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The Amazonian cichlid Astronotus ocellatus is highly tolerant to hypoxia, and is known to reduce its metabolic rate by reducing the activity of energetically expensive metabolic processes when oxygen is lacking in its environment. Our objectives were to determine how protein metabolism is regulated in A. ocellatus during hypoxia. Fish were exposed to a stepwise decrease in air saturation (100%, 20%, 10% and 5%) for 2 h at each level, and sampled throughout the experiment. A flooding dose technique using a stable isotope allowed us to observe an overall decrease in protein synthesis during hypoxia in liver, muscle, gill and heart. We estimate that this decrease in rates of protein synthesis accounts for a 20 to 36% decrease in metabolic rate, which would enable oscars to maintain stable levels of ATP and prolong survival. It was also determined for the first time in fish that a decrease in protein synthesis during hypoxia is likely controlled by signaling molecules (4EBP1 and eIF2-α), and not simply due to a lack of ATP. We could not detect any effects of hypoxia on protein degradation as the levels of NH 4 excretion, indicators of the ubiquitin proteasome pathway, and enzymatic activities of lysosomal and non-lysosomal proteolytic enzymes were maintained throughout the experiment., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

There is a wide interspecific range in plasma glucose levels in teleosts from less than 0.5 to greater than 10 mmol l -1 Here we assessed how glucose availability influences glucose metabolism in hearts of Atlantic cod ( Gadus morhua ), rainbow trout ( Oncorhynchus mykiss ), lumpfish ( Cyclopterus lumpus ) and short-horned sculpin ( Myoxocephalus scorpius ) under normoxic and hypoxic conditions. These species had plasma glucose levels of 5.1, 4.8, 0.9 and 0.5 mmol l -1 , respectively. Rates of glucose metabolism and lactate production were determined in isolated hearts perfused with medium containing physiological levels of glucose. Under normoxic conditions there was no significant difference in rates of either glucose metabolism (average 15 nmol g -1  min -1 ) or lactate production (average 30 nmol g -1  min -1 ) across species. Under hypoxia (12% of air saturation) there were significant increases in rates of glucose metabolism and lactate production in hearts from Atlantic cod (glucose-130; lactate-663 nmol g -1  min -1 ) and rainbow trout (glucose-103; lactate-774 nmol g -1  min -1 ); however, there was no change in rate of glucose metabolism in hearts from either lumpfish or short-horned sculpin and only increases in lactate production to rates much lower than the other species. Furthermore, Atlantic cod hearts perfused with medium containing low non-physiological levels of glucose (0.5 mmol l -1 ) had the same rates of glucose metabolism under normoxic and hypoxic treatments. Anaerobic metabolism supported by extracellular glucose is compromised in fish with low levels of plasma glucose, which in turn may decrease performance under oxygen-limiting conditions at the whole-animal level., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

The gas gland of physoclistous fish utilizes glucose to generate lactic acid that leads to the off-loading of oxygen from haemoglobin. This study addresses characteristics of the first two steps in glucose utilization in the gas gland of Atlantic cod (Gadus morhua). Glucose metabolism by isolated gas gland cells was 12- and 170-fold higher, respectively, than that in heart and red blood cells (RBCs) as determined by the production of (3)H2O from [2-(3)H]glucose. In the gas gland, essentially all of the glucose consumed was converted to lactate. Glucose uptake in the gas gland shows a very high dependence upon facilitated transport as evidenced by saturation of uptake of 2-deoxyglucose at a low extracellular concentration and a requirement for high levels of cytochalasin B for uptake inhibition despite the high efficacy of this treatment in heart and RBCs. Glucose transport is via glucose transporter 1 (GLUT1), which is localized to the glandular cells. GLUT1 western blot analysis from whole-tissue lysates displayed a band with a relative molecular mass of 52 kDa, consistent with the deduced amino acid sequence. Levels of 52 kDa GLUT1 in the gas gland were 2.3- and 33-fold higher, respectively, than those in heart and RBCs, respectively. Glucose phosphorylation is catalysed by hexokinase Ib (HKIb), a paralogue that cannot bind to the outer mitochondrial membrane. Transcript levels of HKIb in the gas gland were 52- and 57-fold more abundant, respectively, than those in heart and RBCs. It appears that high levels of GLUT1 protein and an unusual isoform of HKI are both critical for the high rates of glycolysis in gas gland cells., (© 2016. Published by The Company of Biologists Ltd.)

