Hypoxia Induces the Activation of the Phosphatidylinositol 3-Kinase/Akt Cell Survival Pathway in PC12 Cells PROTECTIVE ROLE IN APOPTOSIS*

Hypoxia is a common environmental stress that influ-ences signaling pathways and cell function. Several cell types, including neuroendocrine chromaffin cells, have evolved to sense oxygen levels and initiate specific adaptive responses to hypoxia. Here we report that under hypoxic conditions, rat pheochromocytoma PC12 cells are resistant to apoptosis induced by serum withdrawal and chemotherapy treatment. This effect is also observed after treatment with deferoxamine, a com-pound that mimics many of the effects of hypoxia. The hypoxia-dependent protection from apoptosis correlates with activation of the phosphatidylinositol 3-ki-nase (PI3K)/Akt pathway, which is detected after 3–4 h of hypoxic or deferoxamine treatment and is sustained while hypoxic conditions are maintained. Hypoxia-in-duced Akt activation can be prevented by treatment with cycloheximide or actinomycin D, suggesting that de novo protein synthesis is required. Finally, inhibition of PI3K impairs both the

Mammalian cell function is critically dependent on a continuous supply of oxygen. Organisms respond to changes in oxygen tension with specific local and systemic adaptations aimed to restore a normal oxygen supply. Several tissues and cell types are responsible for the detection of blood pO 2 and the induction of specific adaptive responses; among them, the chromaffin cells of the adrenal medulla play a critical role by releasing catecholamines in response to hypoxia (1). PC12, a rat pheochromocytoma cell line derived from a tumor of adrenal medulla chromaffin tissue, is an oxygen-sensitive cell type that provides a useful system to study the effects of hypoxia on catecholamine gene expression (2). PC12 cells are exquisitely sensitive to hypoxia, because very small reductions in atmospheric oxygen dramatically induce tyrosine hydroxylase gene expression and mRNA stability (3). In response to hypoxia several transcription factors are activated in PC12 cells. These include the cAMP response element-binding protein, the hypoxia-inducible factor (HIF-2 or EPAS), 1 and c-Fos (2,4,5). PC12 cells also express hypoxia-regulated ion channels, as shown by the finding that PC12 cells depolarize under hypoxia via an oxygen-regulated K ϩ current; as a consequence of depolarization, they secrete dopamine and norepinephrine.
In addition, PC12 cells have been used extensively as a model to study programmed cell death. Programmed cell death, or apoptosis, is an evolutionary conserved mechanism of cellular demise developed by animals to delete damaged, misplaced, or redundant cells during development and tissue homeostasis. Apoptosis was first described as cell death with specific morphologic features (6). In addition to the characteristic morphology, apoptotic cells present specific biochemical alterations including exposure of phosphatidylserine to the extracellular side of the plasma membrane and activation of specific proteases (caspases). Upon activation, caspases cleave several intracellular molecules, leading to the functional and morphological changes observed during apoptosis. Caspase processing also leads to the activation of specific nucleases, which in turn cleave genomic DNA, giving rise to a characteristic pattern of DNA degradation that is considered a hallmark of apoptosis (7).
Cell fate is largely dependent upon extracellular survival signals that prevent the activation of the apoptotic machinery (8). Studies on the survival effect of nerve growth factor (NGF) on PC12 cells provided the first evidence that activation of the enzyme PI3K was critical for its protective effect (9). Upon activation, PI3K phosphorylates membrane phosphoinositides at the D-3 position. These 3Ј-phosphorylated phospholipids act as second messengers that mediate the diverse cellular functions of PI3K. One of the targets of these lipid second messengers is the serine/threonine kinase Akt/protein kinase B (10). The amino terminus of Akt contains a pleckstrin homology domain that is thought to directly bind the phospholipid products of PI3K activation. This binding recruits Akt to the plasma * This work was supported in part by grants from Ministerio de Educación y Cultura (PM 98/1328 and PM 97/0132), from Fondo de Investigaciones Sanitarias (FIS 98/1328), and from Comunidad Autónoma de Madrid (CAM 08.3/0016/99). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a postdoctoral fellowship from the Comunidad de Madrid.
