A deficiency in hydrogen sulfide has been implicated in the development and progression of diabetic chronic kidney disease. The purpose of this study was to determine the effect of diabetes on the H2S system in early-stage diabetic kidney disease. We characterised gene and protein expression profile of the enzymes that regulate H2S production and degradation, and H2S production capacity, in the kidney from 10-week-old C57BL6Jdb/db mice (n = 6), in age-matched heterozygous controls (n = 7), and in primary endothelial cells (HUVECs) exposed to high glucose. In db/db mice, renal H2S levels were significantly reduced (P = 0.009). Protein expression of the H2S production enzymes was differentially affected by diabetes: cystathionine β-synthase (CBS) was significantly lower in both db/db mice and high glucose-treated HUVECs (P < 0.0001; P = 0.0318) whereas 3-mercatopyruvate sulfurtransferase (3-MST) expression was higher in the db/db kidney (P < 0.0001), yet lower in the HUVECs (P = 0.0001). Diabetes had no effect on the expression of cystathionine γ-lyase (CSE) in the db/db kidney (P = ns) but was associated with reduced expression in the HUVECs (P = 0.0004). Protein expression of degradation enzyme sulfide quinone reductase (SQOR) was significantly higher in db/db kidney (P = 0.048) and lower in the high glucose-treated HUVECs (P = 0.008). Immunofluorescence studies revealed differential localisation of the H2S enzymes in the kidney, including both tubular and vascular localisation, suggestive of functionally distinct actions in the kidney. The results of this study provide foundational knowledge for future research looking at the H2S system in both kidney physiology and the aetiology of chronic diabetic kidney disease.

Aberrant extracellular matrix synthesis and remodeling contributes to muscle degeneration and weakness in Duchenne muscular dystrophy (DMD). ADAMTS-5, a secreted metalloproteinase with catalytic activity against versican, is implicated in myogenesis and inflammation. Here, using the mdx mouse model of DMD, we report increased ADAMTS-5 expression in dystrophic hindlimb muscles, localized to regions of regeneration and inflammation. To investigate the pathophysiological significance of this, 4-week-old mdx mice were treated with an ADAMTS-5 monoclonal antibody (mAb) or IgG2c (IgG) isotype control for 3 weeks. ADAMTS-5 mAb treatment did not reduce versican processing, as protein levels of the cleaved versikine fragment did not differ between hindlimb muscles from ADAMTS-5 mAb or IgG treated mdx mice. Nonetheless, ADAMTS-5 blockade improved ex vivo strength of isolated fast extensor digitorum longus, but not slow soleus, muscles. The underpinning mechanism may include modulation of regenerative myogenesis, as ADAMTS-5 blockade reduced the number of recently repaired desmin positive myofibers without affecting the number of desmin positive muscle progenitor cells. Treatment with the ADAMTS-5 mAb did not significantly affect markers of muscle damage, inflammation, nor fiber size. Altogether, the positive effects of ADAMTS-5 blockade in dystrophic muscles are fiber-type-specific and independent of versican processing.

The authors wish to make a change to this published paper [...]

