Chronic rhinosinusitis with nasal polyps is associated with impaired TMEM16A-mediated epithelial chloride secretion.

BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is one of the most common chronic disorders with limited therapeutic options. However, the pathogenesis of CRSwNP remains poorly understood. OBJECTIVE: We sought to determine the role of abnormalities in nasal epithelial ion transport in primary epithelial cultures and patients with CRSwNP. METHODS: We studied epithelial ion transport and transcript levels of the Cl- channels cystic fibrosis transmembrane conductance regulator and transmembrane protein 16A (TMEM16A) in human primary nasal epithelial cultures of patients with CRSwNP and healthy controls. Furthermore, we determined expression levels of proinflammatory cytokines that have been implicated in the regulation of epithelial ion channels (IL-1ß, INF-γ, TNF-α, IL-13) and studied effects of the key TH2 signaling molecule IL-13 in CRSwNP and control nasal epithelial cultures. Finally, we measured in vivo nasal potential difference to compare epithelial ion transport in patients with CRSwNP and controls. RESULTS: Bioelectric studies demonstrated that Ca2+-activated Cl- secretion was reduced in CRSwNP versus control nasal epithelial cultures. Transcript levels of IL-13 and the Ca2+-activated Cl- channel TMEM16A were increased in CRSwNP cultures. Stimulation with IL-13 increased TMEM16A expression further and restored Ca2+-activated Cl- secretion in CRSwNP cultures. Nasal potential difference measurements demonstrated reduced Ca2+-activated Cl- transport in patients with CRSwNP versus controls. CONCLUSIONS: This study demonstrates that TMEM16A-mediated Ca2+-activated Cl- secretion is reduced in primary nasal epithelial cultures and nasal epithelia of patients with CRSwNP. Our data suggest that the Ca2+-activated Cl- channel TMEM16A may be implicated in the pathogenesis and serve as a novel therapeutic target in patients with CRSwNP.

Hypoxia Aggravates Inhibition of Alveolar Epithelial Na-Transport by Lipopolysaccharide-Stimulation of Alveolar Macrophages.

Inflammation and hypoxia impair alveolar barrier tightness, inhibit Na- and fluid reabsorption, and cause edema. We tested whether stimulated alveolar macrophages affect alveolar Na-transport and whether hypoxia aggravates the effects of inflammation, and tested for involved signaling pathways. Primary rat alveolar type II cells (rA2) were co-cultured with rat alveolar macrophages (NR8383) or treated with NR8383-conditioned media after stimulation with lipopolysaccharide (LPS; 1 µg/mL) and exposed to normoxia and hypoxia (1.5% O2). LPS caused a fast, transient increase in TNFα and IL-6 mRNA in macrophages and a sustained increase in inducible nitric oxide synthase (NOS2) mRNA in macrophages and in rA2 cells resulting in elevated nitrite levels and secretion of TNF-α and IL-6 into culture media. In normoxia, 24 h of LPS treated NR8383 decreased the transepithelial electrical resistance (TEER) of co-cultures, of amiloride-sensitive short circuit current (ISCΔamil); whereas Na/K-ATPase activity was not affected. Inhibition was also seen with conditioned media from LPS-stimulated NR8383 on rA2, but was less pronounced after dialysis to remove small molecules and nitrite. The effect of LPS-stimulated macrophages on TEER and Na-transport was fully prevented by the iNOS-inhibitor L-NMMA applied to co-cultures and to rA2 mono-cultures. Hypoxia in combination with LPS-stimulated NR8383 totally abolished TEER and ISCΔamil. These results indicate that the LPS-stimulation of alveolar macrophages impairs alveolar epithelial Na-transport by NO-dependent mechanisms, where part of the NO is produced by rA2 induced by signals from LPS stimulated alveolar macrophages.

Impairment of left atrial mechanics does not contribute to the reduction in stroke volume after active ascent to 4559 m.