Fish exhibit a wide range of species-specific blood glucose levels. How this relates to glucose utilization is yet to be fully realized. Here, we assessed glucose transport and metabolism in myocytes isolated from Atlantic cod (Gadus morhua) and short-horned sculpin (Myoxocephalus scorpius), species with blood glucose levels of 3.7 and 0.57 mmol l(-1), respectively. Glucose metabolism was assessed by the production of (3)H2O from [2-(3)H]glucose. Glucose metabolism was 3.5- to 6-fold higher by myocytes from Atlantic cod than by those from short-horned sculpin at the same level of extracellular glucose. In Atlantic cod myocytes, glucose metabolism displayed what appears to be a saturable component with respect to extracellular glucose, and cytochalasin B inhibited glucose metabolism. These features revealed a facilitated glucose diffusion mechanism that accounts for between 30% and 55% of glucose entry at physiological levels of extracellular glucose. Facilitated glucose diffusion appears to be minimal in myocytes for short-horned sculpin. Glucose entry by simple diffusion occurs in both cell types with the same linear relationship between glucose metabolism and extracellular glucose concentration, presumably due to similarities in membrane composition. Oxygen consumption by myocytes incubated in medium containing physiological levels of extracellular glucose (Atlantic cod 5 mmol l(-1), short-horned sculpin 0.5 mmol l(-1)) was similar in the two species and was not decreased by cytochalasin B, suggesting that these cells have the capability of oxidizing alternative on-board metabolic fuels. Cells produced lactate at low rates but glycogen levels did not change during the incubation period. In cells from both species, glucose utilization assessed by both simple chemical analysis of glucose disappearance from the medium and (3)H2O production was half the rate of lactate production and as such extracellular glucose was not available for oxidative metabolism. Overall, extracellular glucose makes only a minor contribution to ATP production but a sustained glycolysis may be necessary to support Ca(2+) transport mechanisms at either the sarcoplasmic reticulum or the sarcolemmal membrane., (© 2016. Published by The Company of Biologists Ltd.)

Energy metabolism was assessed in red blood cells (RBCs) from Atlantic cod and short-horned sculpin, two species that have markedly different levels of blood glucose. The objective was to determine whether the level of extracellular glucose has an impact on rates of glucose metabolism. The blood glucose level was 2.5 mmol l(-1) in Atlantic cod and 0.2 mmol l(-1) in short-horned sculpin, respectively. Oxygen consumption, lactate production and glucose utilization were measured in whole blood and related to grams of RBCs. Glucose utilization was assessed by measuring both glucose disappearance and the production of (3)H2O from [2-(3)H]-glucose. RBCs from both species have an aerobic-based metabolism. In Atlantic cod, extracellular glucose is sufficient to provide the sum of glucosyl equivalents to support both oxidative metabolism and lactate production. In contrast, extracellular glucose can account for only 10% of the metabolic rate in short-horned sculpin RBCs. In both species, about 70% of glucose enters the RBCs via facilitated transport. The difference in rates of extracellular glucose utilization is related to the extremely low levels of blood glucose in short-horned sculpin. In this species energy metabolism by RBCs must be supported by alternative fuels., (© 2014. Published by The Company of Biologists Ltd.)

The aim of the present study was to assess whether mechanisms of glucose trafficking by red blood cells (RBCs) relate to species-specific extracellular glucose levels. Atlantic cod (Gadus morhua), Atlantic salmon (Salmo salar), cunner (Tautogolabrus adspersus) and short-horned sculpin (Myoxocephalus scorpius) had plasma glucose levels of 4, 4.1, 1.95 and 0.73 mmol l(-1), respectively. Glucose uptake by isolated RBCs was measured by the initial incorporation of [6-(14)C]-glucose and steady-state glucose metabolism was determined by the production of (3)H(2)O from [2-(3)H]-glucose. Saturation kinetics of glucose uptake and inhibition of both glucose uptake and metabolism by cytochalasin B and phloretin revealed that Atlantic cod, cunner and sculpin RBCs all had a facilitated transport component to glucose trafficking. RBCs from Atlantic salmon showed a linear relationship between glucose uptake and extracellular glucose level, but exhibited clear inhibition of glucose metabolism by cytochalasin B and phloretin, suggesting a component of facilitated glucose transport that is more elusive to detect. The production of (3)H(2)O was linear for at least 6 h and as such presents a rigorous approach to measuring glycolytic rate. Steady-state rates of glucose metabolism were achieved at extracellular levels of approximately 1 mmol l(-1) glucose for RBCs from all species, showing that within-species normal extracellular glucose level is not a primary determinant of the basal level of glycolysis. At physiological levels of extracellular glucose, the ratio of initial glucose uptake to glucose metabolism was 1.5 to 4 for all RBCs, suggesting that there is scope to increase metabolic rate without alteration of the basal glucose uptake capacity.

