Search

The pirarucu is one of the very few obligate air-breathing fish, employing a gigantic, highly vascularized air-breathing organ (ABO). Traditionally, the ABO is thought to serve mainly for O 2 uptake (ṀO 2 ), with the gills providing the major route for excretion of CO 2 (ṀCO 2 ) and N-waste. However, under aquatic hypercapnia, a common occurrence in its natural environment, branchial ṀCO 2 to the water may become impaired. Under these conditions, does the ABO become an important route of ṀCO 2 excretion to the air? We have answered this question by measuring ṀCO 2 and ṀO 2 in both air and water phases, as well as the pattern of air-breathing, in pirarucu under aquatic normocapnia and hypercapnia (3% CO 2 ). Indeed, ṀCO 2 to the air phase via the ABO increased 2- to 3-fold during exposure to high water PCO 2 , accounting for 59-71% of the total, with no change in the dominant contribution of the ABO to ṀO 2 (71-75% of the total). These adjustments were quickly reversed upon restoration of aquatic normocapnia. During aquatic hypercapnia, ṀCO 2 via the ABO became more effective over time, and the pattern of air-breathing changed, exhibiting increased frequency and decreased breath volume. Ammonia-N excretion (86-88% of total) dominated over urea-N excretion and tended to increase during exposure to aquatic hypercapnia. We conclude that the ability of the ABO to take on the dominant role in CO 2 excretion when required may have been an important driver in the original evolution of air-breathing, as well as in the functionality of the ABO in modern air-breathing fish., Competing Interests: Declarations. Ethical approval: All procedures were in compliance with Brazilian national animal care regulations, and those of the Brazilian National Institute for Research of the Amazon (INPA) in Manaus, Brazil under protocol 027/2015. Competing interests: Bernd Pelster is an editor of this journal, but had no role in the reviewing or publication process., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The first studies on ion regulation in fish exposed to low pH, which were inspired by the Acid Rain environmental crisis, seemed to indicate that ion transport at the gills was completely and irreversibly inhibited at pH 4.0-4.5 and below. However, work on characid fish native to the Rio Negro, a naturally acidic, blackwater tributary of the Amazon River, found that they possess ion transport mechanisms that are completely insensitive to pHs as low as 3.25. As more species were examined it appeared that pH-insensitive transport was a trait shared by many, if not most, species in the Order Characiformes. Subsequently, a few other species of fish have been shown to be able to transport ions at low pH, in particular zebrafish (Danio rerio), which show rapid recovery of Na + uptake at pH 4.0 after initial inhibition. Measurements of rates of Na + transport during exposure to pharmacological agents that inhibit various transport proteins suggested that characiform fish do not utilize the generally accepted mechanisms for Na + transport that rely on some form of H + extrusion. Examination of zebrafish transport at low pH suggest the rapid recovery may be due to a novel Na + /K + exchanger, but after longer term exposure they may rely on a coupling of Na + /H + exchangers and NH 3 excretion. Further work is needed to clarify these mechanisms of transport and to find other acid-tolerant species to fully gain an appreciation of the diversity of physiological mechansisms involved., (© 2024. The Author(s).)

Tambaqui (Colossoma macropomum) are a model species for tropical fish physiology, but details are lacking about their ionoregulatory response to acid waters. To provide specifics, we measured unidirectional Na + fluxes in low pH waters. Sodium influx ([Formula: see text]) was uninhibited during acute exposure to pH 4.5 and 3.5, and Na efflux ([Formula: see text]) rose only slightly at pH 3.5; net Na + flux ([Formula: see text]) remained positive at all pH. Similarly, during 24 h transfer to pH 3.5 [Formula: see text], [Formula: see text], and [Formula: see text] were unchanged at all times. Taking a closer look at the mechanism of Na + transport in the gills of tambaqui we found that [Formula: see text] was uninhibited by HMA, a Na + /H + -exchanger blocker, and Benzamil, a Na + -channel inhibitor, casting doubt on their role in Na + uptake in this fish. Measurement of Na + /K + -ATPase (NKA) and H + -ATPase (VHA) activity showed that neither changed at low pH compared to measurements at pH 6.5. Western blot analysis of ATPase expression saw no changes in amount of NKA and VHA at low pH, and immunohistochemistry showed expression of both NKA and VHA on lamellae and interlamellar region of tambaqui gills and that both proteins co-localized to the same gill cells. Location of expression also did not change in low pH water. Amazingly, tambaqui seem unaffected by pH 3.5 water, making them one of the most acid-tolerant fish species examined so far. In addition, they appear to share key ionoregulatory traits with other fish of the order Characiformes, which suggest a common origin for the ionoregulatory attributes.

