Published ahead of print on April 2, 2003, doi:10.1164/rccm.200302-159OC
© 2003 American Thoracic Society
Endogenous Nitric Oxide Release by Vasoactive Drugs Monitored in Exhaled AirDepartments of Physiology and Pharmacology, Surgical Sciences, and Thoracic Surgery, Karolinska Institute; and Divisions of Anaesthesiology and Intensive Care and Cardiothoracic Anaesthetics and Intensive Care, Karolinska Hospital, Stockholm, Sweden Correspondence and requests for reprints should be addressed to Eddie Weitzberg, M.D., Ph.D., Department of Surgical Sciences, Karolinska Institute, S-17176 Stockholm, Sweden. E-mail: eddie.weitzberg{at}ks.se
Direct measurements of endogenous nitric oxide (NO) release is of great interest but difficult to perform in vivo. We hypothesized that endogenous NO release from vasoactive substances would be detectable in exhaled air. Exhaled NO was measured after intravenous injections of various endothelium-dependent and endothelium-independent vasoactive drugs, in anesthetized pigs and humans. In pigs, a dose-dependent release of exhaled NO was observed for acetylcholine (ACh), bradykinin, substance P, endothelin (ET)-1, and nitroglycerine. Each compound had an individual and highly reproducible release pattern. Bradykinin-induced NO release was enhanced by angiotensin converting enzyme inhibition. ET receptor antagonism markedly reduced the response in exhaled NO to ET-1, whereas atropin abolished the NO response to ACh. NO synthase inhibition abolished basal levels of exhaled NO as well as the responses in exhaled NO to all compounds except nitroglycerine. In humans, ACh evoked a dose-dependent increase of NO levels in exhaled air. NO release by endogenous vasoactive agonists can be measured online in the exhaled air of pigs and humans. These novel findings may be useful when characterizing NO release from compounds that interfere with NO synthesis or drugs that act as donors of NO. Moreover, the possibility of using exhaled NO as an indicator of pulmonary endothelial dysfunction merits further studies.
Key Words: acetylcholine • endothelin • endothelium • nitric oxide • vasodilation There is a great interest in nitric oxide (NO) biology, but there are difficulties in performing direct in vivo measurements of this pluripotent gas. Most sensors are not sensitive enough and rapid to measure the very small quantities of NO that are transiently released. Moreover, the half-life of free NO in blood is extremely short because of its rapid reaction with proteins, e.g., hemoglobin (1). However, pulmonary NO production represents a unique exception. Because NO is a tiny lipid-soluble molecule that easily crosses biological membranes, this gas can escape into the airway lumen. NO in the gaseous phase is fairly stable, and it is measurable directly by chemiluminescence in exhaled air (2, 3). This allows for direct and noninvasive measurements of NO in different pulmonary disorders affecting the airways and/or the pulmonary vascular endothelium. It has become evident that the vascular endothelium is not merely a passive barrier between the blood and surrounding tissues but in fact a highly active organ possessing many secretory, metabolic, and immunologic functions (4). During the last 2 decades there has been much focus on NO and endothelial function (for review see Reference 5). The capacity of the endothelium to produce NO in response to physiologic or chemical stimulants is crucial for the integrity of this major organ. Many important vasoactive endogenous compounds like prostacyclin, NO, endothelin (ET), angiotensin II, endothelium-derived hyperpolarizing factor, free radicals, and bradykinin (BK) are formed in endothelial cells to control the functions of vascular smooth muscle cells and of circulating blood cells. Many vasodilators, e.g., acetylcholine (ACh) and BK, are known to stimulate endothelial NO release. The pulmonary endothelium represents a very large surface area, the major part of which lies in close contact with the alveoli. Infusion of the NO donor glyceryl trinitrate (GTN) increases exhaled NO both in humans and animal models (6–8). In vitro experiments have shown the possibility of measuring ACh-induced NO release from the lung (9). We hypothesized that endogenous endothelium–dependent vasoactive substances would give rise to a detectable release of NO in exhaled air. In a pig model, we characterized exhaled NO in relation to dosage, vascular responses, and effects of inhibitors. Moreover, using the same technique, we also studied the possibility of monitoring drug-induced endogenous release of NO in humans.
