Inhibition of nitric oxide synthesis augments pulmonary oedema in isolated perfused rabbit lung

A. L. Mundy and K. L. Dorrington*

University Laboratory of Physiology, Parks Road, Oxford OX1 3PT, UK

Accepted for publication: April 10, 2000


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The role of nitric oxide (NO) in precipitating pulmonary oedema in acute lung injury remains unclear. We have investigated the mechanism of involvement of NO in the maintenance of liquid balance in the isolated rabbit lung. Thirty pairs of lungs were perfused with colloid for up to 6 h, during which pulmonary vascular resistance (PVR) and capillary pressure (PCP) were measured frequently, and time to gain 5 g in weight (t5) was recorded. Four protocols with different perfusate additives were studied: (i) none (control, n=11); (ii) 10 mmol NG-nitro-L-arginine methyl ester (L-NAME) (n=6); (iii) 10 mmol L-NAME with 100 µmol lodoxamide, an inhibitor of mast cell degranulation (n=7); (iv) 10 mmol L-NAME with 10 µmol 8-bromo-3',5'-cyclic guanosine monophosphate (8Br-cGMP), an analogue of cGMP that may reduce vascular permeability by relaxing contractile elements in endothelial cells (n=6). Neither PVR nor PCP differed between protocols. L-NAME markedly reduced t5 from 248 (27) min (mean (SEM)) in protocol (i) to 144 (5) min in protocol (ii) (P<0.05). Both lodoxamide (t5=178 (7) min) and 8Br-cGMP (t5=204 (10) min) substantially corrected the effect of L-NAME (P<0.005). Results suggest that maintenance of a low permeability by NO may involve mast cell stabilization and endothelial cell relaxation.

Br J Anaesth 2000; 85: 570–6

Keywords: lung; pharmacology, nitric oxide; rabbit


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Septic shock is associated with a fall in systemic vascular resistance. There is interest in using inhibitors of nitric oxide synthase (NOS) systemically to correct this change, but concern that inhibition of NOS may exacerbate pulmonary hypertension and worsen pulmonary oedema. Hinder and colleagues1 have used an animal model of sepsis to show that nitric oxide (NO) inhalation may protect against pulmonary oedema associated with endotoxaemia plus NOS inhibition. An important issue is whether NO has independent effects upon vascular pressures and microvascular permeability, which separately influence liquid balance across the vascular wall. NO has been shown to participate in the regulation of blood flow in a number of species and vascular beds,26 and is, therefore, capable of changing the microvascular pressure that drives oedema formation. There is, in addition, growing evidence that NO may play a significant role in the maintenance of pulmonary fluid balance independently of its affects on vascular pressures. NO has been seen to protect the lungs from increased permeability during injury in a number of species. Inhaled NO in the isolated perfused rabbit lung reduced pulmonary vascular permeability caused by hydrogen peroxide injury.7 In the rat lung the inhibition of NOS by NG-nitro-L-arginine methyl ester (L-NAME) during hyperoxia potentiated injury, indicating a protective effect of NO.8 Moore and colleagues demonstrated that NO prevented microvascular damage (measured as increases in permeability) after ischaemia-reperfusion injury in the guinea-pig lung.9 There is also evidence that the basal release of NO helps to maintain normal pulmonary vascular fluid balance, while NO released during inflammation or injury increases vascular permeability.1012

The mechanisms by which NO stabilizes microvascular permeability remain unclear. There is evidence for both a direct and an indirect effect of NO on the permeability of the epithelium and endothelium. NO may act directly on intercellular junctions by stimulating increases in 3',5'-cyclic guanosine monophosphate (cGMP) which causes cellular relaxation. This may narrow intercellular junctions and there is evidence to suggest that it reduces permeability.13 Alternatively, or additionally, NO has been shown to have a stabilizing effect on mast cells.14 Mast cells are found at the host–environment interface and their activation results in the release of mediators (e.g. histamine) that have been associated with increased vascular permeability.15

