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Altered ion transporter expression in bronchial epithelium in mountaineers with high-altitude pulmonary edema

¹Division of Sports Medicine, Department of Medicine, University of Heidelberg, Germany; ²Intensive Care Unit, Department of Internal Medicine, University Hospital, Zürich, Switzerland; ³National Heart and Lung Institute, Imperial College School of Science, Technology and Medicine, London, United Kingdom; and ⁴Medical and Research Services, Veterans Affairs Puget Sound Health Care System, University of Washington, Seattle 98195

Submitted 16 December 2002 ; accepted in final form 25 June 2003

	ABSTRACT

TOP ABSTRACT METHODS RESULTS DISCUSSION DISCLOSURES ACKNOWLEDGMENTS REFERENCES

 
Hypoxia inhibits activity and expression of transport proteins of cultured lung alveolar epithelial cells. Here we tested whether hypoxia at high altitude affected the expression of ion transport proteins in tissues obtained from controls and mountaineers with high-altitude pulmonary edema (HAPE) at the Capanna Margherita (4,559 m). Expression was determined by RT-PCR and Western blots from brush biopsies of bronchial epithelium and from leukocytes obtained before and during the stay at high altitude. At low altitude, amounts of mRNAs were not different between control and HAPE-susceptible subjects. At high altitude, the amount of mRNA of Na-K-ATPase, CFTR, and -actin of brush biopsies did not change in controls but decreased significantly (-60%) in HAPE-susceptible subjects. There was no change in Na channel mRNAs at high altitude in controls and HAPE. No statistically significant correlation was found between the expression of Na transporters and PO₂ and O₂ saturation. In leukocytes, 28S-rRNA and Na-K-ATPase decreased at altitude in control and HAPE-susceptible subjects, but no significant change in Na-K-ATPase protein was found. Hypoxia-inducible factor-1 mRNA and GAPDH mRNA tended to increase in leukocytes obtained from HAPE-susceptible subjects at high altitude but did not change in controls. These results show that hypoxia induces differences in mRNA expression of ion transport-related proteins between HAPE-susceptible and control subjects but that these changes may not necessarily predict differences in protein concentration or activity. It is therefore unclear whether these differences are related to the pathophysiology of HAPE.

nasal potential; alveolar fluid clearance; airway Na-K-ATPase; epithelial Na channels

Alveolar edema occurs when fluid filtration driven by high alveolar capillary pressure exceeds the rate of fluid removal (4). Alveolar fluid reabsorption is dependent on the reabsorption of Na, which generates the osmotic gradient required for movement of water across the alveolar epithelium. Hypoxia inhibits fluid reabsorption in cultured alveolar epithelial cells (10, 12, 18) and fluid-filled lungs (24) by decreasing activity and expression of Na-transport proteins such as the Na-K-ATPase and epithelial Na channels (ENaC) (17, 25). If mountaineers susceptible to HAPE have a low preexisting activity and/or expression of Na-transport proteins, hypoxic transport inhibition might further diminish the reabsorption of water from the alveolar space and subsequently result in insufficient removal of fluid filtered into alveoli. This hypothesis is supported by results obtained in mice genetically engineered to have reduced expression of ENaC. These mice maintain normal alveolar fluid balance in normoxia but develop alveolar edema when exposed to hypoxia (7). In accordance with the above hypothesis and results on mice, there are functional differences in epithelial Na transport between HAPE-susceptible and nonsusceptible individuals, as demonstrated by nasal potential measurements (11, 20). Also, salmeterol, a -adrenergic agent that stimulates alveolar Na transport, reduces the occurrence of HAPE (20).

Active Na transport is a process vital to virtually all cells in the body because it controls the Na gradients required for transmembrane transport of various substrates, neuronal and muscular excitability, and a variety of other cellular functions. Therefore, decreased expression of Na-K-ATPase and ENaC will not only affect alveolar fluid reabsorption but also might depolarize smooth muscle cells of small pulmonary arteries and veins, increase their excitability, and thus contribute to the development of exaggerated pulmonary vasoconstriction and increased pulmonary capillary pressure. Therefore, any preexisting generalized defect in the expression of Na transporters in combination with hypoxia might contribute to HAPE and other hypoxia-associated symptoms by disturbing cellular ion transport.

