Hypoxia regulates expression and activity of Kv1.3 channels in T lymphocytes: a possible role in T cell proliferation

T lymphocytes are exposed to hypoxia during their development and also when they migrate to hypoxic pathological sites such as tumors and wounds. Although hypoxia can affect T cell development and function, the mechanisms by which immune cells sense and respond to changes in O(2)-availability are poorly understood. K(+) channels encoded by the Kv1.3 subtype of the voltage-dependent Kv1 gene family are highly expressed in lymphocytes and are involved in the control of membrane potential and cell function. In this study, we investigate the sensitivity of Kv1.3 channels to hypoxia in freshly isolated human T lymphocytes and leukemic Jurkat T cells. Acute exposure to hypoxia (20 mmHg, 2 min) inhibits Kv1.3 currents in both cell types by 20%. Prolonged exposure to hypoxia (1% O(2) for 24 h) selectively decreases Kv1.3 protein levels in Jurkat T cells by 47%, but not Kvbeta2 and SK2 Ca-activated K(+) channel subunit levels. The decrease in Kv1.3 protein levels occurs with no change in Kv1.3 mRNA expression and is associated with a significant decrease in K(+) current density. A decrease in Kv1.3 polypeptide levels similar to that obtained during hypoxia is produced by Kv1.3 channel blockage. Our results indicate that hypoxia produces acute and long-term inhibition of Kv1.3 channels in T lymphocytes. This effect could account for the inhibition of lymphocyte proliferation during hypoxia. Indeed, we herein present evidence showing that hypoxia selectively inhibits TCR-mediated proliferation and that this inhibition is associated with a decrease in Kv1.3 proteins.     

Chronic hypoxia impairs the differentiation of 3T3-L1 fibroblast in culture: Role of sustained protein kinase C activation

The effect of hypoxia on 3T3-L1 cell differentiation was examined in confluent cultures incubated with differentiation medium (DM) followed by incubation in growth medium (GM). Control cultures remained in GM throughout the incubation period. Eight days after the incubation, cells were assessed either for changes in morphology by staining with Oil Red O/hematoxylin or harvested to measure protein kinase C activity. Morphological examination of stained cells showed almost complete differentiation of normoxic cells to adipocytes when exposed to DM. By contrast hypoxia caused a dramatic inhibition of differentiation under similar media conditions with only 34 ± 4% of cells accumulating fat deposits. Cultures sustained in GM under normoxic or hypoxic conditions were devoid of any fat deposits, reflecting an undifferentiated phenotype. Normoxic cells exposed to DM exhibited a significantly lower membrane to cytosolic ratio of protein kinase C in comparison with cells maintained in GM, which is consistent with differentiated and undifferentiated phenotypes, respectively. In comparison with normoxic cells incubated in DM, cells exposed to hypoxia under similar media conditions exhibited a significantly higher membrane to cytosolic ratio of protein kinase C, indicating sustained activation of the enzyme. In addition, cells in differentiation medium exposed to hypoxia in the presence of the protein kinase C inhibitors staurosporine or H₇ exhibited a significant increase in the number of fat accumulating cells when compared with hypoxic controls. These studies indicate that chronic hypoxia impairs the differentiation of 3T3-L1 cells to adipocytes in association with the sustained activation of protein kinase C, which appears to play a role in mediating this process. © 1994 Wiley-Liss, Inc.     