The PI3K/Akt pathway is activated in response to a large number of stimuli (13). In addition to many different agonists, it has been described that Akt is also activated in response to several types of stress including oxidative stress (14). Importantly, the activation of this pathway, even by stress signals, results in an antiapoptotic effect. Finally, it has recently been shown that hypoxia activates Akt in pten-deficient glioma cells (15).
In the present study we show that hypoxia results in the activation of the PI3K/Akt pathway in PC12 cells by a novel mechanism that involves de novo protein synthesis. The activation of this pathway by hypoxia results in the protection against apoptosis induced by different stimuli.

EXPERIMENTAL PROCEDURES
Cell Culture-Undifferentiated PC12 cells were maintained in RPMI 1640 medium with GLUTAMAX-I (Life Technologies, Inc.) supplemented with 10% horse serum (Life Technologies, Inc.), 5% fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin in a humidified atmosphere containing 5% CO 2 at 37°C. Hypoxia (1% O 2 ) was induced by culture of cells inside an air-tight chamber with inflow and outflow valves that was infused with a mixture of 1% O 2 , 5% CO 2 , 94% N 2 (S.E. Carburos Metalicos S.A., Madrid, Spain); in those experiments using mild hypoxia (5% O 2 ) the mixture used was 5% O 2 , 5% CO 2 , 90% N 2 (S.E. Carburos Metalicos S.A.). Prior to transferring to the hypoxia chamber, the medium was removed from the plates, cells were washed, and fresh (complete or serum-free) medium was added to the plates. In experiments in which drugs were used, the compounds were added immediately prior to transfer to hypoxia/normoxia conditions. For experiments using kinase inhibitors, cells were pretreated during 1 h with the different inhibitors or vehicle and then placed in hypoxia chambers.
Detection of Apoptosis-Apoptosis was determined by flow cytometry analysis of the cell cycle after DNA staining with propidium iodide (PI) as described (16). Cells were grown in 6-well plates until they reached ϳ50% confluence (typically 24 -48 h). Medium was then replaced with complete or serum-free medium (where indicated, specific compounds were included in culture medium), and cells were placed in normoxic or hypoxic conditions for the indicated periods of time. After treatment, cells that remained attached to plates were harvested in PBS/EDTA (5 mM EDTA in PBS) and collected together with those floating (detached) dead cells. Cells were then centrifuged for 5 min at ϳ200 ϫ g, the supernatant was discarded, and the cell pellet was suspended in 0.5 ml of PBS by pipetting thoroughly to avoid cell clumping. The cell suspension was transferred to 4.5 ml of 70% cold ethanol and kept in this fixative for a minimum of 2 h. Ethanol-fixed cells were centrifuged for 5 min at ϳ200 ϫ g and washed once with 10 ml of PBS. Finally, the cell pellet was suspended in 1 ml of PI/Triton X-100 staining solution (0.1% Trixon X-100, 20 g/ml PI, 0.2 mg/ml RNase in PBS) and incubated for 15 min at 37°C. Samples were analyzed by flow cytometry (FACSCalibur, Beckton Dickinson). The analysis of samples included a first selection (gate 1) in which events with the appropriate size (forward scatter) and complexity (side scatter) were selected. Then selected events were analyzed to discard doublets using a PI intensity-width versus PI intensity-area dot plot (gate 2). Finally, events (cells) that were contained in gates 1 and 2 were plotted in a histogram representing the number of events (cells) containing a specific PI intensity-area (e.g. specific amount of DNA). Apoptosis was measured as the percentage of cells with a sub G 0 /G 1 DNA content in the PI intensity-area histogram plot (16).
Viability Assay-Viability was measured by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method as described (17). Briefly, cells were plated on poly-L-lysine pre-coated 96-well plates and grown in complete medium until they reached 30 -50% confluence (usually 12-24 h). Then medium was replaced with complete or serumfree medium, and cells were placed under normoxic (21% O 2 ) or hypoxic conditions (1% O 2 ) for 18 -24 h. After treatments MTT (Sigma) diluted in PBS was added to the culture medium to a final concentration of 1.5 mg/ml, and all samples were incubated at 37°C for 2-4 h under normoxic conditions. Finally, medium was removed, and precipitated MTT salts were solubilized in 0.04 N HCl diluted in isopropanol. Product formation was monitored by reading absorbance at 550 nm using a microplate reader.
