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Many species exhibit social living which offers ecological advantages such as increased foraging opportunities, more efficient locomotion and reduced predation risk. Additionally, exposure to multiple individuals of the same species can decrease an individual's stress and metabolic demand, termed social buffering. If disruption to an animal's social structure occurs and prevents social buffering, an elevated metabolic rate and thus ventilation frequency and gill permeability are likely. A potential consequence of this physiological response could be the increased accumulation of toxicants. The objective of this study was to investigate whether inducing social stress in marine threespine stickleback (Gasterosteus aculeatus) through social isolation during a sublethal water-borne copper (Cu) exposure would affect Cu uptake and whether that would translate to differences in behaviour and biochemical functioning. We hypothesized that isolating threespine stickleback during a Cu exposure would increase Cu uptake and sublethal toxicity compared to a grouped exposure. Wild-caught fish were exposed to control, low Cu or high Cu conditions (0 - 150 µg/L of Cu, nominally), either in isolation or groups of six for 96 h. Isolated stickleback travelled three times less distance, took six times longer to consume food and exhibited moderately increased gill sodium-potassium ATPase activity than group exposed fish, with no effect of Cu. Isolated stickleback also demonstrated significantly higher Cu levels in their gill and liver tissue compared to the group exposed fish. However, this Cu distribution was also present within the control fish, which had not been exposed to Cu, suggesting that the social context affects endogenous Cu distribution under stressful conditions. Our results illustrate the differences in physiology and behaviour that can arise when social contexts are manipulated and stress the importance of considering sociality when conducting toxicity tests with social organisms., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The fish gill serves many physiological functions, among which is the excretion of ammonia, the primary nitrogenous waste in most fishes. Although it is the end-product of nitrogen metabolism, ammonia serves many physiological functions including acting as an acid equivalent and as a counter-ion in mechanisms of ion regulation. Our current understanding of the mechanisms of ammonia excretion have been influenced by classic experimental work, clever mechanistic approaches, and modern molecular and genetic techniques. In this review, I will overview the history of the study of ammonia excretion by the gills of fishes, highlighting the important advancements that have shaped this field with a nearly 100-year history. The developmental and evolutionary implications of an ammonia and gill-dominated nitrogen regulation strategy in most fishes will also be discussed. Throughout the review, I point to areas in which more work is needed to push forward this field of research that continues to produce novel insights and discoveries that will undoubtedly shape our overall understanding of fish physiology., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Ammonia excretion in fish excretory epithelia is a complex interplay of multiple membrane transport proteins and mechanisms. Using the model system of zebrafish (Danio rerio) larvae, here we identified three paralogues of a novel ammonia transporter, hippocampus-abundant transcript 1 (DrHiat1), also found in most vertebrates. When functionally expressed in Xenopus laevis oocytes, DrHiat1a and DrHiat1b promoted methylamine uptake in a competitive manner with ammonia. In situ hybridization experiments showed that both transporters were expressed as early as the 4-cell stage in zebrafish embryos and could be identified in most tissues 4 days post-fertilization. Larvae experiencing morpholino-mediated knockdown of DrHiat1b exhibited significantly lower whole-body ammonia excretion rates compared with control larvae. Markedly decreased site-specific total ammonia excretion of up to 85% was observed in both the pharyngeal region (site of developing gills) and the yolk sac (region shown to have the highest NH4+ flux). This study is the first to identify DrHiat1b/DrHIAT1 in particular as an important contributor to ammonia excretion in larval zebrafish. Being evolutionarily conserved, these proteins are likely involved in multiple other general ammonia-handling mechanisms, making them worthy candidates for future studies on nitrogen regulation in fishes and across the animal kingdom., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

We hypothesized that the skin acts as an extrabranchial route for ammonia excretion in adult rainbow trout (Oncorhynchus mykiss) following high environmental ammonia (HEA) exposure. Trunks of control or HEA-exposed trout were perfused with saline containing 0 or 1 mmol l -1 NH 4 + . Cutaneous ammonia excretion rates increased 2.5-fold following HEA exposure, however there was no difference in rates between trunks perfused with 0 or 1 mmol l -1 NH 4 + . The skin is therefore capable of excreting its own ammonia load, but it does not clear circulating ammonia from the plasma., (© 2021 Fisheries Society of the British Isles.)

Alterations in water chemistry can challenge resident fish species. More specifically, chemical changes that disrupt ion balance will negatively affect fish health and impact physiological and ecological performance. However, our understanding of which species and populations are at risk from ionoregulatory disturbances in response to changing freshwater environments is currently unclear. Therefore, we propose a novel framework for incorporating ionoregulatory physiology into conservation management of inland fishes. This framework introduces the concepts of fundamental chemical niche, which is the tolerable range of chemical conditions for a given species based on laboratory experiments, and realized chemical niche, which is the range of chemical conditions in which a species resides based on distribution surveys. By comparing these two niches, populations that may be at risk from ionoregulatory disturbances and thus require additional conservation considerations can be identified. We highlight the potential for commonly measured ionoregulatory traits to predict fundamental and realized chemical niches but caution that some traits may not serve as accurate predictors despite being important for understanding ionoregulatory mechanisms. As a sample application of our framework, the minimum pH distribution (realized niche) and survival limit pH (fundamental niche) of several North American fishes were determined by systematic review and were compared. We demonstrate that ionoregulatory capacity is significantly correlated with a realized niche for many species, highlighting the influence of ionoregulatory physiology on fish distribution patterns along chemical gradients. Our aim is that this framework will stimulate further research in this field and result in a broader integration of physiological data into conservation management decisions for inland waters., (© The Author(s) 2021. Published by Oxford University Press and the Society for Experimental Biology.)

The mechanisms underlying ionoregulation in fishes have been studied for nearly a century, and reductionist methods have been applied at all levels of biological organization in this field of research. The complex nature of ionoregulatory systems in fishes makes them ideally suited to reductionist methods and our collective understanding has been dramatically shaped by their use. This review provides an overview of the broad suite of techniques used to elucidate ionoregulatory mechanisms in fishes, from the whole-animal level down to the gene, discussing some of the advantages and disadvantages of these methods. We provide a roadmap for understanding and appreciating the work that has formed the current models of organismal, endocrine, cellular, molecular, and genetic regulation of ion balance in fishes and highlight the contribution that reductionist techniques have made to some of the fundamental leaps forward in the field throughout its history., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In fishes, branchial cytosolic carbonic anhydrase (CA) plays an important role in ion and acid-base regulation. The Ca17a isoform in zebrafish ( Danio rerio ) is expressed abundantly in Na + -absorbing/H + -secreting H + -ATPase-rich (HR) cells. The present study aimed to identify the role of Ca17a in ion and acid-base regulation across life stages using CRISPR/Cas9 gene editing. However, in preliminary experiments, we established that ca17a knockout is lethal with ca17a -/- mutants exhibiting a significant decrease in survival beginning at ∼12 days postfertilization (dpf) and with no individuals surviving past 19 dpf. Based on these findings, we hypothesized that ca17a -/- mutants would display alterations in ion and acid-base balance and that these physiological disturbances might underlie their early demise. Na + uptake rates were significantly increased by up to 300% in homozygous mutants compared with wild-type individuals at 4 and 9 dpf; however, whole body Na + content remained constant. While Cl - uptake was significantly reduced in ca17a -/- mutants, Cl - content was unaffected. Reduction of CA activity by Ca17a morpholino knockdown or ethoxzolamide treatments similarly reduced Cl - uptake, implicating Ca17a in the mechanism of Cl - uptake by larval zebrafish. H + secretion, O 2 consumption, CO 2 excretion, and ammonia excretion were generally unaltered in ca17a -/- mutants. In conclusion, while the loss of Ca17a caused marked changes in ion uptake rates, providing strong evidence for a Ca17a-dependent Cl - uptake mechanism, the underlying causes of the lethality of this mutation in zebrafish remain unclear.

