Hypoxia exposure and B-type natriuretic peptide release from Langendorff heart of rats.

AIM: We studied whether available oxygen without induced mechanical stretch regulates the release of the biologically active B-type natriuretic peptide (BNP) from Langendorff heart. METHODS: Rat hearts were isolated and perfused with a physiological Krebs-Henseleit solution at a constant hydrostatic pressure in Langendorff set-up. The basal O2 level of perfusate (24.4 ± 0.04 mg L-1 ) was gradually lowered to 3.0 ± 0.01 mg L-1 over 20 min using N2 gas (n = 7). BNP and O2 level were measured from coronary flow. During control perfusions (n = 5), the O2 concentration was kept at 26.6 ± 0.3 mg L-1 . RESULTS: A low oxygen concentration in the perfusate was associated with a significant increase in BNP release (F = 40.4, P < 0.001). Heart rate decreased when the oxygen concentration in the perfusate reached 9.1 ± 0.02 mg L-1 and continued to fall in lower oxygen concentrations (F = 14.8, P < 0.001). There was also a significant but inverse correlation between BNP and oxygen in the coronary flow (R2  = 0.27, P < 0.001). CONCLUSION: In the spontaneously beating Langendorff rat heart, a decreasing concentration of oxygen in the ingoing perfusion increased the secretion of BNP. The effect of oxygen was independent of mechanical stretch of the heart as it occurred even when the heart rate decreased but the pressure conditions remained constant. The difference in the oxygen capacitance of blood and Krebs-Henseleit solution appears to be a major factor affecting secretion of BNP, which is correlated with the oxygen tension of myocardial cells and affected both by the oxygen concentration and capacitance of solution perfusing the heart and by the coronary flow.

Resistance to oxidative damage but not immunosuppression by organic tin compounds in natural populations of Daubenton's bats (Myotis daubentonii).

The acute toxicity of organic tin compounds (OTCs) has been studied in detail. However, due to their complex nature, very little is known about species-specific methods of accumulation and consequences for food-webs. Chironomids, on which e.g. Daubenton's bats feed, may act as vectors for the transport of organic tin compounds from aquatic to terrestrial ecosystems. Bats are prone to environmental toxins because of their longevity and their ecological role as top predators. Organic tin compounds are associated with increased formation of reactive oxygen species and associated oxidative damage as well as suppression of immune function. The present paper investigates whether the OTC, tributyltin (TBT) and its metabolite, dibutyltin (DBT), accumulate in natural populations of Daubenton's bats and whether TBT-associated effects are seen in general body condition, redox balance, redox enzyme activities, associated oxidative damage of red blood cells and complement function. We discovered the concentration of bat fur DBT correlated with local marine sediment TBT concentrations. However, we did not find a correlation between the explanatory factors, bat fur DBT and marine sediment TBT concentrations, and several physiological and physical response variables apart from complement activity. Higher DBT concentrations resulted in weaker complement activity and thus a weaker immune response. Although the observed physiological effects in the present study were not strongly correlated to butyltin concentrations in fur or sediment, the result is unique for natural populations so far and raises interesting questions for future ecotoxicological studies.

Transcription and redox enzyme activities: comparison of equilibrium and disequilibrium levels in the three-spined stickleback.

Evolutionary and acclimatory responses require functional variability, but in contrast with mRNA and protein abundance data, most physiological measurements cannot be obtained in a high-throughput manner. Consequently, one must either rely on high-throughput transcriptomic or proteomic data with only predicted functional information, or accept the limitation that most physiological measurements can give fewer data than those provided by transcriptomics or proteomics. We evaluated how transcriptional and redox enzyme activity data agreed with regard to population differentiation (i.e. a system in steady state in which any time lag between transcription, translation and post-translational effects would be irrelevant) and in response to an acute 6°C increase in temperature (i.e. a disequilibrium state wherein translation could not have caught up with transcription) in the three-spined stickleback (Gasterosteus aculeatus). Transcriptional and enzyme activity data corresponded well with regard to population differentiation, but less so with regard to acute temperature increase. The data thus suggest that transcriptional and functional measurements can lead to similar conclusions when a biological system is in a steady state. The responses to acute changes must, as has been demonstrated earlier, be based on changes in cellular conditions or properties of existing proteins without significant de novo synthesis of new gene products.