The effects of temperature on protein metabolism have been studied mostly with respect to protein synthesis. Temperature generally has a parabolic effect on protein synthesis with a maximum rate being observed at optimal growth temperature. The effect of temperature on protein degradation is poorly understood. The 20S proteasome is mainly responsible for the degradation of short-lived and oxidatively modified proteins and has been recently identified as a potentially good proxy for protein degradation in fish. The aim of this experiment was to examine the relationships between the rate of protein synthesis, activity of the 20S proteasome, oxidative stress markers and antioxidant capacity in white muscle of juvenile spotted wolffish (Anarhichas minor) acclimated at three temperatures (4, 8 and 12 degrees C). The rate of protein synthesis was lower at 4 degrees C than at 8 degrees C while it was intermediate at 12 degrees C. Despite the decrease of protein synthesis at low temperature, the activity of 20S proteasome activity was maintained high in fish acclimated at lower temperature (4 degrees C), reaching levels 130% of that of fish acclimated at 8 degrees C when measured at a common temperature. The oxidative stress markers TBARS and protein-carbonyl content did not change among temperature groups, but reduced glutathione concentration was higher in cold-acclimated fish, suggesting a higher antioxidant capacity in this group. Our data suggest that lower growth rate in cold temperature results from both high 20S proteasome activity and a reduced rate of protein synthesis.

Rainbow smelt Osmerus modax accumulate glycerol in winter that serves as an antifreeze. Fish were held at 8 degrees C, or subjected to a decrease in water temperature to -1 degrees C over a 19 day period, and subsequently maintained at -1 degrees C from 15 January to 11 May 2004. Starved fish did not survive the challenge of temperature decrease, with death ensuing above the typical freeze point for marine teleosts (-0.8 degrees C). A decrease in temperature activates the glycerol accumulation mechanism at about 5 degrees C with peak plasma levels exceeding 300 micromol ml(-1). Glycerol levels begin to decrease in late February even at water temperatures below -1 degrees C, suggesting either an inherent circannual or photoperiod trigger, possibly in association with sufficiently high levels of antifreeze protein. Glycogen levels in liver did not change significantly in starved fish maintained at 8 degrees C. However, liver glycogen was depleted in fish subjected to the low-temperature challenge and at a faster rate in starved than in fed fish. Stored glycogen in liver and other tissues can account for only a small amount of the total glycerol production, suggesting a strong requirement for food during accelerated glycerol production. Liver glycogen levels increased in April and May in association with the decrease in glycerol. Levels of glycerol in liver, kidney, spleen, gill, intestine, heart, muscle and brain follow the same pattern as that in plasma. During the early part of the glycerol accumulation phase, all tissues except for liver have lower levels of glycerol in the intracellular space than the levels in plasma. In liver, glycerol is in equilibrium between the two compartments.

Oxygen consumption, as an indicator of routine metabolic rate (RoMR), and tissue-specific changes in protein synthesis, as measured by (3)H-labelled phenylalanine incorporation rates, were determined in Astronotus ocellatus to investigate the cellular mechanisms behind hypoxia-induced metabolic depression and recovery. RoMR was significantly depressed, by approximately 50%, when dissolved oxygen levels reached 10% saturation (0.67+/-0.01 mg l(-1) at 28+/-1 degrees C). This depression in RoMR was accompanied by a 50-60% decrease in liver, heart and gill protein synthesis, but only a 30% decrease in brain protein synthesis. During recovery from hypoxia, an overshoot in RoMR to 270% of the normoxic rate was observed, indicating the accumulation of an oxygen debt during hypoxia. This conclusion was consistent with significant increase in plasma lactate levels during the hypoxic exposure, and the fact that lactate levels rapidly returned to pre-hypoxic levels. In contrast, a hyperactivation of protein synthesis did not occur, suggesting the overshoot in oxygen consumption during recovery is attributed to an increase in cellular processes other than protein synthesis.