As a key endocrine axis involved in responding to stress, the hypothalamic-pituitary-interrenal axis plays dual roles in mobilizing energy and maintaining ionic/osmotic balance in fishes. Although these roles have been examined independently in detail in adult fishes, less attention has been paid to the effects of an endogenous stress response during early life, particularly with respect to its potential effects on ionic/osmotic balance. The present study tested the hypothesis that exposure of zebrafish to stress during early development would alter ion balance later in life. Zebrafish at three developmental stages (4, 7, or 15 days post-fertilization, dpf) were subjected to an air-exposure stressor twice a day for 2 days, causing elevation of whole-body cortisol levels. Individuals stressed early in life exhibited decreased survival and growth, altered cortisol responses to a subsequent air-exposure stressor, and increased whole-body Na + and Ca 2+ concentrations. Changes in whole-body Ca 2+ concentrations were accompanied by increased ionocyte abundance at 7 dpf and increased rates of Ca 2+ uptake from the environment. Differences in whole-body ion concentrations at 15 and 35 dpf were not accompanied by altered ion uptake rates. Across all ages examined, air-exposure stress experienced at 7 dpf was particularly effective at eliciting phenotypic changes, suggesting a critical window at this age for a stress response to influence development. These findings demonstrate that early-life stress in zebrafish triggers developmental plasticity, with age-dependent effects on both the cortisol stress axis and ion balance.

Fishes living in fresh water counter the passive loss of salts by actively absorbing ions through specialized cells termed ionocytes. Ionocytes contain ATP-dependent transporters and are enriched with mitochondria; therefore ionic regulation is an energy-consuming process. The purpose of this study was to assess the aerobic costs of ion transport in larval zebrafish ( Danio rerio ). We hypothesized that changes in rates of Na + uptake evoked by acidic or low Na + rearing conditions would result in corresponding changes in whole-body oxygen consumption ( Ṁ O 2 ) and/or cutaneous oxygen flux ( J O 2 ), measured at the ionocyte-expressing yolk sac epithelium using the scanning micro-optrode technique (SMOT). Larvae at 4 days post-fertilization (dpf) that were reared under low pH (pH 4) conditions exhibited a higher rate of Na + uptake compared with fish reared under control conditions (pH 7.6), yet they displayed a lower Ṁ O 2  and no difference in cutaneous J O 2 Despite a higher Na + uptake capacity in larvae reared under low Na + conditions, there were no differences in Ṁ O 2  and J O 2  at 4 dpf. Furthermore, although Na + uptake was nearly abolished in 2 dpf larvae lacking ionocytes after morpholino knockdown of the ionocyte proliferation regulating transcription factor foxi3a , Ṁ O 2   and J O 2  were unaffected. Finally, laser ablation of ionocytes did not affect cutaneous J O 2 Thus, we conclude that the aerobic costs of ion uptake by ionocytes in larval zebrafish, at least in the case of Na + , are below detection using whole-body respirometry or cutaneous SMOT scans, providing evidence that ion regulation in zebrafish larvae incurs a low aerobic cost., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

In zebrafish (Danio rerio), the ammonia-transporting Rhesus glycoprotein Rhcgb is implicated in mechanisms of ammonia excretion and Na + uptake. In particular, Rhcgb is thought to play an important role in maintaining ammonia excretion in response to alkaline conditions and high external ammonia (HEA) exposure, in addition to facilitating Na + uptake via a functional metabolon with the Na + /H + -exchanger Nhe3b, specifically under low Na + conditions. In the present study, we hypothesized that CRISPR/Cas9 knockout of rhcgb would reduce ammonia excretion and Na + uptake capacity, particularly under the conditions listed above that have elicited increases in Rhcgb-mediated ammonia excretion and/or Na + uptake. Contrary to this hypothesis, however, larval and juvenile rhcgb knockout (KO) mutants showed no reductions in ammonia excretion or Na + uptake under any of the conditions tested in our study. In fact, under control conditions, rhcgb KO mutants generally displayed an increase in ammonia excretion, potentially due to increased transcript abundance of another rh gene, rhbg. Under alkaline conditions, rhcgb KO mutants were also able to maintain ammonia excretion, similar to wild-type fish, and stimulation of ammonia excretion after HEA exposure also was not affected by rhcgb KO. Surprisingly, ammonia excretion and Na + uptake were unaffected by rhcgb or nhe3b KO in juvenile zebrafish acclimated to normal (800 μmol/L) or low (10 μmol/L) Na + conditions. These results demonstrate that Rhcgb is expendable for ammonia excretion and Na + uptake in zebrafish, highlighting the plasticity and flexibility of these physiological systems in this species., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020. Published by Elsevier Inc.)

The H + -ATPase-rich (HR) cells of zebrafish larvae are a sub-type of ion-transporting cell located on the yolk sac epithelium that are responsible for Na + uptake and H + extrusion. Current models of HR cell ion transport mechanisms in zebrafish larvae are well established, but little is known about the involvement of the various ion transport pathways in regulating intracellular acid-base status. Here, a ratiometric imaging technique was developed and validated to monitor intracellular pH (pHi) continuously in larval zebrafish HR cells in vivo Gene knockdown or CRISPR/Cas9 knockout approaches were used to evaluate the roles of the two principal apical membrane acid excretory pathways, the Na + /H + exchanger (NHE3b; slc9a3.2 ) and the H + -ATPase ( atpv1aa ). Additionally, the role of HR cell cytosolic carbonic anhydrase (CAc) was investigated because of its presumed role in providing H + for Na + /H + exchange and H + -ATPase. The temporal pattern and extent of intracellular acidification during exposure of fish to 1% CO 2 and the extent of post-CO 2 alkalisation were altered markedly in fish experiencing knockdown/knockout of CAc, NHE3b or H + -ATPase. Although there were slight differences among the three knockdown/knockout experiments, the typical response was a greater degree of intracellular acidification during CO 2 exposure and a reduced capacity to restore pHi to baseline levels post-hypercapnia. The metabolic alkalosis and subsequent acidification associated with 20 mmol l -1 NH 4 Cl exposure and its washout were largely unaffected by gene knockdown. Overall, the results suggest markedly different mechanisms of intracellular acid-base regulation in zebrafish HR cells depending on the nature of the acid-base disturbance., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Freshwater fishes absorb Na + from their dilute environment using ion-transporting cells. In larval zebrafish ( Danio rerio ), Na + uptake is coordinated by (1) Na + /H + exchanger 3b (Nhe3b) and (2) H + -ATPase-powered electrogenic uptake in H + -ATPase-rich (HR) cells and by (3) Na + -Cl - -cotransporter (Ncc) expressed in NCC cells. The present study aimed to better understand the roles of these three proteins in Na + uptake by larval zebrafish under 'normal' (800 µmol l -1 ) and 'low' (10 µmol l -1 ) Na + conditions. We hypothesized that Na + uptake would be reduced by CRISPR/Cas9 knockout (KO) of slc9a3.2 (encoding Nhe3b), particularly in low Na + where Nhe3b is believed to play a dominant role. Contrary to this hypothesis, Na + uptake was sustained in nhe3b KO larvae under both Na + conditions, which led to the exploration of whether compensatory regulation of H + -ATPase or Ncc was responsible for maintaining Na + uptake in nhe3b KO larvae. mRNA expression of the genes encoding H + -ATPase and Ncc was not altered in nhe3b KO larvae. Moreover, morpholino knockdown of H + -ATPase, which significantly reduced H + flux by HR cells, did not reduce Na + uptake in nhe3b KO larvae, nor did rearing larvae in chloride-free conditions, thereby eliminating any driving force for Na + -Cl - -cotransport via Ncc. Finally, simultaneously treating nhe3b KO larvae with H + -ATPase morpholino and chloride-free conditions did not reduce Na + uptake under normal or low Na + These findings highlight the flexibility of the Na + uptake system and demonstrate that Nhe3b is expendable to Na + uptake in zebrafish and that our understanding of Na + uptake mechanisms in this species is incomplete., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Convective water flow across respiratory epithelia in water-breathing organisms maintains transcutaneous oxygen (O 2 ) partial pressure (Po 2 ) gradients that drive O 2 uptake. Following hatch, larval fishes lack a developed gill and the skin is the dominant site of gas transfer, yet few studies have addressed the contribution of convective water flow to cutaneous O 2 uptake in larvae. We hypothesized that the pectoral fins, which can generate water flow across the skin in larvae, promote transcutaneous O 2 transfer and thus aid in O 2 uptake. In zebrafish ( Danio rerio ), the frequency of pectoral fin movements increased in response to hypoxia at 4 days postfertilization (dpf), but the response was blunted by 15 dpf, when the gills become the dominant site of O 2 uptake, and was absent by 21 dpf. In rainbow trout ( Oncorhynchus mykiss ), Po 2 measured at the skin surface of ventilating larvae was lower when the pectoral fins had been surgically removed, directly demonstrating that fins contribute to convective flow that dissipates cutaneous Po 2 boundary layers. Lack of pectoral fins compromised whole animal O 2 consumption in trout during hypoxia, but this effect was absent in zebrafish. Overall, our findings support a respiratory role of the pectoral fins in rainbow trout, but their involvement in zebrafish remains equivocal.

