Published ahead of print on January 9, 2003, doi:10.1164/rccm.200208-864OC
© 2003 American Thoracic Society
Nasal Epithelium Potential Difference at High Altitude (4,559 m)Evidence for SecretionDivision of Sports Medicine, Department of Medicine, University of Heidelberg, Heidelberg, Germany; Medical and Research Services, Veterans Affairs Puget Sound Health Care System, University of Washington, Seattle, Washington; Intensive Care Unit, Department of Internal Medicine, University Hospital, Zürich, Switzerland; National Heart and Lung Institute, Imperial College School of Science, Technology and Medicine, London, United Kingdom Correspondence and requests for reprints should be addressed to Heimo Mairbäurl, Division of Sports Medicine, Department of Medicine, University of Heidelberg, Luisenstraße 5, Geb. 4100, 69115 Heidelberg, Germany. E-mail: heimo_mairbaeurl{at}med.uni-heidelberg.de
Hypoxia inhibits activity and expression of ion transport proteins of cultured lung alveolar epithelial cells. Here we tested, whether in vivo hypoxia at high altitude (4,559 m) also inhibits lung ion transport. Transepithelial nasal potentials (NP) were determined as a surrogate measure of lung ion transport activity before and during the stay at altitude. In normoxia, total NP was approximately 20% higher in control subjects than in susceptibles to high-altitude pulmonary edema, but there was no difference between groups in amiloride-inhibitable NPs. At high altitude total NP increased 250% in both groups, whereas amiloride-sensitive NP decreased in control subjects only (-80%), and the chloride ion (Cl-)–sensitive portion of NP almost doubled. Because many mountaineers suffer from nasal dryness at high altitude, a control study was performed in normobaric hypoxia (12% oxygen, 6 hours) at a controlled humidity of 50%. In this study, no change in total NP or its amiloride- and Cl-sensitive portions was observed. The increased Cl- secretion at high altitude but no such change in normobaric hypoxia suggests that nasal dryness may stimulate local active Cl- and fluid secretion in the upper respiratory tract. It is therefore uncertain whether similar changes also occur at the alveolar epithelium.
Key Words: hypoxia • nasal potential • sodium ion transport • chloride ion secretion • nasal dryness Active sodium ion (Na+) and water reabsorption is essential in maintaining alveolar lining fluid as thin as possible to allow rapid diffusion of respiratory gases, which is of critical importance in situations of low inspired oxygen (O2). Transepithelial Na+ reabsorption generates the osmotic driving force for removal of alveolar fluid (1, 2). It is well documented that hypoxia inhibits Na+ transport in cultured alveolar epithelial cells (3, 4–6) by decreasing activity and expression of transport proteins such as Na/potassium adenosine triphosphatase (ATPase), epithelial Na channel and Na/potassium/2Cl cotransport (NKCC) (4, 7, 8). Exposure to hypoxia at altitudes above 3,000 m may cause high-altitude pulmonary edema (HAPE) with a high recurrence rate in those who had developed HAPE on past occasions (9). Alveolar edema occurs when fluid filtration exceeds the rate of fluid removal by active reabsorption and when the capacity of fluid removal from alveolar space is limited (10). Exaggerated pulmonary hypertension, which increases fluid filtration into the interstitial and alveolar space (for review see Reference 9) is thought to be the main cause of HAPE. However, a decreased capacity for the removal of alveolar fluid might contribute to the persistence of alveolar edema. This notion is supported by results obtained in mice genetically engineered to express a reduced number of epithelial Na channels, in which exposure to hypoxia causes mild pulmonary edema (11, 12). Indirect evidence for a similar situation in mountaineers has recently been presented by Sartori and coworkers (13), who reported decreased potential differences and a decreased Na+ transport activity of the nasal mucosa in HAPE-susceptibles. Nasal potential measurement is an established tool to evaluate ion transport activity in airway epithelia (14). It has also been used to interpret alveolar epithelial function (13). Because alveolar transport activity cannot be assessed directly, in this study the nasal potential (NP) difference was measured to ascertain whether hypoxic inhibition of alveolar ion transport also occurs in the human lung in vivo and whether any evidence can be derived that might link hypoxic modulation of ion transport to HAPE. It was also of interest whether these differences in ion transport activity between HAPE-susceptibles and nonsusceptible control subjects exist not only in normoxia (13) but also in hypoxia. Preliminary results of these studies have been published in abstract form (15, 16).