Enzymatic removal of oxidized protein aggregates from erythrocyte membranes

Erythrocytes oxidized or aged in the circulation undergo membrane protein aggregation and anti-band 3 autoantibody binding to the cell surface. When human erythrocytes were mildly oxidized in vitro with 0.1 mM Fe(III) at 37 degrees C for 3 h, the aggregation of nonionic detergent C(12)E(8)-insoluble membrane protein and the binding of anti-band 3 IgG to the cell surface were increased. Incubation of membranes isolated from the oxidized cells increased the amount of protein aggregates by 5-fold after 6 h, while incubation for a further 12 h sharply decreased the amount of aggregates. In the presence of diisopropyl fluorophosphate (DFP), however, the increased amount of aggregates was maintained in the subsequent incubation. Western blot analysis of the aggregates using rabbit anti-band 3 showed that band 3 protein aggregates increased in the initial stage of incubation and decreased upon subsequent incubation, whereas the increased band 3 protein aggregates did not subsequently decrease when membranes were incubated in the presence of DFP. Incubation of the oxidized cells at 37 degrees C for 18 h caused reduction of the membrane protein aggregates and the (125)I-anti-band 3 IgG binding to the cell surface, while incubation in the presence of DFP did not cause these reductions. The results suggest that the oxidation-induced cell membrane protein aggregates were probably removed by 80-kDa serine protease, namely, oxidized protein hydrolase (OPH), in the oxidized cell membranes [Fujino et al. (1998) Biochim. Biophys. Acta 1374, 47-54; (1998) J. Biochem. 124, 1077-1085; (2000) Biochim. Biophys. Acta 1478, 102-112], and as a result the increased anti-band 3 binding to the cell surface was reduced.     

Effects of hypoxia, anoxia, and metabolic inhibitors on KATP channels in rat femoral artery myocytes

Vascular ATP-sensitive potassium (KATP) channels have an important role in hypoxic vasodilation. Because KATP channel activity depends on intracellular nucleotide concentration, one hypothesis is that hypoxia activates channels by reducing cellular ATP production. However, this has not been rigorously tested. In this study we measured KATP current in response to hypoxia and modulators of cellular metabolism in single smooth muscle cells from the rat femoral artery by using the whole cell patch-clamp technique. KATP current was not activated by exposure of cells to hypoxic solutions (Po2 approximately 35 mmHg). In contrast, voltage-dependent calcium current and the depolarization-induced rise in intracellular calcium concentration ([Ca2+]i) was inhibited by hypoxia. Blocking mitochondrial ATP production by using the ATP synthase inhibitor oligomycin B (3 microM) did not activate current. Blocking glycolytic ATP production by using 2-deoxy-D-glucose (5 mM) also did not activate current. The protonophore carbonyl cyanide m-chlorophenylhydrazone (1 microM) depolarized the mitochondrial membrane potential and activated KATP current. This activation was reversed by oligomycin B, suggesting it occurred as a consequence of mitochondrial ATP consumption by ATP synthase working in reverse mode. Finally, anoxia induced by dithionite (0.5 mM) also depolarized the mitochondrial membrane potential and activated KATP current. Our data show that: 1) anoxia but not hypoxia activates KATP current in femoral artery myocytes; and 2) inhibition of cellular energy production is insufficient to activate KATP current and that energy consumption is required for current activation. These results suggest that vascular KATP channels are not activated during hypoxia via changes in cell metabolism. Furthermore, part of the relaxant effect of hypoxia on rat femoral artery may be mediated by changes in [Ca2+]i through modulation of calcium channel activity.     

Quercetin suppresses hypoxia-induced accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) through inhibiting protein synthesis

Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells and induce the accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) in normoxia. In this study, under hypoxic conditions (1% O(2)), we examined the effect of quercetin on the intracellular level of HIF-1alpha and extracellular level of vascular endothelial growth factor (VEGF) in a variety of human cancer cell lines. Surprisingly, we observed that quercetin suppressed the HIF-1alpha accumulation during hypoxia in human prostate cancer LNCaP, colon cancer CX-1, and breast cancer SkBr3 cells. Quercetin treatment also significantly reduced hypoxia-induced secretion of VEGF. Suppression of HIF-1alpha accumulation during treatment with quercetin in hypoxia was not prevented by treatment with 26S proteasome inhibitor MG132 or PI3K inhibitor LY294002. Interestingly, hypoxia (1% O(2)) in the presence of 100 microM quercetin inhibited protein synthesis by 94% during incubation for 8 h. Significant quercetin concentration-dependent inhibition of protein synthesis and suppression of HIF-1alpha accumulation were observed under hypoxic conditions. Treatment with 100 microM cycloheximide, a protein synthesis inhibitor, replicated the effect of quercetin by inhibiting HIF-1alpha accumulation during hypoxia. These results suggest that suppression of HIF-1alpha accumulation during treatment with quercetin under hypoxic conditions is due to inhibition of protein synthesis.     