Hypoxia challenges left ventricular (LV) function due to reduced energy supply. Conflicting results exist whether high-altitude exposure impairs LV diastolic function and thus contributes to the high altitude-induced increase in systolic pulmonary artery pressure (sPAP) and reduction in stroke volume (SV). This study aimed to assess LV diastolic function, LV end-diastolic pressure (LVEDP), and LA mechanics using comprehensive echocardiographic imaging in healthy volunteers at 4559 m. Fifty subjects performed rapid (<20 hours) and active ascent from 1130 m to 4559 m (high). All participants underwent echocardiography during baseline examination at 424 m (low) as well as 7, 20 and 44 hours after arrival at high altitude. Heart rate (HR), sPAP, and comprehensive volumetric- and Doppler- as well as speckle tracking-derived LA strain parameters were obtained to assess LV diastolic function, LA mechanics, and LVEDP in a multiparametric approach. Data for final analyses were available in 46 subjects. HR (low: 64 ± 11 vs high: 79 ± 14 beats/min, P < 0.001) and sPAP (low: 24.4 ± 3.8 vs high: 38.5 ± 8.2 mm Hg, P < 0.001) increased following ascent and remained elevated at high altitude. Stroke volume (low: 64.5 ± 15.0 vs high: 58.1 ± 16.4 mL, P < 0.001) and EDV decreased following ascent and remained decreased at high altitude due to decreased LV passive filling volume, whereas LA mechanics were preserved. There was no case of LV diastolic dysfunction or increased LVEDP estimates. In summary, this study shows that rapid and active ascent of healthy individuals to 4559 m impairs passive filling and SV of the LV. These alterations were not related to changes in LV and LA mechanics.

Effects of Lumacaftor-Ivacaftor Therapy on Cystic Fibrosis Transmembrane Conductance Regulator Function in Phe508del Homozygous Patients with Cystic Fibrosis.

RATIONALE: The combination of the CFTR (cystic fibrosis transmembrane conductance regulator) corrector lumacaftor with the potentiator ivacaftor has been approved for the treatment of patients with cystic fibrosis homozygous for the Phe508del CFTR mutation. The phase 3 trials examined clinical outcomes but did not evaluate CFTR function in patients. OBJECTIVES: To examine the effect of lumacaftor-ivacaftor on biomarkers of CFTR function in Phe508del homozygous patients with cystic fibrosis aged 12 years and older. METHODS: This prospective observational study assessed clinical outcomes including FEV1% predicted and body mass index, and CFTR biomarkers including sweat chloride concentration, nasal potential difference, and intestinal current measurement before and 8-16 weeks after initiation of lumacaftor-ivacaftor. MEASUREMENTS AND MAIN RESULTS: A total of 53 patients were enrolled in the study, and 52 patients had baseline and follow-up measurements. After initiation of lumacaftor-ivacaftor sweat chloride concentrations were reduced by 17.8 mmol/L (interquartile range [IQR], -25.9 to -6.1; P < 0.001), nasal potential difference showed partial rescue of CFTR function in nasal epithelia to a level of 10.2% (IQR, 0.0-26.1; P < 0.011), and intestinal current measurement showed functional improvement in rectal epithelia to a level of 17.7% of normal (IQR, 10.8-29.0; P < 0.001). All patients improved in at least one CFTR biomarker, but no correlations were found between CFTR biomarker responses and clinical outcomes. CONCLUSIONS: Lumacaftor-ivacaftor results in partial rescue of Phe508del CFTR function to levels comparable to the lower range of CFTR activity found in patients with residual function mutations. Functional improvement was detected even in the absence of short-term improvement of FEV1% predicted and body mass index. Clinical trial registered with www.clinicaltrials.gov (NCT02807415).

The Hen or the Egg: Impaired Alveolar Oxygen Diffusion and Acute High-altitude Illness?