It is unclear whether endogenous NO plays a role in the maintenance of basal pulmonary fluid balance, and if it does, by what mechanism. We used L-NAME, an inhibitor of NOS, to examine whether basal NO release helps to maintain a normal (control) rate of weight gain in the isolated perfused rabbit lung. The potential role of NO as a mast cell stabilizer was investigated with L-NAME and lodoxamide (a mast cell stabilizer). To examine the possible direct effect of NO on cGMP we utilized L-NAME and 8-bromo-cGMP (8Br-cGMP), an analogue of cGMP. Fortunately, in this preparation, changes in the rate of weight gain are observed in the absence of changes in microvascular pressure, thereby eliminating potential effects on weight gain of pressure changes alone.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Lung isolation and perfusion
This study was approved by the UK Home Office (Licence 30/01030). Male New Zealand White rabbits (body weight range 2.5–3.5 kg) were anaesthetized with 4–6 ml h–1 i.v. of a mixture of ketamine (30 mg ml–1; Willows Francis Veterinary, Crawley, West Sussex, UK) and xylazine (6 mg ml–1; Bayer, Bury St Edmunds, Suffolk, UK). A pneumothorax was effected by opening the diaphragm via a laparotomy and a midline thoracotomy was performed. The rabbit was ventilated via a tracheal cannula at 30 b.p.m. with a tidal volume of 15–20 ml and an inspiration:expiration ratio of 1:1. The rabbit was anticoagulated with 1000 u. i.v. heparin and exsanguinated via the carotid artery. The pulmonary artery was then isolated and cannulated and a non-recirculating flow of perfusate (5°C) with a flow of 10–20 ml min–1 was begun. The perfusate contained 137 mmol NaCl, 5.37 mmol KCl, 0.57 mmol KH2PO4 1.67 mmol CaCl2, 0.81 mmol MgSO4, 0.34 mmol Na2HPO4, 1.0 g litre–1 glucose (Hanks Balanced Salts, Sigma, Poole, Dorset, UK), plus 40 g litre–1 dextran and 20 g litre–1 bovine serum albumin. The lungs were excised from the thorax and suspended freely from a force transducer via the trachea (Pioden Controls Ltd, Lectromed, Letchworth, Hertfordshire, UK). Between 800 and 1000 ml of perfusate were allowed to flow through the lungs to wash out red blood cells. The left atrium was then cannulated via an incision in the left ventricle, and the perfusate recirculated.

The perfusion rate was slowly increased to 122–125 ml min–1, the temperature was brought up to 38–40°C, the tidal volume was increased to 10 ml min–1 kg body weight–1 and the ventilation gas was changed from 100 to 21% oxygen, 5% carbon dioxide, balance N2 (euoxic gas). The lungs were ventilated with a positive end-expiratory pressure of 2.0 mm Hg and a valve was inserted in the ventilator circuit to prevent inspiratory pressures from rising above 18 mm Hg. The pH of the perfusate was adjusted to the range of 7.37–7.42 with NaHCO3. The left atrial pressure was set at 2.0–3.0 mm Hg (referred to zero pressure at the hilum of the lung) to ensure zone III conditions through much of the lung at end-expiration. The perfusate flowed through a gas exchanger prior to entry into the lung in order to maintain gas tension in the perfusate entering the lung within 4.7±3.6 mm Hg of that exiting the lung. Perfusate pH, PO2 and PCO2 were measured every 15 min in perfusate samples taken from the pulmonary arterial cannula (Instrumentation Laboratory 1306 Blood Gas Analyzer, Warrington, Cheshire, UK). Pulmonary arterial, left atrial, and airway pressures were measured via small-bore tubing threaded into the perfusion cannulae and the endotracheal tube, and connected to pressure transducers. Pulmonary vascular resistance (PVR) was calculated by dividing the difference between pulmonary arterial pressure and left atrial pressure by perfusate flow. Capillary pressures were measured using the double-vascular occlusion technique of Dawson and colleagues.16 With this method, in-flow to the lung and out-flow from the lung are occluded simultaneously (the perfusate continues to flow around a bypass circuit), and the resulting equilibrium pressure in the lung at end-expiration is taken to be equal to the pulmonary capillary pressure. The perfusate was exchanged hourly. Experiments were terminated at 300 min or earlier if complete alveolar and airway oedema was present at end expiration, as indicated by a trachea full of liquid.

Protocol (i): controls (n=11)
Lungs were perfused for 300 min and ventilated with euoxic gas in order to establish the stability of the preparation.

