help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ahmad, S.
Right arrow Articles by White, C. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ahmad, S.
Right arrow Articles by White, C. W.
American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 179-187, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0004OC

Hypoxia Protects Human Lung Microvascular Endothelial and Epithelial-like Cells against Oxygen Toxicity

Role of Phosphatidylinositol 3-Kinase

Shama Ahmad*, Aftab Ahmad*, Evgenia Gerasimovskaya, Kurt R. Stenmark, Corrie B. Allen and Carl W. White

Department of Pediatrics, National Jewish Medical and Research Center; and Developmental Lung Biology Laboratory, University of Colorado Health Sciences Center, Denver, Colorado.

Address correspondence to: Carl W. White, M.D., 1400 Jackson Street, Room B109, Denver, CO 80206. E-mail: whitec{at}njc.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hypoxic preconditioning is protective against oxidant-related damage in various organs, such as the heart. We previously showed that rats exposed to hypoxia also exhibit resistance to lethal pulmonary oxygen toxicity. The underlying mechanism and whether similar preconditioning is applicable to cellular models is unknown. In the present study, it was found that hypoxic pre-exposure induces a significant protective effect against hyperoxia-induced cell death in human lung microvascular endothelial cells (HLMVECs) and epithelial type II-like A549 cells. This effect of hypoxia is mediated by the phosphatidylinositol 3-kinase (PI3-K) signaling pathway because the presence of the PI3-K inhibitors, LY294002 and wortmannin, during pre-exposure to hypoxia completely blocks subsequent protection. Further, the hypoxia-dependent protection from hyperoxia was found to be associated with a 2-fold increase in PI3-K activity in hypoxia. Transient overexpression of a catalytically active class IA PI3-K p110{alpha} isoform also enhanced survival of A549 cells 2-fold compared with the empty vector control. These results indicate that hypoxia-induced activation of PI3-K is an important event in the acquisition of resistance against subsequent hyperoxic toxicity.

Abbreviations: bronchopulmonary dysplasia, BPD • green fluorescent protein, GFP • hexokinase, HK-II • human lung microvascular endothelial cells, HLMVECs • 5-hydroxytryptamine, 5-HT • phosphatidylinositol 3-kinase, PI3-K • phosphatidylinositol 3-phosphate, PI3-P


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Supplemental oxygen is often indispensable for life support in the preterm newborn. For the premature at birth, even room air is sensed as hyperoxia in comparison to relatively hypoxic fetal life. The very premature can have decreased levels of some endogenous antioxidants and may suffer impaired nutrition. Exposure to elevated oxygen concentrations causes acute oxidative stress in their lungs, leading to inflammation. This inflammation is an early marker, strongly associated with the development of bronchopulmonary dysplasia (BPD) (1), a common chronic lung disease in prematurely born babies.

Impaired alveolar and vascular development are key elements in BPD (2, 3). Alveolar development is closely linked to vascular development, which, in turn, is dependent upon survival of the cells in the alveolar-capillary membrane. This suggests that factors which regulate lung endothelial survival and growth are likely to be involved in the pathogenesis of BPD. Hyperoxia can induce injury and death of pulmonary capillary endothelial, as well as epithelial cells (4, 5).

Endothelial cells thrive in hypoxic environments in which they can better maintain their integrity, survive, replicate, and form capillary networks (6, 7). Paradoxically, exposures to hypoxia can precondition various organs, including the heart, brain, kidney, liver, and skeletal muscle, protecting them against subsequent oxidative stress such as that caused by ischemia-reperfusion. Therefore, it is reasonable to anticipate that such preconditioning in cultured lung cells might be protective against hyperoxic injury. Previously, our studies with rats acclimated to hypoxia have shown consistent (100%) and prolonged survival during subsequent hyperoxic exposure which is almost uniformly fatal to 21% oxygen–pre-exposed control animals (8). Protection against oxidative stress by hypoxic preconditioning has not been described previously in cultured lung cells. In this study, pre-exposure to hypoxia was protective against cell death due to subsequent hyperoxic exposure, both in human lung microvascular endothelial cells (HLMVEC) and epithelial type II-like A549 cells.

The molecular basis of hypoxic preconditioning has not been well characterized. Studies in our laboratory have established that increased messenger RNA and activity of one isoform of hexokinase (HK-II), the rate-limiting enzyme of glycolysis in lungs, is an adaptative response which occurs uniformly in lungs of hyperoxia-exposed primate and rodent models, as well as in lung cells (9–11). In addition, our recent studies have indicated that overexpression of HK-II provides protection against hyperoxic exposure in A549 cells (11). We also have shown that pre-exposure to hypoxia causes increased expression of HK-II in lung epithelial-like (A549) and small airway epithelial cells (12). These findings indicate that some similar pathways might be activated during adaptation to hypoxia and to hyperoxia.

Survival of cells depends in part upon the availability of growth factors and growth factor-dependent cell survival signaling. Phosphatidylinositol 3-kinase (PI3-K) is an important mediator of growth factor-dependent signaling pathways. It is a heterodimeric enzyme consisting of a p85 regulatory subunit and a p110 catalytic subunit. It phosphorylates phosphoinositides at the D-3 position, which in turn, can activate various downstream effectors involved in cell survival signaling (13–15). Involvement of PI3-K in cellular proliferation has been documented in airway smooth muscle cells where PI3-kinase appears to regulate transcription from cyclin D1 promoter and DNA synthesis (16). Recently, it has also been demonstrated that neuregulin, a polypeptide growth factor, protects neuronal PC12 cells from H2O2-induced apoptosis (17). The protection was completely abolished by the addition of LY 294002, a specific inhibitor of PI3-K, suggesting the involvement of PI3-K–mediated signaling in this protection.

On this basis, the effect of hypoxia on the activity of PI3-K was evaluated. In addition to PI3-K activity studies, work with PI3-K inhibitors confirmed the possible involvement of a PI3-K–dependent mechanism. To further elucidate the protective role of PI3-K against hyperoxia, p110{alpha}-overexpressing A549 cells were established by transient transfection using a full-length active form of p110{alpha} complimentary DNA. PI3-K p110{alpha}-overexpression was found to confer protection against lethal hyperoxic exposure in a manner comparable to that observed with protection by hypoxic pre-exposure.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and Culture
The human epithelial-like lung carcinoma cell line A549 was obtained from the American Type Culture Collection (Rockville, MD). Cells were grown in 100 mm Falcon polystyrene tissue culture dishes in 10 ml of F12K growth medium (Life Technologies Inc. Rockville, MD) containing 10% fetal calf serum, penicillin (100 units/ml), streptomycin (100 mg/ml), 20 mM glucose incubated at 37°C under a humidified atmosphere of air containing 5% CO2. A549 cultures were routinely passaged by trypsinization and subcultured at an initial plating density of 0.5 million cells per plate. HLMVEC were purchased as frozen primary cultures from Clonetics Ltd (San Diego, CA). They were cultured in 10 ml endothelial cell basal medium (EBM-2) supplemented with vascular endothelial growth factor (VEGF), human fibroblast growth factor, human epidermal growth factor, hydrocortisone, ascorbic acid, insulin like growth factor-1, GA1000 (gentamycin/amphotericin-B), and fetal bovine serum, as per the manufacturer's protocol, in 100 mm tissue culture dishes.

