- [Show abstract] [Hide abstract] ABSTRACT: Nitric oxide (NO) modulates epithelial ion transport pathways in mammals, but this remains largely unexamined in fish. We explored the involvement of NO in controlling NaCl secretion by the opercular epithelium of seawater killifish using an Ussing chamber approach. Pharmacological agents were used to explore the mechanism(s) triggering NO action. A modified Biotin-switch technique was used to investigate S-nitrosation of proteins. Stimulation of endogenous NO production via the nitric oxide synthase (NOS) substrate L-arginine (2.0 mmol l(-1)), and addition of exogenous NO via the NO donor SNAP (10(-6) mol l(-1) to 10(-4) mol l(-1)), decreased the epithelial short-circuit current (Isc). Inhibition of endogenous NO production by the NOS inhibitor L-NAME (10(-4) mol l(-1)) increased Isc and revealed a tonic control of ion transport by NO in unstimulated opercular epithelia. The NO scavenger PTIO (10(-5) mol l(-1)) supressed the NO-mediated decrease in Isc, and confirmed that the effect observed was elicited by release of NO. The effect of SNAP on Isc was abolished by inhibitors of the soluble guanylyl cyclase (sGC), ODQ (10(-6) mol l(-1)) and methylene blue (MB; 10(-4) mol l(-1)), revealing NO signalling via the sGC/cGMP pathway. Incubation of opercular epithelium and gill tissues with SNAP (10(-4) mol l(-1)) led to S-nitrosation of proteins, including Na(+)/K(+)-ATPase. Blocking of NOS with L-NAME (10(-6) mol l(-1)) or scavenging of NO with PTIO during hypotonic shock suggested an involvement of NO in the hypotonic-mediated decrease in Isc Yohimbine (10(-4) mol l(-1)), an inhibitor of α2-adrenoceptors, did not block NO effects suggesting that NO is not involved in the α-adrenergic control of NaCl secretion.
- [Show abstract] [Hide abstract] ABSTRACT: Striped catfish (Pangasianodon hypophthalmus) are farmed intensively at high stocking densities in Vietnam where they are likely to encounter environmental hypercapnia as well as occasional high levels of aquatic nitrite. Nitrite competes with Cl(-) for uptake at the branchial HCO3(-)/Cl(-) exchanger, causing a drastic reduction in the blood oxygen carrying capacity through the formation of methaemoglobin and nitrosylhaemoglobin. Environmental hypercapnia induces a respiratory acidosis where the branchial HCO3(-)/Cl(-) exchange activity is reduced in order to retain HCO3(-) for pH recovery, which should lead to a reduced nitrite uptake. To assess the effect of hypercapnia on nitrite uptake, fish were cannulated in the dorsal aorta, allowing repeated blood sampling for measurements of haemoglobin derivatives, plasma ions and acid-base status during exposure to 0.9mM nitrite alone and in combination with acute and 48h acclimated hypercapnia over a period of 72h. Nitrite uptake was initially reduced during the hypercapnia-induced acidosis, but after pH recovery the situation was reversed, resulting in higher plasma nitrite concentrations and lower functional haemoglobin levels that eventually caused mortality. This suggests that branchial HCO3(-)/Cl(-) exchange activity is reduced only during the initial acid-base compensation, but subsequently increases with the greater availability of internal HCO3(-) counter-ions as pH is compensated. The data further suggest that branchial Na(+)/H(+) exchange plays a significant role in the initial phase of acid-base compensation. Overall, longer term environmental hypercapnia does not protect against nitrite uptake in P. hypophthalmus, but instead enhances it. In addition, we observed a significant size effect in nitrite accumulation, where large fish attained plasma [nitrite] above the ambient concentration, while small fish did not. Small P. hypophthalmus instead had significantly higher plasma [nitrate], and haemoglobin concentrations, revealing greater capacity for detoxifying nitrite by oxidising it to nitrate.
