Abstract
In the lung, nitric oxide synthase (NOS) has been found in both alveolar epithelial and vascular endothelial cells. Nitric oxide (NO) in the exhaled air stemming from the lower respiratory tract has been claimed to represent a marker of the vascular endothelial NO production. Experimental evidence for this concept, however, is lacking. We compared, in eight healthy volunteers, effects on exhaled NO of epithelial NOS inhibition by N G-monomethyl-l-arginine (l-NMMA) inhalation (6 mg/kg over 15 min) with those of endothelial NOS inhibition by l-NMMA infusion (25 μ g/kg/min for 30 min). We also measured blood pressure, heart rate, and l-NMMA plasma concentration. The major new findings were that l-NMMA inhalation which did not have any detectable effect on hemodynamics and l-NMMA plasma concentration, decreased the pulmonary exhaled NO by almost 40%. In contrast, l-NMMA infusion that inhibited endothelial NOS, as evidenced by an increase in blood pressure and a decrease in heart rate, had only a barely detectable effect on exhaled NO ( − 11 ± 4% from baseline). Pulmonary exhaled NO is mostly of epithelial rather than endothelial origin, and does not provide a marker for vascular endothelial NO production and/or endothelial function in healthy humans.
Over the past few years, nitric oxide (NO) has emerged as a major regulator mechanism of the cardiovascular system (1). The measurement of the activity of the vascular endothelial nitric oxide synthase (NOS) and its final product NO in vivo still represents a major challenge, because the small amount of NO produced by the endothelium is rapidly captured by hemoglobin and then inactivated. So far, the contribution of NO to a particular process was assessed by indirect methods, examining specific physiological responses to NOS inhibitors and NO donors, and/or measuring NO metabolites or cyclic guanosine monophosphate (cGMP) (2).
Recently, NO has been found in the exhaled air of many animal species and humans (3), but its origin and physiological function are incompletely understood. In the respiratory tract, NOS is found in several cell types, including respiratory epithelial and vascular endothelial cells, macrophages, bacteria, and neuronal synapses (4). Virtually all of these cells may contribute to the exhaled NO. It is now well established that an important part of the exhaled NO is produced by the epithelial cells of the upper respiratory tract, located mainly in the nasal and paranasal cavities (5, 6). The lower respiratory tract, however, also contributes to the exhaled NO, because in tracheotomized patients breathing purified air, the analysis of the exhaled air reveals small amounts of NO (5, 7). The origin of the lower respiratory tract exhaled NO is unknown. It has been postulated that, despite its great affinity for hemoglobin, the NO produced by the vascular endothelial cells diffuses into the alveolar space and makes up an important part of the lower respiratory tract NO. Even though experimental evidence for this concept is lacking, the lower respiratory tract has been used as a marker of endothelial NO production and vascular endothelial function (8). To provide such evidence, we compared effects of systemic (intravenous infusion), and local (inhalation) inhibition of NO synthesis by N G-monomethyl-l-arginine (l-NMMA) administration on the lower and upper respiratory tract exhaled NO in healthy subjects.
We studied eight healthy male subjects (mean [± SD] age 26 ± 1 yr). All the subjects were normotensive, were taking no medications, and had no evidence of cardiopulmonary, or allergic disease at the time of the study. Two of the subjects were light smokers (< 10 cigarettes/ day). The experimental protocol was approved by the Institutional Review Board on Human Investigation and all subjects provided written informed consent.
The subjects were studied in the semisupine position. Blood pressure and heart rate (Finapres blood pressure monitor; Ohmeda, Englewood, CO) were recorded continuously on a personal computer. An intravenous catheter was inserted in an antecubital vein for drug infusion and blood sampling.