In the pirarucu (Arapaima gigas), gill surface area and thus gas exchange capacity of the gills are reduced with proceeding development. It, therefore, is expected that A. gigas, starting as a water breather, progressively turns into an obligate air-breathing fish using an air-breathing organ (ABO) for gas exchange. We assessed the air-breathing activity, O 2 and CO 2 exchange into air and water, ammonia-N and urea-N excretion, ion flux rates, and activities of ion transport ATPases in large versus small pirarucu. We found that even very young A. gigas (4-6 g, 2-3 weeks post-hatch) with extensive gills are air-breathers (18.1 breaths*h -1 ) and cover most (63%) of their O 2 requirements from the air whereas 600-700-g animals (about 3-4 months post-hatch), with reduced gills, obtain 75% of their O 2 from the air (10.8 breaths*h -1 ). Accordingly, the reduction in gill surface area hardly affected O 2 uptake, but development had a significant effect on aerial CO 2 excretion, which was very low (3%) in small fish and increased to 12% in larger fish, yielding a hyper-allometric scaling coefficient (1.12) in contrast to 0.82-0.84 for aquatic and total CO 2 excretion. Mass-specific ammonia excretion decreased in approximate proportion to mass-specific O 2 consumption as the fish grew, but urea-N excretion dropped from 18% (at 4-6 g) to 8% (at 600-700 g) of total N-excretion; scaling coefficients for all these parameters were 0.70-0.80. Mass-specific sodium influx and efflux rates, as well as potassium net loss rates, departed from this pattern, being greater in larger fish; hyper-allometric scaling coefficients were > 1.0. Gill V-type H + ATPase activities were greater than Na + , K + -ATPase activities, but levels were generally low and comparable in large and small fish, and similar activities were detected in the ABO. A. gigas is a carnivorous fish throughout its lifecycle, and, despite fasting, protein oxidation accounted for the major portion (61-82%) of aerobic metabolism in both large and small animals. ABO PO 2 and PCO 2 (measured in 600-700-g fish) were quite variable, and aerial hypoxia resulted in lower ABO PO 2 values. Under normoxic conditions, a positive correlation between breath volume and ABP PO 2 was detected, and on average with a single breath more than 50% of the ABO volume was exchanged. ABO PCO 2 values were in the range of 1.95-3.89 kPa, close to previously recorded blood PCO 2 levels. Aerial hypoxia (PO 2 down to 12.65 kPa) did not increase either air-breathing frequency or breath volume.

Dissolved organic carbon (DOC) represents a heterogeneous group of naturally-occurring molecules in aquatic environments, and recent studies have evidenced that optically dark DOCs can exert some positive effects on ionoregulatory homeostasis of aquatic organisms in acidic waters. We investigated the effects of Luther Marsh DOC, a dark allochthonous DOC, on ion regulation and N-waste excretion of zebrafish acutely exposed to either neutral or low pH in ion-poor water. In the first experiment, simultaneous exposure to pH 4.0 and DOC greatly attenuated the stimulation of Na + diffusive losses (J out Na ), and prevented the blockade of Na + uptake (J in Na ) seen in zebrafish exposed to pH 4.0 alone, resulting in much smaller disturbances in Na + net losses (J net Na ). DOC also attenuated the stimulation of net Cl - losses (J net Cl ) and ammonia excretion (J net Amm ) during acidic challenge. In the second experiment, zebrafish acclimated to DOC displayed similar regulation of J in Na and J out Na , and, therefore, reduced J net Na at pH 4.0, effects which persisted even when DOC was no longer present. Protective effects of prior acclimation to DOC on J net Cl and J net Amm at pH 4.0 also occurred, but were less marked than those on Na + balance. Urea fluxes were unaffected by the experimental treatments. Overall, these effects were clearly beneficial to the ionoregulatory homeostasis of zebrafish at low pH, and were quite similar to those seen in a recent parallel study using darker DOC from the upper Rio Negro. This suggests that dark allochthonous DOCs share some chemical properties that render fish tolerant to ionoregulatory disturbances during acidic challenge.