Freshwater (FW) fishes inhabit dilute environments and must actively absorb ions in order to counteract diffusive salt loss. Neuroendocrine control of ion uptake in FW fishes is an important feature of ion homeostasis and several important neuroendocrine factors have been identified. The role of serotonin (5-HT), however, has received less attention despite several studies pointing to a role for 5-HT in the control of ion balance. Here, we used a gene knockout approach to elucidate the role of 5-HT in regulating Na + and Ca 2+ uptake rates in larval zebrafish. Tryptophan hydroxylase (TPH) is the rate-limiting step in 5-HT synthesis and we therefore hypothesized that ion uptake rates would be altered in zebrafish larvae carrying knockout mutations in tph genes. We first examined the effect of tph1b knockout (KO) and found that tph1bKO larvae, obtained from Harvard University, had reduced rates of Na + and Ca 2+ uptake compared to wild-type (WT) larvae from our institution (uOttawa WT), lending support to our hypothesis. However, further experiments controlling for differences in genetic background demonstrated that WT larvae from Harvard University (Harvard WT) had lower ion uptake rates than those of uOttawa WT, and that ion uptake rate between Harvard WT and tph1bKO larvae were not significantly different. Therefore, our initial observation that tph1bKO larvae (Harvard source) had reduced ion uptake rates relative to uOttawa WT was a function of genetic background and not of knockout itself. These data provide a cautionary tale of the importance of controlling for genetic background in gene knockout experiments., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In water-breathing fishes, the hypoxic ventilatory response (HVR) represents an increase in water flow over the gills during exposure to lowered ambient O 2 levels. The HVR is a critical defense mechanism that serves to delay the negative consequences of hypoxia on aerobic respiration. However, the physiological significance of the HVR in larval fishes is unclear as they do not have a fully developed gill and rely primarily on cutaneous gas transfer. Using larval zebrafish (4, 7, 10 and 15 days post-fertilization; dpf), we examined HVR under three levels of hypoxia (25, 45 and 60 mmHg). The larvae exhibited widely different HVRs as a function of developmental age and level of the hypoxia. Yet, critical O 2 tensions ( P crit ) remained constant (30-34 mmHg) over the same period of development. Micro-optrode O 2 sensors were used to measure a significant decrease in buccal cavity water O 2 tensions in 4 and 7 dpf larvae compared with the water they inspired, demonstrating significant extraction of O 2 from the buccal cavity. To assess the physiological significance of the HVR, ventilatory water flow was prevented in larvae at 4 and 7 dpf by embedding their heads in agar. An increase in P crit was observed in larvae at 7 dpf but not 4 dpf, suggesting that buccal ventilation is important for O 2 extraction by 7 dpf. Combined, these data indicate that branchial/buccal gas transfer plays a significant role in O 2 uptake during hypoxia, and supports a physiological benefit of the HVR in early life stages of zebrafish., (© 2019. Published by The Company of Biologists Ltd.)

Loss-of-function technologies, such as morpholino- and RNAi-mediated gene knockdown, and TALEN- and CRISPR/Cas9-mediated gene knockout, are widely used to investigate gene function and its physiological significance. Here, we provide a general overview of the various knockdown and knockout technologies commonly used in comparative physiology and discuss the merits and drawbacks of these technologies with a particular focus on research conducted in zebrafish. Despite their widespread use, there is an ongoing debate surrounding the use of knockdown versus knockout approaches and their potential off-target effects. This debate is primarily fueled by the observations that, in some studies, knockout mutants exhibit phenotypes different from those observed in response to knockdown using morpholinos or RNAi. We discuss the current debate and focus on the discrepancies between knockdown and knockout phenotypes, providing literature and primary data to show that the different phenotypes are not necessarily a direct result of the off-target effects of the knockdown agents used. Nevertheless, given the recent evidence of some knockdown phenotypes being recapitulated in knockout mutants lacking the morpholino or RNAi target, we stress that results of knockdown experiments need to be interpreted with caution. We ultimately argue that knockdown experiments should not be discontinued if proper control experiments are performed, and that with careful interpretation, knockdown approaches remain useful to complement the limitations of knockout studies (e.g. lethality of knockout and compensatory responses)., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Purely diffusive O 2 transport typically is insufficient to sustain aerobic metabolism in most multicellular organisms. In animals that are small enough, however, a high surface-to-volume ratio may allow passive diffusion alone to supply sufficient O 2 transfer. The purpose of this study was to explore the impacts of internal convection on respiratory gas transfer in a small complex organism, the larval zebrafish ( Danio rerio). Specifically, we tested the hypothesis that internal convection is required for the normal transfer of the respiratory gases O 2 and CO 2 and maintenance of resting aerobic metabolic rate in larvae at 4 days postfertilization (dpf). Morpholino knockdown of the vascular endothelial growth factor (VEGF) or cardiac troponin T (TNNT2) proteins allowed an examination of gas transfer in two independent models lacking internal convection. With the use of a scanning micro-optrode technique to measure regional epithelial O 2 fluxes ( Jo 2 ), it was demonstrated that larvae lacking convection exhibited reduced Jo 2 in regions spanning the head to the trunk. Moreover, the acute loss of internal convection caused by heart stoppage resulted in reduced rates of cutaneous Jo 2 , an effect that was reversed upon the restoration of internal convection. With the use of whole body respirometry, it was shown that loss of internal convection was associated with reduced resting rates of O 2 consumption and CO 2 excretion in larvae at 4 dpf. The results of these experiments clearly demonstrate that internal convection is required to maintain resting rates of respiratory gas transfer in larval zebrafish.