- [Show abstract] [Hide abstract] ABSTRACT: Recent research suggest that anoxia-tolerant fish transfer extracellular nitrite into the tissues, where it is used for nitric oxide (NO) generation, iron-nitrosylation and S-nitrosation of proteins as part of the cytoprotective response towards prolonged oxygen lack and subsequent re-oxygenation. We hypothesized that crucian carp take up ambient nitrite and utilize it as a source of cellular NO availability during hypoxia. Fish were exposed for 1 day to normoxia (Po2 > 140 mmHg) and deep hypoxia (Po2< 3 mmHg) at both low (<0.2 µM) and moderately elevated (10 µM) ambient [nitrite] in order to decipher NO metabolites in plasma and several tissues. We also compared NO metabolite changes during acute (10 min) and chronic (1 day) exposures to three different O2 levels. Plasma [nitrite] decreased with decreasing [O2], while the cellular concentrations of nitrite and nitros(yl)ated compounds either increased or stayed constant, depending on O2 level and tissue type. Nitrite was notably increased in the heart during deep hypoxia, and the increase was amplified by elevated ambient [nitrite]. Raised nitrite also increased gill [nitrite] and decreased mRNA expression of an inducible nitric oxide synthase-2 gene variant. The data support that ambient nitrite is taken up across the gills to be distributed via the blood to tissues, particularly the heart, where it assists in cytoprotection. Cardiac nitrite was not elevated in acutely exposed fish, revealing that the response requires time. NO metabolite levels were higher during acute than chronic exposures, possibly caused by increased swimming activity and stress in acutely exposed fish.
- [Show abstract] [Hide abstract] ABSTRACT: Nitrite secures essential nitric oxide (NO) bioavailability in hypoxia at low endogenous concentrations, whereas it becomes toxic at high concentrations. We exposed brown trout to normoxic and hypoxic water in the absence and presence of added ambient nitrite to decipher the cellular metabolism and effects of nitrite at basal and elevated concentrations under different oxygen regimes. We also tested hypotheses concerning influences of nitrite on branchial nitric oxide synthase (NOS), Na(+)/K(+)-ATPase (nka) and heat shock protein (hsp70) mRNA expression. Basal plasma and erythrocyte nitrite levels were higher in hypoxia than normoxia, suggesting increased NOS activity. Nitrite exposure strongly elevated nitrite concentrations in plasma, erythrocytes, heart tissue and white muscle, which was associated with an extensive metabolism of nitrite to nitrate and to iron-nitrosylated and S-nitrosated compounds. Nitrite uptake was slightly higher in hypoxia than normoxia, and high internal nitrite levels extensively converted blood hemoglobin to methemoglobin and nitrosylhemoglobin. Hypoxia increased inducible NOS (iNOS) mRNA levels in gills, which was overruled by a strong inhibition of iNOS expression by nitrite in both normoxia and hypoxia, suggesting negative feedback regulation of iNOS gene expression by nitrite. A similar inhibition was absent for neuronal NOS. Branchial NKA activity stayed unchanged, but mRNA levels of the NKA α1a subunit increased with hypoxia and nitrite, which may have countered an initial NKA inhibition. Nitrite also increased hsp70 gene expression, probably contributing to cytoprotective effects of nitrite at low concentrations. Nitrite displays a concentration-dependent switch between positive and negative effects resembling other signaling molecules. © 2015. Published by The Company of Biologists Ltd.
