., AND ROTH, R. A. (1978). Effects of monocrotaline pretreatment of rats on removal of 5-hydroxytryptamine and noradrenaline by perfused lung. kit. J. Pharmaco~. 63, 435443.
HANDIN, R. I., KARABIN, R., AND BOXER, G. J. (1977). Enhancement of platelet function by superoxide anion. J. Clin. invest. 59, 959-965. HILLIKER, K. S., AND ROTH, R. A. (1980). The effect of monocrotaline (MCT) on 5-hydroxytryptamine (5HT) uptake by rat lung slices. Pharmacologist 22, 172. IWASAWA, Y., GILLIS, C. N., AND AGHAJANIAN, G. (1973). Hypothermic inhibition of 5-hydroxytryptamine and norepinephrine uptake by lung: Cellular location of amines after uptake. J. Pharmacol. Exp. Ther. 186,498-507. IWASAWA, Y., AND GILLIS, C. N. (1974). Pharmacological analysis of norepinephrine and 5-hydroxytryptamine removal from the pulmonary circulation: Differentiation of uptake sites for each amine. J. Pharmacol. Exp. Ther. 188, 386-393. JUNOD, A. F. (1972). Uptake, metabolism and efflux of ‘%-5-hydroxytryptamine in isolated perfused rat lungs. J. Pharmacol. Exp. Ther. 183, 341-355. JUNOD, A. F., AND ODY, C. (1977). Amine uptake and metabolism by endothelium of pig pulmonary artery. Amer. J. Physiol. 232, C88-C94. KIND, L. S., BURKHALTER, A., AND SHERINS, R. (1961). Increased lung serotonin and pulmonary edema in mice injected with antiserum to Ehrlich’s; ascites tumor. Proc. Sot. Exp. Biol. Med. 108, 735737.
KUNG, H. C., AND WILSON, A. G. E. (1979). Characterization of rat pulmonary monoamine oxidase. Life Sei. 24, 425-432. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (19.51). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275.
MAIS, D. E., LAHR, P. D., AND BOSIN, T. R. (1981). Determination of serotonin, its precursors, metabolites and [ 3H] serotonin in lung by high performance liquid chromatography with fluorescence detection. J. Chromatogr. Biomed. Appt. 225, 27-35. MUSTAFA, M. G., HACKER, A. D., OSPITAL, J. J., HusSAIN, M. Z., AND LEE, S. D. (1977). Biochemical effects of environmental oxidant pollutants in animal lungs. In Biochemical Eflects of Environmental PO!lutants (S. D. Lee, ed.), pp. 59-96, Ann Arbor Science Publ. Ann Arbor, Mich. MUSTAFA, M. G., AND TIERNY, D. F. (1978). Biochemical and metabolic changes in the lung with oxygen, ozone, and nitrogen dioxide toxicity. Amer. Rev. Req. Dis. 118, 1061-1090.
EFFECT
OF HYPEROXIA
Nc, K. K. F., AND VANE, J. R. (1967). Conversion of angiotensin I to angiotensin II. Nature (London) 216, 762-766.
Report of Workshop on the Pulmonary Endothelial Cell. (1979). Divisions of Lung Diseases, NHLBI, U.S. Department of Health, Education, and Welfare Publication No. (NIH) 79-1524, Washington, D.C. RICHARDS, G. M. (1974). Modification of the diphenylamine reaction giving increased sensitivity and simplicity in the estimation of DNA. Anal. Biochem. 57, 369-376.
ROTH, R. A., WALLACE, K. B., ALPER, R. H., AND BAILIE, M. D. (1979). Effect of paraquat treatment of rats on disposition of S-hydroxytryptamine and angiotensin I by perfused lung. Biochem. Pharmacol. 28, 2349-2355.
RYAN, U. S., RYAN, J. W., WHITAKER, C., AND CHIN, A. ( 1976). Localization of angiotensin converting enzyme (kininase II). II. Immunocytochemistry and immunofluorescence. Tissue Cell 8, 125- 145. SHASBY, D. M., Fox, R. B., HARADA, R. N., AND REPINE, J. E. ( 198 1). Mechanisms of pulmonary oxygen toxicity: Neutropenia protects against acute lung injury from hyperoxia. Amer. J. Respir. Dis. 121, 25x.