Selenoprotein S (Seps1) is an endoplasmic reticulum (ER) resident antioxidant implicated in ER stress and inflammation. In human vastus lateralis and mouse hindlimb muscles, Seps1 localization and expression were fiber-type specific. In male Seps1(+/-) heterozygous mice, spontaneous physical activity was reduced compared with wild-type littermates (d = 1.10, P = 0.029). A similar trend was also observed in Seps1(-/-) knockout mice (d = 1.12, P = 0.051). Whole body metabolism, body composition, extensor digitorum longus (EDL), and soleus mass and myofiber diameter were unaffected by genotype. However, in isolated fast EDL muscles from Seps1(-/-) knockout mice, the force frequency curve (FFC; 1-120 Hz) was shifted downward versus EDL muscles from wild-type littermates (d = 0.55, P = 0.002), suggestive of reduced strength. During 4 min of intermittent, submaximal (60 Hz) stimulation, the genetic deletion or reduction of Seps1 decreased EDL force production (d = 0.52, P < 0.001). Furthermore, at the start of the intermittent stimulation protocol, when compared with the 60-Hz stimulation of the FFC, EDL muscles from Seps1(-/-) knockout or Seps1(+/-) heterozygous mice produced 10% less force than those from wild-type littermates (d = 0.31, P < 0.001 and d = 0.39, P = 0.015). This functional impairment was associated with reduced mRNA transcript abundance of thioredoxin-1 (Trx1), thioredoxin interacting protein (Txnip), and the ER stress markers Chop and Grp94, whereas, in slow soleus muscles, Seps1 deletion did not compromise contractile function and Trx1 (d = 1.38, P = 0.012) and Txnip (d = 1.27, P = 0.025) gene expression was increased. Seps1 is a novel regulator of contractile function and cellular stress responses in fast-twitch muscles.

Light environments critically impact species that rely on vision to survive and reproduce. Animal visual systems must accommodate changes in light that occur from minutes to years, yet the mechanistic basis of their response to spectral (color) changes is largely unknown. Here, we used a laboratory experiment where replicate guppy populations were kept under three different light environments for up to 8-12 generations to explore possible differences in the expression levels of nine guppy opsin genes. Previous evidence for opsin expression-light environment "tuning" has been either correlative or focused exclusively on the relationship between the light environment and opsin expression over one or two generations. In our multigeneration experiment, the relative expression levels of nine different guppy opsin genes responded differently to light environment changes: some did not respond, while others differed due to phenotypic plasticity. Moreover, for the LWS-1 opsin we found that, while we observed a wide range of plastic responses under different light conditions, common plastic responses (where the population replicates all followed the same trajectory) occurred only after multigenerational exposure to different light environments. Taken together this suggests that opsin expression plasticity plays an important role in light environment "tuning" in different light environments on different time scales, and, in turn, has important implications for both visual system function and evolution.

Nitrite ([Formula: see text]) causes vasodilation in mammals due to the formation of (nitric oxide) NO by endogenous [Formula: see text] reduction in the vascular wall. In this study, we determined if a similar mechanism operates in amphibians. Dual-wire myography of the iliac artery from Rhinella marina showed that applied [Formula: see text] caused a concentration-dependent vasodilation in normoxia (21% O2; EC50: 438 µM). Hypoxia (0.63% O2) significantly increased the maximal dilation to [Formula: see text] by 5% ( P = 0.0398). The addition of oxyhemoglobin significantly increased the EC50 ( P = 0.0144; EC50: 2,236 µM) but did not affect the maximal vasodilation. In contrast, partially deoxygenated hemoglobin (90% desaturation) did not affect the EC50 ( P = 0.1189) but significantly ( P = 0.0012) increased the maximal dilation to [Formula: see text] by 11%. The soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) completely abolished the response to [Formula: see text] ( P < 0.0001), and of the nitric oxide synthase inhibitors, only N5-(1-imino-3-butenyl)-l-ornithine (vinyl-l-NIO; P = 0.0028) significantly reduced the [Formula: see text] vasodilation. The xanthine oxidoreductase inhibitor allopurinol ( P = 0.927), the nitric oxide-scavenger 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide (C-PTIO; P = 0.478), and disruption of the endothelium ( P = 0.094) did not affect the [Formula: see text] vasodilation. Incubation of iliac arteries with 1 mM [Formula: see text] did not a cause a change in the cGMP concentration (P = 0.407). Plasma [Formula: see text] was found to be 0.86 ± 0.20 µmol/l, while nitrate ([Formula: see text]) was 19.55 ± 2.55 µmol/l. Both cygb and ngb mRNAs were expressed in the iliac artery, and it is possible that these globins facilitate [Formula: see text] reduction in hypoxia. In addition, [Formula: see text] intracellular disproportionation processes could be important in the generation of NO from [Formula: see text].