The Ethics Committees for animal and human experiments, Stockholm, Sweden, approved the experimental protocols for this study.
Anesthesia and Surgical Preparation
Measurements of Exhaled NO
Experimental Protocol
Human Study After a stabilization period and measurements of baseline parameters, intravenous injections of ACh (17–210 nmol/kg) and GTN (6 nmol/kg) were given via the proximal port of the pulmonary artery catheter into the superior caval vein. Cardiovascular parameters and exhaled NO were measured at each dose of ACh and GTN, and values were allowed to return to baseline between each injection.
Calculations
Drugs
Vascular Responses in the Pig Basal MAP, Ppa, heart rate (HR), and  were 79 ± 7 mm Hg, 21 ± 1 mm Hg, 95 ± 8 beats/minute, and 3.3 ± 0.2 L/minute, respectively (n = 14). ACh, BK, SP, terbutaline, papaverine, prostacyclin, and GTN caused dose-dependent reduction of PVR and MAP and increase in . Sodium nitroprusside decreased PVR and MAP without significantly altering . ET-1 caused dose-dependent elevation of MAP with only marginal effects on and PVR. Vascular responses observed at the highest dose of each substance are given in Table 1
. Phenylephrine increased PVR and MAP, while lowering , at the single dose given (Table 1).
Captopril (1 mg/kg) lowered MAP (by 22 ± 7%) with only marginal effects on other parameters per se. Captopril markedly enhanced and prolonged the vascular response to BK (1 nmol/kg, MAP reduced by 60 ± 5% vs. control 47 ± 4%, the duration of this response was 430 ± 20 second vs. control 90 ± 10 second, both p < 0.05 vs. control, n = 5). Vascular responses to ACh (15 nmol/kg) and GTN (40 nmol/kg) were not affected by captopril (data not shown). Atropine (0.5 mg/kg) elevated HR and (by 14 ± 5% and 12 ± 5%, respectively) without significant effects on other basal parameters per se. Atropine completely abolished the vascular response to ACh (50 nmol/kg, n = 4) but did not affect the response to BK (3 nmol/kg, e.g., MAP reduced by 62 ± 5% vs. control 67 ± 2%, not significant n = 4) and GTN (40 nmol/kg, data not shown). Tezosentan (3 mg/kg) lowered PVR and MAP (both by 16 ± 5%) and elevated HR and (by 10 ± 3% and 9 ± 2%, respectively) per se. Tezosentan largely abolished the vascular response to ET-1 (150 pmol/kg, n = 4) but did not affect responses to ACh (15 nmol/kg) and BK (3 nmol/kg) (data not shown).
L-NAME (10 mg/kg) elevated PVR and MAP (by 75 ± 16% and 28 ± 7%, respectively) while lowering HR and
Exhaled NO in the Pig There was a dose-dependent increase in exhaled NO by the endothelium-dependent vasodilators ACh (Figure 1) , BK, and SP, as well as by ET-1 (Table 2) . This was also seen with the endothelium-independent substances GTN and sodium nitroprusside (Table 2). In contrast, the vasodilators papaverine, terbutaline, and prostacyclin did not evoke any significant release of NO in exhaled air, as was the case also with the vasoconstrictor phenylephrine (Figure 2 , Table 2). The agents showed a varied profile in their release of NO in exhaled air. ET-1 gave by far the most prolonged release of NO in exhaled air, whereas the response to ACh was more rapid and of quite short duration (Figure 2). The peak exhaled NO levels for the substances are shown in Table 2. On a molar basis, the order of potency in release of NO in exhaled air of the administered endothelium-dependent compounds was ET-1 (3,914) more than SP (527) more than BK (10.4) more than ACh (1). In this context, the potency of GTN was 2.9 (Table 3) . The NO release caused by ACh (15 nmol/kg) in the pig was highly reproducible and did not change during the course of the experiments.
