RESUMEN

Teleost fishes constitute 95% of extant aquatic vertebrates, and we suggest that this is related in part to their unique mode of tissue oxygenation. We propose the following sequence of events in the evolution of their oxygen delivery system. First, loss of plasma-accessible carbonic anhydrase (CA) in the gill and venous circulations slowed the Jacobs-Stewart cycle and the transfer of acid between the plasma and the red blood cells (RBCs). This ameliorated the effects of a generalised acidosis (associated with an increased capacity for burst swimming) on haemoglobin (Hb)-O2 binding. Because RBC pH was uncoupled from plasma pH, the importance of Hb as a buffer was reduced. The decrease in buffering was mediated by a reduction in the number of histidine residues on the Hb molecule and resulted in enhanced coupling of O2 and CO2 transfer through the RBCs. In the absence of plasma CA, nearly all plasma bicarbonate ultimately dehydrated to CO2 occurred via the RBCs, and chloride/bicarbonate exchange was the rate-limiting step in CO2 excretion. This pattern of CO2 excretion across the gills resulted in disequilibrium states for CO2 hydration/dehydration reactions and thus elevated arterial and venous plasma bicarbonate levels. Plasma-accessible CA embedded in arterial endothelia was retained, which eliminated the localized bicarbonate disequilibrium forming CO2 that then moved into the RBCs. Consequently, RBC pH decreased which, in conjunction with pH-sensitive Bohr/Root Hbs, elevated arterial oxygen tensions and thus enhanced tissue oxygenation. Counter-current arrangement of capillaries (retia) at the eye and later the swim bladder evolved along with the gas gland at the swim bladder. Both arrangements enhanced and magnified CO2 and acid production and, therefore, oxygen secretion to those specialised tissues. The evolution of ß-adrenergically stimulated RBC Na(+)/H(+) exchange protected gill O2 uptake during stress and further augmented plasma disequilibrium states for CO2 hydration/dehydration. Finally, RBC organophosphates (e.g. NTP) could be reduced during hypoxia to further increase Hb-O2 affinity without compromising tissue O2 delivery because high-affinity Hbs could still adequately deliver O2 to the tissues via Bohr/Root shifts. We suggest that the evolution of this unique mode of tissue O2 transfer evolved in the Triassic/Jurassic Period, when O2 levels were low, ultimately giving rise to the most extensive adaptive radiation of extant vertebrates, the teleost fishes.

Asunto(s)

Evolución Biológica , Peces/fisiología , Oxígeno/metabolismo , Anaerobiosis , Animales , Transporte Biológico , Conducta Alimentaria , Natación

RESUMEN

Teleost fishes and mammalian lineages diverged 400 million years ago, and environmental requirements (water versus air) have resulted in marked differences in cardiovascular function between fish and mammals. Suggestions that the fish secondary vascular system (SVS) could be used as a model for the mammalian lymphatic system should be taken with caution. Despite molecular markers indicating similar genetic origin, functions of the SVS in teleost fish are probably different from those of the mammalian lymphatic system. We determined that, in resting glass catfish (Kryptopterus bicirrhis), plasma moves from the primary vascular system (PVS) to the SVS through small connecting vessels less than 10 µm in diameter, smaller than the red blood cells (RBCs). During and following hypoxia or exercise, flow increases and RBCs enter the SVS, possibly via ß-adrenoreceptor-mediated dilation of the connecting vessels. The volume of the SVS can be large and, as RBCs flow into the SVS, the haematocrit of the PVS falls by as much as 50% of the resting value. Possible functions of the SVS, including skin respiration, ionic and osmotic buffering, and reductions in heart work and RBC turnover, are discussed.

Asunto(s)

Bagres/fisiología , Sistema Linfático/fisiología , Condicionamiento Físico Animal , Antagonistas Adrenérgicos/farmacología , Agonistas Adrenérgicos beta/farmacología , Anaerobiosis , Animales , Fluorescencia , Hematócrito , Sistema Linfático/efectos de los fármacos , Microscopía por Video , Microesferas

RESUMEN

Hypoxia induced apoptosis has been studied extensively in many mammalian cell lines but there are only a few studies using whole animal models. We investigated the response of the intact liver to hypoxia in a hypoxia tolerant fish, the carp (Cyprinus carpio, L). We exposed carp to hypoxia for up to 42 days, using oxygen level (0.5 mgO(2)/L) that were slightly higher than the critical oxygen level of carp. There was extensive DNA damage in liver cells, especially during the first week of exposure, indicated by a massive TUNEL signal. However there was no change in cell proliferation, cell number or size, no increase in caspase-3 activity, no increase in single stranded DNA and this, combined with a number of other observations, led us to conclude there was no increase in apoptosis in the liver during hypoxia. There was up-regulation of some anti-apoptotic genes and proteins (Bcl-2, HSP70, p27) and down-regulation of some pro-apoptotic genes (Tetraspanin 5 and Cell death activator). The cells appeared to enter cell cycle arrest, presumably to allow repair of damaged DNA. As there was no change in cell proliferation and cell number, the damaged cells were not entering apoptosis and must have recovered during prolonged hypoxia.

