Localization of NADPH diaphorase in central nervous system of the chiton Leptochiton assimilis in normal conditions and during hypoxia

By light and electron microscope histochemical and cytochemical methods, the localization and activity of NADPH-diaphorase (NADPH-d) were studied in the central nervous system (CNS) of the chiton in control and after hypoxia. After acute hypoxia, the enzymatic activity increased in all regions of CNS. At a chronic hypoxia, the activity of NADPH-d decreased to remain, however, higher than in control. Ultrastructural studies confirmed the availability of structural changes in neurons, and shifts in the activity of NADPH-d in control and in experimental mollusks. The elevated enzymatic activity revealed in this study may be due to the fact that these mollusks have been evolutionary adapted to a periodical oxygen deficiency.     

Hypoxia-inducible factor 1α under rapid enzymatic hypoxia: Cells sense decrements of oxygen but not hypoxia per se

HIF1 (hypoxia-inducible factor 1α) is considered a central oxygen-threshold sensor in mammalian cells. In the presence of oxygen, HIF1 is marked by prolyl hydroxylases (PHDs) at the oxygen-dependent degradation (ODD) domain for ubiquitination followed by rapid proteasomal degradation. However, the actual mechanisms of oxygen sensing by HIF1 are still controversial. Thus, HIF1 expression correlates poorly with tissue oxygen levels, and PHDs are themselves target genes of HIF1 considered to readjust to new oxygen thresholds. In contrast to hypoxia chambers, we here establish an enzymatic model that allows both the rapid induction of stable hypoxia and independent control of H₂O₂. Rapid enzymatic hypoxia only transiently induced HIF1 in various cell types and the HIF1 was completely degraded within 8–12 h despite sustained hypoxia. HIF1 degradation under sustained hypoxia could be blocked by a competitive ODD–GFP construct and PHD siRNA, but also by cobalt chloride and micromolar H₂O₂ levels. Concomitant induction of PHDs further confirmed their role in degrading HIF1 under enzymatic hypoxia. The rapid and complete degradation of HIF1 under enzymatic hypoxia suggests that, in addition to hypoxia sensing, the HIF1/PHD loop may rather compensate for fluctuations of tissue oxygen staying tuned to other, e.g., metabolic, signals. In addition to hypoxia chambers, enzymatic hypoxia provides a valuable tool for independently studying the regulatory functions of hypoxia and oxidative stress in vitro.     

Temporal dynamics of dissolved oxygen in a floating–leaved macrophyte bed

1. Oxygen concentrations in shallow vegetated areas of aquatic systems can be extremely dynamic. In these waters, characterizing the average oxygen content or frequency of low oxygen events (hypoxia) may require high frequency measurements that span seasons and even years. In this study, moored sondes were used to collect 15‐min interval dissolved oxygen (DO) readings in an embayment of the tidal Hudson River with dense coverage by an invasive floating leaved plant (Trapa natans) and in adjacent open waters. Measurements were made from late spring to summer over a 2‐year period (2005, 2006). 2. Oxygen concentrations were far more dynamic in the vegetated embayment than in the adjacent open waters and while hypoxic conditions never occurred in the open waters, they occurred frequently in the vegetated site. Overall the vegetated site was hypoxic (DO < 2.5 mg L^?1) 30% of the time and had an average oxygen concentration of 5.1 mg L^?1. Oxygen concentration was significantly (P < 0.0001, anova ) related to season, year and tide. Low tide periods during summer of 2006 had the lowest average oxygen concentration and the highest frequency of hypoxia. 3. The greater hypoxia in summer than spring is related to changes in plant morphology. In the spring and early summer when plants are submersed hypoxia occurs at lower frequency and duration than in the summer when dense floating vegetation covers the water. The tidal pattern in oxygen is related to hydrologic exchange with the non‐vegetated open waters. Year‐to‐year variation may be related to relatively small changes in plant biomass between years. 4. Oxygen concentrations in aquatic systems can be critical to habitat quality and can have cascading impacts on redox sensitive nutrient and metal cycling. For some systems with dynamic oxygen patterns neither weekly spot sampling nor short‐duration, high‐frequency measurements may be sufficient to characterize oxygen conditions of the system.     