Individuals ascending rapidly to altitudes >2500 m may develop symptoms of acute mountain sickness (AMS) within a few hours of arrival and/or high-altitude pulmonary edema (HAPE), which occurs typically during the first three days after reaching altitudes above 3000-3500 m. Both diseases have distinct pathologies, but both present with a pronounced decrease in oxygen saturation of hemoglobin in arterial blood (SO2). This raises the question of mechanisms impairing the diffusion of oxygen (O2) across the alveolar wall and whether the higher degree of hypoxemia is in causal relationship with developing the respective symptoms. In an attempt to answer these questions this article will review factors affecting alveolar gas diffusion, such as alveolar ventilation, the alveolar-to-arterial O2-gradient, and balance between filtration of fluid into the alveolar space and its clearance, and relate them to the respective disease. The resultant analysis reveals that in both AMS and HAPE the main pathophysiologic mechanisms are activated before aggravated decrease in SO2 occurs, indicating that impaired alveolar epithelial function and the resultant diffusion limitation for oxygen may rather be a consequence, not the primary cause, of these altitude-related illnesses.

Identification of a Prognostic Hypoxia-Associated Gene Set in IDH-Mutant Glioma.

Glioma growth is often accompanied by a hypoxic microenvironment favorable for the induction and maintenance of the glioma stem cell (GSC) phenotype. Due to the paucity of cell models of Isocitrate Dehydrogenase 1 mutant (IDH1mut) GSCs, biology under hypoxic conditions has not been sufficiently studied as compared to IDH1 wildtype (IDH1wt) GSCs. We therefore grew well-characterized IDH1mut (n = 4) and IDH1wt (n = 4) GSC lines under normoxic (20%) and hypoxic (1.5%) culture conditions and harvested mRNA after 72 h. Transcriptome analyses were performed and hypoxia regulated genes were further analyzed using the expression and clinical data of the lower grade glioma cohort of The Cancer Genome Atlas (LGG TCGA) in a confirmatory approach and to test for possible survival associations. Results show that global expression changes were more pronounced in IDH1wt than in IDH1mut GSCs. However, when focusing on known hypoxia-regulated gene sets, enrichment analyses showed a comparable regulation in both IDH1mut and IDH1wt GSCs. Of 272 significantly up-regulated genes under hypoxic conditions in IDH1mut GSCs a hypoxia-related survival score (HRS-score) of five genes (LYVE1, FAM162A, WNT6, OTP, PLOD1) was identified by the Least Absolute Shrinkage and Selection Operator (LASSO) algorithm which was able to predict survival independent of age, 1p19q co-deletion status and WHO grade (II vs. III) in the LGG TCGA cohort and in the Rembrandt dataset. Altogether, we were able to identify and validate a novel hypoxia-related survival score in IDH1mut GSCs consisting of five hypoxia-regulated genes which was significantly associated with patient survival independent of known prognostic confounders.

Factor inhibiting HIF-1 (FIH-1) modulates protein interactions of apoptosis-stimulating p53 binding protein 2 (ASPP2).

The asparaginyl hydroxylase factor inhibiting HIF-1 (FIH-1) is an important suppressor of hypoxia-inducible factor (HIF) activity. In addition to HIF-α, FIH-1 was previously shown to hydroxylate other substrates within a highly conserved protein interaction domain, termed the ankyrin repeat domain (ARD). However, to date, the biological role of FIH-1-dependent ARD hydroxylation could not be clarified for any ARD-containing substrate. The apoptosis-stimulating p53-binding protein (ASPP) family members were initially identified as highly conserved regulators of the tumour suppressor p53. In addition, ASPP2 was shown to be important for the regulation of cell polarity through interaction with partitioning defective 3 homolog (Par-3). Using mass spectrometry we identified ASPP2 as a new substrate of FIH-1 but inhibitory ASPP (iASPP) was not hydroxylated. We demonstrated that ASPP2 asparagine 986 (N986) is a single hydroxylation site located within the ARD. ASPP2 protein levels and stability were not affected by depletion or inhibition of FIH-1. However, FIH-1 depletion did lead to impaired binding of Par-3 to ASPP2 while the interaction between ASPP2 and p53, apoptosis and proliferation of the cancer cells were not affected. Depletion of FIH-1 and incubation with the hydroxylase inhibitor dimethyloxalylglycine (DMOG) resulted in relocation of ASPP2 from cell-cell contacts to the cytosol. Our data thus demonstrate that protein interactions of ARD-containing substrates can be modified by FIH-1-dependent hydroxylation. The large cellular pool of ARD-containing proteins suggests that FIH-1 can affect a broad range of cellular functions and signalling pathways under certain conditions, for example, in response to severe hypoxia.