Protocol (ii): L-NAME exposure (n=6)
As in protocol (i) but with 10 mmol L-NAME added into the perfusion circuit from 15 min.

Protocol (iii): L-NAME and lodoxamide exposure (n=6)
As in protocol (i) but with 10 mmol L-NAME and 10 µM lodoxamide added simultaneously from 15 min.

Protocol (iv): L-NAME and 8Br-cGMP (n=6)
As in protocol (i) but with 10 mmol L-NAME and 10 µmol 8Br-cGMP added simultaneously from 15 min.

Statistical analysis
Analysis of variance was used (SPSS for Windows, version 7.5) to compare the hourly rates of weight gain between the four groups. The pulmonary vascular resistance, capillary pressures, pH, PO2 and PCO2 were compared using analysis of variance with the Bonferoni correction.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Control of perfusate gas composition and pH
Table 1 shows the perfusate PO2, PCO2 and pH for the duration of the experiment averaged from measurements taken every 15 min during each of the four protocols. Analysis of variance showed no significant differences between the four conditions.


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Table 1 Perfusate PO2 and PCO2 (mm Hg), perfusate pH, and pulmonary vascular resistance (PVR, mm Hg ml–1 min–1) averaged for the duration of the experiments from measurements every 15 min; also the time (t5, min) from the beginning of the experiment to a cumulative weight gain of 5 g. Values are mean (SEM). * Denotes a significant difference from control (protocol (i)) (P<0.009). {dagger} Denotes a significant difference from L-NAME treated lungs (protocol (ii)) (P<0.003). {ddagger} Denotes a significant difference from L -NAME and lodoxamide treated lungs (protocol (iii)) (P<0.045)
 
Pulmonary vascular resistance
Table 1 shows the pulmonary vascular resistance averaged for the duration of each experiment, for the four groups. Analysis of variance showed no significant differences between the four conditions. Only one of the L-NAME-treated lungs (protocols (ii)–(iv)) completed the 300 min protocol; the other experiments were terminated prior to 300 min due to complete alveolar and airway oedema.

Capillary pressure
Figure 1 shows capillary pressure for each of the four protocols. Hourly mean values for each of the experimental protocols are given in Table 2. Analysis of variance showed no significant differences between the four conditions.



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Fig 1 Capillary pressures measured by vascular occlusion at 15-min intervals in the four groups of isolated perfused rabbit lungs. Error bars represent SEM.

 

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Table 2 Capillary pressures averaged from measurements taken every 15 min via the double occlusion method (mm Hg). Values are mean (SEM). * Denotes a significant difference from protocol (i) (P<0.03)
 
Weight gain
Figure 2 shows weight gain for the four protocols. Figure 2A compares protocols (i) and (ii); Figure 2B compares protocols (ii) and (iii); Figure 2C compares protocols (ii) and (iv); and Figure 2D compares protocols (iii) and (iv).



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Fig 2 Weight gain in four groups of isolated rabbit lungs perfused at constant flow. (A) Control (n=11) and 10 mmol L-NAME; (B) 10 mmol L-NAME and 10 mmol L-NAME with 10 µmol lodoxamide (n=7); (C) 10 mmol L-NAME and 10 mmol L-NAME with 10 µmol 8Br-cGMP (n=6); (D) 10 mmol L-NAME with 10 µmol lodoxamide and 10 mmol L-NAME with 10 µmol 8Br-cGMP. Symbols above or below data indicate drop-out of experiments from a given group; {dagger}, one experiment, {ddagger}, two experiments. Error bars represent SEM.

 
The addition of 10 mmol L-NAME into the perfusion circuit caused a dramatic and significant increase in the rate of weight gain in the third and fourth hour of the experiment (P<0.016 and P<0.002, respectively) as compared with control lungs; none of the L-NAME treated lungs survived into the fifth hour of the experiment due to oedema.

