Increased phosphatidylcholine production but disrupted glycogen metabolism in fetal type II cells of mice that overexpress CTP:phosphocholine cytidylyltransferase

CTP:phosphocholine cytidylyltransferase (CCT) is a rate-determining enzyme in the de novo synthesis of phosphatidylcholine (PtdCho). Alveolar type II cells synthesize large quantities of disaturated PtdCho, the surface-active agent of pulmonary surfactant, particularly at late gestation when the lung prepares itself for postnatal air breathing. To clarify the role of CCTalpha in lung surfactant maturation, we overexpressed CCTalpha(1-367) using the surfactant protein-C promoter. Lungs of transgenic mice were analyzed at day 18 of gestation (term = 19 days). Overexpression of CCTalpha(1-367) increased the synthesis and content of PtdCho in fetal type II cells isolated from the transgenic mice. Also, PtdCho content of fetal lung fluid was increased. No changes in surfactant protein content were detected. Interestingly, fetal type II cells of transgenic mice contained more glycogen than control cells. Incorporation studies with [U-(14)C]glucose demonstrated that overexpression of CCTalpha(1-367) in fetal type II cells increased glycogen synthesis without affecting glycogen breakdown. To determine which domain contributes to this glycogen phenotype, two additional transgenes were created overexpressing either CCTalpha(1-239) or CCTalpha(239-367). Glycogen synthesis and content were increased in fetal type II cells expressing CCTalpha(239-367) but not CCTalpha(1-239)(.) We conclude that overexpression of CCTalpha increases surfactant PtdCho synthesis without affecting surfactant protein levels but that it disrupts glycogen metabolism in differentiating type II cells via its regulatory domain.     

Immunological dysregulation of lung cells in response to vitamin E deficiency

Vitamin E supplementation exhibits anti-inflammatory properties. In the lung, the beneficial effects of vitamin E supplementation on inflammation and infections are well documented, but potential consequences of alimentary vitamin E deficiency to the immunological status of lung cells are not known. It is unclear if temporary vitamin E deficiency exhibits deleterious consequences or can be compensated for by other cellular antioxidants. To address this question, the alimentary vitamin E supply to rats was modified. We then investigated the effects on major histocompatibility molecule (MHC) class II, cell adhesion molecules, interleukin (IL)10, tumor necrosis factor (TNF)alpha in various lung cells. The constitutive expression of MHC class II, intercellular adhesion molecule (ICAM)-1, L-selectin, alpha5-integrin, and CD 166, was demonstrated by flow cytometry on type II pneumocytes, alveolar macrophages, and on co-isolated lymphocytes. Vitamin E depletion increased ICAM-1 and CD166 on type II cells and macrophages, whereas the expression of L-selectin increased only on macrophages. Furthermore, the vitamin E depletion increased the cellular content and secretion of IL10 in type II cells, but decreased the content and secretion of TNFalpha. Vitamin E depletion decreased the cellular vitamin E content, but did not change the activity of antioxidant enzymes (catalase, superoxide dismutase) and the glutathion (GSH)/oxidized glutathion (GSSG) ratio in alveolar type II cells. The shift of protein kinase C (PKC) from the cytosol to membranes indicates that a PKC-dependent signaling pathway may be involved in the change of the immunological status of type II cells. All these effects were reversed by vitamin E repletion. In summary, these results are clearly compatible with the view that a temporary vitamin E deficiency induces a reversible immunological dysregulation in alveolar type II cells and lung macrophages. This deficiency might predispose the lung to develop acute or chronic inflammation.     

Pulmonary-specific expression of tumor necrosis factor-alpha alters surfactant lipid metabolism