Pre-exposure to severe hypoxia (0% O2, 24 h) or normoxia (21% O2, 24 h) was performed at Denver atmospheric pressure (635 mm Hg). This hypoxic exposure system has recently been characterized (18) and allows gradual equilibration to severe hypoxia over ~ 90 min for a cell-containing system. Hyperoxic exposures following the pre-exposures were performed at sea level atmospheric pressure as described previously (10, 18). The cells were 70–80% confluent when exposed to hyperoxia. Fresh media was supplied daily during hyperoxic exposures.

Assay for PI3-Kinase Activity
PI3-kinase activity was assessed by the incorporation of [32P]ATP into exogenous phosphoinositide resulting in the production of PI3-phosphate (PI3-P) (19). HLMVEC were lysed in buffer (20 mM Tris-HCl, pH 7.5, containing 137 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 0.2 mM Na3VO4, protease inhibitor cocktail (Calbiochem, La Jolla, CA), 1 mM PMSF, 10 mM sodium fluoride, 1% NP-40, and 1 mM EDTA after treatments and the PI3-K enzyme immunoprecipated with antiphospho Tyr agarose conjugate or anti-p85 agarose conjugate. The enzyme was then incubated with phosphotidylinositol and [32P]ATP in kinase buffer for 15 min at 37°C. The reaction was terminated by the addition of 1 M HCl, and the lipids were then extracted by a mixture of chloroform and methanol (1:1). The lower chloroform phase containing the resulting PI3-P formed was separated by TLC and visualized by autoradiography. The product was identified by comparing its relative mobility value with the relative mobility value of the standard PI3-P, which was visualized by exposure of TLC plates to phospholipid-specific molybdenum blue spray reagent.

Transfection of A549 Cells with p110{alpha} to Examine Its Effect on Hyperoxic Injury
Human lung epithelial-like A549 cells, grown on 100 mm plates, were cotransfected with 8 µg of p110{alpha}-CAAX containing the CAAX motif (CKCVLS) (20) or the empty vector pSG5 (Stratagene, La Jolla, CA) and 2 µg of the green fluorescent protein (GFP)-expressing vector, pEGFP-C1 (Stratagene, La Jolla, CA) using Fugene-6 (Roche, Indianapolis, IN) as the transfection reagent. Transfection was performed with a DNA:Fugene-6 ratio of 1:3 as per the manufacturer's protocol. The GFP-positive cells were sorted 24 h after transfection using a flow cytometer (MoFlo; Dako Cytomation, Fort Collins, CO) and replated. At 48 h after transfection, the cells were exposed to either 21% O2-5% CO2-balance N2 (Air) or 95% O2-5% CO2 (hyperoxia) for 6 d. Cell death was evaluated with propidium iodide using a flow cytometer as described above.

Immunoblot
Anti-p85{alpha}, anti-p110ß (Santa Cruz Biotechnology, Inc, Santa Cruz, CA.) and anti-p110{alpha} (Upstate Biotechnology, Lake Placid, NY) antibodies were used as primary antibodies. Total protein lysate (20 µg) was suspended in reduced 5x SDS sample buffer and boiled for 5 min. Protein lysates were subjected to SDS-PAGE (4–15%), and the separated proteins were transferred to nitrocellulose membranes (HyBond, ECL; Amersham Pharmacia Biotech Inc, Buckinghamshire, UK) by electrophoretic blotting (BioRad, Hercules, CA). Nonspecific binding was prevented by blocking the membrane with Tris-buffered saline containing 0.1% Tween 20 plus 5% nonfat dry milk overnight at 4°C. Immunoblotting was performed in the following manner. Briefly, membranes were washed four times (10 min/wash) with Tween 20, incubated with the primary antibody in the same buffer for 1 h at room temperature and washed four times (15 min/wash). Membranes were then incubated with the secondary antibody conjugated with peroxidase in PBS-T containing 5% nonfat dry milk for 1 h at room temperature. After washing with PBS-T four times (15 min/wash), immunodetection was performed by using Super Signal West Pico staining kit (Pierce, Rockford, IL). For the detection of p85, immunoprecipitation was performed using p110{alpha} followed by Western blotting.

Other Biochemical Assays
For the propidium iodide (Molecular Probes, Eugene, OR) staining of nonviable cells, ~ 106 cells were suspended in 1 ml PBS, and propidium iodide (2 µg/ml, final concentration) was added. After incubating for 5 min on ice in the dark, the flow cytometric analysis was performed as described earlier (21). Cell death in HLMVEC, which become permeant to propidium iodide after trypsinization, was assessed by using YOYO-1 (Molecular Probes, Eugene, OR). YOYO-1, also is cell-permeable, but, unlike propidium iodide, binds to DNA irreversibly. This dye was applied to adherent cells before trypsinization. Briefly, the media and suspended cells overlying the adherent cells were gently pipetted to a 15 ml polystyrene centrifuge tube. YOYO-1 was added to a final concentration of 300 nM. The media was gently returned to the plate of origin and allowed to incubate for an hour. The media and the suspended cells were then pipetted to another 15 ml tube, and the adherent cells also were added to the same tube after trypsinization. Dead cells which stained positive for YOYO-1 were analyzed using a flow cytometer.

Trypan blue exclusion was performed by adding 25 µL of 0.1% trypan blue solution to 100 µL of cells suspended in PBS, and the cells which excluded the dye were counted on a hemocytometer (AO Scientific Instruments, Buffalo, NY).

Protein concentration in the cell lysate was determined using the BioRad DC protein assay kit in a 96-well plate with bovine serum albumin (BioRad, Hercules, CA) as a standard. The absorbance was recorded using Spectramax 340 microtiter plate reader (Molecular Devices, Sunnyvale, CA.). Data analysis was performed with Softmax Pro 1.2 software (Molecular Devices).

For ATP analysis, cells were harvested, the extract was prepared as described previously (1), and the total cellular ATP content was estimated with a luciferase-luciferin kit (Analytical Luminescence Laboratory, Sparks, MD).

Cellular oxygen consumption was measured in a custom built 6-place respirometer as described previously (21).

Serotonin (5-hydroxytryptamine, 5-HT) uptake was measured according to the method of Lee and coworkers (22). The cell monolayer was washed three times with PBS-dextrose (dextrose [15 mM], pH 7.4) and incubated 30 min with 0.1 mM iproniazid (Sigma, St. Louis, MO), [3H]5-HT (NEN Lifesciences Products, Inc., Boston, MA) (10–40 µCi/ml) (1 nM–4 nM) was added in 1.5 ml PBS-dextrose containing 10 µg/ml EDTA and 10 µg/ml ascorbic acid and 0.1 mM iproniazid for 30 min. The cells were washed three times with PBS-dextrose and then the cells were treated with 0.2 N NaOH. A total of 1.5 ml of cells in NaOH were added to 15 ml of scintillation liquid and counted in a ß counter.

