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Release of eicosanoids from isolated lungs of guinea-pigs exposed to pure oxygen: effect of dexamethasone
European Journal of Pharmacology, 126 (1986) 11-20 Elsevier
11
R E L E A S E OF E I C O S A N O I D S F R O M I S O L A T E D L U N G S OF G U I N E A - P I G S E X P O S E D T O P U R E OXYGEN: E F F E C T OF D E X A M E T H A S O N E GILBERTO DE NUCCI *, PAUL ASTBURY, NICHOLAS READ, JOHN A. SALMON and SALVADOR MONCADA WellcomeResearch Laboratories, Langley Court, Beckenham, Kent, BR3 3BS, U.K.
Received 18 November 1985, revised MS received 25 March 1986, accepted 8 April 1986
G. DE NUCCI, P. ASTBURY, N. READ, J.A. SALMON and S. MONCADA, Release of eicosanoids from isolated lungs of guinea-pigs exposed to pure oxygen: effect of dexamethasone, European J. Pharmacol. 126 (1986) 11-20. The effect of dexamethasone on bradykinin-induced release of eicosanoids from isolated lungs of guinea-pigs exposed to pure oxygen (02) is described. Pathological changes were induced in the lungs of guinea-pigs exposed to pure 02 for 72 and 96 h. Bradykinin-induced release of 6-oxo-PGF1, , and thromboxane Bz (TXB2) was increased in lungs from guinea-pigs exposed to 72 and 96 h of 02. Removal of PGI 2 and PGE 2 was not affected by the exposure to 02, but the removal of bradykinin was significantly reduced after 96 h of 02 exposure. Dexamethasone did not reduce bradykinin-induced release of eicosanoids from lungs of control animals, but it did inhibit the release from lungs of guinea-pigs exposed to O z. Dexamethasone had no effect on the metabolism of Bk by the inflamed lungs. Dexamethasone
Hyperoxia
Bradykinin
Eicosanoids
1. Introduction The release of eicosanoids from guinea-pig isolated lungs is dependent on the stimulus employed, for example, bradykinin releases more 6oxo-PGFl~than thromboxane B2 (TXB2) whereas other stimuli, like the calcium ionophore A23187, arachidonic acid (AA) and ovalbumin in sensitised guinea-pigs, release more TXB 2 than 6-oxo-PGFl, (Bakhle et al., 1985a). This suggests that bradykinin might act on a different population of cells from other stimuli. These may be the vascular endothelial cells since prostacyclin (PGI2) is the major cyclo-oxygenase product generated by these cells. Oxygen (O2) toxicity induces non-specific lesions in the lungs consisting of oedema, alveolar haemorrhage, inflammation, fibrin deposition and thickening and hyalinisation of alveolar membranes (Clark and Lambertsen, 1971). However, it has been suggested (in the mouse, Bowden et al., 1968; in the rat, Klister et al., 1967 and in non-hu* To whom all correspondence should be addressed. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
man primates, Kapanci et al., 1969) that the earliest and most characteristic lesion of 02 acute toxicity is the injury to endothelial cells. We have now investigated the release of 6-oxo-PGFt~, TXB 2 and leukotriene B4 (LTB4) induced by bradykinin from isolated lungs obtained from guinea-pigs exposed to pure 0 2 for different periods. In addition we have studied the metabolism of exogenous prostaglandins and bradykinin and the effect of dexamethasone on the release of eicosanoids from these lungs. Furthermore we have investigated, by light and transmission electron microscopy (T.E.M.), the morphological changes occurring in the lungs during the period of study.
2. Materials and methods 2.1. Method of exposure to 02
Male Dunkin-Hartley guinea-pigs (350-400 g body weight) were placed in a plexiglass chamber supplied continuously with pure 02 at a pressure of one atmosphere and a flow rate of 4 1. min-1
12 A CO 2 absorber (Carbosorb, BDH Chemicals, Poole, England) was kept inside the chamber. The animals were exposed to 02 for periods of 24, 48, 72 and 96 h. Control animals breathed laboratory air. 02 tension was measured by passing samples of gas obtained from the inside of the chamber into a radiometer 02 electrode assembly coupled to a radiometer PHM 71 Mark lI acid-base analyser. The gas samples were saturated with water vapour at ambient room temperature (circa 20°C). The device was calibrated by setting zero 02 tension with a radiometer Oz-free calibration solution and setting a high O 2 tension using pure 02 and correcting for saturated water vapour pressure and barometric pressure at the time of measurements. Linearity was checked with gas mixtures of intermediate composition.
2.2. Isolated lungs After the exposure period, the guinea-pigs were anaesthetised with pentobarbitone (60 rag. kg -~ i.p.). Following mid-thoracotomy the pulmonary artery was cannulated and perfused for 5 rain with 25 ml of heparinised (10 U . m 1 - 1 ) Krebs bicarbonate solution. The trachea was cannulated and the lungs removed and suspended in a heated chamber. The lungs were perfused via the pulmonary artery with warmed (37°C) and gassed (95% 02-5% CO2) Krebs bicarbonate solution at a constant rate of 5 ml. min -1, and left to stabilise for 30 min (Bakhle et al., 1969).
2.3. Collection of lung effluent and drug administration Bradykinin (250 ng. m1-1) was given in 5 min infusions at 0.1 ml. min -1. For radioimmunoassay (RIA) of eicosanoids, the lung effluent was collected in 3 min fractions. Each pair of lungs was used for a single infusion of a single stimulus (Bakhle et al., 1985a). Dexamethasone (2 /Lg. m1-1) was infused for 1 h at 0.1 ml • min -~ before challenge with bradykinin.
2.4. Radioimmunoassay of the lung effluent The concentrations of TXB 2, 6-oxo-PGFl~ and LTB 4 in the lung effluent were determined by specific radioimmunoassay after suitable dilutions (1 : 2-1 : 100) in RIA buffer but without prior extraction or purification. The specificity of the antisera used in these RIA has been established previously (Salmon, 1978; Salmon et al., 1982).
2.5. Histological examination of the lungs For histological studies the trachea was cannulated and the lungs inflated with 10 ml of air and then perfused in situ through the pulmonary artery with 25 ml of heparinised (10 U . m1-1) Krebs bicarbonate solution at 5 ml. rain-1 followed by 25 ml of a storage fixative (4% formaldehyde, 1% glutaraldehyde in phosphate buffer pH 7.2; McDowell and Trump, 1976) for 5 min. The trachea was then ligated and the lungs removed, placed in containers of storage fixative and prepared for light and transmission microscopy (Humphrey and Pittman, 1974). Following fixation transverse slices of both caudal lobes, from a point just proximal to the middle of each lobe, were removed from each guinea-pig. One slice from each caudal lobe was processed for routine paraffin histology and 4 /~ sections cut and stained with haematoxylin and eosin and examined by light microscopy. A further tissue block, 4-5 mm square and 2 mm thick, was taken from each caudal lobe. The area selected included a portion of the pulmonary artery, visceral pleura and associated parenchyma. The tissue blocks were post-fixed in 1% osmium tetroxide in 0.1 M cacodylate buffer, pH 7.2, dehydrated in a graded series of acetone and water mixtures and embedded in Spurr epoxy resin using a Polaron E9100 (Polaron Equipment Ltd., Watford) tissue processor. Semi-thin sections (1 /~m) were cut using a Reichert OMU4 ultramicrotome (Reichert-Jung Ltd., Slough) and stained with methylene blue-azure II-basic fuchsin (Humphrey and Pittman, 1974) and examined by light microscopy. After examination of the semi-thin sections, the specimen blocks were trimmed and thin sections (70-90 nm) of selected areas were cut. The
13 sections were collected on copper/palladium G200 grids, stained with aqueous uranyl acetate and lead citrate. 2.6. Metabolism of bradykinin
The metabolism of bradykinin in the isolated lungs was measured by bioassay. The effluent from the lungs superfused longitudinal strips of cat jejunum (Ferreira and Vane, 1967). The tissues received infusions (0.1 ml. rain-l) of a combination of antagonists (Gilmore et al., 1968) and indomethacin 5.6 ~M (Eckenfels and Vane, 1972). Contractions of the assay tissues were recorded with auxotonic levers attached to Harvard isotonic transducers (Paton, 1957), and displayed on a six channel Watanabe recorder (type WTR 281). The metabolism of bradykinin was calculated by comparing the contractions of the assay tissues in response to 5 min infusions of bradykinin given directly over the tissues (O.T.), with those in response to bradykinin infused into the pulmonary artery (T.L.) as previously described (Ferreira and Vane, 1967; Alabaster and Bakhle, 1972) and expressed as % of survival.