Stock-specific variation of trophic position, diet and environmental stress markers in Atlantic salmon Salmo salar during feeding migrations in the Baltic Sea.

This study investigated stock-specific variation in selected ecophysiological variables during the feeding migrations of Atlantic salmon Salmo salar in the Baltic Sea. Oxidative stress biomarkers and EROD (ethoxyresorufin-O-deethylase, Cyp1A enzyme) activity were used as indicators of possible environmental stress and stable isotopes as determinants of diet and trophic position. Latvian S. salar stocks Daugava and Gauja had distinct stable-isotope signatures compared to the other stocks, indicating differences in migration patterns, residency or arrival times, or dietary specialization among stocks. Salmo salar originating from Daugava and Gauja also had lower catalase enzyme activity than the other stocks. Post-smolts originating from rivers of the Gulf of Finland had elevated EROD activities compared to fish of the same age from Bothnian Bay rivers, which could indicate exposure to organochlorine pollutants. No other stock-specific differences in oxidative stress biomarkers were found. The study demonstrates how genetic, oxidative stress biomarker, EROD and stable-isotope data may be combined to study trophic position, prey prevalence and environmental stress of mixed S. salar stocks foraging in the sea.

Responses of threespine stickleback (Gasterosteus aculeatus, L) transcriptome to hypoxia.

Hypoxia is a naturally occurring phenomenon in aquatic systems. Its occurrence is potentiated by eutrophication caused by human actions and it may be made even more severe as a result of increasing temperatures due to climate change. Threespine stickleback (Gasterosteus aculeatus) has previously been used by ecologists and evolutionary biologists, but has great potential also for physiological studies. We subjected threespine sticklebacks to hypoxia (air saturation 24-28%) or normoxia for 3 and 48 h. To study changes in the transcriptome, microarray determinations were carried out for the 48 h treatments and complementary real-time quantitative PCR was run on selected transcripts at both time points. The microarray results suggest downregulation of genes encoding proteins with functions typically inhibited by hypoxia, i.e., cell proliferation, DNA replication and repair, and protein degradation, and upregulation of transcripts with products having oxygenase and oxidase activities including two 2-oxoglutarate-deoxygenases. These transcripts encode for JmjC domain containing proteins JMJD6 and JMJD2C. JMJD6 transcription has not earlier been characterized to change in hypoxia. Cyp1A2 mRNA was also increased in the microarray and the upregulation could be confirmed on protein level by measuring ethoxyresorufin-O-deethlyase (EROD)-activity.

Oxygen-dependent ion transport in erythrocytes.

The present contribution reviews current knowledge of apparently oxygen-dependent ion transport in erythrocytes and presents modern hypotheses on their regulatory mechanisms and physiological roles. In addition to molecular oxygen as such, reactive oxygen species, nitric oxide, carbon monoxide, regional variations of cellular ATP and hydrogen sulphide may play a role in the regulation of transport, provided that they are affected by oxygen tension. It appears that the transporter molecules themselves do not have direct oxygen sensors. Thus, the oxygen level must be sensed elsewhere, and the effect transduced to the transporter. The possible pathways involved in the regulation of transport, including haemoglobin as a sensor, and phosphorylation/dephosphorylation reactions both in the transporter and its upstream effectors, are discussed.

Effects of seasonal and latitudinal cold on oxidative stress parameters and activation of hypoxia inducible factor (HIF-1) in zoarcid fish.

Acute, short term cooling of North Sea eelpout Zoarces viviparus is associated with a reduction of tissue redox state and activation of hypoxia inducible factor (HIF-1) in the liver. The present study explores the response of HIF-1 to seasonal cold in Zoarces viviparus, and to latitudinal cold by comparing the eurythermal North Sea fish to stenothermal Antarctic eelpout (Pachycara brachycephalum). Hypoxic signalling (HIF-1 DNA binding activity) was studied in liver of summer and winter North Sea eelpout as well as of Antarctic eelpout at habitat temperature of 0 degrees C and after long-term warming to 5 degrees C. Biochemical parameters like tissue iron content, glutathione redox ratio, and oxidative stress indicators were analyzed to see whether the cellular redox state or reactive oxygen species formation and HIF activation in the fish correlate. HIF-1 DNA binding activity was significantly higher at cold temperature, both in the interspecific comparison, polar vs. temperate species, and when comparing winter and summer North Sea eelpout. Compared at the low acclimation temperatures (0 degrees C for the polar and 6 degrees C for the temperate eelpout) the polar fish showed lower levels of lipid peroxidation although the liver microsomal fraction turned out to be more susceptible to lipid radical formation. The level of radical scavenger, glutathione, was twofold higher in polar than in North Sea eelpout and also oxidised to over 50%. Under both conditions of cold exposure, latitudinal cold in the Antarctic and seasonal cold in the North Sea eelpout, the glutathione redox ratio was more oxidised when compared to the warmer condition. However, oxidative damage parameters (protein carbonyls and thiobarbituric acid reactive substances (TBARS) were elevated only during seasonal cold exposure in Z. viviparus. Obviously, Antarctic eelpout are keeping oxidative defence mechanisms high enough to avoid accumulation of oxidative damage products at low habitat temperature. The paper discusses how HIF could be instrumental in cold adaptation in fish.