The discovery of hydrogen sulfide (H 2 S) as an important biological signalling molecule has resulted in the elucidation of many complex biochemical and physiological functions of the molecule. One of its key physiological functions is the modulation of vascular tone. Our lab has shown potent effects of hypoxia on blood vessels from a wide range of craniates that is mimicked almost exactly by the application of exogenous H 2 S. Based on these data, we propose that H 2 S is a key transducer of hypoxia signalling and may be the elusive “oxygen sensor”. However, we hypothesise that some of the responses attributed to H 2 S may in fact be due to polysulfides (S n ) which form spontaneously upon dissolution. We hypothesised that vascular responses to H 2 S and polysulfides would mimic hypoxia responses in vascular smooth muscle. In the current study, we aimed to test this hypothesis by examining the effects of H 2 S, the mitochondrial H 2 S donor AP39 and polysulfides on isolated bovine pulmonary arteries and porcine coronary arteries. Paired vascular segments (with or without endothelium) were mounted inside Radnotti organ baths filled with Krebs‐Hanseleit Ringer (21% O 2 /5% CO 2 ; 37°C). Vascular responses were recorded in response to hypoxia (1%) or cumulative doses of H 2 S (Na 2 S), K 2 Sn, H 2 S 2 , H 2 S 3 , H 2 S 4 and AP39. Hypoxia caused a rapid and potent two‐phase constriction (50% of max) followed by dilation (25% of Max) of bovine pulmonary arteries. In contrast, hypoxia caused a small transient constriction (10% of max) followed by sustained dilation (30% of max) of porcine coronary arteries. Consistent with the effects of exogenous H 2 S application, K 2 S n , H 2 S 2 , H 2 S 3 , H 2 S 4 caused a constriction‐dilation in bovine pulmonary arteries of a similar magnitude to hypoxia responses. In contrast, AP39 caused a potent dilation only of pulmonary arteries. Application of all sulfides dilated porcine coronary arteries in a similar manner to hypoxia with responses also of a similar magnitude. Removal of the endothelium did not affect vascular responses in any preparation (P>0.05, F‐test). The current data support the hypothesis that H 2 S, and possibly polysulfides, act as an “oxygen‐sensor” in oxygen‐sensitive vessels since the physiological responses observed to hypoxia were mimicked by sulfide treatments. The data also suggest that polysulfides may be responsible for vasoactivity in response to exogenous H 2 S application. Future work will elucidate intracellular signalling events common to hypoxia and sulfide treatments on exposure to both H 2 S and polysulfides.

Hydrogen sulfide (H 2 S) has emerged as an important regulator of physiological processes in multiple organ systems. One of the key functions of H 2 S is the regulation of vasoactivity and consequently blood‐flow. Our lab has shown potent effects of H 2 S and other sulfides on the vasculature of numerous vertebrate species, including vasoconstriction and vasodilation of both systemic and pulmonary arteries. Remarkably we have also shown that these effects are mimicked by hypoxia in every vertebrate class, a response which appears to be endothelium independent. Based on these observations, we hypothesised that changes in gene transcription of H 2 S production and degradation pathway genes by H 2 S and polysulfides would mimic hypoxia responses in vascular smooth muscle. In the current study we aimed to determine the effects of hypoxia and sulfides on H 2 S production ( cse, cth, mpst, got1 and got2 ) and degradation ( sqrdl, suox, tst, cdo1, tstd1 and ethe1 ) pathway genes in vascular smooth muscle using RT‐qPCR. Briefly, cultured primary bovine pulmonary artery smooth muscle cells (BPASMC) were grown until 70% confluent (passage 5) and exposed to either normoxia (21%O 2 /5%CO 2 ) or hypoxia (1%O 2 /5%CO 2 ) for 12 h with or without supplemental cysteine or sulfides (H 2 S, K 2 Sn, H 2 S 2 , H 2 S 4 ) and the cells harvested for molecular analysis. Changes in gene expression in response to hypoxia were mimicked by H 2 S and polysulfides. In general, the presence of cysteine potentiated the responses to hypoxia. In the presence of cysteine, hypoxia caused a downregulation of mRNA transcript abundance of cbs and cth (P<0.001 ANOVA). In a similar way, H 2 S and H 2 S 2 treatments resulted in a downregulation of cbs . In contrast, cth and mpst were unaffected by any treatment, although the intermediaries got1 and got2 were downregulated in both hypoxia and sulfide treated BPASMC. Degradation pathway genes ( sqrdl, suox, tst ) were downregulated in response to both hypoxia and sulfides, while cdo1 was upregulated in cysteine treatments (P<0.05 ANOVA). The housekeeping gene rn28s was not affected by any treatment. We conclude that the current data support our hypothesis that hypoxia and sulfides initiate gene transcription responses which mimic each other. Future work will include examining changes in H 2 S production and degradation pathway gene expression over an acute time‐course during hypoxia.