- [Show abstract] [Hide abstract] ABSTRACT: Air-breathing fishes represent interesting organisms in terms of understanding the physiological changes associated with the terrestrialization of vertebrates, and are further of great socio-economic importance for aquaculture in South East Asia. In order to understand how environmental factors such as high temperature affect O2 transport in air-breathing fishes, this study assessed the effects of temperature on O2 binding of blood and hemoglobin (Hb) in the economically important air-breathing fish Pangasianodon hypophthalmus. To determine blood O2 binding properties, blood was drawn from resting cannulated fishes and O2 binding curves made at 25 and 35(o)C. To determine the allosteric regulation and thermodynamics of Hb O2 binding, Hb was purified and O2 equilibria recorded at 5 temperatures in the absence and presence of adenosine triphosphate (ATP) and Cl(-). Whole blood had a high O2 affinity (O2 tension at half saturation P50= 4.6 mmHg at extracellular pH 7.6 and 25(o)C), a high temperature sensitivity of O2 binding (apparent heat of oxygenation ΔHapp=-28.3 kcal mol(-1)), and lacked a Root effect. Further, the data on Hb revealed weak ATP binding and a complete lack of Cl(-) binding to Hb, which in part explains the high O2 affinity and high temperature sensitivity of blood O2 binding. This study demonstrates how a potent mechanism for increasing O2 affinity is linked to increased temperature sensitivity of O2 transport, and provides a basic framework for a better understanding of how hypoxia-adapted species will react to increasing temperatures. Copyright © 2014, American Journal of Physiology - Regulatory, Integrative and Comparative Physiology.
- [Show abstract] [Hide abstract] ABSTRACT: Among vertebrates able to tolerate periods of oxygen deprivation, the painted and red-eared slider turtles (Chrysemys picta and Trachemys scripta) and the crucian carp (Carassius carassius) are the most extreme and can survive even months of total lack of oxygen during winter. The key to hypoxia survival resides in concerted physiological responses, including strong metabolic depression, protection against oxidative damage and-in air-breathing animals-redistribution of blood flow. Each of these responses is known to be tightly regulated by nitric oxide (NO) and during hypoxia by its metabolite nitrite. The aim of this review is to highlight recent work illustrating the widespread roles of NO and nitrite in the tolerance to extreme oxygen deprivation, in particular in the red-eared slider turtle and crucian carp, but also in diving marine mammals. The emerging picture underscores the importance of NO and nitrite signaling in the adaptive response to hypoxia in vertebrate animals. ©2015 Int. Union Physiol. Sci./Am. Physiol. Soc.
- [Show abstract] [Hide abstract] ABSTRACT: Goldfish tolerate prolonged and severe hypoxia, thus representing a well-suited model to study the maintenance of cardiac function when O2 availability represents a limiting factor. Using a working heart preparation, we explored the role of the intracardiac nitric oxide synthase (NOS)-derived Nitric Oxide (NO) under normoxic and hypoxic conditions. Cardiac performance was examined both under basal (constant preload and afterload) and loading conditions, i.e. preload-induced increases in stroke volume (SV) and hence cardiac output at constant afterload (the Frank-Starling response). Hypoxic hearts showed an increased basal mechanical performance compared to the normoxic counterpart. Under basal conditions, in both normoxic and hypoxic hearts, NOS and soluble guanylyl cyclase (sGC) inhibition increased SV, while exogenous NO supply decreased it. The normoxic heart was very sensitive to filling pressure increases; the maximum SV=1.08±0.09 mL/kg body mass was obtained at 0.4 kPa. Acute hypoxia increased this sensitivity, SV reaching the maximum value (1,45±0,12 mL/kg body mass) at 0.25 kPa. NOS inhibition by L-NMMA reduced the Frank-Starling response under normoxia, but was ineffective under acute hypoxia, where NO may come from nitrite reduction. In both conditions, sGC inhibition induced a reduction of the cardiac response to preload. Moreover, under acute hypoxia, NO scavenging significantly reduced the Frank-Starling response. The hypoxia-induced hemodynamic patterns were complemented by western blotting analysis which revealed increased expressions of NOS and hypoxia inducible factor α(HIF-1α). In conclusion, we demonstrated that intracardiac NO/NOS enhances goldfish heart performance, remarkably expanding its hypoxic tolerance.
