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BACKGROUND: The Richalet hypoxia sensitivity test (RT), which quantifies the cardiorespiratory response to acute hypoxia during exercise at an intensity corresponding to a heart rate of ~130 bpm in normoxia, can predict susceptibility of altitude sickness. Its ability to predict exercise performance in hypoxia is unknown. OBJECTIVES: Investigate: (1) whether cerebral blood flow (CBF) and cerebral tissue oxygenation (O2Hb; oxygenated hemoglobin, HHb; deoxygenated hemoglobin) responses during RT predict time-trial cycling (TT) performance in severe hypoxia; (2) if subjects with blunted cardiorespiratory responses during RT show greater impairment of TT performance in severe hypoxia. STUDY DESIGN: Thirteen men [27 ± 7 years (mean ± SD), Wmax: 385 ± 30 W] were evaluated with RT and the results related to two 15 km TT, in normoxia and severe hypoxia (FIO2 = 0.11). RESULTS: During RT, mean middle cerebral artery blood velocity (MCAv: index of CBF) was unaltered with hypoxia at rest (p > 0.05), while it was increased during normoxic (+22 ± 12 %, p < 0.05) and hypoxic exercise (+33 ± 17 %, p < 0.05). Resting hypoxia lowered cerebral O2Hb by 2.2 ± 1.2 μmol (p < 0.05 vs. resting normoxia); hypoxic exercise further lowered it to -7.6 ± 3.1 μmol below baseline (p < 0.05). Cerebral HHb, increased by 3.5 ± 1.8 μmol in resting hypoxia (p < 0.05), and further to 8.5 ± 2.9 μmol in hypoxic exercise (p < 0.05). Changes in CBF and cerebral tissue oxygenation during RT did not correlate with TT performance loss (R = 0.4, p > 0.05 and R = 0.5, p > 0.05, respectively), while tissue oxygenation and SaO2 changes during TT did (R = -0.76, p < 0.05). Significant correlations were observed between SaO2, MCAv and HHb during RT (R = -0.77, -0.76 and 0.84 respectively, p < 0.05 in all cases). CONCLUSIONS: CBF and cerebral tissue oxygenation changes during RT do not predict performance impairment in hypoxia. Since the changes in SaO2 and brain HHb during the TT correlated with performance impairment, the hypothesis that brain oxygenation plays a limiting role for global exercise in conditions of severe hypoxia remains to be tested further.

Intermittent hypoxia is known to elicit adaptive changes that increase tissue oxygen delivery. We investigated the effects of intermittent hypoxic exposure (IHE) in normal skin (course study) and evaluated whether preoperative IHE-preconditioning alleviates acute ischemic injury in skin flaps (flap study). For 4 weeks, 55 rats were exposed to a daily IHE-session (6 hypoxic cycles, 9% O(2), 6 to 10 min) separated by 3 min of reoxygenation (20.9% O(2)). The time course of intracutaneous PO(2) was measured at weekly intervals. VEGF/VEGF-receptor-mRNA and vascular density were measured in normal skin before and after 1, 2, 3, and 4 weeks of the IHE course (20 rats). In 35 rats, skin flaps were elevated after IHE and similar studies were undertaken in samples from 3 flap areas at postoperative days 0.5, 2, and 4. Additionally, flap survival and cutaneous blood flow were quantitated. In normal skin, PO(2) was initially significantly decreased during the hypoxic cycles (PO(2) 4.1 +/- 1.5 mmHg). After 4 weeks of IHE, PO(2) (34.5 +/- 5.8 mmHg) was maintained even under hypoxic conditions. In flaps, IHE led to a 2.6-fold increase in PO(2), increased flap survival (+ 19.4%, day 7), and blood flow (+ 19.1%, day 10) (p < 0.01). In neither study did IHE-preconditioning increase VEGF/VEGF-receptor-mRNA expression or vascular density. We conclude that (1) IHE induces pronounced hypoxia in the skin, but progressive adaptation occurs within 4 weeks, (2) preoperative IHE-preconditioning leads to significantly higher PO(2), blood flow, and tissue survival in ischemic skin flaps, and (3) the transient hypoxia induced by IHE does not alter expression of VEGF or its receptors nor does it trigger angiogenesis.