The Tambaqui is a model neotropical teleost which is of great economic and cultural importance in artisanal fisheries and commercial aquaculture. It thrives in ion-poor, often acidic Amazonian waters and exhibits excellent regulation of physiology down to water pH 4.0. Curiously, however, it is reported to perform poorly in aquaculture at pH 8.0, an only slightly alkaline pH which would be benign for most freshwater fish. In initial experiments with Tambaqui of intermediate size (30-50 g), we found that ammonia excretion rate was unchanged at pH 4, 5, 6, and 7, but elevated after 20-24 h at pH 8, exactly opposite the pattern seen in most teleosts. Subsequent experiments with large Tambaqui (150-300 g) demonstrated that only ammonia, and not urea excretion was increased at pH 8.0, and that the elevation was proportional to a general increase in MO 2 . There was an accompanying elevation in net acidic equivalent excretion and/or basic equivalent uptake which occurred mainly at the gills. Net Na + balance was little affected while Cl - balance became negative, implicating a disturbance of Cl - versus base exchange rather than Na + versus acid exchange. Arterial blood pH increased by 0.2 units at pH 8.0, reflecting combined metabolic and respiratory alkaloses. Most parameters recovered to control levels by 18-24 h after return to pH 6.0. With respect to large Tambaqui, we conclude that a physiology adapted to acidic pH performs inappropriately at moderately alkaline pH. In small Tambaqui (4-15 g), the responses were very different, with an initial inhibition of ammonia excretion rate at pH 8.0 followed by a subsequent restoration of control levels. Elevated ammonia excretion rate occurred only after return to pH 6.0. Furthermore, MO 2 , plasma cortisol, and branchial vH + ATPase activities all declined during pH 8.0 exposure in small Tambaqui, in contrast to the responses in larger fish. Overall, small Tambaqui appear to cope better at pH 8.0, a difference that may correlate with their natural history in the wild.

The jeju Hoplerythrinus unitaeniatus and the traira Hoplias malabaricus are two neighboring genera from the family of erythrinid fish, both possessing a two-chambered physostomous swimbladder. In the jeju the anterior section of the posterior bladder is highly vascularized, and the swimbladder is used for aerial respiration; the traira, in turn, is a water-breather that uses the swimbladder as a buoyancy organ and not for aerial oxygen uptake. Measurement of swimbladder oxygen partial pressure (PO 2 ) of fish kept at 26 °C in normoxic, hyperoxic (28-32 mg O 2 L - 1 ) or hypoxic (1-1.5 mg O 2 L - 1 ) water revealed constant values in traira swimbladder. Under normoxic conditions in the jeju swimbladder PO 2 was higher than in traira, and the PO 2 significantly increased under hyperoxic conditions, even in the absence of air breathing. In jeju, air-breathing activity increased significantly under hypoxic conditions. Hypoxic air-breathing activity was negatively correlated to swimbladder PO 2 , indicating that the swimbladder was intensely used for gas exchange under these conditions. In traira, the capacity of the ROS defense system, as assessed by measurement of activities of enzymes involved in ROS degradation and total glutathione (GSH + GSSG) concentration, was elevated after 4 h of hyperoxic and/or hypoxic exposure, although swimbladder PO 2 was not affected. In jeju, experiencing a higher variability in swimbladder PO 2 due to the air-breathing activity, only a reduced responsiveness of the ROS defense system to changing environmental PO 2 was detected.

Air breathing in fish is commonly believed to have arisen as an adaptation to aquatic hypoxia. The effectiveness of air breathing for tissue O 2 supply depends on the ability to avoid O 2 loss as oxygenated blood from the air-breathing organ passes through the gills. Here, we evaluated whether the armoured catfish (Hypostomus aff. pyreneusi)-a facultative air breather-can avoid branchial O 2 loss while air breathing in aquatic hypoxia, and we measured various other respiratory and metabolic traits important for O 2 supply and utilization. Fish were instrumented with opercular catheters to measure the O 2 tension (PO 2 ) of expired water, and air breathing and aquatic respiration were measured during progressive stepwise hypoxia in the water. Armoured catfish exhibited relatively low rates of O 2 consumption and gill ventilation, and gill ventilation increased in hypoxia due primarily to increases in ventilatory stroke volume. Armoured catfish began air breathing at a water PO 2 of 2.5 kPa, and both air-breathing frequency and hypoxia tolerance (as reflected by PO 2 at loss of equilibrium, LOE) was greater in individuals with a larger body mass. Branchial O 2 loss, as reflected by higher PO 2 in expired than in inspired water, was observed in a minority (4/11) of individuals as water PO 2 approached that at LOE. Armoured catfish also exhibited a gill morphology characterized by short filaments bearing short fused lamellae, large interlamellar cell masses, low surface area, and a thick epithelium that increased water-to-blood diffusion distance. Armoured catfish had a relatively low blood-O 2 binding affinity when sampled in normoxia (P 50 of 3.1 kPa at pH 7.4), but were able to rapidly increase binding affinity during progressive hypoxia exposure (to a P 50 of 1.8 kPa). Armoured catfish also had low activities of several metabolic enzymes in white muscle, liver, and brain. Therefore, low rates of metabolism and gill ventilation, and a reduction in branchial gas-exchange capacity, may help minimize branchial O 2 loss in armoured catfish while air breathing in aquatic hypoxia.