SK&F Stevenage, U.K.), methysergide bimaleate (Sandoz Producs Ltd., Leeds, U.K.), mepyramine maleate (May & Baker, Dagenham, U.K.), propranolol hydrochloride and atropine sulphate (Sigma) and dexamethasone sodium phosphate (Decadron, Merck, Sharp & Dohme Ltd.). 5,6,8,9,11,12,14,1513H]TXB2, specific activity (s.a.) 140 Ci/mmol; 6-oxo-5,6,8,9,11,14,1513H]PGFl~, s.a. 150 Ci/mmol and 5,6,8,9,11,12,14,1513H] LTB4, s.a. 221 Ci/mmol were purchased from Amersham International (Amersham, Bucks., U.K.). PGE 2 and prostacyclin were obtained from the Upjohn Co. (Kalamazoo, USA). 25% glutaraldehyde (EMScope Laboratories Ltd., Kent, U.K.), 40% formaldehyde (BDH Chemicals Ltd., Poole, U.K.) and Spurr epoxy resin (Polaron Equipment Ltd., Watford, U.K.) were also used. 2.9. Statistics
Results are shown as mean values + S.E.M. for n experiments. The Student's unpaired t-test was used to determine the significance of differences between means and a P value of < 0.05 was taken as significant.
2. 7. Inactivation of prostacyclin and PGE e
The survival of prostacyclin when infused through the isolated lungs was also measured by bioassay. The effluent from the lungs superfused spiral strips of rabbit coeliac artery (RbCA) and rabbit mesenteric artery (RbMesA) (Bunting et al., 1976). The removal of PGE 2 was studied using the rat stomach strip (RSS) (Vane, 1957). 2.8. Materials
The Krebs bicarbonate solution had the following composition (mM): NaC1 118, KC1 4.7, KH2PO 4 1.2, MgSOa.7H20 1.17, CaClz.6H20 2.5, NaHCO 3 25 and glucose 8.4. Bradykinin triacetate salt (Sigma Chemical Company, Poole, U.K.) was dissolved in saline. Indomethacin (Merck, Sharp & Dohme Ltd., Hertfordshire, U.K.) was dissolved in 5% NaHCO 3 and diluted in Krebs solution for use. Other drugs used were: phenoxybenzamine hydrochloride (Dibenyline,
3. Results
3.1. Survival
The 0 2 tension in the chamber was always above 95%. CO 2 tension was not monitored. Under these conditions, the survival of the guinea-pigs exposed to O z was 100% up to 48 h, 85 + 9% at 72 h and 70_+ 11% at 96 h (n = 24). 3.2. Histological examination 3.2.1. Light microscopy All the lungs examined from the 24 h group (n = 5) were morphologically indistinguishable from the control groups (n = 11). The earliest evidence of pathological changes was seen in the 48 h group. However, these changes were only present in 2 of the 5 animals in the group. Alveolar oedema was the most prominent feature of the
14 lesion, however, increased cellularity of the alveolar septal walls, extravasated red blood cells and increased numbers of macrophages in the alveolar spaces were common findings. Pathological changes were seen in all (n = 10) the animals from the 72 h group. Considerable variation was seen in the extent and degree of pathology between individual animals. Despite this individual variation of response to the toxic action of 0 2 the underlying nature of the lesion was consistent. The lesions were typically focal and areas of essentially normal parenchyma could be observed adjacent to areas undergoing extensive changes. In the focal areas the alveolar (septal) walls were thickened by increased numbers of mononuclear cells and polymorphonuclear leucocytes (PMNs). In addition, the adventitia of the large blood vessels in the focal areas was oedematous and the lymphatic vessels dilated. Pathological changes in the lungs of animals from the 96 h group (n = 10) were more advanced than those observed at 72 h. The oedema of the adventitia of the large blood vessels and dilatation of the lymphatics were widespread and marked. As well as the alveolar oedema, interstitial oedema
and fibrosis of the alveolar septa were apparent. Hyaline membrane formation and markedly increased numbers of macrophages were observed throughout the proteinaceous exudate within the alveoli.
3.2.2. Transmission electron microscopy The ultrastructure of the control tissue, as observed by transmission electron microscopy, appeared normal (fig. 1). The earliest changes seen in the alveolar regions from animals exposed to pure O z involved both the endothelial cells and membranous (type I) pneumocytes. The changes in the endothelial cells were usually degenerative, however, adaptive changes were seen sometimes. Degenerative changes included swelling of the cytoplasm and the formation of large blebs and projections protruding into the capillary lumens which significantly reduced the luminal cross-sectional area. Within the cytoplasm, swollen mitochondria were found along with enlarged vesicles and the occasional myelin fibre (fig. 2). The nuclei often appeared enlarged and had deeply indented and irregular profiles. The adaptive changes included
Fig. 1. Transmission electron micrograph of an alveolar region from a control guinea-pig lung. Note the patent capillary lumens (C), the capillary endothelium (En), the alveolar type I pneumocytes (Epl) which form thin delicate sheets, and the type II pneumocytes (Ep2) with characteristic cytoplasmic granules. Fibrocyte (F) (bar = 2 /xm). Inset: detail of the air blood barrier, formed from thin sheets of type I pneumocytes (Epl) and endothelial cells (En) separated by a common basal lamina (bar = 20 nm).
15
Fig. 2. Transmission electron micrograph of the alveolar septum from a guinea-pig exposed to pure 02 for 72 h illustrating the interstitial oedema (*), degenerative changes to the endothelium (En) and type I pneumocytes (Epl). Instead of forming a thin sheet, the outline of the endothelium is irregular and the cytoplasm contains vacuoles. The cytoplasm of the type I pneumocytes appears swollen and rarefied. Note the junctions of both cell types ("~) appear intact (bar = 1 /~m).
prominent nucleoli and increased amounts of r o u g h e n d o p l a s m i c r e t i c u l u m . T h e n e c r o t i c cells were usually swollen with rarefied cytoplasm and few o r g a n e l l e s w h i l e s m a l l e r n u m b e r s a p p e a r e d
shrunken with condensed granular cytoplasm and p y k n o t i c nuclei. D e s p i t e large n u m b e r s of n e c r o t i c e n d o t h e l i a l cells, o n l y the o c c a s i o n a l a r e a of exp o s e d b a s e m e n t m e m b r a n e was o b s e r v e d . Loss of
Fig. 3. Transmission electron micrograph of the alveolar region from a guinea-pig exposed to pure 02 for 96 h. Extensive alveolar oedema (*) and thickening of the alveolar septum is evident (bar = 4 ~m).