Atropine (0.5 mg/kg, n = 4) abolished the NO response to ACh (50 nmol/kg) (Figure 3) , whereas the NO release on administering BK (3 nmol/kg) and GTN (40 nmol/kg) was unaffected (data not shown). Tezosentan (3 mg/kg) inhibited (by 80 ± 17%, p < 0.05, n = 4) exhaled NO induced by ET-1 (150 pmol/kg) (Figure 3) but did not affect responses to ACh (15 nmol/kg) and BK (3 nmol/kg) (data not shown). Captopril (1 mg/kg) increased (by 75 ± 25%, p < 0.05, n = 5) the NO release detected on injecting BK (1 nmol/kg) (Figure 3) without affecting the NO release on injecting ACh (15 nmol/kg) and GTN (40 nmol/kg) (data not shown). Neither atropine, tezosentan, or captopril had any effect on basal levels of exhaled NO. In contrast, L-NAME (10 mg/kg, n = 5) led to a total loss of detectable NO in exhaled air within 1 minute, and NO release was abolished for all endothelium-dependent vasodilators tested. However, the response to GTN was unchanged after L-NAME (data not shown). N  -nitro-D-arginine methyl ester (10 mg/kg, n = 3) did not affect either basal or ACh-evoked NO release (data not shown).
Human Study Inhaled NO was below 1 ppb, and basal exhaled NO levels were 2.9 ± 0.6 ppb. Baseline exhaled NO was unchanged during the course of the experiments. Basal MAP, HR, , and PVRI were 90 ± 6 mm Hg, 42 ± 2 beats/minute, 3.8 ± 0.4 L/minute, and 350 ± 50 dyne second cm-5 m2. ACh (17–210 nmol/kg, intravenous) injections dose-dependently increased NO in exhaled air (Figures 4 and 5)
. ACh (140 nmol/kg) decreased PVRI by a moderate 28 ± 13% due to small changes in Ppa and pulmonary capillary wedge pressure. No clear dose–response relationship could be observed for ACh on PVRI. MAP, HR, and were not, or only marginally, affected by ACh at any dose given. In one patient, GTN (6 nmol/kg, intravenous) decreased MAP and PVRI by 20 and 13%, respectively, whereas HR was elevated by 19% and was unaffected. In the following patients, because of these negative circulatory effects, an infusion of norepinephrine (50–200 ng/kg/minute, intravenous) was administered simultaneously as GTN was given, thereby precluding relevant measurements of the cardiovascular effects of GTN per se. GTN (6 nmol/kg) elevated levels of exhaled NO by 2.3 ± 0.2 ppb (n = 4).
We show here that intravenous administration of several endothelium-dependent vasoactive substances cause dose-dependent release of exhaled NO in a highly reproducible manner. There was a marked difference between the compounds with regard to the profile and total release of NO. These findings demonstrate the possibility of using exhaled measurements to detect endogenous NO release induced by vasoactive substances. The inhibitory effects of atropine and tezosentan clearly demonstrated receptor-mediated stimulation of NO release for ACh and ET, respectively. Furthermore, the inhibition of L-NAME showed the enzymatic origin of the NO release caused by endothelium-dependent vasodilators ACh, BK, and SP, as well as ET, Moreover, by interfering with the degradation of BK (with captopril), both exhaled NO and vascular responses were enhanced in parallel. It has been very difficult to directly measure the minute amounts of NO transiently released by the endothelium on stimulation with, e.g., ACh. Therefore, one is most often confined to indirect measurements of NO reaction products such as nitrite, nitrate, and S-nitrosothiols. However, such measurements are less sensitive and unable to display the rapid dynamics in NO release. In addition, analysis has to be done ex vivo and is time consuming. Direct measurement of NO in the circulation has been described by Malinski and coworkers using intravascular chemical sensors (10). Although somewhat invasive, this method may be useful in certain scenarios but is to date not applicable in the pulmonary vasculature. The anatomic origin of exhaled NO in this study was not investigated. Several methods based on mathematic two-compartment models of the airways and alveoli have been used in awake humans to try to locate the origin of exhaled NO. They are based on controlled exhalations with plateau NO levels at different flow rates. In humans, NO released from the airway wall is highly flow-dependent (11, 12), whereas NO from the alveolar region is not (13). These techniques are difficult to perform in anesthetized, nonparticipating pigs and humans. Nevertheless, there are some other indications that a major part of the NO measured here is in fact derived from a distal source of the airways (alveolar epithelium) or the pulmonary vascular endothelium. The animals were tracheotomized, which excludes any contribution from the upper airways (14). Also, as mentioned in METHODS, inflation of the pulmonary artery catheter balloon led to an instant increase in exhaled NO. This could have been the effect of a stopped blood flow in a part of the lung leading to diminished scavenging