RESUMEN

The gills are the major site of acid-base regulation in most fish. Acid-base transfer across fish gills is dominated by carbon dioxide and ammonia excretion, especially the former. Bicarbonate buffering in the blood is less than that found in mammals; regulation of ventilation has little effect on CO(2) levels in the blood and control of ventilation is not used to regulate body pH in fish. Proton ATPase (freshwater fish), Na(+)/H(+) exchangers (marine fish) and anion exchangers (marine and freshwater fish) are located in the gills. These transporters contribute to the regulation of internal pH, but little is known about how this is done in fish. Fish kept in confined water volumes acidify their environment, largely due to CO(2). This acidification augments ammonia excretion and reduces ammonia toxicity. The possible involvement of ammonia recycling in acid excretion is also discussed.

Asunto(s)

Equilibrio Ácido-Base , Cipriniformes/metabolismo , Branquias/metabolismo , Carpa Dorada/metabolismo , Amoníaco/metabolismo , Animales , Antiportadores/metabolismo , Bicarbonatos/metabolismo , Transporte Biológico , Dióxido de Carbono/sangre , Concentración de Iones de Hidrógeno , ATPasas de Translocación de Protón/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo

RESUMEN

The potent vasomodulator adenosine (AD), thanks to the interaction with by A(1) and A(2) receptors, dilates systemic, coronary and cerebral vasculatures but exert a constrictor action in several vessels of respiratory organs. Recent investigations suggest that nitric oxide (NO) contributes to AD effects. In fish, both NO and AD induce atypical effects compared to mammals. Since there is very little information on the role of NO and its involvement in mediating the actions of AD in fish, we have analysed this question in the branchial vasculature of the elasmobranch Squalus acanthias and the teleost Anguilla anguilla using an isolated perfused head and a branchial basket preparation, respectively. In both dogfish and eel, AD dose-response curves showed a biphasic effect: vasoconstriction (pico to nanomolar range) and vasodilation (micromolar range). Both effects were abolished by the classic xanthine inhibitor theophylline (Theo) and also by specific antagonists of A(1) and A(2) receptor subtypes. To analyse the involvement of the NO/cGMP system in the AD responses, we tested a NOS inhibitor, l-NIO, and a specific soluble guanylate cyclase (sGC) blocker, ODQ. In both dogfish and eel preparations l-NIO abrogated all vasomotor effects of AD, whereas ODQ blocked the AD-mediated vasoconstriction without affecting the vasorelaxant response. This indicates that only AD-induced vasoconstriction is mediated by a NO-cGMP-dependent mechanism. By using the NO donor SIN-1, we showed a dose-dependent vasoconstrictory effect which was completely blocked by ODQ. These results provide compelling evidence that the vasoactive role of AD in the branchial circulation of S. acanthias and A. anguilla involves a NO signalling.

Asunto(s)

Adenosina/química , Anguilla/metabolismo , Óxido Nítrico/química , Squalus acanthias/metabolismo , Adenosina/metabolismo , Animales , GMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Hipoxia , Técnicas In Vitro , Masculino , Molsidomina/análogos & derivados , Molsidomina/farmacología , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/química , Perfusión , Presión , Transducción de Señal , Factores de Tiempo , Vasodilatación