cDNAs of putative glucose transporters, GLUT4 and GLUT2, were cloned from Atlantic cod (Gadus morhua). The GLUT4 cDNA encodes a 503 amino acid and the GLUT2 cDNA a 506 amino acid protein. Phylogenetic analysis, amino acid sequence alignment, and tissue distribution support categorizing them as homologues of mammalian GLUT4 and 2. GLUT4 clusters with GLUT4s from fish and other vertebrates. It shows 84% amino acid identity to GLUT4 from coho salmon and brown trout and 65% identity with other vertebrates. It is most highly expressed in heart, strongly expressed in red and white skeletal muscle and present at lower levels in gill, gonad, intestine, and kidney. GLUT2 clusters with GLUT2 from rainbow trout and other vertebrates. It shows 75% amino acid identity with rainbow trout and 62% identity with chicken GLUT2. In Atlantic cod, GLUT2 is most highly expressed in liver with lower levels noted in intestine and kidney. Food deprivation for 2 months was used as a vehicle to monitor GLUT expression at different blood glucose levels. Starvation resulted in a decrease in blood glucose and liver glycogen that recovered following 20 days of re-feeding. GLUT4 expression in heart was decreased with starvation and increased with re-feeding. GLUT4 mRNA level in heart correlated with blood glucose. It is suggested that this relationship is related to insulin responsiveness. GLUT4 expression in white muscle increased with starvation and decreased with re-feeding. It is proposed that this is due to the necessity to maintain high levels of the glucose transporter protein in the face of starvation-associated proteolysis. GLUT2 expression in liver correlated with blood glucose, consistent with higher rates of glucose transport from liver to blood in the fed state than in the food-deprived state. Glycerol-3-phosphate dehydrogenase (GPDH) cDNA was also cloned. It encodes a 351 amino acid protein, which is 73-90% identical to GPDH from numerous other fish species. GPDH is ubiquitously expressed. Expression in heart decreased with starvation and increased with refeeding, whereas expression in liver did not change with starvation. In other studies, gene expression was monitored at nine time points from fertilization of eggs to larval development. GLUT4 is detectable in fertilized eggs and is fully expressed by the halfway to hatching point. GLUT2 is not evident at fertilization, is detectable at halfway to hatching, and fully expressed at hatching. GPDH expression was evident from fertilization.

In recent years, there has been a great deal of interest in how growth hormone (GH) transgenesis affects fish physiology. However, the results of these studies are often difficult to interpret because the transgenic and non-transgenic fish had very different environmental/rearing histories. This study used a stable line of size-matched GH Atlantic salmon (Salmo salar) that were reared in a shared tank with controls (at 10 degrees C, for approximately 9 months) to perform a comprehensive examination of the cardiorespiratory physiology of GH transgenic salmon, and serves as a novel test of the theory of symmorphosis. The GH transgenic salmon had a 3.6x faster growth rate, and 21 and 25% higher values for mass-specific routine and standard oxygen consumption (M(O(2))), respectively. However, there was no concurrent increase in their maximum M(O(2)), which resulted in them having an 18% lower metabolic scope and a 9% reduction in critical swimming speed. This decreased metabolic capacity/performance was surprising given that the transgenics had a 29% larger heart with an 18% greater mass-specific maximum in situ cardiac output, a 14% greater post-stress blood haemoglobin concentration, 5-10% higher red muscle and heart aerobic enzyme (citrate synthase or cytochrome oxidase) activities, and twofold higher resting and 1.7x higher post-stress, catecholamine levels. However, gill surface area was the only cardiorespiratory parameter that was not enhanced, and our data suggest that gill oxygen transfer may have been limiting. Overall, this research: (1) shows that there are significant metabolic costs associated with GH transgenesis in this line of Atlantic salmon; (2) provides the first direct evidence that cardiac function is enhanced by GH transgenesis; (3) shows that a universal upregulation of post-smolt (adult) GH transgenic salmon cardiorespiratory physiology, as suggested by symmorphosis, does not occur; and (4) supports the idea that whereas differences in arterial oxygen transport (i.e. cardiac output and blood oxygen carrying capacity) are important determinants of inter-specific differences in aerobicity, diffusion-limited processes must be enhanced to achieve substantial intra-specific improvements in metabolic and swimming performance.