Neuronal autophagy is increased in numerous excitotoxic conditions including neonatal cerebral hypoxia-ischemia (HI). However, the role of this HI-induced autophagy remains unclear. To clarify this role we established an in vitro model of excitotoxicity combining kainate treatment (Ka, 30 µM) with hypoxia (Hx, 6% oxygen) in primary neuron cultures. KaHx rapidly induced excitotoxic death that was completely prevented by MK801 or EGTA. KaHx also stimulated neuronal autophagic flux as shown by a rise in autophagosome number (increased levels of LC3-II and punctate LC3 labeling) accompanied by increases in lysosomal abundance and activity (increased SQSTM1/p62 degradation, and increased LC3-II levels in the presence of lysosomal inhibitors) and fusion (shown using an RFP-GFP-LC3 reporter). To determine the role of the enhanced autophagy we applied either pharmacological autophagy inhibitors (3-methyladenine or pepstatinA/E64) or lentiviral vectors delivering shRNAs targeting Becn1 or Atg7. Both strategies reduced KaHx-induced neuronal death. A prodeath role of autophagy was also confirmed by the enhanced toxicity of KaHx in cultures overexpressing BECN1 or ATG7. Finally, in vivo inhibition of autophagy by intrastriatal injection of a lentiviral vector expressing a Becn1-targeting shRNA increased the volume of intact striatum in a rat model of severe neonatal cerebral HI. These results clearly show a death-mediating role of autophagy in hypoxic-excitotoxic conditions and suggest that inhibition of autophagy should be considered as a neuroprotective strategy in HI brain injuries.

OBJECTIVES The aim of this study was to test whether adenosine triphosphate sensitive K(+) (KATP) channel expression relates to mechanical and hypoxic stress within the left human heart. BACKGROUND The KATP channels play a vital role in preserving the metabolic integrity of the stressed heart. However the mechanisms that govern the expression of their subunits (e.g. potassium inward rectifier [Kir] 6.2) in adult pathologies are mostly unknown. METHODS We collected biopsies from the 4 cardiac chambers and 50 clinical parameters from 30 surgical patients with severe mitral dysfunction. Proteins and messenger ribonucleic acids (mRNAs) of KATP pore subunits and mRNAs of their known transcriptional regulators (forkhead box [FOX] F2 FOXO1 FOXO3 and hypoxia inducible factor [HIF] 1a) were measured respectively by Western blotting immunohistochemistry and quantitative real time polymerase chain reaction and submitted to statistical analysis. RESULTS In all heart chambers Kir6.2 mRNA correlated with HIF 1a mRNA. Neither Kir6.1 nor Kir6.2 proteins positively correlated with their respective mRNAs. The HIF 1a mRNA related in the left ventricle to aortic pressure in the left atrium to left atrial pressure and in all heart chambers to a decreased Kir6.2 protein/mRNA ratio. Interestingly in the left heart Kir6.2 protein and its immunohistochemical detection in myocytes were maximal at low venous PO(2). In the left ventricle the Kir6.2 protein/mRNA ratio was also significantly higher at low venous PO(2) suggesting that tissue hypoxia might stabilize the Kir6.2 protein. CONCLUSIONS Results suggest that post transcriptional events determine Kir6.2 protein expression in the left ventricle of patients with severe mitral dysfunction and low venous PO(2). Mechanical stress mainly affects transcription of HIF 1a and Kir6.2. This study implies that new therapies could aim at the proteasome for stabilizing the left ventricular Kir6.2 protein.

Erythropoietin (EPO) production in response to hypoxic hypoxia is known to be attenuated by simultaneous hypercapnia. This study aimed to investigate whether this inhibitory effect of hypercapnia is 1) a direct effect of carbon dioxide or mediated by changes in pH or bicarbonate, 2) affects also carbon monoxide hypoxia, and 3) influences either the synthesis and release of EPO or the mechanisms by which hypoxia triggers an increase in EPO production rate. We found that EPO formation in mice exposed to normobaric hypoxia (8% O2) or to carbon monoxide (0.1%) was reduced by 30 and 42% when animals were simultaneously exposed to hypercapnia (7% CO2), by 35 and 38% when subjected to metabolic acidosis (NH4Cl), and unchanged when subjected to metabolic alkalosis (NaHCO3). In animals exposed to brief hypoxia (15 min) and subsequent normoxia (2 h), metabolic acidosis did not affect EPO levels when initiated after the hypoxic period. The results indicate that acidosis inhibits hypoxia-induced triggering of EPO formation independently of PCO2 and HCO3 levels. Because this inhibitory effect is also present during carbon monoxide hypoxia, it appears not solely due to potentiated hyperpnea. Alternatively, it may result from a facilitated intrarenal oxygen release or a direct effect at the EPO production sites.