Tumor necrosis factor (TNF)-alpha is a major cytokine implicated in inducing acute and chronic lung injury, conditions associated with surfactant phosphatidylcholine (PtdCho) deficiency. Acutely, TNF-alpha decreases PtdCho synthesis but stimulates surfactant secretion. To investigate chronic effects of TNF-alpha, we investigated PtdCho metabolism in a murine transgenic model exhibiting lung-specific TNF-alpha overexpression. Compared with controls, TNF-alpha transgenic mice exhibited a discordant pattern of PtdCho metabolism, with a decrease in PtdCho and disaturated PtdCho (DSPtdCho) content in the lung, but increased levels in alveolar lavage. Transgenics had lower activities and increased immunoreactive levels of cytidylyltransferase (CCT), a key PtdCho biosynthetic enzyme. Ceramide, a CCT inhibitor, was elevated, and linoleic acid, a CCT activator, was decreased in transgenics. Radiolabeling studies revealed that alveolar reuptake of DSPtdCho was significantly decreased in transgenic mice. These observations suggest that chronic expression of TNF-alpha results in a complex pattern of PtdCho metabolism where elevated lavage PtdCho may originate from alveolar inflammatory cells, decreased surfactant reuptake, or altered surfactant secretion. Reduced parenchymal PtdCho synthesis appears to be attributed to CCT enzyme that is physiologically inactivated by ceramide or by diminished availability of activating lipids.     

Maternal loading with very low-density lipoproteins stimulates fetal surfactant synthesis

We examined whether administration of very low-density lipoproteins (VLDL) to pregnant rats increases surfactant phosphatidylcholine (PtdCho) content in fetal pre-type II alveolar epithelial cells. VLDL-triglycerides are hydrolyzed to fatty acids by lipoprotein lipase (LPL), an enzyme activated by heparin. Fatty acids released by LPL can incorporate into the PtdCho molecule or activate the key biosynthetic enzyme cytidylyltransferase (CCT). Dams were given BSA, heparin, VLDL, or VLDL with heparin intravenously. Radiolabeled VLDL given to the pregnant rat crossed the placenta and was distributed systemically in the fetus and incorporated into disaturated PtdCho (DSPtdCho) in pre-type II cells. Maternal administration of VLDL with heparin increased DSPtdCho content in cells by 45% compared with control (P < 0.05). VLDL produced a dose-dependent, saturable, and selective increase in CCT activity. VLDL did not significantly alter immunoreactive CCT content but increased palmitic, stearic, and oleic acids in pre-type II cells. Furthermore, hypertriglyceridemic apolipoprotein E knockout mice contained significantly greater levels of DSPtdCho content in alveolar lavage and CCT activity compared with either LDL receptor knockout mice or wild-type controls that have normal serum triglycerides. Thus the nutritional or genetic modulation of serum VLDL-triglycerides provides specific fatty acids that stimulate PtdCho synthesis and CCT activity thereby increasing surfactant content.     

Leptin mediates the parathyroid hormone-related protein paracrine stimulation of fetal lung maturation

Developing rat lung lipofibroblasts express leptin beginning on embryonic day (E) 17, increasing 7- to 10-fold by E20. Leptin and its receptor are expressed mutually exclusively by fetal lung fibroblasts and type II cells, suggesting a paracrine signaling "loop." This hypothesized mechanism is supported by the following experimental data: 1) leptin stimulates the de novo synthesis of surfactant phospholipid by both fetal rat type II cells (400% x 100 ng(-1) x ml(-1) x 24 h(-1)) and adult human airway epithelial cells (85% x 100 ng(-1) x 24 h(-1)); 2) leptin is secreted by lipofibroblasts in amounts that stimulate type II cell surfactant phospholipid synthesis in vitro; 3) epithelial cell secretions such as parathyroid hormone-related protein (PTHrP), PGE(2), and dexamethasone stimulate leptin expression by fetal rat lung fibroblasts; 4) PTHrP or leptin stimulate the de novo synthesis of surfactant phospholipid (2- to 2.5-fold/24 h) and the expression of surfactant protein B (SP-B; >25-fold/24 h) by fetal rat lung explants, an effect that is blocked by a leptin antibody; and 5) a PTHrP receptor antagonist inhibits the expression of leptin mRNA by explants but does not inhibit leptin stimulation of surfactant phospholipid or SP-B expression, indicating that PTHrP paracrine stimulation of type II cell maturation requires leptin expression by lipofibroblasts. This is the first demonstration of a paracrine loop that functionally cooperates to induce alveolar acinar lung development.     