- [Show abstract] [Hide abstract] ABSTRACT: During winter hibernation, brown bears (Ursus arctos) lie in dens for half a year without eating while their basal metabolism is largely suppressed. To understand underlying mechanisms of metabolic depression in hibernation, we measured type and content of blood metabolites of two ubiquitous inhibitors of mitochondrial respiration, hydrogen sulfide (H2S) and nitric oxide (NO), in winter hibernating and summer active free-ranging Scandinavian brown bears. We found that levels of sulfide metabolites were overall similar in summer active and hibernating bears but their composition in the plasma differed significantly, with a decrease of bound sulfane sulfur in hibernation. High levels of unbound free sulfide correlated with high levels of cysteine (Cys) and with low levels of bound sulfane sulfur, indicating that during hibernation H2S, besides being formed enzymatically from the substrate Cys, may also be regenerated from its oxidation products, including thiosulfate and polysulfides. In the absence of any dietary intake, this shift in the mode of H2S synthesis would help preserve free Cys for synthesis of glutathione (GSH), a major antioxidant found at high levels in the RBCs of hibernating bears. In contrast, circulating nitrite and erythrocytic S-nitrosation of the glyceraldehyde 3-phosphate dehydrogenase, taken as markers of NO metabolism, did not change appreciably. Our findings reveal that remodeling of H2S-metabolism and enhanced intracellular GSH levels are hallmarks of the aerobic metabolic suppression of hibernating bears.
- [Show abstract] [Hide abstract] ABSTRACT: Moderate elevations of nitrite and nitric oxide (NO) protect mammalian tissues against ischemia (anoxia)-reperfusion damage by inhibiting mitochondrial electron transport complexes and reducing the formation of reactive oxygen species (ROS) upon reoxygenation. Crucian carp appears to exploit this mechanism by up-regulating nitrite and other nitrite/NO metabolites (S-nitroso and iron-nitrosyl compounds) in several tissues when exposed to anoxia. We investigated whether this is a common strategy amongst anoxia-tolerant vertebrates by evaluating NO metabolites in red-eared slider turtles during long-term (9 days) anoxia and subsequent reoxygenation at low temperature, a situation naturally encountered by turtles in ice-covered ponds. We also measured glutathione in selected tissues and assessed the impact of anoxia on electrolyte status. Anoxia induced major increases in [nitrite] in the heart, pectoral muscle and red blood cells, while [nitrite] was maintained unaltered in brain and liver. Concomitantly, the concentrations of S-nitroso and iron-nitrosyl compounds increased, showing that nitrite was used to produce NO and to S-nitrosate cellular molecules during anoxia. The changes were gradually reversed during reoxygenation (1h and 24h), testifying that the processes were reversible. The increased NO bioavailability occurred in the absence of nitric oxide synthase activity (due to global anoxia) and may involve mobilization of internal/external nitrite reservoirs. Our data supports that anoxic up-regulation of nitrite and other NO metabolites could be a general cytoprotective strategy amongst anoxia-tolerant vertebrates. The possible mechanisms of nitrite-derived NO and S-nitrosation in protecting cells from destructive Ca(2+) influx during anoxia and in limiting ROS formation during reoxygenation are discussed.
- [Show abstract] [Hide abstract] ABSTRACT: Turtles of the genus Trachemys show a remarkable ability to survive prolonged anoxia. This is achieved by a strong metabolic depression, redistribution of blood flow and high levels of antioxidant defence. To understand whether nitric oxide (NO), a major regulator of vasodilatation and oxygen consumption, may be involved in the adaptive response of Trachemys to anoxia, we measured NO metabolites (nitrite, S-nitroso, Fe-nitrosyl and N-nitroso compounds) in the plasma and red blood cells of venous and arterial blood of Trachemys scripta turtles during normoxia and after anoxia (3 h) and reoxygenation (30 min) at 21°C, while monitoring blood oxygen content and circulatory parameters. Anoxia caused complete blood oxygen depletion, decrease in heart rate and arterial pressure, and increase in venous pressure, which may enhance heart filling and improve cardiac contractility. Nitrite was present at high, micromolar levels in normoxic blood, as in some other anoxia-tolerant species, without significant arterial-venous differences. Normoxic levels of erythrocyte S-nitroso compounds were within the range found for other vertebrates, despite very high measured thiol content. Fe-nitrosyl and N-nitroso compounds were present at high micromolar levels under normoxia and increased further after anoxia and reoxygenation, suggesting NO generation from nitrite catalysed by deoxygenated haemoglobin, which in turtle had a higher nitrite reductase activity than in hypoxia-intolerant species. Taken together, these data indicate constitutively high circulating levels of NO metabolites and significant increases in blood NO after anoxia and reoxygenation that may contribute to the complex physiological response in the extreme anoxia tolerance of Trachemys turtles.