of endothelial or alveolar NO in that pulmonary region. Finally, if the NO release on administering ACh was derived from the airway wall, one would expect that other muscarinic receptor agonists, e.g., metacholine, would also result in increased NO when administered locally in the airways. However, this is not the case as has been shown in bronchial provocation studies in humans (15). Further studies, e.g., using multiple flow techniques, could probably help to better locate the origin of exhaled NO after agonist stimulation. Interestingly, ET-1 was by far the most potent mediator in increasing exhaled NO compared with both endothelium-dependent endogenous vasodilators as well as exogenous NO donors. ET-1 is considered to be one of the most potent endogenous vasoconstrictors in humans acting on at least two different receptor subtypes. Activation of the ETA receptor subtype located on the vascular smooth muscle leads to prolonged vasoconstriction, whereas ETB receptor subtype activation on the vascular endothelium leads to vasodilation through release of NO and/or prostacyclin (16, 17). There are also ETB receptors on the vascular smooth muscle that induce vasoconstriction. Endogenous ET is considered to act in a paracrine fashion on the underlying vascular smooth muscle. However, after bolus injections of exogenous ET, the response is often a short initial vasodilation followed by a prolonged vasoconstriction. Clearance of circulating ET is mediated by the ETB receptors in the lung (16). In the pulmonary vasculature, the overall net effects of ET-1 depend on dose, pre-existing vascular tone, and the distribution of receptor subtypes. In the pigs, ET-1 did not evoke any major pulmonary vascular changes independent of dose because the vasoconstrictory properties of this peptide were effectively counteracted by the concomitant release of NO. However, L-NAME unmasked this ETB receptor–mediated counteraction, and a marked pulmonary vasoconstriction was evident. It is fascinating to note that behind the vascular response to this extremely potent vasoconstrictor, NO is released in amounts greatly outnumbering that of all the pure vasodilators tested here (including GTN). L-NAME abolished the increases in exhaled NO observed at endothelium-dependent vasoactive substances. In contrast, it became evident that L-NAME, with the exception of the ET response, exerted, at most, very marginal inhibition of the vascular responses to the endothelium-dependent vasodilators ACh, BK, and SP. These latter substances, however, were given at rather high doses (high enough to yield measurable levels of NO in exhaled air). The moderate inhibitory effect exerted by L-NAME on the vascular responses to these doses was likely a dose-related phenomenon. Thus, a higher dose of L-NAME significantly inhibited vascular responses to these high doses of ACh (that yielded measurable increases in exhaled NO before L-NAME treatment) as well as abolished vascular responses to lower doses of ACh. Thus, it seems that inhibition of NO-synthase in vivo with L-NAME is not effective enough to completely antagonize stimulated NO production on administering high doses of endothelium-dependent vasodilators. Because L-NAME abolished the exhaled levels of NO on administering these high doses of endothelium-dependent vasodilators, this detectable NO release may represent a small spillover from a much larger release within the tissue, the rest of which is possibly buffered by hemoglobin or inactivated in other ways. Interestingly, L-NAME markedly altered the vascular response to ET. Thus, when NO counteracts vasoconstriction, as for ET, the involvement of NO seems crucial for the outcome of the vascular response. That L-NAME in comparison affected the ET response more than the endothelium-dependent vasodilators may also be related to the doses given. Evidently, ET was a much more potent stimulator of NO release than the other substance studied, and it also exerted quite a moderate vascular response at the dose given, thus becoming more susceptible to NOS inhibition. In agreement with previous in vivo studies, the effects of GTN were slightly augmented after treatment with L-NAME. In a study by Moncada and coworkers, this was explained by a removal of the basal NO-mediated vasodilator tone leading to hypersensitivity to nitrovasodilators at the level of the soluble gunalylyl cyclase (18). Accordingly, the actual release of NO, as measured in exhaled air, to GTN was not affected after L-NAME in the present study. There was an obvious difference between pigs and humans in the response to ACh in this study. First, approximately 10-fold higher doses were needed in humans to give a similar release in exhaled NO and reduction in PVRI. Second, no systemic response (decrease in MAP) was seen in humans, whereas a marked fall in MAP was evident at all the three doses in the