RESUMEN

Most tropical fishes are ammonotelic, producing ammonia and excreting it as NH3 by diffusion across the branchial epithelia. Hence, those air-breathing tropical fishes that survive on land briefly or for an extended period would have difficulties in excreting ammonia when out of water. Ammonia is toxic, but some of these air-breathing fishes adopt special biochemical adaptations to ameliorate the toxicity of endogenous ammonia accumulating in the body. The amphibious mudskipper Periophthalmodon schlosseri, which is very active on land, reduces ammonia production by suppressing amino acid catabolism (strategy 1) during aerial exposure. It can also undergo partial amino acid catabolism, leading to the accumulation of alanine (strategy 2) to support locomotory activities on land. In this case, alanine formation is not an ammonia detoxification process but reduces the production of endogenous ammonia. The snakehead Channa asiatica, which exhibits moderate activities on land although not truly amphibious, accumulates both alanine and glutamine in the muscle, with alanine accounting for 80% of the deficit in reduction in ammonia excretion during air exposure. Unlike P. schlosseri, C. asiatica apparently cannot reduce the rates of protein and amino acid catabolism and is incapable of utilizing partial amino acid catabolism to support locomotory activities on land. Unlike alanine formation, glutamine synthesis (strategy 3) represents an ammonia detoxification mechanism that, in effect, removes the accumulating ammonia. The four-eyed sleeper Bostrichyths sinensis, which remains motionless during aerial exposure, detoxifies endogenous ammonia to glutamine for storage. The slender African lungfish Protopterus dolloi, which can aestivate on land on a mucus cocoon, has an active ornithine-urea cycle and converts endogenous ammonia to urea (strategy 4) for both storage and subsequent excretion. Production of urea and glutamine are energetically expensive and appear to be adopted by fishes that remain relatively inactive on land. The Oriental weatherloach Misgurnus anguillicaudatus, which actively burrows into soft mud during drought, manipulates the pH of the body surface to facilitate NH3 volatilization (strategy 5) and develops high ammonia tolerance at the cellular and subcellular levels (strategy 6) during aerial exposure. Hence, with regard to excretory nitrogen metabolism, modern tropical air-breathing fishes exhibit a variety of strategies to survive on land, and they represent a spectrum of specimens through which we may examine various biochemical adaptations that would have facilitated the invasion of the terrestrial habitat by fishes during evolution.

Asunto(s)

Adaptación Fisiológica , Amoníaco/metabolismo , Ambiente , Peces/fisiología , Consumo de Oxígeno/fisiología , Aminoácidos/metabolismo , Amoníaco/toxicidad , Animales , Peces/metabolismo , Nitrógeno/metabolismo , Especificidad de la Especie , Clima Tropical , Urea/metabolismo

RESUMEN

The giant mudskipper, Periophthalmodon schlosseri, is an amphibious, obligate, air-breathing teleost fish. It uses its buccal cavity for air breathing and for taking and holding large gulps of air. These fish live in mud burrows at the top of the intertidal zone of mangrove mudflats; the burrow water may be hypoxic and hypercapnic and have high ammonia levels. The buccal epithelium is highly vascularized, with small diffusion distances between air and blood. The gill epithelium is densely packed with mitochondria-rich cells. Periophthalmodon schlosseri can maintain tissue ammonia levels in the face of high ammonia concentrations in the water. This is probably achieved by active ammonium ion transport across the mitochondria-rich cells via an apical Na/H+(NH4+) exchanger and a basolateral Na/K+(NH4+) ATPase. When exposed to air, the animal reduces ammonia production, but there is some increase in tissue ammonia levels after 24 h. There is no detoxification by increased production of glutamine or urea, but there is partial amino acid catabolism, leading to the accumulation of alanine. CO2 production and proton excretion cause acidification of the burrow water to reduce ammonia toxicity. The skin has high levels of cholesterol and saturated fatty acids decreasing membrane fluidity and gas, and therefore ammonia, permeability. Exposure to elevated environmental ammonia further decreases membrane permeability. Acidification of the environment and having a skin with a low NH3 permeability reduces ammonia influx, so that the fish can maintain tissue ammonia levels by active ammonium ion excretion, even in water containing high levels of ammonia.

Asunto(s)

Adaptación Fisiológica , Amoníaco/metabolismo , Ambiente , Modelos Biológicos , Consumo de Oxígeno/fisiología , Perciformes/fisiología , Aminoácidos/metabolismo , Animales , Transporte Biológico Activo , Epitelio/fisiología , Branquias/metabolismo , Branquias/fisiología , Oxígeno/metabolismo , Perciformes/metabolismo