The Increase in Hemoglobin Concentration With Altitude Differs Between World Regions and Is Less in Children Than in Adults.

To compensate for decreased oxygen partial pressure, high-altitude residents increase hemoglobin concentrations [Hb]. The elevation varies between world regions, posing problems in defining cutoff values for anemia or polycythemia. The currently used altitude adjustments (World Health Organization [WHO]), however, do not account for regional differences. Data from The Demographic and Health Survey (DHS) Program were analyzed from 32 countries harboring >4% of residents at altitudes above 1000 m. [Hb]-increase, (ΔHb/km altitude) was calculated by linear regression analysis. Tables show 95% reference intervals (RIs) for different altitude ranges, world regions, and age groups. The prevalence of anemia and polycythemia was calculated using regressions in comparison to WHO adjustments. The most pronounced Δ[Hb]/km was found in East Africans and South Americans while [Hb] increased least in South/South-East Asia. In African regions and Middle East, [Hb] was decreased in some altitude regions showing inconsistent changes in different age groups. Of note, in all regions, the Δ[Hb]/km was lower in children than in adults, and in the Middle East, it was even negative. Overall, the Δ[Hb]/km from our analysis differed from the region-independent adjustments currently suggested by the WHO resulting in a lower anemia prevalence at very high altitudes. The distinct patterns of Δ[Hb] with altitude in residents from different world regions imply that one single, region-independent correction factor for altitude is not be applicable for diagnosing abnormal [Hb]. Therefore, we provide regression coefficients and reference-tables that are specific for world regions and altitude ranges to improve diagnosing abnormal [Hb].

MicroRNA-20a inhibits stress-induced cardiomyocyte apoptosis involving its novel target Egln3/PHD3.

Excessive stress, e.g. due to biomechanical overload or ischemia/reperfusion is a potent inductor of cardiomyocyte apoptosis, which contributes to maladaptive remodeling. Despite substantial progress in the understanding of the molecular pathophysiology, many components of the signaling pathways underlying remodeling in general and apoptosis in particular still remain unknown. Recent evidence suggests that microRNAs (miRs) play an important role in the heart's response to increased cardiac stress. To identify novel modulators of stress-dependent remodeling, we conducted a genome-wide miR-screen of mechanically stretched neonatal rat cardiomyocytes (NRCM). Out of 351 miRs, eight were significantly regulated by biomechanical stress, including microRNA-20a, which is part of the miR17-92 cluster. Interestingly, further expression analyses also revealed upregulation of microRNA-20a in an in vitro hypoxia/"reperfusion" model. Given the potential apoptosis-modulating properties of the miR17-92 cluster, we subjected NRCM to hypoxia and subsequent reoxygenation. AdmiR-20a significantly inhibited hypoxia-mediated apoptosis in a dose-dependent fashion, while targeted knockdown of miR-20a in NRCM induced cardiomyocyte apoptosis. Mechanistically, the antiapoptotic effect of miR-20a appears to be mediated through direct targeting and subsequent downregulation of the proapoptotic factor Egln3. Thus, miR-20a is upregulated in acute biomechanical stress as well as hypoxia and inhibits apoptosis in cardiomyocytes. These properties reveal miR-20a as a cardioprotective micro-RNA and a potential target for novel therapeutic strategies to prevent cardiac remodeling.