In Duchenne muscular dystrophy (DMD), a dysregulated extracellular matrix (ECM) directly exacerbates pathology. Glucocorticoids are beneficial therapeutics in DMD, and have pleiotropic effects on the composition and processing of ECM proteins in other biological contexts. The synthesis and remodelling of a transitional versican-rich matrix is necessary for myogenesis; whether glucocorticoids modulate this transitional matrix is not known. Here, versican expression and processing were examined in hindlimb and diaphragm muscles from mdx dystrophin-deficient mice and C57BL/10 wild type mice. V0/V1 versican (Vcan) mRNA transcripts and protein levels were upregulated in dystrophic compared to wild type muscles, especially in the more severely affected mdx diaphragm. Processed versican (versikine) was detected in wild type and dystrophic muscles, and immunoreactivity was highly associated with newly regenerated myofibres. Glucocorticoids enhanced C2C12 myoblast fusion by modulating the expression of genes regulating transitional matrix synthesis and processing. Specifically, Tgfβ1, Vcan and hyaluronan synthase-2 (Has2) mRNA transcripts were decreased by 50% and Adamts1 mRNA transcripts were increased three-fold by glucocorticoid treatment. The addition of exogenous versican impaired myoblast fusion, whilst glucocorticoids alleviated this inhibition in fusion. In dystrophic mdx muscles, versican upregulation correlated with pathology. We propose that versican is a novel and relevant target gene in DMD, given its suppression by glucocorticoids and that in excess it impairs myoblast fusion, a process key for muscle regeneration.

