Responses to hypoxia and recovery: Repayment of oxygen debt is not associated with compensatory protein synthesis in the Amazonian cichlid, Astronotus ocellatus

Oxygen consumption, as an indicator of routine metabolic rate (RoMR), and tissue-specific changes in protein synthesis, as measured by 3H-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°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

Enzymes of cardiac energy metabolism in Amazonian teleosts and the fresh-water stingray (Potamotrygon hystrix)

The maximal in vitro activity of key enzymes of energy metabolism was determined in heart from three Amazonian teleosts, matrinchã (Brycon cephalus), acará açu (Astronotus ocellatus), and tambaqui (Collossoma macropomum), as well as an elasmobranch, the fresh-water stingray (Potamotrygon hystrix). All species are obligate water breathers. Hearts of Amazonian teleosts have activity levels of the glycolytic enzymes hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), and lactate dehydrogenase (LDH) similar to north temperate and Antarctic species when comparisons are made within the usual body temperature range. In contrast, activity level of enzymes required for aerobic oxidation of fatty acids, citrate synthase (CS), carnitine palmitoyl transferase (CPT), and 3-hydroxyacyl CoA dehydrogenase (HOAD) were all substantially lower in the Amazonian teleosts compared to other teleosts. The enzyme profile suggests that 1) activity levels of enzymes of carbohydrate metabolism are conserved over a wide range of body temperatures, and 2) Amazonian teleosts have a much greater reliance upon anaerobic metabolism from glucose than aerobic metabolism to sustain energy production. The heart of fresh-water stingray has high levels of CS, HK, PFK, and PK, implying an aerobic metabolism which is glucose based. In contrast to marine elasmobranchs, the fresh-water stingray has detectable levels of CPT and HOAD, suggestive of a capacity for low-level fatty acid catabolism. As such, the inability of muscle of marine elasmobranchs to utilize fatty acids as an energy source may not be a common feature of all elasmobranchs. © 1996 Wiley-Liss, Inc

Osmotic pressure-adaptive responses in the eye tissues of rainbow smelt (Osmerus mordax)

Purpose: The rainbow smelt (Osmerus mordax), is a teleost fish, which avoids freezing by becoming virtually isosmotic with seawater. The effects that such massive changes in osmolarity have on both its visual system and its highly evolved and specialized circulation are not known. New knowledge about the osmotic adaptation of the rainbow smelt eye is highly relevant to the adaptation and survival of this species and to its ability to feed as a visual predator in the face of environmental pressures. Moreover, the molecular physiologic response of the smelt to osmotic stress might provide valuable insights into understanding and managing mammalian pathological hyperosmolarity conditions, such as diabetes. We undertook the present study to provide an initial assessment of gene expression in ocular vasculature during osmotic adaptation in rainbow smelt. Methods: Immunohistochemistry with species cross reactive antibodies was used to assess blood vessel protein expression in paraffin sections. Western blotting was used to further verify antibody specificity for orthologs of mammalian blood vessel proteins in rainbow smelt. Thermal hysteresis and the analysis of glycerol concentrations in vitreous fluid were used to assess the physiologic adaptive properties of cold stressed eyes. Results: Glycerol levels and osmotic pressure were significantly increased in the vitreal fluid of smelt maintained at <0.5 °C versus those maintained at 8–10 °C. Compared to the 8–10 °C adapted specimens, the rete mirabile blood vessels and connecting regions of the endothelial linings of the choroidal vessels of the <0.5 °C adapted specimens showed a higher expression level of Tubedown (Tbdn) protein, a marker of the endothelial transcellular permeability pathway. Expression of the zonula occludens protein ZO-1, a marker of the endothelial paracellular permeability pathway showed a reciprocal expression pattern and was downregulated in rete mirabile blood vessels and connecting regions in the endothelial linings of choroidal vessels in <0.5 °C adapted specimens. Smelt orthologs of the mammalian Tbdn and zoluna occludens protein 1 (ZO-1) proteins were also detected by western blotting using anti-mammalian antibodies raised against the same epitopes as those used for immunohistochemistry. Conclusions: This work provides the first evidence that molecules known to play a role in ocular vascular homeostasis are expressed and may be differentially regulated during anti-freezing cold adaptation in smelt eyes. We propose a hypothesis that in a state of cold-induced hyperosmolarity, changes in ZO-1 expression are associated with the passage of small solutes from the plasma space to ocular fluid, while changes in Tbdn expression regulate the passage of proteins between the ocular fluid and plasma space. This work also provides fundamental insight into the mechanisms underlying the adaptation of the blood-retinal barrier to metabolically relevant compounds such as glycerol