The total rate of N-waste excretion (M N ) in juvenile tambaqui living in ion-poor Amazonian water comprised 85 % ammonia-N (M Amm-N ) and 15 % urea-N (M Urea-N ). Both occurred mainly across the gills with only ~5 % of M Amm-N and ~39 % of M Urea-N via the urine. Tambaqui were not especially tolerant to high environmental ammonia (HEA), despite their great resistance to other environmental factors. Nevertheless, they were able to maintain a continued elevation of M Amm-N during and after 48-h exposure to 2.5 mmol L -1 HEA. The normally negative transepithelial potential (-18 mV) increased to -9 mV during the HEA period, which would help to reduce branchial NH 4 + entry. During 3 h of acute environmental hypoxia (30 % saturation), M Amm-N declined, and recovered thereafter, similar to the response seen in other hypoxia-tolerant teleosts; M Urea-N did not change. However, during gradual hypoxia, M Amm-N remained constant, but M Urea-N eventually fell. The acute temperature sensitivities of M Amm-N and M N were low from 28 °C (acclimation) to 33 °C (Q10 ~1.5), but high (~3.8) from 33 to 38 °C, relative to [Formula: see text] (~1.9 throughout). In contrast, M Urea-N exhibited a different pattern over these temperature ranges (Q10 2.6 and 2.1, respectively). The nitrogen quotient (NQ = 0.16-0.23) was high at all temperatures, indicating a 60-85 % reliance on protein to fuel aerobic metabolism in these fasting animals. During steady-state aerobic exercise, [Formula: see text] and M Urea-N increased in parallel with velocity (up to 3.45 body lengths s -1 ), but M Amm (and thus M N ) remained approximately constant. Therefore, the NQ fell progressively, indicating a decreasing reliance on protein-based fuels, as work load increased. In group feeding trials using 45 % protein commercial pellets, tambaqui excreted 82 % (range 39-170 %) of the dietary N within 24 h; N-retention efficiency was inversely related to the ration voluntarily consumed. M Amm-N peaked at 4-6 h, and M Urea-N at 6-9-h post-feeding, with an additional peak in M Amm-N only at 21 h. During subsequent fasting, M N stabilized at a high endogenous rate from 2 through 8 days post-feeding. Possible reasons for the high wasting of protein-N during both fasting and feeding are discussed.

The jeju Hoplerythrinus unitaeniatus and the traira Hoplias malabaricus are two closely related erythrinid fish, both possessing a two-chambered physostomous swimbladder. In the jeju the anterior section of the posterior bladder is highly vascularized and the swimbladder is used for aerial respiration; the traira, in turn, is a water-breather that uses the swimbladder as a buoyancy organ and not for aerial oxygen uptake. Observation of the breathing behavior under different levels of water oxygenation revealed that the traira started aquatic surface respiration only under severe hypoxic conditions and did not breathe air. In the jeju air-breathing behavior was observed under normoxic conditions, and the frequency of air-breathing was significantly increased under hypoxic conditions. Unexpectedly, even under hyperoxic conditions (30 mg O2 L(-1)) the jeju continued to take air breaths, and compared with normoxic conditions the frequency was not reduced. Because the frequently air-exposed swimbladder tissue faces higher oxygen partial pressures than normally experienced by other fish tissues, it was hypothesized that in the facultative air-breathing jeju, swimbladder tissue would have a higher antioxidative capacity than the swimbladder tissue of the water breathing traira. Measurement of total glutathione (GSSG/GSH) concentration in anterior and posterior swimbladder tissue revealed a higher concentration of this antioxidant in swimbladder tissue as compared to muscle tissue in the jeju. Furthermore, the GSSG/GSH concentration in jeju tissues was significantly higher than in traira tissues. Similarly, activities of enzymes involved in the breakdown of reactive oxygen species were significantly higher in the jeju swimbladder as compared to the traira swimbladder. The results show that the jeju, using the swimbladder as an additional breathing organ, has an enhanced antioxidative capacity in the swimbladder as compared to the traira, using the swimbladder only as a buoyancy organ.