H YDROGEN sulfide (H2S) is a potentially toxic gas with an obnoxious smell. It is a common cause of gas–related fatalities and is notorious for its use to commit suicide. Despite these properties, scientists have readily embraced H2S ever since its discovery as the third gaseous signaling molecule after carbon monoxide and nitric oxide. Exogenously H2S or H2S-donating compounds were reported to yield beneficial effects in numerous biologic systems. As such, H2S has been quickly targeted for its therapeutic potential in a variety of diseases. However, concerns of toxicity remain. In particular, pulmonary toxicity is reported, underlining the need for a thorough analysis of this compound. In this issue of ANESTHESIOLOGY, Francis RC et al. shed light on this matter by comparing the route of administration of H2S in a murine model of ventilator-induced lung injury (VILI). Although bolus injection of H2S-donating compound sodium sulfide exerted a protective effect, inhalation of high doses of H2S was found to aggravate lung injury. The mechanism of sodium sulfide-induced protection included a favorable balance in the antioxidant levels by up-regulation of genes involved in this process. Presumably, toxicity of H2S is because of inhibition of the respiratory complex in mitochondria, resulting in an inability of cells to use oxygen for oxidative metabolism. The authors hypothesize that because hypoxia (low arterial oxygen tension) is associated with pulmonary vasoconstriction and pulmonary edema, this may have been the mechanism underlying the detrimental effect of inhaling H2S in VILI. However, in their study there was no difference in arterial oxygenation between animals treated with a high dose of H2S compared with controls, while the fraction of inspired oxygen was unchanged. There may have been “functional hypoxia” after inhalation of H2S, i.e., an inability to use available oxygen, but to our knowledge, there is no known association between cytopathic hypoxia and pulmonary vasoconstriction. In addition, beneficial effects of H2S inhalation on lung injury have previously been found in another model of VILI. Thereby, the question remains what the mechanism is of the observed toxicity of inhaled H2S in the present study. The dose is likely to be of importance. The toxicity of H2S is correlated with its concentration and depends less upon the duration of exposure, suggesting that H2S is rapidly eliminated, with concomitant low levels of free H2S. This begs the questions as to what “physiologic” concentrations of H2S are. Measuring H2S concentrations is difficult, however, as the various techniques measure not only the biologic active form but also its intermediates. An explanation for discrepant results of routes of administration on toxicity could be that parenteral H2S is metabolized and exhaled within seconds, whereas continuous inhalation may expose the alveoli to high H2S concentrations. Also, the murine nasal cavity has an enormous surface area, thereby enabling deposition efficiency of inhaled H2S in the nose. Mechanical ventilation via a tracheotomy exposes the lungs to high H2S concentration as compared with spontaneously breathing animals. Of note is that only a high dose of H2S aggravated lung injury, whereas the low dose of inhaled H2S had no effect. This high dose nearly corresponds to a dose that has been shown to induce a “suspended animation like” state, characterized by a reduction of oxygen consumption and a concomitant decrease in body temperature to ambient temperature. In their study, Francis RC et al. notably kept body temperature constant. If H2S exerts protection against VILI via reducing energy expenditure, it can be hypothesized that eliminating oxygen utilization with the use of “hibernating” doses of H2S, while preventing a drop in body temperature that lowers oxygen consumption, can be toxic.

In mammals, nitric oxide (NO) produced by nitric oxide synthase 3 (NOS3) localised in vascular endothelial cells is an important vasodilator but the presence of NOS3 in the endothelium of amphibians has been concluded to be absent, based on physiological studies. In this study, a nos3 cDNA was sequenced from the toad, Rhinella marina. The open reading frame of R. marina nos3 encoded an 1170 amino acid protein that showed 81 % sequence identity to the recently cloned Xenopus tropicalis nos3. Rhinella marina nos3 mRNA was expressed in a range of tissues and in the dorsal aorta and pulmonary, mesenteric, iliac and gastrocnemius arteries. Furthermore, nos3 mRNA was expressed in the aorta of Xenopus laevis and X. tropicalis. Quantitative real-time PCR showed that removal of the endothelium of the lateral aorta of R. marina significantly reduced the expression of nos3 mRNA compared to control aorta with the endothelium intact. However, in situ hybridisation was not able to detect any nos3 mRNA in the dorsal aorta of R. marina. Immunohistochemistry using a homologous R. marina NOS3 antibody showed immunoreactivity (IR) within the basal region of many endothelial cells of the dorsal aorta and iliac artery. NOS3-IR was also observed in the proximal tubules and collecting ducts of the kidney but not within the capillaries of the glomeruli. This is the first study to demonstrate that vascular endothelial cells of an amphibian express NOS3.

Nitric oxide is one of the most important signalling molecules involved in the regulation of physiological function. It first came to prominence when it was discovered that the vascular endothelium of mammals synthesises and releases nitric oxide (NO) to mediate a potent vasodilation. Subsequently, it was shown that NO is synthesised in the endothelium by a specific isoform of nitric oxide synthase (NOS) called NOS3. Following this discovery, it was assumed that an endothelial NO/NOS3 system would be present in all vertebrate blood vessels. This review will discuss the latest genomic, anatomical and physiological evidence which demonstrates that an endothelial NO/NOS3 signalling is not ubiquitous in non-mammalian vertebrates, and that there have been key evolutionary steps that have led to the endothelial NO signalling system being a regulatory system found only in reptiles, birds and mammals. Furthermore, the emerging role of nitrite as an endocrine source of NO for vascular regulation is discussed.

