Exploring pathways of NO and H2S signaling in metabolic depression: The case of anoxic turtles.

In contrast to most vertebrates, freshwater turtles of the genera Trachemys and Chrysemys survive total oxygen deprivation for long periods of time. This remarkable tolerance makes them ideal August Krogh's model animals to study adaptions to survive oxygen deprivation. The gasotransmitters nitric oxide (NO) and hydrogen sulfide (H2S) and their metabolic derivatives are central in regulating the physiological responses to oxygen deprivation. Here, we explore the role of these signaling molecules in the anoxia tolerance of the freshwater turtle, including metabolic suppression and protection against oxidative damage with oxygen deprivation. We describe the interaction of NO and H2S with protein thiols and specifically how this regulates the function of central metabolic enzymes. These interactions contribute both to metabolic suppression and to prevent oxidative damage with oxygen deprivation. Furthermore, NO and H2S interact with ferrous and ferric heme iron, respectively, which affects the activity of central heme proteins. In turtles, these interactions contribute to regulate oxygen consumption in the mitochondria, as well as vascular tone and blood flow during oxygen deprivation. The versatile biological effects of NO and H2S underscore the importance of these volatile signaling molecules in the remarkable tolerance of freshwater turtles to oxygen deprivation.

Dynamic changes in nitric oxide synthase expression are involved in seawater acclimation of rainbow trout Oncorhynchus mykiss.

Recent research has shown that nitric oxide (NO) produced by nitric oxide synthases (NOS) is an inhibitor of ion transporter activity and a modulator of epithelial ion transport in fish, but little is known on changes in the NOS/NO system during osmotic stress. We hypothesized that the NOS/NO system responds to salinity changes as an integrated part of the acclimation process. Expression and localization of nos1/Nos1 and nos2/Nos2 were investigated in gill, kidney, and intestine of freshwater (FW)- and seawater (SW)-transferred trout using quantitative PCR, Western blotting, and immunohistochemistry, along with expressional changes of major ion transporters in the gill. The classical branchial ion transporters showed expected expressional changes upon SW transfer, there among a rapid decrease in Slc26a6 mRNA, coding a branchial Cl-/[Formula: see text] exchanger. There was a major downregulation of nos1/ nos2/Nos2 expression in the gill during SW acclimation. A significant decrease in plasma nitrite supported an overall decreased Nos activity and NO production. In the middle intestine, Nos1 was upregulated during SW acclimation, whereas no changes in nos/Nos expression were observed in the posterior intestine and the kidney. Nos1 was localized along the longitudinal axis of the gill filament, beneath smooth muscle fibers of the intestine wall and in blood vessel walls of the kidney. Nos2 was localized within the epithelium adjacent to the gill filament axis and in hematopoietic tissues of the kidney. We conclude that downregulation of branchial NOS is integrated to the SW acclimation process likely to avoid the inhibitory effects of NO on active ion extrusion.

Methemoglobin reductase activity in intact fish red blood cells.

Red blood cells (RBCs) possess methemoglobin reductase activity that counters the ongoing oxidation of hemoglobin (Hb) to methemoglobin (metHb), which in circulating blood is caused by Hb autoxidation or reactions with nitrite. We describe an assay for determining metHb reductase activity in intact RBCs in physiological saline at normal Pco2 and pH. After initial loading of oxygenated RBCs with nitrite (partly oxidizing Hb to metHb), the nitrite is removed by three washes of the RBCs in nitrite-free physiological saline to enable the detection of RBC metHb reductase activity in the absence of counteracting oxidation. This assay was used to compare metHb reduction in rainbow trout and carp RBCs under both oxygenated and deoxygenated conditions. Washing resulted in effective wash-out of nitrite to low and safe values (~2µM). The subsequent decline in [metHb] with time followed first-order kinetics, allowing characterization of metHb reductase activity through the first order rate constant k. In oxygenated RBCs at 25°C, the k values for rainbow trout and carp were slightly below or above 0.01min-1, respectively; which is double the value reported for mammals at 37°C. We conclude the higher metHb reductase activity in fish offsets their higher Hb autoxidation and higher likelihood of encountering elevated nitrite. Deoxygenation significantly raised the rates of RBC metHb reduction, and more so in rainbow trout than in carp. The temperature sensitivity of metHb reduction in rainbow trout RBCs was high (Q10 ~2.8) and instrumental in handling increased Hb autoxidation with temperature.

Interspecific variation and plasticity in hemoglobin nitrite reductase activity and its correlation with oxygen affinity in vertebrates.