This study was designed to examine whether the expression of transport proteins involved in alveolar fluid reabsorption is altered by exposure to high-altitude hypoxia and whether differences in the expression of transporters in normoxia and/or hypoxia are associated with susceptibility to HAPE. Because the alveolar epithelium cannot be assessed in vivo, we studied the expression of Na transporters in brush biopsies of bronchial epithelium of control subjects and mountaineers susceptible to HAPE in normoxia and hypoxia. Airway epithelial characteristics may not totally resemble those of alveolar epithelial cells, but similarities in the expression of Na-transport proteins have been shown (19). We also studied transporter expression in white blood cells to find out whether effects of hypoxia are tissue specific. The results indicate that at low altitude the expression of Na transporters was not different in both groups of subjects, but that hypoxia at 4,559 m affected mRNA expression differently in controls and HAPE. Preliminary results have been published in abstract form (8, 9).

	METHODS

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Twenty-two nonacclimatized, experienced mountaineers aged 24–52 participated in the high-altitude study after giving written, informed consent. The study protocol was approved by the ethics committees of the University of Zürich, Switzerland, and of the University of Heidelberg, Germany. Ten subjects developed HAPE (HAPE group; 3 women, 7 men; age 42.4 ± 8.4 yr) during the stay at the Capanna Regina Margherita (4,559 m). HAPE was assessed by chest radiography (TRS, Siemens, Stockholm, Sweden) and clinical evaluation (23). Twelve subjects without a previous history of HAPE (controls; 40.5 ± 8.6 yr; 4 women, 8 men) remained well at altitude. Study protocol and subjects were the same as described in a recent publication (23).

Prealtitude measurements were performed in Zürich, Switzerland (450 m). Within 3 wk after baseline values were obtained, the subjects ascended to the Monte Rosa starting from Alagna, Italy (1,100 m) by cable car to Punta Indren (3,200 m) and climbed to the Capanna Gnifetti (3,600 m), where they spent one night. On the following morning, they climbed to the Capanna Regina Margherita (4,559 m, barometric pressure 440 mmHg), where the research laboratory has been established.

In the Capanna Margherita (4,559 m), oxygen saturation was measured by pulse oximetry, and arterial blood samples were analyzed with a blood-gas analyzer (model 278 and CO-oximeter; Ciba-Corning, Dietikon, Switzerland) to evaluate the degree of hypoxemia. About 3 h after arrival (M1) as well as on the mornings of days 2 and 3 at the Capanna Margherita (M2 and M3, respectively), blood samples (heparin; 10 U/ml) were obtained from antecubital veins and were centrifuged (3,000 rpm, 4°C, 15 min). The buffy coat was collected and washed three times with lysis medium composed of (in mM) 155 NH₄Cl, 10 KHCO₃, 0.1 EDTA, pH 7.4, to remove contaminating erythrocytes. Buffy coat from 4 ml of whole blood was lysed with 1.5 ml of TriStar reagent (Hybaid-AGS, Heidelberg, Germany) and stored frozen in liquid nitrogen.

Brush biopsies of bronchial epithelium were obtained by bronchoscopy in prealtitude tests and on M2 (23). During bronchoscopy, subjects were monitored by electrocardiography. Atropine sulfate (0.4 mg) was given intravenously. Some subjects were slightly sedated by intravenous application of midazolam (1–2 mg). Local anesthesia of upper airways was achieved by application of 2% lidocaine. A flexible fiber optic bronchoscope (Pentax, Tokyo, Japan) was passed through the mouth into the left lung lobe for bronchoalveolar lavage (23). After the lavage, the bronchoscope was moved to a second-generation bronchus, cytological brushes (Boston Scientific, Ratingen, Germany) were inserted, and samples of airway epithelium were obtained by brushing the bronchial surface. Optical evaluation indicated that brushing caused no bleeding. After retraction, the brushes were cut and the tips holding the epithelium were washed into 1.5 ml of TriStar reagent (Hybaid-AGS).

Samples of brush biopsies and buffy coat were frozen in liquid nitrogen and were transported to Heidelberg, where they were stored in a freezer at -80°C until further use.