RESUMEN

Teleost fish possess discrete blocks of oxidative red muscle (RM) and glycolytic white muscle, whereas tetrapod skeletal muscles are mixed oxidative/glycolytic. It has been suggested that the anatomy of RM in teleost fish could lead to higher intramuscular O2 partial pressures (PO2) than in mammalian skeletal muscles. This study provides the first direct experimental support for this suggestion by using novel optical fibre sensors to discover a mean (+/- S.E.M., N=6) normoxic steady-state red muscle PO2 (PrmO2) of 61+/-10 mmHg (1 mmHg=133.3 Pa) in free-swimming rainbow trout Oncorhynchus mykiss. This is significantly higher than literature reports for mammalian muscles, where the PO2 never exceeds 40 mmHg. Aerobic RM powers sustained swimming in rainbow trout. During graded incremental exercise, PrmO2 declined from 62+/-5 mmHg at the lowest swim speed down to 45+/-3 mmHg at maximum rates of aerobic work, but then rose again to 51+/-5 mmHg at exhaustion. These measurements of PrmO2 during exercise indicated, therefore, that O2 supply to the RM was not a major limiting factor at exhaustion in trout. The current study found no evidence that teleost haemoglobins with a Root effect cause extremely elevated O2 tensions in aerobic tissues. Under normoxic conditions, PrmO2 was significantly lower than arterial PO2 (119+/-5 mmHg), and remained lower when the arterial to tissue PO2 gradient was reduced by exposure to mild hypoxia. When two sequential levels of mild hypoxia (30 min at a water PO2 of 100 mmHg then 30 min at 75 mmHg) caused PaO2 to fall to 84+/-2 mmHg then 61+/-3 mmHg, respectively, this elicited simultaneous reductions in PrmO2,to 51+/-6 mmHg then 41+/-5 mmHg, respectively. Although these hypoxic reductions in PrmO2 were significantly smaller than those in PaO2, the effect could be attributed to the sigmoid shape of the trout haemoglobin-O2 dissociation curve.

Asunto(s)

Hipoxia/fisiopatología , Fibras Musculares de Contracción Rápida/metabolismo , Músculo Esquelético/metabolismo , Oncorhynchus mykiss/fisiología , Oxígeno/metabolismo , Esfuerzo Físico/fisiología , Análisis de Varianza , Animales , Factores de Tiempo

RESUMEN

In the tropics, air-breathing fishes can be exposed to environmental ammonia when stranded in puddles of water during the dry season, during a stay inside a burrow, or after agricultural fertilization. At low concentrations of environmental ammonia, NH(3) excretion is impeded, as in aerial exposure, leading to the accumulation of endogenous ammonia. At high concentrations of environmental ammonia, which results in a reversed NH(3) partial pressure gradient (DeltaP(NH3)), there is retention of endogenous ammonia and uptake of exogenous ammonia. In this review, several tropical air-breathing fishes (giant mudskipper, African catfish, oriental weatherloach, swamp eel, four-eyed sleeper, abehaze and slender African lungfish), which can tolerate high environmental ammonia exposure, are used as examples to demonstrate how eight different adaptations can be involved in defence against ammonia toxicity. Four of these adaptations deal with ammonia toxicity at branchial and/or epithelial surfaces: (1) active excretion of NH(4)(+); (2) lowering of environmental pH; (3) low NH(3) permeability of epithelial surfaces; and (4) volatilization of NH(3), while another four adaptations ameliorate ammonia toxicity at the cellular and subcellular levels: (5) high tolerance of ammonia at the cellular and subcellular levels; (6) reduction in ammonia production; (7) glutamine synthesis; and (8) urea synthesis. The responses of tropical air-breathing fishes to high environmental ammonia are determined apparently by behavioural adaptations and the nature of their natural environments.

Asunto(s)

Aire , Amoníaco/envenenamiento , Peces/fisiología , Respiración , Clima Tropical , Animales , Exposición a Riesgos Ambientales , Intoxicación/prevención & control

RESUMEN

The oriental weatherloach Misgurnus anguillicaudatus is an extremely ammonia-tolerant fish. Many ammonia-protection mechanisms have been reported in this fish. Six strategies used by this fish to deal with the problem of excess ammonia are described. The fish can (1) reduce ammonia production through reduction in protein and/or amino acid catabolism; (2) reduce ammonia production and obtain energy through partial amino acid catabolism leading to alanine formation; (3) detoxify ammonia to glutamine; (4) tolerate very high ammonia levels in its tissues; (5) get rid of ammonia as NH(3) gas and, probably, (6) possesses background K(+) channels that are impermeable to NH(4)(+). The effects of extracellular ammonia on the contraction performance of the heart from this fish were found to be the same as in rainbow trout, an ammonia-sensitive fish. It suggests that the hearts of most, if not all, fish species are protected against ammonia. MK-801, an NMDA receptor blocker, was found to have a protective effect against ammonia intoxication in the oriental weatherloach, which suggests that the NMDA receptor, as in mammals, is involved in ammonia toxicity.