Statistical Analysis-Where indicated, experimental data were analyzed using the Prism TM GraphPad (version 2.0) software. Data were analyzed by the analysis of variance (ANOVA) test followed by the Newman-Keuls test; the p values obtained in this analysis are indicated in the text and figures. Similar results (statistical significance) were obtained when using the Tukey or Bonferroni test after ANOVA or when comparing relevant pairs of data by the Student's t test.

RESULTS
Hypoxia Protects PC12 Cells from Apoptosis-To assess the effect of hypoxia on apoptosis, PC12 cells were treated with different combinations of either complete or serum-free medium and normoxic (21% O 2 ) or hypoxic conditions (1% O 2 ) for various periods of time (Fig. 1A). After treatments, apoptosis was measured as the percentage of cells with a content of DNA lower than G 0 /G 1 by analysis of the cell cycle by flow cytometry (16). Fig. 1A shows that hypoxia itself can induce apoptosis in PC12 cells, as previously reported (20), but its importance is only significant after longer than 36 h in low oxygen conditions (Fig. 1A, inset). In contrast, serum withdrawal, which is a potent apoptotic signal for PC12 cells (21), results in a significant percentage of cells undergoing apoptosis as early as 9 h (Fig. 1A). Interestingly, the induction of apoptosis upon serum withdrawal was greatly reduced when cells were cultured under hypoxic conditions (Fig. 1A). The reduction of apoptosis was statistically significant at 12 (p Ͻ 0.001), 24 (p Ͻ 0.001), and 36 (p Ͻ 0.01) h after serum removal (57, 68, and 62% inhibition, respectively). Fig. 1B shows the cell cycle profile of PC12 cells 24 h after the indicated treatments in a representative experiment. The effect of hypoxia on cellular viability was confirmed using the MTT assay (17,22). Whereas hypoxia (1% O 2 for 20 h) itself had little effect on cell viability (14% reduction in viability as compared with cells in normoxia), serum starvation for 20 h reduced viability to 53% as compared with control cells (Fig.  1C). In agreement with flow cytometry data, the reduction of cell viability caused by serum withdrawal was decreased under hypoxic conditions (21% reduction of cell viability as compared with cells in normoxia) (Fig. 1C). The cellular responses to hypoxia can be observed at higher concentrations of oxygen, although with less efficiency. Consistently, a significant reduction in apoptosis, triggered by serum withdrawal, was also observed at 5% O 2 ( Fig. 2A), which corresponds to a pO 2 (36 torr) within the physiological range found in venous blood in vivo. Moreover, treatment with deferoxamine or cobalt chloride (CoCl 2 ), 2 two compounds widely used to mimic many of the effects of hypoxia (23), resulted in a reduction of apoptosis ( Fig. 2A).
We next tested whether hypoxia was able to protect against apoptotic stimuli other than serum withdrawal. Treatment of PC12 cells with the anti-cancer drugs taxol or fluorouracil (5-fluoro-2,4(1H,3H)-pyrimidinedione) for 12-18 h induced a low, but significant, percentage of apoptosis (Fig. 2B). As seen for serum withdrawal, under hypoxic conditions (1% O 2 ) PC12 cells were significantly protected against apoptosis (p Ͻ 0.05 for taxol and p Ͻ 0.001 for fluorouracil) induced by the chemotherapeutic drugs (Fig. 2B). Thus, hypoxic conditions induced by low oxygen or the drug deferoxamine are able to prevent apoptosis induced by different stimuli in PC12 cells.
Hypoxia Induces Akt Activation in PC12 Cells-To identify the mechanism responsible for the pro-survival effect of hy-poxia, we decided to study the effect of hypoxia on the protooncogenic serine/threonine kinase Akt, a molecule involved in the transduction of antiapoptotic signals (13,24). Activation of Akt can be easily detected by Western blotting with antibodies that specifically recognize Akt molecules phosphorylated at serine 473, because this phosphorylation correlates with Akt activity (13).
Hypoxia treatment resulted in phosphorylation of Akt on serine 473 (Fig. 3A). The hypoxia-induced phosphorylation of Akt was observed regardless of the presence or absence of serum or the type of culture substrate used (Fig. 3A). Phosphorylation of Akt on serine 473 reflects its activation as demonstrated by increased kinase activity of immunoprecipitated Akt from hypoxia-treated cells (Fig. 3B).