We compared levels of the major organic osmolytes in the muscle of elasmobranchs, including the methylamines trimethylamine oxide (TMAO), betaine and sarcosine as well as the beta-amino acids taurine and beta-alanine, and the activities of enzymes of methylamine synthesis (betaine and TMAO) in species with a wide range of urea contents. Four marine, a euryhaline in freshwater (Dasyatis sabina), and two freshwater species, one that accumulates urea (Himantura signifer) and one that does not (Potamotrygon motoro), were analyzed. Urea contents in muscle ranged from 229-352 micromol g-1 in marine species to 2.0 micromol g-1 in P. motoro. Marine elasmobranchs preferentially accumulate methylamines, possibly to counteract urea effects on macromolecules, whereas the freshwater species with lower urea levels accumulate the beta-amino acid taurine as the major non-urea osmolyte. A strong correlation (r2=0.84, P<0.001) with a slope of 0.40 was found between muscle urea content and the combined total methylamines plus total beta-amino acids, supporting the hypothesis that ;non-urea' osmolytes are specifically maintained at an approximately 2:1 ratio with urea in the muscle of elasmobranchs. All species examined had measurable synthetic capacity for betaine in the liver but only one species had detectable TMAO synthetic capacity. We propose a phylogenetic explanation for the distribution of TMAO synthesis in elasmobranchs and suggest that activation of liver betaine aldehyde dehydrogenase, relative to choline dehydrogenase, coincides with betaine accumulation in elasmobranchs. The latter relationship may be important in maintaining methylamine levels during periods of low dietary TMAO intake for species lacking TMAO synthesis.

Rainbow smelt (Osmerus mordax) accumulate high levels of glycerol in winter that serves as an antifreeze. Fish were subjected to controlled decreases in water temperature and levels of plasma glycerol, liver metabolites and liver enzymes were determined in order to identify control mechanisms for the initiation of glycerol synthesis. In two separate experiments, decreases in temperature from 8 degrees C to 0 degrees C over a period of 10-11 days resulted in increases in plasma glycerol from levels of less than 4 mmol l(-1) to approximate mean levels of 40 (first experiment) and 150 mmol l(-1) (second experiment). In a third experiment, decreases in temperature to -1 degrees C resulted in plasma glycerol levels approaching 500 mmol l(-1). The accumulation of glycerol could be driven in either December or March, thus eliminating decreasing photoperiod as a necessary cue for glycerol accumulation. Glycerol accumulation in plasma was associated with changes in metabolites in liver leading to increases in the mass action ratio across the reactions catalyzed by glycerol-3-phosphate dehydrogenase (GPDH) and glycerol-3-phosphatase (G3Pase). The maximal, in vitro activity of GPDH, increased twofold in association with a sharp increase in plasma glycerol level. The metabolite levels and enzyme activities provide complementary evidence that GPDH is a regulatory site in the low temperature triggered synthesis of glycerol. Indirect evidence, based on calculated rates of in vivo glycerol production by liver, suggests that G3Pase is a potential rate-limiting step. As well, transient increases in glyceraldehyde-3-phosphate dehydrogenase and alanine aminotransferase suggest that these sites are components of a suite of responses, in rainbow smelt liver, induced by low temperature.

A putative glucose transporter, GLUT1, is reported for Atlantic cod Gadus morhua. A combination of RT-PCR, RLM-RACE and genome walking were used to articulate a 4560 bp cDNA (GenBank accession number AY526497). It contains a 149 bp 5' UTR, a 1470 bp open reading frame and a 2941 bp 3' UTR. At the nucleotide level, the cod GLUT1 ORF shares 78.2% sequence identity to human GLUT1 and the deduced amino acid sequence clusters with GLUT1s from rainbow trout and carp. GLUT1 transcript is highly expressed in brain, gill, heart and kidney and expressed to a lower level in at least six other tissues. Expression is evident immediately upon fertilization of eggs. Six hours of hypoxia at 40% DO(2) did not alter expression levels in brain, gill, heart or kidney. The level of expression is not substantially altered in heart during low temperature challenge, although there is a suggestion that colder temperature could lead to lower levels of expression, consistent with the concept that the cold-acclimated heart has a reduced dependence upon glucose as a metabolic fuel. Two months of starvation did not significantly alter the level of expression of GLUT1 in heart. This is in marked contrast to the rat heart where fasting leads to a substantial decrease in GLUT1 levels. Overall, there is a ubiquitous tissue distribution of GLUT1, consistent with other species, and the level of gene expression, especially in heart, is relatively constant over a range of physiological conditions.