Expression of transporters in samples of bronchial epithelium and white blood cells from the buffy coat was measured by RT-PCR. Total RNA was prepared from the Tristar-dissolved samples according to the manufacturer's instructions. cDNAs were prepared from 1 µg of total RNA in a thermocycler (T-Gradient; Biometra, Göttingen, Germany) by using random hexamere primers [pd(N)₆; Roche, Mannheim, Germany] and Superscript II reverse transcriptase (Life Technologies, Karlsruhe, Germany). Dilutions of cDNAs with water (1:25) were used in subsequent amplifications by real-time PCR using SybrGreen for detection (Lightcycler, Roche) and conventional PCR (see below). Primers for human sequences used in both types of PCR were ₁-Na-K-ATPase: ATG GGG AAG GGG GTT GGA CGT GAT AA and TTC TCA CCA TTT CGA ATC ACA AGG GCT T; ₁-Na-K-ATPase: GAG ACT TTA ATC ATG AAC GAG GAG A GGG and CTG CAG GGA GTT TGC CAT AGT ACG G; epithelial Na channel (ENaC): GCT AAT GAG ATT CCT GTC GCT TCC ATC C and CTC TGC CCC CTT CCT TTG GTC TTC TTC C; ENaC: CTT GTC TCA GGA GCG GGA CCA and AGG CTG GAA GCC AAA GTT GGT G; ENaC: CAG TGC GCC CTC CTC GTC TCC TCC TTC and CCC ATG CAT CGG GTG GTG AAA AAG CGT; cystic fibrosis transmembrane regulator (CFTR): GGC CAA ATG ACT GTC AAA GA and ATG GAA TCG TAC TGC CGC AC; hypoxia-inducible factor-1 (HIF-1): ACA AGT CAC CAC AGG ACA G and AGG GAG AAA ATC AAG TCG; GAPDH: ACC ACA GTC CAT GCC ATC AC and TCC ACC ACC CTG TTG CTG TA; 28S-rRNA: TTG AAA ATC CGG GGG AGA G and ACA TTG TTC CAA CAT GCC AG.

Conventional PCR was used for the evaluation of samples from brush biopsies. In this case, a multiplex PCR protocol was applied with -actin used as an internal standard to control for the efficiency of reverse transcription and for loading of the gels. Because of the high abundance of -actin in the samples, a kit containing nucleotides to quench the -actin signal was used according to the manufacturer's instructions [Quantum RNA -actin (commercial, no primer sequence available); Ambion, Austin, TX]. Amplified samples were separated on agarose gels and stained with GelStar (BioWhittaker Molecular Applications, Taufkirchen, Germany). Digitized images of the stained gels were obtained, and band densities were measured by using the DC120 digital camera and the 1D image analysis software (Kodak, Rochester, NY). PCR of all samples was repeated two times. Band density evaluation was repeated by two independent researchers. Figure 1 shows an image of a representative agarose gel of multiplex PCR products, which indicates that PCR resulted in single bands of the expected size of the product of interest. The specificity of PCR was validated by commercial sequencing of eluted bands (MWG-Biotech).

View larger version (65K): [in this window] [in a new window]   Fig. 1. Typical pictures of agarose gels of products obtained by multiplex PCR. Total RNA from brush biopsies of bronchial epithelium was reverse transcribed. cDNAs of 1- and 1-Na-K-ATPase (Na/K), of the -, -, and -subunits of epithelial Na channels (ENaC), and of the cystic fibrosis transmembrane regulator (CFTR) were enhanced by multiplex PCR using -actin as an internal standard and the primers listed in Table 1.

Comparisons between groups and between low and high altitude were performed by two-way analysis of variance for repeated measures followed by Tukey's post hoc tests for multiple comparisons. For the evaluation of results from brush biopsies, no post hoc tests were performed because of the small sample size. Results are shown as mean values ± SD. P < 0.05 was used to indicate statistical significance.

	RESULTS

TOP ABSTRACT METHODS RESULTS DISCUSSION DISCLOSURES ACKNOWLEDGMENTS REFERENCES

 
Blood gases and arterial oxygen saturation were measured to quantify the degree of hypoxemia during the stay at the Capanna Margherita. Table 1 shows the results obtained on those participants whose brush biopsies were evaluated. The results confirm earlier findings at this altitude by showing that HAPE-susceptible subjects had decreased values of PO₂ and O₂ saturation relative to controls (23). No differences between groups were found in pH and PCO₂. It should be noted that the data presented here comprise a subgroup of the subjects whose data are presented by Swenson et al. (23).

Expression of transport proteins in airway epithelium. The results of changes in mRNA of Na transporters in airway epithelium during exposure to high altitude are shown in Fig. 2 and indicate that in control subjects the mRNA expression of ₁ and ₁ subunits of the Na-K-ATPase remained unchanged at high altitude. In contrast, in HAPE-susceptible individuals, mRNA levels of both subunits of the Na-K-ATPase had decreased significantly at high altitude. There was great variability in mRNA expression of ENaC subunits among subjects (Fig. 2). Therefore, the changes were statistically not significant. In controls, CFTR mRNA did not change on ascent to high altitude, whereas in HAPE levels of CFTR mRNA had decreased significantly (Fig. 3).