Whereas previous studies have shown that opening of the mitochondrial ATP-sensitive K+ (mitoKATP) channel protects the adult heart against ischemia-reperfusion injury, it remains to be established whether this mechanism also operates in the developing heart. Isolated spontaneously beating hearts from 4-day-old chick embryos were subjected to 30 min of anoxia followed by 60 min of reoxygenation. The chrono-, dromo-, and inotropic disturbances, as well as alterations of the electromechanical delay (EMD), reflecting excitation-contraction (E-C) coupling, were investigated. Production of reactive oxygen species (ROS) in the ventricle was determined using the intracellular fluorescent probe 2′,7′-dichlorofluorescin (DCFH). Effects of the specific mitoKATP channel opener diazoxide (Diazo, 50 μM) or the blocker 5-hydroxydecanoate (5-HD, 500 μM), the nitric oxide synthase (NOS) inhibitor NG-nitro-l-arginine methyl ester (l-NAME, 50 μM), the antioxidant N-(2-mercaptopropionyl)glycine (MPG, 1 mM), and the PKC inhibitor chelerythrine (Chel, 5 μM) on oxidative stress and postanoxic functional recovery were determined. Under normoxia, the baseline parameters were not altered by any of these pharmacological agents, alone or in combination. During the first 20 min of postanoxic reoxygenation, Diazo doubled the peak of ROS production and, interestingly, accelerated recovery of ventricular EMD and the PR interval. Diazo-induced ROS production was suppressed by 5-HD, MPG, or l-NAME, but not by Chel. Protection of ventricular EMD by Diazo was abolished by 5-HD, MPG, l-NAME, or Chel, whereas protection of the PR interval was abolished by l-NAME exclusively. Thus pharmacological opening of the mitoKATP channel selectively improves postanoxic recovery of cell-to-cell communication and ventricular E-C coupling. Although the NO-, ROS-, and PKC-dependent pathways also seem to be involved in this cardioprotection, their interrelation in the developing heart can differ markedly from that in the adult myocardium.

Angiopoietins are ligands for the endothelial cell tyrosine kinase receptor Tie-2. Ang-1, the major physiological activator of Tie-2, promotes blood vessel maturation and stability. Ang-2 counteracts this effect by competitively inhibiting the binding of Ang-1 to Tie-2. Using a combined RNase protection/semiquantitative reverse transcriptase-polymerase chain reaction approach, we demonstrate that hypoxia up-regulates Ang-2 mRNA levels by up to 3.3-fold in two human endothelial cell lines. In bovine microvascular endothelial (BME) cells, the flavoprotein oxidoreductase inhibitor diphenylene iodonium (DPI) and the related compound iodonium diphenyl mimic induction of Ang-2 but not vascular endothelial growth factor (VEGF) by hypoxia; in combination with hypoxia, DPI further increases Ang-2 expression but has no effect on the induction of VEGF by hypoxia. Neither Ang-2 or VEGF was increased by cyanide or rotenone, suggesting that failure in mitochondrial electron transport is not involved in the oxygen-sensing system that controls their expression. In ischemic rat dorsal skin flaps or in the brain of rats maintained for 12 hours under conditions of hypoxia, Ang-2 mRNA was up-regulated 7.5- or 17.6- fold, respectively. VEGF was concomitantly increased, whereas expression of Ang-1, Tie-2, and the related receptor Tie-1 was unaltered. In situ hybridization localized Ang-2 mRNA to endothelial cells in hypoxic skin. These findings 1) show that up-regulation of Ang-2 by hypoxia occurs widely in endothelial cells in vitro and in vivo; 2) suggest that induction of Ang-2, but not VEGF, by hypoxia in BME cells is controlled by a flavoprotein oxidoreductase that is sensitive to iodonium compounds; and 3) point to Ang-2 and VEGF as independently regulated and selective effectors of hypoxia-induced vascular sprouting.