Statistical Analysis
All statistical calculations were performed with JMP software (SAS Institute, Cary, NC [23]). Means were compared by two-tailed t test for comparison between two groups and one-way ANOVA followed by the Tukey-Kramer test for multiple comparisons. A P value of < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of Hypoxic Pre-Exposure on Survival and Proliferation of HLMVECs in Hyperoxia
To determine the protective effect of hypoxia in HLMVECs, cells were exposed to 6 h or 24 h of severe hypoxia (0% O2). After replacing the spent media with fresh media, the hypoxia–pre-exposed, as well as air (21% O2)–pre-exposed control cells, were then exposed to hyperoxia (95% O2/5% O2) at sea level pressure for 8 d. After exposure, the extent of cell death was determined using YOYO-1 staining. As shown in Figure 1A , 58.0 ± 3.0% cell death was detected upon completion of hyperoxic exposure of air–pre-exposed HLMVECs. The extent of cell death was similar (55.0 ± 3.0%) in those cells, which were exposed to hypoxia for 6 h before being exposed to hyperoxia. In addition, there was no reduction in cell death in hyperoxia among cells pre-exposed to hypoxia for 12 h (not shown). However, there was a significant decrease in cell death to 31.0 ± 5.2% in cells which initially were exposed to 24 h of hypoxia. These data reflect the percentage of surviving cells among those which remained adherent after hyperoxic exposure.



View larger version (37K):
[in this window]
[in a new window]
  		Figure 1. Effect of hypoxic pre-exposure on the viability of human lung microvascular endothelial cells (HLMVECs) in hyperoxia. Cells were plated on a 100 mm dish at a density of 0.5 x 106 cells and were exposed to hypoxia/hyperoxia as described in the text. (A) Dead cells were examined by YOYO-1 staining (details in METHODS AND MATERIALS) at day 8 of hyperoxic exposure. This assay quantitates the percentage of cells which remain viable among those which remain adherent following hyperoxic exposure. This study was repeated five times, and representative results are shown. (B) Viable cells were examined for their ability to exclude trypan blue at each time interval (2–8 d) and compared with their respective 21% O2 control cells. This assay quantitates absolute numbers of surviving cells remaining adherent following hyperoxic exposure. This study was repeated three times, and representative results are shown. Closed squares represent 21% O2 control, Open squares represents hyperoxia-exposed (no hypoxic pre-exposure) cells and open diamonds represents cells pre-exposed to hypoxia and then exposed to hyperoxia. (C) Cellular morphology of HLMVECs after hypoxic/hyperoxic exposure. This study was repeated twice. Representative illustration of cell appearance by phase contrast microscopy after 8 d of hyperoxic exposure. 21% O2-exposed (Air) cells were grown under normal oxygen tension. Magnification x25. Values are means ± SEM. n = 3 determinations per condition per time point. ‘*’ indicates significant difference from simultaneous normoxic cells. ‘#’ indicates significant difference from hyperoxia-exposed (Oxy) (no hypoxic pre-exposure) cells, P <= 0.05 by analysis of variance. Cells denoted PE were pre-exposed to hypoxia and then exposed to hyperoxia.

 
HLMVECs conditioned by hypoxic pre-exposure also had increased survival in hyperoxia as evidenced by the numbers of cells remaining adherent. As seen in Figure 1B, these cells were able to survive better starting from the sixth day of hyperoxic exposure where the 21% O2–pre-exposed cells began to detach and die. This figure demonstrates the absolute numbers of viable cells surviving exposures of various intervals. The total number of hypoxia–pre-exposed cells which survived (remained adherent and excluded trypan blue) 8 d of exposure to hyperoxia were 2-fold greater than those pre-exposed to 21% oxygen (Figure 1B).

Figure 1C shows the morphology of the 21% oxygen-exposed control cells, oxygen-exposed cells with prior hypoxic treatment, and of oxygen-exposed cells without hypoxic pretreatment. Some of the dead cells can be seen as highly reflective, rounded cells floating freely in the medium. These were uncommon among the cells that were hypoxia-preconditioned before hyperoxic treatment. This further confirmed the viability measurements obtained by YOYO-1 staining and trypan blue exclusion in Figure 1A and 1B.

Effect of Hyperoxic Exposure on Serotonin (5-hydroxytryptamine, 5-HT) Uptake by HLMVECs
5-HT uptake is important both as a physiologic function of the pulmonary circulation and as a measure of endothelial cell metabolic function. To determine the effect of hyperoxia on endothelial cell function and whether hypoxic pre-exposure enhances that functional capacity, 5-HT uptake experiments were performed. Hyperoxic (95% O2/5% O2 at sea level for 24 h) exposure significantly decreased the uptake of 5-HT in the endothelial cells as compared with the control cells in 21% O2 (see Table 1). A 2-fold increase (24,139 ± 3,300 cpm/mg protein in air versus 48,049 ± 1,913 cpm/mg protein in hypoxia) in the 5-HT uptake was observed in cells that were exposed to 0% O2 for 24 h. Hypoxia–pre-exposed endothelial cells, when subsequently exposed to hyperoxia for 24 h, maintained their 5-HT uptake (28,264 ± 3,310 cpm/mg protein) to a level which was significantly higher than 5-HT uptake (17,795 ± 287 cpm/mg protein) in 21% O2-exposed control cells. Hypoxia did not alter the level of expression of serotonin translocator, as detected by Western blot analysis (data not shown). In addition, the serotonin 1D receptor could not be detected in HLMVECs (data not shown).


View this table:
[in this window]
[in a new window]
  		TABLE 1 Effect of hyperoxic pre-exposure on serotonin uptake of human lung microvascular endothelial cells

 
Effect of Hypoxic Pre-Exposure on Survival of Lung Epithelial-like A549 Cells in Hyperoxia
As seen in Table 2, hyperoxic exposure of A549 cells caused 29.0 ± 2.6% (positive nuclear propidium iodide staining) cell death within 6 d of exposure. Even 4 d of hyperoxic exposure caused 22.0 ± 2.0% cell death. Hypoxic pre-exposure for 24 h also was found to be protective in these cells, resulting in a decrease of ~ 40–50% in death of these cells (12.4 ± 2% at 4 d and 16.6 ± 3.0% at 6 d) compared with those that were not exposed to hypoxia before being exposed to hyperoxia.