Deoxygenated hemoglobin (Hb) is a nitrite reductase that reduces naturally occurring nitrite to nitric oxide (NO), supplying physiological relevant NO under hypoxic conditions. The nitrite reductase activity is modulated by the allosteric equilibrium between the R and T structures of Hb that also determines oxygen affinity. In the present study we investigated nitrite reductase activity and O2 affinity in Hbs from ten different vertebrate species under identical conditions to disclose interspecific variations and allow an extended test for a correlation between the rate constant for nitrite reduction and O2 affinity. We also tested plastic changes in Hb properties via addition of T-structure-stabilizing organic phosphates (ATP and GTP). The decay in deoxyHb during its reaction with nitrite was exponential-like in ectotherms (Atlantic hagfish, carp, crucian carp, brown trout, rainbow trout, cane toad, Indian python and red-eared slider turtle), while it was sigmoid in mammals (harbor porpoise and rabbit). Typically, hypoxia-tolerant species showed a faster reaction than intolerant species. Addition of ATP and GTP decreased O2 affinity and slowed the rate of nitrite reduction in a concentration-dependent manner. The initial second order rate constant of the deoxyHb-mediated nitrite reduction showed a strong curvilinear correlation with oxygen affinity among all ectothermic vertebrates, and the relationship also applied to plastic variations of Hb properties via organic phosphates. The relationship predicts high nitrite reductase activity in hypoxic tolerant species with high Hb-O2 affinity and reveals that the decrease in erythrocyte ATP and/or GTP during acclimation to hypoxia in ectotherms increases the erythrocyte NO generating capacity.

A comparison of blood nitric oxide metabolites and hemoglobin functional properties among diving mammals.

The ability of marine mammals to hunt prey at depth is known to rely on enhanced oxygen stores and on selective distribution of blood flow, but the molecular mechanisms regulating blood flow and oxygen transport remain unresolved. To investigate the molecular mechanisms that may be important in regulating blood flow, we measured concentration of nitrite and S-nitrosothiols (SNO), two metabolites of the vasodilator nitric oxide (NO), in the blood of 5 species of marine mammals differing in their dive duration: bottlenose dolphin, South American sea lion, harbor seal, walrus and beluga whale. We also examined oxygen affinity, sensitivity to 2,3-diphosphoglycerate (DPG) and nitrite reductase activity of the hemoglobin (Hb) to search for possible adaptive variations in these functional properties. We found levels of plasma and red blood cells nitrite similar to those reported for terrestrial mammals, but unusually high concentrations of red blood cell SNO in bottlenose dolphin, walrus and beluga whale, suggesting enhanced SNO-dependent signaling in these species. Purified Hbs showed similar functional properties in terms of oxygen affinity and sensitivity to DPG, indicating that reported large variations in blood oxygen affinity among diving mammals likely derive from phenotypic variations in red blood cell DPG levels. The nitrite reductase activities of the Hbs were overall slightly higher than that of human Hb, with the Hb of beluga whale, capable of longest dives, having the highest activity. Taken together, these results underscore adaptive variations in circulatory NO metabolism in diving mammals but not in the oxygenation properties of the Hb.

Cortisol regulates nitric oxide synthase in freshwater and seawater acclimated rainbow trout, Oncorhynchus mykiss.

Cortisol and nitric oxide (NO) are regulators of ion transport and metabolic functions in fish. In the gill, they show opposite effects on Na+/K+-ATPase (NKA) activity: cortisol stimulates NKA activity while NO inhibits NKA activity. We hypothesized that cortisol may impact NO production in osmoregulatory tissues by regulating NO synthase (NOS) expression. We evaluated the influence of cortisol treatment on mRNA expression of Nos1 and Nos2 in gill, kidney and middle intestine of both freshwater (FW) and seawater (SW) acclimated rainbow trout and found both tissue- and salinity-dependent effects. Nos2 expression was down-regulated in the gill by cortisol injection in both FW and SW trout. This was substantiated by incubating gill tissue with cortisol ex vivo. Similarly, cortisol injection significantly down-regulated Nos2 expression in kidney of SW fish but not in FW fish. In the middle intestine, Nos2 expression was up-regulated by cortisol injection in FW but unchanged in SW fish. Nos1 expression was up-regulated by cortisol injection in FW kidney and down-regulated in SW kidney, whereas it was unaffected in gill and middle intestine of FW and SW fish. Our data provide the first evidence that cortisol may influence NO production in fish by regulating Nos expression. Indeed, the down-regulation of Nos2 expression by cortisol in the gill may prevent the inhibitory effect of NO on NKA activity thereby furthering the stimulatory effect of cortisol on ion-transport.