16 endothelial cells resulted in the collapse of the capillaries. The degenerative changes in the type I pneumocytes involved the rarefaction of the cytoplasm along with mitochondrial swelling (fig. 2). In advanced stages of degeneration breaks in the plasma membrane were seen with organelles spilling into the alveolar space. Areas of exposed basement membrane were also observed. Interstitial oedema was an early event in the evolution of the lesion, involving the extracellular matrix of the thick region (Pietra, 1984) of the interstitial space (fig. 2). This was followed by the progressive invasion of cells (fibrocytes, PMNs and mononuclear cells) and the deposition of collagen and fibrinoid material (fig. 3). The thin region of the interstitial space, where the basement membranes of the type I pneumocyte and endothelium are fused, appeared unaffected when either of the bordering cells remained viable. The interstitial oedema could not be attributed to any specific lesion of the endothelial cells, no breaks were observed in the cell wall and the tight junctions appeared intact.
3.3. Basal release of eicosanoids
[ ] 6-°x°-PGFIo~ [ ] TXB2 L *p'~O.O01
30
== 2
15
L
20
10
i.:
i
10
t~ 0
0
24 48
72
96
0
24 48
72
96
Time of exposure to 100% 0 2 (h)
Fig. 4. Release of 6-oxo-PGFl, and TXB/ induced by an infusion of bradykinin (250 ng-ml-l) into isolated lungs of guinea-pigs exposed to 100% 02 for various time intervals. The mean concentration_+S.E.M. of 6-oxo-PGFl,, is represented by the hatched bars, and the mean concentration__+S.E.M, of TXB2 is represented by the open bars. The numbers inside the bars indicate the numbers of animals employed.
3.5. Metabolism of eicosanoids
There was no significant difference in the basal release of TXB 2 (control 0.49 + 0.08 n g - m l - l , n = 22; 72-96 h 02 0.62 + 0.09 ng. m1-1, n = 23) and 6-oxo-PGFl~ (less than 0.2 ng. m1-1) in the effluent of control lungs and of lungs of guineapigs exposed to 72 or 96 h 02.
Prostacyclin was poorly removed either by control or inflamed guinea-pig lungs (control 91 + 9%, 96 h 02 93 + 7%, n = 5, values expressed as % of survival). There was no significant difference either in the removal of P G E 2 from the pulmonary circulation of control lungs or lungs of guinea-pigs exposed to 96 h of 02 (control 11 + 5%, 96 h 02 6 _ 3%, n = 5, values expressed as % of survival).
3.4. Bradykinin-induced release of eicosanoids
3.6. Metabolism of bradykinin
There was a significant increase in the amounts of 6-oxo-PGFt~ and TXB 2 released by bradykinin (250 ng. ml - t ) infusions into the lungs from guinea-pigs exposed to 72 and 96 h 02 when compared to the amounts released from control lungs (fig. 4), however, the ratio of eicosanoids was not significantly changed. LTB 4 was not detected (less than 0.05 n g - m 1 - 1 ) in the effluent from either control lungs or from lungs of guineapigs exposed to 02 .
There was an increase in the survival of bradykinin from the lungs of guinea-pigs exposed to 96 h of 02 when compared to control lungs (fig. 5). The survival of bradykinin in control lungs was 6 + 1% (n = 8) and in lungs of guinea-pigs exposed to 96 h of 02 the survival was significantly (P < 0.001) increased to 16 + 3% (n---7). No significant changes were observed at earlier stages (24 h, 9_.+ 2%, n = 7; 48 h, 7 + 1%, n = 4 and 72 h, 6+2%,n=4).
17
CONTROL LUNG
V"
10.T.
0.50.T.
20 T.L.
BK (ng ml "1)
HOURS 0 2 LUNG 96
50.T.
20 T.L.
3,50.T.
BK (ng m1-1 )
Fig. 5. Effect of exposure t o 0 2 (96 h) on the survival of bradykinin by the isolated lungs. The effluent from control lungs (upper panel) and O2-exposed lungs (lower panel) superfuses longitudinal strips of cat jejunum (CJ). In the upper panel the response of the tissue tO an infusion of bradykinin (1 ng. ml-1) is shown followed by the response to 0.5 ng. ml-1 over the tissues (O.T.) and 20 ng-m1-1 through the lung (T.L.). In the lower panel the response of the tissue to 5 ng.m1-1 of bradykinin O.T. is followed by the response to 20 ng. m l - l T.L. and 3.5 ng-ml-1 O.T. The survival of bradykinin in the control lungs is less than 5% while the survival in the exposed lungs is greater than 15%. The same pattern of responses was observed in another 6 experiments.
3. 7. Effect of dexamethasone on eicosanoid release induced by bradykinin Dexamethasone (2 /~g-m1-1, 60 min) did not inhibit release of 6-oxo-PGFl~ induced by brady-
~'~ ]
[ ] Control [ ] Oexamethasone 0.05
30 a. I
20 j
I
0
kinin in normal guinea-pig-isolated lungs even when large concentrations of bradykinin (2.5/~g. ml - 1) were employed (control 24.4 + 1.6 ng. m l - 1; dexamethasone 17.6 + 4.8 ng- m1-1, n = 5). However, it did have an inhibitory effect on the bradykinin-induced release of 6-oxo-PGFl~ from lungs of guinea-pigs exposed to 96 h of 02 (control 36.5 + 6.9 ng- ml-1; dexamethasone 20.2 + 2 ng. ml-1, n = 5, fig. 6). Dexamethasone (2 btg. m1-1, 60 min) did not affect the metabolism of bradykinin in the lungs from animals exposed to 02 for 96 h (not shown), nor did it affect the w e t / d r y weight ratio (control 12.44 _+ 1.78; dexamethasone-treated 13.06 ___0.87, n = 5).
4. Discussion
~ o
Time of
exposure
to 02 (hi
Fig. 6. Effect of dexamethasone (2 ~g. m l - l , 60 min) infusion
on bradykinin-induced release of 6-oxo-PGFl= from isolated lungs of guinea-pigs. The hatched bars represent the lungs treated with dexamethasone for 1 h before the challenge with bradykinin (250 ng-ml- ~). The height of the bars represents the mean value of 5 experiments with 1 S.E.M.