SKILLEN, R. G., THIENES, C. H., CANGELOSI, J., AND STRAIN, L. (1961). Lung 5-hydroxytryptamine and ozone induced pulmonary edema in rats. Proc. Sot. Exp. Biol. Med. 107, 1788180. SMITH, L. L., LOCK, E. A., AND ROSE, M. S. (1976). The relationship between 5-hydroxytryptamine and
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paraquat accumulation into rat lung. Biochem. macol.
229 Phar-
25, 2485-2487.
STEEL, R. G., AND TORRIE, J. H. (1960). Principles and Procedures of Statistics. McGraw--Hill, New York. STEINBERG, H., BASSETT, D. J. P., AND FISHER, A. B. ( 1975). Depression of pulmonary 5-hydroxytryptamine uptake by metabolic inhibitors. Amer. J. Physiol. 228, I298- 1303. STRUM, J. M., AND JUNOD, A. F. (1972). Radioautographic demonstration of 5-hydroxytryptamine-‘H uptake by pulmonary endothelial cells. J. Cell Bin/. 54,456-467.
WEKBERG, J. E. S., AND HEDE, A. R. (1981). Characterization of 5-hydroxytryptamine uptake in sliced rat lung. Acta Pharmacol. Toxicol. 48, 151-l 56. WITSCHI, H. (1975). Exploitable biochemical approaches for the evaluation of toxic lung damage. E.7.f. Toxicol. 6, I 25 I9 1. WOOD, C. D., SEAGER, L. D., AND PERKINS, G. ( 1967 ). Blood pressure changes and pulmonary edema in the rat associated with hyperbaric oxygen. Aerospace Med.
38, 479-48
1.
WOOD, C. D., PERKINS, G. F., SMITH, A. G., ,XND REAUX, J. M. (1972). Response of the cardiovascular system in oxygen toxicity. Aerospace Med. 43, I26 167. YAMADA, O., MOLDOW, C. F., AND JACOB, H. S. ( 1979). Immunologic model for atherogenesis: Evidence that platelets and platelet products augment neutrophil (PMN) mediated endothelial cell injury. Chin. Res. 27, 466A.
TOXICOLOGYANDAPPLIEDPHARMACOLOGY
Oxygen-induced
DALE Pharmacology
Lung Toxicity: Effect on Serotonin and Metabolism
E. MAIS, Section,
PATRICIA
Medical
Received
D. LAHR, AND TALMAGE
Sciences Program, Indiana Bloomington, Indiana 47405
January
25, 1981;
accepted
University
March
Disposition
R. BOSIN School
of Medicine,
9, 1982
Oxygen-Induced Lung Toxicity: Effect on Serotonin Disposition and Metabolism. MAIS, E., LAHR, P. D., AND BOSIN, T. R. (1982). Toxicol. Appl. Pharmacol. 64, 221-229. The effect of oxygen-induced lung injury on serotonin (5-hydroxytryptamine, 5-HT) disposition and metabolism was studied in the mouse. Animals were exposed to 100% oxygen under normobaric conditions, and the levels of endogenous 5-HT and exogenous 13H]S-HT were assessedafter 24 to 96 hr of exposure. By determining both 5-HT and [)H]S-HT, information concerning changes occurring over each 24-hr interval and at specific time points was obtained. After only 48 hr of oxygen exposure, lung contents of 5-HT and [3H]5-HT were significantly elevated (164 and 147%, respectively, of air-exposed controls) and continued to increase as a function of oxygen exposure time (96 hr, 225 and 2066, respectively). The opposite trend was observed in the plasma levels of 5-HT and [‘HIS-HT. The observed changes in 5-HT/ [)H]S-HT occurred prior to pulmonary edema formation and the earliest reported morphological changes. Lung slices obtained from animals exposed to 100% oxygen did not significantly differ from air-exposed controls in their ability to accumulate [14C]5-HT. Lung monoamine oxidase (MAO) activity was significantly decreased (24 to 37%) at 24 hr of oxygen exposure but returned to and occasionally exceeded control levels with continued exposure. It was shown that lung levels of 5-HT and [‘H]5-HT correlated with the degree of oxygeninduced injury to the pulmonary vasculature and suggested that 5-HT may be a factor in the pathogenesis of the vasculature injury and/or pulmonary edema formation. D.