The aim of this study was to test at maximal exercise the hypothesis of the temperature-dependence of the kinetics of O2 consumption (VO2), which predicts a greater O2 deficit as muscle temperature is decreased. Six male subjects underwent 3 min exercise bouts at the minimum power eliciting maximum O2 consumption (VO2max), at normal temperature (A) and after cooling the thigh muscles by water immersion (C). Breath-by-breath VO2 was measured together with muscle blood flow (Qm), blood lactate accumulation ("early lactate", eLa), heart rate and muscle temperature (Tm). The O2 deficit was calculated by standard procedure. Net VO2max was 2.92 +/- 0.85 (SD) and 3.19 +/- 0.71 l center dot min-1 in C and A respectively (P < 0.05). Correspondingly, maximum power was 20 W lower in C than in A. At exercise start, Tm was 35.0 +/- 1.2 and 27.5 +/- 1.8 degrees C in A and C respectively. O2 deficit was 2.25 +/- 0.53 and 3.05 +/- 1.12 l in A and C respectively. The corresponding eLa was 7.7 +/- 2.5 and 13.8 +/- 2.5 mM, (P < 0.05) while Qm was 376 +/- 92 and 290 +/- 50 ml center dot kg-1 center dot min-1 (P < 0.05) in A and C, respectively. The eLa increase in C is associated with an impaired muscle blood flow and decreased muscle O2 unloading, and does not completely explain the greater O2 deficit in C. The unexplained fraction of the latter is perhaps accounted for by a greater net alactic O2 deficit, in agreement with a temperature-dependent decrease of the velocity constants of oxidative reactions, as suggested by the tested hypothesis.

Since in juvenile rats the liver is the predominant site of erythropoietin (EPO) gene expression, we have used primary cultures of juvenile rat hepatocytes to establish an in vitro system for investigation of oxygen-dependent EPO formation. When isolated hepatocytes were incubated at reduced oxygen tensions for 18–48 h, we found increased secretion of EPO protein and elevated levels of EPO mRNA, as determined by RNase protection. This increase was maximal at 3% O2, where EPO mRNA levels after 18 h were approximately 15fold higher than at 20% O2. The increase in EPO mRNA at low oxygen tensions was specific insofar as [3H]uridine incorporation, as a measure of total RNA synthesis, was reduced by approximately 50% at 3% O2, and it appeared to involve gene transcription since it was abolished in the presence of actinomycin D (35 μM). Significant increases in EPO mRNA were also observed in cells kept at 20% oxygen in the presence of cobalt chloride (50 μM) and nickel chloride (400 μM), but EPO mRNA levels achieved under these conditions were less than 7% of those in cells incubated at 3% oxygen. No increase in EPO mRNA levels was observed in cultures incubated at 20% O2 in the presence of cyclic dibutyrylAMP (10 μM-3 mM), cyclic 8-bromoGMP (10 μM1 mM), cyclohexyladenosine (1 μM), 5′-N-ethylcarboxamidoadenosine (1 μM) and phorbol 12-myristate 13acetate (3 nM). In the presence of 10% carbon monoxide, used to block haem proteins in their oxy conformation, EPO mRNA levels in hepatocytes incubated at low oxygen tensions were reduced to 63%. Taken together, these findings indicate that oxygen-dependent control of the EPO gene in hepatocytes operates via intrinsic cellular oxygen-sensing mechanisms. Their signal transduction does not seem to occur via classical “second-messenger” pathways. A haem protein may be involved in oxygen sensing, but no conclusive evidence was obtained as to whether it is essential.

Serum erythropoietin (EPO) levels in response to hypoxia are known to decline before an increase in blood oxygen carrying capacity. To define the possible mechanisms underlying this phenomenon, we have investigated 1) how renal EPO mRNA content and EPO production rate underlying the early kinetics of serum EPO levels change under different degrees of normobaric hypoxia, and 2) if a feedback inhibition of either EPO formation or EPO survival in the circulation exists by the hormone itself. We found that serum immunoreactive EPO levels in rats peaked after 12-h exposure to 7.5 or 9% oxygen (2,949 +/- 600 and 756 +/- 108 mU/ml, respectively, mean +/- SE) and declined to 29 and 64% of peak levels, respectively, after 36 h of hypoxia. EPO levels in response to 11.5% oxygen showed no consistent change between 12 (122 +/- 21 mU/ml, mean +/- SE) and 36 h (182 +/- 35 mU/ml) of hypoxia. The decline in EPO levels under severe hypoxia (7.5% O2) was paralleled by a marked reduction in renal EPO mRNA content, indicating that it was primarily a result of diminished hormone production. The observed reductions in serum EPO after 36 h corresponded to preceding declines of calculated EPO production rates from 163- to 62-fold (7.5% O2) and 36- to 25-fold (9% O2) basal values. Application of 50 IU recombinant human EPO to rats 12 h, 6 h, or immediately before hypoxic exposure to mimic the early increase in EPO levels did not affect endogenous EPO formation during a subsequent hypoxic exposure of 12 h.(ABSTRACT TRUNCATED AT 250 WORDS)