NO from the lower respiratory tract was measured in the mixed orally exhaled gas during spontaneous oral tidal breathing using chemiluminescence analysis. The rise time of the analyzer (TR 780; ECO Physics, Dürnten, Switzerland) was 0.3 s, and the detection limit was 0.05 part per billion (ppb) during an integration time of 1 min. Admixing of nasally released NO into the respiratory gas was prevented by a continuous suction (flow rate of 1.3 L/min) which was directed out of the nose via a tightly fitting nasal mask (9). Nasal NO was measured in the aspirated gas. Before inhalation, air was purified from NO (NO concentration less than 0.2 ppb) by a zero air generator (PAG003; ECO Physics). Respiratory gas flow was determined using a pneumotachograph (PT36; Jäger, Würzburg, Germany). Measured values of nitric oxide concentration and respiratory flow rate were recorded on line on a data acquisition system.
l-NMMA plasma concentration was measured with an automated amino acid analyzer (Beckman Instruments, Fullerton, CA).
l-NMMA, d-, and l-arginine were obtained from Clinalfa (Läufelfingen, Switzerland), and were dissolved in normal saline immediately before use. The doses of l-NMMA infusion used in these studies had been shown previously to increase arterial pressure and decrease heart rate in healthy humans (10-12), whereas those used for inhalation had been reported to have no hemodynamic effects (13).
Protocols 1 and 2 were performed in random order on two different days separated by at least 24 h.
Protocol 1: Effects of systemic inhibition of the endothelial NOS (l-NMMA infusion) on exhaled NO. After at least 15 min of rest, baseline values for exhaled NO, arterial pressure, and heart rate were obtained. Thereafter, a continuous infusion of low-dose l-NMMA (25 μg/kg/min for 30 min) was started. To examine whether the effects of l-NMMA were related specifically to NOS inhibition, d-arginine (50 mg/kg for 10 min), followed by l-arginine (50 mg/kg for 10 min) was infused. Hemodynamic and exhaled NO measurements were obtained during the last 5 min of l-NMMA infusion, and 5 min after the end of the d-, and the l-arginine infusion, respectively.
Protocol 2: Effects of selective inhibition of the epithelial NOS (l-NMMA inhalation) on exhaled NO. After baseline measurements were completed, l-NMMA (6 mg/kg dissolved in 2 ml of normal saline) was inhaled over 15 min. Hemodynamic values and exhaled NO were measured immediately after the end of the inhalation (13).
Protocol 3: Effects of high-dose l-NMMA infusion and inhalation on exhaled NO. The aim of this protocol was to examine the effects of larger doses of l-NMMA infusion and inhalation on exhaled NO. Four of the subjects returned on two additional occasions for this protocol. The protocols were identical to Protocols 1 and 2, except that larger doses of l-NMMA were infused (50 μg/kg/min for 30 min, followed by 100 μg/kg/min for another 30 min), and inhaled (12 mg/kg over 15 min). The l-NMMA infusion was followed by sequential 10-min infusions of d- (50 mg/kg), and l-arginine (50 mg/kg).
The values of NO concentration and respiratory flow rate were averaged over a time interval of 1 min. Pulmonary and nasal NO excretion rates were calculated as the product of the corresponding NO concentration with the respiratory, or suction flow rate, respectively. The measurements of heart rate and blood pressure that were collected over 5-min periods were averaged to a single value. The statistical analysis was performed with an analysis of variance for repeated measures and paired two-tailed t tests. Unless stated otherwise, data are given as mean ± SE. A p value of less than 0.05 was considered to indicate statistical significance.
Measured baseline NO concentration was 4.7 ± 0.6 ppb in the orally expired gas, and 294 ± 35 ppb in the gas aspirated from the nose.
l-NMMA infusion which increased the l-NMMA plasma concentration from 0 to 7.8 ± 2.5 μmol/L, and inhibited the endothelial NO synthesis, as evidenced by an increase of the mean arterial pressure by 6 ± 2 mm Hg (p < 0.05), and a decrease of the heart rate by 5 ± 2 beats/min, had only a barely detectable, albeit statistically significant, effect on the pulmonary exhaled NO (Figure 1). Pulmonary exhaled NO decreased by roughly 10% from 40 ± 5 to 36 ± 5 nl/min. The relative magnitude of this decrease was very similar to the one of the nasal NO, which decreased from 341 ± 43 to 305 ± 37 nl/min (Figure 2).
Fig. 1. Effects of l-NMMA infusion (25 μ/kg/min for 30 min), and inhalation (6 mg/kg over 10 min), in eight subjects on mean arterial pressure, l-NMMA plasma concentration, and pulmonary exhaled NO. l-NMMA infusion, but not inhalation, had significant effects on hemodynamics and l-NMMA plasma concentration. l-NMMA inhalation, however, had a roughly 3 times larger effect on pulmonary exhaled NO than infusion. *p < 0.05, **p < 0.01, versus corresponding baseline; §p < 0.05, l-NMMA infusion versus inhalation.