- [Show abstract] [Hide abstract] ABSTRACT: Harbor porpoises are active divers that exchange O(2) and CO(2) with the environment during a fast single breath upon surfacing. We investigated blood O(2)-transporting properties, buffer characteristics, Cl(-) transport via the erythrocyte anion exchanger (AE1), circulating nitric oxide metabolites and hemoglobin nitrite reduction in harbor porpoises with the aim to evaluate traits that are adaptive for diving behavior. Blood O(2) affinity was higher in harbor porpoises than in similar sized terrestrial mammals, as supported by our parallel recordings of O(2) equilibria in sheep and pig blood. Further, O(2) affinity tended to increase with increasing body mass. A high O(2) affinity favors O(2) extraction from the lungs, but a normal Bohr effect (ΔlogP(50)/ΔpH=-0.46) gradually lowers O(2) affinity during dives (where CO(2) accumulates) to assist O(2) off-loading to perfused tissues. The true plasma non-bicarbonate buffer value was moderately higher than in terrestrial mammals and increased upon deoxygenation. Plasma bicarbonate was also relatively high, contributing to increase the overall buffer capacity. The apparent Cl(-) permeability of harbor porpoise erythrocytes was similar to the human value at 37°C, showing absence of a comparative increase in the velocity of erythrocyte HCO(-)(3)/Cl(-) exchange to aid CO(2) excretion. The Q(10) for AE1-mediated Cl(-) transport in harbor porpoises was lower than in humans and seemed to match the Q(10) for metabolism (Q(10)≈2). Plasma nitrite, plasma nitrate and hemoglobin-mediated nitrite reduction were elevated compared with mammalian standards, suggesting that increased nitric oxide bioavailability and nitrite-derived nitric oxide could play important roles in diving physiology.
- [Show abstract] [Hide abstract] ABSTRACT: The tolerance and effects of nitrite on ion balance and haematology were investigated in the striped snakehead, Channa striata Bloch 1793, which is an air-breathing fish with reduced gills of importance for aquaculture in South East Asia. C. striata was nitrite tolerant with a 96 h LC50 of 4.7 mM. Effects of sub-lethal exposures to nitrite (0mM, 1.4mM, and 3.0mM) were determined during a 7-day exposure period. Plasma nitrite increased, but the internal concentration remained well below ambient levels. Extracellular nitrate rose by several mM, indicating that a large proportion of the nitrite taken up was converted to nitrate. Nitrite reacted with erythrocyte haemoglobin (Hb) causing methaemoglobin (metHb) to increase to 30% and nitrosylhaemoglobin (HbNO) to increase to 10% of total Hb. Both metHb and HbNO stabilised after 4 days, and functional Hb levels accordingly never fell below 60% of total Hb. Haematocrit and total Hb were unaffected by nitrite. Although the effects of nitrite exposure seemed minor in terms of plasma nitrite and metHb increases, ion balance was strongly affected. In the high exposure group, total osmolality decreased from 320 mOsm to 260 mOsm, and plasma sodium from 150 mM to 120 mM, while plasma chloride fell from 105 mM to 60mM and plasma bicarbonate rose from 12 mM in controls to 20mM in exposed fish. The extreme changes in ion balance in C. striata are different from the response reported in other fish, and further studies are needed to investigate the mechanism behind the observed changes in regulation.