Anoxic cardiac performance in amazonian and north-temperate-zone teleosts

Amazonian fish face periodic conditions of hypoxic water not commonly encountered by most other species. We examined the impact of simulated anoxia on cardiac performance of Amazonian species relative to north-temperate-zone species to assess whether the ability to maintain anoxic heart performance is related to either respiratory strategy or geographic location. Four Amazonian species (acara-acu (Astronotus ocellatus), acari-bodo (Lipossarcus pardalis), tambaqui (Colossoma macropomum), and tamoata (Hoplosternum littorale)) and four north-temperate-zone species (American eel (Anguilla rostrata), bullhead (Ictalurus punctatus), yellow perch (Perca flavescens), and rainbow trout (Oncorhynchus mykiss)) were studied. The Amazonian species display a range of respiratory modes from obligate gill-breather to facultative air-breather. The performance of isolated ventricular strips under conditions of impaired oxidative phosphorylation was monitored under the same conditions of temperature (25°C) and contraction frequency (30 bpm) for all species. In all cases, ventricular strips showed a decrease in the force of contraction with sodium cyanide (NaCN) treatment. Ventricular strips from two Amazonian and one north-temperate-zone species showed signs of recovery from NaCN treatment. There was considerable diversity in the ability of ventricular strips to develop force under conditions of impaired oxidative phosphorylation that was not related to the ability to obtain oxygen from the air or to geographic location. However, there was a major difference between Amazonian and north-temperate-zone species in the ability of ventricular strips to maintain resting tension under NaCN treatment. Resting tension increased under conditions of simulated anoxia in ventricular strips from all north-temperate-zone species, but there was no change in resting tension in ventricular strips from Amazonian species. We propose that hearts from the Amazonian species are better able to reduce cytoplasmic calcium levels, possibly through a more highly developed sarcoplasmic reticulum

Cloning of GLUT3 cDNA from Atlantic cod (Gadus morhua) and expression of GLUT1 and GLUT3 in response to hypoxia

A putative facilitative glucose transporter, GLUT3, cDNA was cloned from Atlantic cod. It is ubiquitously expressed, with substantial levels in kidney. The 519 aa protein has the highest sequence identity (66.3%) to grass carp GLUT3. Atlantic cod were exposed to a hypoxic challenge (45% DO2) for 24 h and the effects on GLUT1 and GLUT3 expression assessed. GLUT1 expression in gill is upregulated; however, in spleen, there is a significant decrease in both GLUT1 and GLUT3 expression. The increase in GLUT1 mRNA is considered to be associated with an increased energy demand on gill, whereas, the decrease in gene expression in spleen potentially reflects a general decrease in rates of transcription.Peer reviewed: YesNRC publication: Ye

The freeze-avoidance response of smelt Osmerus mordax : initiation and subsequent suppression of glycerol, trimethylamine oxide and urea accumulation

Smelt (Osmerus mordax) were maintained at either ambient water temperature or approximately 5\ub0C 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.Peer reviewed: YesNRC publication: Ye

Changes in ventilation, metabolism, and behaviour, but not bradycardia, contribute to hypoxia survival in two species of Amazonian armoured catfish

Amazonian armoured catfishes exhibit substantial cardiac hypoxia tolerance, but little is known concerning organismal cardiorespiratory, metabolic, and behavioural responses to low oxygen levels. This study assessed the general mechanisms used by two species of armoured catfish, Glyptoperichthyes gibbceps and Liposarcus pardalis, to survive the frequent periods of hypoxia encountered in the Amazon River. The gill ventilation rate (fv) and heart rate (fh) were studied under controlled hypoxia in aquaria and under natural hypoxia in a simulated pond. Glyptoperichthyes gibbceps were fitted with radiotelemetry tags and held in field cages to study their habits of depth selection and air breathing. When denied aerial respiration under hypoxia in aquaria, G. gibbceps increased fv, but neither they nor L. pardalis exhibited alterations in fh. An increase in fv was initially observed in G. gibbceps during pond hypoxia before aerial respiration was initiated and fv declined. Glyptoperichthyes gibbceps were hyperglycaemic under normoxia, and extremely large increases in plasma glucose and lactate concentrations were observed under hypoxia. Field studies confirmed their nocturnal behaviour and showed that air breathing increased at night, regardless of dissolved oxygen concentration. Our results show that armoured catfishes preferentially up-regulate fv and anaerobic metabolism and exhibit no bradycardia during hypoxia