The pulmonary toxicity of high partial pressures of oxygen is well known and results in alterations of both structure and function (Clark and Lambertsen, 197 1). These alterations lead to progressive respiratory distress and hypoxemia which may culminate in the death of the exposed animal. The earliest structural lesions in lungs of mice exposed to oxygen involve damage to capillary endothelial cells followed by granulocyte migration and interstitial and perivascular edema (Bowden et al., 1968; Adamson et al., 1970). Furthermore, changes in circulatory hemodynamics have been frequently reported as manifestations of oxygen toxicity (Wood et al., 1967, 1972). Since it has been
shown that the pulmonary endothelium plays a primary role in the regulation of the arterial concentration of several vasoactive agents (Fishman and Pietra, 1974; Gillis and Roth, 1976), a relationship may exist between oxygen-induced lung damage and the pathophysiology of altered hemodynamics. For example, circulating angiotensin I is rapidly converted to angiotensin II (Ng and Vane, 1967) by angiotensin converting enzyme which is associated with the luminal membrane of capillary endothelial cells (Ryan et al., 1976). The pulmonary endothelium is also involved in regulating the circulating levels of several biogenic amines, including serotonin (5hydroxytryptamine. 221
0041-008X/82/080221-09$02.00/0 Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
222
MAIS,
LAHR,
5-HT). Using isolated perfused lungs from various species it has been shown that 5-HT is removed from the pulmonary circulation by an active process and is metabolized by monoamine oxidase to 5-hydroxyindole-3acetic acid (5HIAA) (Alabaster and Bakhle, 1970; Junod, 1972; Iwasawa et al., 1973; Iwasawa and Gillis, 1974; Steinberg et al., 1975). The pulmonary endothelium has been identified as the site of 5-HT uptake by histofluorescence and autoradiography (Strum and Junod, 1972; Iwasawa et al., 1973; Cross et al., 1974). Since 5-HT is a potent vasoconstrictor (Gilbert et al., 1958; Brody and Stemmler, 1968), interference with its removal as a result of damage to the pulmonary endothelium could seriously affect the pulmonary and systemic circulation. Indeed, toxic agents known to injure pulmonary endothelial cells have been shown to decrease the clearance of 5-HT in isolated perfused lungs (Block and Fisher, 1977; Gillis et al., 1978; Roth et al., 1979; Fisher et al., 1980; Block and Schoen, 1981). For example, exposure of rats on 100% oxygen for 18 to 48 hr resulted in a 20 to 35% decrease in 5-HT clearance by isolated perfused lungs (Block and Fisher, 1977). Since previous work (Buckpitt et al., 1978; Bosin and Lahr, 198 1) had shown significant differences in 5-HT disposition in vivo versus the isolated lung (differences which may be due in part to the different techniques), the present work was done in the intact animal and involved the study of the effects of oxygen-induced lung injury on 5-HT and [ 3H]5-HT disposition and metabolism. METHODS Materials. Male, Swiss-Webster mice weighing 20 to 25 g were purchased from Laboratory Supply Company (Indianapolis, Ind.). All animals were fed Purina Rodent Chow, 5012 (Ralston Purina Co., St. Louis, MO.) and water ad libitum, maintained on a 12/12-hr light/dark cycle, and rested for 4 days prior to use. The following radiochemicals were purchased from New England Nuclear Corporation (Boston, Mass.): 5-[ 1,2-
AND
BOSIN