Killifish (Fundulus heteroclitus) has been extensively used as a model for ion regulation by euryhaline fishes. Na + and Cl - dynamics have been well studied in killifish, but few studies have addressed that of Ca 2+ . Therefore, this study aimed to characterize Ca 2+ fluxes in freshwater (FW) and seawater (SW)-acclimated killifish, their response to salinity transfer, and to elucidate the mechanisms of Ca 2+ influx in FW and SW. SW killifish displayed a significantly higher Ca 2+ influx rate than that of FW fish, while Ca 2+ efflux rates were comparable in both salinities. Ca 2+ influx was saturable in FW (K m  = 78 ± 19 µmol/L; J max  = 53 ± 3 nmol/g/h) and influx by SW killifish was linear up to 7 mmol/L Ca 2+ . In SW-acclimated fish, 36% of Ca 2+ influx was attributed to "intestinal Ca 2+ intake", likely caused by drinking, whereas intestinal Ca 2+ intake in FW contributed to < 2% of total. Throughout the study, results suggested that "cation competition" in SW modulates Ca 2+ influx. Therefore, we hypothesized that SW-acclimated fish actually have a higher affinity Ca 2+ influx system than FW-acclimated fish but that it is competitively inhibited by competing SW cations. In agreement with this cation competition hypothesis, we demonstrated for the first time that "extra-intestinal" Ca 2+ influx was inhibited by Mg 2+ in both FW and SW-acclimated killifish. Following acute salinity transfer, extra-intestinal Ca 2+ influx was rapidly regulated within 12-24 h, similar to Na + and Cl - . Ca 2+ influx in FW was inhibited by La 3+ , an epithelial Ca 2+ channel blocker, whereas La 3+ had no significant effect in SW.

Na + uptake in larval zebrafish (Danio rerio) is coordinated by three mechanisms: Na + /H + -exchanger 3b (NHE3b) expressed in H + -ATPase-rich (HR) cells, an unidentified Na + channel coupled to electrogenic H + -ATPase expressed in HR cells, and Na + -Cl - -cotransporter (NCC) expressed in NCC cells. Recently, acid-sensing ion channels (ASICs) were proposed to be the putative Na + channel involved in H + -ATPase-mediated Na + uptake in adult zebrafish and rainbow trout. In the present study, we hypothesized that ASICs also play this role in Na + uptake in larval zebrafish. In support of this hypothesis, immunohistochemical analyses revealed that ASIC4b was expressed in HR cells on the yolk sac skin at 4 days post-fertilization (dpf). However, neither treatment with the ASIC-specific blocker 4,6-diamidino-2-phenylindole (DAPI) nor morpholino knockdown of ASIC4b reduced Na + uptake in circumneutral conditions at 4 dpf. However, because ASIC4b knockdown led to significant increases in the mRNA expression of nhe3b and ncc and a significant increase in HR cell density, it is possible that Na + influx was sustained by increased participation of non-ASIC4b pathways. Moreover, when fish were reared in acidic water (pH = 4), ASIC4b knockdown led to a stimulation of Na + uptake at 3 and 4 dpf, results which also were inconsistent with an essential role for ASIC-mediated Na + uptake, even under conditions known to constrain Na + uptake via NHE3b. Thus, while ASIC4b clearly is expressed in HR cells, the current functional experiments cannot confirm its involvement in Na + uptake in larval zebrafish., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Nitrogen metabolism in fishes has been a focus of comparative physiologists for nearly a century. In this Review, we focus specifically on early life stages of fishes, which have received considerable attention in more recent work. Nitrogen metabolism and excretion in early life differs fundamentally from that of juvenile and adult fishes because of (1) the presence of a chorion capsule in embryos that imposes a limitation on effective ammonia excretion, (2) an amino acid-based metabolism that generates a substantial ammonia load, and (3) the lack of a functional gill, which is the primary site of nitrogen excretion in juvenile and adult fishes. Recent findings have shed considerable light on the mechanisms by which these constraints are overcome in early life. Perhaps most importantly, the discovery of Rhesus (Rh) glycoproteins as ammonia transporters and their expression in ion-transporting cells on the skin of larval fishes has transformed our understanding of ammonia excretion by fishes in general. The emergence of larval zebrafish as a model species, together with genetic knockdown techniques, has similarly advanced our understanding of ammonia and urea metabolism and excretion by larval fishes. It has also now been demonstrated that ammonia excretion is one of the primary functions of the developing gill in rainbow trout larvae, leading to new hypotheses regarding the physiological demands driving gill development in larval fishes. Here, we highlight and discuss the dramatic changes in nitrogen handling that occur over early life development in fishes., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Copper (Cu) is a persistent environmental contaminant that elicits several physiological disturbances in aquatic organisms, including a disruption in ammonia regulation. We hypothesized that exposure to Cu in a model crustacean (blue crab, Callinectes sapidus) acclimated to brackish water (2 ppt) would lead to hyperammonemia by stimulating an increase in ammonia production and/or by inhibiting ammonia excretion. We further hypothesized that urea production would represent an ammonia detoxification strategy in response to Cu. In a pilot experiment, exposure to 0, 100, and 200 µg/L Cu for 6 h caused significant concentration-dependent increases in ammonia excretion (J amm ). Based on these results, an acute 24-h 100 µg/L Cu exposure was conducted and this similarly caused an overall stimulation of J amm during the 24-h period, indicative of an increase in ammonia production. Terminal haemolymph total ammonia content (T amm ) was unchanged, suggesting that while ammonia production was increased, there was no inhibition of the excretion mechanism. In support of our second hypothesis, urea excretion (J urea ) increased in response to Cu exposure; haemolymph [urea] was unaffected. This suggested that urea production also was increased. To further test the hypothesis that J urea increased to prevent hyperammonemia during Cu exposure, crabs were exposed to high environmental ammonia (HEA; 2.5 mmol/L NH 4 HCO 3 ) for 12 h in a separate experiment. This led to a fourfold increase in haemolymph T amm , whereas J urea increased only transiently and haemolymph [urea] was unchanged, indicating that urea production likely does not contribute to the attenuation of hyperammonemia in blue crabs. Overall, Cu exposure in blue crabs led to increased ammonia and urea production, which were both eliminated by excretion. These results may have important implications in aquaculture systems where crabs may be exposed to elevated Cu and/or ammonia.

In rainbow trout, the dominant site of Na + uptake ( J Na,in ) and ammonia excretion ( J amm ) shifts from the skin to the gills over development. Post-hatch (PH; 7 days post-hatch) larvae utilize the yolk sac skin for physiological exchange, whereas by complete yolk sac absorption (CYA; 30 days post-hatch), the gill is the dominant site. At the gills, J Na,in and J amm occur via loose Na + /NH 4 + exchange, but this exchange has not been examined in the skin of larval trout. Based on previous work, we hypothesized that, contrary to the gill model, J Na,in by the yolk sac skin of PH trout occurs independently of J amm Following a 12 h exposure to high environmental ammonia (HEA; 0.5 mmol l -1 NH 4 HCO 3 ; 600 µmol l -1 Na + ; pH 8), J amm by the gills of CYA trout and the yolk sac skin of PH larvae, which were isolated using divided chambers, increased significantly. However, this was coupled to an increase in J Na,in across the gills only, supporting our hypothesis. Moreover, gene expression of proteins involved in J Na,in [Na + /H + -exchanger-2 (NHE2) and H + -ATPase] increased in response to HEA only in the CYA gills. We further identified expression of the apical Rhesus (Rh) proteins Rhcg2 in putative pavement cells and Rhcg1 (co-localized with apical NHE2 and NHE3b and Na + /K + -ATPase) in putative peanut lectin agglutinin-positive (PNA + ) ionocytes in gill sections. Similar Na + /K + -ATPase-positive cells expressing Rhcg1 and NHE3b, but not NHE2, were identified in the yolk sac epithelium. Overall, our findings suggest that the mechanisms of J Na,in and J amm by the dominant exchange epithelium at two distinct stages of early development are fundamentally different., (© 2017. Published by The Company of Biologists Ltd.)