Twenty-two nonacclimatized mountaineers who had never experienced HAPE during previous exposures to comparable altitudes (control subjects; 40.5 ± 8.6 years; 4 women, 8 men) and mountaineers with a history of HAPE (HAPE-susceptibles; 42.4 ± 8.4 years; 3 women, 7 men) participated in this high-altitude study after written informed consent (see also Reference 17). The study protocol was approved by the Ethics Committee of the University of Zürich, Switzerland and the University of Heidelberg, Germany. All control subjects remained well at altitude, 10 subjects developed HAPE, which was assessed by chest radiography (TRS; Siemens, Stockholm, Sweden) and clinical evaluation as described elsewhere (17). Prealtitude measurements were performed in Zürich, Switzerland (490 m; low altitude [LA]). Subjects ascended from Alagna, Italy (1,100 m), spent one night in the Capanna Gnifetti (3,600 m), and climbed to the Capanna Regina Margherita (4,559 m) on the next morning. Measurements at 4,559 m were made within 3 hours after arrival (M1) and on the morning of the second day, 18 hours after arrival (M2) (17). Due to the unexpected results of the transepithelial NP measurements at 4,559 m, a control study was performed in Heidelberg (105 m). Seventeen male subjects (26.7 ± 3.6 years), after giving written informed consent, were exposed to 6 hours of normobaric hypoxia (12% O2; corresponding to an altitude of 4,500 m) in a room equipped with an air-conditioning system controlling the O2 concentration by admixing nitrogen. The study protocol was approved by the Ethics Committee of the Medical Faculty, University of Heidelberg, Germany. Measurements were performed 1 hour before exposure to hypoxia as well as after 1 and 6 hours in hypoxia. Control measurements were also performed in normoxia 2 to 3 days before hypoxic exposure. One subject dropped out due to severe symptoms of mountain sickness. Four other subjects reported moderate to severe headaches and nausea but completed the study. In both studies, O2 saturation was monitored by pulse oximetry (Biox 3700; Ohmeda, Denver, CO). Arterial blood samples were analyzed for pH, PCO2, PO2 and O2 saturation in the high-altitude study only (model 278 Ciba-Corning Diagnostics Analyzer and Co-oximeter; Ciba-Corning, Dietlikon, Switzerland). Nasal potential differences were determined according to Knowles and coworkers (14) and Middleton and coworkers (18). Subjects were seated comfortably in chair with their head leaning on a headrest. An umbilical vessel catheter (Sherwood Medical, Tullamore, Ireland) cut to a length of approximately 20 cm was placed on the nasal mucosa for superfusion (100 µl/minute) with the measuring electrode (WPI, Berlin, Germany) in line. An intravenous infusion line connected with the reference electrode was placed into an antecubital vein and perfused with Ringer solution (100 µl/minute). The potential difference was measured with a high-impedance mV-meter (W. Nagel, Munich, Germany) and recorded on a computer.
The total NP (NPtot) was measured during superfusion with Ringer solution. Na+ transport was assessed as the change in potential in presence of apical amiloride (100 µM; NP
Concentrations of norepinephrine and epinephrine were measured by HPLC according to Weicker and coworkers (19) in plasma from samples obtained after subjects rested for 20 minutes. Transepithelial NP differences are shown as positive values (mean values ± SD). Comparison between groups and between normoxia and hypoxia was performed by two-way analysis of variance for repeated measures. A p value of less than 0.05 indicates statistical significance.