Vertebrate metabolic homeostasis is integrally linked to the maintenance of blood flow, a process arising from a complex interplay between neural and humoral factors. A key effector in this process is nitric oxide (NO). The constitutive nitric oxide synthase (NOS) isoforms, NOS1 and NOS3, are involved in NO production in mammalian blood vessels, but this is not the case in vertebrates in general. Previous research has shown that the NO control of vertebrate blood vessels has undergone a series of key evolutionary steps. For example, NO vasodilation first appears in some teleost fish but is ubiquitous in tetrapods. Interestingly, NOS 1 is expressed in perivascular nerves of many vertebrates including fish. However, the NOS3 gene first appears in amphibians but is not expressed in the vascular endothelium. Furthermore, NOS3 can be identified in reptilian and avian genomes. Thus, the question then arises as to when did the endothelium first express the NOS protein. The aim of this research is determine if reptiles were the first vertebrates to express NOS3 in the vascular endothelium. We have obtained a full clone of a nos3 cDNA and a partial clone of nos1 from the green anole, Anolis carolinensis. The deduced NOS3 protein from the cDNA clone varies from the NOS3 protein predicted from the Anolis genome. We will present in situ hybridisation data using fully homologous constitutive NOS riboprobes and immunohistochemical data using a custom antibody for the A. carolinensis NOS3 protein.

Although spiny rock lobster (Jasus edwardsii) is a wholly sub-littoral species, they show a considerable ability to survive prolonged emersion, a fact exploited during the commercial export of this species. Yet, despite this remarkable hardiness, basic information on how this species responds physiologically to emersion is somewhat lacking. Using flow-through respirometry and electrophysiological techniques, we identified that J. edwardsii undergoes marked physiological changes during rest, emersion and recovery over a broad range of temperatures (3.7–17.8 °C). Under resting conditions, routine metabolic rates (RMR) were 22.57 ± 2.39, 9.69 ± 0.55 and 8.09 ± 0.27 mL O2 h−1, average heart rates (Hr) were 54.72 ± 4.46, 37.68 ± 2.86 and 29.67 ± 0.59 BPM, and ventilation frequencies were 83.71 ± 5.86, 45.34 ± 2.91 and 41.62 ± 0.65 BPM at 15.0, 7.5 and 3.7 °C, respectively. Notably, the surgical implantation of electrodes elevated RMR compared with non-surgical treatments. In surgery and non-surgery groups, Q 10 was calculated to be ca. 3.0. Upon emersion, rate of oxygen consumption and Hr decreased below resting rates in a temperature-dependent manner, but, along with rate of CO2 production, increased steadily during 24-h emersion. Ventilation frequencies upon emersion showed a contrasting response and increased significantly above resting rates. When returned to flow-through sea water for recovery, elevated respiration rates provided clear evidence of an O2 debt, and near-complete recovery was observed after 17 h at both 15.0 and 7.5 °C, but close to no debt was recovered at 3.7 °C. In addition, J. edwardsii was observed to undergo marked diurnal and periodic ventilation cycles, characterised by synchronous changes in RMR, Hr and ventilation frequency.

The key roles the cardiovascular system play in the complex distribution of blood, and consequently oxygen, have been extensively studied in vertebrates. Numerous studies have also revealed the complex and varied ways in which tissues cope with compromised oxygen supply. The links between these two processes are the subject of much current research. This article aims to review how blood supply influences tissue oxygenation and affects metabolism, and how this might have played a role in the evolution of the complex muscle arrangements which characterise vertebrates. Muscle tissue is the greatest proportion of body mass in most vertebrates and undergoes dramatic alterations in metabolism and associated oxygen flux. Special attention is given to the myotome of fishes, in which the partitioning of the fibre types contrasts with the mosaic arrangement of tetrapods. This gives us the opportunity to study pure whole vascularised muscle blocks, rather than single fibres, and further explore the interrelationship between oxygen supply and tissue energetics.