Asunto(s)

Adaptación Fisiológica , Amoníaco/metabolismo , Cipriniformes/metabolismo , Alanina/metabolismo , Aminoácidos/metabolismo , Amoníaco/toxicidad , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Maleato de Dizocilpina/metabolismo , Glutamina/biosíntesis , Contracción Miocárdica/efectos de los fármacos , Canales de Potasio/metabolismo , Proteínas/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores

RESUMEN

We have isolated a 1586-bp full-length CITED3 cDNA from grass carp which specifies for a cAMP-responsive element-binding protein/p300-interacting transactivator with glutamic acid (E)/aspartic acid (D)-rich C-terminal domain protein. The cDNA, designated as gcCITED3, has an open reading frame of 762 bp and encodes a protein of 253 amino acids with a predicted molecular mass of 28.3 kDa and pI of 6.4. Pairwise comparison showed that gcCITED3 shares high sequence identity with the CITED3 of zebrafish (94%), chicken (72%) and Xenopus (59%). Northern blot analysis indicated that gcCITED3 is most highly expressed and responsive to hypoxia in the carp kidney. Hypoxic induction was also observed in heart, albeit at a lower level. This is the first report on the isolation of a hypoxia-responsive CITED3 gene from fish.

Asunto(s)

Carpas/genética , Proteínas de Peces/genética , Hipoxia/genética , Transactivadores/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Clonación Molecular , Proteínas de Peces/química , Perfilación de la Expresión Génica , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Datos de Secuencia Molecular , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido

RESUMEN

Experiments were performed to evaluate the effects of alkaline environmental pH on urea and ammonia excretion rates and on tissue urea, ammonia, and free amino acid concentrations in two mudskippers, Periophthalmodon schlosseri and Boleophthalmus boddaerti. Periophthalomodon schlosseri is known to be capable of actively excreting ammonia. The rate of ammonia excretion in B. boddaerti exposed to 50% seawater (brackish water, BW) at pH 9 decreased significantly during the first 2 d of exposure when compared with that of specimens exposed to pH 7 or 8. This suggested that B. boddaerti was dependent on NH(3) diffusion for ammonia excretion, as in most fishes. It was incapable of detoxifying the accumulating endogenous ammonia to urea but could store and tolerate high concentrations of ammonia in the muscle, liver, and plasma. It did not undergo reductions in proteolysis and/or amino acid catabolism in alkaline water, probably because the buildup of endogenous ammonia was essential for the recovery of the normal rate of ammonia excretion by the third day of exposure to a pH 9 medium. Unlike B. boddaerti, P. schlosseri did not accumulate ammonia in the body at an alkaline pH (i.e., pH 9) because it was capable of actively excreting ammonia. Periophthalmodon schlosseri did not undergo partial amino acid catabolism (no accumulation of alanine) either, although there might be a slight reduction in amino acid catabolism in general. The significant decrease in blood pCO(2) in B. boddaerti at pH 9 might lead to respiratory alkalosis in the blood. In contrast, P. schlosseri was able to maintain its blood pH in BW at pH 9 despite a decrease in pCO(2) in the blood. With 8 mM NH(4)Cl in BW at pH 7, both mudskippers could actively excrete ammonia, although not to the same extent. Only P. schlosseri could sustain ammonia excretion against 8 mM NH(4)Cl in BW at pH 8. In BW containing 8 mM NH(4)Cl at pH 9, both mudskippers died within a short period of time. Boleophthalmus boddaerti consistently died faster than did P. schlosseri. This indicates that the body surfaces of these mudskippers were permeable to NH(3), but the skin of P. schlosseri might be less permeable to NH(3) than that of B. boddaerti. Both mudskippers excreted acid (H(+)) to alter the pH of the alkaline external medium. Such a capability, together with modifications in gill morphology and morphometry as in P. schlosseri, might be essential to the development of an effective mechanism for the active excretion of NH+4.

Asunto(s)

Amoníaco/metabolismo , Cloruro de Amonio/metabolismo , Branquias/metabolismo , Perciformes/metabolismo , Agua de Mar/química , Equilibrio Ácido-Base , Adaptación Fisiológica , Álcalis/química , Animales , Exposición a Riesgos Ambientales , Femenino , Concentración de Iones de Hidrógeno , Masculino , Fuerza Protón-Motriz/fisiología , Especificidad de la Especie , Urea/metabolismo , Equilibrio Hidroelectrolítico/fisiología