Adenosine is a product of adenylate phosphate breakdown that can exert protective effects on tissues during energy limitation. Accumulation of cardiac adenosine under hypoxia is well documented in mammals but has not been shown in fish. Adenosine content was measured in heart and brain tissue from short-horned sculpin Myoxocephalus scorpius L. exposed to acute hypoxia and to graded hypoxia and reoxygenation at 8 degrees C. Cardiorespiratory parameters were recorded along with plasma lactate, K(+), Ca(2+) and Na(+) levels and their relationship to adenosine levels investigated. Sculpin exhibited a large bradycardia during hypoxia, with a concomitant drop in cardiac output that recovers fully with reoxygenation. Ventilation rate also declined with hypoxia, suggesting a depression of activity. Plasma lactate concentration was significantly elevated after 4 h at 2.0 mg l(-1) dissolved oxygen while K(+) levels increased during acute hypoxia. Adenosine levels were maintained in heart under acute and graded hypoxia. Brain levels fluctuated under hypoxia and showed no change with reoxygenation. It is concluded that a depression of cardiac activity in conjunction with an adequate anaerobic metabolism allow sculpin to avoid excessive adenosine accumulation under conditions of moderate hypoxia. Cardiac adenosine levels decreased and plasma K(+) levels and heart rate increased significantly at reoxygenation.

This study investigated the regulation of glucose uptake in Atlantic cod (Gadus morhua) hearts. Isolated hearts were perfused with or without glucose in the medium, under either normoxic or severely hypoxic conditions. Working at basal levels, hearts did not require extracellular glucose to maintain power under aerobic conditions. However, cardiac performance was significantly reduced without exogenous glucose under oxygen-limiting conditions. The addition of the glucose transporter inhibitor cytochalasin B caused hypoxic hearts to fail early, and hearts perfused with a glucose analogue, 2-deoxyglucose (2-DG), increased glucose uptake 3-fold under hypoxia. The uptake of 2-DG was only partially inhibited when cytochalasin B was added to the medium. Isolated ventricle strips were also incubated in the presence of 2-DG and the extracellular marker mannitol. Glucose uptake (glucose transport plus intracellular phosphorylation) was assessed by measuring the initial rate of 2-deoxyglucose-6-phosphate (2-DG-6-P) accumulation. At 1 mmol l(-1) 2-DG, the rate of 2-DG uptake remained linear for 60 min, and 2-DG-6-P, but not free 2-DG, accumulation was increased. The fact that intracellular 2-DG did not increase indicates that glucose transport is the rate-limiting step for glucose utilization in non-stimulated cardiac tissue. Replacement of Na(+) by choline in the incubation medium did not affect 2-DG uptake, providing evidence that Na(+)-coupled glucose transport is absent in cod cardiac tissue. Similar to cytochalasin B, glucose uptake was also inhibited by phloridzin, suggesting that facilitated, carrier-mediated glucose transport occurs in cod hearts. Under the conditions employed in these experiments, it is clear that (1) activation of glucose transport is required to support hypoxic performance, (2) the rate-limiting step for glucose utilization is glucose transport rather than glucose phosphorylation, (3) 2-DG uptake accurately reflects glucose transport activity and (4) glucose uptake in cod hearts does not involve an Na(+)-dependent mechanism.

The influence of ATP-sensitive K+ channels (K(ATP) channels) on cardiac performance during anoxia and reoxygenation was investigated in two species of fish showing different cardiac responses to anoxia. Force production in isometrically contracting ventricular muscle preparations from yellowtail flounder is potentiated at the onset of anoxia, while force immediately declines in Atlantic cod preparations. Glibenclamide, a general K(ATP) blocker, impaired oxygenated force development in yellowtail flounder heart but was without effect on cod preparations. The mitochondrial K(ATP) (mK(ATP))-specific blocker 5-hydroxydecanoic acid (5HD) improved oxygenated force production in yellowtail flounder heart without influencing contractility during anoxia or reoxygenation. The specific mK(ATP) agonist diazoxide preserved resting tension and eliminated anoxic force potentiation in yellowtail flounder heart preparations. Neither 5HD nor diazoxide affected contractility in cod ventricle preparations. Results indicate that K(ATP) channels can modulate contractility in yellowtail flounder heart and are potentially important in cardiac hypoxia survival in this species.