Vitamin E deficiency sensitizes alveolar type II cells for apoptosis

Pre-term neonates and neonates in general exhibit physiological vitamin E deficiency and are at increased risk for the development of acute lung diseases. Apoptosis is a major cause of acute lung damage in alveolar type II cells. In this paper, we evaluated the hypothesis that vitamin E deficiency predisposes alveolar type II cells to apoptosis. Therefore, we measured markers of apoptosis in alveolar type II cells isolated from control rats, vitamin E deficient rats and deficient rats that were re-fed a vitamin E-enriched diet. Bax and cytosolic cytochrome c increased, and the mitochondrial transmembrane potential and Hsp25 expression was reduced in vitamin E deficiency. Furthermore, increased DNA-fragmentation and numbers of early and late apoptotic cells were seen, but caspases 3 and 8 activities and expression of Fas, Bcl-2, Bcl-x and p53 remained unchanged. Vitamin E depletion did not change the GSH/GSSG ratio and the activities of antioxidant enzymes. Thus, vitamin E deficiency may induce a reversible pro-apoptotic response in lung cells and sensitise them for additional insult. In agreement with this hypothesis, we demonstrate that in vivo hyperoxia alone does not induce apoptosis in type II cells of control rats but reversibly increases DNA-fragmentation and numbers of early apoptotic type II cells in vitamin E-depleted cells.     

High-throughput screening of bacteriorhodopsin mutants in whole cell pastes

Pre-term neonates and neonates in general exhibit physiological vitamin E deficiency and are at increased risk for the development of acute lung diseases. Apoptosis is a major cause of acute lung damage in alveolar type II cells. In this paper, we evaluated the hypothesis that vitamin E deficiency predisposes alveolar type II cells to apoptosis. Therefore, we measured markers of apoptosis in alveolar type II cells isolated from control rats, vitamin E deficient rats and deficient rats that were re-fed a vitamin E-enriched diet. Bax and cytosolic cytochrome c increased, and the mitochondrial transmembrane potential and Hsp25 expression was reduced in vitamin E deficiency. Furthermore, increased DNA-fragmentation and numbers of early and late apoptotic cells were seen, but caspases 3 and 8 activities and expression of Fas, Bcl-2, Bcl-x and p53 remained unchanged. Vitamin E depletion did not change the GSH/GSSG ratio and the activities of antioxidant enzymes. Thus, vitamin E deficiency may induce a reversible pro-apoptotic response in lung cells and sensitise them for additional insult. In agreement with this hypothesis, we demonstrate that in vivo hyperoxia alone does not induce apoptosis in type II cells of control rats but reversibly increases DNA-fragmentation and numbers of early apoptotic type II cells in vitamin E-depleted cells.     

Lysophosphatidylcholine metabolism in Saccharomyces cerevisiae: the role of P-type ATPases in transport and a broad specificity acyltransferase in acylation

We recently described a new route for the synthesis of phosphatidylethanolamine (PtdEtn) from exogenous lyso-PtdEtn, which we have termed the exogenous lysolipid metabolism (ELM) pathway. The ELM pathway for lyso-PtdEtn requires the action of plasma membrane P-type ATPases Dnf1p and Dnf2p and their requisite beta-subunit, Lem3p, for the active uptake of lyso-PtdEtn. In addition, the acyl-CoA-dependent acyltransferase, Ale1p, mediates the acylation of the imported lysolipid to form PtdEtn. We now report that these components of the lyso-PtdEtn ELM pathway are also active with lyso-1-acyl-2-hydroxyl-sn-glycero-3-phosphocholine (PtdCho) as a substrate. Lyso-PtdCho supports the growth of a choline auxotrophic pem1Delta pem2Delta strain. Uptake of radiolabeled lyso-PtdCho was impaired by the dnf2Delta and lem3Delta mutations. Introduction of a lem3Delta mutation into a pem1Delta pem2Delta background impaired the ability of the resulting strain to grow with lyso-PtdCho as the sole precursor of PtdCho. After import of lyso-PtdCho, the recently characterized lyso-PtdEtn acyltransferase, Ale1p, functioned as the sole lyso-PtdCho acyltransferase in yeast. A pem1Delta pem2Delta ale1Delta strain grew with lyso-PtdCho as a substrate but showed a profound reduction in PtdCho content when lyso-PtdCho was the only precursor of PtdCho. Ale1p acylates lyso-PtdCho with a preference for monounsaturated acyl-CoA species, and the specific LPCAT activity of Ale1p in yeast membranes is >50-fold higher than the basal rate of de novo aminoglycerophospholipid biosynthesis from phosphatidylserine synthase activity. In addition to lyso-PtdCho, lyso-PtdEtn, and lyso-phosphatidic acid, Ale1p was also active with lysophosphatidylserine, lysophosphatidylglycerol, and lysophosphatidylinositol as substrates. These results establish a new pathway for the net synthesis of PtdCho in yeast and provide new tools for the study of PtdCho synthesis, transport, and remodeling.     