RESUMEN

Ammonia is present in the aquatic environment due to agricultural run-off and decomposition of biological waste. Ammonia is toxic to all vertebrates causing convulsions, coma and death, probably because elevated NH4+ displaces K+ and depolarizes neurons, causing activation of NMDA type glutamate receptor, which leads to an influx of excessive Ca2+ and subsequent cell death in the central nervous system. Present ammonia criteria for aquatic systems are based on toxicity tests carried out on, starved, resting, non-stressed fish. This is doubly inappropriate. During exhaustive exercise and stress, fish increase ammonia production and are more sensitive to external ammonia. Present criteria do not protect swimming fish. Fish have strategies to protect them from the ammonia pulse following feeding, and this also protects them from increases in external ammonia, as a result starved fish are more sensitive to external ammonia than fed fish. There are a number of fish species that can tolerate high environmental ammonia. Glutamine formation is an important ammonia detoxification strategy in the brain of fish, especially after feeding. Detoxification of ammonia to urea has also been observed in elasmobranches and some teleosts. Reduction in the rate of proteolysis and the rate of amino acid catabolism, which results in a decrease in ammonia production, may be another strategy to reduce ammonia toxicity. The weather loach volatilizes NH3, and the mudskipper, P. schlosseri, utilizes yet another unique strategy, it actively pumps NH4+ out of the body.

Asunto(s)

Amoníaco/toxicidad , Peces/fisiología , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Contaminantes del Agua/toxicidad , Adaptación Fisiológica , Aminoácidos/metabolismo , Amoníaco/metabolismo , Animales , Encéfalo/fisiología , Muerte Celular , Dieta , Condicionamiento Físico Animal , Temperatura , Pruebas de Toxicidad , Volatilización , Agua/química , Contaminantes del Agua/metabolismo

RESUMEN

Short-term exposure of coho salmon smolts (Oncorhynchus kisutch) to a gradual increase in salinity over 2 d (0 per thousand -32 per thousand ) resulted in a decrease in proton pump abundance, detected as changes in immunoreactivity with a polyclonal antibody against subunit A of bovine brain vacuolar H(+)-ATPase. N-ethylmaleimide (NEM)-sensitive H(+)-ATPase activities in gill homogenates remained unchanged over 8 d to coincide with a 3.5-fold increase in Na(+)/K(+)-ATPase activities. A transient increase in plasma [Na(+)] and [Cl(-)] levels over the 8-d period was preceded by a 10-fold increase in plasma cortisol levels, which peaked after 12 h. Long-term (1 mo) acclimation to seawater resulted in the loss of apical immunoreactivity for vH(+)-ATPase and band 3-like anion exchanger in the mitochondria-rich cells identified by high levels of Na(+)/K(+)-ATPase immunoreactivity. The polyclonal antibody Ab597 recognized a Na(+)/H(+) exchanger (NHE-2)-like protein in what appears to be an accessory cell (AC) type. Populations of these ACs were found associated with Na(+)/K(+)-ATPase rich chloride cells in both freshwater- and seawater-acclimated animals.

Asunto(s)

Adaptación Fisiológica , Epitelio/metabolismo , Branquias/metabolismo , Salmón/metabolismo , Agua de Mar/química , Animales , Cloruros/metabolismo , Agua Dulce , Transporte Iónico , Sodio/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Factores de Tiempo , Distribución Tisular , Equilibrio Hidroelectrolítico

RESUMEN

This study tested the hypothesis that swimming exacerbates ammonia toxicity in fish. Both sub-lethal and acute toxicity testing was conducted in a swim tunnel on swimming and resting coho salmon and rainbow trout, respectively. The sub lethal tests on coho salmon also considered the compartmentalization of ammonia within the fish. Coho salmon showed a significant linear decrease in U(crit) both with increasing water ammonia (0, 0.02, 0.04 and 0.08 mg per l NH3) and increasing plasma ammonia. Data collected included plasma pH and ammonia, muscle pH and ammonia and muscle membrane potential. Based on results found in these experiments it was concluded that the reduction in swimming performance was due to both metabolic challenges as well as depolarization of white muscle. Acute toxicity testing on swimming and resting rainbow trout revealed that swimming at (60% U(crit) or approximately 2.2 body lengths/s) decreased the LC50 level from 207+/-21.99 mg N per l in resting fish to 32.38+/-10.81. The LC50 for resting fish was significantly higher than that for swimming fish. The acute value set forth by the US EPA at the same pH is 36.1 mg N per l and may not protect swimming fish. In addition the effect of water hardness on ammonia toxicity was considered. It was found that increased water calcium ameliorates ammonia toxicity in fish living in high pH water.