View this table:
[in this window]
[in a new window]
  		TABLE 2 Enhancement of survival of A549 cells in hyperoxia by hypoxic pre-exposure

 
Evaluation of trypan blue-excluding adherent cells further revealed the enhanced survival capacity of hypoxia–pre-exposed A549 cells in subsequent hyperoxia. As seen in Figure 2 , the total number of hypoxia–pre-exposed cells which survived 4 and 6 d of hyperoxia was 2-fold greater than those pre-exposed to 21% oxygen.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 2. Effect of hypoxic pre-exposure on the viability of A549 cells in hyperoxia. Cells were plated on a 100 mm dish at a density of 0.5 x 106 cells and were exposed to hypoxia/hyperoxia as described in the text. Viable cells were examined by their ability to exclude trypan blue at each time interval (2–6 d) and compared with their respective 21% O2 controls. This study was repeated more than 3 times and representative results are shown. Closed squares represent 21%O2 control, open squares represent hyperoxia-exposed (no hypoxic pre-exposure) cells and open diamonds represent cells pre-exposed to hypoxia and then exposed to hyperoxia. Values are means ± SEM. n = 6 determinations per condition per time point. ‘#’ indicates significant difference from hyperoxia-exposed (no hypoxic pre-exposure) cells, P <= 0.05 by analysis of variance.

 
Hypoxic Protection is Mediated by Phosphotidylinositol 3-Kinase in A549 and HLMVE Cells
To further elucidate the mechanism of protection, cells were treated with the specific inhibitors of PI3-K, wortmannin (20 nM) and LY 294002 (30 µM) during hypoxic exposure. Treatment of endothelial cells with the PI3-K inhibitors for 24 h was not toxic as there was no increased cell death when compared with control cells in 21% O2 (Figure 3A) . Importantly, addition of wortmannin and LY 294002 at the above concentrations did not cause any additional cell death in 24 h hypoxia (data not shown). However, when wortmannin and LY 294002 were present during the course of hypoxic exposure, the protective effect of hypoxia in subsequent hyperoxic exposure was totally abolished (63.0 ± 2.0% cell death in non–pre-exposed, 34.0 ± 2.2% in hypoxia–pre-exposed, 62.0 ± 3.0% in hypoxia–pre-exposed+wortmannin and 66.0 ± 5.0% cell death in hypoxia–pre-exposed+LY 294002; Figure 3A). Inclusion of low concentrations of wortmannin (1 nM) throughout the 8 d exposures was equally deleterious in both A549 cells (30.0 ± 4.0% cell death in nonpre-exposed versus 28.0 ± 7.2% in hypoxia–pre-exposed+wortmannin) and HLMVEC (60.0 ± 6.0% cell death in nonpre-exposed versus 62.0 ± 5.0% in hypoxia–pre-exposed+wortmannin; P > 0.05 for both comparisons) in hyperoxia. The results were somewhat different in 21% oxygen (6.0 ± 3.0% cell death in absence of wortmannin versus 15.0 ± 4.0% in presence of wortmannin in A549 cells; 8.0 ± 2.0% in absence of wortmannin versus 14.0 ± 1.0% in the presence of wortmannin in HLMVECs; P < 0.05 for both comparisons), further indicating the importance of PI3-K in the survival of these cells.



View larger version (24K):
[in this window]
[in a new window]
  		Figure 3. (A) Protection of HLMVECs by hypoxic pre-exposure is mediated by PI3-K. HLMVECs were plated on 100 mm dishes and grown until they reached 60–70% confluence. The culture media was replaced with fresh media, or media containing wortmannin (20 nM) or LY294002 (30 µM) where indicated, and exposed to normoxia or hypoxia for 24 h. After hypoxic pre-exposure, fresh media was added and cells were exposed to hyperoxia at sea level pressure for 8 d. Values are means ± SEM. n = 3 determinations per group. ‘*’ indicates significant difference from simultaneous normoxic cells. ‘#’ indicates significant difference from hyperoxia-exposed (no hypoxic pre-exposure) cells, P <= 0.05 by analysis of variance. The experiment was repeated three times and representative results shown. (B) Exposure to hypoxia induces PI3-K activity in immunoprecipitates. HLMVE cells were incubated under normoxic (21% O2) or hypoxic (0% O2) conditions for 2 h or 24 h. Cell lysates were prepared and ~ 500 µg of protein was incubated with 20 µl of anti-p85–conjugated agarose (200 µg anti p85/200 µl agarose), and the PI3-K activity was measured in the immunoprecipitate as described in METHODS. The experiment was repeated several times. The upper panel of B shows a representative autoradiogram demonstrating PI3-K–mediated phosphorylation of phosphoinositides. Conditions were: lane 1: air 2 h; lane 2: hypoxia 2 h; lanes 3 and 4: air 24 h; lanes 5 and 6; hypoxia 24 h; lane 7: LY294002 (30 µM), 24 h; and lane 8: wortmannin (20 nM), 24 h. The lower panel of B shows a quantitative representation of PI3-K activity as assessed by the densitometry on the scans of spots obtained by autoradiography. Results are quantitated in arbitrary densitometric units.

 
Hypoxia Induces PI3-K Activity in HLMVE Cells
To assess the potential involvement of PI3-K directly, PI3-K was immunoprecipitated from cell lysates prepared after hypoxic exposures for various time intervals. PI3-K activity was increased (1.7-fold) within 2 h of hypoxic exposure (2.64 ± 0.01 arbitrary units in 21% versus 4.52 ± 0.04 arbitrary units in 0% O2) (Figure 3B). The increased levels of PI3-K were maintained throughout the 24 h experiment at 1.8-fold higher than that in the normoxic control cells (3.53 ± 0.46 arbitrary units in 21% O2 versus 6.27 ± 0.51 arbitrary units in 0% O2) at the termination of the 24 h hypoxic exposure. Addition of LY294002 (30 µM) decreased the PI3-K activity by 70%, and wortmannin (20 nM) caused a loss of more than 85% of the activity (Figure 3B).

Relative PI3-K Activity and Its Isoforms in A549 Cells
Immunoprecipitation and Western blot results in these cells suggested that a class IA PI3-kinase containing both the p110{alpha} catalytic subunit and p85 adapter subunit was the major isoform present (Figure 4A) . Hypoxic or hyperoxic exposures did not cause an obvious change in the protein expression of these isoforms. Exposure to 0% O2 for 2 h resulted in a similar increase in PI3-K activity in A549 cells (Figure 4B).



View larger version (32K):
[in this window]
[in a new window]
  		Figure 4. (A) Western blot detection of PI3-K isoforms and measurement of PI3-K activity in A549 cells. For the Western blot analysis, 70–80% confluent A549 cells cultured on 100 mm dishes were exposed 0% or 21% oxygen for 24 h. Lysates were prepared and immunoblotting was performed as described in METHODS AND MATERIALS. p85 was immunoprecipitated from the cell lysate using anti-p110{alpha}. (B) PI3-K activity also was determined in A549 cells exposed to hypoxia for 2 h as described for endothelial cells in METHODS AND MATERIALS. Two examples for each group are shown (lanes 1 and 2 represent 21% O2 for 2 h; and 3 and 4 represent 0% oxygen for 2 h). This study was conducted twice.