The roles of tissue nitrate reductase activity and myoglobin in securing nitric oxide availability in deeply hypoxic crucian carp.

In mammals, treatment with low doses of nitrite has a cytoprotective effect in ischemia/reperfusion events, as a result of nitric oxide formation and S-nitrosation of proteins. Interestingly, anoxia-tolerant lower vertebrates possess an intrinsic ability to increase intracellular nitrite concentration during anoxia in tissues with high myoglobin and mitochondria content, such as the heart. Here, we tested the hypothesis that red and white skeletal muscles develop different nitrite levels in crucian carp exposed to deep hypoxia and assessed whether this correlates with myoglobin concentration. We also tested whether liver, muscle and heart tissue possess nitrate reductase activity that supplies nitrite to the tissues during severe hypoxia. Crucian carp exposed to deep hypoxia (1

Nitric oxide inhibition of NaCl secretion in the opercular epithelium of seawater-acclimated killifish, Fundulus heteroclitus.

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 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 (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.

The effect of environmental hypercapnia and size on nitrite toxicity in the striped catfish (Pangasianodon hypophthalmus).

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.

Nitric oxide availability in deeply hypoxic crucian carp: acute and chronic changes and utilization of ambient nitrite reservoirs.

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 toward prolonged hypoxia and subsequent reoxygenation. We hypothesized that crucian carp take up ambient nitrite and use it as a source of cellular NO availability during hypoxia. Fish were exposed for 1 day to normoxia (Po2 > 140 mmHg) and deep hypoxia (1 < Po2 < 3 mmHg) at both low (< 0.2 µM) and moderately elevated (10 µM) ambient [nitrite] 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 the tissues, particularly the heart, where it assists in cytoprotection and other functions. 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.

Metabolic fates and effects of nitrite in brown trout under normoxic and hypoxic conditions: blood and tissue nitrite metabolism and interactions with branchial NOS, Na+/K+-ATPase and hsp70 expression.

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 the influence 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 the 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 nkaα1a subunit increased with hypoxia and nitrite, which may have countered an initial NKA inhibition. Nitrite also increased hsp70 gene expression, probably contributing to the cytoprotective effects of nitrite at low concentrations. Nitrite displays a concentration-dependent switch between positive and negative effects similar to other signaling molecules.

High affinity and temperature sensitivity of blood oxygen binding in Pangasianodon hypophthalmus due to lack of chloride-hemoglobin allosteric interaction.

Air-breathing fishes represent interesting organisms in terms of understanding the physiological changes associated with the terrestrialization of vertebrates, and, further, are of great socio-economic importance for aquaculture in Southeast Asia. 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 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°C and 35°C. To determine the allosteric regulation and thermodynamics of Hb O2 binding, Hb was purified, and O2 equilibria were recorded at five temperatures in the absence and presence of 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°C), a high temperature sensitivity of O2 binding (apparent heat of oxygenation ΔH(app) = -28.3 kcal/mol), 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.

Hypoxia tolerance, nitric oxide, and nitrite: lessons from extreme animals.

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.

Hydrogen sulfide and nitric oxide metabolites in the blood of free-ranging brown bears and their potential roles in hibernation.

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 the 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 in 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, in addition to 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 red blood cells of hibernating bears. In contrast, circulating nitrite and erythrocytic S-nitrosation of 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.

Nitric oxide metabolites during anoxia and reoxygenation in the anoxia-tolerant vertebrate Trachemys scripta.

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 appear to exploit this mechanism by upregulating 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 (1 h and 24 h), testifying that the processes were reversible. The increased NO bioavailability occurred in the absence of NO synthase activity (due to global anoxia) and may involve mobilization of internal/external nitrite reservoirs. Our data support the theory that anoxic upregulation 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.

Circulating nitric oxide metabolites and cardiovascular changes in the turtle Trachemys scripta during normoxia, anoxia and reoxygenation.

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.

Respiratory properties of blood in the harbor porpoise, Phocoena phocoena.

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.

Haematological and ion regulatory effects of nitrite in the air-breathing snakehead fish Channa striata.

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.

Integrating nitric oxide, nitrite and hydrogen sulfide signaling in the physiological adaptations to hypoxia: A comparative approach.

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.

Dramatic increase of nitrite levels in hearts of anoxia-exposed crucian carp supporting a role in cardioprotection.

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.