‘H(N)]hydroxytryptamine binoxalate (specific activity, 27.0 Ci/mmol); 5-[2-‘VZlhydroxytryptamine (spe51.5 mCi/mmol); @-[ethyl-lcific activity, ‘“Clphenylethylamine hydrochloride (specific activity, 50.0 mCi/mmol); [ I-i4C]tyramine (specific activity, 51.8 mCi/mmol). The purity of all labeled compounds was obtained by thin-layer chromatography on 250-Frn silica gel G plates purchased from Analtech, Inc. (Newart, Del.), in a solvent system composed of acetone2-propanol-water-ammonium hydroxide (50:40:7:3). The thin-layer plates were scanned on a Packard Model 7200 radiochromatogram scanner. Radiochemical purity was >98%. The following chemicals were purchased from Sigma Chemical Company (St. Louis, MO.): 5hydroxytryptamine binoxalate, phenylethylamine hydrochloride, and tyramine hydrochloride. Liquid scintillation supplies and hyamine hydroxide were purchased from Research Products International (Downers Grove, Ill.). oxygen exposure. Groups (8 to 10) of mice were exposed in 45-liter Plexiglas chambers to 100% oxygen or compressed air (3 to 4 liters/min) for varying periods of time. The chambers contained a carbon dioxide absorbent, and food and water were provided ad libitum. The concentrations of oxygen and carbon dioxide were monitored with an oxygen analyzer (Applied Technology Products, Denver, Colo.) and a carbon dioxide analyzer (Beckman Instruments Co., Fullerton, Calif.). During oxygen exposure the chamber concentration of oxygen was greater than 97% and the carbon dioxide less than 0.4%. The chamber temperature range was 22 to 25°C and the relative humidity varied from 50 to 70%. Lung indole analyses. After specific exposure times, a solution containing 0.2 &i [‘HIS-HT and 75 pmol of 5-HT binoxlate/jd was injected (1 PI/g) into the tail vein of mice. The mice were loosely restrained under an inverted loo-ml blackened beaker with the tail protruding from the lip of the beaker. Previous work has shown [‘HIS-HT accumulation by lung to be independent of 5-HT binoxalate concentration over the range 0.1 to 100 pmol/rl; the uptake of 5-HT was dose dependent whereas the percentage of the dose taken up by the lung was constant (Bosin and Lahr, 1981). Fifteen min after the administration of [‘HIS-HT, the animals were killed, and the lungs prepared (Mais et al., 1981). Blood was collected from the decapitation site in glass tubes previously rinsed with heparin dissolved in 0.9% saline (1800 units/ml) and dried. Hematocrits were routinely determined and were comparable to values previously reported (Friedman, 1959). Plasma samples were prepared by centrifugation for 0.5 hr at 750g in a Beckman J-6B centrifuge cooled to 4°C which produced essentially platelet-poor plasma. A 100~~1 aliquot of each plasma sample was transferred to a 125 X 15mm glass tube and stored at -80°C for analysis. The analysis of lung and plasma indole compounds was done
EFFECT
OF HYPEROXIA
as previously described (Mais et al., 1981). No difference in lung S-HT/[‘H]S-HT levels was observed between heparin-in-saline perfused and nonperfused lungs. Lung biochemical analyses. After specific exposure times, animals were killed by decapitation, and the lungs prepared (Mais et al., 198 1). The lungs were homogenized (1:20) in 0.25 M sucrose, the homogenate was centrifuged for 10 min/4”C at 3OOOg, and the supernatant fraction was removed and stored at -80°C for analyses. Lung protein (Lowry et al, 1951) and DNA (Richards, 1974) contents were determined on the homogenate. Pulmonary monomine oxidase activity was determined for three enzyme substrates: 5-hydroxytryptamine (5HT), tyramine (TYR), and phenethylamine (PEA). The reaction mixture consisted of 50 ~1 of 0.2 M phosphate buffer (pH 7.2) 50 pl H20, and 100 ~1 of lung homogenate and was preincubated for 5 min at 37°C prior to the addition of 100 ~1 of the specific substrate (60 pM and 100 nCi/lOO ~1). The final substrate concentration was 20 /IM. The incubation was continued for 20 min at 37°C and the reaction was terminated by the addition of 200 pl of 2 N HCl. The reaction for each substrate was linear with both time and protein concentration. Blank samples were prepared by adding 200 &cl of 2 N HCI prior to the addition of substrate. The S-HT and