 
Effect of Overexpression of p110{alpha}-CAAX in A549 Cells
To confirm the involvement of PI3-K further, A549 cells were cotransfected with p110{alpha}-CAAX, or the empty vector pSG5, and the GFP-expressing vector EGFP-C1. Overexpression of p110{alpha} was confirmed by western blot. Cells which were transfected, sorted, and grown for 24 h had a 3–4-fold higher level of p110{alpha}, as detected by Western blot, compared with the untransfected cells (Figure 5A) . After hyperoxic exposure for 6 d, the p110{alpha} overexpressors had enhanced survival as compared with the untransfected cells or the empty vector controls (Figure 5B).



View larger version (25K):
[in this window]
[in a new window]
  		Figure 5. Western blot detection of p110{alpha} protein and quantitation of relative cell death in p110{alpha}-CAAX-transfected A549 cells exposed to hyperoxia. A shows the expression of p110{alpha} protein in the transfected A549 cells. In B, propidium iodide staining was used to determine the percentage of cell death among adherent control (A549), empty vector control (pSG5) and p110{alpha}-CAAX-transfected (p110{alpha}) A549 cells. Values are mean ± SEM. n = 3 determinations per group. ‘*’ indicates significant difference from simultaneous normoxic cells. ‘#’ indicates significant difference from hyperoxia-exposed (empty vector control) cells, P <= 0.05 by analysis of variance. This study was conducted twice. Where the SEM is not apparent, it is too small to be distinguished from the upper margin of the column.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Hyperoxic exposure causes increased intracellular generation of oxidants in lungs, which can damage cellular components and contribute to severe pathologic conditions like BPD. Lung epithelium and endothelium are subject to considerable injury following exposure to elevated oxygen concentrations (4). In vitro models can provide a simplified system in which to establish biochemical mechanisms and test potential therapeutic interventions for such complex human disorders.

Hypoxic preconditioning has been found to have protective effects against oxidant injury in various organs, including the heart, kidney, liver, and skeletal muscle. Our laboratory and others previously have determined that rats acclimated to hypoxia uniformly survive in subsequent hyperoxia (8, 24, 25). To evaluate the potential role of hypoxia as a protective intervention against oxygen toxicity in lung cellular models, we studied parameters including cell death, cellular morphology, and metabolic function in microvascular endothelial cells and A549 cells. In this study, we report for the first time that hypoxic pre-exposure confers resistance to subsequent hyperoxic exposure in cultured lung endothelial, as well as epithelial, cells and that this protection occurs through a PI3-K–dependent mechanism.

Hypoxic pre-exposure for 24 h caused a substantial decrease in cell death in HLMVECs. Interestingly, the level of cell death after hyperoxic exposure in cells pre-exposed to hypoxia for only 6 h was almost identical to that seen in the 21% oxygen–pre-exposed cells, indicating that a greater duration of hypoxic exposure is necessary for the acquisition of resistance. That the hypoxia–pre-exposed cells are better survivors in hyperoxia was confirmed further by the trypan blue exclusion studies where the viable cells were counted at each time interval. During early exposure (2–4 d), there was no difference between surviving cell numbers among hypoxia–pre-exposed and non–pre-exposed cells. Decreases in cell numbers beginning on day 6 and continuing on day 8 of exposure in the non–pre-exposed cells indicated their greater susceptibility to hyperoxia. Morphologic examination confirmed protection of hypoxia–pre-exposed cells in hyperoxia.

5-HT uptake is an energy-dependent, physiologically important index of endothelial cell metabolic function (26). Decreased serotonin uptake reflects compromised endothelial cell integrity upon hyperoxic exposure (27, 28). In an effort to evaluate endothelial cell function, 5-HT uptake studies were performed. Hypoxia increased 5-HT uptake by endothelial cells 2-fold. A similar increase in 5-HT uptake by endothelial cells under hypoxic conditions has been documented previously by others (22). It was possible that this increased uptake is due to enhanced expression of the serotonin receptor or translocator. However, no significant change in expression of serotonin translocator was observed in hypoxia. There are a number of isoforms of serotonin receptors, including type 1D, which are reported to be commonly present in some types of endothelial cells (29). This isoform was not detected in HLMVEC under our experimental conditions. Hyperoxic exposure for 24 h caused a significant decrease in 5-HT uptake in 21% oxygen–pre-exposed cells. During their hyperoxic exposure, the hypoxia–pre-exposed cells maintained their 5-HT uptake close to that of normoxic cells, indicating that hypoxic preconditioning may improve or preserve this endothelial cell metabolic function. Pulmonary oxygen toxicity also decreases the activity of lung capillary angiotensin-converting enzyme. Using angiotensin-converting enzyme as a marker of oxygen toxicity, Jackson and coworkers (30) previously demonstrated the protection of rat lung endothelial metabolic function in hyperoxia by hypoxic pre-adaptation (10% O2 for 4 d) in vivo.

As with endothelial cells, lung epithelial type II-like A549 cells also acquired resistance to O2 toxicity conferred by pre-exposure to hypoxia. A549 cell death caused by hyperoxic exposure has been previously reported (48, 49). Considerable differences in cell survival have been noted by different groups using A549 cells at near sea level atmospheric pressures (48, 49). Here it would be important to note that confluency of cells and frequency of media changes are important factors in hyperoxic toxicity. When specifically examined in A549 cells, subconfluent cells appeared to be more resistant to cell death in hyperoxia than were confluent cells (49). Our studies employed subconfluent cells, and this and other experimental conditions may explain their relative resistance to cell death caused by hyperoxia in our studies. Hypoxic pre-exposure decreased the cell death in hyperoxia in A549 cells by ~ 50%. This finding indicated that protection of cells against hyperoxia by pre-exposure to hypoxia is not cell-type–specific, and that such protection can be conferred to transformed as well as primary cultured lung cells.

To further elucidate the mechanism of protection, endothelial cells were treated with the specific inhibitors of PI3-K, wortmannin and LY 294002, during hypoxic exposure. Interestingly, addition of inhibitors of PI3-K totally abrogated the protection provided by hypoxia, suggesting the involvement of a PI3-K–mediated signaling pathway. A substantial increase in PI3-K activity was found within 2 h of the onset of hypoxic exposure in both the cell types and was maintained even after 24 h of exposure. Our studies on PI3-K activity had the limitation of having 5% serum, which is essential for endothelial cell survival, present throughout the exposure. In many cell types, serum causes a substantial background expression of PI3-K activity. Contrasting findings obtained in the absence of serum compared with stimulation caused by growth factors in this milieu often results in more impressive induction of PI3-K activities. However, our findings in the presence of serum are relevant since resistance to hyperoxia conferred by pre-exposure to hypoxia also occurred in the presence of serum.