Our results demonstrate that 02 toxicity in guinea-pigs shares some of the histopathological characteristics reported for other species. Interstitial oedema was an early event in the evolution of the lesion and although it could not be attributed to any specific lesion of the endothelial
18 cells, it should be taken as evidence of endothelial cell damage. Klister et al. (1967) studying hyperoxia-induced alterations in rat lungs demonstrated, by morphometric analysis, an enlargement of the interstitial compartment related to the accumulation of oedema fluid, but they also found it difficult to determine the distribution of endothelial lesions among pulmonary vessels of different types. The finding that areas of essentially normal parenchyma could be observed adjacent to areas undergoing extensive changes is not surprising since Bowden et al. (1968), studying hyperoxia in mice, also found that capillary ultrastructural changes were focal and that many capillaries showed no structural alterations. The absence of platelet thrombi in the pulmonary microvasculature has also been reported previously in rabbits (Dobuler et al., 1982). Isolated lungs from guinea-pigs exposed to 02 for 72 and 96 h release increased amounts of 6-oxo-PGF~ and TXB 2 when stimulated with bradykinin. Several mechanisms could be responsible for such an increase: (i) an increase in phospholipase A 2 activity, (ii) a decrease in the metabolism of the eicosanoids, (iii) a decrease in the metabolism of bradykinin or (iv) a combination of all the above factors. The first possibility seems unlikely since the basal levels of eicosanoid released in the Oz-treated lung effluent were not significantly higher than those in control lung effluent. As has been demonstrated in other species (Dusting et al., 1978), prostacyclin is not metabolised during passage through the pulmonary circulation of the guinea-pig. No changes were observed in the removal of exogenous PGE 2 or prostacyclin by the inflamed lungs and therefore the increased release is unlikely to be due to changes in eicosanoid inactivation. We cannot rule out the possibility that the intracellular prostaglandin inactivation system was affected. Prostaglandin inactivation in the lung consists of two phases, transport of prostaglandins into the lung cells and intracellular metabolism by prostaglandin dehydrogenase (Anderson and Eling, 1976), and in this study we have only investigated the former. Chaudhari et al. (1979), however, have suggested that O 2 exposure in guinea-pigs leads to an inhibition of the dehydrogenase enzyme. Exposure to
high tensions of oxygen caused a reduction in the activity of angiotensin converting enzyme (ACE) in rat lungs in vitro (Toivonen et al., 1981) measured as reduced conversion of angiotensin I into angiotensin II. These authors have also reported reduction in the inactivation of bradykinin, although that occurred at a later stage (60 h of exposure). In rabbits Dobuler et al. (1982) observed the same effect. Since ACE is also responsible for the metabolism of bradykinin (Ferreira and Vane, 1967), we investigated whether isolated lungs of guinea-pigs exposed to O 2 would also show decreased ACE activity and consequently increased survival of bradykinin. The lungs of guinea-pigs exposed to 02 for 96 h did exhibit a decreased inactivation of bradykinin, suggesting that this could be the mechanism for the increased eicosanoid release observed in lungs from these animals. The fact that we did not observe any change in the metabolism of bradykinin after shorter periods of exposure to 02, although there was a significant increase in the eicosanoid levels, could be due to the difference in the amounts of substrate used to assess metabolism of bradykinin (20 ng. m1-1) and the amount needed to induce release of eicosanoids (250 ng. ml-1). It might be that at 72 h the system of inactivation of bradykinin is already affected, but it behaves normally at low amounts of substrate. The decrease observed in the metabolism of bradykinin could be the consequence of the damage induced by the 02 exposure in the endothelial cells, since ACE is located at the plasma membranes of the endothelial cells (Ryan et al., 1976). The release of eicosanoids induced by bradykinin from guinea-pig isolated lungs is insensitive to corticosteroid treatment (Blackwell et al., 1978; Robinson and Hoult, 1980). This we have confirmed in normal lungs. However it is interesting that dexamethasone had an inhibitory effect on the bradykinin-induced release of 6-oxo-PGFl~ from the inflamed lungs. Since dexamethasone induces the synthesis of new kinases, such as ACE (Ialenti et al., 1986), it would be possible for the observed effect to be achieved through an enhanced metabolism of bradykinin. However, our experiments show that this is an unlikely mecha-
19
nism since dexamethasone failed to increase bradykinin metabolism in lungs of guinea-pigs exposed to 96 h of 02. This inhibitory action of dexamethasone could therefore be due to bradykinin reaching during the inflammatory process cells whose phospholipases are sensitive to lipocortin (Di Rosa et al., 1984). Those cells could be either migrating cells such as neutrophils or macrophages, or resident cells in t h e lung. We demonstrated the presence of mononuclear cell infiltration in the lungs after 96 h of 02 exposure and it is known that macrophages can release lipocortin (Blackwell et al., 1980). The second possibility is that a different lung cell population, due to damage of the endothelium, is now being stimulated either with bradykinin or dexamethasone. Indeed, we have shown that administration of antigen via the airways in guinea-pig isolated lungs induced a release of vasoactive materials which was different from that induced if the antigen was injected through the pulmonary artery (Bakhle et al., 1985b). We have also shown that the release of lyso-PAF can be induced by stimulating the lungs via the airways with calcium ionophore. However, unless the lungs are inflamed (exposed to 02), this is not possible if the ionophore is injected through the pulmonary artery (Parente et al., 1985). These results suggest that the population of cells which participates in the inflammatory process might be located closer to the alveolar side of the lung and therefore might be reached more easily from that side than from the circulation. Whether the effect of dexamethasone is due to an effect on a population of cells in the lungs which is not easily reached in non-inflamed lungs or to the migration during the inflammatory process of cells which are sensitive to the action of the steroid remains to be investigated.
Acknowledgements
The authors wish to thank Dr. Y.S. Bakhle for discussing the manuscript, Dr. R. White for measuring the 0 2 tension and Mr. A. Kotecha for technical assistance.
References Alabaster, V.A. and Y.S. Bakhle, 1972, The inactivation of bradykinin in the pulmonary circulation of isolated lungs, Br. J. Pharmacol. 45, 299. Anderson, M.W. and T.E. Eling, 1976, Prostaglandin removal and metabolism by isolated perfused rat lung, Prostaglandins 11,645. Bakhle, Y.S., S. Moncada, G. De Nucci and J.A. Salmon, 1985a, Differential release of eicosanoids by bradykinin, arachidonic acid and calcium ionophore A23187 in guineapig isolated perfused lung, Br. J. Pharmacol. 86, 55. Bakhle, Y.S., S. Moncada, G. De Nucci and J.A. Salmon, 1985b, Ovalbumin-induced release of eicosanoids from guinea-pig lungs perfused via the pulmonary artery and via the trachea, Br. J. Pharmacol. 86, 423P. Bakhle, Y.S., A.M. Reynard and J.R. Vane, 1969, Metabolism of the angiotensins in isolated perfused tissues, Nature (London) 222, 956. BlackweU, G., R. Flower, F. Nijkamp and J.R. Vane, 1978, Phospholipase A 2 activity of guinea-pig isolated perfused lungs: stimulation, and inhibition by anti-inflammatory steroids, Br. J. Pharmacol. 62, 79. Blackwell, G.J., R. Carnuccio, M. Di Rosa, R.J. Flower, L. Parente and P. Persico, 1980, Macrocortin: a polypeptide causing the anti-phospholipase effect of glucocorticoids, Nature (London) 287, 147. Bowden, D.H., I.Y.R. Adamson and J.P. Wyatt, 1968, Reaction of the lung cells to a high concentration of oxygen, Arch. Pathol. 86, 671. Bunting, S., S. Moncada and J.R. Vane, 1976, The effects of prostaglandin endoperoxides and thromboxane A 2 on strips of rabbit coeliac artery and certain other smooth muscle preparations, Br. J. Pharmacol. 57, 462P. Chaudhari, A., K. Sivarajah, R. Warnock, T.E. Eling and M.W. Anderson, 1979, Inhibition of pulmonary prostaglandin metabolism by exposure of animals to oxygen or nitrogen dioxide, Biochem. J. 184, 51. Clark, J.M. and C.J. Lambertsen, 1971, Pulmonary oxygen toxicity: a review, Pharmacol. Rev. 23, 38. Di Rosa, M., R.J. Flower, F. Hirata, L. Parente and F. Russo-Marie, 1984, Antiphospholipase proteins, Prostaglandins 28, 441. Dobuler, K.J., J.D. Catravas and C.N. Gillis, 1982, Early detection of oxygen-induced lung injury in conscious rabbits, Am. Rev. Respir. Dis. 126, 534. Dusting, D.J., D.J. Chapple, R. Hughes, S. Moncada and J.R. Vane, 1978, Prostacyclin (PGI 2) induces coronary vasodilatation in anaesthetised dogs, Cardiovasc. Res. XII, 720. Eckenfels, A. and J.R. Vane, 1972, Prostaglandins, oxygen tension and smooth muscle tone, Br. J. Pharmacol. 45, 451. Ferreira, S.H. and J.R. Vane, 1967, The disappearance of bradykinin and eledoisin in the circulation and vascular beds of the cat, Br. J. Pharmacol.Chemother. 30, 417. Gilmore, N., J.R. Vane and J.H. Wyllie, 1968, Prostaglandins released by the spleen, Nature (London) 218, 1135. Humphrey, C.D. and F.E. Pittman, 1974, A simple methylene
20 blue-azure II-basic fuchsin stain for epoxy-embedded tissue sections, Stain Technol. 49, 9. Ialenti, A., A. Calignano, R. Carnuccio and M. Di Rosa, 1986, Glucocorticoid induction of angiotensin converting enzyme, Agents Actions 17, 294. Kapanci, Y., E.R. Weibel, H.P. Kaplan and F.R. Robinson, 1969, Pathogenesis and reversibility of the pulmonary lesions of oxygen toxicity in monkeys, Lab. Invest. 20, 101. Klister, G.S., P.R.B. Caldwell and E. Weibel, 1967, Development of fine structural damage to alveolar and capillary lining cells in oxygen-poisoned rat lungs, J. Cell Biol. 32, 605. McDowell, E.M. and B.F. Trump, 1976, Histological fixatives suitable for diagnostic light and electron microscopy, Arch. Pathol. Lab. Med. 100, 405. Parente, L., M. Fitzgerald, G. De Nucci and S. Moncada, 1985, The release of lyso-PAF from guinea-pig lungs, European J. Pharmacol. 112, 281. Paton, W.D.M., 1957, A pendulum auxotonic lever, J. Physiol. (London) 137, 35P.