Adeno-Associated Virus-Mediated Gene Transfer of Inducible Nitric Oxide Synthase to an Animal Model of Pulmonary Hypertension.

Pulmonary hypertension (PH) is characterized by progressive obstruction of pulmonary arteries owing to inflammatory processes, cellular proliferation, and extracellular matrix deposition and vasoconstriction. As treatment options are limited, we studied gene transfer of an inducible nitric oxide synthase (iNOS) using adeno-associated virus (AAV) vectors specifically targeted at endothelial cells of pulmonary vessels in a murine model of PH. Adult mice were intravenously injected with AAV vectors expressing iNOS. Mice were subjected to hypoxia for 3 weeks and killed afterward. We found elevated levels of iNOS both in lung tissue and pulmonary endothelial cells in hypoxic controls that could be further increased by AAV-mediated iNOS gene transfer. This additional increase in iNOS was associated with decreased wall thickness of pulmonary vessels, less macrophage infiltration, and reduced molecular markers of fibrosis. Taken together, using a tissue-targeted approach, we show that AAV-mediated iNOS overexpression in endothelial cells of the pulmonary vasculature significantly decreases vascular remodeling in a murine model of PH, suggesting upregulation of iNOS as promising target for treatment of PH.

In Vitro Erythropoiesis at Different pO2 Induces Adaptations That Are Independent of Prior Systemic Exposure to Hypoxia.

Hypoxia is associated with increased erythropoietin (EPO) release to drive erythropoiesis. At high altitude, EPO levels first increase and then decrease, although erythropoiesis remains elevated at a stable level. The roles of hypoxia and related EPO adjustments are not fully understood, which has contributed to the formulation of the theory of neocytolysis. We aimed to evaluate the role of oxygen exclusively on erythropoiesis, comparing in vitro erythroid differentiation performed at atmospheric oxygen, a lower oxygen concentration (three percent oxygen) and with cultures of erythroid precursors isolated from peripheral blood after a 19-day sojourn at high altitude (3450 m). Results highlight an accelerated erythroid maturation at low oxygen and more concave morphology of reticulocytes. No differences in deformability were observed in the formed reticulocytes in the tested conditions. Moreover, hematopoietic stem and progenitor cells isolated from blood affected by hypoxia at high altitude did not result in different erythroid development, suggesting no retention of a high-altitude signature but rather an immediate adaptation to oxygen concentration. This adaptation was observed during in vitro erythropoiesis at three percent oxygen by a significantly increased glycolytic metabolic profile. These hypoxia-induced effects on in vitro erythropoiesis fail to provide an intrinsic explanation of the concept of neocytolysis.

Hemoglobin is an oxygen-dependent glutathione buffer adapting the intracellular reduced glutathione levels to oxygen availability.

Fast changes in environmental oxygen availability translate into shifts in mitochondrial free radical production. An increase in intraerythrocytic reduced glutathione (GSH) during deoxygenation would support the detoxification of exogenous oxidants released into the circulation from hypoxic peripheral tissues. Although reported, the mechanism behind this acute oxygen-dependent regulation of GSH in red blood cells remains unknown. This study explores the role of hemoglobin (Hb) in the oxygen-dependent modulation of GSH levels in red blood cells. We have demonstrated that a decrease in Hb O2 saturation to 50% or less observed in healthy humans while at high altitude, or in red blood cell suspensions results in rising of the intraerythrocytic GSH level that is proportional to the reduction in Hb O2 saturation. This effect was not caused by the stimulation of GSH de novo synthesis or its release during deglutathionylation of Hb's cysteines. Using isothermal titration calorimetry and in silico modeling, we observed the non-covalent binding of four molecules of GSH to oxy-Hb and the release of two of them upon deoxygenation. Localization of the GSH binding sites within the Hb molecule was identified. Oxygen-dependent binding of GSH to oxy-Hb and its release upon deoxygenation occurred reciprocally to the binding and release of 2,3-bisphosphoglycerate. Furthermore, noncovalent binding of GSH to Hb moderately increased Hb oxygen affinity. Taken together, our findings have identified an adaptive mechanism by which red blood cells may provide an advanced antioxidant defense to respond to oxidative challenges immediately upon deoxygenation.