The physiological effects of high environmental ammonia (HEA) exposure have been well documented in many aquatic species. In particular, it has recently been demonstrated that exposure to ammonia in fish leads to a similar hyperventilatory response as observed during exposure to hypoxia. In littoral crabs, such as the green crab (Carcinus maenas), exposure to severe hypoxia triggers an emersion response whereby crabs escape hypoxia to breathe air. We hypothesized that exposure to HEA in green crabs would lead to a similar behavioural response which is specific to ammonia. Using an experimental arena containing a rock bed onto which crabs could emerse, we established that exposure to HEA (4mmol/l NH 4 HCO 3 ) for 15min triggers emersion in crabs. In experiments utilizing NaHCO 3 controls and NH 4 HCO 3 injections, we further determined that emersion was triggered specifically by external ammonia and was independent of secondary acid-base or respiratory disturbances caused by HEA. We then hypothesized that emersion from HEA provides a physiological benefit, similar to emersion from hypoxia. Exposure to 15min of HEA without emersion (no rock bed present) caused significant increases in arterial haemolymph total ammonia (T amm ), pH, and [HCO 3 - ] . When emersion was allowed, arterial haemolymph T amm and [HCO 3 - ] increased, but no alkalosis developed. Moreover, emersion decreased haemolymph partial pressure of NH 3 relative to crabs which could not emerse. Overall, we demonstrate a novel behavioural response to HEA exposure in crabs which we propose may share similar mechanistic pathways with the emersion response triggered by hypoxia., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Hagfish skin has been reported as an important site for ammonia excretion and as the major site of systemic oxygen acquisition. However, whether cutaneous O 2 uptake is the dominant route of uptake remains under debate; all evidence supporting this hypothesis has been derived using indirect measurements. Here, we used partitioned chambers and direct measurements of oxygen consumption and ammonia excretion to quantify cutaneous and branchial exchanges in Pacific hagfish (Eptatretus stoutii) at rest and following exhaustive exercise. Hagfish primarily relied on the gills for both O 2 uptake (81.0%) and ammonia excretion (70.7%). Following exercise, both O 2 uptake and ammonia excretion increased, but only across the gill; cutaneous exchange was not increased. When branchial O 2 availability was reduced by exposure to anteriorly localized hypoxia (∼4.6 kPa O 2 ), cutaneous O 2 consumption was only slightly elevated on an absolute basis. These results refute a major role for cutaneous O 2 acquisition in the Pacific hagfish., (© 2016. Published by The Company of Biologists Ltd.)

All teleost fish produce ammonia as a metabolic waste product. In embryos, ammonia excretion is limited by the chorion, and fish must detoxify ammonia by synthesizing urea via the ornithine urea cycle (OUC). Although urea is produced by embryos and larvae, urea excretion (J(urea)) is typically low until yolk sac absorption, increasing thereafter. The aim of this study was to determine the physiological and molecular characteristics of J(urea) by posthatch rainbow trout (Oncorhynchus mykiss). Following hatch, whole body urea concentration decreased over time, while J(urea) increased following yolk sac absorption. From 12 to 40 days posthatch (dph), extra-branchial routes of excretion accounted for the majority of J(urea), while the gills became the dominant site for J(urea) only after 55 dph. This represents the most delayed branchial ontogeny of any process studied to date. Urea transporter (UT) gene expression in the gills and skin increased over development, consistent with increases in branchial and extra-branchial J(urea). Following exposure to 25 mmol/l urea, the accumulation and subsequent elimination of exogenous urea was much greater at 55 dph than 12 dph, consistent with increased UT expression. Notably, UT gene expression in the gills of 55 dph larvae increased in response to high urea. In summary, there is a clear increase in urea transport capacity over posthatch development, despite a decrease in OUC activity., (Copyright © 2016 the American Physiological Society.)

Objective: To identify factors associated with excessive bleeding (ExB) after cardiac surgery in adults., Background: Excessive bleeding after cardiac surgery must be anticipated for implementation of timely interventions., Methods: A prospective cohort study with 323 adults requiring open-chest cardiac surgery. Potential factors associated with ExB were investigated through univariate analysis and logistic regression. The accuracy of the relationship between the independent variables and the outcome was depicted through the receiver-operating characteristic (ROC) curve., Results: The factors associated with ExB included gender, body mass index (BMI), preoperative platelet count, intraoperative heparin doses and intraoperative platelet transfusion. The ROC curve cut-off points were 26.35 for the BMI; 214,000 for the preoperative platelet count, and 6.25 for intraoperative heparin dose. This model had an accuracy = 77.3%, a sensitivity = 81%, and a specificity = 62%., Conclusions: Male gender, BMI, preoperative platelet count, dose of intraoperative heparin >312.5 mg without subsequent platelet transfusion, are factors associated with ExB., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: To identify predictors of red blood cell transfusion (RBCT) after cardiac surgery., Method: A prospective cohort study performed with 323 adults after cardiac surgery, from April to December of 2013. A data collection instrument was constructed by the researchers containing factors associated with excessive bleeding after cardiac surgery, as found in the literature, for investigation in the immediate postoperative period. The relationship between risk factors and the outcome was assessed by univariate analysis and logistic regression., Results: The factors associated with RBCT in the immediate postoperative period included lower height and weight, decreased platelet count, lower hemoglobin level, higher prevalence of platelet count <150x10(3)/mm (3), lower volume of protamine, longer duration of anesthesia, higher prevalence of intraoperative RBCT, lower body temperature, higher heart rate and higher positive end-expiratory pressure. The independent predictor was weight <66.5Kg., Conclusion: Factors associated with RBCT in the immediate postoperative period of cardiac surgery were found. The independent predictor was weight.

Larval rainbow trout (Oncorhynchus mykiss) were reared from hatch under control ([Na(+)]=0.60 mmol l(-1)) or high NaCl ([Na(+)]=60 mmol l(-1)) conditions to elucidate the driving force for the ontogeny of branchial Na(+)/NH4 (+) exchange, one of the earliest gill functions. We hypothesized that if Na(+) uptake is the driving force, then in high NaCl there would be a delay in the skin-to-gill shift in ammonia excretion (Jamm) and/or an elevation in whole-body total ammonia (Tamm). In both groups, however, the skin-to-gill shift for Jamm, determined using divided chambers, occurred at the same time (13 days post-hatch; dph) and whole-body Tamm was unchanged. Moreover, high NaCl larvae displayed elevated whole-body [Na(+)] relative to controls by 18 dph, suggesting that maintaining branchial Jamm occurs at the expense of Na(+) balance. Overall, these results support the 'ammonia hypothesis', which posits that ammonia excretion, probably as Na(+)/NH4 (+) exchange, is the primary function of the early fish gill., (© 2015. Published by The Company of Biologists Ltd.)