Pietra, G.G., 1984, Biology of disease: New insights into mechanisms of pulmonary edema, Lab. Invest. 51,489. Robinson, C. and J. Hoult, 1980, Evidence for functionally distinct pools of phospholipase responsible for prostaglandin release from the perfused guinea-pig lung, European J. Pharmacol. 64, 333. Ryan, U.S., J.W. Ryan and A.T. Chiu, 1976, Localization of angiotensin converting enzyme (kininase II). II. Immunocytochemistry and immunofluorescence, Tissue Cell 8, 125. Salmon, J.A., 1978, A radioimmunoassay for 6-keto-prostaglandin F1~,, Prostaglandins 15, 383. Salmon, J.A., P.M. Simmons and R.M.J. Palmer, 1982, A radioimmunoassay for LTB4, Prostaglandins 24, 225. Toivonen, H., J. Hartiala and Y.S. Bakhle, 1981, Effects of high oxygen tension on the metabolism of vasoactive hormones in isolated perfused rat lungs, Acta Physiol. Scand. 111, 185. Vane, J.R., 1957, A sensitive method for the assay of 5-hydroxytryptamine, Br. J. Pharmacol. Chemother. 12, 344.
11
R E L E A S E OF E I C O S A N O I D S F R O M I S O L A T E D L U N G S OF G U I N E A - P I G S E X P O S E D T O P U R E OXYGEN: E F F E C T OF D E X A M E T H A S O N E GILBERTO DE NUCCI *, PAUL ASTBURY, NICHOLAS READ, JOHN A. SALMON and SALVADOR MONCADA WellcomeResearch Laboratories, Langley Court, Beckenham, Kent, BR3 3BS, U.K.
Received 18 November 1985, revised MS received 25 March 1986, accepted 8 April 1986
G. DE NUCCI, P. ASTBURY, N. READ, J.A. SALMON and S. MONCADA, Release of eicosanoids from isolated lungs of guinea-pigs exposed to pure oxygen: effect of dexamethasone, European J. Pharmacol. 126 (1986) 11-20. The effect of dexamethasone on bradykinin-induced release of eicosanoids from isolated lungs of guinea-pigs exposed to pure oxygen (02) is described. Pathological changes were induced in the lungs of guinea-pigs exposed to pure 02 for 72 and 96 h. Bradykinin-induced release of 6-oxo-PGF1, , and thromboxane Bz (TXB2) was increased in lungs from guinea-pigs exposed to 72 and 96 h of 02. Removal of PGI 2 and PGE 2 was not affected by the exposure to 02, but the removal of bradykinin was significantly reduced after 96 h of 02 exposure. Dexamethasone did not reduce bradykinin-induced release of eicosanoids from lungs of control animals, but it did inhibit the release from lungs of guinea-pigs exposed to O z. Dexamethasone had no effect on the metabolism of Bk by the inflamed lungs. Dexamethasone
Hyperoxia
Bradykinin
Eicosanoids
1. Introduction The release of eicosanoids from guinea-pig isolated lungs is dependent on the stimulus employed, for example, bradykinin releases more 6oxo-PGFl~than thromboxane B2 (TXB2) whereas other stimuli, like the calcium ionophore A23187, arachidonic acid (AA) and ovalbumin in sensitised guinea-pigs, release more TXB 2 than 6-oxo-PGFl, (Bakhle et al., 1985a). This suggests that bradykinin might act on a different population of cells from other stimuli. These may be the vascular endothelial cells since prostacyclin (PGI2) is the major cyclo-oxygenase product generated by these cells. Oxygen (O2) toxicity induces non-specific lesions in the lungs consisting of oedema, alveolar haemorrhage, inflammation, fibrin deposition and thickening and hyalinisation of alveolar membranes (Clark and Lambertsen, 1971). However, it has been suggested (in the mouse, Bowden et al., 1968; in the rat, Klister et al., 1967 and in non-hu* To whom all correspondence should be addressed. 0014-2999/86/$03.50 © 1986 Elsevier Science Publishers B.V.
man primates, Kapanci et al., 1969) that the earliest and most characteristic lesion of 02 acute toxicity is the injury to endothelial cells. We have now investigated the release of 6-oxo-PGFt~, TXB 2 and leukotriene B4 (LTB4) induced by bradykinin from isolated lungs obtained from guinea-pigs exposed to pure 0 2 for different periods. In addition we have studied the metabolism of exogenous prostaglandins and bradykinin and the effect of dexamethasone on the release of eicosanoids from these lungs. Furthermore we have investigated, by light and transmission electron microscopy (T.E.M.), the morphological changes occurring in the lungs during the period of study.
2. Materials and methods 2.1. Method of exposure to 02
Male Dunkin-Hartley guinea-pigs (350-400 g body weight) were placed in a plexiglass chamber supplied continuously with pure 02 at a pressure of one atmosphere and a flow rate of 4 1. min-1
12 A CO 2 absorber (Carbosorb, BDH Chemicals, Poole, England) was kept inside the chamber. The animals were exposed to 02 for periods of 24, 48, 72 and 96 h. Control animals breathed laboratory air. 02 tension was measured by passing samples of gas obtained from the inside of the chamber into a radiometer 02 electrode assembly coupled to a radiometer PHM 71 Mark lI acid-base analyser. The gas samples were saturated with water vapour at ambient room temperature (circa 20°C). The device was calibrated by setting zero 02 tension with a radiometer Oz-free calibration solution and setting a high O 2 tension using pure 02 and correcting for saturated water vapour pressure and barometric pressure at the time of measurements. Linearity was checked with gas mixtures of intermediate composition.