ß2-Adrenergics in hypoxia desensitize receptors but blunt inhibition of reabsorption in rat lungs.

Alveolar edema and decreased inspired Po(2) decrease the oxygen supply to alveolar epithelia, impairing ß(2)-adrenergic receptor (ß2AR) signaling and alveolar reabsorption. ß2AR agonists potently stimulate alveolar reabsorption. Thus, hypoxia impairs a major defense mechanism that provides protection from alveolar edema. Because in vivo data on the combined effects of prolonged hypoxia and ß2AR agonist treatment on ß2AR signaling are sparse, we tested whether in vivo hypoxia augments the inactivation of ß2AR during prolonged stimulation. Rats were exposed to normoxia (N) and hypoxia (8% O(2); H), and were also treated with terbutaline (T; 2.5 mg/kg, intraperitoneal, twice daily) or saline (S) for 4 days. ß2AR signaling was studied in alveolar epithelial (ATII) cells and in whole-lung tissue from treated rats. The terbutaline-stimulated formation of cyclic adenosine monophosphate was decreased by approximately 40% in whole lung and in ATII cells of NT, HS, and HT. The effects were not additive. The ß2AR number was increased in HS, but decreased in NT and HT. Treatment increased the G-protein-coupled receptor kinase 2 protein in the plasma membranes of ATII cells, but did not affect G proteins. In vivo hypoxia significantly decreased total and amiloride-sensitive alveolar fluid reabsorption, which was prevented by acute alveolar treatment and 4 days of systemic terbutaline treatment. The αENaC (subunit of epithelial Na channels) protein in plasma membranes was increased in HT, without effects on mRNA. These results indicate that prolonged alveolar hypoxia and treatment with terbutaline impaired ß2AR signaling in alveolar epithelia and in whole lungs, and this signaling was not further impaired by hypoxia. Despite impaired ß2AR signaling, treatment with terbutaline for 4 days prevented the inhibition of alveolar reabsorption caused by in vivo hypoxia.

Expression and regulation of AC133 and CD133 in glioblastoma.

The biological significance of CD133 in glioblastoma is controversial. Above all, there is disagreement concerning the proper approach, the appropriate (cell) model and the suitable microenvironment to study this molecule, often leading to inconsistent experimental results among studies. In consideration of a primary need to dissect and to understand the CD133 phenotype in glioblastoma we performed a comprehensive analysis of CD133 expression and regulation in a large set of glioblastoma cell lines (n = 20) as well as in tumor xenografts. Our analysis considered alternatively spliced mRNA transcripts, different protein epitopes as well as varying sub-cellular localizations of CD133 and explored its regulation under pertinent micro-environmental conditions. CD133 mRNA and CD133 protein could be detected in all relevant types of glioblastoma cell lines. In addition, we detected frequent intracellular CD133 protein accumulations located to the ER and/or Golgi apparatus but seemingly unrelated to particular CD133 splice variants or protein epitopes. In contrast, membrane-bound expression of CD133 was restricted to tumor cells bearing the extracellular CD133 epitope AC133. Only in these cells, differentiation and oxygen levels clearly impacted on AC133 expression and to some extent also influenced CD133 mRNA and protein expression. Most importantly, however, modulation of AC133 levels could occur independently of changes in CD133 mRNA transcription, CD133 protein translation, protein retention or protein shedding. Our results suggest that the AC133 epitope, rather than CD133 mRNA or protein, mirrors malignancy-related tumor traits such as tumor differentiation and local oxygen tension levels, and thus corroborate its role as a biologically relevant cancer marker.

Loss or silencing of the PHD1 prolyl hydroxylase protects livers of mice against ischemia/reperfusion injury.