In vitro gut sac experiments were performed on freshwater and 60% seawater acclimated trout (Oncorhynchus mykiss) under treatments designed to discern possible mechanisms of intestinal ammonia transport. Seawater acclimation increased ammonia flux rate into the serosal saline (Jsamm) in the anterior intestine, however it did not alter Jsamm in the mid- or posterior intestine suggesting similar mechanisms of ammonia handling in freshwater and seawater fish. Both fluid transport rate (FTR) and Jsamm were inhibited in response to basolateral ouabain treatment, suggesting a linkage of ammonia uptake to active transport, possibly coupled to fluid transport processes via solvent drag. Furthermore, decreases in FTR and Jsamm caused by low Na(+) treatment indicated a Na(+) linked transport mechanism. Mucosal bumetanide (10(-4) M) had no impact on FTR, yet decreased Jsamm in the anterior and mid-intestine, suggesting NH4(+) substitution for K(+) on an apical NKCC, and at least a partial uncoupling of ammonia transport from fluid transport. Additional treatments (amiloride, 5-(N-ethyl-N-isopropyl)amiloride (EIPA), phenamil, bafilomycin, 4',6-diamidino-2-phenylindole (DAPI), high sodium) intended to disrupt alternative routes of Na(+) uptake yielded no change in FTR or Jsamm, suggesting the absence of direct competition between Na(+) and ammonia for transport. Finally, [(14)C]methylamine permeability (PMA) measurements indicated the likely presence of an intestinal Rh-mediated ammonia transport system, as increasing NH4Cl (0, 1, 5 mmol l(-1)) concentrations reduced PMA, suggesting competition for transport through Rh proteins. Overall, the data presented in this paper provide some of the first insights into mechanisms of teleost intestinal ammonia transport., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In freshwater fish, exposure to sub-lethal concentrations of waterborne copper (Cu) results in inhibitions of ammonia excretion (Jamm) and Na(+) uptake (J(Na)in), yet the mechanisms by which these occur are not fully understood. In the present study, rainbow trout (Oncorhynchus mykiss) fry exposed to 50μg/l Cu for 24h displayed a sustained 40% decrease in Jamm and a transient 60% decrease in J(Na)in. Previously, these effects have been attributed to inhibitions of gill Na(+)/K(+)-ATPase and/or carbonic anhydrase (CA) activities by Cu. Trout fry did not display significant reductions in the branchial activities of these enzymes or H(+)-ATPase over 24h Cu exposure. Recently, Rhesus (Rh) glycoproteins, bi-directional NH3 gas channels, have been implicated in the mechanism of Cu toxicity. Juvenile trout were exposed to nominal 0, 50, and 200μg/l Cu for 3-6h under control conditions (ammonia-free water) followed by 6h exposure to high environmental ammonia (HEA; 1.5mmol/l NH4HCO3). HEA led to significant ammonia uptake in control fish (0μg/l Cu), and exposure to 50 and 200μg/l Cu resulted in significant reductions of ammonia uptake during HEA exposure. This is the first evidence that Cu inhibits both the excretion and uptake of ammonia, implicating bi-directional Rh glycoproteins as a target for Cu toxicity. We propose a model whereby Rh blockade by Cu causes the sustained inhibition of Jamm and transient inhibition of J(Na)in, with H(+)-ATPase potentially aiding in J(Na)in recovery. More work is needed to elucidate the role of Rh proteins in sub-lethal Cu toxicity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: To describe the nursing clinical judgment as a basis for ND identification and development of a NIC treatment plan for a child after cardiac surgery under intensive care., Method: A case study with data retrospectively collected from charts., Results: Three nurses identified NANDA-I diagnoses and NIC interventions. A 6-month-old child submitted to cardiac surgery, requiring extracorporeal membrane oxygenation in the postoperative period. Four main nursing diagnoses were identified, towards which ten interventions were directed. The proposal of interventions to respond to the priority human responses of the child was optimized by the use of standard terminologies. Every nursing diagnosis was supported by diagnostic indicators; every intervention was scientifically supported., Conclusion: There must be an expectation that nurses address not only physiological responses, but also those within psychosocial domains.

Post-hatch fishes lack a functional gill and use cutaneous surfaces for exchange with the surrounding environment. The ionoregulatory hypothesis posits that ionoregulation is the first physiological process to be limited by cutaneous exchange, necessitating its shift to the gills. We hypothesized that the ontogeny of branchial ammonia excretion (J amm) is coupled to Na(+) uptake (J Na in) in accordance with the current model for Na+/NH4+ in exchange in freshwater. Using divided chambers, branchial and cutaneous J amm, J Na in and oxygen consumption (MO2) by larval rainbow trout were assessed. Following hatch, the skin accounted for 97% and 86% of total J amm and J Na in, respectively. J amm and J Na in shifted to the gills simultaneously at 15 days post-hatch (dph) and were highly correlated (R(2) = 0.951) at the gills, but not the skin, over development. Contrastingly, MO2 shifted significantly later at 27 dph, in agreement with the ionoregulatory hypothesis. Moreover, the mRNA expression and/or enzymatic activity of Rhesus proteins, Na(+)/H(+)-exchanger, H(+)-ATPase, Na(+)/K(+)-ATPase and carbonic anhydrase, all key components of the Na+/NH4+-exchange system, increased in the gills over larval development. We propose that the ontogeny of branchial J Na in occurs as Na+/NH4+ exchange and provide evidence for a novel element to the ionoregulatory hypothesis, the excretion of potentially lethal metabolic ammonia., (© 2014 The Author(s) Published by the Royal Society. All rights reserved.)

The effects of acute moderate (20% air O2 saturation; 6-h exposure) and severe (5% air O2 saturation; 4-h exposure) hypoxia on N-waste, acid-base, and ion balance in dogfish sharks (Squalus acanthias suckleyi) were evaluated. We predicted that the synthesis and/or retention of urea, which are active processes, would be inhibited by hypoxia. Exposure to moderate hypoxia had negligible effects on N-waste fluxes or systemic physiology, except for a modest rise in plasma lactate. Exposure to severe hypoxia led to a significant increase in urea excretion (Jurea), while plasma, liver, and muscle urea concentrations were unchanged, suggesting a loss of urea retention. Ammonia excretion (Jamm) was elevated during normoxic recovery. Moreover, severe hypoxia led to disruptions in acid-base balance, indicated by a large increase in plasma [lactate] and substantial decreases in arterial pHa and plasma [Formula: see text], as well as loss of ionic homeostasis, indicated by increases in plasma [Mg(2+)], [Ca(2+)], and [Na(+)]. We suggest that severe hypoxia in dogfish sharks leads to a reduction in active gill homeostatic processes, such as urea retention, acid-base regulation and ionoregulation, and/or an osmoregulatory compromise due to increased functional gill surface area. Overall, the results provide a comprehensive picture of the physiological responses to a severe degree of hypoxia in an ancient fish species.

Objective: to identify the relationship between different presentations of acute coronary syndrome and cardiovascular risk factors among hospitalized individuals., Method: cross-sectional study performed in a teaching hospital in São Paulo, in the State of São Paulo (SP). Socio-demographic, clinical and anthropometric data of 150 individuals hospitalized due to acute coronary syndrome were collected through interviews and review of clinical charts. Association between these data and the presentation of the syndrome were investigated., Results: there was a predominance of ST segment elevation acute myocardial infarction. There was significant association of systemic hypertension with unstable angina and high values of low density lipoprotein with infarction, without influence from socio-demographic characteristics., Conclusion: arterial hypertension and high levels of low-density lipoprotein were associated with different presentations of coronary syndrome. The results can provide support for health professionals for secondary prevention programs aimed at behavioural changing.