2.2. Isolated lungs After the exposure period, the guinea-pigs were anaesthetised with pentobarbitone (60 rag. kg -~ i.p.). Following mid-thoracotomy the pulmonary artery was cannulated and perfused for 5 rain with 25 ml of heparinised (10 U . m 1 - 1 ) Krebs bicarbonate solution. The trachea was cannulated and the lungs removed and suspended in a heated chamber. The lungs were perfused via the pulmonary artery with warmed (37°C) and gassed (95% 02-5% CO2) Krebs bicarbonate solution at a constant rate of 5 ml. min -1, and left to stabilise for 30 min (Bakhle et al., 1969).
2.3. Collection of lung effluent and drug administration Bradykinin (250 ng. m1-1) was given in 5 min infusions at 0.1 ml. min -1. For radioimmunoassay (RIA) of eicosanoids, the lung effluent was collected in 3 min fractions. Each pair of lungs was used for a single infusion of a single stimulus (Bakhle et al., 1985a). Dexamethasone (2 /Lg. m1-1) was infused for 1 h at 0.1 ml • min -~ before challenge with bradykinin.
2.4. Radioimmunoassay of the lung effluent The concentrations of TXB 2, 6-oxo-PGFl~ and LTB 4 in the lung effluent were determined by specific radioimmunoassay after suitable dilutions (1 : 2-1 : 100) in RIA buffer but without prior extraction or purification. The specificity of the antisera used in these RIA has been established previously (Salmon, 1978; Salmon et al., 1982).
2.5. Histological examination of the lungs For histological studies the trachea was cannulated and the lungs inflated with 10 ml of air and then perfused in situ through the pulmonary artery with 25 ml of heparinised (10 U . m1-1) Krebs bicarbonate solution at 5 ml. rain-1 followed by 25 ml of a storage fixative (4% formaldehyde, 1% glutaraldehyde in phosphate buffer pH 7.2; McDowell and Trump, 1976) for 5 min. The trachea was then ligated and the lungs removed, placed in containers of storage fixative and prepared for light and transmission microscopy (Humphrey and Pittman, 1974). Following fixation transverse slices of both caudal lobes, from a point just proximal to the middle of each lobe, were removed from each guinea-pig. One slice from each caudal lobe was processed for routine paraffin histology and 4 /~ sections cut and stained with haematoxylin and eosin and examined by light microscopy. A further tissue block, 4-5 mm square and 2 mm thick, was taken from each caudal lobe. The area selected included a portion of the pulmonary artery, visceral pleura and associated parenchyma. The tissue blocks were post-fixed in 1% osmium tetroxide in 0.1 M cacodylate buffer, pH 7.2, dehydrated in a graded series of acetone and water mixtures and embedded in Spurr epoxy resin using a Polaron E9100 (Polaron Equipment Ltd., Watford) tissue processor. Semi-thin sections (1 /~m) were cut using a Reichert OMU4 ultramicrotome (Reichert-Jung Ltd., Slough) and stained with methylene blue-azure II-basic fuchsin (Humphrey and Pittman, 1974) and examined by light microscopy. After examination of the semi-thin sections, the specimen blocks were trimmed and thin sections (70-90 nm) of selected areas were cut. The
13 sections were collected on copper/palladium G200 grids, stained with aqueous uranyl acetate and lead citrate. 2.6. Metabolism of bradykinin
The metabolism of bradykinin in the isolated lungs was measured by bioassay. The effluent from the lungs superfused longitudinal strips of cat jejunum (Ferreira and Vane, 1967). The tissues received infusions (0.1 ml. rain-l) of a combination of antagonists (Gilmore et al., 1968) and indomethacin 5.6 ~M (Eckenfels and Vane, 1972). Contractions of the assay tissues were recorded with auxotonic levers attached to Harvard isotonic transducers (Paton, 1957), and displayed on a six channel Watanabe recorder (type WTR 281). The metabolism of bradykinin was calculated by comparing the contractions of the assay tissues in response to 5 min infusions of bradykinin given directly over the tissues (O.T.), with those in response to bradykinin infused into the pulmonary artery (T.L.) as previously described (Ferreira and Vane, 1967; Alabaster and Bakhle, 1972) and expressed as % of survival.
SK&F Stevenage, U.K.), methysergide bimaleate (Sandoz Producs Ltd., Leeds, U.K.), mepyramine maleate (May & Baker, Dagenham, U.K.), propranolol hydrochloride and atropine sulphate (Sigma) and dexamethasone sodium phosphate (Decadron, Merck, Sharp & Dohme Ltd.). 5,6,8,9,11,12,14,1513H]TXB2, specific activity (s.a.) 140 Ci/mmol; 6-oxo-5,6,8,9,11,14,1513H]PGFl~, s.a. 150 Ci/mmol and 5,6,8,9,11,12,14,1513H] LTB4, s.a. 221 Ci/mmol were purchased from Amersham International (Amersham, Bucks., U.K.). PGE 2 and prostacyclin were obtained from the Upjohn Co. (Kalamazoo, USA). 25% glutaraldehyde (EMScope Laboratories Ltd., Kent, U.K.), 40% formaldehyde (BDH Chemicals Ltd., Poole, U.K.) and Spurr epoxy resin (Polaron Equipment Ltd., Watford, U.K.) were also used. 2.9. Statistics
Results are shown as mean values + S.E.M. for n experiments. The Student's unpaired t-test was used to determine the significance of differences between means and a P value of < 0.05 was taken as significant.
2. 7. Inactivation of prostacyclin and PGE e
The survival of prostacyclin when infused through the isolated lungs was also measured by bioassay. The effluent from the lungs superfused spiral strips of rabbit coeliac artery (RbCA) and rabbit mesenteric artery (RbMesA) (Bunting et al., 1976). The removal of PGE 2 was studied using the rat stomach strip (RSS) (Vane, 1957). 2.8. Materials
The Krebs bicarbonate solution had the following composition (mM): NaC1 118, KC1 4.7, KH2PO 4 1.2, MgSOa.7H20 1.17, CaClz.6H20 2.5, NaHCO 3 25 and glucose 8.4. Bradykinin triacetate salt (Sigma Chemical Company, Poole, U.K.) was dissolved in saline. Indomethacin (Merck, Sharp & Dohme Ltd., Hertfordshire, U.K.) was dissolved in 5% NaHCO 3 and diluted in Krebs solution for use. Other drugs used were: phenoxybenzamine hydrochloride (Dibenyline,
3. Results
3.1. Survival
The 0 2 tension in the chamber was always above 95%. CO 2 tension was not monitored. Under these conditions, the survival of the guinea-pigs exposed to O z was 100% up to 48 h, 85 + 9% at 72 h and 70_+ 11% at 96 h (n = 24). 3.2. Histological examination 3.2.1. Light microscopy All the lungs examined from the 24 h group (n = 5) were morphologically indistinguishable from the control groups (n = 11). The earliest evidence of pathological changes was seen in the 48 h group. However, these changes were only present in 2 of the 5 animals in the group. Alveolar oedema was the most prominent feature of the
14 lesion, however, increased cellularity of the alveolar septal walls, extravasated red blood cells and increased numbers of macrophages in the alveolar spaces were common findings. Pathological changes were seen in all (n = 10) the animals from the 72 h group. Considerable variation was seen in the extent and degree of pathology between individual animals. Despite this individual variation of response to the toxic action of 0 2 the underlying nature of the lesion was consistent. The lesions were typically focal and areas of essentially normal parenchyma could be observed adjacent to areas undergoing extensive changes. In the focal areas the alveolar (septal) walls were thickened by increased numbers of mononuclear cells and polymorphonuclear leucocytes (PMNs). In addition, the adventitia of the large blood vessels in the focal areas was oedematous and the lymphatic vessels dilated. Pathological changes in the lungs of animals from the 96 h group (n = 10) were more advanced than those observed at 72 h. The oedema of the adventitia of the large blood vessels and dilatation of the lymphatics were widespread and marked. As well as the alveolar oedema, interstitial oedema
and fibrosis of the alveolar septa were apparent. Hyaline membrane formation and markedly increased numbers of macrophages were observed throughout the proteinaceous exudate within the alveoli.