A Novel Prolyl Hydroxylase Inhibitor Protects Against Cell Death After Hypoxia

Hypoxia-inducible factor 1 (HIF-1) is regulated by the oxygen-dependent hydroxylation of proline residues by prolyl hydroxylases (PHDs). We recently developed a novel PHD inhibitor, TM6008, that suppresses the activity of PHDs, inducing continuous HIF-1α activation. In this study, we investigated how TM6008 affects cell survival after hypoxic conditions capable of inducing HIF-1α expression and how TM6008 regulates PHDs and genes downstream of HIF-1α. After SHSY-5Y cells had been subjected to hypoxia, TM6008 was added to the cell culture medium under normoxic conditions. Apoptotic cell death was significantly augmented just after the hypoxic conditions, compared with cell death under normoxic conditions. Notably, when TM6008 was added to the media after the cells had been subjected to hypoxia, the expression level of HIF-1α increased and the number of cell deaths decreased, compared with the results for cells cultured in media without TM6008 after hypoxia, during the 7-day incubation period under normoxic conditions. Moreover, the protein expression levels of heme oxygenase 1, erythropoietin, and glucose transporter-3, which were genes downstream of HIF-1α, were elevated in media to which TM6008 had been added, compared with media without TM6008, during the 7-day incubation period under normoxic conditions. However, the protein expression levels of PHD2 and p53 which suppressed cell proliferation were suppressed in the media to which TM6008 had been added. Thus, TM6008, which suppresses the protein expressions of PHD2 and p53, might play an important role in cell survival after hypoxic conditions, with possible applications as a new compound for treatment after ischemic stroke.     

70-kDa heat shock protein expression in cultured rat astrocytes after hypoxia: regulatory effect of almitrine

Induction of heat shock proteins (Hsps), especially the 70-kDa family, is well observed in nervous tissues in response to various stressful conditions. By using rat astrocytes in primary culture, the expression of the inducible (Hsp70) and the constitutive (Hsc70) 70-kDa Hsps immunoreactivity of cells exposed to hypoxic conditions has been investigated. We observed that exposure of astroglial cells to an hypoxic-normoxic sequence induces a significant decrease of Hsc70 immunoreactivity. The presence of the heat inducible stress protein Hsp70 is never observed in hypoxic cells not in control. Hsc 70 lowering is associated with ultrastructural alterations characterized by mitochondria swelling, formation of vacuoles and accumulation of dense material in the cell cytoplasm. The effects of addition of almitrine to the culture medium before and during hypoxia on Hsps immunoreactivity have been examined. The presence of the drug prevents the decrease of Hsc 70 immunoreactivity induced by hypoxia. Furthermore, some ultrastructural improvement is observed in astroglial cells treated with almitrine suggesting some protecting role of Hsc70 on cell damage induced by hypoxia.     

Differential inhibition of myoblast fusion.

The aggregation and fusion of myoblasts in the presence of either metabolic inhibitors or alterations in the incubation medium or under conditions which result in structural changes in the cells was studied using previously described assays for the intercellular interactions of myoblasts in suspension [Knudsen, K. A., and Horwitz, A. F. (1977). Develop. Biol.58, 328]. These perturbations inhibit myoblast fusion differently. For example, energy poisons, prior trypsin or glutaraldehyde treatment, and inhibitors of protein or cholesterol synthesis all inhibit the Ca²⁺-mediated myoblast aggregation. In contrast, whereas myoblasts aggregate in the presence of 20 mM Mg²⁺, these aggregates are dispersed, even after 1–2 hr, with EDTA or trypsin. Furthermore, enriching the fatty acyl chains in elaidate or prior incubation of the myoblasts in the presence of cytochalasin B or colchicine results in aggregates which, after 1–2 hr, are dispersed by trypsin but not by EDTA. Aggregates of unaltered, control myoblasts, on the other hand, begin to show resistance to dispersion by trypsin after these times. These observations support the suggestion that multinucleate cell formation results from a sequence of events. The influence of these perturbations on cellular aggregation also provides some initial, tentative insight into the molecular mechanism of myoblast fusion. Recognition (calcium-mediated aggregate formation) appears to be mediated by a protein(s) that is turning over during the period of fusion competence, while membrane union (formation of aggregates resistant to dispersion by trypsin) most likely involves the direct participation of membrane lipid.     