[More] [Minimize]
Fig. 2. Effects of l-NMMA infusion (25 μ/kg/min for 30 min) in eight subjects on pulmonary and nasal exhaled NO. l-NMMA infusion had comparable effects on the pulmonary and nasal (produced almost exclusively by epithelial cells) exhaled NO.
[More] [Minimize]The effects of l-NMMA infusion on exhaled NO were reversed by l-, but not by d-arginine infusion. Compared with baseline values, the relative changes at the end of the l-arginine infusion for the exhaled pulmonary and nasal NO were −0.9 ± 4.2%, and −0.9 ± 3.4%, respectively.
l-NMMA inhalation had no detectable hemodynamic effect (mean arterial pressure remained unchanged at 88 ± 4 mm Hg, and heart rate was 61 ± 3 and 60 ± 3 beats/min, before and during l-NMMA inhalation, respectively), and did not result in measurable l-NMMA plasma concentration at the end of the inhalation. Inhalation had, however, a more than 3 times larger effect on the pulmonary exhaled NO than systemic l-NMMA infusion (p < 0.01, inhalation versus infusion, Figure 1), and decreased the exhaled NO from 38 ± 8 to 24 ± 5 nl/min.
Inhalation of the higher dose of l-NMMA had no detectable systemic hemodynamic effects, and did not result in detectable l-NMMA plasma levels. High-dose inhalation decreased the exhaled pulmonary and nasal NO similarly to low-dose inhalation, from 26 ± 5 to 15 ± 3, and from 241 ± 22 to 199 ± 7 nl/ min, respectively. Pulmonary exhaled NO decreased by 41 ± 3% during the high-dose, and by 37 ± 3% during the low-dose l-NMMA inhalation; the nasal exhaled NO decreased by 19 ± 8% during the high-dose, and by 12 ± 5% during the low-dose l-NMMA inhalation (p > 0.1, low- versus high-dose inhalation, for both pulmonary and nasal exhaled NO).
High-dose l-NMMA infusion, as expected had more pronounced effects on hemodynamics (at the end of the infusion mean arterial pressure had increased by 13 ± 2 mm Hg, and heart rate had decreased by 7 ± 2 beats/min), and on l-NMMA plasma concentration (which increased to 16.0 ± 1.6 μmol/L, p = 0.02, versus low-dose infusion) than low-dose infusion. It also caused a roughly 4.5 times larger (p < 0.05) decrease of both the nasal (−39 ± 3 versus −9 ± 2%) and the pulmonary exhaled NO (−52 ± 5 versus −11 ± 4%) than low-dose l-NMMA infusion. During high-dose l-NMMA infusion, the nasal and the pulmonary exhaled NO decreased from 249 ± 45 to 150 ± 26 nl/min, and from 33 ± 7 to 16 ± 5 nl/min, respectively.
To examine the respective contribution of respiratory epithelial and vascular endothelial cells to the lower respiratory tract exhaled NO, we separately measured upper and lower respiratory tract exhaled NO during pharmacological interventions designed to inhibit selectively either the respiratory epithelial, or the vascular endothelial NO production. We found that l-NMMA inhalation at a dose that did not have any detectable hemodynamic effect, decreased the pulmonary (lower respiratory tract) exhaled NO by roughly 40%, whereas systemic l-NMMA infusion which increased arterial pressure, had only a barely detectable effect. These findings provide the first evidence that epithelial, rather than endothelial NO makes up for most of the pulmonary exhaled NO, and indicate that, in contrast to what has been suggested (8), exhaled NO does not provide a marker of vascular NO production, and/or vascular endothelial dysfunction in healthy humans.
This interpretation is predicated on the assumption that the NO measured in the orally expired gas was pulmonary in origin, and that the pharmacological interventions selectively inhibited either the epithelial or the endothelial NOS. The successful separation of pulmonary and nasal NO in the present study is evidenced by the observation that the NO concentration in the orally expired gas obtained during nasal suction was in the same range as the one reported in endotracheal intubated patients (5, 14, 15). Consistent with this interpretation, nasal suction at a constant high flow rate has been shown to remove all the NO containing air, regardless of the volume of the nasal cavity (16). l-NMMA inhalation at both doses used in the present studies did not reach the systemic circulation, as indicated by the lack of detectable l-NMMA in the plasma and/or hemodynamic effects. On the other hand, when infused at the lower dose, l-NMMA inhibited the endothelial NO synthesis, as evidenced by the increase in blood pressure and the decrease in heart rate. This effect was related specifically to NOS inhibition, because it was reversed by l-arginine (but not by d-arginine) infusion.