The addition of 10 mmol L-NAME significantly shortened the time it took for the isolated lungs to gain 5 g (see Table 1) (P<0.009) as compared with control lungs. The addition of 10 µmol lodoxamide with 10 mmol L-NAME (protocol (iii)) significantly delayed the time it took to increase lung weight by 5 g (P<0.003) as compared with lungs treated with L-NAME alone. The addition of 10 µmol 8Br-cGMP to L-NAME treated lungs (protocol (iv)) appeared to slow the time to 5 g even more effectively than the addition of lodoxamide (P<0.0005). Lungs treated with L-NAME and 8Br-cGMP took significantly less time to gain 5 g than did lungs treated with L-NAME and lodoxamide (P<0.045). Although lungs treated with L-NAME and lodoxamide reached 5 g significantly more quickly than control lungs (P<0.043), those treated with L-NAME and 8Br-cGMP were not significantly different from control. These data show 8Br-cGMP to delay the onset of L-NAME-induced weight gain more effectively than lodoxamide at the doses used.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
This study indicates that inhibition of NO with L-NAME dramatically enhances weight gain in isolated perfused rabbit lungs. This effect is not due to hydrostatic pulmonary oedema, as the inhibition of NO does not raise pulmonary vascular resistance, and more importantly, it does not change the capillary pressures from those found in control lungs. This study thus suggests a protective role for NO in the maintenance of normal pulmonary vascular permeability and further raises the possibility that NO has both a direct affect on vascular endothelial cells via cGMP as well an indirect effect of stabilizing mast cells.

The inhibition of NO with 10-mmol L-NAME did not change pulmonary vascular resistance significantly in this study. There is conflicting evidence in the literature as to the importance of endogenously released NO in maintaining a normal PVR. Inhibition of NO in isolated buffer-perfused rabbit-lungs as well as in open-chest rabbits raised the pulmonary arterial pressure.17 18 L-NAME also caused increased PVR in the lungs of pigs, sheep, and humans.19 However, there is evidence that the inhibition of NO does not affect pulmonary vascular tone in euoxic lungs. Lindeborg and colleagues found that L-NAME did not alter baseline pulmonary pressure in either blood or buffer perfused rabbit lungs.20 Neither L-NAME nor L-NMMA affected baseline euoxic pulmonary arterial pressure in isolated blood perfused rat lungs.21 Our study indicates that the baseline pulmonary vascular pressures seen do not result from production of NO. It may be that the disparate findings amongst the studies are due to species and preparation differences. Indeed a number of studies have advocated the importance of the red blood cell for the action of NO;22 23 as we did not use erythrocytes in our preparation it could be that the lack of response to L-NAME was due to their absence.

An important finding of our study was that capillary pressures were not affected by the addition of 10 mmol L-NAME into the perfusion circuit. There is some disagreement as to the effects of NO on pulmonary capillary pressure. Some studies indicate that capillary pressure is lowered by NO while others find that NO has little or no effect. It has been shown in the isolated, perfused rabbit lung that inhaled NO, only affects the pre-capillary segment of the vasculature without significantly changing capillary pressure. Inhibition of NO production with L-NAME did not significantly affect capillary pressures as measured with double occlusion.20 24 Benzing and colleagues saw that inhaled NO lowers capillary pressure in patients with adult respiratory distress syndrome.25 L-NAME administration in thoroughbred horses caused a significant rise in pulmonary capillary pressures.26 It has been suggested that NO serves a potential protective role in pulmonary liquid balance in that it acts to lower microvascular pressures, thereby reducing fluid filtration.27 In our study, however, capillary pressures were unaffected by L-NAME, while the isolated lungs showed a dramatic increase in rate of weight gain. This implies that the increased weight gain seen in our experiments is not due to increased microvascular pressures causing hydrostatic oedema.