The effects of hypoxia and temperature on metabolic aspects of embryonic development in the annual killifish Austrofundulus limnaeus

Embryos of Austrofundulus limnaeus are exceptional in their ability to tolerate prolonged bouts of complete anoxia. Hypoxia and anoxia are a normal part of their developmental environment. Here, we exposed embryos to a range of PO₂ levels at two different temperatures (25 and 30 °C) to study the combined effects of reduced oxygen and increased temperature on developmental rate, heart rate, and metabolic enzyme capacity. Hypoxia decreased overall developmental rate and caused a stage-specific decline in heart rate. However, the rate of early development prior to the onset of organogenesis is insensitive to PO₂. Increased incubation temperature caused an increase in the developmental rate at high PO₂s, but hindered developmental progression under severe hypoxia. Embryonic DNA content in pre-hatching embryos was positively correlated with PO₂. Citrate synthase, lactate dehydrogenase, and phosphoenolpyruvate carboxykinase capacity were all reduced in embryos developing under hypoxic conditions. Embryos of A. limnaeus are able to develop normally across a wide range of PO₂s and contrary to most other vertebrates severe hypoxia is not a teratogen. Embryos of A. limnaeus do not respond to hypoxia through an increase in the capacity for enzymatic activity of the metabolic enzymes lactate dehydrogenase, citrate synthase, or phosphoenolpyruvate carboxykinase. Instead they appear to adjust whole-embryo metabolic capacity to match oxygen availability. However, decreased DNA content in hypoxia-reared embryos suggests that cellular enzymatic capacity may remain unchanged in response to hypoxia, and the reduced capacity may rather indicate reduced cell number in hypoxic embryos.     

Hypoxia and cold: influence on cellular oxygen consuming systems in guinea pig liver

Traditionally any biochemical changes found in animals exposed to high altitude have been interpreted solely in relation to hypobaric hypoxia. The present work has been carried out to study the influence of cold and hypoxia in guinea pig native to high altitude.The three major oxygen consuming systems of the liver were measured using cytochrome oxidase as a mitochondrial marker, catalase as a peroxisomal marker, and the o-demethylation of p-nitroanisole and the hydroxilation of hexobarbital as markers for microsomal activity. Serum levels of thyroid hormones (T₃ and T₄) were also determined.Evidence is presented showing that cold produces a dramatic increase of liver catalase and cytochrome oxidase activities, and of serum T₃ and T₄.Interestingly, the increase in thyroid hormones did not precede the increase of the two enzyme activities in the liver of guinea pig exposed to 4°C.On the other hand, it was found that hypoxia appears to have no significant influence upon any of the three major oxygen consuming systems of the liver.     

Intrasystemic and Intersystemic Rearrangements of Physiological Parameters in Experimental Acute Hypoxia

Simultaneous computer recording of the parameters of external respiration, systemic and cerebral blood circulation, arterial pressure, oxygen saturation of hemoglobin, and tissue oxygen tension has been used to study the intrasystemic and intersystemic rearrangements at different stages of acute hypoxia caused by breathing hypoxic oxygen–nitrogen mixtures containing 6.8–8.0% oxygen. It has been found that all main vital systems of the body are involved in the response to hypoxia; however, the degree of their involvement and the changes in individual parameters vary considerably in different subjects. The functional strain of some systems may remain almost constant throughout the period of hypoxia, whereas the strain of others may gradually increase or decrease. It has been found that each stage of hypoxia is characterized by certain limits of the strain (involvement) of the functional reserves of oxygen supply systems; if the functional strain goes beyond these limits, then either intrasystemic and intersystemic relationships are disorganized or compensation reserves are overspent and the time of tolerance to hypoxia is decreased.     