Interestingly, the activation of PI3K/Akt does not seem to be a general cellular response to hypoxia, because Akt phosphorylation was not observed in other cell lines tested 2 including human vascular endothelial cells, neuroblastoma-69 cells, human embryo kidney 293 cells, HeLa cells, Chinese hamster ovary cells, COS cells, and Hepa cells. However, it seems to be a response restricted to specific cell types, because, in addition to the PC12 cell line, Akt phosphorylation was observed in primary chromaffin cells from cow adrenal gland and neuro-2A cells under hypoxic conditions. 2 Akt phosphorylation was also observed in PC12 cells that were differentiated to a neuronal phenotype by treatment with NGF for 3-6 days, and it correlated with protection against apoptosis. 2 Kinetic studies showed that Akt phosphorylation was detected 4 -6 h after initiation of hypoxic treatment (Fig. 4A). The time course of Akt activation in response to hypoxia was dis- tinct from that observed after challenge with NGF; whereas hypoxia requires several hours to induce Akt activation, stimulation with NGF activates Akt within minutes (Fig. 4B). Moreover, hypoxia-induced Akt phosphorylation was sustained, in contrast with the transient effect found after NGF stimulation (Fig. 4B). The activation of hypoxia-induced signaling pathways was monitored by the stabilization of the hypoxia-inducible factor, EPAS, one of the best characterized intracellular responses to oxygen deprivation (Fig. 4B). Interestingly, hypoxia-activated transcription, assessed by EPAS stabilization, preceded Akt activation (Fig. 4, A and B). The activation of Akt by deferoxamine, a chemical that mimics hypoxia, followed a similar time course to that observed under hypoxia (Fig. 4B). The difference in the kinetics of EPAS stabilization and Akt activation was particularly evident after deferoxamine treatment (Fig. 4B). In addition, activation of Akt by deferoxamine was only observed at doses capable of activating hypoxia-like signaling pathways, as measured by EPAS stabilization (Fig. 4C). Moderate hypoxia (5% O 2 ) also resulted in Akt phosphorylation. 2 Finally, Akt phosphorylation was also observed by treatment with the hypoxia-mimetic CoCl 2 (Fig. 4C).
Hypoxia-induced Akt Activation Requires Protein Synthesis and PI3K Activity-Next, we studied the molecular mechanism by which hypoxia induced Akt activation. Because Akt activation, in most instances, is dependent on PI3K activation (13), we first analyzed whether PI3K activity was affected by hypoxia. As shown in Fig. 5A, PI3K was activated by hypoxia in PC12 cells. Interestingly, the PI3K activity shown in Fig. 5A was detected in anti-phosphotyrosine immunoprecipitates from hypoxic cells, suggesting that hypoxia resulted in the activation of a tyrosine kinase upstream of PI3K/Akt. To confirm the involvement of PI3K, we tested the effect of two structurally nonrelated PI3K inhibitors, LY294002 and wortmannin. Both inhibitors completely prevented Akt phosphorylation induced by hypoxia (Fig. 5B).
The delayed time course of Akt activation (Fig. 4), together with the observation that it occurred after the induction of hypoxia-responsible transcription factors such as EPAS (Fig.  4), suggested that Akt activation by hypoxia required gene expression. Thus, treatment of cells with inhibitors of RNA (actinomycin D) or protein (cycloheximide) synthesis com- A, PC12 cells were plated in 6-well plates, some of which were precoated with poly-L-lysine (PLL), and grown until they reached 40 -50% confluence. Thereafter, the culture medium was replaced with fresh complete (ϩ) or serum-free medium (Ϫ), and cells were transferred to a normoxic (N, 21% O 2 ) or hypoxic (H, 1% O 2 ) atmosphere for 7 h. Cells were collected and lysed, and proteins were detected by Western blotting as described. The experiment was repeated once with similar results. B, PC12 cells were plated in 10-cm culture dishes and grown until they reached ϳ50 -70% confluence. Then the medium was replaced with serum-free medium, and cells were incubated under normoxic (21% O 2 , Nx) or hypoxic (1% O 2 , Hx) conditions for 17 h. Where indicated, cells were stimulated with 150 ng/ml NGF for 10 min. Akt was immunoprecipitated from cells, and its activity was assessed in vitro using H2B as substrate (Kinase React.). The total amount of immunoprecipitated Akt is shown (Immunopr.). Phosphorylation of Akt at serine 473 is also shown (Lysate). WB, Western blot. pletely prevented hypoxia-induced Akt activation (Fig. 5B). The effect of actinomycin or cycloheximide was not due to a toxic or nonspecific effect, because pretreatment with these compounds for several hours did not affect NGF-induced Akt phosphorylation. 2 Finally, we tested whether other signaling pathways contributed to Akt phosphorylation. The calmodulin-regulated and mitogen-activated protein kinase (MAPK) cascades are activated by hypoxia and mediate some of its effects in PC12 cells (25)(26)(27). Inhibition of MAPK/extracellular signal-regulated kinase kinase (MEK), one of the upstream kinases in the MAPK cascade, with PD59098 or treatment with antagonists of calmodulin signaling (W12 and W13 2 ; calmidazolium) had no effect on Akt phosphorylation induced by hypoxia (Fig. 5B). The lack of effect of calmodulin antagonists is particularly relevant because the Ca 2ϩ /calmodulin-dependent protein kinase was recently shown to be able to phosphorylate and activate Akt (13).