Altered lung phospholipid metabolism in mice with targeted deletion of lysosomal-type phospholipase A2

Lung surfactant dipalmitoylphosphatidylcholine (DPPC) is endocytosed by alveolar epithelial cells and degraded by lysosomal-type phospholipase A2 (aiPLA2). This enzyme is identical to peroxiredoxin 6 (Prdx6), a bifunctional protein with PLA2 and GSH peroxidase activities. Lung phospholipid was studied in Prdx6 knockout (Prdx6-/-) mice. The normalized content of total phospholipid, phosphatidylcholine (PC), and disaturated phosphatidylcholine (DSPC) in bronchoalveolar lavage fluid, lung lamellar bodies, and lung homogenate was unchanged with age in wild-type mice but increased progressively in Prdx6-/- animals. Degradation of internalized [3H]DPPC in isolated mouse lungs after endotracheal instillation of unilamellar liposomes labeled with [3H]DPPC was significantly decreased at 2 h in Prdx6-/- mice (13.6 +/- 0.3% vs. 26.8 +/- 0.8% in the wild type), reflected by decreased dpm in the lysophosphatidylcholine and the unsaturated PC fractions. Incorporation of [14C]palmitate into DSPC at 24 h after intravenous injection was decreased by 73% in lamellar bodies and by 54% in alveolar lavage surfactant in Prdx6-/- mice, whereas incorporation of [3H]choline was decreased only slightly. Phospholipid metabolism in Prdx6-/- lungs was similar to that in wild-type lungs treated with MJ33, an inhibitor of aiPLA2 activity. These results confirm an important role for Prdx6 in lung surfactant DPPC degradation and synthesis by the reacylation pathway.     

The Lung Surfactant and Immune System Response to Intratracheal Administration of “Empty” Liposomes

Abstract

After intratracheal administration of “empty” lecithin-cholesterol liposomes to rats it was found out twofold enhancement of the surfactant content with maximum on the 2nd-3rd day and with normalization to the control level by the 7th day. Phagocytic index of the alveolar macrophages was also increased. It was shown the change of the blast-transformation reaction of bronchoalveolar lavage and blood lymphocytes. Immune complexes content in bronchoalveolar lavage at different period of time after liposomes administration increased 1.5-2-fold. The natural killers (NK) activity of cells obtained from bronhoalveolar lavage and blood was enhanced 10 times and 2 times respectively. It is supposed that enhancement of lung surfactant phospholipid content is caused by substrate stimulation of type II alveolocytes activity. The stimulation of immunocompetent cells might be connected with imitation of bacterial attack by liposomes with proteins adsorbed on their surface.     

Mainstream and sidestream cigarette smoke exposure increases Ca2+-dependent phospholipid binding proteins in guinea pig alveolar type II cells

We have earlier identified the presence of a 36 kDa Ca²⁺-dependent phospholipid-binding protein (PLBP) in guinea pig alveolar type II cells. PLBP has been suggested to act as a mediator in facilitating and regulating intracellular surfactant assembly and delivery to the plasma membrane of type II cells for secretion into alveolar space. It has been reported that cigarette smoke exposure (CSE) causes a decrease in the surfactant activity in bronchial washings. We have also reported earlier that mainstream (MS) and sidestream (SS) CSE causes desensitization of -adrenoreceptors in guinea pig alveolar type II cells. Since both Ca²⁺ and -adrenoreceptors are involved in surfactant secretion and PLBP is involved in surfactant delivery, it is important to know whether CSE causes any change in the PLBP level in alveolar type II cells. In the present study, we have demonstrated that MS and SS CSE causes a significant increase in the levels of PLBP in alveolar type II cells (107 and 150%, respectively) and in lung lavage (42 and 125%, respectively) in comparison to that in sham control (430 ng/mg protein in alveolar type II cells and 780 ng/mg protein in lung lavage). The mechanism by which smoke exposure causes an elevation in the levels of PLBP in alveolar type II cells and lung lavage remains to be investigated.     