3.2.2. Transmission electron microscopy The ultrastructure of the control tissue, as observed by transmission electron microscopy, appeared normal (fig. 1). The earliest changes seen in the alveolar regions from animals exposed to pure O z involved both the endothelial cells and membranous (type I) pneumocytes. The changes in the endothelial cells were usually degenerative, however, adaptive changes were seen sometimes. Degenerative changes included swelling of the cytoplasm and the formation of large blebs and projections protruding into the capillary lumens which significantly reduced the luminal cross-sectional area. Within the cytoplasm, swollen mitochondria were found along with enlarged vesicles and the occasional myelin fibre (fig. 2). The nuclei often appeared enlarged and had deeply indented and irregular profiles. The adaptive changes included
Fig. 1. Transmission electron micrograph of an alveolar region from a control guinea-pig lung. Note the patent capillary lumens (C), the capillary endothelium (En), the alveolar type I pneumocytes (Epl) which form thin delicate sheets, and the type II pneumocytes (Ep2) with characteristic cytoplasmic granules. Fibrocyte (F) (bar = 2 /xm). Inset: detail of the air blood barrier, formed from thin sheets of type I pneumocytes (Epl) and endothelial cells (En) separated by a common basal lamina (bar = 20 nm).
15
Fig. 2. Transmission electron micrograph of the alveolar septum from a guinea-pig exposed to pure 02 for 72 h illustrating the interstitial oedema (*), degenerative changes to the endothelium (En) and type I pneumocytes (Epl). Instead of forming a thin sheet, the outline of the endothelium is irregular and the cytoplasm contains vacuoles. The cytoplasm of the type I pneumocytes appears swollen and rarefied. Note the junctions of both cell types ("~) appear intact (bar = 1 /~m).
prominent nucleoli and increased amounts of r o u g h e n d o p l a s m i c r e t i c u l u m . T h e n e c r o t i c cells were usually swollen with rarefied cytoplasm and few o r g a n e l l e s w h i l e s m a l l e r n u m b e r s a p p e a r e d
shrunken with condensed granular cytoplasm and p y k n o t i c nuclei. D e s p i t e large n u m b e r s of n e c r o t i c e n d o t h e l i a l cells, o n l y the o c c a s i o n a l a r e a of exp o s e d b a s e m e n t m e m b r a n e was o b s e r v e d . Loss of
Fig. 3. Transmission electron micrograph of the alveolar region from a guinea-pig exposed to pure 02 for 96 h. Extensive alveolar oedema (*) and thickening of the alveolar septum is evident (bar = 4 ~m).
16 endothelial cells resulted in the collapse of the capillaries. The degenerative changes in the type I pneumocytes involved the rarefaction of the cytoplasm along with mitochondrial swelling (fig. 2). In advanced stages of degeneration breaks in the plasma membrane were seen with organelles spilling into the alveolar space. Areas of exposed basement membrane were also observed. Interstitial oedema was an early event in the evolution of the lesion, involving the extracellular matrix of the thick region (Pietra, 1984) of the interstitial space (fig. 2). This was followed by the progressive invasion of cells (fibrocytes, PMNs and mononuclear cells) and the deposition of collagen and fibrinoid material (fig. 3). The thin region of the interstitial space, where the basement membranes of the type I pneumocyte and endothelium are fused, appeared unaffected when either of the bordering cells remained viable. The interstitial oedema could not be attributed to any specific lesion of the endothelial cells, no breaks were observed in the cell wall and the tight junctions appeared intact.
3.3. Basal release of eicosanoids
[ ] 6-°x°-PGFIo~ [ ] TXB2 L *p'~O.O01
30
== 2
15
L
20
10
i.:
i
10
t~ 0
0
24 48
72
96
0
24 48
72
96
Time of exposure to 100% 0 2 (h)
Fig. 4. Release of 6-oxo-PGFl, and TXB/ induced by an infusion of bradykinin (250 ng-ml-l) into isolated lungs of guinea-pigs exposed to 100% 02 for various time intervals. The mean concentration_+S.E.M. of 6-oxo-PGFl,, is represented by the hatched bars, and the mean concentration__+S.E.M, of TXB2 is represented by the open bars. The numbers inside the bars indicate the numbers of animals employed.
3.5. Metabolism of eicosanoids
There was no significant difference in the basal release of TXB 2 (control 0.49 + 0.08 n g - m l - l , n = 22; 72-96 h 02 0.62 + 0.09 ng. m1-1, n = 23) and 6-oxo-PGFl~ (less than 0.2 ng. m1-1) in the effluent of control lungs and of lungs of guineapigs exposed to 72 or 96 h 02.
Prostacyclin was poorly removed either by control or inflamed guinea-pig lungs (control 91 + 9%, 96 h 02 93 + 7%, n = 5, values expressed as % of survival). There was no significant difference either in the removal of P G E 2 from the pulmonary circulation of control lungs or lungs of guinea-pigs exposed to 96 h of 02 (control 11 + 5%, 96 h 02 6 _ 3%, n = 5, values expressed as % of survival).
3.4. Bradykinin-induced release of eicosanoids
3.6. Metabolism of bradykinin
There was a significant increase in the amounts of 6-oxo-PGFt~ and TXB 2 released by bradykinin (250 ng. ml - t ) infusions into the lungs from guinea-pigs exposed to 72 and 96 h 02 when compared to the amounts released from control lungs (fig. 4), however, the ratio of eicosanoids was not significantly changed. LTB 4 was not detected (less than 0.05 n g - m 1 - 1 ) in the effluent from either control lungs or from lungs of guineapigs exposed to 02 .
There was an increase in the survival of bradykinin from the lungs of guinea-pigs exposed to 96 h of 02 when compared to control lungs (fig. 5). The survival of bradykinin in control lungs was 6 + 1% (n = 8) and in lungs of guinea-pigs exposed to 96 h of 02 the survival was significantly (P < 0.001) increased to 16 + 3% (n---7). No significant changes were observed at earlier stages (24 h, 9_.+ 2%, n = 7; 48 h, 7 + 1%, n = 4 and 72 h, 6+2%,n=4).
17
CONTROL LUNG
V"
10.T.
0.50.T.
20 T.L.
BK (ng ml "1)
HOURS 0 2 LUNG 96
50.T.
20 T.L.
3,50.T.
BK (ng m1-1 )
Fig. 5. Effect of exposure t o 0 2 (96 h) on the survival of bradykinin by the isolated lungs. The effluent from control lungs (upper panel) and O2-exposed lungs (lower panel) superfuses longitudinal strips of cat jejunum (CJ). In the upper panel the response of the tissue tO an infusion of bradykinin (1 ng. ml-1) is shown followed by the response to 0.5 ng. ml-1 over the tissues (O.T.) and 20 ng-m1-1 through the lung (T.L.). In the lower panel the response of the tissue to 5 ng.m1-1 of bradykinin O.T. is followed by the response to 20 ng. m l - l T.L. and 3.5 ng-ml-1 O.T. The survival of bradykinin in the control lungs is less than 5% while the survival in the exposed lungs is greater than 15%. The same pattern of responses was observed in another 6 experiments.