Blood gasses and O2 saturation show changes typical of exposure to high altitude. Results are listed in Table 1 . In both groups there was a decrease in arterial PCO2 and a concomitant increase in arterial pH. Changes were not different between control subjects and HAPE-susceptibles. Arterial PO2 and SaO2 decreased significantly, and the change was more pronounced in HAPE-susceptibles.
Nasal Potentials at High Altitude Nasal potentials were measured to assess whether hypoxia at high altitude affects ion transport activity in vivo and to find any possible differences in transport activity between control subjects and subjects suffering from HAPE that might correlate with hypoxic edema formation. The results shown in Figure 2A indicate that in normoxia NPtot was lower (-20%) in subjects suffering from HAPE than in control subjects. The lower potential might indicate a decreased basal reabsorptive transport activity in those subjects. In all subjects NPtot increased at high altitude, but values in HAPE-susceptibles were always lower than in control subjects, although this difference was statistically not significant at altitude (p < 0.2).
The amiloride-inhibitable portion of NP (NP  amil) is a measure of apical Na uptake by epithelial cells mediated by transporters such as epithelial Na channels, nonselective cation channels, and Na/H exchange. In normoxia, there was no difference in NPamil between control subjects and HAPE-susceptibles (Figure 2A). In control subjects, at high altitude NPamil decreased significantly reaching nearly undetectable values the next day (M2). In HAPE-susceptibles no statistically significant decrease in NPamil was found at high altitude.
Because the nasal epithelium is also capable of active Cl--driven fluid secretion, it was important to obtain a measure of Cl- transport. Figure 2B shows that at low altitude NPamil-IS was lower in HAPE-susceptibles than in control subjects. In both groups of subjects, NPamil-IS increased on arrival at high altitude thus paralleling changes in NPtot. No statistically significant difference was found at altitude. By generating a Cl- gradient from blood to the apical surface by superfusion with a medium low in chloride in the presence of amiloride and isoproterenol to stimulate Cl- transport to the apical surface, a Cl--sensitive portion of the NP (NP
It was of interest to determine whether changes in the transepithelial NP differences at altitude correlate with the level of oxygenation. Plots of NPtot as a function of arterial PO2 (Figure 3A)
and SaO2 (Figure 3B) indicate that no correlations exist between these parameters. This is also true for NP
Nasal Potentials in Normobaric Hypoxia Nasal potentials were difficult to measure at high altitude. Many subjects reported dry, encrusted and even bloody noses most likely due to the dry air. To control for climate conditions in the Margherita hut other than decreased PO2, a different set of subjects in a separate study was exposed to normobaric hypoxia (12% O2) matching the degree of hypoxia experienced at the Capanna Margherita, but at a constant comfortable humidity of approximately 50%. The results of this study are summarized in Figure 4 . The figure shows no clear diurnal variation in measured components of NP differences. In contrast to the high-altitude results, the 6-hours exposure to normobaric hypoxia did not alter NPtot, NP amil, and NPCl.
Figure 5 summarizes the results of measurements of norepinephrine and epinephrine in normoxia and during normobaric hypoxia. On exposure to hypoxia for one hour plasma norepinephrine tended to increase (p < 0.064), but levels were not different from the 1-hour time point on the control day (normoxia). There was no difference in normoxic values after 6 hours of normobaric hypoxia. Although plasma epinephrine was significantly decreased at the 1-hour time point on the control day, no statistically significant change was observed in hypoxia.