From critters to cancers: bridging comparative and clinical research on oxygen sensing, HIF signaling, and adaptations towards hypoxia.

The objective of this symposium at the First International Congress of Respiratory Biology (ICRB) was to enhance communication between comparative biologists and cancer researchers working on O(2) sensing via the HIF pathway. Representatives from both camps came together on August 13-16, 2006, in Bonn, Germany, to discuss molecular adaptations that occur after cells have been challenged by a reduced (hypoxia) or completely absent (anoxia) supply of oxygen. This brief "critters-to-cancer" survey discusses current projects and new directions aimed at improving understanding of hypoxic signaling and developing therapeutic interventions.

Hypoxic responses of Na+/K+ ATPase in trout hepatocytes.

Reduction in oxygenation induces inhibition of Na+/K+ ATPase in a number of cells and tissues, including hepatocytes. When not reversed, decrease in Na+/K+ pump activity leads to a gradual Na+ accumulation, cell swelling and death. However, when accompanied by suppression of dissipative cation pathways, it has also been shown to be a beneficial adaptive strategy used by some hypoxia-tolerant species to reduce ATP consumption during prolonged periods of anoxia. This study aims to investigate acute hypoxic responses of the Na+/K+ ATPase in primary cultures of trout hepatocytes. Gradual decrease in oxygenation was followed by an instantaneous transient dose-dependent downregulation of the Na+/K+ ATPase transport activity, but was without an effect on hydrolytic function of the enzyme. Hypoxia-induced inhibition of active K+ influx was reversed spontaneously when hypoxic incubation time exceeded 20 min. The stimulating effect of prolonged hypoxic exposure on the Na+/K+ pump is most probably secondary to hypoxia-induced activation of the Na+/H+ exchanger with the following Na+ accumulation leading to Na+/K+ ATPase activation. Hypoxia-induced inhibition of the Na+/K+ pump was not caused by ATP depletion or global oxidative stress. However, local controlled production of reactive oxygen species seems to play an important role in hypoxia-induced regulation of the Na+/K+ ATPase. Treatment of cells with mercaptopropionyl glycine (MPG), a scavenger of OH*-, abolished hypoxia-induced inhibition of the Na+/K+ ATPase. Earlier on we have shown that activation of Na+/H+ exchanger under hypoxic conditions can be opposed by MPG treatment as well. Taken together our results suggest that regulation of both oxygen-sensitive transporters may be accomplished by local changes in free radical production.

Intracellular pH regulation in rainbow trout (Oncorhynchus mykiss) hepatocytes: the activity of sodium/proton exchange is oxygen-dependent.

We studied pH regulation in freshly isolated rainbow trout hepatocytes using microspectrofluorometry with the fluorescent dye BCECF. In accordance with earlier data on rainbow trout hepatocytes, ion substitution (N-methyl D-glucamine for sodium and gluconate for chloride) and transport inhibitor [10 microM M methyl isobutyl amiloride (MIA) to inhibit sodium/proton exchange and 100 microM DIDS to inhibit bicarbonate transport] studies in either Hepes-buffered or bicarbonate/carbon dioxide-buffered media (extracellular pH 7.6) indicated a role for sodium/proton exchange, sodium-dependent bicarbonate transport, and sodium-independent anion exchange in the regulation of hepatocyte pH. In Hepes-buffered medium, the activity of the sodium/proton exchanger (i.e. proton extrusion inhibited by MIA) was greater at 1% than at 21% oxygen. The oxygen dependency of the sodium/proton exchange is not caused by hydroxyl radicals, which appear to mediate the oxygen sensitivity of potassium-chloride cotransport in erythrocytes.