TYR samples were extracted with 6 ml of a I:1 mixture of ethyl acetate/benzene and PEA samples were extracted with 6 ml of toluene. The extraction was done by vigorously mixing for 0.5 min; extraction efficiencies were >90%. After centrifugation, a 3-ml aliquot of the organic layer was added to 10 ml of a liquid scintillation mixture [5 g PPO/POPOP (98/2), and 100 g naphthalene per liter p-dioxane] and counted for 10 min. Blank values for 5-HT, TYR, and PEA were 0.6, 1.0, and 1.3 mmol product/lung/20 min, respectively. Counts were corrected for quench by internal standardization with [‘4C]toluene. [“C]S-HT uptake and acrumulation into lung slices. After specific exposure times, animals were killed by decapitation, and the lungs prepared (Maiset al., 1981). A Mcllwain tissue slicer (H. Mickel, Surrey, England) was employed to obtain 0.75mm slices, and only slices with two cut surfaces were used. Approximately 30 mg of tissue was placed in 2.45 ml of incubation medium (pH 7.4) consisting of NaCl (129 mM), KC1 (5 mM), CaCI, (1 .O mM), MgSO, (1.3 mM), Na,HPO, (10 mM), glucose (11 mM), and pargyline hydrochloride (0.4 mM). Pargyline effectively inhibited the metabolism of [ “‘C]5-HT by monoamine oxidase. Analysis of lung slice samples and medium by high-performance liquid chromatography (Mais et al.. 198 1) indicated a complete absence of metabolism. The slices were preincubated for 5 min at 37°C in a 95% 02/5% CO2 atmosphere prior to the addition of 50 ~1 (100 nCi) of [14C]5-HT (final 5-HT concentration, I FM). The slices were incubated for 15 min, washed
ON
LUNG
60 ’
SEROTONIN
24
48
72
96
TIME iHOUF6l
FIG. 1. Changes in lung parameters as a function of oxygen exposure. Data are expressed as percentage of control (air exposed) group and are X + SEM; asterisks indicate significant differences (p < 0.05) from control group. S f SEM (N) control group values were: lung wet weight, 0.202 f 0.007 g (64); percentage lung wet weight/body weight, 0.85 f 0.05 (64); DNA, CO.75 +0.06 mg/lung (16). twice with cold medium, blotted dry, weighed, and placed in liquid scintillation vials containing 0.5 ml of hyamine hydroxide. Solubilization was achieved by heating the samples (50°C) overnight. The hyamine hydroxide was neutralized with 32 pl of glacial acetic acid prior to the addition of 10 ml of liquid scintillation fluid. Samples were allowed to stand for 2 hr in the dark to diminish the chemiluminescence before being counted in Beckman LS-8000 liquid scintillation counter (Beckman Instrument Co.). Statistics. Problems associated with expressing data derived from damaged lung have been reviewed, and it has been suggested that the most appropriate means of expressing such data is per total lung (Witschi, 1975). In the present study, the lung data have been expressed as units per whole lung and the plasma data as units per milliliter. Student’s t test (Steel and Torrie, 1960) was used for statistical analysis of air-exposed versus oxygen-exposed groups, and a significance level of p < 0.05 (two-tailed) was selected for rejection of the null hypothesis.
RESULTS The time course of select pulmonary changes resulting from oxygen-induced lung injury is shown in Fig. 1. During the first 72 hr of oxygen exposure the animals appeared normal, while at later exposure times the toxic effects of oxygen became rapidly apparent (LT50 - 108 hr). At 48 hr of oxygen exposure no change in lung wet weights or
224
600
MAIS, LAHR, AND BOSIN
TIME biO”RS;2
FIG. 2. Changes in lung indole compounds as a function of oxygen exposure. Data are expressed as percentage of control (air exposed) group and are X f SEM; asterisks indicate significant differences (p < 0.05) from control group. X f SEM (N) control group values were: TRP, 1.85 f 0.24 pg/lung (32); 5-HT, 0.58 + 0.04 fig/ lung (64); r3H]5-HT, 277 + 16 nCi/Iung (64).