There is a general agreement that in healthy subjects, the upper respiratory tract exhaled NO almost exclusively originates from the respiratory epithelial cells located in the nose and in the paranasal sinuses (5, 6). When infused at the higher dose, l-NMMA, as expected, had larger hemodynamic effects, and caused a comparable additional decrease in both the nasal and the pulmonary exhaled NO. This observation suggests that when infused at a higher dose, l-NMMA loses its specificity for inhibiting the endothelial NOS, and exerts inhibitory effects on both the endothelial and the epithelial NOS. The observation that when infused at the lower dose, l-NMMA had small, but statistically significant, effects on both the nasal and the pulmonary exhaled NO, could be consistent with the hypothesis that even at this low dose, l-NMMA was not completely specific and exerted effects on the epithelial NOS. Alternatively, it is possible that a barely detectable fraction of both the nasal and the pulmonary exhaled NO originates from the vasculature. Taken together, these observations support the concept that the endothelial NOS barely, if at all, contributes to the exhaled NO in healthy humans.
It has been postulated that, despite its great affinity for hemoglobin, the NO produced by the vascular endothelial cells diffuses into the alveolar space and makes up an important part of the lower respiratory tract NO (8). Based on this concept, augmented concentrations of exhaled NO found in patients with hepatic cirrhosis have been used to indicate augmented vascular endothelial NO production (17-19), whereas decreased concentrations in patients with pulmonary (20), and systemic (21) arterial hypertension, and mitral valve disease (22), have been used to indicate endothelial dysfunction. Although further studies are needed to determine the exact cause for altered exhaled NO in these disorders, our findings in normal subjects suggest that such extrapolations may not be valid. In line with this hypothesis, in patients with sepsis and acute respiratory distress syndrome (ARDS), conditions thought to be associated with augmented endothelial NO synthesis, exhaled NO was reported to be lower than in control subjects (21, 23).
The present data should not be used, however, to indicate that there exists no relationship between exhaled NO and the regulation of vascular tone. It is indeed conceivable that the NO synthesized in the respiratory tract diffuses into the vasculature where it may exert hemodynamic effects. A recent study in humans, showing a lower pulmonary vascular resistance during nasal breathing (high endogenous NO content) than during oral breathing (low endogenous NO content) has provided experimental evidence consistent with this concept (24).
The authors are indebted to Dr. Erik R. Swenson and Markus Schuster for help with some of the studies.
Supported by grants from the Swiss National Science Foundation (32.46797.96, 3238-051157.97), the International Olympic Committee, the Placide Nicod Foundation, and the Deutsche Forschungsgemeinschaft (Ba 1368, Fa 139 /4-1/2).