This study was carried out to investigate the early changes in erythropoietin (EPO) formation in humans in response to hypoxia. Six volunteers were exposed to simulated altitudes of 3,000 and 4,000 m in a decompression chamber for 5.5 h. EPO was measured by radioimmunoassay in serum samples withdrawn every 30 min during altitude exposure and also in two subjects after termination of hypoxia (4,000 m). EPO levels during hypoxia were significantly elevated after 114 and 84 min (3,000 and 4,000 m), rising thereafter continuously for the period investigated. Mean values increased from 16.0 to 22.5 mU/ml (3,000 m) and from 16.7 to 28.0 mU/ml (4,000 m). This rise in EPO levels corresponds to 1.8-fold (3,000 m) and 3.0-fold (4,000 m) increases in the calculated production rate of the hormone. After termination of hypoxia, EPO levels continued to rise for approximately 1.5 h and after 3 h declined exponentially with an average half-life time of 5.2 h.

In order to test the hypothesis that the early cessation of erythropoietin (Ep) production during hypobaric hypoxia is induced by lowered food intake, we have compared the plasma Ep titer of rats after exposure to continuous hypoxia (42.6 kPa = 7000 m altitude) for 4 days with that in fed or fasted rats after exposure to discontinuous hypoxia. We found that plasma Ep was rather low after 4 days of continuous hypoxia. However, the Ep titer significantly rose again, when rats were maintained normoxic for 18 h and then exposed to repeated hypoxia for 6 h. Because this was also found in rats which were deprived of food during the normoxic interval and the second hypoxic period, we conclude that the fall of the Ep titer during continuous hypoxia is not primarily due to reduced food intake. In addition, our findings show that fasting per se lowers the Ep-response to hypoxia in normal rats but not exhypoxic rats., Rechteverwertung 1966-1994

In view of recent findings which suggest that renal prostaglandins mediate the effect of hypoxia on erythropoietin production, we have studied whether hypoxia is a stimulus for in vitro prostaglandin synthesis. Studies were carried out in rat renal mesangial cell cultures which produce erythropoietin in an oxygen-dependent manner. Production rates of PGE2 and in specified samples also of 6-keto-PGF1 alpha, as a measure of PGI2, and PGF2 alpha were determined by radioimmunoassay after incubation at either 20% O2 (normoxic) or 2% O2 (hypoxic) in gas permeable dishes for 24 hrs. Considerable variation in PGE2 production was noted among independent cell lines. PGE2 production appeared to be inversely correlated to the cellular density of the cultures. In addition, PGE2 production was enhanced in hypoxic cell cultures. The mean increase was 50 to 60%. PGF2 alpha and 6-keto-PGF1 alpha increased by about the same rate. These results indicate that hypoxia is a stimulus for in vitro prostaglandin production.

We investigated whether erythropoietin (Ep) can be extracted from renal glomeruli of hypoxic rats. Hypoxia was induced in a hypobaric chamber at 0.42 atmospheres for usually 6 h. Glomeruli were isolated with a sieving technique from kidneys that were flushed free of blood. Ep activities in glomeruli homogenates were determined in the fetal mouse liver cell assay and in the hypoxia exposed mouse assay. We found in vitro erythropoietic activity of glomerular extracts, which increased about 25-fold during hypoxia. Incubation of the extracts with anti-Ep serum abolished the activity. Its binding characteristics on Wheat germ lectin. Ricinus communis lectin and DEAE-cellulose suggested that glomeruli contained Ep precursor(s) that lacked terminal sugar residues. Such an incomplete carbohydrate portion seems also compatible with the finding that glomerular extracts did not stimulate erythropoiesis in hypoxia exposed mice.