Respiratory control in aquatic insects dictates their vulnerability to global warming

Forecasting species responses to climatic warming requires knowledge of how temperature impacts may be exacerbated by other environmental stressors, hypoxia being a principal example in aquatic systems. Both stressors could interact directly as temperature affects both oxygen bioavailability and ectotherm oxygen demand. Insufficient oxygen has been shown to limit thermal tolerance in several aquatic ectotherms, although, the generality of this mechanism has been challenged for tracheated arthropods. Comparing species pairs spanning four different insect orders, we demonstrate that oxygen can indeed limit thermal tolerance in tracheates. Species that were poor at regulating oxygen uptake were consistently more vulnerable to the synergistic effects of warming and hypoxia, demonstrating the importance of respiratory control in setting thermal tolerance limits.     

Effects of thermal stress on respiratory responses to hypoxia of a South American Prochilodontid fish, Prochilodus scrofa

Oxygen consumption (o₂) and respiratory variables were measured in the Prochilodontid fish, Prochilodus scrofa exposed to graded hypoxia after changes in temperature. The measurements were performed on fish acclimated to 25°C and in four further groups also acclimated to 25°C and then changed to 15, 20, 30 and 35°C. An increase in o₂ occurred with rising temperature, but at each temperature o₂ was kept constant over a wide range of O₂ tensions of inspired water ( Pi o₂). The critical oxygen tensions ( Pc o₂) were Pi o₂= 22 mmHg for 25°C acclimated specimens and after transfer from 25°C to 15, 20, 30 and 35°C the Pc o₂ changed to Pi o₂= 28, 22, 24 and 45 mmHg, respectively. Gill ventilation ( _G ) increased or decreased following the changes in o₂ as the temperature changed and was the result of an accentuated increase in breath frequency. During hypoxia the increases in _G were characterized by larger increases in breath volume. Oxygen extraction was kept almost constant at about 63% regardless of temperature and ambient oxygen tensions in normoxia and moderate hypoxia ( P o₂∼70 mmHg). P. scrofa showed high tolerance to hypoxia after abrupt changes in temperature although its survival upon transfer to 35°C could become limited by the capacity of ventilatory mechanisms to alleviate hypoxic stress.     

Differences in oxygen-dependent regulation of enzymes between tumor and normal cell systems in culture

Metabolic studies in tumor cells have indicated that bioenergetic regulatory mechanisms geared to acute changes in oxygen availability are abnormal. In the present studies we have examined bioenergetic adaptations to chronic oxygen depletion in culture maintained tumor cells in comparison to normal cell lines. Activities of two key glycolytic enzymes (pyruvate kinase (PyKI) and phosphofructokinase (PFK)) were measured in two tumor cell lines (fibrosarcoma (FS) and Hela) and two normal cell lines (rat lung fibroblasts (RLF) and WI-38) maintained in culture for up to 96 hours under aerobic (PO2 approximately 140) and hypoxic PO2 approximately 15) conditions. Exposure to low O2 tensions for 96 hours resulted in significant increases in PyKi and PFK in both RLF and WI-38, ut did not alter activities of these enzymes in either FS or HeLa cell systems. Activities of two enzymes involved in O2 metabolism (cytochrome oxidase (CyOx) and superoxide dismutase (SOD) were also measured in the two tumor cell lines and in RLF. chronic hypoxia significantly decreased the activities of CyOx and SOD in RLF cell systems but did not alter the activities of these enzymes in the tumor cells. In these studies, the tumor-derived cell lines do not demonstrate specific enzymatic responses to sustained oxygen depletion in vitro noted in normal cell systems, suggesting significant abnormalities in regulatory mechanisms geared to chronic changes in molecular O2.     