In freshwater rainbow trout (Oncorhynchus mykiss), two common responses to acute waterborne copper (Cu) exposure are reductions in ammonia excretion and Na(+) uptake at the gills, with the latter representing the likely lethal mechanism of action for Cu in adult fish. Larval fish, however, lack a functional gill following hatch and rely predominantly on cutaneous exchange, yet represent the most Cu-sensitive life stage. It is not known if Cu toxicity in larval fish occurs via the skin or gills. The present study utilized divided chambers to assess cutaneous and branchial Cu toxicity over larval development, using disruptions in ammonia excretion (Jamm) and Na(+) uptake (Jin(Na)) as toxicological endpoints. Early in development (early; 3 days post-hatch; dph), approximately 95% of Jamm and 78% of Jin(Na) occurred cutaneously, while in the late developmental stage (late; 25 dph), the gills were the dominant site of exchange (83 and 87% of Jamm and Jin(Na), respectively). Exposure to 50 μg/l Cu led to a 49% inhibition of Jamm in the late developmental stage only, while in the early and middle developmental (mid; 17 dph) stages, Cu had no effect on Jamm. Jin(Na), however, was significantly inhibited by Cu exposure at the early (53% reduction) and late (47% reduction) stages. Inhibition at the early stage of development was mediated by a reduction in cutaneous uptake, representing the first evidence of cutaneous metal toxicity in an intact aquatic organism. The inhibitions of both Jamm and Jin(Na) in the late developmental stage occurred via a reduction in branchial exchange only. The differential responses of the skin and gills to Cu exposure suggest that the mechanisms of Jamm and Jin(Na) and/or Cu toxicity differ between these tissues. Exposure to 20μg/l Cu revealed that Jamm is the more Cu-sensitive process. The results presented here have important implications in predicting metal toxicity in larval fish. The Biotic Ligand Model (BLM) is currently used to predict metal toxicity in aquatic organisms. However, for rainbow trout this is based on gill binding constants from juvenile fish. This may not be appropriate for post-hatch larval fish where the skin is the site of toxic action of Cu. Determining Cu binding constants and lethal accumulation concentrations for both skin and gills in larval fish may aid in developing a larval fish-specific BLM. Overall, the changing site of toxic action and physiology of developing larval fish present an interesting and exciting avenue for future research., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The widespread plant volatile beta-caryophyllene (BCP) was recently identified as a natural selective agonist of the peripherally expressed cannabinoid receptor 2 (CB₂). It is found in relatively high concentrations in many spices and food plants. A number of studies have shown that CB₂ is critically involved in the modulation of inflammatory and neuropathic pain responses. In this study, we have investigated the analgesic effects of BCP in animal models of inflammatory and neuropathic pain. We demonstrate that orally administered BCP reduced inflammatory (late phase) pain responses in the formalin test in a CB₂ receptor-dependent manner, while it had no effect on acute (early phase) responses. In a neuropathic pain model the chronic oral administration of BCP attenuated thermal hyperalgesia and mechanical allodynia, and reduced spinal neuroinflammation. Importantly, we found no signs of tolerance to the anti-hyperalgesic effects of BCP after prolonged treatment. Oral BCP was more effective than the subcutaneously injected synthetic CB₂ agonist JWH-133. Thus, the natural plant product BCP may be highly effective in the treatment of long lasting, debilitating pain states. Our results have important implications for the role of dietary factors in the development and modulation of chronic pain conditions., (© 2013 Published by Elsevier B.V. and ECNP.)

Ammonia transport and metabolism were investigated in the intestinal tract of freshwater rainbow trout which had been either fasted for 7 days, or fasted then fed a satiating meal of commercial trout pellets. In vivo, total ammonia concentrations (T amm) in the chyme were approximately 1 mmol L(-1) across the entire intestine at 24 h after the meal. Highest chyme pH and P NH3 values occurred in the posterior intestine. In vitro gut sac experiments examined ammonia handling with mucosal (Jmamm) and serosal (Js amm) fluxes under conditions of fasting and feeding, with either background (control ≤ 0.013 mmol L(-1)) or high luminal ammonia concentrations (HLA = 1 mmol L(-1)), the latter mimicking those seen in chyme in vivo. Feeding status (fasted or fed) appeared to influence ammonia handling by each individual section. The anterior intestine exhibited the greatest Jm amm and Js amm values under fasted control conditions, but these differences tended to disappear under typical post-feeding conditions when total endogenous ammonia production (Jt amm = Js amm - Jm amm, signs considered) was greatly elevated in all intestinal sections. Under fasted conditions, glutamate dehydrogenase (GDH) and glutaminase (GLN) activities were equal across all sections, but the ammonia-trapping enzyme glutamine synthetase (GS) exhibited highest activity in the posterior intestine, in contradiction to previous literature. Feeding clearly stimulated the total rate of endogenous ammonia production (Jt amm), even in the absence of a high luminal ammonia load. This was accompanied by an increase in GDH activity of the anterior intestine, which was also the site of the largest Jt amm. In all sections, during HLA exposure, either alone or in combination with feeding, there were much larger increases in endogenous Jt amm, most of which was effluxed to the serosal solution. This is interpreted as a response to avoid potential cytotoxicity due to overburdened detoxification mechanisms in the face of elevated mucosal ammonia. Thus T amm of the intestinal tissue remained relatively constant regardless of feeding status and exposure to HLA. Ammonia production by the gut may explain up to 18 % of whole-body ammonia excretion in vivo under fasting conditions, and 47 % after feeding, of which more than half originates from endogenous production rather than from absorption from the lumen.

Recent molecular evidence points towards a capacity for ammonia transport across the skin of adult rainbow trout. A series of in vivo and in vitro experiments were conducted to understand the role of cutaneous ammonia excretion (J amm) under control conditions and after 12-h pre-exposure to high environmental ammonia (HEA; 2 mmol/l NH4HCO3). Divided chamber experiments with bladder-catheterized, rectally ligated fish under light anesthesia were performed to separate cutaneous J amm from branchial, renal, and intestinal J amm. Under control conditions, cutaneous J amm accounted for 4.5 % of total J amm in vivo. In fish pre-exposed to HEA, plasma total ammonia concentration increased 20-fold to approximately 1,000 μmol/l, branchial J amm increased 1.5- to 2.7-fold, and urinary J amm increased about 7-fold. Urinary J amm still accounted for less than 2 % of total J amm. Cutaneous J amm increased 4-fold yet amounted to only 5.7 % of total J amm in these fish. Genes (Rhcg1, Rhcg2, Rhbg, NHE-2, v-type H(+)-ATPase) known to be involved in ammonia excretion at the gills of trout were all expressed at the mRNA level in the skin, but their expression did not increase with HEA pre-exposure. In vitro analyses using [(14)C] methylamine (MA), an ammonia analog which is transported by Rh proteins, demonstrated that MA permeability in isolated skin sections was higher in HEA pre-exposed fish than in control fish. The addition of basolateral ammonia (1,000 μmol/l) to this system abolished this increase in permeability, suggesting ammonia competition with MA for Rh-mediated transport across the skin of HEA pre-exposed trout; this did not occur in skin sections from control trout. Moreover, in vitro J amm by the skin of fish which had been pre-exposed to HEA was also higher than in control fish in the absence of basolateral ammonia, pointing towards a possible cutaneous ammonia loading in response to HEA. In vitro MA permeability was reduced upon the addition of amiloride (10(-4) mol/l), but not phenamil (10(-5) mol/l) suggesting a role for a Na/H-exchanger (NHE) in cutaneous ammonia transport, as has been previously described in the skin of larval fish. Overall, it appears that under control conditions and in response to HEA pre-exposure, the skin makes only a very minor contribution to total J amm, but the observed increases in cutaneous J amm in vivo and in cutaneous J amm and MA permeability in vitro demonstrate the capacity for ammonia transport in the skin of adult trout. It remains unclear if this capacity may become significant under certain environmental challenges or if it is merely a remnant of cutaneous transport capacity from early life stages in these fish.