Post-irradiation hypoxic incubation of X-irradiated MOLT-4 cells reduces apoptotic cell death by changing the intracellular redox state and modulating SAPK/JNK pathways

To elucidate radiobiological effects of hypoxia on X-ray-induced apoptosis, MOLT-4 cells were treated under four set of conditions: (1) both X irradiation and incubation under normoxia, (2) X irradiation under hypoxia and subsequent incubation under normoxia, (3) X irradiation under normoxia and subsequent incubation under hypoxia, and (4) both X irradiation and incubation under hypoxia, and the induction of apoptosis was examined by fluorescence microscopy. About 28–33% apoptosis was observed in cells treated under conditions 1 and 2, but this value was significantly reduced to around 18–20% in cells treated under conditions 3 and 4, suggesting that post-irradiation hypoxic incubation rather than hypoxic irradiation mainly caused the reduction of apoptosis. The activation and expression of apoptosis signal-related molecules SAPK/JNK, Fas and caspase-3 were also suppressed by hypoxic incubation. Effects of hypoxic incubation were canceled when cells were treated under conditions 3 and 4 with an oxygen-mimicking hypoxic cell radiosensitizer, whereas the addition of N-acetyl-L-cysteine again reduced the induction of apoptosis. From these results it was concluded that hypoxia reduced the induction of apoptosis by changing the intracellular redox state, followed by the regulation of apoptotic signals in X-irradiated MOLT-4 cells.     

Hypoxia increases transepithelial electrical conductance and reduces occludin at the plasma membrane in alveolar epithelial cells via PKC-ζ and PP2A pathway

During pulmonary edema, the alveolar space is exposed to a hypoxic environment. The integrity of the alveolar epithelial barrier is required for the reabsorption of alveolar fluid. Tight junctions (TJ) maintain the integrity of this barrier. We set out to determine whether hypoxia creates a dysfunctional alveolar epithelial barrier, evidenced by an increase in transepithelial electrical conductance (G(t)), due to a decrease in the abundance of TJ proteins at the plasma membrane. Alveolar epithelial cells (AEC) exposed to mild hypoxia (Po(2) = 50 mmHg) for 30 and 60 min decreased occludin abundance at the plasma membrane and significantly increased G(t). Other cell adhesion molecules such as E-cadherin and claudins were not affected by hypoxia. AEC exposed to hypoxia increased superoxide, but not hydrogen peroxide (H(2)O(2)). Overexpression of superoxide dismutase 1 (SOD1) but not SOD2 prevented the hypoxia-induced G(t) increase and occludin reduction in AEC. Also, overexpression of catalase had a similar effect as SOD1, despite not detecting any increase in H(2)O(2) during hypoxia. Blocking PKC-ζ and protein phosphatase 2A (PP2A) prevented the hypoxia-induced occludin reduction at the plasma membrane and increase in G(t). In summary, we show that superoxide, PKC-ζ, and PP2A are involved in the hypoxia-induced increase in G(t) and occludin reduction at the plasma membrane in AEC.     