Environmental influences on the development of the cardiac system in fish and amphibians

In poikilothermic animals body temperature varies with environmental temperature, and this results in a change in metabolic activity (Q10 of enzymatic reactions typically is around 2-3). Temperature changes also modify gas transport in body fluids. While the diffusion coefficient increases with increasing temperatures, physical solubility and also hemoglobin oxygen affinity decrease. Therefore, an increase in temperature typically requires adjustments in cardiac activity because ventilatory and convectional transport of respiratory gases usually are tightly coupled in adults in order to meet the oxygen demand of body tissues. Hypoxic conditions also provoke adaptations in the central circulatory system, like the hypoxic bradycardia, which has been described for many adult lower vertebrates, combined with an increase in stroke volume and peripheral resistance. In embryos and larvae the situation is much more complicated, because nervous control of the heart is established only late during development, and because the site of gas exchange changes from mainly cutaneous gas exchange during early development to mainly pulmonary or branchial gas exchange in late stages. In addition, recent studies in amphibian and fish embryos and larvae reveal, that at least in very early stages convectional gas transport of the hemoglobin is not essential, which means that in these early stages ventilatory and convectional gas transport are not yet coupled. Accordingly, in early stages of fish and amphibians the central cardiac system often does not respond to hypoxia, although in some species behavioral adaptations indicate that oxygen sensors are functional. If a depression of cardiac activity is observed, it most likely is a direct effect of oxygen deficiency on the cardiac myocytes. Regulated cardiovascular responses to hypoxia appear only in late stages and are similar to those found in adult species.     

The effect of progressive hypoxia on respiration in the dogfish (scyliorhinus canicula) at different seasonal temperatures.

1. Dogfish were acclimated to 7, 12 or 17 degrees C and exposed to progressive hypoxia at the temperature to which they had been acclimated. During normoxia, the Q10 values for oxygen uptake, heart rate, cardiac output and respiratory frequency over the full 10 degrees C range were: 2.1, 2.1, 2.1 and 2.5 respectively. Increased acclimation temperature had no effect on cardiac stroke volume or systemic vascular resistance, although there was a decrease in branchial vascular resistance, pHa and pHv. 2. Progressive hypoxia had no effect on heart rate or oxygen uptake at 7 degrees C, whereas at 12 degrees C and 17 degrees C there was bradycardia, and a reduction in O2 uptake, with the critical oxygen tension for both variables being higher at the higher temperature. Cardiac stroke volume increased during hypoxia at each temperature, such that cardiac output did not change significantly at 12 and 17 degrees C. Neither pHa nor pHv changed significantly during hypoxia at any of the three temperatures. 3. The influence of acclimation temperatures on experimental results from poikilotherms is pointed out. Previously-published results show quantitative differences. 4. The significance of the present results with respect to the functioning and location of oxygen receptors is discussed. It is argued that as the metabolic demand and critical oxygen tension of the whole animal are increased at high acclimation temperatures the same must be the case with the oxygen receptor. This would raise the stimulation threshold and could account for the bradycardia seen during hypoxia becoming manifest at higher values of PI,O2, Pa,O2 and Pv,O2 as the acclimation temperature is raised.     

Metabolic and cardiorespiratory responses of summer flounder Paralichthys dentatus to hypoxia at two temperatures

To quantify the tolerance of summer flounder Paralichthys dentatus to episodic hypoxia, resting metabolic rate, oxygen extraction, gill ventilation and heart rate were measured during acute progressive hypoxia at the fish's acclimation temperature (22° C) and after an acute temperature increase (to 30° C). Mean ±s.e. critical oxygen levels (i.e. the oxygen levels below which fish could not maintain aerobic metabolism) increased significantly from 27 ± 2% saturation (2·0 ± 0·1 mg O(2) l(-1) ) at 22° C to 39 ± 2% saturation (2·4 ± 0·1 mg O(2) l(-1) ) at 30° C. Gill ventilation and oxygen extraction changed immediately with the onset of hypoxia at both temperatures. The fractional increase in gill ventilation (from normoxia to the lowest oxygen level tested) was much larger at 22° C (6·4-fold) than at 30° C (2·7-fold). In contrast, the fractional decrease in oxygen extraction (from normoxia to the lowest oxygen levels tested) was similar at 22° C (1·7-fold) and 30° C (1·5-fold), and clearly smaller than the fractional changes in gill ventilation. In contrast to the almost immediate effects of hypoxia on respiration, bradycardia was not observed until 20 and 30% oxygen saturation at 22 and 30° C, respectively. Bradycardia was, therefore, not observed until below critical oxygen levels. The critical oxygen levels at both temperatures were near or immediately below the accepted 2·3 mg O(2) l(-1) hypoxia threshold for survival, but the increase in the critical oxygen level at 30° C suggests a lower tolerance to hypoxia after an acute increase in temperature.     