A mechanism by which NO may help to maintain normal pulmonary vascular permeability is by a direct action on endothelial cells. It has been known for some time that endothelial cells have the ability to contract.28 They have been shown to possess actin and myosin, which play a central role in regulating the width of intercellular clefts.29 Hence, the contraction of endothelial cells could increase vascular permeability. Calcium has been demonstrated to play a role in vascular integrity in the lung. In 1971, Nicolaysen30 found that reducing intravascular calcium in perfused rabbit lungs resulted in pulmonary oedema. This role of calcium in endothelial cell contraction has been investigated more closely in recent years. In porcine pulmonary arterial and human umbilical vein endothelial cells it was shown that the formation of intercellular gaps was dependent on a rise in intracellular calcium and that the chelation of extracellular calcium caused cellular retraction.28 29 Stimulation of calcium influx causes endothelial cell contraction and this is prevented by the elevation of cGMP (via 8Br-cGMP, atrial natriuretic factor or sodium nitroprusside).31 NO has been shown to raise cGMP in endothelial cells by stimulating guanylate cyclase,32 and NO has been found to decrease permeability in monolayers of bovine aortic endothelial cells.13 The inhibition of NO synthesis by L-NAME also caused an increase in permeability in thrombin-stimulated human aortic and umbilical vein endothelial cells.31 Hence, NO stimulates production of cGMP which, in turn, suppresses the elevation of intracellular calcium via cGMP-dependent protein kinase and elevates cAMP, which helps to regulate permeability.31 In our study we found that the inhibition of NO production with L-NAME caused a significant increase in the rate of weight gain and that the addition of 8Br-cGMP attenuated this increase. In light of the previous research our findings may be interpreted as demonstrating a role for endogenous NO in the relaxation of endothelial cells; this relaxation is related to the elevation of cGMP levels.

NO may also be acting indirectly to help prevent pulmonary oedema by stabilizing mast cells. We have used lodoxamide to investigate this possibility. In isolated rat peritoneal mast cells33 and human lung mast cells34 lodoxamide has been shown to inhibit degranulation, as measured by release of histamine and prostaglandin D2. Other studies have used mast cell protease activity14 or tryptase release35 to demonstrate inhibition of mast cells by lodoxamide. Other actions of lodoxamide in the lung that may contribute to its anti-inflammatory behaviour include inhibition of xanthine oxidase36 and prevention of neutrophil accumulation.37 Of considerable clinical interest, in the context of lung transplantation, has been the finding that lodoxamide reduces lung injury after ischaemia and reperfusion38 and after re-expansion from a state of atelectasis.37 The details of the mechanism of action of lodoxamide in preventing lung injury in these experiments remain uncertain.

There is evidence that NO stabilizes mast cells in the gut of various animal preparations. In the rat jejunum,14 the feline small ileum,39 and the rat duodenum40 41 inhibition of NO synthesis caused a dramatic increase in permeability. In the rat jejunum, this increase was attenuated by the addition of either of two mast cell stabilizers (lodoxamide or doxantrazole), and identified as having a rapid component due to mast cell release of platelet activating factor, and a slower component due to mast cell release of histamine.14 It has also been found that constitutive NO may play a role in mucosal protection in the jejunum.42 43 Salvemini and co-workers have found that NO has an inhibitory effect on mast cells. 44 In our study we found that lodoxamide attenuates the increase in weight gain seen in isolated lungs exposed to L-NAME. This finding can be interpreted, in light of the cited evidence, to show that endogenous NO may stabilize mast cells in the isolated perfused rabbit lung. However, the beneficial effect of lodoxamide would also be consistent with its ability to inhibit xanthine oxidase, and thereby reduce oxidative damage to the lung. It would have been preferable in our own study to have performed additional experiments to identify whether mast cell degranulation had been inhibited by lodoxamide.

In conclusion, our study found that L-NAME caused a significant increase in rate of weight gain in the isolated perfused rabbit lung. This increase was attenuated by both lodoxamide and 8Br-cGMP, although 8Br-cGMP was slightly more effective at the dose used. All of these effects were independent of changes in pulmonary vascular resistance and capillary pressure. Future work could usefully examine the possibility that lodoxamide and 8Br-cGMP act synergistically. Indeed, Kimura and colleagues hypothesized that a reduction of cGMP could result in the degranulation of mast cells,45 and it is known that mast cells themselves contain a constitutive calcium–calmodulin-dependent isoform of NOS.46 Hence it may be that L-NAME in the isolated rabbit lung is reducing cGMP in endothelial cells and mast cells effecting both a constriction of the endothelial cell and a degranulation of the mast cell.


    Acknowledgements
 
We thank Mr David O’Connor for technical assistance. We thank the Upjohn Company (Kalamazoo, Michigan, USA) for supplying lodoxamide tromethamine. This work was supported by a grant from the Herbert Dunhill Medical Trust.


    Footnotes
 
* Corresponding author Back


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 Abstract
 Introduction
 Methods
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 Discussion
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