Macrophage and type II cell catabolism of SP-A and saturated phosphatidylcholine in mouse lungs

Type II cells and macrophages are the major cells involved in the alveolar clearance and catabolism of surfactant. We measured type II cell and macrophage contributions to the catabolism of saturated phosphatidylcholine and surfactant protein A (SP-A) in mice. We used intratracheally administered SP-A labeled with residualizing (125)I-dilactitol-tyramine, radiolabeled dipalmitoylphosphatidylcholine ([(3)H]DPPC), and its degradation-resistant analog [(14)C]DPPC-ether. At 15 min and 7, 19, 29, and 48 h after intratracheal injection, the mice were killed; alveolar lavage was then performed to recover macrophages and surfactant. Type II cells and macrophages not recovered by the lavage were subsequently isolated by enzymatic digestion of the lung. Radioactivity was measured in total lung, lavage fluid macrophages, alveolar washes, type II cells, and lung digest macrophages. Approximately equal amounts of (125)I-dilactitol-tyramine-SP-A and [(14)C]DPPC-ether associated with the macrophages (lavage fluid plus lung digest) and type II cells when corrected for the efficiency of type II cell isolation. Eighty percent of the macrophage-associated radiolabel was recovered from lung digest macrophages. We conclude that macrophages and type II cells contribute equally to saturated phosphatidylcholine and SP-A catabolism in mice.     

Effects of pulmonary surfactant and surfactant protein A on phagocytosis of fractionated alveolar macrophages: relationship to starvation.

Pulmonary surfactant isolated from bronchoalveolar lavage fluid of rat lung contained a high content of surfactant protein A (SP-A) in starved for 2 days compared to fed controls, but this phenomena returned to baseline following more than 4 days starvation. As determined by immunoperoxidase staining of lung sections using SP-A antibody, SP-A could be consistently observed in nonciliated bronchiolar (Clara) cells, alveolar type II cells and some alveolar macrophages (AM). Fc receptor-mediated phagocytosis of AM was enhanced by SP-A, which was dependent on the dosis and reached a maximum at 10 micrograms of SP-A/ml. Antibody to SP-A completely inhibited the enhanced response of phagocytosis. When exposed AM subpopulations, separated into four fractions (I, II, III and IV) by discontinuous Percoll gradient, to SP-A or pulmonary surfactant prepared from rats fed and starved for 2 days enhanced their phagocytic activity in high dense cells (III and IV), particularly to SP-A and pulmonary surfactant from rats starved for 2 days. Whereas little change in lower dense fractions (I and II) were seen in all exposures except for SP-A that enhanced the cells of fraction II. These results supported the concept that pulmonary surfactant and its apoprotein, SP-A, are a factor to regulate lung defense system including activation of AM that undergo different processes following starvation.     

Association of chlorphentermine with phospholipids in rat alveolar lavage materials, alveolar macrophages and type II cells

Administration of chlorphentermine to rats leads to an increase in the phospholipid content of pulmonary surfactant materials and alveolar macrophages. It is known that this drug binds to pure phospholipids and prevents their degradation by phospholipases. Therefore, experiments were carried out to determine if chlorphentermine binds to surfactant phospholipids in vitro and to measure the in vivo association of drug with phospholipids in alveolar lavage materials from rats injected with [14C]chlorphentermine. The presence of chlorphentermine in alveolar macrophages, type II cells and other small pneumocytes (a population of lung cells which does not include alveolar macrophages or type II cells) from treated animals was also assessed. Binding of the drug to surfactant phospholipids, as measured with the fluorescent probe, 1-anilino-8-naphthalene sulfonate, occurs in vitro and does not differ in various subfractions of alveolar lavage materials isolated by differential centrifugation. Following daily administration of chlorphentermine to rats for 3 days, the drug appears to be associated with surfactant phospholipids such that the molar ratio is 1:100 (chlorphentermine/phospholipid). Chlorphentermine is also associated with alveolar macrophages (molar ratio, 1:18) and type II cells (molar ratio, 1:33). Not much drug is associated with the population of other lung cells (molar ratio, 1:333). In alveolar macrophages, approx. 70% of the drug seems to be bound to phospholipid and/or sequestered in subcellular organelles. However, only 20% of the chlorphentermine is bound and/or sequestered in type II cells. The results of these experiments suggest that following chlorphentermine administration, the drug is associated with phospholipids in acellular pulmonary lavage materials, alveolar macrophages and type II cells. This drug-phospholipid interaction may impair phospholipid degradation and lead to a phospholipidosis in surfactant materials and alveolar macrophages.     