percentage lung wet weight/body weight was observed; however, significant increases in these values, indicative of pulmonary edema formation, were observed at 72 and 96 hr. Lung deoxyribonucleic acid (DNA) content, which has been suggested as an appropriate basis for expressing lung data (Cronin and Giri, 1974), was markedly elevated 48 hr after initiation of oxygen exposure. In order to determine if these oxygen-induced lung changes alter the pulmonary disposition of endogenous 5-HT as well as exogenously administered [ 3H]5-HT, lung and plasma levels of these compounds were determined after select periods of oxygen exposure (Figs. 2 and 3). By determining both endogenous 5-HT and exogenous [ 3H]5-HT levels it is possible to obtain information concerning the effect of exposure on 5-HT disposition over an entire 24-hr time period as well as its effect on [3H]5-HT disposition over a discrete time period. The levels of tryptophan (TRP), a serotonin precursor, were also measured. Although the analytical procedure (Mais et al., 198 1) was capable
of measuring the immediate 5-HT precursor, 5-hydroxytryptophan (5-HTP), and primary metabolite, 5-hydroxyindole-5-acetic acid (5-HIAA), these compounds were not routinely detected. The levels of 5-HIAA-0 conjugate were determined however, and did not significantly differ from control values. During the oxygen exposure period there was a significant increase in lung levels of both 5-HT and [3H]5-HT as early as 48 hr after initiation of exposure (Fig. 2) and these levels continued to increase with increasing exposure time. Similarly, the lung levels of tryptophan were elevated as a function of exposure time, being significant at the 72- and 96-hr time points. In contrast, the plasma levels of these compounds showed a different time course. After 72 hr of oxygen exposure, the plasma levels of TRP, 5-HT, and [3H]5-HT were decreased by 34,76, and 78%, respectively. Thus, oxygen-induced changes in lung TRP, 5-HT, and [3H]5-HT levels are reflected by opposite changes in the plasma levels of these compounds. The parallel changes observed in lung and plasma levels of 5-HT and [3H]5-HT in oxygen and
FIG. 3. Changes in plasma indole compounds as a function of oxygen exposure. Data are expressed as percentage of control (air exposed) group and are 2 k SEM; asterisks indicate significant differences (p < 0.05). i + SEM (N) control group values were: TRP, 14.8 + 2.5 pg/ml(32); 5-HT, 1.56 f 0.22 rg/ml(32); [‘HIS-HT, 595 k 26 nCi/ml(32).
EFFECT
OF HYPEROXIA
air-exposed animals produced no change in [ 3H]5-HT specific activity. During the entire oxygen exposure the lung/plasma ratios for 5-HT and [3H]5-HT ranged from 1.8 to 23, reflecting its avid pulmonary accumulation, while the corresponding ratios for TRP were always less than one. These results suggest that the elevated lung levels of 5-HT and [ 3H]5-HT produced by oxygen exposure could result from stimulation of the transcellular uptake and accumulation of 5-HT and/or the depression of its intracellular metabolism by monoamine oxidase (MAO). When the uptake and accumulation of [14C]5-HT into lung slices obtained from air and oxygen-exposed animals were examined, no significant difference was observed. Since increased 5-HT uptake and accumulation were not responsible for the observed changes, altered metabolism was examined. The activity of pulmonary MAO was measured in the lungs of oxygen and air-exposed animals (Table 1). Three substrates, which have known specificities for the two forms of the enzyme, types A and B (Kung and Wilson, 1979), were evaluated: 5-HT (type A), tyramine (TYR, types A and B), and phenylethylamine (PEA, type B). The effect of oxygen exposure on pulmonary MAO occurred very early in the course of exposure. At 24 hr of oxygen exposure the activity of both forms of the enzyme was reduced by 27 and 37% respectively. With continued oxygen exposure the activity of the two forms of MAO returned to and occasionally exceeded air control levels. DISCUSSION The pathophysiology of oxygen-induced lung damage has been well characterized (Clark and Lambertsen, 1971) and can be divided into two phases; an immediate exudative phase and a delayed proliferative phase. The exudative phase is characterized by destruction of capillary endothelial and
ON
LUNG
‘25
SEROTONIN TABLE
EFFECT
Treatment
1
OF OXYGEN EXPOSURE ON MOUSE LUNG MONOAMINE OXIDASE ACTIVITY’ Time (hr)
Substrate
MAO activityb (nmol product/ lung/20 min)
Air
24
S-HT TYR PEA
41.5 f 91.2i 129.2 +
3.8 76 5.7
Oxygen
24
S-HT TYR PEA
30.9 + 59.8 + 94.6 +
I 9’ 3.8’ 3.8’
Air
48
5-HT TYR PEA
59.4 -fr 91.8 k 104.4 C
I 8 7.2 2.6
Oxygen
48
5-HT TYR PEA
67.0 -+ 3.0 99.5 F 3.0 134.0 -+ 7 1’
Air
72
5-HT TYR PEA
78.8 f 9.0 128.3 + 12.4 160.0 -+ 13.5
Oxygen
12
5-HT TYR PEA
83.2 It 107.6 + 136.0 +-
4.0 7.1 2.0
Air
96
5-HT TYR PEA
61.7 f 93.5 +123.4 rt
4.7 6.6 9.8
Oxygen
96
5-HT TYR PEA
76.7 c 102.2 f 125.7 f
1.5‘ 1.5 1.5
a Mice (20 to 25 g) were exposed to air or 100%’ oxygen. b X f SEM; four animals per time point. c Significantly different from air control group (p < 0.05).