View larger version (29K): [in this window] [in a new window]   Fig. 2. mRNA expression of subunits of Na-K-ATPase and of ENaC in bronchial epithelium in controls and high-altitude pulmonary edema (HAPE)-susceptible subjects at 4,559 m. Total mRNA was prepared from brush biopsies of bronchial epithelium. Relative changes in levels of mRNA were measured as changes in band densities after multiplex PCR amplification. Only samples obtained in both locations, Zürich (450 m) and in the Capanna Margherita (4,559 m), with levels of total mRNA in the detectable range were used for evaluation. Results are mean values ± SD from 4 HAPE subjects and 6 controls. *P < 0.05 relative to values obtained in Zürich.

View larger version (25K): [in this window] [in a new window]   Fig. 3. Transcription of CFTR and of -actin in bronchial epithelium in controls and HAPE-susceptible subjects at 4,559 m. Multiplex PCR was performed as described in the legend to Fig. 2. Results are mean values ± SD from 4 HAPE subjects and 6 controls. *P < 0.05 relative to values obtained in Zürich.

To control for differences in the efficiency of reverse transcription and gel loading, multiplex PCR was performed with the use of -actin mRNA as an internal standard. Interestingly, in hypoxia, band densities of -actin did not change in controls but decreased significantly in HAPE (Fig. 3).

In this study, nasal potentials also were determined before ascent and on several occasions after arrival at the Margherita hut (11). It is therefore of interest to correlate transport activity with mRNA expression of transport proteins. Figure 4 shows plots of the total nasal potential at low and high altitudes as a function of relative amounts of ₁-Na-K-ATPase mRNA and of the amiloride-sensitive portion of the nasal potential with -ENaC mRNA/-actin. Likewise, as the plots depicted in this figure show, none of the other possible plots of mRNA data resulted in statistically significant correlations.

View larger version (12K): [in this window] [in a new window]   Fig. 4. Lack of correlation between nasal potential difference and mRNA expression of Na-K-ATPase (A) and ENaC (B). Nasal potentials shown here were also reported in a recent publication (11) but are from a subgroup of subjects for whom also brush biopsies of bronchial epithelium were available from Zürich (450 m) and from day 2 at the Capanna Margherita (4,559 m). Band densities of mRNAs from the 1-Na-K-ATPase and -ENaC were normalized to the band density of -actin obtained by multiplex PCR.

Expression in cells from buffy coat. We wanted to know whether changes in mRNA expression of transporters reflect changes in amounts of transport proteins and whether changes observed in airway epithelium also occurred in nonepithelial, non-lung-derived tissues such as white blood cells. No further purification of leukocyte subtypes was attempted. In this series of experiments, amounts of cDNAs obtained by reverse transcription were measured quantitatively by real-time PCR using 28S-rRNA to control for efficiency of reverse transcription and known standards for quantification. Results are shown as ratios of amounts of the cDNA of interest and 28S-rRNA. The ₁ and ₁ subunits of the Na-K-ATPase were well detectable. In accordance with Bubien et al. (3), we were able to detect all three subunits of ENaC by PCR, but levels were too low for quantification. In control subjects, ₁-Na-K-ATPase expression had decreased slightly on M3 (-25%; P < 0.05), whereas in HAPE levels were decreased at M2 (-50%; P < 0.05) and M3 (-25%; P < 0.09) (Fig. 5A). 28S-rRNA was decreased on M3 in HAPE only (Fig. 5B).

View larger version (25K): [in this window] [in a new window]   Fig. 5. 1-Na-K-ATPase mRNA and 28S rRNA in leukocytes from control and HAPE-susceptible subjects at 4,559 m. Blood samples were obtained in Zürich within 3 h after arrival at the Capanna Regina Margherita (4,559 m; M1), and on mornings of the following days (M2, M3). Buffy coat was prepared by centrifugation of heparinized blood. cDNAs were prepared by random hexamere priming. Real-time PCR was performed for quantification. A: amounts of 1-Na-K-ATPase relative to amounts of 28S-rRNA. B: absolute amounts of 28S-rRNA. Results are mean values ± SD from 10 HAPE and 12 controls. *P < 0.05 relative to values obtained in Zürich; #P < 0.05 between controls and HAPE-susceptible subjects on the respective day.

Figure 6 shows a great variability of band densities of Western blots of ₁-Na-K-ATPase throughout the study even when band densities were normalized to prealtitude values. Therefore, changes in ₁-Na-KATPase protein were statistically not significant.