pig. This is most likely due to differences in efficacy of the circulating ACh esterases between the two species. Indeed, the plasma esterase activity in the pig seems lower than in many other species (19). Furthermore, plasma esterase activity in pigs increases with age (20), and it should be noted that in the present study pigs at ages between 2 and 3 months were studied. In humans, breakdown of circulating ACh is highly efficient, and the site of injection strongly determinates the effect (21). Moreover, there was no effect on heart rate in the patients, which speaks against any remaining ACh entering the left side of the heart. This is satisfying because ACh reaching the coronary circulation might cause vasoconstriction in patients with coronary artherosclerosis and damaged endothelium (22). Therefore, ACh seems to be a suitable agonist to induce endothelium-dependent, NO-releasing vasodilation in the pulmonary circulation without causing negative effects on cardiac performance and the systemic circulation. Much effort has been put to try to evaluate the function of the vascular endothelium to monitor treatment or predict future cardiovascular disease. Several tests have been proposed such as forearm blood flow response and coronary artery diameter response to ACh and brachial artery flow–mediated dilatory response to postischemic hyperemia (23). Also, the function of the pulmonary vascular endothelium has been studied but with more invasive techniques that involve measurements of pulmonary vasoreactivity to various agonists (24). Clearly NO is important in endothelial dysfunction, but the exact mechanism is yet to be elucidated. A decreased synthesis or an increased breakdown of NO has been suggested. In addition, impaired cellular responses to NO have also been discussed (25). If future studies demonstrate that, e.g., ACh-evoked exhaled NO release truly is derived from the pulmonary vascular endothelium, an exhaled NO test could possibly be used to reveal endothelial dysfunction. Naturally, the nature of the underlying defect in the NO pathway in endothelial dysfunction will determine this. Endothelial dysfunction is present at an early stage in many disorders of the cardiovascular system, including hypertension, cardiac failure, hypercholesterolemia, sepsis, and diabetes (25). However, whether the pulmonary endothelium is also affected in any of these conditions is less certain but of interest because the present methodology is evidently limited to the pulmonary circulation. If ACh-evoked release of exhaled NO reflects endothelial function, it may be of use in disorders affecting the pulmonary circulation. The pulmonary endothelium is clearly affected in acute lung injury, chronic obstructive pulmonary disease, acute respiratory distress syndrome, and sepsis. It will be interesting to study if the increase in exhaled NO evoked by, e.g., ACh is attenuated in such patients with pulmonary endothelial dysfunction. Nitroglycerine and nitroprusside were used in the present study as endothelium-independent NO releasing agents. They are obviously also needed as "positive controls" in situations where altered NO release on administering endothelium-dependent agonists may occur. We conclude that endogenous NO release induced by vasoactive substances can be monitored online in exhaled air of pigs and humans. Furthermore, exhaled NO may be useful to characterize NO release from compounds that interfere with NO synthesis or drugs that act as donors of NO. Once the exact source of agonist-evoked release of NO in exhaled air is determined, the usefulness of this procedure to detect pulmonary endothelial dysfunction can be evaluated.
The authors thank Ms. M. Stensdotter for expert technical assistance.
Supported by the Swedish Medical Research Council (projects 12,585 and 12,586), Harald and Greta Jeanssons Foundation, the Swedish Heart-Lung Foundation and the Swedish Society of Medicine, Karolinska Institutet, and the Vårdal Foundation. Received in original form February 4, 2003; accepted in final form April 1, 2003
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ABSTRACT
METHODS
, FIO2, 0.30; Servo 900 ventilator; Siemens Elema, Umeå, Sweden). The respiratory frequency was set to 16 to 18 breaths/minute, and the 
). For measurement of mean arterial blood pressure (MAP), a catheter was introduced into the abdominal aorta via a femoral artery. A continuous infusion of isotonic saline with glucose (2.5 mg/ml) at a rate of 10 ml/kg/hour was maintained throughout the experiment via a catheter inserted in a femoral vein with its tip in the caval vein. A midline laparatomy was performed and a catheter was placed in the urinary bladder to collect urine. A left-sided thoracotomy was performed, and an ultrasonic flow probe (Transonic Systems, Ithaca, NY) was placed around the pulmonary artery for continuous registration of 