The evolutionary transition from water-breathing to air-breathing involved not only a change in function of the organs of respiratory gas exchange and N-waste excretion, but also in the organs of ion uptake from the environment. A combination of in vivo and in vitro techniques was used to look at the relative importance of the gills versus the gut in Na(+), Cl(-), and K(+) balance in two closely related erythrinid species: a facultative air-breather, the jeju (Hoplerythrinus unitaeniatus) and an obligate water-breather, the traira (Hoplias malabaricus). The jeju has a well-vascularized physostomous swimbladder, while that in the traira is poorly vascularized, but the gills are much larger. Both species are native to the Amazon and are common in the ion-poor, acidic blackwaters of the Rio Negro. Under fasting conditions, the traira was able to maintain positive net Na(+) and Cl(-) balance in this water, and only slightly negative net K(+) balance. However, the jeju was in negative net balance for all three ions and had lower plasma Na(+) and Cl(-) concentrations, despite exhibiting higher branchial Na(+), K(+)ATPase and v-type H(+)ATPase activities. In the intestine, activities of these same enzymes were also higher in the jeju, and in vitro measurements of net area-specific rates of Na(+), Cl(-), and K(+) absorption, as well as the overall intestinal absorption capacities for these three ions, were far greater than in the traira. When acutely exposed to disturbances in water O2 levels (severe hypoxia ~15% or hyperoxia ~420% saturation), gill ionoregulation was greatly perturbed in the traira but less affected in the jeju, which could "escape" the stressor by voluntarily air-breathing. We suggest that a shift of ionoregulatory capacity from the gills to the gut may have occurred in the evolutionary transition to air-breathing in jeju, and in consequence branchial ionoregulation, while less powerful, is also less impacted by variations in water O2 levels.

In the traditional osmorespiratory compromise, fish increase their effective gill permeability to O2 during exercise or hypoxia, and in consequence suffer unfavorable ionic and osmotic fluxes. However oscars, which live in the frequently hypoxic ion-poor waters of the Amazon, actually decrease ionic fluxes across the gills during acute hypoxia without changing gill paracellular permeability, and exhibit rapid paving over of the mitochondrial-rich cells (MRCs). But what happens during prolonged exercise? Gill paracellular permeability, ionic fluxes, and gill morphology were examined in juvenile oscars at rest and during aerobic swimming. Initial validation tests with urinary catheterized fish quantified drinking, glomerular filtration, and urinary flow rates, and confirmed that measurements of gill paracellular permeability as [(3)H]PEG-4000 clearances were the same in efflux and influx directions, but far lower than previously measured in comparably sized trout. Although the oscars achieved a very similar proportional increase (90%) in oxygen consumption (MO2) to trout during steady-state swimming at 1.2 body lengths sec(-1), there was no increase in gill paracellular permeability, in contrast to trout. However, oscars did exhibit increased unidirectional Na(+) efflux and net K(+) rates during exercise, but no change in drinking rate. There were no changes in MRC numbers or exposure, or other alterations in gill morphology during exercise. A substantial interlamellar cell mass (ILCM) that covered the lamellae to a depth of 30% was unchanged by 4 h of swimming activity. We conclude that a low branchial paracellular permeability which can be dissociated from changes in O2 flux, as well as the presence of the ILCM, may be adaptive in limiting ionoregulatory costs for a species endemic to ion-poor, frequently hypoxic waters.