Criteria of individual variations in the reactivity of the respiratory system

Individual variations of the respiratory system reactivity have been studied in experiments on rats. It is shown expedient to estimate reactivity of the respiratory system to hypoxic hypoxia by the pattern of changes in the total oxygen uptake. Animals demonstrating no essential changes in the oxygen uptake in response to hypoxia (11% O2) are referred to individuals with high reactivity of the respiratory system; those responding by a drastic decrease in the oxygen uptake--to animals with low reactivity of the respiratory system. A strong correlation is determined between the respiratory system reactivity and individual resistance of organism to acute hypoxic hypoxia.     

Effect of intermittent high altitude hypoxia on the structure and enzymatic activity of cardiac myosin

The time course of structural and enzymatic changes in cardiac myosin was studied in the right and left ventricle of rats exposed to intermittent high altitude (IHA) hypoxia. In the controls, ATPase activity and myosin structure in both ventricles was the same. After the third exposure to simulated high altitude (2 600 m), myosin enzymatic activity rose significantly in the left ventricle and a significant right-left difference appeared. In the next phase of adaptation (11 exposures, 6 000 m), myosin ATPase activity fell in both ventricles and the right-left difference disappeared. After the 16th exposure (7 000 m), enzymatic activity increased again in both ventricles and attained control values. IHA also produced significant structural changes in cardiac myosin, particularly in the rigaht ventricle. The changes were characterized by the formation of myosin aggregates with significantly lower ATPase activity that the myosin monomer. The time course and localization of structural and enzymatic changes in cardiac myosin corresponded to the morphological damage to the heart fibres.     

Effects of hypoxia on heme oxygenase expression in human chorionic villi explants and immortalized trophoblast cells

Although hypoxia induces heme oxygenase (HO)-1 protein and mRNA expression in many cell types, hypoxia has also been shown to decrease HO-1 mRNA and protein expression. We tested the hypothesis that 24-h preexposure to hypoxia in human placental preparations suppresses HO protein expression and enzymatic function. Immortalized HTR-8/SVneo first-trimester trophoblast cells and explants of normal human chorionic villi (CV) from term placentas were cultured for 24 h in 1%, 5%, or 20% O(2). HO protein levels were determined by Western blot analysis, and microsomal HO activity was measured. HO-2 protein content was decreased by 17% and 5% in human trophoblast cells after 24-h exposure to 1% and 5% O(2), respectively, versus 20% O(2). In contrast, HO-2 protein content in CV explants was unaffected by changes in oxygenation. HO-1 protein content, which was barely detectable in both biological systems, was not affected by changes in oxygenation. Similarly, HO enzymatic activity was unchanged in both preparations after 24-h exposure to 1%, 5%, or 20% O(2). The above data do not support the hypothesis that hypoxia in the human placenta suppresses both HO protein content and HO protein function. The present observations reinforce the necessity to determine both HO protein expression and function.     