Carbohydrate management, anaerobic metabolism, and adenosine levels in the armoured catfish, Liposarcus pardalis (Castelnau), during hypoxia

The armoured catfish, Liposarcus pardalis, tolerates severe hypoxia at high temperatures. Although this species can breathe air, it also has a strong anaerobic metabolism. We assessed tissue to plasma glucose ratios and glycogen and lactate in a number of tissues under "natural" pond hypoxia, and severe aquarium hypoxia without aerial respiration. Armour lactate content and adenosine in brain and heart were also investigated. During normoxia, tissue to plasma glucose ratios in gill, brain, and heart were close to one. Hypoxia increased plasma glucose and decreased tissue to plasma ratios to less than one, suggesting glucose phosphorylation is activated more than uptake. High normoxic white muscle glucose relative to plasma suggests gluconeogenesis or active glucose uptake. Excess muscle glucose may serve as a metabolic reserve since hypoxia decreased muscle to plasma glucose ratios. Mild pond hypoxia changed glucose management in the absence of lactate accumulation. Lactate was elevated in all tissues except armour following aquarium hypoxia; however, confinement in aquaria increased armour lactate, even under normoxia. A stress-associated acidosis may contribute to armour lactate sequestration. High plasma lactate levels were associated with brain adenosine accumulation. An increase in heart adenosine was triggered by confinement in aquaria, although not by hypoxia alone. © 2006 Wiley-Liss, Inc

Mitochondrial KATP channels and sarcoplasmic reticulum influence cardiac force development under anoxia in the Amazonian armored catfish Liposarcus pardalis

The contribution of alterations in mitochondrial KATP channel activity and the sarcoplasmic reticulum (SR) to anaerobic cardiac function in the anoxia tolerant armored catfish Liposarcus pardalis were assessed. KATP channels contribute to hypoxic cardioprotection in mammals, but little is known of their action in more hypoxia tolerant animals. Anoxia resulted in a decrease in force in isometrically contracting ventricle strips to approximately 40% of the pre-anoxic level. This was maintained for at least 2 h. Upon reoxygenation, hearts recovered to the same level as control preparations. Treatment with 5-hydroxydecanoic acid (5HD), a specific mitochondrial KATP blocker significantly increased force in preparations during anoxia and caused hypercontracture at reoxygenation. Ryanodine, a specific inhibitor of SR function, significantly increased force loss in ventricle preparations under anoxia. Results show that mitochondrial KATP channel activity and SR function are important in anaerobic and post-anaerobic contractility in armored catfish heart. © 2002 Elsevier Science Inc. All rights reserved

Acclimation to hypercarbia protects cardiac contractility and alters tissue carbohydrate metabolism in the Amazonian armored catfish Pterygoplichthys pardalis

The armored catfish Pterygoplichthys pardalis tolerates environmental hypercarbia, high partial pressures of CO2 (PCO2), by preferentially protecting intracellular pH (pHi) in the face of extracellular acidosis. This response is associated with ionic changes which may disrupt contractility in cardiac muscle, and it is not known whether acclimation to hypercarbia provides protection against these changes. We studied the influence of different PCO2 acclimation histories on cardiac muscle function using isometrically contracting ventricular strip preparations. Fish were held for &gt;4 months at 21 mmHg PCO2 and then exposed to normocarbia (6 mmHg PCO2) for either 15 h or 5–6 days. Acclimation to chronic hypercarbia eliminated the negative inotropic effects of in vitro hypercarbia, decreased extracellular Ca2+ sensitivity, and reduced maximum pacing frequency in ventricular strip preparations. Fish acclimated to chronic hypercarbia also exhibited hepatic glycogen and plasma glucose accumulation, and lower plasma lactate levels compared to fish acclimated to normocarbia for 5–6 days. We suggest chronic hypercarbia may induce cardiac remodeling to protect contractility and reduce the energetic demands of pHi regulation. The activation of HCO3 − synthesis pathways may decrease glucose utilization and enhance carbohydrate stores, potentially providing protection against hypoxia, a stressor frequently encountered in conjunction with hypercarbia in the Amazon. © 2015, Springer International Publishing Switzerland