We demonstrate that two commonly used chilled storage temperatures (4.15 and 0.3°C) differ in their effects on whole rested harvested Australasian snapper (Pagrus auratus) stored in refrigerated seawater (RSW) with respect to white muscle biochemistry and skin and fillet colour. White muscle pH decreased from 7.64 to ca. 6.4 over 48h at both temperatures, but remained significantly (P<0.05) elevated at 4.15°C compared to 0.3°C until 24h post-mortem. This corresponded with significantly elevated potential energy and concentrations of ATP, ADP and glycogen. Depletion of these metabolites occurred by 24h at both temperatures and was mirrored by rises in lactate and inosine monophosphate (IMP). At 24h snapper stored at 4.15°C remained in partial rigor, whereas animals at 0.3°C were in firm rigor. After 72h significantly higher inosine and hypoxanthine concentrations were present in the 4.15°C group, demonstrating temperature related mass action. Tissue biochemistry significantly correlated with changes in fillet colour, particularly redness and yellowness, and we propose that fillet colour can be used as a non-destructive indicator of muscle biochemistry. We link these data with muscle ultrastructure and translucency through histology and with use of a novel laser penetration method.

Isoeugenol residues in Australasian snapper (Pagrus auratus) white muscle, blood plasma and seawater were accurately and precisely quantified after extraction with acetonitrile using fluorometric detection (ex. 260 nm, em. 340 nm) without chromatographic separation. Isoeugenol residues in Australasian snapper (P. auratus) muscle tissue following 30 min exposure to 58.2 μmol L−1 isoeugenol (ca. 20 ppm of the aquatic anaesthetic AQUI-S™) reached a maximum of 134.37 ± 8.13 μmol kg−1 (±SEM; n = 6). Blood plasma isoeugenol concentrations following this harvesting regime were 253.2 ± 25.1 μmol L−1. After 7 h recovery, fillet isoeugenol residues reduced to 7.89 ± 1.67 μmol kg−1. Storage of fillets from fish harvested with AQUI-S™ at 3.87 ± 0.54 °C for 5 days resulted in a rate of isoeugenol decay in the fillets of 6.51 ± 1.19 μmol kg−1 day−1. The method reported can be used for measuring isoeugenol residues in food products or to further study the physiological and biological effects of isoeugenol in fish.

We present evidence that oxygen consumption (VO2) is oxygen partial pressure (PO2)dependent in striated muscles and PO2-independent in the vasculature in representatives of three craniate taxa: two teleost fish, a hagfish and a rat. Blood vessel VO2 displayed varying degrees of independence in a PO2 range of 15-95 mmHg, while VO2 by striated muscle tissue slices from all species related linearly to PO2 between 0 and 125 mmHg, despite VO2 rates varying greatly between species and muscle type. In salmon red muscle, lactate concentrations fell in slices incubated at a PO2 of either 30 or 100 mmHg, suggesting aerobic rather than anaerobic metabolism. Consistent with this finding, potential energy, a proxy of ATP turnover, was PO2-dependent. Our data suggest that the reduction in VO2 with falling PO2 results in a decrease in ATP demand, suggesting that the hypoxic signal is sensed and cellular changes effected. Viability and diffusion limitation of the preparations were investigated using salmon cardiac and skeletal muscles. Following the initial PO2 depletion, reoxygenation of the Ringer bathing salmon cardiac muscle resulted in VO2S that was unchanged from the first run. VO2 increased in all muscles uncoupled with p-trifluoromethoxylphenyl-hydrazone (FCCP) and 2,4-dinitrophenol (DNP). Mitochondrial succinate dehydrogenase activity, quantified by reduction of 3-(4,5-dimethylthiazol)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to formazan, was constant over the course of the experiment. These three findings indicate that the tissues remained viable over time and ruled out diffusion-limitation as a constraint on VO2.