- [Show abstract] [Hide abstract] ABSTRACT: Hydrogen sulfide (H(2)S), nitric oxide (NO) and nitrite (NO(2)(-)) are formed in vivo and are of crucial importance in the tissue response to hypoxia, particularly in the cardiovascular system, where these signaling molecules are involved in a multitude of processes including the regulation of vascular tone, cellular metabolic function and cytoprotection. This report summarizes current advances on the mechanisms by which these signaling pathways act and may have evolved in animals with different tolerance to hypoxia, as presented and discussed during the scientific sessions of the annual meeting of the Society for Experimental Biology in 2011 in Glasgow. It also highlights the need and potential for a comparative approach of study and collaborative effort to identify potential link(s) between the signaling pathways involving NO, nitrite and H(2)S in the whole-body responses to hypoxia.
- [Show abstract] [Hide abstract] ABSTRACT: Nitrite (NO(2)(-)) functions as an important nitric oxide (NO) donor under hypoxic conditions. Both nitrite and NO have been found to protect the mammalian heart and other tissues against ischemia (anoxia)-reoxygenation injury by interacting with mitochondrial electron transport complexes and limiting the generation of reactive oxygen species upon reoxygenation. The crucian carp naturally survives extended periods without oxygen in an active state, which has made it a model for studying how evolution has solved the problems of anoxic survival. We investigated the role of nitrite and NO in the anoxia tolerance of this fish by measuring NO metabolites in normoxic, anoxic, and reoxygenated crucian carp. We also cloned and sequenced crucian carp NO synthase variants and quantified their mRNA levels in several tissues in normoxia and anoxia. Despite falling levels of blood plasma nitrite, the crucian carp showed massive increases in nitrite, S-nitrosothiols (SNO), and iron-nitrosyl (FeNO) compounds in anoxic heart tissue. NO(2)(-) levels were maintained in anoxic brain, liver, and gill tissues, whereas SNO and FeNO increased in a tissue-specific manner. Reoxygenation reestablished normoxic values. We conclude that NO(2)(-) is shifted into the tissues where it acts as NO donor during anoxia, inducing cytoprotection under anoxia/reoxygenation. This can be especially important in the crucian carp heart, which maintains output in anoxia. NO(2)(-) is currently tested as a therapeutic drug against reperfusion damage of ischemic hearts, and the present study provides evolutionary precedent for such an approach.
- [Show abstract] [Hide abstract] ABSTRACT: In this study we investigated nitrite (NO₂⁻) effects in striped catfish, a facultative air-breather. Fish were exposed to 0, 0.4, and 0.9 mM nitrite for 0, 1, 2, 4, and 7 days, and levels of functional haemoglobin, methaemoglobin (metHb) and nitrosyl haemoglobin (HbNO) were assessed using spectral deconvolution. Plasma concentrations of nitrite, nitrate, chloride, potassium, and sodium were also measured. Partitioning of oxygen consumption was determined to reveal whether elevated metHb (causing functional hypoxia) induced air-breathing. The effects of nitrite on maximum oxygen uptake (MO(2max)) and critical swimming speed (U(crit)) were also assessed. Striped catfish was highly tolerant to nitrite exposure, as reflected by a 96 h LC₅₀ of 1.65 mM and a moderate nitrite uptake into the blood. Plasma levels of nitrite reached a maximum after 1 day of exposure, and then decreased, never exceeding ambient levels. MetHb, HbNO and nitrate (a nitrite detoxification product) also peaked after 1 day and then decreased. Only high levels of nitrite and metHb caused reductions in MO(2max) and U(crit). The response of striped catfish contrasts with that seen in most other fish species and discloses efficient mechanisms of combating nitrite threats. Furthermore, even though striped catfish is an efficient air-breather, this species has the ability to sustain aerobic scope and swimming performance without air-breathing, even when faced with nitrite-induced reductions in blood oxygen carrying capacity. Our study is the first to confirm that high levels of nitrite and metHb reduce MO(2max) and thereby aerobic scope, while more moderate elevations fail to do so. Further studies are needed to elucidate the mechanisms underlying the low nitrite accumulation in striped catfish.