Organotypic cultures of diploid type II alveolar pneumonocytes: surfactant associated esterase activity

Organotypic cultures, established from enzymatically dispersed day 19 fetal rat lung, are comprised primarily of cells which are morphologically similar to type II alveolar pneumonocytes, the cells involved in surfactant synthesis. To further characterize these cultures, the nonspecific esterase pool was examined to determine if these cultures contained certain nonspecific esterases previously shown to be enzyme markers for the surfactant system. The results of biochemical, electrophoretic and cytochemical studies indicate that these organotypic cultures contain the same nonspecific esterases already demonstrated in surface active fractions derived from rat and mouse lung homogenates and pulmonary lavage fluid. As in whole lung, the major site of esterase activity in the organotypic cultures is the type II cell lamellar body, the putative site of surfactant synthesis and storage. These findings support the concept that the organotypic cultures derived from fetal rat lung are comprised predominantly of type II cells which retain surfactant associated functions in vitro.     

Surfactant protein D influences surfactant ultrastructure and uptake by alveolar type II cells

Surfactant protein D (SP-D) is a member of the collectin family of the innate host defense proteins. In the lung, SP-D is expressed primarily by type II cells. Gene-targeted SP-D-deficient [SP-D(-/-)] mice have three- to fivefold higher surfactant lipid pool sizes. However, surfactant synthesis and secretion by type II cells and catabolism by alveolar macrophages are normal in SP-D(-/-) mice. Therefore, we hypothesized that SP-D might regulate surfactant homeostasis by influencing surfactant structure, thereby altering its uptake by type II cells. Large (LA) and small aggregate (SA) surfactant were isolated from bronchoalveolar lavage fluid (BALF) from SP-D(-/-), wild-type [SP-D(+/+)], and transgenic mice in which SP-D was expressed under conditional control of doxycycline in alveolar type II cells. Uptake of both LA and SA isolated from SP-D(-/-) mice by normal type II cells was decreased. Abnormally dense lipid forms were observed by electron microscopy of LA from SP-D(-/-) mice. SA from SP-D(-/-) mice consisted of atypical multilamellated small vesicles. Abnormalities in surfactant uptake by type II cells and in surfactant ultrastructure were corrected by conditional expression of SP-D in vivo. Preincubation of BALF from SP-D(-/-) mice with SP-D changed surfactant ultrastructure to be similar to that of SP-D(+/+) mice in vitro. The rapid changes in surfactant structure, increased uptake by type II cells, and decreased pool sizes normally occurring in the postnatal period were not seen in SP-D(-/-) mice. SP-D regulates uptake and catabolism by type II cells and influences the ultrastructure of surfactant in the alveolus.     

Platelet-activating factor (PAF)-acetylhydrolase and PAF-like compounds in the lung: effects of hyperoxia.

Platelet-activating factor (PAF)-acetylhydrolase is the enzyme modulating in tissues and biological fluids the concentration of the proinflammatory factors PAF and PAF-like oxidation products of phospholipids (PAF-like compounds). We investigated whether there is a relation between PAF-acetylhydrolase activity and the concentration of PAF-like compounds in bronchoalveolar lavage (BAL). We found that alveolar type II cells are an additional source of PAF-acetylhydrolase in BAL beside macrophages. Secretion of PAF-acetylhydrolase was stimulated by phorbol ester in alveolar type II cells but not in macrophages. Studies in BAL suggested that secreted PAF-acetylhydrolase was bound to alveolar surfactant. Exposure of rats to high oxygen concentration reduced the activity of PAF-acetylhydrolase in BAL and macrophages, but not in plasma or alveolar type II cells. In contrast, hyperoxia increased the concentration of PAF-like-compounds, lipid hydroperoxides and malonedialdehyde in plasma but not in BAL. Therefore, we conclude that neither the oxidant-induced decrease of the PAF-acetylhydrolase activity nor the direct peroxidation of surfactant lipids in the alveoli provide a likely mechanism for hyperoxia-induced lung injury. Instead, lung injury is apparently caused by lipid peroxidation in plasma rather than by high oxygen pressure in the alveoli.