View larger version (34K): [in this window] [in a new window]   Fig. 6. Western blots of 1-Na-K-ATPase in leukocytes from control and HAPE-susceptible subjects at 4,559 m. Blood samples were prepared as described in the legend to Fig. 4. Buffy coat was prepared by centrifugation of blood anticoagulated with heparin and lysis of contaminating red cells. Total cell protein was prepared from a whole cell lysate by using the TriStar reagent (Hybaid-AGS, Heidelberg, Germany). Inset shows a typical Western blot (subject 19,Zürich) using the 6H monoclonal antibody (15). Results are mean values ± SD from 10 HAPE-susceptible subjects and 12 controls. *P < 0.05 relative to values obtained in Zürich.

It was also tested whether exposure to high altitude changed the levels of mRNA of GAPDH and HIF-1, both of which have been shown to increase in hypoxia in a variety of tissues and cultured cells. The results shown in Fig. 7 indicate that immediate exposure to high-altitude hypoxia did not increase levels of mRNA of GAPDH and HIF-1. Whereas HIF-1 mRNA was increased significantly (P < 0.05) at the end of the stay at the Capanna Margherita (M3), no statistically significant change in the mRNA expression of GAPDH was found (P < 0.12).

View larger version (26K): [in this window] [in a new window]   Fig. 7. Amounts of GAPDH (A) and hypoxia-inducible factor-1 (HIF-1; B) mRNA in leukocytes from control and HAPE-susceptible subjects at 4,559 m. Blood sampling and measurements were performed as described in legend to Fig. 4. Results are mean values ± SD from 10 HAPE and 12 controls. *P < 0.05 relative to values obtained in Zürich; #P < 0.05 between control and HAPE-susceptible subjects on the respective day.

Despite the differences described above in patterns of changes in mRNAs between control and HAPE-susceptible individuals at high altitude, the mRNA expression of transporters did not correlate with arterial PO₂, neither in brush biopsies nor in leukocytes. There was also no correlation between PO₂ and amounts of ₁-Na-K-ATPase measured by Western blot. However, in leukocytes, an inverse relation existed between arterial PO₂ and HIF-1 mRNA (P < 0.05) when the results from the last day at the Margherita hut (M3) were analyzed (Fig. 8).

View larger version (13K): [in this window] [in a new window]   Fig. 8. Inverse correlation between HIF-1 mRNA expression and arterial PO2 in leukocytes at high altitude (4,559 m). Samples from day 3 (M3) at the Capanna Regina Margherita are shown. , Control; , HAPE. There was no statistically significant correlation between GAPDH mRNA and PO2 (A; r = 0.272) but a statistically significant correlation between HIF-1 mRNA and arterial PO2 (B; r = 0.431, P < 0.05).

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION DISCLOSURES ACKNOWLEDGMENTS REFERENCES

 
Our results indicate that in normoxia no differences existed in the amounts of mRNA of transporters in airway epithelium as well as in leukocytes between controls and HAPE-susceptible subjects. On exposure to high-altitude hypoxia mRNAs of the Na-K pumps, CFTR and -actin decreased significantly in bronchial epithelium of HAPE-susceptible subjects only. After 3 days at the Capanna Regina Margherita, HIF-1 mRNA expression was increased in leukocytes of HAPE-susceptible subjects. These changes might be a consequence of the greater degree of hypoxemia in this group of mountaineers.

The goal of the study was to find out whether HAPE susceptibility is associated with a diminished expression of transporters involved in alveolar fluid clearance. This hypothesis seemed reasonable because mice with a reduced expression of ENaC in normoxia are able to maintain alveolar fluid balance in normoxia but develop pulmonary edema when exposed to hypoxia (21). Recent results of decreased transepithelial nasal potential difference in HAPE-susceptible individuals in normoxia (11, 20) support the notion, although it is not clear whether transport activity across the nasal epithelium actually reflects transport across the alveolar epithelium (11). Because the alveolar epithelium could not be assessed in vivo, in the present study the expression of transport proteins was measured in airway epithelium. This approach seems justified on the basis of reported similarities in ENaC expression along the respiratory tract (19). The lack of differences in mRNA expression of Na transporters between controls and mountaineers who later experienced HAPE in the Capanna Regina Margherita indicates that in normoxia HAPE susceptibility is not correlated with differences in mRNA expression in airway epithelium. This result indicates furthermore that differences in airway ion transport activity measured as nasal potential between control and HAPE-susceptible subjects in normoxia cannot be explained by differences in mRNA expression and therefore must relate to differences in translation and posttranslational modifications.