Type I alveolar epithelial cells, interstitial and alveolar edema, and hyaline membrane formation. During the proliferative phase there is a proliferation of Type II alveolar and interstitial cells, infiltration of blood cells and fibroblasts, and interstitial fibrosis. In the mouse lung, the capillary endothelium has been identified as the distinctive cellular target of oxygen toxicity and similar phases have been described (Bowden et al., 1968; Adamson et al., 1970). Since the pulmonary
226
MAIS,
LAHR,
endothelium is a prominent site of oxygen damage and is intimately involved in the regulation of circulating vasoactive substances (Gillis and Roth, 1976), we undertook to study the effect of oxygen-induced endothelial cell damage on the disposition and metabolism of one such vasoactive substance, 5-HT. Earlier work has shown that pretreatment of animals with toxic compounds which damage the pulmonary capillary endothelium such as the pyrrolizidine alkaloid, monocrotaline (Gillis et al., 1978), the rat poison, a-naphthylthiourea (Block and Schoen, 198 1 ), or the herbicide, paraquat (Roth et al., 1979), resulted in a decreased clearance of 5-HT by the isolated perfused lung. Furthermore, Block and Fisher (1977) have shown that exposure of rats to 1 atm of 100% oxygen for as little as 18 hr resulted in a 20% decrease in 5-HT clearance when the lungs were isolated and perfused. The depressed 5-HT clearance was directly related to oxygen exposure time (35% at 48 hr) and provided an early indication of pulmonary endothelium change. In the present study, the effect of oxygeninduced lung injury on 5-HT disposition and metabolism was studied in the intact animal. Previous work (Buckpitt et al., 1978; Bosin and Lahr, 198 1) had indicated some significant differences between the results obtained in vivo and those obtained using isolated perfused lungs, differences which may in part reflect the differences in the techniques. Thus, the marked increases in both endogenous 5-HT and exogenous [ 3H]5-HT levels (Fig. 2) reported herein, contrast sharply with the reported decrease in 5-HT clearance using the isolated perfused lung (Block and Fisher, 1977). In an attempt to understand the mechanisms responsible for these changes in lung 5-HT content, oxygen-induced alterations in 5-HT uptake, accumulation, and metabolism were evaluated. The uptake of 5-HT is an active process and represents the rate-
AND
BOSIN
limiting step in the clearance of 5-HT by the isolated perfused lung (Junod, 1972). Uptake and accumulation of 5-HT by lung slices which allow 5-HT access to cells other than endothelium cells closely resemble the isolated perfused lung (Smith et al., 1976; Junod and Ody, 1977; Wekberg and Hede, 198 1). Since the endothelium accounts for approximately 40% of the lung cells (Fisher, 1978) and oxygen toxicity can reduce their number by 47% (Report of Workshop on the Pulmonary Endothelial Cell, 1979), significant changes therein should be reflected in changes in 5-HT uptake and accumulation. Indeed, monocrotaline, which damages the endothelium and depresses 5-HT clearance in the perfused lung (Gillis et al., 1978), also significantly decreases 5-HT uptake and accumulation into lung slices (Hilliker and Roth, 1980). When 5-HT uptake and accumulation were examined in lung slices obtained from oxygen-exposed animals, no significant difference from controls was observed. The lung monoamine oxidase (MAO) system is affected by pulmonary toxicants (Mustafa et al,, 1977; Roth et al., 1979). Acute exposure to high concentrations of ozone, which primarily affects the pulmonary epithelium, resulted in decreased MAO activity in lung homogenates, mitochondria, and microsomes, while chronic exposure to low ozone concentrations was associated with increased MAO activity. In addition, paraquat decreased pulmonary MAO activity (Roth et al., 1979). In this study, we found that exposure of mice to 100% oxygen caused an initial 25 to 35% decrease in lung MAO activity followed by recovery and occasional stimulation with continued exposure (Table 1). The time course of these oxygen-induced changes in pulmonary MAO activity correlated with the described (Mustafa and Tierney, 1978) morphological and metabolic changes in lung mitochondria, initial depression followed by stimulation. Although the lung homogenate data do not to-