- [Show abstract] [Hide abstract] ABSTRACT: Atlantic salmon (Salmo salar) presmolts, smolts, and postsmolts compensate for a respiratory acidosis associated with 96 h of exposure to hyperoxia (100% O2; hO2), hypercapnia (2% CO2 and 98% air; hCO2), and combined hO2/hCO2 in freshwater (FW) by increasing strong ion difference, predominantly through a reduction in plasma [Cl-] (presumably via branchial Cl-/HCO3- exchange). In smolts, compensation during hO2 or hCO2 occurred within 24 h, whereas that in combined hO2/hCO2 was much slower, resulting in 33% mortality by 96 h. FW hO2 and combined hO2/hCO2 appeared to impair gill function, likely through oxidative cell damage. This resulted in reduced hypoosmoregulatory ability following subsequent transfer to seawater (SW), as indicated by changes in plasma ion levels, osmolality, and muscle water content, resulting in considerable mortalities. Interestingly, FW hCO2 appeared to enhance hypoosmoregulatory ability during subsequent SW transfer. Smolts are often transported from FW to a subsequent SW release site, and these data indicate that care should be taken to minimize the degree of hyperoxia experienced by the smolts. Hypercapnia, which results from metabolic CO2 production and inadequate water aeration, does not impair SW transfer, provided it does not occur in conjunction with hyperoxia.
- [Show abstract] [Hide abstract] ABSTRACT: Nitrite is a physiologically important nitric oxide donor at low concentrations but becomes toxic at high concentrations, as develops in freshwater fish exposed to environmental nitrite. We hypothesized that nitrite uptake across the gills differs between normoxic and hypoxic fish and that nitrite accumulation causes excess nitric oxide formation and nitrosative stress. Nitrite and its metabolites were measured via chemiluminescence in normoxic and hypoxic goldfish in control conditions and after 1 day of nitrite exposure. Exposure to nitrite produced much higher nitrite levels in plasma, red blood cells (RBCs) and muscle tissue of normoxic than hypoxic goldfish, suggesting that nitrite uptake was augmented by normoxia in spite of a predictable lower gill surface area. Elevation of nitrite was associated with increased concentrations of S-nitroso, N-nitroso and Fe-nitrosyl compounds in both extracellular and intracellular compartments, revealing nitrosative stress with extensive nitros(yl)ation of thiols, amines and heme groups. The degree of nitrosative stress correlated with nitrite load. Nitrate levels increased in all compartments, reflecting that a significant fraction of the nitrite taken up was converted to non-toxic nitrate. The generation of methemoglobin and nitrosylhemoglobin (assessed by spectral deconvolution) was more pronounced during normoxic nitrite exposure than during hypoxic nitrite exposure, in agreement with the higher nitrite load in normoxic fish. However, at any given nitrite load inside RBCs, the formation of S-nitroso compounds was augmented by hypoxia. We conclude that ambient oxygen conditions have profound influence on branchial nitrite uptake and that nitrosative stress is an integral part of nitrite toxicity at high nitrite concentrations.
- [Show abstract] [Hide abstract] ABSTRACT: Th e extant lungfish – that comprise the predominantly water-breathing Neoceratodus forsteri from Australia and the obligate air - breathing Protopterus and Lepidosiren from Africa and South America, respectively – face extraordinary variations in exogenous factors (O2 and water availability and temperature) and endogenous constraints (bimodal breathing and estivation in moist or dried mud). These circumstances predictably result in inordinate variations in factors (blood levels of pH, O2 and CO2 tensions, urea and lactate levels and osmolality) that mandatorily affect O2 and CO2 binding by the circulating hemoglobin (Hb). This treatise focuses on the distinctive, compensatory adaptations in the gas-transporting functions of lungfish blood and Hb and the underlying molecular mechanisms that support aerobic metabolism in lungfish under harsh conditions.