High-altitude hypoxia caused a decrease in the expression of ₁ and ₁ subunits of the Na-K-ATPase and of CFTR-mRNA only in HAPE-susceptible subjects but not in controls. There was, however, also a decrease in the expression of -actin, a housekeeping gene whose mRNA expression has been shown to change in hypoxia in different cell lines (26). Therefore, the decrease in mRNA expression of Na-K-ATPase and CFTR may not be caused by hypoxia-specific effects on transport proteins but rather by a more general inhibition of transcription typical for adaptation to severe hypoxia such as occurs in HAPE (6). However, no statistically significant change in mRNA levels of ENaC was found at high altitude, indicating that in hypoxic airway epithelium ENaC mRNA expression is controlled by mechanisms different from those that control Na-KATPase, CFTR, and -actin. These data contrast with those obtained in cultured alveolar epithelial cells in which hypoxic downregulation of ENaC and Na-KATPase mRNA and protein expression has been observed (17, 25). Because -adrenergic agents have been shown to increase Na transport of alveolar epithelium (24) by stimulation of the expression of Na channels (14), increased levels of endogenous catecholamines during exposure to high altitude of HAPE-susceptible mountaineers (2) might limit hypoxic inhibition of ENaC mRNA expression.

The functional consequences of altered protein expression at high altitude are unclear. In cultured alveolar epithelial cells, a parallel decrease in ENaC activity and mRNA expression was found (17). Similarly, a decrease in amiloride-sensitive nasal potential has been found in mountaineers exposed to high altitude (11, 13), although in the present study no change in ENaC mRNA expression in airway epithelium of mountaineers could be demonstrated. In contrast, in lungs of hypoxic rats, an increase in transport protein expression was found despite inhibition of alveolar fluid reabsorption and amiloride-sensitive transport (24). These results indicate that transport activity in hypoxia may be affected not only by altered mRNA expression (in case of Na-K-ATPase) but also by posttranslational handling of transport proteins, regulation of the activity of transporters located in the plasma membrane (12), and/or internalization of membrane-associated transport proteins (5, 16, 25).

We also wanted to know whether differences in mRNA expression between control and HAPE-susceptible subjects existed in non-lung-derived cells such as leukocytes, which would provide a rather simple method to screen for susceptibility if a difference existed. Similar to our lung cell data, leukocyte housekeeping genes such as -actin and 28S-rRNA decreased at high altitude, but only in HAPE-susceptible subjects. In accordance with a previous report (3), we were able to detect ENaC mRNA in white blood cells from the buffy coat, but levels were too low to quantify changes even by real-time PCR. In normoxia, there was no statistically significant difference in levels of ₁-Na-K-ATPase mRNA (normalized for 28S-rRNA) between control and HAPE-susceptible individuals, indicating that on the basis of Na-K-ATPase mRNA expression no distinction between both groups can be made in normoxia. At high altitude, Na-K-ATPase mRNA expression decreased somewhat in both HAPE-susceptible and control subjects, whereas amounts of whole cell ₁-Na-K-ATPase protein were increased on the second day at high altitude in controls only. This indicates that whole cell Na-K-ATPase protein does not parallel changes in mRNA levels.

To test whether the degree of hypoxia at high altitude was sufficient to induce typically oxygen-sensitive genes (22), we measured amounts of mRNA of GAPDH and HIF-1 in leukocytes. We found no upregulation of mRNA expression in the first 2 days at high altitude. This contrasts with results obtained on a variety of different cell types cultured under hypoxic conditions (for review, see Ref. 22). On day 3 at 4,559 m, a statistically significant increase HIF-1 mRNA was seen (Fig. 6) in HAPE-susceptible mountaineers but not in controls, whereas no statistically significant change in GAPDH mRNA was found (P < 0.12). The results of hypoxia-dependent stimulation (HIF-1) and inhibition (Na-K-ATPase, CFTR, -actin, 28S-rRNA) of mRNA expression in leukocytes may represent a threshold phenomenon in which the pronounced changes found in HAPE-susceptible subjects relate to the higher degree of hypoxemia (Table 1 and Ref. 23).