The role of PI3-K in protection against oxidative stress is not well established. Protection of neuronal PC12 cells from oxidant (H2O2)-induced apoptosis by neuregulin, a transmembrane polypeptide growth factor, recently has been reported (17). This protection was eliminated by the addition of LY 294002, suggesting the involvement of PI3-K mediated signaling in the protection. Similarly, another study has established that hypoxic preconditioning of PC12 cells protects them from H2O2-induced cell death and injury and that the protection was due to PI3-K-mediated signaling (31).

In this study, involvement of PI3-K in the protection afforded by hypoxia was further confirmed by the transient transfection of A549 cells with p110{alpha}-CAAX. Exposure of p110{alpha}-CAAX-overexpressing cells, empty vector control cells, and untransfected A549 cells to hyperoxia revealed that the overexpressors had enhanced survival in hyperoxia as compared with empty vector control and untransfected A549 cells. Overexpression of the active catalytic subunit of PI3-kinase using the same vector (p110 {alpha}-CAAX) in airway smooth muscle cells and resulting in the induction of cyclin D1 promoter activity and DNA synthesis has already been described (16).

The PI3-K pathway could act either by regulating toxic levels of reactive oxygen species generated in hyperoxia or by activating other factors that enhance cell survival. Various downstream effectors of the PI3-K pathway could be involved. Recently, expression of a constitutively active form of Akt, a serine/threonine kinase and important downstream effector of PI3-K action, has been shown to protect mice from hyperoxic pulmonary damage (32). In another study, the antiapoptotic activity of Akt was shown to be related to the initial phosphorylation of glucose catalyzed by hexokinase (33). Potential downstream targets of Akt include VEGF (34), a proapoptotic Bcl-2 family member BAD, glycogen synthase kinase-3ß, and the transcription factor Forkhead, among others (13–15, 35). In previous studies, increases in glucose uptake in hypoxia were directly related to increased levels of PI3-K in cardiac mycocytes H9c2 cells (36). In that study, increased glucose utilization caused by prolonged (48 h) exposure to hypoxia caused acidosis and was associated with necrotic cell death. By contrast, another study by Malhotra and Brosius (37) demonstrated that hypoxia-induced apoptosis of isolated cardiomycocytes could be prevented by addition of glucose to the medium. PI3-K–induced stabilization of HIF (38, 39), which is known to induce glycolytic enzymes and VEGF production during hypoxia (40–44), also was reported recently. Hence, HIF and other downstream targets of HIF, including VEGF and glycolytic enzymes such as hexokinase, warrant further investigation in this model. It is noteworthy that other stimuli which can confer resistance to hyperoxic lung damage, such as bacterial LPS, tumor necrosis factor, and interleukin-1, also are capable of activating PI3-K–dependent survival pathways in alveolar macrophages (45, 46) and endothelial cells (47), respectively. Hence, the potential involvement of a complex variety of downstream actions and effectors, including Akt, HIF, VEGF, glycolytic enzymes, glycogen synthase kinase-3ß, a proapoptotic Bcl-2 family member BAD, Forkhead, and other transcription factors which could be involved in protection by hypoxia, is suggested by the role of PI3-K documented in this study.

On the basis of the results of this study, early adaptation could be an advantage for cells in enduring subsequent hyperoxic injury. Total cellular ATP content and oxygen consumption also were evaluated. Following hyperoxic exposure, the total ATP content and oxygen consumption in hypoxia–pre-exposed cells (HLMVEC and A549) were similar to those that were not pre-exposed to hypoxia. This indicated that the protection conferred by pre-exposure to hypoxia may be more complex than simple preservation or enhancement of glycolytic and/or mitochondrial function. However, it also should be noted that cells appear to detach from the plate rapidly once the death process is initiated. Since ATP analysis and respiration studies were performed in cells which remained attached, abnormalities which were present in the subpopulation which had begun the cell-death process likely were excluded from these studies.

Endothelial cells and epithelial cells that were preconditioned with hypoxia had enhanced survival and enhanced metabolic function. Like pre-exposure to hypoxia, overexpression of p110{alpha}–CAAX protected cells against hyperoxia-induced death, and PI3-K inhibitors prevented protection by hypoxia. Hence, in this study, PI3-K activation by hypoxia protects lung epithelial and endothelial cells against hyperoxia. Further studies involving other potential downstream targets of PI3-K are needed to fully understand the mechanism of resistance to hyperoxia present in this model.


   Acknowledgments
 
The authors are grateful to Dr. Julian Downward for the kind gift of active PI3-kinase expression vector (p110 {alpha}-CAAX). Supported by National Institute of Health Grants HL52732 (C.W.), HL57144 (K.S. and C.W.), and HL56263 (C.W.). The authors are grateful to Dr. Wayne Zundel for his critical reading of the manuscript and to Gretchen Czapla for its preparation.


   Footnotes
 
* The first two authors contributed equally to the work presented in this article. Back