This study was performed to determine whether differences in ion transport at the nasal epithelium exist between individuals susceptible and not susceptible to HAPE. Our results show a difference in NPtot but not in NP amil in normoxia between control subjects and HAPE-susceptibles. At high altitude, NPamil decreased significantly in control subjects only. In both groups, NPtot, NPamil-IS and NPCl increased greatly at high altitude but not during shorter exposure to normobaric hypoxia indicating that this response may be dependent on other factors of the mountain environment such as the cold, dry air. Alveolar fluid reabsorption cannot be assessed noninvasively in humans in vivo. On the basis of similarities in the expression of epithelial Na channels, Na/K-ATPase, and other ion transporters between nasal mucosa, airways, and alveolar epithelium, the potential difference across the nasal mucosa might reflect transport activity in the alveolar epithelium (20), although there seems to be some variation in the expression of subunits of epithelial Na channel along the respiratory tract (21). On the other hand, the nasal and alveolar epithelia are considerably different. The nasal epithelium is a composite tissue similar to airway epithelium, which, in addition to mucus secretion, has secretory and reabsorptive functions. In contrast, the adult alveolar epithelium, besides surfactant secretion by ATII-cells, is thought to be strictly reabsorptive, and transport is mediated both by ATI- (22) and ATII-cells (1).
Total Nasal Epithelial Potential Difference
On ascent to high altitude, NPtot increases. Similar results have been found by Mason and coworkers on ascent to an altitude of 3,800 m (23). The highest values were found on the day of arrival at 4,559 m (M1). On the next day (M2) NPtot had increased further. The increase appears to be due to stimulated secretion of Cl- as indicated by the increase in NPamil-IS and NP
Evidence for Stimulated Cl- Secretion in the Nasal Mucosa The question arises whether hypoxia-stimulated Cl- secretion indicated by the altered transport activity of the nasal mucosa might also occur in the alveolar epithelium. Evidence comes from the fetal lung, whose distal lung epithelium secretes Cl- and water (25) and functions at a level of oxygenation similar to that experienced by mountaineers at an altitude of 4,559 m. When fetal alveolar epithelial cells cultured at their physiological (intrauterine) PO2 of approximately 40 mm Hg are switched to 21% O2, expression and activity of Na+ transporters are stimulated (6). Stimulation of Na+ transport by oxygenation can be reversed by exposure to hypoxia. However, it has not been demonstrated yet whether these maneuvers also alter Cl- secretion. If exposure to high-altitude hypoxia inhibits Na+ reabsorption and at the same time stimulates Cl- secretion by the alveolar epithelium, one might speculate that hypoxia might actually stimulate fluid secretion into the alveolar space and contribute to the formation of alveolar edema. However, adult alveolar epithelial cells lack the cAMP-activated Cl- channels (26) that in airway epithelium mediate Cl- secretion (2). Therefore, the surprising hypothesis of hypoxia-induced alveolar secretion requires experimental support. In a more general sense, these data suggest caution in extending the results from transport events in the nasal mucosa as measured by changes in potential difference to active ion transport at the alveolar level.
Hypoxic Inhibition of Na+ Transport
In mountaineers who developed HAPE, no statistically significant inhibition of Na+ transport (NP
An inverse relation has been found between the cystic fibrosis transmembrane regulator, a major pathway for airway epithelial Cl- secretion, and Na+ reabsorption (35, 36). It is therefore conceivable that the decrease in NP In conclusion, our results indicate that an increased chloride secretion compensating for drying of the nasal mucosa underlies the increment in transepithelial NP at high altitude. Unchanged NP in normobaric hypoxia in a comfortably humid environment support this hypothesis. Therefore, the decrease in the amiloride-inhibitable portion of NP, although in accordance with hypoxic transport inhibition at the alveolar epithelium, appears to be due to inhibition of epithelial Na+ channels subsequent to the stimulation of Cl- secretion rather than to hypoxia directly. These results suggest that changes in NP during high-altitude exposure occur as a specific response of the nasal mucosa to the environment at high altitude and, most likely, do not reflect processes at the alveolar epithelium.
The authors are most grateful to the subjects participating in both studies as well as to the hut keepers and the Sezione Varallo of the Club Alpino Italiano for providing an excellent research facility at the Capanna Regina Margherita.
Supported by departmental money and grants from the German Research Foundation (DFG) Ma 1503/11-1, Ma 1503/14-1. Received in original form August 15, 2002; accepted in final form January 7, 2003
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