| 1. | Palmer R. M. J., Ferrige A. G., Moncada S.Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature3271987524526 |
| 2. | Vallance P., Patton S., Bhagat K., MacAllister R., Radomski M., Moncada S., Malinski T.Direct measurement of nitric oxide in human beings. Lancet3451995153154 |
| 3. | Gustafsson L. E., Leone A. M., Persson M. G., Wiklund N. P., Moncada S.Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs, and humans. Biochem. Biophys. Res. Commun.1811991852857 |
| 4. | Barnes P. J.Nitric oxide and airways. Eur. Respir. J.61993163165 |
| 5. | Gerlach H., Rossaint R., Pappert D., Knorr M., Falke K. J.Autoinhalation of nitric oxide after endogenous synthesis in nasopharynx. Lancet3431994518519 |
| 6. | Lundberg J. N., Weitzberg E., Nordvall S. L., Kuylenstierna R., Lundberg J. M., Alving K.Primarily nasal origin of exhaled nitric oxide and absence in Kartagener's syndrome. Eur. Respir. J.8199415011504 |
| 7. | Schedin U., Frostell C., Persson M. G., Jakobsson J., Andersson G., Gustafsson L. E.Contribution from upper and lower airways to exhaled endogenous nitric oxide in humans. Acta Anaesthesiol. Scand.391995327332 |
| 8. | Borland C., Cox Y., Higenbottam T.Measurement of exhaled nitric oxide in man. Thorax48199311601162 |
| 9. | Busch T., Knorr M., Kuhlen R., Gerlach H., Rossaint R., Falke K. J.Exhaled nitric oxide in smokers and non-smokers (abstract). Am. J. Respir. Crit. Care Med.1531996A102 |
| 10. | Haynes W., Noon J., Walker B., Webb D.Inhibition of nitric oxide synthesis increases blood pressure in healthy humans. J. Hypertens.11199313751380 |
| 11. | Owlya R., Vollenweider L., Trueb L., Sartori C., Lepori M., Nicod P., Scherrer U.Cardiovascular and sympathetic effects of nitric oxide inhibition at rest and during static exercise in humans. Circulation96199738973903 |
| 12. | Lepori M., Sartori C., Trueb L., Owlya R., Nicod P., Scherrer U.Haemodynamic and sympathetic effects of inhibition of nitric oxide synthase by systemic infusion of NG-monomethyl-L-arginine into humans are dose dependent. J. Hypertens.161998519523 |
| 13. | Yates D. H., Kharitonov S. A., Robbins R. A., Thomas P. S., Barnes P. J.Effect of a nitric oxide synthase inhibitor and a glucocorticosteroid on exhaled nitric oxide. Am. J. Respir. Crit. Care Med.1521995892896 |
| 14. | Dillon W. C., Hampl V., Schultz P. J., Rubins J. B., Archer S. L.Origins of breath nitric oxide in humans. Chest1101996930938 |
| 15. | Kimberly B., Nejadnik B., Giraud G. D., Holden W. E.Nasal contribution to exhaled nitric oxide at rest and during breathholding in humans. Am. J. Respir. Crit. Care Med.1531996829836 |
| 16. | Lundberg J. O. N., Rinder J., Weitzberg E., Alving K., Lundberg J. M.Heavy physical exercise decreases nitric oxide levels in the nasal airways in humans. Acta Physiol. Scand.15919975157 |
| 17. | Sogni P., Garnier P., Gadano A., Moreau R., Dall'Ava-Santucci J., Dinh-Xuan A. T., Lebrec D.Endogenous pulmonary nitric oxide production measured from exhaled air is increased in patients with severe cirrhosis. J. Hepatol.231995471473 |
| 18. | Matsumoto A., Ogura K., Hirata Y., Kakoki M., Watanabe F., Takenaka K., Shiratori Y., Momomura S., Omata M.Increased nitric oxide in the exhaled air of patients with decompensated liver cirrhosis. Ann. Intern. Med.1231995110113 |
| 19. | Martin P., Ginès P., Schrier R. W.Nitric oxide as a mediator of hemodynamic abnormalities and sodium and water retention in cirrhosis. N. Engl. J. Med.3391998533541 |
| 20. | Cailes J. B., Kharitonov S. A., Yates D., Barnes P. J., Du Bois R. M.Decreased endogenous nitric oxide in the exhaled air of systemic sclerosis patients. Thorax501995452P |
| 21. | Schilling J., Holzer P., Guggenbach M., Gyurech D., Marathia K., Geroulanos S.Reduced endogenous nitric oxide in the exhaled air of smokers and hypertension. Eur. Respir. J.71994467471 |
| 22. | Bucca C., Colagrande P., Brussino L., Chiavassa G., Del Piano L., Dutto L., Polizzi S., Rolla G.Exhaled nitric oxide in patients with mitral valve disease (abstract). Am. J. Respir. Crit. Care Med.1571998A227 |
| 23. | Brett S. J., Evans T. W.Measurement of endogenous nitric oxide in the lungs of patients with the acute respiratory distress syndrome. Am. J. Respir. Crit. Care Med.1571998993997 |
| 24. | Settergren G., Angdin M., Astudillo R., Gelinder S., Liska J., Lundberg J. O., Weitzberg E.Decreased pulmonary vascular resistance during nasal breathing: modulation by endogenous nitric oxide from the paranasal sinuses. Acta Physiol. Scand.1631998235239 |
Presented in part at the International Conference of the American Thoracic Society, Chicago, April 25–29, 1998.