Smelt (Osmerus mordax) were maintained at either ambient water temperature or approximately 5 degrees C and various aspects of their freeze-avoidance response were examined from early winter until early spring. Plasma levels of glycerol, trimethylamine oxide (TMAO) and urea were elevated by December 15 and continued to increase in fish held in ambient conditions. In contrast, fish held under warm conditions exhibited decreased glycerol and urea content in plasma, muscle and liver. Plasma and liver TMAO levels also decreased in these fish while muscle TMAO did not vary from the initial values. The activity of liver enzymes involved with the production of glycerol did not differ significantly between groups and had decreased by the end of the study. Antifreeze protein (AFP) expression increased over the duration of the experiment. In January samples, AFP activity (thermal hysteresis) did not vary significantly between groups but mRNA levels were significantly lower in the smelt held at warm temperatures.

Throughout the life cycle of the rainbow trout (Oncorhynchus mykiss), the heart exhibits periods of enhanced growth. Two such instances are cardiac enlargement associated with sexual maturity in males and heart growth at seasonally low environmental temperatures. Heart growth includes a parallel increase in the number of mitochondria. These natural models of heart growth have been exploited to study protein synthesis directed by the mitochondrial genome. Methods were developed to assess protein synthesis in mitochondria isolated from the heart of rainbow trout. Protein synthesis was assessed by tracking the incorporation of l-[2,6-(3)H]phenylalanine into trichloracetic-acid-precipitable protein. Amino acid incorporation into mitochondrial protein was linear with respect to time and was inhibited by chloramphenicol. Radiolabel was selectively enhanced in molecular mass fractions over the same size range as polypeptides known to be encoded by the mitochondrial genome. Protein synthesis was measured in mitochondria isolated from sexually mature animals and from animals subjected to different thermal regimes. The relative ventricular mass of sexually mature male rainbow trout was significantly greater than that of sexually mature females (0. 104+/-0.004 versus 0.087+/-0.002; mean +/- s.e.m.). Mitochondria isolated from the heart of males synthesized protein at a faster rate than mitochondria isolated from the heart of females (0.22+/-0. 02 versus 0.11+/-0.02 pmol phenylalanine mg(-)(1 )protein min(-)(1)). That is, 'male' mitochondria are inherently predisposed to synthesize protein at faster rates. We speculate that the difference may result from higher levels of mitochondrial RNA in males than in females. Mitochondria isolated from the heart of sexually immature rainbow trout acclimated to 13 degrees C synthesized protein at the same rate at 25 degrees C (0.456+/-0.075 pmolphenylalanine mg(-)(1 )protein min(-)(1)) and 15 degrees C (0.455+/-0.027 pmol phenylalanine mg(-)(1 )protein min(-)(1)). However, the rate of protein synthesis was severely impaired at 5 degrees C (0.125+/-0.02 pmol phenylalanine mg(-)(1 )protein min(-)(1)). Since the rate of state 3 respiration by isolated mitochondria decreased in a linear fashion over the temperature range 25 to 5 degrees C, the rate of mitochondrial protein synthesis is not directly coupled to the rate of respiration. Thermal acclimation to 5 degrees C did not result in positive thermal compensation in either the rate of protein synthesis or the rate of oxygen consumption by isolated mitochondria. In a further series of experiments, total protein synthesis and oxygen consumption were measured in isolated myocytes. The rate of oxygen consumption by myocytes remained constant over the temperature range 25 to 5 degrees C. There was no difference in the rate of total cell protein synthesis between 25 degrees C (1.73+/-0. 29 pmol phenylalanine 10(6 )cells(-)(1 )h(-)(1)) and 15 degrees C (2. 12+/-0.19 pmol phenylalanine 10(6 )cells(-)(1 )h(-)(1)), but at 5 degrees C protein synthesis was substantially impaired to approximately one-sixth of the level observed at 15 degrees C. As such, rates of total cell protein synthesis were not directly coupled to rates of respiration and were curtailed at low temperature. In vitro studies show that mitochondria isolated from the heart of sexually mature male rainbow trout are inherently different from mitochondria isolated from the heart of females such that the former are able to synthesize protein at a faster rate. The rate of mitochondrial protein synthesis does not correlate with the greater than twofold changes in rates of oxygen consumption induced by acute changes in assay temperature, suggesting that protein synthesis is not directly coupled to rates of ATP or GTP synthesis.