In patients with heart failure, reactivation of a fetal gene program, including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), is a hallmark for maladaptive remodeling of the LV. The mechanisms that regulate this reactivation are incompletely understood. Histone acetylation and methylation affect the conformation of chromatin, which in turn governs the accessibility of DNA for transcription factors. Using human LV myocardium, we found that, despite nuclear export of histone deacetylase 4 (HDAC4), upregulation of ANP and BNP in failing hearts did not require increased histone acetylation in the promoter regions of these genes. In contrast, di- and trimethylation of lysine 9 of histone 3 (H3K9) and binding of heterochromatin protein 1 (HP1) in the promoter regions of these genes were substantially reduced. In isolated working murine hearts, an acute increase of cardiac preload induced HDAC4 nuclear export, H3K9 demethylation, HP1 dissociation from the promoter region, and activation of the ANP gene. These processes were reversed in hearts with myocyte-specific deletion of Hdac4. We conclude that HDAC4 plays a central role for rapid modifications of histone methylation in response to variations in cardiac load and may represent a target for pharmacological interventions to prevent maladaptive remodeling in patients with heart failure.

Inhibition of ammonia excretion (J(amm)) is a common response to Cu exposure in freshwater (FW) and seawater (SW) organisms. To determine the mechanism of this response, a euryhaline species of guppy (Poecilia vivipara) was exposed to 20 μg Cu/l in FW (0 ppt) and SW (25 ppt) for 96 h. In both salinities, Cu transiently inhibited ammonia excretion (J(amm)) followed by a full recovery by the end of the 96 h exposure. The activities of Na(+)/K(+)-ATPase, H(+)-ATPase, and carbonic anhydrase (CA) were examined in the gills at 12 and 96 h of Cu exposure. In both salinity acclimations, CA activity was significantly inhibited following 12h of Cu exposure in P. vivipara, marking the first in vivo evidence of Cu-induced inhibition of CA in fish. Moreover, the inhibition and recovery of this enzyme were correlated with the inhibition and recovery of J(amm) in both salinity acclimations. The blockade of CA potentially acts as a common mechanism of J(amm) inhibition in FW and SW. There were no significant effects on Na(+)/K(+)-ATPase or H(+)-ATPase activity at either time point or salinity. However, H(+)-ATPase activity was upregulated at 96 h relative to the 12h time point, potentially involving this enzyme in re-establishing J(amm)., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Under cold acclimated conditions, goldfish (Carassius auratus) express an interlamellar cell mass (ILCM) which limits diffusive ion loss but may also impede branchial ammonia excretion (J(amm)). In the present study, goldfish were subjected to a 2-week 5 or 25 °C acclimation in order to modulate the degree of ILCM gill coverage and determine potential effects on J(amm). 25 °C-fish displayed gill coverage which was significantly lower than the 5 °C-fish, though the ILCM was not completely absent in these fish. 5 °C-fish demonstrated J(amm) values approximately 60% lower than those of 25 °C-fish. The magnitude of anterior (branchial) J(amm) strongly correlated with gill coverage (r(2)=0.83), suggesting that the ILCM may impede branchial J(amm). Divided chamber experiments demonstrated that relative to the 25 °C-fish, 5 °C-fish relied more upon posterior routes of excretion. In response to high external ammonia (HEA; 1.5mM NH(4)HCO(3)) exposures, 25 °C-fish displayed ammonia uptake while 5 °C-fish maintained excretion against HEA, suggesting that the ILCM may act as a barrier preventing ammonia uptake. In summary, the ILCM appears to impede branchial J(amm), such that 5 °C-rely more on extra-branchial routes of excretion. We hypothesize that gill remodeling in these fish may be intimately tied to physiological adjustments on the whole-body scale., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The objective of this cross-sectional, descriptive study was to identify the activities of the Nursing Intervention Classification considered as priorities for an Ineffective Breathing Pattern and not performed for elderly inpatients of a teaching hospital in the state of Goiás. The study participants were 43 nursing professionals, and data collection was performed in the period spanning October to December 2008, after receiving approval from the Ethics Committee. It was observed that among the 67 activities considered to be priorities for the referred diagnosis, only seven were performed by all of the participants; the other activities, with a varied frequency, were not performed, with the main reason cited being that a professional from a different area completed the activity. It is understood that the fact that the nursing staff does not perform these activities can cause lack of complete coverage in nursing care; therefore there is a need for a legal apparatus to describe the activities that comprise professional practice exclusive to nursing personnel and those activities that have an interdisciplinary nature.

Recently, a "Na(+)/NH(4)(+) exchange complex" model has been proposed for ammonia excretion in freshwater fish. The model suggests that ammonia transport occurs via Rhesus (Rh) glycoproteins and is facilitated by gill boundary layer acidification attributable to the hydration of CO(2) and H(+) efflux by Na(+)/H(+) exchanger (NHE-2) and H(+)-ATPase. The latter two mechanisms of boundary layer acidification would occur in conjunction with Na(+) influx (through a Na(+) channel energized by H(+)-ATPase and directly via NHE-2). Here, we show that natural ammonia loading via feeding increases branchial mRNA expression of Rh genes, NHE-2, and H(+)-ATPase, as well as H(+)-ATPase activity in juvenile trout, similar to previous findings with ammonium salt infusions and high environmental ammonia (HEA) exposure. The associated increase in ammonia excretion occurs in conjunction with a fourfold increase in Na(+) influx after a meal. When exposed to HEA (1.5 mmol/l NH(4)HCO(3) at pH 8.0), both unfed and fed trout showed differential increases in mRNA expression of Rhcg2, NHE-2, and H(+)-ATPase, but H(+)-ATPase activity remained at control levels. Unfed fish exposed to HEA displayed a characteristic reversal of ammonia excretion, initially uptaking ammonia, whereas fed fish (4 h after the meal) did not show this reversal, being able to immediately excrete ammonia against the gradient imposed by HEA. Exposure to HEA also led to a depression of Na(+) influx, demonstrating that ammonia excretion can be uncoupled from Na(+) influx. We suggest that the efflux of H(+), rather than Na(+) influx itself, is critical to the facilitation of ammonia excretion.

Cannabinoid CB1 receptor is abundantly expressed throughout the CNS and is implicated in numerous physiological and behavioral functions, including appetite and feeding. In the present study, wild-type and CB1 heterozygous and homozygous knockout mice were tested on an instrumental outcome-selective devaluation task to assess changes in acquired instrumental response levels for a distinct food reward following selective satiation. Deletion of CB1 receptor, as well as reduction in CB1 expression (HET), produced deficits in outcome-selective instrumental devaluation. These results identify a critical role for CB1 receptor in the ability of animals to represent, update, and/or use sensory-specific outcome representations to alter appetitive behaviors.