Asunto(s)

Amoníaco/toxicidad , Oncorhynchus kisutch/metabolismo , Oncorhynchus mykiss/metabolismo , Amoníaco/efectos adversos , Animales , Calcio/farmacología , Relación Dosis-Respuesta a Droga , Exposición a Riesgos Ambientales , Oncorhynchus kisutch/fisiología , Oncorhynchus mykiss/fisiología , Natación , Factores de Tiempo

RESUMEN

Present fresh water ammonia standards have been established using data collected from toxicity tests on unfed fish. Ammonia, however, is an unusual toxicant as it is produced as a metabolic waste following protein catabolism. The present research was conducted to investigate the relationship between feeding and ammonia toxicity in rainbow trout, Oncorhynchus mykiss. Results from these studies revealed that some fish fed to satiation have plasma ammonia levels greater than 30 microg/ml. This level was similar to the plasma ammonia levels in rainbow trout at the ammonia LC50 value calculated in the present experiments. Even though plasma ammonia in fed fish was elevated there was no significant difference between the 96 h LC50 values for fed and unfed fish (174 mg N per l) at pH 7.2. Feeding rates during these experiments decreased during the first 48 h of ammonia exposure, but increased again in the second 48 h at all but the highest ammonia level. Feeding rate never increased to the control level in ammonia exposed fish. In a second set of experiments feeding fish had a significantly higher 24 h LC50 level, 177 mg N per l, than fish fasted for 5 or 10 days, 135-143 mg N per l. No significant difference was noted however, between the 48 h LC50 values for fed and fasted fish. It was evident from these studies that feeding protects rainbow trout from ammonia toxicity during the first 24 h of exposure and that fasting exacerbates ammonia toxicity.

Asunto(s)

Amoníaco/toxicidad , Oncorhynchus mykiss/fisiología , Amoníaco/sangre , Animales , Glucemia/efectos de los fármacos , Calcio/sangre , Relación Dosis-Respuesta a Droga , Ingestión de Alimentos/fisiología , Privación de Alimentos/fisiología , Oncorhynchus mykiss/metabolismo , Sodio/sangre

RESUMEN

Many species of fishes have evolved mechanisms for coping with ammonia caused by either high ammonia environments or an inability to excrete nitrogenous wastes. Rainbow trout (Oncorhynchus mykiss), have not been known to have such a mechanism. The present study investigated whether rainbow trout can use amino acid synthesis and storage to cope with ammonia. Experiments were performed on fed and unfed rainbow trout under both control and elevated ammonia conditions (0 and 10 mgN/l (total ammonia nitrogen), pH 7.2). The results indicate that both feeding and ammonia exposure increased plasma ammonia significantly 6 h postprandial and post ammonia exposure. After 48 h the fed/ammonia exposed fish had plasma ammonia levels that were not significantly different than the fed/control fish. Plasma ammonia was reduced by more than 50%, attributable to ammonia being converted to glutamine in brain, liver and muscle tissue. Feeding alone also increased glutamine levels in brain tissue. Activity of glutamine synthetase in brain and liver was increased corresponding to an increase in glutamine concentrations when fish were exposed to ammonia. This is the first report showing that rainbow trout can detoxify endogenous and exogenous ammonia.

Asunto(s)

Amoníaco/metabolismo , Amoníaco/farmacología , Privación de Alimentos/fisiología , Glutamina/metabolismo , Oncorhynchus mykiss/metabolismo , Amoníaco/administración & dosificación , Amoníaco/sangre , Animales , Encéfalo/enzimología , Encéfalo/metabolismo , Glutamato-Amoníaco Ligasa/metabolismo , Glutaminasa/metabolismo , Glutamina/análisis , Hidrocortisona/sangre , Concentración de Iones de Hidrógeno , Hígado/enzimología , Hígado/metabolismo , Músculos/enzimología , Músculos/metabolismo , Oncorhynchus mykiss/sangre , Periodo Posprandial , Temperatura , Factores de Tiempo

RESUMEN

Information about the presence and effects of nitric oxide (NO) in fish vasculature is scant and contradictory. We have studied the NO/cGMP system in the branchial circulation of the teleost Anguilla anguilla using a branchial basket preparation under basal conditions and cholinergic stimulation. The effects of endogenous and exogenous NO were tested with L-arginine, the nitric oxide synthase (NOS) substrate, and the NO donors 3-morpholinosydnonimine (SIN-1) and sodium nitroprusside (SNP), respectively. L-arginine (from 10(-11) to 10(-6) M) and the NO donors (starting from 10(-14) M) caused dose-dependent vasoconstriction. Conversely, in the ACh-pre-contracted preparations both donors elicited vasodilation. SIN-1-induced vasoconstriction was due to NO generation: it was increased by superoxide dismutase (SOD) and blocked by NO scavenger hemoglobin. Pre-treatment with sGC inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) inhibited the effects of SIN-1 and SNP. The stable cGMP analogue 8-bromo-guanosine 3',5'-cyclic monophosphate (8-Br cGMP) induced dose-dependent vasoconstriction. Unexpectedly, three NOS inhibitors, N(G)-nitro-L-arginine methyl ester (L-NAME), N(G)-monomethyl-L-arginine (L-NMMA), L-N(5)-(1-iminoethyl) ornithine (L-NIO), caused mild vasoconstriction. ACh caused vasoconstriction, but at pico- and nanomolar concentrations it caused mild but significant vasodilation in 40% of the preparations. Both responses, blocked by atropine and pirenzepine, required an intact endothelium. The ACh-induced vasoconstriction was substantially independent of a NO-cGMP mechanism.