Possible limitations of this study must be addressed. The major limitation is certainly the lack of direct data on the alveolar epithelium because changes in mRNA expression in airway epithelium and blood cells may not be the same as in alveolar epithelium despite similarities in the distribution of ENaC subunits along the airways (19). It is therefore unclear whether results on leukocytes and airway epithelial cells justify a conclusion of impaired alveolar cell function and blunted edema clearance in hypoxia. Changes in mRNA expression of transport proteins may not reflect changes in actual transport activity as indicated by a lack of correlation between nasal potential differences (11), changes in mRNA, and HAPE susceptibility (Fig. 4). This discrepancy may be explained partly on the basis of differences in posttranslational modification. These limitations make it difficult to derive a coherent mechanistic explanation for the relation between the occurrence of HAPE and the observed changes in mRNA expression. Because of these limitations, our results cannot be used to discriminate HAPE-susceptible from nonsusceptible individuals. Only if these changes also occurred at the alveolar epithelium, which needs to be demonstrated, they might indicate a limited ability of HAPE-susceptible mountaineers to clear alveolar fluid.

	DISCLOSURES

TOP ABSTRACT METHODS RESULTS DISCUSSION DISCLOSURES ACKNOWLEDGMENTS REFERENCES

 
This study was supported by grants from the German Research Foundation (DFG) Ma 1503/11-1 and Ma 1503/14-1.

	ACKNOWLEDGMENTS

TOP ABSTRACT METHODS RESULTS DISCUSSION DISCLOSURES ACKNOWLEDGMENTS REFERENCES

 
We are most grateful to the subjects participating in this study 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. The expert technical assistance of Sonja Engelhardt and Christiane Herth is gratefully acknowledged.

	FOOTNOTES

 

Address for reprint requests and other correspondence: H. Mairbäurl, Division of Sports Medicine, Dept. of Medicine, Univ. of Heidelberg, Luisenstrae 5, Geb. 4100, 69115 Heidelberg, Germany (E-mail: heimo.mairbaeurl{at}med.uni-heidelberg.de).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

TOP ABSTRACT METHODS RESULTS DISCUSSION DISCLOSURES ACKNOWLEDGMENTS REFERENCES

 