- [Show abstract] [Hide abstract] ABSTRACT: Nitric oxide (NO), produced by nitric oxide synthases (NOS enzymes), regulates multiple physiological functions in animals. NO exerts its effects by binding to iron (Fe) of heme groups (exemplified by the activation of soluble guanylyl cyclase) and by S-nitrosylation of proteins - and it is metabolized to nitrite and nitrate. Nitrite is used as a marker for NOS activity but it is also a NO donor that can be activated by various cellular proteins under hypoxic conditions. Here, we report the first systematic study of NO metabolites (nitrite, nitrate, S-nitroso, N-nitroso and Fe-nitrosyl compounds) in multiple tissues of a non-mammalian vertebrate (goldfish) under normoxic and hypoxic conditions. NO metabolites were measured in blood (plasma and red cells) and heart, brain, gill, liver, kidney and skeletal muscle, using highly sensitive reductive chemiluminescence. The severity of the chosen hypoxia levels was assessed from metabolic and respiratory variables. In normoxic goldfish, the concentrations of NO metabolites in plasma and tissues were comparable with values reported in mammals, indicative of similar NOS activity. Exposure to hypoxia [at P(O₂) (partial pressure of O₂) values close to and below the critical P(O₂)] for two days caused large decreases in plasma nitrite and nitrate, which suggests reduced NOS activity and increased nitrite/nitrate utilization or loss. Tissue NO metabolites were largely maintained at their tissue-specific values under hypoxia, pointing at nitrite transfer from extracellular to intracellular compartments and cellular NO generation from nitrite. The data highlights the preference of goldfish to defend intracellular NO homeostasis during hypoxia.
- [Show abstract] [Hide abstract] ABSTRACT: The inverse relationship between temperature and hemoglobin-O(2) affinity resulting from the exothermic nature of heme oxygenation favors O(2) unloading from blood to warm, metabolically active tissues. However, this temperature sensitivity is maladaptive, and commonly countered in regional heterotherms, where it may hamper unloading (e.g. in cold extremities of arctic mammals) or increase the diffusive arterio-venous short-circuiting of O(2) (e.g. in counter-current heat exchangers of warm swimming muscles of tuna). We hypothesized analogous blood specializations in heterothermic billfish, whose warm eyes and brains increase the temporal resolution of vision, and measured hemoglobin-O(2) binding properties in three species over a wide pH range, at two temperatures, and in the absence and presence of the major red cell effector, ATP, permitting detailed assessment of overall oxygenation enthalpies (DeltaH') and contributions from oxygenation-linked proton and ATP dissociation. Billfish express multiple isohemoglobins with similar O(2) affinities and pronounced sensitivities to pH and ATP. Compared with the moderate effects associated with proton dissociation upon oxygenation, dissociation of ATP and coupled extra Bohr protons virtually obliterates the temperature sensitivities. At pH 7.4, where this effect is maximal, ATP changes DeltaH' values of blue marlin, striped marlin and shortbill spearfish hemoglobins from -39, -49 and -44 kJ mol(-1) O(2), respectively, to +26, +4 and -7 kJ mol(-1). Thus in addition to allosterically modulating hemoglobin-O(2) affinity, ATP diminishes its temperature sensitivity, reducing deleterious arterio-venous short-circuiting of oxygen in the cranial billfish heat exchangers. The mechanism underlying this reduction in oxygenation enthalpy differs fundamentally from that in tuna, supporting independent evolution of this trait in these scombroid lineages.
University of Southern Denmark
Odense, South Denmark, Denmark
- Department of Biology
Aarhus, Central Jutland, Denmark
- Department of Zoophysiology
Odense University HospitalOdense, South Denmark, Denmark
University of Helsinki
Helsinki, Uusimaa, Finland
- Department of Physiology