The physiological integrity of the in situ perfused heart of the ocean pout was established by its ability to maintain cardiac output (Q) over a range of work loads, and by the dependence of Q upon the filling pressure of the heart. Similar observations have been reported previously for the in situ perfused heart of the sea raven. Physiological levels of extracellular acidosis (pH 7.6/1% CO2 and pH 7.4/2% CO2) significantly depressed cardiac performance in sea raven and ocean pout hearts in situ. Negative chronotropic and inotropic responses were observed. Adrenaline (AD; 10(-7) M) under control conditions (pH 7.9/0.5% CO2) produced a sustained tachycardia. The tachycardia reduced filling time of the ventricle and stroke volume was compromised because of the constant preload to the heart. Consequently, AD produced only an initial, transient increase in stroke volume and Q. Thereafter, stroke volume was reduced in proportion with the increase in heart rate, and Q remained unchanged. The combined challenge of extracellular acidosis and AD demonstrated interactive effects between AD and acidosis in situ. Q and power output were maintained in both species at both levels of extracellular acidosis during the combined challenge. Thus AD alone can maintain (but not improve upon) basal Q during extracellular acidosis. The effects of extracellular acidosis, circulating catecholamines and venous return pressure to the heart are discussed in relation to the regulation of Q following exhaustive exercise.

Common carp (Cyprinus carpio L.), 1 kg body weight, were acclimated for 1-2 months to water temperatures of either 7-8 degrees C (cold-acclimated group) or 23-24 degrees C (warm-acclimated group). Single fast fibres and small bundles of slow fibres were isolated from the myotomal muscles and chemically skinned. Force-velocity (P-V) characteristics were determined at 7 degrees C and 23 degrees C. The contractile properties of carp muscle fibres are dependent on acclimation temperature. In the warm-acclimated group maximum isometric tensions (P0, kN m-2) are 47 +/- 6 and 64 +/- 5 for slow muscle fibres and 76 +/- 10 and 209 +/- 21 for fast muscle fibres at 7 degrees C and 23 degrees C, respectively. Maximum contraction velocities (Vmax, muscle lengths-1), are 0.4 +/- 0.05 and 1.5 +/- 0.1 at 7 degrees C (slow fibres) and 0.6 +/- 0.04 and 1.9 +/- 0.4 at 23 degrees C (fast fibres). All values represent mean +/- S.E. P0 and Vmax at 7 degrees C are around 1.5-2.0 times higher for slow and fast muscle fibres isolated from the cold-acclimated group. Fibres from 7 degrees C-acclimated carp fail to relax completely following maximal activations at 23 degrees C. The resulting Ca-insensitive force component (50-70% P0) is associated with the development of abnormal crossbridge linkages and very slow contraction velocities. Activities of enzymes associated with energy metabolism were determined at a common temperature of 15 degrees C. Marker enzymes of the electron transport system (cytochrome oxidase), citric acid cycle (citrate synthase), fatty acid metabolism (carnitine palmitoyl transferase, beta-hydroxyacyl CoA dehydrogenase) and aerobic glucose utilization (hexokinase) have 30-60% higher activities in slow muscle from cold-acclimated than from warm-acclimated fish. Activities of cytochrome oxidase and citrate synthase in fast muscle are also elevated following acclimation to low temperature. It is concluded that thermal compensation of mechanical power output by carp skeletal muscle is matched by a concomitant increase in the potential to supply aerobically-generated ATP at low temperatures.

Cardiac myoglobin plays a role in oxygen consumption and has a protective effect during periods of hypoxia, but little is known about the role of myoglobin during periods of ischaemia. Myoglobin-rich sea raven hearts and myoglobin-poor ocean pout hearts were isolated and perfused at varying flow rates and under conditions of low and high oxygen demand to assess the role of myoglobin in oxygen extraction. In the myoglobin-rich hearts, oxygen extraction remained constant over the flow range. In the myoglobin-poor hearts, oxygen extraction was significantly elevated, relative to controls, at the lower flow rates but decreased as the flow rate increased. In hearts where myoglobin was inactivated by an oxidizing agent, oxygen extraction was similar to that observed in myoglobin-poor hearts. Under conditions of high oxygen demand, myoglobin-rich hearts again showed a constant oxygen extraction over the flow range. Myoglobin-inactivated hearts had a significantly elevated oxygen extraction at low flows, and this decreased as flow rate increased. These data suggest that myoglobin renders oxygen extraction by fish hearts independent of the rate of perfusion.