1. Bärtsch P. High altitude pulmonary edema. Respiration 64: 435-443, 1997.[ISI][Medline]
2. Bärtsch P, Shaw S, Franciolli M, Gnädinger MP, and Weidmann P. Atrial natriuretic peptide in acute mountain sickness. J Appl Physiol 65: 1929-1937, 1988.[Abstract/Free Full Text]
3. Bubien JK, Watson B, Khan MA, Langloh AL, Fuller CM, Berdiev B, Tousson A, and Benos DJ. Expression and regulation of normal and polymorphic epithelial sodium channel by human lymphocytes. J Biol Chem 276: 8557-8566, 2001.[Abstract/Free Full Text]
4. Crandall ED and Matthay MA. Alveolar epithelial transport. Basic science to clinical medicine. Am J Respir Crit Care Med 163: 1021-1029, 2001.[Free Full Text]
5. Dada L, Chandel NS, Ridge KM, Bertorello AM, and Sznajder JI. Hypoxia regulates Na,K-ATPase endocytosis via mitochondrial reactive oxygen species (ROS) in alveolar epithelial cells (Abstract). FASEB J 16: A461, 2002.
6. Hochachka PW. Defence strategies against hypoxia and hypothermia. Science 231: 234-241, 1986.[Abstract/Free Full Text]
7. Lepori M, Hummler E, Feihl F, Sartori C, Nicod P, Rossier B, and Scherrer U. Amiloride sensitive sodium transport dysfunction augments susceptibility to hypoxia-induced lung edema (Abstract). FASEB J 12: 231, 1998.[Abstract/Free Full Text]
8. Mairbäurl H, Höschele S, Schwöbel F, Swenson E, Maggiorini M, Gibbs S, and Bärtsch P. Altered expression of Na-transporters in HAPE-susceptibles in high altitude hypoxia. High Alt Med Biol 2: 88, 2001.
9. Mairbäurl H, Höschele S, Schwöbel F, Weymann J, Swenson ER, Maggiorini M, Gibbs JSR, and Bärtsch P. Expression and activity of ion transporters of mountaineers susceptible to high altitude pulmonary edema. FASEB J 16: A65/A112.8, 2002.
10. Mairbäurl H, Mayer K, Kim KJ, Borok Z, Bärtsch P, and Crandall ED. Hypoxia decreases active Na transport across primary rat alveolar epithelial cell monolayers. Am J Physiol Lung Cell Mol Physiol 282: L659-L665, 2002.[Abstract/Free Full Text]
11. Mairbäurl H, Weymann J, Möhrlein A, Swenson ER, Maggiorini M, Gibbs JSR, and Bärtsch P. Nasal epithelium potential difference at high altitude (4,559 m): evidence for secretion. Am J Respir Crit Care Med 167: 862-867, 2003.[Abstract/Free Full Text]
12. Mairbäurl H, Wodopia R, Eckes S, Schulz S, and Bärtsch P. Impairment of cation transport in A549 cells and rat alveolar epithelial cells by hypoxia. Am J Physiol Lung Cell Mol Physiol 273: L797-L806, 1997.[Abstract/Free Full Text]
13. Mason NP, Petersen M, Melot C, Imanov B, Matveykine O, Gautier MT, Sarybaev A, Aldashev A, Mirrakhimov MM, Brown BH, Leathard AD, and Naeije R. Serial changes in nasal potential difference and lung electrical impedance tomography at high altitude. J Appl Physiol 94: 2043-2050, 2003.[Abstract/Free Full Text]
14. Minakata Y, Suzuki S, Grygorczyk C, Dagenais A, and Berthiaume Y. Impact of -adrenergic agonist on Na⁺ channel and Na⁺-K⁺ -ATPase expression in alveolar type II cells. Am J Physiol Lung Cell Mol Physiol 275: L414-L422, 1998.[Abstract/Free Full Text]
15. Pietrini G, Matteoli M, Banker G, and Caplan MJ. Isoforms of the Na,K-ATPase are present in both axons and dendrites of hippocampal neurons in culture. Proc Natl Acad Sci USA 89: 8414-8418, 1992.[Abstract/Free Full Text]
16. Planes C, Blot-Chabaud M, Matthay MA, Couette S, Uchida T, and Clerici C. Hypoxia and ₂-agonists regulate cell surface expression of the epithelial sodium channel in native alveolar epithelial cells. J Biol Chem 277: 47318-47324, 2002.[Abstract/Free Full Text]
17. Planes C, Escoubet B, Blot-Chabaud M, Friedlander G, Farman N, and Clerici C. Hypoxia downregulates expression and activity of epithelial sodium channels in rat alveolar epithelial cells. Am J Respir Cell Mol Biol 17: 508-518, 1997.[Abstract/Free Full Text]
18. Planes C, Friedlander G, Loiseau A, Amiel C, and Clerici C. Inhibition of Na-K-ATPase activity after prolonged hypoxia in an alveolar epithelial cell line. Am J Physiol Lung Cell Mol Physiol 271: L70-L78, 1996.[Abstract/Free Full Text]
19. Rochelle LG, Li DC, Ye H, Lee E, Talbot CR, and Boucher RC. Distribution of ion transport mRNAs throughout murine nose and lung. Am J Physiol Lung Cell Mol Physiol 279: L14-L24, 2000.[Abstract/Free Full Text]
20. Sartori C, Allemann Y, Duplain H, Lepori M, Egli M, Lipp E, Hutter D, Turini P, Hugli O, Cook S, Nicod P, and Scherrer U. Salmeterol for the prevention of high-altitude pulmonary edema. N Engl J Med 346: 1631-1636, 2002.[Abstract/Free Full Text]
21. Scherrer U, Sartori C, Lepori M, Allemann Y, Duplain H, Trueb L, and Nicod P. High-altitude pulmonary edema: from exaggerated pulmonary hypertension to a defect in transepithelial sodium transport. Adv Exp Med Biol 474: 93-107, 1999.[ISI][Medline]
22. Semenza GL. HIF-1: mediator of physiological and pathophysiological responses to hypoxia. J Appl Physiol 88: 1474-1480, 2000.[Abstract/Free Full Text]
23. Swenson ER, Maggiorini M, Mongovin S, Gibbs JSR, Greve I, Mairbäurl H, and Bärtsch P. Pathogenesis of high-altitude pulmonary edema: inflammation is not an etiologic factor. JAMA 287: 2228-2235, 2002.[Abstract/Free Full Text]
24. Vivona ML, Matthay MA, Chabaud MB, Friedlander G, and Clerici C. Hypoxia reduces alveolar epithelial sodium and fluid transport in rats: reversal by -adrenergic agonist treatment. Am J Respir Cell Mol Biol 25: 554-561, 2001.[Abstract/Free Full Text]
25. Wodopia R, Ko HS, Billian J, Wiesner R, Bärtsch P, and Mairbäurl H. Hypoxia decreases proteins involved in transepithelial electrolyte transport of A549 cells and rat lung. Am J Physiol Lung Cell Mol Physiol 279: L1110-L1119, 2000.[Abstract/Free Full Text]
26. Zhong H and Simons JW. Direct comparison of GAPDH, -actin, cyclophilin, and 28S rRNA as internal standards for quantifying RNA levels under hypoxia. Biochem Biophys Res Commun 259: 523-526, 1999.[ISI][Medline]

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