Oxygen dependency of the adrenergic Na/H exchange in rainbow trout erythrocytes is diminished by a hydroxyl radical scavenger.

AIM: Potassium transport via the potassium chloride cotransporter in rainbow trout erythrocytes is increased by high oxygen tension. It appears that the effect of oxygen is mediated by reactive oxygen species, especially hydroxyl radicals. In contrast, the activity of adrenergically stimulated sodium proton exchange decreases with increasing oxygen tension. As available data suggest that the two transporters are regulated reciprocally, the present study was undertaken to evaluate, if hydroxyl radicals may inhibit sodium transport via the adrenergically stimulated sodium proton exchanger. METHODS: The effects of the hydroxyl radical scavenger, 2 mm mercaptopropionyl glycine (MPG), on the activity of the adrenergically activated sodium proton exchange in rainbow trout erythrocytes were examined by measuring unidirectional sodium flux, using radioactive isotope, and cellular water content. RESULTS: The activity of the sodium proton exchange increased with decreasing oxygen tension after adrenergic stimulation. When MPG was present during incubation, there was no statistically significant effect of oxygen tension on the adrenergically stimulated sodium proton exchange, whereby the activity of the transporter at atmospheric oxygen tension was markedly higher in the presence than in the absence of MPG. In the absence of adrenergic stimulation, MPG did not influence the transporter activity significantly at any oxygen tension. CONCLUSION: The data suggest that hydroxyl radicals are involved in the inhibition of the adrenergically stimulated sodium proton exchange at elevated oxygen tensions.

Haemoglobin function in vertebrates: evolutionary changes in cellular regulation in hypoxia.

The evolution of erythrocytic hypoxia responses is reviewed by comparing the cellular control of haemoglobin-oxygen affinity in agnathans, teleost fish and terrestrial vertebrates. The most ancient response to hypoxic conditions appears to be an increase in cell volume, which increases the haemoglobin-oxygen affinity in lampreys. In teleost fish, an increase of cell volume in hypoxic conditions is also evident. The volume increase is coupled to an increase in erythrocyte pH. These changes are caused by an adrenergic activation of sodium/proton exchange across the erythrocyte membrane. The mechanism is important in acute hypoxia and is followed by a decrease in cellular adenosine triphosphate (ATP) and guanosine triphosphate (GTP) concentrations in continued hypoxia. In hypoxic bird embryos, the ATP levels are also reduced. The mechanisms by which hypoxia decreases cellular ATP and GTP concentrations remains unknown, although at least in bird embryos cAMP-dependent mechanisms have been implicated. In mammals, hypoxia responses appear to occur mainly via modulation of cellular organic phosphate concentrations. In moderate hypoxia, 2,3-diphosphoglycerate levels are increased as a result of alkalosis caused by increased ventilation.

Characterization of a hypoxia-inducible factor (HIF-1alpha ) from rainbow trout. Accumulation of protein occurs at normal venous oxygen tension.

The mammalian hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that controls the induction of several genes involved in glycolysis, erythropoiesis, and angiogenesis when cells are exposed to hypoxic conditions. Until now, the expression and function of HIF-1alpha have not been studied in fish, which experience wide fluctuations of oxygen tensions in their natural environment. Using electrophoretic mobility shift assay, we have ascertained that a hypoxia-inducible factor is present in rainbow trout cells. We have also cloned the full-length cDNA (3605 base pairs) of the HIF-1alpha from rainbow trout with a predicted protein sequence of 766 amino acids that showed a 61% similarity to human and mouse HIF-1alpha. Polyclonal antibodies against the N-terminal part (amino acids 12-363) and the C-terminal part (amino acids 330-730) of rainbow trout HIF-1alpha protein recognized rainbow trout and chinook salmon HIF-1alpha protein in Western blot analysis. Also, the human and mouse HIF-1alpha proteins were recognized by the N-terminal rainbow trout anti-HIF-1alpha antibody but not by the C-terminal HIF-1alpha antibody. The accumulation of HIF-1alpha was studied by incubating rainbow trout and chinook salmon cells at different oxygen concentrations from 20 to 0.2% O(2) for 1 h. The greatest accumulation of HIF-1alpha protein occurred at 5% O(2) (38 torr), a typical oxygen tension of venous blood in normoxic animals. The protein stability experiments in the absence or presence of a proteasome inhibitor, MG-132, demonstrated that the inhibitor is able to stabilize the protein, which normally is degraded via the proteasome pathway both in normoxia and hypoxia. Notably, the hypoxia response element of oxygen-dependent degradation domain is identical in mammalian, Xenopus, and rainbow trout HIF-1alpha proteins, suggesting a high degree of evolutionary conservation in degradation of HIF-1alpha protein.