BACKGROUND & AIMS: Liver ischemia/reperfusion (I/R) injury is a frequent cause of organ dysfunction. Loss of the oxygen sensor prolyl hydroxylase domain enzyme 1 (PHD1) causes tolerance of skeletal muscle to hypoxia. We assessed whether loss or short-term silencing of PHD1 could likewise induce hypoxia tolerance in hepatocytes and protect them against hepatic I/R damage. METHODS: Hepatic ischemia was induced in mice by clamping of the portal vessels of the left lateral liver lobe; 90 minutes later livers were reperfused for 8 hours for I/R experiments. Hepatocyte damage following ischemia or I/R was investigated in PHD1-deficient (PHD1(-/-)) and wild-type mice or following short hairpin RNA-mediated short-term inhibition of PHD1 in vivo. RESULTS: PHD1(-/-) livers were largely protected against acute ischemia or I/R injury. Among mice subjected to hepatic I/R followed by surgical resection of all nonischemic liver lobes, more than half of wild-type mice succumbed, whereas all PHD1(-/-) mice survived. Also, short-term inhibition of PHD1 through RNA interference-mediated silencing provided protection against I/R. Knockdown of PHD1 also induced hypoxia tolerance of hepatocytes in vitro. Mechanistically, loss of PHD1 decreased production of oxidative stress, which likely relates to a decrease in oxygen consumption as a result of a reprogramming of hepatocellular metabolism. CONCLUSIONS: Loss of PHD1 provided tolerance of hepatocytes to acute hypoxia and protected them against I/R-damage. Short-term inhibition of PHD1 is a novel therapeutic approach to reducing or preventing I/R-induced liver injury.

Acute in vitro hypoxia and high-altitude (4,559 m) exposure decreases leukocyte oxygen consumption.

Hypoxia impairs metabolic functions by decreasing activity and expression of ATP-consuming processes. To separate hypoxia from systemic effects, we tested whether hypoxia at high altitude affects basal and PMA-stimulated leukocyte metabolism and how this compares to acute (15 min) and 24 h of in vitro hypoxia. Leukocytes were prepared at low altitude and ∼24 h after arrival at 4559 m. Mitochondrial oxygen consumption (JO2) was measured by respirometry, oxygen radicals by electron spin resonance spectroscopy, both at a Po2 = 100 mmHg (JO2,100) and 20 mmHg (JO2,20). Acute hypoxia of leukocytes decreased JO2 at low altitude. Exposure to high altitude decreased JO2,100, whereas JO2,20 was not affected. Acute hypoxia of low-altitude samples decreased the activity of complexes I, II, and III. At high altitude, activity of complexes I and III were decreased when measured in normoxia. Stimulation of leukocytes with PMA increased JO2,100 at low (twofold) and high altitude (five-fold). At both locations, PMA-stimulated JO2 was decreased by acute hypoxia. Basal and PMA-stimulated reactive oxygen species (ROS) production were unchanged at high altitude. Separate in vitro experiments performed at low altitude show that ∼75% of PMA-induced increase in JO2 was due to increased extra-mitochondrial JO2 (JO2(,res); in the presence of rotenone and antimycin A). JO2(,res) was doubled by PMA. Acute hypoxia decreased basal JO2(,res) by ∼70% and PMA-stimulated JO2(,res) by about 50% in cells cultured in normoxia and hypoxia (1.5% O2; 24 h). Conversely, 24 h in vitro hypoxia decreased mitochondrial JO2,100 and JO2,20, extra-mitochondrial, basal, and PMA-stimulated JO2 were not affected. These results show that 24 h of high altitude but not 24 h in vitro hypoxia decreased basal leukocyte metabolism, whereas PMA-induced JO2 and ROS formation were not affected, indicating that prolonged high-altitude hypoxia impairs mitochondrial metabolism but does not impair respiratory burst. In contrast, acute hypoxia impairs respiratory burst at either altitude.