EFFECT
OF
HYPEROXIA
ON
LUNG
SEROTONIN
127
cell tally reflect the nature of MAO in lung alveoli is consistent with endothelial mitochondria, the mitochondrial fraction damage. Since PMN can release potent oxaccounts for greater than 77% of the total idants which can damage endothelial cells lung MAO activity (Kung and Wilson, (Yamada et al., 1979) and which can also 1979). Thus changes in the mitochondrial cause the release of platelet 5-HT (Handin enzyme would be expected to result in et al., 1977) they may significantly contribchanges in lung homogenate activity. Due ute to the pathophysiology of oxygen toxto differences in the time course and extent icity. Indeed, Shasby et al. ( 198 1) have of inhibition, these data indicate that altered shown that depletion of PMN protects ani5-HT metabolism by MAO appears not to mals from oxygen-induced lung injury. be a significant mechanism responsible for Platelets have been frequently implicated the elevated lung 5-HT levels observed dur- in lung microvascular injury. Not only are ing oxygen exposure. they components of microemboli, but they Earlier work has demonstrated a relationare capable of accumulating and releasing ship between pulmonary vasculature injury, a number of vasoactive substances, including edema, and increased lung 5-HT levels 5-HT. Recently, Yamada et al. (1979) dem(Kind et al., 1961; Skillen et al., 1961). onstrated that platelets and platelet products Acute exposure of animals to ozone pro- (5-HT) potentiate PMN-mediated endotheduced significant pulmonary edema and el- lial cell injury. They showed that platelets evated levels of 5-HT (Skillen et al., 1961). augment PMN adherence to endothelial Such increases in lung 5-HT content followcells and strikingly amplify PMN-induced ing oxygen- or ozone-induced injury may endothelial cell damage. Furthermore, they represent either a consequence of the injury found that 5-HT could completely mimic the or may be a factor in the response of pul- platelet effect, and they concluded that 5monary microvasculature to injury. It is easy HT functions as an effector of PMN-endothelial cell interactions. Since platelets to envision how damage to the pulmonary endothelium might allow 5-HT access to contain most of the circulating 5-HT, oxyother lung cells capable of accumulating the gen-induced alterations of platelet 5-HT upvasoactive amine, and how lung 5-HT con- take, storage, or release could result in intent might then parallel the extent of injury. creased levels of the free amine which could Alternatively, several lines of evidence potentiate endothelial cell damage by the suggest a possible role for 5-HT and other mechanisms described above. blood-borne substances in the response of the pulmonary microvasculature to injury. Teleologically, one might expect the pulmonary ACKNOWLEDGMENTS microvasculature to be insensitive to bloodborne mediators of inflammation, since all This work was supported by a grant-in-aid from the the systemic venous blood flows through the American Heart Association, Indiana Affiliate, and by lungs and concentrations of injurious sub- a Young Investigator Pulmonary Research Grant (HL 19573) from the National Institute of Heart, Lung. and stances would be expected to be greatest Blood. there. Recent data, however, indicate that this may not be the case. Fox et al. (198 1) REFERENCES have correlated the number of alveolar polymorphonuclear leukocytes (PMN) with the ADAMSON, 1. Y. R., BOWDEN, D. H., AND WYATT. degree of pulmonary edema in animals exJ. P. (1970). Oxygen poisoning in mice. Uhrastrucposed to 100% oxygen. This correlation of tural and surfactant studies during exposure and recovery. Arch. Pathol. 90, 463-472. pulmonary edema with influx of PMN into
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