Reactive oxygen species regulate oxygen-sensitive potassium flux in rainbow trout erythrocytes.

In the present study, we have investigated if reactive oxygen species are involved in the oxygen-dependent regulation of potassium-chloride cotransport activity in trout erythrocyte membrane. An increase in the oxygen level caused an increase in chloride-sensitive potassium transport (K(+)-Cl(-) cotransport). 5 mM hydrogen peroxide caused an increase in K(+)-Cl(-) cotransport at 5% oxygen. The increase in flux could be inhibited by adding extracellular catalase in the incubation. Pretreatment of the cells with mercaptopropionyl glycine (MPG), a scavenger of reactive oxygen species showing preference for hydroxyl radicals, abolished the activation of the K(+)-Cl(-) cotransporter by increased oxygen levels. The inhibition by MPG was reversible, and MPG could not inhibit the activation of transporter by the sulfhydryl reagent, N-ethylmaleimide, indicating that the effect of MPG was due to the scavenging of reactive oxygen species and not to the reaction of MPG with the cotransporter. Copper ions, which catalyze the production of hydroxyl radicals in the Fenton reaction, activated K(+)-Cl(-) cotransport significantly at hypoxic conditions (1% O(2)). These data suggest that hydroxyl radicals, formed from O(2) in close vicinity to the cell membrane, play an important role in the oxygen-dependent activation of the K(+)-Cl(-) cotransporter.

Beta-adrenergic stimulation of volume-sensitive chloride transport in lamprey erythrocytes.

We measured the effects of a beta-adrenergic agonist, isoproterenol, on chloride transport and volume regulation of lamprey (Lampetra fluviatilis) erythrocytes in isotonic (288 mosm L(-1)) and hypotonic (192 mosm L(-1)) medium. Isoproterenol at a high concentration (10(-5) M) did not influence chloride transport in isotonic medium but markedly increased chloride fluxes in hypotonic conditions: unidirectional flux increased from 100 mmol kg dcw(-1) h(-1) in the absence to 350 mmol kg dcw(-1) h(-1) (dcw=dry cell weight) in the presence of isoproterenol. Simultaneously, the half-time for volume recovery decreased from 27 to 9 min. Isoproterenol caused an increase in cellular cyclic AMP (cAMP) concentration. The stimulation of chloride transport in hypotonic conditions could be induced by application of the permeable cAMP analogue, 8-bromo-cyclic AMP, suggesting that the effect of beta-adrenergic stimulation on chloride transport occurs downstream of cAMP production. As isoproterenol did not affect unidirectional rubidium fluxes in hypotonic conditions, the transport pathway influenced by beta-adrenergic stimulation is most likely the swelling-activated chloride channel. Because the beta-adrenergic agonist only influenced the transport in hypotonic conditions despite the fact that cAMP concentration also increased in isotonic conditions, the activation may involve a volume-dependent conformational change in the chloride channel.

The adrenergic volume changes of immature and mature rainbow trout (Oncorhynchus mykiss) erythrocytes.

In this study, we examined whether the adrenergic volume response of teleost erythrocytes is related to cell maturity. Rainbow trout (Oncorhynchus mykiss) were made anaemic by reducing their haematocrit to approximately 50 % of the original value. After 3-4 weeks, small, young erythrocytes were seen in the circulation. By measuring the volume distribution of blood samples from anaemic fish before and after noradrenaline stimulation (10 min, 10(-5)mol l(-1) final concentration), we were able to show that the volume response of young, immature erythrocytes to catecholamine stimulation was greater than that of mature erythrocytes. In addition, the membrane fluidity, measured using the steady-state fluorescence polarisation method, was greater in anaemic fish after 24 days of recovery from bleeding than in control fish. Since blood from anaemic fish contained a large fraction of immature erythrocytes, this result indicates that the fluidity of the membrane of immature erythrocytes is greater than that of mature erythrocytes.