Hence, activation of Akt by hypoxia required de novo synthesis of proteins and was dependent on PI3K activity but not MAPK cascade or calmodulin signaling. Importantly, a conditioned medium from hypoxia-treated PC12 cells was unable to induce Akt activation in normoxic cells, 2 suggesting that the effect of hypoxia was not due to the synthesis of a soluble factor able to activate the PI3K/Akt pathway in an autocrine or paracrine fashion.
Inhibition of Apoptosis by Hypoxia Requires PI3K Activi-ty-We have shown that hypoxia prevents apoptosis in response to several stimuli and that this effect correlates with the activation of the PI3K/Akt pathway. To investigate whether there was a functional link between these two phenomena, we investigated the effect of PI3K inhibitors on the protective effect of hypoxia. As shown in Fig. 6, the reduction of apoptosis, upon serum removal, observed under hypoxia was completely prevented by treatment of cells with the PI3K inhibitors LY294002 or wortmannin. These results indicate that the protection against apoptosis induced by hypoxia depends on the activation of the PI3K/Akt survival pathway. In addition to the PI3K/Akt pathway, the MAPK cascade has an important role in survival of PC12 cells (28). However, the MEK inhibitor (PD98059) had no effect on the protective effect of hypoxia (Fig.  6), despite increasing the percentage of cells undergoing apoptosis upon serum withdrawal under normoxic conditions (Fig.  6). In agreement with these results, the protective effects of deferoxamine and CoCl 2 treatment on apoptosis upon serum withdrawal ( Fig. 2A) were also abrogated by inhibition of PI3K. 2

DISCUSSION
Reduction of oxygen supply has deleterious effects on many tissues. Neurons and cardiac muscle cells are particularly sensitive because they suffer both necrotic and apoptotic cell death when deprived of oxygen, which occurs during ischemia (29,30). However, hypoxia is not always lethal. Faced with this FIG. 4. Akt is phosphorylated in response to hypoxia and deferoxamine at late times. In all cases PC12 cells were plated in 6-well plates and grown until they reached 40 -50% confluence prior to treatments. After treatments, cell lysates were processed for Western blotting using the indicated antibodies. A, the culture medium was replaced with fresh complete medium, and cells were cultured for the indicated periods of time (h) in a 21% O 2 (Nx) or 1% O 2 (Hypoxia) atmosphere. Similar results were obtained in at least two more experiments. B, cells were exposed to 1% stress, some cell types undergo cell cycle arrest but remain viable (31), and others remain unaltered. Moreover, the induction of apoptosis observed after long periods of hypoxia could be due to acidosis rather than to a direct effect of hypoxia (31). In addition, organisms are able to respond to both acute and chronic reductions in oxygen tension with specific adaptive responses aimed to restore appropriate oxygen supply. Some of these responses, such as induction of angiogenesis, are local, whereas others are systemic. The systemic responses include hyperventilation, increase in heart output, increase in erythropoiesis, pulmonary vasoconstriction, and carotid body hypertrophy, among others. The induction of these responses is under the control of specific cell types that have evolved to sense oxygen levels. These cell types include the glomus cells of the carotid body, the cells of the neuroepithelial bodies in the lung, and the chromaffin cells of the medulla of the adrenal gland, among others. In particular, the chromaffin cells are critical players in the response to oxygen deprivation in fetuses and neonates (1). Thus, it is feasible that these cell types have developed specific mechanisms ensuring their viability and functionality during hypoxia.