Effects of hypoxia on potassium homeostasis and pigment epithelial cells in the cat retina

Intracellular recordings show that light-evoked hyperpolarizations of the apical and basal membranes of the cat retinal pigment epithelium (RPE) are altered by mild hypoxia. RPE cells, like glia, have a high K+ conductance, and measurements with K+-sensitive microelectrodes show that the hypoxic changes in the RPE cell are largely the result of changes in extracellular [K+] in the subretinal space [( K+]o) rather than direct effects on RPE cells. During hypoxia, light-evoked [K+]o responses and membrane responses have longer times to peak, slower and less complete recovery during illumination, and larger amplitudes. In addition to the effects on light-evoked responses, hypoxia causes a depolarization of first the apical and then the basal membranes of RPE cells under dark-adapted conditions. The basal depolarization is accompanied by a decrease in basal membrane resistance. These depolarizations appear to be caused by a rapid increase in [K+]o at the onset of hypoxia, which is maximal in dark adaptation, and smaller if the retina is subjected to maintained illumination. All of the effects are graded with the severity of hypoxia and can be observed at arterial oxygen tensions as high as 65 mmHg, although the threshold may be even higher. We argue that the origin of hypoxic [K+]o changes is probably an inhibition of the photoreceptors' Na+/K+ pump. This work then suggests that photoreceptors are more sensitive to hypoxia than previously believed, and that the high oxygen tension normally provided by the choroidal circulation is necessary for normal photoreceptor function.     

Chronic hypoxia alters the physiological and morphological trajectories of developing chicken embryos

Chicken embryos were chronically exposed to hypoxia (P(O(2)) approximately 110 mmHg) during development, and assessed for detrimental metabolic and morphological effects. Eggs were incubated in one of four groups: control (i.e. 151 mmHg), or treated with continuous 110 mmHg (15% O(2)) during days 1-6 (H1-6), 6-12 (H6-12), or 12-18 (H12-18) with normoxia during the remaining incubation. Metabolism (V(O(2))), body mass, hemoglobin (Hb) and hematocrit (Hct) were measured in embryos on days 12 and 18 of incubation and in day-old hatchlings. Ability to maintain V(O(2)) was acutely measured during a step-wise decrease in P(O(2)) from normoxia to hypoxia (55 mmHg). On day 12, V(O(2)) of H1-6 eggs were significantly lower than in the control and H6-12 eggs. P(crit) in H6-12 eggs was lower than in control and H1-6 eggs. Body mass of H1-6 and H6-12 embryos on day 12 was significantly lower than in control embryos, while in H6-12 embryos, Hct and Hb were higher. On day 18, H6-12 embryos had significantly lower V(O(2)) than control eggs. Body mass of H6-12 and H12-18 embryos was significantly lower than control embryos. Hct and Hb did not differ between treatments. In hatchlings, mass, Hb and Hct had returned to values statistically identical to controls. However, H6-12 embryos had significantly lower V(O(2)). Long-term hypoxia altered V(O(2)) when hypoxic incubation occurred during the middle third of incubation, but not during earlier or later incubation. Thus, chronic hypoxic exposure during critical periods in development altered the developmental physiological trajectories and modified the phenotypes of the developing embryos.     

Activation of AMP-activated protein kinase mediates acute and severe hypoxic injury to pancreatic beta cells

In islet transplantation, a substantial part of the graft becomes nonfunctional for several reasons including hypoxia. AMP-activated protein kinase (AMPK) in mammalian cells is a regulator of energy homeostasis, and is activated by metabolic stresses such as hypoxia. However, the role of AMPK in hypoxic injury to pancreatic beta cells is not clear. When a rat beta cell line, INS-1 cell, was incubated in an anoxic chamber, phosphorylation of both AMPK and its downstream protein, acetyl-CoA carboxylase 2 increased with time. Adenovirus-mediated expression of constitutively active form of AMPK under normoxic conditions increased caspase-3 activation, suggesting induction of apoptosis. Reactive oxygen species production also increased with time during hypoxia. Pretreatment with compound C, an AMPK inhibitor, or N-acetyl-l-cysteine, an antioxidant, significantly lowered hypoxia-mediated cell death. These results suggest that AMPK, in association with oxidative stress, plays an important role in acute and severe hypoxic injury to pancreatic beta cells.     