3. 7. Effect of dexamethasone on eicosanoid release induced by bradykinin Dexamethasone (2 /~g-m1-1, 60 min) did not inhibit release of 6-oxo-PGFl~ induced by brady-
~'~ ]
[ ] Control [ ] Oexamethasone 0.05
30 a. I
20 j
I
0
kinin in normal guinea-pig-isolated lungs even when large concentrations of bradykinin (2.5/~g. ml - 1) were employed (control 24.4 + 1.6 ng. m l - 1; dexamethasone 17.6 + 4.8 ng- m1-1, n = 5). However, it did have an inhibitory effect on the bradykinin-induced release of 6-oxo-PGFl~ from lungs of guinea-pigs exposed to 96 h of 02 (control 36.5 + 6.9 ng- ml-1; dexamethasone 20.2 + 2 ng. ml-1, n = 5, fig. 6). Dexamethasone (2 btg. m1-1, 60 min) did not affect the metabolism of bradykinin in the lungs from animals exposed to 02 for 96 h (not shown), nor did it affect the w e t / d r y weight ratio (control 12.44 _+ 1.78; dexamethasone-treated 13.06 ___0.87, n = 5).
4. Discussion
~ o
Time of
exposure
to 02 (hi
Fig. 6. Effect of dexamethasone (2 ~g. m l - l , 60 min) infusion
on bradykinin-induced release of 6-oxo-PGFl= from isolated lungs of guinea-pigs. The hatched bars represent the lungs treated with dexamethasone for 1 h before the challenge with bradykinin (250 ng-ml- ~). The height of the bars represents the mean value of 5 experiments with 1 S.E.M.
Our results demonstrate that 02 toxicity in guinea-pigs shares some of the histopathological characteristics reported for other species. Interstitial oedema was an early event in the evolution of the lesion and although it could not be attributed to any specific lesion of the endothelial
18 cells, it should be taken as evidence of endothelial cell damage. Klister et al. (1967) studying hyperoxia-induced alterations in rat lungs demonstrated, by morphometric analysis, an enlargement of the interstitial compartment related to the accumulation of oedema fluid, but they also found it difficult to determine the distribution of endothelial lesions among pulmonary vessels of different types. The finding that areas of essentially normal parenchyma could be observed adjacent to areas undergoing extensive changes is not surprising since Bowden et al. (1968), studying hyperoxia in mice, also found that capillary ultrastructural changes were focal and that many capillaries showed no structural alterations. The absence of platelet thrombi in the pulmonary microvasculature has also been reported previously in rabbits (Dobuler et al., 1982). Isolated lungs from guinea-pigs exposed to 02 for 72 and 96 h release increased amounts of 6-oxo-PGF~ and TXB 2 when stimulated with bradykinin. Several mechanisms could be responsible for such an increase: (i) an increase in phospholipase A 2 activity, (ii) a decrease in the metabolism of the eicosanoids, (iii) a decrease in the metabolism of bradykinin or (iv) a combination of all the above factors. The first possibility seems unlikely since the basal levels of eicosanoid released in the Oz-treated lung effluent were not significantly higher than those in control lung effluent. As has been demonstrated in other species (Dusting et al., 1978), prostacyclin is not metabolised during passage through the pulmonary circulation of the guinea-pig. No changes were observed in the removal of exogenous PGE 2 or prostacyclin by the inflamed lungs and therefore the increased release is unlikely to be due to changes in eicosanoid inactivation. We cannot rule out the possibility that the intracellular prostaglandin inactivation system was affected. Prostaglandin inactivation in the lung consists of two phases, transport of prostaglandins into the lung cells and intracellular metabolism by prostaglandin dehydrogenase (Anderson and Eling, 1976), and in this study we have only investigated the former. Chaudhari et al. (1979), however, have suggested that O 2 exposure in guinea-pigs leads to an inhibition of the dehydrogenase enzyme. Exposure to
high tensions of oxygen caused a reduction in the activity of angiotensin converting enzyme (ACE) in rat lungs in vitro (Toivonen et al., 1981) measured as reduced conversion of angiotensin I into angiotensin II. These authors have also reported reduction in the inactivation of bradykinin, although that occurred at a later stage (60 h of exposure). In rabbits Dobuler et al. (1982) observed the same effect. Since ACE is also responsible for the metabolism of bradykinin (Ferreira and Vane, 1967), we investigated whether isolated lungs of guinea-pigs exposed to O 2 would also show decreased ACE activity and consequently increased survival of bradykinin. The lungs of guinea-pigs exposed to 02 for 96 h did exhibit a decreased inactivation of bradykinin, suggesting that this could be the mechanism for the increased eicosanoid release observed in lungs from these animals. The fact that we did not observe any change in the metabolism of bradykinin after shorter periods of exposure to 02, although there was a significant increase in the eicosanoid levels, could be due to the difference in the amounts of substrate used to assess metabolism of bradykinin (20 ng. m1-1) and the amount needed to induce release of eicosanoids (250 ng. ml-1). It might be that at 72 h the system of inactivation of bradykinin is already affected, but it behaves normally at low amounts of substrate. The decrease observed in the metabolism of bradykinin could be the consequence of the damage induced by the 02 exposure in the endothelial cells, since ACE is located at the plasma membranes of the endothelial cells (Ryan et al., 1976). The release of eicosanoids induced by bradykinin from guinea-pig isolated lungs is insensitive to corticosteroid treatment (Blackwell et al., 1978; Robinson and Hoult, 1980). This we have confirmed in normal lungs. However it is interesting that dexamethasone had an inhibitory effect on the bradykinin-induced release of 6-oxo-PGFl~ from the inflamed lungs. Since dexamethasone induces the synthesis of new kinases, such as ACE (Ialenti et al., 1986), it would be possible for the observed effect to be achieved through an enhanced metabolism of bradykinin. However, our experiments show that this is an unlikely mecha-
19
nism since dexamethasone failed to increase bradykinin metabolism in lungs of guinea-pigs exposed to 96 h of 02. This inhibitory action of dexamethasone could therefore be due to bradykinin reaching during the inflammatory process cells whose phospholipases are sensitive to lipocortin (Di Rosa et al., 1984). Those cells could be either migrating cells such as neutrophils or macrophages, or resident cells in t h e lung. We demonstrated the presence of mononuclear cell infiltration in the lungs after 96 h of 02 exposure and it is known that macrophages can release lipocortin (Blackwell et al., 1980). The second possibility is that a different lung cell population, due to damage of the endothelium, is now being stimulated either with bradykinin or dexamethasone. Indeed, we have shown that administration of antigen via the airways in guinea-pig isolated lungs induced a release of vasoactive materials which was different from that induced if the antigen was injected through the pulmonary artery (Bakhle et al., 1985b). We have also shown that the release of lyso-PAF can be induced by stimulating the lungs via the airways with calcium ionophore. However, unless the lungs are inflamed (exposed to 02), this is not possible if the ionophore is injected through the pulmonary artery (Parente et al., 1985). These results suggest that the population of cells which participates in the inflammatory process might be located closer to the alveolar side of the lung and therefore might be reached more easily from that side than from the circulation. Whether the effect of dexamethasone is due to an effect on a population of cells in the lungs which is not easily reached in non-inflamed lungs or to the migration during the inflammatory process of cells which are sensitive to the action of the steroid remains to be investigated.
Acknowledgements
The authors wish to thank Dr. Y.S. Bakhle for discussing the manuscript, Dr. R. White for measuring the 0 2 tension and Mr. A. Kotecha for technical assistance.
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