Received in original form January 17, 2002

Received in final form July 1, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Jobe, A. H., and E. Bancalari. 2001. Bronchopulmonary dysplasia. Am. J. Respir. Crit. Care Med. 163:1723–1729.[Free Full Text]
  2. Jobe, A. J. 1999. The new BPD: an arrest of lung development. Pediatr. Res. 46:614–643.
  3. Coalson, J. J., V. T. Winter, T. Siler-Khodr, and B. A. Yoder. 1999. Neonatal chronic lung disease in extremely immature baboons. Am. J. Respir. Crit. Care Med. 160:1333–1346.[Abstract/Free Full Text]
  4. Crapo, J. D., B. E. Barry, H. A. Foscue, and J. Shelburne. 1980. Structural and biochemical changes in rat lungs occurring during exposures to lethal and adaptive doses of oxygen. Am. Rev. Respir. Dis. 122:123–143.[Medline]
  5. Mantell, L. L., and P. J. Lee. 2000. Signal transduction pathways in hyperoxia-induced lung cell death. Mol. Genet. Metab. 71:359–370.[CrossRef][Medline]
  6. Phillips, P. G., L. M. Birnby, and A. Narendran. 1995. Hypoxia induces capillary network formation in cultured bovine pulmonary microvessel endothelial cells. Am. J. Physiol. 268:L789–L800.[Abstract/Free Full Text]
  7. Berra, E., J. Milanini, D. E. Richard, M. Le Gall, F. Vinals, E. Gothie, D. Roux, G. Pages, and J. Pouyssegur. 2000. Signaling angiogenesis via p42/p44 MAP kinase and hypoxia. Biochem. Pharmacol. 60:1171–1178.[CrossRef][Medline]
  8. White, C. W., J. H. Jackson, I. F. McMurtry, and J. E. Repine. 1988. Hypoxia increases glutathione redox cycle and protects rat lungs against oxidants. J. Appl. Physiol. 65:2607–2616.[Abstract/Free Full Text]
  9. Allen, C. B., X. L. Guo, and C. W. White. 1998. Changes in pulmonary expression of hexokinase and glucose transporter mRNAs in rats adapted to hyperoxia. Am. J. Physiol. 274:L320–L329.[Abstract/Free Full Text]
  10. Allen, C. B., and C. W. White. 1998. Glucose modulates cell death due to normobaric hyperoxia by maintaining cellular ATP. Am. J. Physiol. 274:L159–L164.[Abstract/Free Full Text]
  11. Ahmad, A., S. Ahmad, B. K. Schneider, C. B. Allen, L.-Y. Chang, and C. W. White. 2002. Elevated expression of hexokinase-II protects human lung epithelial-like A549 cells against oxidative injury. Am. J. Physiol. Lung Cell. Mol. Physiol. 283:L573–L584.[Abstract/Free Full Text]
  12. Riddle, S. R., A. Ahmad, S. Ahmad, S. S. Deeb, M. Malkki, B. K. Schneider, C. B. Allen, and C. W. White. 2000. Hypoxia induces hexokinase II gene expression in human lung cell line A549. Am. J. Physiol. Lung Cell. Mol. Physiol. 278:L407–L416.[Abstract/Free Full Text]
  13. Toker, A., and L. C. Cantley. 1997. Signaling through the lipid products of phosphoinositide-3-OH kinase. Nature 387:673–676.[CrossRef][Medline]
  14. Toker, A., and A. C. Newton. 2000. Cellular signaling: pivoting around PDK-1. Cell 103:185–188.[CrossRef][Medline]
  15. Toker, A. 2000. Protein kinases as mediators of phosphoinositide 3-kinase signaling. Mol. Pharmacol. 57:652–658.[Free Full Text]
  16. Page, K., J. Li, Y. Wang, S. Kartha, R. G. Pestell, and M. B. Hershenson. 2000. Regulation of cyclin D(1) expression and DNA synthesis by phosphatidylinositol 3-kinase in airway smooth muscle cells. Am. J. Respir. Cell Mol. Biol. 23:436–443.[Abstract/Free Full Text]
  17. Goldshmidt, Y., S. Erlich, and R. Pinkas-Kramarski. 2001. Neuregulin rescues PC12-ErbB4 cells from cell death induced by H2O2: regulation of reactive oxygen species levels by phosphatidyl inositol 3-kinase. J. Biol. Chem. 276:46379–46385.[Abstract/Free Full Text]
  18. Allen, C. B., B. K. Schneider, and C. W. White. 2001. Limitations to oxygen diffusion and equilibration in in vitro cell exposure systems in hyperoxia and hypoxia. Am. J. Physiol. Lung Cell. Mol. Physiol. 281:L1021–L1027.[Abstract/Free Full Text]
  19. Ward, S. G. 2000. Measurement of phosphoinositide 3-kinase activity. Methods Mol. Biol. 138:163–172.[Medline]
  20. Khwaja, A., P. Rodriguez-Viciana, S. Wennstrom, P. H. Warne, and J. Downward. 1997. Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway. EMBO J. 16:2783–2793.[CrossRef][Medline]
  21. Ahmad, S., C. W. White, L. Y. Chang, B. K. Schneider, and C. B. Allen. 2001. Glutamine protects mitochondrial structure and function in oxygen toxicity. Am. J. Physiol. Lung Cell. Mol. Physiol. 280:L779–L791.[Abstract/Free Full Text]
  22. Lee, S. L., and B. L. Fanburg. 1987. Glycolytic activity and enhancement of serotonin uptake by endothelial cells exposed to hypoxia/anoxia. Circ. Res. 60:653–658.[Abstract/Free Full Text]
  23. SAS Institute. 2000. JMP User's Guide, Version 4. SAS Institute, Cary, NC. pp. 1–281. 2001 Updated version JMP New Features, Version 4.0.4. SAS Institute, Cary, NC, pp. 1–42.
  24. Frank, L. 1982. Protection from O2 toxicity by preexposure to hypoxia: lung antioxidant enzyme role. J. Appl. Physiol. 53:475–482.[Abstract/Free Full Text]
  25. Sjostrom, K., and J. D. Crapo. 1983. Structural and biochemical adaptive changes in rat lungs after exposure to hypoxia. Lab. Invest. 48:68–79.[Medline]
  26. Steinberg, H., D. J. Bassett, and A. B. Fisher. 1975. Depression of pulmonary 5-hydroxytryptamine uptake by metabolic inhibitors. Am. J. Physiol. 228:1298–1303.[Abstract/Free Full Text]
  27. Sheridan, N. P., and E. R. Block. 1988. Serotonin transport and fluidity in plasma membrane vesicles: effect of hyperoxia. Am. J. Physiol. 254:C781–C787.[Abstract/Free Full Text]
  28. Allen, M. C., and S. J. Watt. 1993. Effect of hyperbaric and normobaric oxygen on pulmonary endothelial cell function. Undersea Hyperb. Med. 20:39–48.[Medline]
  29. Cohen, Z., I. Bouchelet, A. Olivier, J. G. Villemure, R. Ball, D. B. Stanimirovic, and E. Hamel. 1999. Multiple microvascular and astroglial 5-hydroxytryptamine receptor subtypes in human brain: molecular and pharmacologic characterization. J. Cereb. Blood Flow Metab. 19:908–917.[CrossRef][Medline]
  30. Jackson, R. M., H. S. Ann, and S. Oparil. 1988. Hypoxia-induced oxygen tolerance: maintenance of endothelial metabolic function. Exp. Lung Res. 14:887–896.
  31. Alvarez-Tejado, M., S. Naranjo-Suarez, C. Jimenez, A. C. Carrera, M. O. Landazuri, and L. del Peso. 2001. Hypoxia induces the activation of the phosphatidylinositol 3-kinase/Akt cell survival pathway in PC12 cells: protective role in apoptosis. J. Biol. Chem. 276:22368–22374.[Abstract/Free Full Text]
  32. Lu, Y., L. Parkyn, L. E. Otterbein, Y. Kureishi, K. Walsh, A. Ray, and P. Ray. 2001. Activated Akt protects the lung from oxidant-induced injury and delays death of mice. J. Exp. Med. 193:545–549.[Abstract/Free Full Text]