Chronic hypoxia promotes an aggressive phenotype in rat prostate cancer cells

In general, tumors cells that are resistant to apoptosis and increase angiogenesis are a result of the hypoxic responses contributing to the malignant phenotype. In this study, we developed a chronic hypoxic cell model (HMLL), by incubating the prostate cancer MatLyLu cells in a hypoxic chamber (1% O(2)) over 3 weeks. Surviving cells were selected through each cell passage and were grown in the hypoxic condition up to 8 weeks. This strategy resulted in survival of only 5% of the cells. The surviving hypoxic cells displayed a greater stimulation on hypoxic adaptive response, including a greater expression of glucose transporter1 (Glut1) and VEGF secretion. In addition, higher invasion activity was observed in the chronic hypoxic HMLL cells as compared to MatLyLu cells exposed to acute hypoxia (1% O(2), 5 h) using the matrigel assay. To further examine the role of HIF-1alpha in tumor progression, both MatLyLu and HMLL cells were transfected with dominant-negative form of HIF-1alpha (DNHIF-1alpha). The Matrigel invasion activity induced by chronic hypoxia was significantly attenuated by DNHIF-1alpha. These results suggest that signaling pathways leading to hypoxic response may be differentially regulated in chronic hypoxic cells and acute hypoxic cells. Chronic hypoxia may play a greater role than acute hypoxia in promoting the aggressive phenotype of tumor cells. This observation mimics the clinical scenario where tumor cells following treatment with radiation are subjected to hypoxic conditions. The reemergence of tumor following treatment usually results in tumor cells that are more aggressive and metastatic.     

Secretion Pores in Human Endothelial Cells during Acute Hypoxia

Weibel-Palade bodies (WPB) are endothelial vesicles that store von Willebrand factor (vWF), involved in the early phase of hemostasis. In the present study we investigated the morphodynamics of single WPB plasma membrane fusion events upon hypoxic stimulation by using atomic force microscopy (AFM). Simultaneously, we measured vWF release from endothelial cells to functionally confirm WPB exocytosis. Exposing human endothelial cells to hypoxia (pO2 = 5 mm Hg) we found an acute (within minutes) release of vWF. Despite acute vWF release, potential cellular modulators of secretion, such as intracellular pH and cell volume, remained unchanged. We only detected a slight instantaneous increase of cytosolic Ca2+ concentration. Although overall cell morphology remained virtually unchanged, high resolution AFM images of hypoxic endothelial cells disclosed secretion pores, most likely the loci of WPB exocytosis on luminal plasma membrane. We conclude that short-term hypoxia barely alters overall cell morphology and intracellular milieu. However, at nanometer scale, hypoxia instantaneously switches the smooth luminal plasma membrane to a rough activated cell surface, covered with secretion pores that release vWF to the luminal cell surface.     

Chronic hypoxia promotes an aggressive phenotype in rat prostate cancer cells

In general, tumors cells that are resistant to apoptosis and increase angiogenesis are a result of the hypoxic responses contributing to the malignant phenotype. In this study, we developed a chronic hypoxic cell model (HMLL), by incubating the prostate cancer MatLyLu cells in a hypoxic chamber (1% O₂) over 3 weeks. Surviving cells were selected through each cell passage and were grown in the hypoxic condition up to 8 weeks. This strategy resulted in survival of only 5% of the cells. The surviving hypoxic cells displayed a greater stimulation on hypoxic adaptive response, including a greater expression of glucose transporter1 (Glut1) and VEGF secretion. In addition, higher invasion activity was observed in the chronic hypoxic HMLL cells as compared to MatLyLu cells exposed to acute hypoxia (1% O₂, 5 h) using the matrigel assay. To further examine the role of HIF-1α in tumor progression, both MatLyLu and HMLL cells were transfected with dominant-negative form of HIF-1α (DNHIF-1α). The Matrigel invasion activity induced by chronic hypoxia was significantly attenuated by DNHIF-1α. These results suggest that signaling pathways leading to hypoxic response may be differentially regulated in chronic hypoxic cells and acute hypoxic cells. Chronic hypoxia may play a greater role than acute hypoxia in promoting the aggressive phenotype of tumor cells. This observation mimics the clinical scenario where tumor cells following treatment with radiation are subjected to hypoxic conditions. The reemergence of tumor following treatment usually results in tumor cells that are more aggressive and metastatic.