Mechanisms of Na(+) uptake, ammonia excretion, and their potential linkage were investigated in three characids (cardinal, hemigrammus, moenkhausia tetras), using radiotracer flux techniques to study the unidirectional influx (J in), efflux (J out), and net flux rates (J net) of Na(+) and Cl(-), and the net excretion rate of ammonia (J Amm). The fish were collected directly from the Rio Negro, and studied in their native "blackwater" which is acidic (pH 4.5), ion-poor (Na(+), Cl(-) ~20 µM), and rich in dissolved organic matter (DOM 11.5 mg C l(-1)). J in (Na) , J in (Cl) , and J Amm were higher than in previous reports on tetras obtained from the North America aquarium trade and/or studied in low DOM water. In all three species, J in (Na) was unaffected by amiloride (10(-4) M, NHE and Na(+) channel blocker), but both J in (Na) and J in (Cl) were virtually eliminated (85-99 % blockade) by AgNO3 (10(-7) M). A time course study on cardinal tetras demonstrated that J in (Na) blockade by AgNO3 was very rapid (<5 min), suggesting inhibition of branchial carbonic anhydrase (CA), and exposure to the CA-blocker acetazolamide (10(-4) M) caused a 50 % reduction in J in (Na) .. Additionally, J in (Na) was unaffected by phenamil (10(-5) M, Na(+) channel blocker), bumetanide (10(-4) M, NKCC blocker), hydrochlorothiazide (5 × 10(-3) M, NCC blocker), and exposure to an acute 3 unit increase in water pH. None of these treatments, including partial or complete elimination of J in (Na) (by acetazolamide and AgNO3 respectively), had any inhibitory effect on J Amm. Therefore, Na(+) uptake in Rio Negro tetras depends on an internal supply of H(+) from CA, but does not fit any of the currently accepted H(+)-dependent models (NHE, Na(+) channel/V-type H(+)-ATPase), or co-transport schemes (NCC, NKCC), and ammonia excretion does not fit the current "Na(+)/NH4 (+) exchange metabolon" paradigm. Na(+), K(+)-ATPase and V-type H(+)-ATPase activities were present at similar levels in gill homogenates, Acute exposure to high environmental ammonia (NH4Cl, 10(-3) M) significantly increased J in (Na) , and NH4 (+) was equally or more effective than K(+) in activating branchial Na(+),(K(+)) ATPase activity in vitro. We propose that ammonia excretion does not depend on Na(+) uptake, but that Na(+) uptake (by an as yet unknown H(+)-dependent apical mechanism) depends on ammonia excretion, driven by active NH4 (+) entry via basolateral Na(+),(K(+))-ATPase.

Preferential intracellular pH (pHi) regulation, where pHi is tightly regulated in the face of a blood acidosis, has been observed in a few species of fish, but only during elevated blood PCO2. To determine whether preferential pHi regulation may represent a general pattern for acid-base regulation during other pH disturbances we challenged the armoured catfish, Pterygoplichthys pardalis, with anoxia and exhaustive exercise, to induce a metabolic acidosis, and bicarbonate injections to induce a metabolic alkalosis. Fish were terminally sampled 2-3 h following the respective treatments and extracellular blood pH, pHi of red blood cells (RBC), brain, heart, liver and white muscle, and plasma lactate and total CO2 were measured. All treatments resulted in significant changes in extracellular pH and RBC pHi that likely cover a large portion of the pH tolerance limits of this species (pH 7.15-7.86). In all tissues other than RBC, pHi remained tightly regulated and did not differ significantly from control values, with the exception of a decrease in white muscle pHi after anoxia and an increase in liver pHi following a metabolic alkalosis. Thus preferential pHi regulation appears to be a general pattern for acid-base homeostasis in the armoured catfish and may be a common response in Amazonian fishes.

We examined the metabolic and ionoregulatory responses of the Amazonian cichlid, Astronotus ocellatus, to 20 h exposure to severe hypoxia (0.37 +/- 0.19 mg O(2)/l; 4.6% air saturation) or 8 h severe hypoxia followed by 12 h recovery in normoxic water. During 20 h exposure to hypoxia, white muscle [ATP] was maintained at normoxic levels primarily through a 20% decrease in [creatine phosphate] (CrP) and an activation of glycolysis yielding lactate accumulation. Muscle lactate accumulation maintained cytoplasmic redox state ([NAD(+)]/[NADH]) and was associated with an inactivation of the mitochondrial enzyme pyruvate dehydrogenase (PDH). The inactivation of PDH was not associated with significant changes in cytoplasmic allosteric modulators ([ADP(free)], redox state, or [pyruvate]). Hypoxia exposure caused an approximately 65% decrease in gill Na(+)/K(+) ATPase activity, which was not matched by changes in Na(+)/K(+) ATPase alpha-subunit protein abundance indicating post-translational modification of Na(+)/K(+) ATPase was responsible for the decrease in activity. Despite decreases in gill Na(+)/K(+) ATPase activity, plasma [Na(+)] increased, but this increase was possibly due to a significant hemoconcentration and fluid shift out of the extracellular space. Hypoxia caused an increase in Na(+)/K(+) ATPase alpha-subunit mRNA abundance pointing to either reduced mRNA degradation during exposure to hypoxia or enhanced expression of Na(+)/K(+) ATPase alpha-subunit relative to other genes.