  33. Gottlob, K., N. Majewsky, S. Kennedy, E. Kandel, R. B. Robey, and N. Hay. 2001. Inhibition of early apoptotic events by Akt/PKB is dependent on the first committed step of glycolysis and mitochondrial hexokinase. Genes & Development: 1406–1418.
  34. Mazure, N. M., E. Y. Chen, K. R. Laderoute, and A. J. Giaccia. 1997. Induction of vascular endothelial growth factor by hypoxia is modulated by a phosphatidylinositol 3-kinase/Akt signaling pathway in Ha-ras-transformed cells through a hypoxia inducible factor-1 transcriptional element. Blood 90:3322–3331.[Abstract/Free Full Text]
  35. Zundel, W., C. Schindler, D. Haas-Kogan, A. Koong, F. Kaper, E. Chen, A. R. Gottschalk, H. E. Ryan, R. S. Johnson, A. B. Jefferson, D. Stokoe, and A. J. Giaccia. 2000. Loss of PTEN facilitates H1F–1-mediated gene expression. Genes Dev. 14:391–396.[Abstract/Free Full Text]
  36. Aki, T., Y. Mizukami, Y. Oka, K. Yamaguchi, K. Uemura, T. Fujimiya, and K. Yoshida. 2001. Phosphoinositide 3-kinase accelerates necrotic cell death during hypoxia. Biochem. J. 358:481–487.[CrossRef][Medline]
  37. Malhotra, R., and F. C. Brosius III. 1999. Glucose uptake and glycolysis reduce hypoxia-induced apoptosis in cultured neonatal rat cardiac myocytes. J. Biol. Chem. 274:12567–12575.[Abstract/Free Full Text]
  38. Jiang, B. H., G. Jiang, J. Z. Zheng, Z. Lu, T. Hunter, and P. K. Vogt. 2001. Phosphatidylinositol 3-kinase signaling controls levels of hypoxia-inducible factor 1. Cell Growth Differ. 12:363–369.[Abstract/Free Full Text]
  39. Zhong, H., K. Chiles, D. Feldser, E. Laughner, C. Hanrahan, M. M. Georgescu, J. W. Simons, and G. L. Semenza. 2000. Modulation of hypoxia-inducible factor 1 alpha expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics. Cancer Res. 60: 1541–1545.[Abstract/Free Full Text]
  40. Ebert, B. L., J. D. Firth, and P. J. Ratcliffe. 1995. Hypoxia and mitochondrial inhibitors regulate expression of glucose transporter-1 via distinct Cis-acting sequences. J. Biol. Chem. 270: 29083–29089.[Abstract/Free Full Text]
  41. Firth, J. D., B. L. Ebert, and P. J. Ratcliffe. 1995. Hypoxic regulation of lactate dehdrogenase A. Interaction between hypoxia-inducible factor 1 and cAMP response elements. J. Biol. Chem. 270:21021–21027.[Abstract/Free Full Text]
  42. Semenza, G. L., P. H. Roth, H. M. Fang, and G. L. Wang. 1994. Transcriptional regulation of genes encoding glycolytic enzymes by hypoxia-inducible factor 1. J. Biol. Chem. 269:23757–23763.[Abstract/Free Full Text]
  43. Zaman, K., H. Ryu, D. Hall, K. O'Donovan, K. I. Lin, M. P. Miller, J. C. Marquis, J. M. Baraban, G. L. Semenza, and R. R. Ratan. 1999. Protection from oxidative stress-induced apoptosis in cortical neuronal cultures by iron chelators is associated with enhanced DNA binding of hypoxia-inducible factor-1 and ATF-1/CREB and increased expression of glycolytic enzymes, p21 (waf1/cip1), and erythropoietin. J. Neurosci. 19:9821–9830.[Abstract/Free Full Text]
  44. Forsythe, J. A., B. H. Jiang, N. V. Iyer, F. Agani, S. W. Leung, R. D. Koos, and G. L. Semenza. 1996. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol. Cell. Biol. 16:4604–4613.[Abstract]
  45. Monick, M. M., A. B. Carter, P. K. Robeff, D. M. Flaherty, M. W. Peterson, and G. W. Hunninghake. 2001. Lipopolysaccharide activates Akt in human alveolar macrophages resulting in nuclear accumulation and transcriptional activity of beta- catenin. J. Immunol. 166:4713–4720.[Abstract/Free Full Text]
  46. Monick, M. M., R. K. Mallampalli, A. B. Carter, D. M. Flaherty, D. McCoy, P. K. Robeff, M. W. Peterson, and G. W. Hunninghake. 2001. Ceramide regulates lipopolysaccharide-induced phosphatidylinositol 3-kinase and akt activity in human alveolar macrophages. J. Immunol. 167:5977–5985.[Abstract/Free Full Text]
  47. Madge, L. A., and J. S. Pober. 2000. A phosphatidylinositol 3-kinase/Akt pathway, activated by tumor necrosis factor or interleukin-1, inhibits apoptosis but does not activate NFkappaB in human endothelial cells. J. Biol. Chem. 275:15458–15465.[Abstract/Free Full Text]
  48. Narula, P., J. Xu, J. A. Kazzaz, C. G. Robbins, J. M. Davis, and S. Horowitz. 1998. Synergistic cytotoxicity from nitric oxide and hyperoxia in cultured lung cells. Am. J. Physiol. 274:L411–L416.[Abstract/Free Full Text]
  49. Jyonouchi, H., S. Sun, T. Abiru, S. Chareancholvanich, and D. H. Ingbar. 1998. The effects of hyperoxic injury and antioxidant vitamins on death and proliferation of human small airway epithelial cells. Am. J. Respir. Cell Mol. Biol. 19:426–436.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
Y. Zhang, T. S. Park, and J. M. Gidday
Hypoxic preconditioning protects human brain endothelium from ischemic apoptosis by Akt-dependent survivin activation
Am J Physiol Heart Circ Physiol, June 1, 2007; 292(6): H2573 - H2581.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
D. Xu, J. R. Guthrie, S. Mabry, T. M. Sack, and W. E. Truog
Mitochondrial aldehyde dehydrogenase attenuates hyperoxia-induced cell death through activation of ERK/MAPK and PI3K-Akt pathways in lung epithelial cells
Am J Physiol Lung Cell Mol Physiol, November 1, 2006; 291(5): L966 - L975.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
T. Nyunoya, M. M. Monick, L. S. Powers, T. O. Yarovinsky, and G. W. Hunninghake
Macrophages Survive Hyperoxia via Prolonged ERK Activation Due to Phosphatase Down-regulation
J. Biol. Chem., July 15, 2005; 280(28): 26295 - 26302.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. Lei, C. N. Mariash, and D. H. Ingbar
3,3',5-Triiodo-L-thyronine Up-regulation of Na,K-ATPase Activity and Cell Surface Expression in Alveolar Epithelial Cells Is Src Kinase- and Phosphoinositide 3-Kinase-dependent
J. Biol. Chem., November 12, 2004; 279(46): 47589 - 47600.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Ahmad, A. Ahmad, M. Ghosh, C. C. Leslie, and C. W. White
Extracellular ATP-mediated Signaling for Survival in Hyperoxia-induced Oxidative Stress
J. Biol. Chem., April 16, 2004; 279(16): 16317 - 16325.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
C. S. Powell, M. M. Wright, and R. M. Jackson
p38mapk and MEK1/2 inhibition contribute to cellular oxidant injury after hypoxia
Am J Physiol Lung Cell Mol Physiol, April 1, 2004; 286(4): L826 - L833.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Ahmad, S.
Right arrow Articles by White, C. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Ahmad, S.
Right arrow Articles by White, C. W.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Crit. Care Med.
Copyright © 2003 American Thoracic Society.