In this study, we have shown that under hypoxic conditions PC12 cells are protected against apoptosis triggered by different stimuli. The protection against apoptosis is observed at pO 2 levels within the physiopathological range, and it can be induced by pharmacological agents that mimic hypoxia. This effect is paralleled by activation of the pro-survival PI3K/Akt pathway through a mechanism that requires de novo protein synthesis. Moreover, inhibition of PI3K not only prevents Akt activation but also the antiapoptotic effect of hypoxia. Thus, it is likely that the activation of the PI3K/Akt pathway by hypoxia is the mechanism responsible for its antiapoptotic effect.
To our knowledge, this is the first report showing that, at least in specific cell types, hypoxia is able to promote survival. Our data concur with a previous report describing deferoxamine treatment as being able to prevent apoptosis (32). Whether the phenomenon of ischemic preconditioning (33), the partial resistance to ischemia-induced damage found after a previous episode of moderate ischemia, is due to a mechanism similar to the one described here will require further work. However, it is intriguing that preconditioning, similar to activation of Akt, requires de novo protein synthesis (29). In addition, we have shown that hypoxia renders cells resistant to apoptosis induced by the chemotherapeutic drugs taxol and fluorouracil, an effect that might contribute to the partial resistance to therapy observed in the hypoxic regions of tumors as compared with the normoxic areas of the same tumors (34). Nevertheless, this is not the only example in which proapoptotic stimuli induce an antiapoptotic response. Exposure of cells to H 2 O 2 results in the activation of the PI3K/Akt pathway and resistance to induction of apoptosis (14). Hence, activation of the PI3K/Akt survival pathway could be a general cellular response to cell and tissue injury.
The activation of Akt by hypoxia in cells derived from ptenϪ/Ϫ tumors has recently been described (15). Here we provide evidence that the activation of the PI3K/Akt pathway by hypoxia also occurs in some specific cells that are apparently normal for pten (35), including PC12 cells and primary chromaffin cells from cow adrenal gland medulla. This seems to be a specific response, restricted to at least cells of chromaffin lineage, rather than a general effect of hypoxia, because we did not detect any significant phosphorylation of Akt in other cell types tested. The lack of Akt activation in these cell lines was not the result of defective responses to hypoxia, because in all cases hypoxia-inducible factors were stabilized in response to hypoxia. 2 In addition, the mechanism, involving de novo protein synthesis, and the sustained activation of Akt have not been described previously. One remaining question about this novel mechanism of Akt activation is the nature of the protein whose synthesis is required for the activation of Akt. Most likely it is not a soluble factor, at least not a stable one, released by hypoxic cells, because conditioned medium from hypoxiatreated cells failed to induce Akt activation in PC12 cells grown in normoxia. 2 It is still possible that the effect is mediated by the induction of a nonsecreted membrane-bound ligand or by the induction of both a soluble factor and its receptor. One further possibility is that the synthesized molecule acts in a cell-autonomous manner by direct activation of the PI3K/Akt pathway from inside the cell. Further work will be required to differentiate among these possibilities. The identification of such a molecule could explain why the effect of hypoxia on Akt is restricted to some cell types.
Hypoxia treatment results in changes in gene expression that are mediated by the activation of different transcription factors. Hypoxia-inducible factors are the best characterized; others include cAMP response element-binding protein (4), NF-B (36), and c-Fos (2). Although we cannot formally rule out that hypoxia-inducible transcription factors are involved in Akt activation, indirect evidence suggests that it is not the case, because both calmodulin antagonists and the MEK inhibitor PD98059 prevent EPAS activity in PC12 cells (25) without affecting hypoxia-induced Akt activation.
We suggest that the activation of survival pathways by hypoxia, at least in chromaffin cells, ensures that the cells remain viable and able to trigger the responses required for the adaptation to varying oxygen tension. Finally, activation of Akt has many other effects, in addition to promoting survival. Among these effects are changes in gene expression, induction of cell proliferation, and increasing glucose uptake (13). It is thus possible that many other effects of hypoxia in cell biology, in this cell type, could be mediated by activation of the PI3K/Akt pathway.