Hypoxia reduced upper thermal limits causing cellular and nuclear abnormalities of erythrocytes in Nile tilapia,Oreochromis niloticus

Global warming is a threat across the world that leads to estimates of the upper thermal limits of ectothermic species. Increased water temperature up-regulates oxygen consumption and metabolic rates, and alters the physiological processes. In this study, we identified the critical thermal maxima (CTmax) and physiological responses under normoxia and hypoxia in Nile tilapia, Oreochromis niloticus. CTmax was 41.25 °C under hypoxia and 44.50 °C under normoxia. Compared to normoxia, lower values of hemoglobin (Hb) and red blood cells (RBCs) were observed at the CTmax under hypoxia. In contrast, higher values of white blood cells (WBCs) and blood glucose (Glu) levels were observed at the CTmax under hypoxia. Consequently, higher frequencies of micronucleus, cellular and nuclear abnormalities of erythrocytes were observed at the CTmax under hypoxia. These results suggest that high temperature tolerance and subsequent physiology are significantly affected by the oxygen supply in Nile tilapia. As climate vulnerability is intensifying day by day, this data will be helpful in successful management practice for the aquatic environment having low oxygen content.     

Human Systemic Reactions to a Dosed Exposure to Hypoxia: A Multiparameter Study

Human physiological reactions to acute hypoxic hypoxia were studied. Analysis of simultaneously recorded parameters of various physiological systems showed the following: activation of the general antihypoxic defense system is based on the formation of an intricate structure of intra- and intersystemic relations of specific and nonspecific elements of adaptation that support vital body functions during environmental oxygen deficit. These specific elements become more important in more severe hypoxia, which suppresses metabolism in some organs and tissues because of redistribution of blood flow. These factors allow the body to function at a lower oxygen tension in its tissues owing to an increased efficiency of mitochondria as a result of changes in the kinetics of enzymes of the mitochondrial respiratory chain. In acute hypoxia, the structure of intra- and intersystemic relations is rather intricate; its functional hierarchy is maintained by stronger individual amplitude-related controlling factors and by modulation of their phase- and time-related links. Advanced stages of hypoxia are associated with disintegration of central regulatory mechanisms, which is manifested by disturbances in amplitude-frequency and spatiotemporal parameters of the brain bioelectrical activity, changes in phasic interactions between elements of regulatory mechanisms, and signs of deregulation and decompensation of vital functions. The interpretation of the results is based on the general theory of adaptation, Medvedev's idea of adaptation as a successive involvement of genetically predetermined and newly-formed regulatory programs of the brain, Anokhin's theory of functional systems, and modern concepts of molecular and biochemical mechanisms of hypoxia. It was concluded that artificial normobaric hypoxia is a unique, biologically adequate model that makes it possible to study the rearrangements in systemic and autonomic regulatory mechanisms in response to strictly determined changes in the environmental concentration of oxygen as a principal factor supporting life.     

Metabolic Responses of the Dopaminergic System during Hypoxia in Newborn Brain

The purpose of this review is to describe the relationship between the dopamine and amino acid neurotransmitter systems and cortical oxygen pressure during different levels of cerebral hypoxia using newborn piglets as an animal model, adding new data from our laboratory. The extracellular dopamine increases as the oxygen pressure in the cortex decreases. The relationship between oxygen pressure and dopamine levels is the same whether the hypoxia is induced by reduced F_iO₂ (high-flow hypoxia) or by hypocapnia-induced cerebral vasoconstriction (low-flow hypoxia). Thus it appears that, particularly in mild hypoxia, the extracellular level of dopamine depends primarily on the oxygen concentration in the tissue with minimal influence of parameters such as blood flow and pH. There is no "oxygen reserve" in the brain of newborn piglets and the extracellular levels of dopamine in the striatum increase almost linearly with decrease in oxygen pressure, with even small decreases in oxygen pressure resulting in increased dopamine levels. In contrast, the changes in extracellular concentrations of the excitatory amino acids glutamate and aspartate are variable and transient. In a majority of 2- to 5 day-old piglets even very low oxygen pressures in the brain did not result in significant alterations in the extracellular levels of glutamate and aspartate. These changes in the dopaminergic system may contribute directly and indirectly to the neuronal damage that occurs during hypoxic/ischemic insult and reoxygenation in newborn brain, particularly in the striatum. A variety of mechanisms are discussed by which dopamine, in particular extracellular dopamine, can increase cellular toxicity.