Asunto(s)

Acetilcolina/farmacología , Región Branquial/irrigación sanguínea , Región Branquial/efectos de los fármacos , GMP Cíclico/metabolismo , Anguilas/fisiología , Molsidomina/análogos & derivados , Óxido Nítrico/metabolismo , Vasoconstricción/efectos de los fármacos , Animales , Arginina/farmacología , Región Branquial/enzimología , Región Branquial/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Relación Dosis-Respuesta a Droga , Endotelio Vascular/efectos de los fármacos , Femenino , Branquias/irrigación sanguínea , Branquias/efectos de los fármacos , Branquias/enzimología , Branquias/metabolismo , Hemoglobinas/farmacología , Técnicas In Vitro , Masculino , Molsidomina/farmacología , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/metabolismo

RESUMEN

The weather loach Misgurnus anguillicaudatus inhabits rice fields that experience drought in summer and ammonia loading during agricultural fertilisation. Exposure of specimens to ammonia led to the accumulation of ammonia in muscle, liver and blood. The level of ammonia reached in the plasma was the highest reported among fishes. Ammonia was not detoxified to urea, and urea excretion rate was unaffected by ammonia exposure. Fish acidified the water to reduce ammonia loading. Ammonia loading, unlike aerial exposure, did not induce glutamine synthesis, and there was no accumulation of glutamine. This is a unique observation different from those reported for other fishes in the literature. An initial switch to partial amino acid catabolism led to the accumulation of alanine and was probably associated with a decreased rate of ammonia production. Aerial exposure led to decreases in rates of ammonia and urea excretion, as well as the accumulation of tissue ammonia. As the internal ammonia levels increased, M. anguillicaudatus was able to excrete some ammonia in the gaseous form (NH(3)). The percentage of ammonia excreted as NH(3) increased with time of exposure and with increasing temperature. It appears that air-breathing through the gut is involved, with the anterior portion of the digestive tract playing a central role: it became significantly more alkaline in fish exposed to air or to environmental ammonia. The skin, which also became more alkaline during air exposure, may also be involved in ammonia volatilization in air-exposed fish. This represents the first report of a fish using volatilization of NH(3) as part of a defence against ammonia toxicity. It can be concluded that the main strategy adopted by M. anguillicaudatus confronted with ammonia loading or air exposure is to tolerate high ammonia levels in the tissues. During periods of elevated tissue ammonia levels, some ammonia is lost by volatilization via air-breathing using the gut. In addition, some ammonia may be lost across the skin during air exposure.

Asunto(s)

Amoníaco/metabolismo , Cipriniformes/fisiología , Aire , Aminoácidos/metabolismo , Amoníaco/sangre , Animales , Dióxido de Carbono/sangre , Femenino , Concentración de Iones de Hidrógeno , Hígado/metabolismo , Masculino , Músculo Esquelético/metabolismo , Especificidad de Órganos , Oxígeno/sangre , Presión Parcial , Urea/metabolismo , Tiempo (Meteorología)

RESUMEN

When the mudskipper Periophthalmodon schlosseri was exposed to terrestrial conditions under a 12h:12h dark:light regime the fish could be very active, and levels of total free amino acids increased significantly in the muscle and plasma. Alanine levels increased threefold in the muscle, fourfold in the liver and twofold in the plasma. Similar phenomena were not observed in the more aquatic mudskipper, Boleophthalmus boddaerti. From these results, we concluded that P. schlosseri was capable of partial catabolism of certain amino acids to support activity on land. The amino groups of these amino acids were transferred directly or indirectly to pyruvate to form alanine. The resulting carbon chain was fed into the Krebs cycle and partially oxidized to malate, which could replenish pyruvate through the function of malic enzyme. This favourable ATP yield from partial amino acid catabolism was not accompanied by a net release of ammonia. Such an adaptation would be advantageous to P. schlosseri confronted with the problem of ammonia excretion during aerial exposure. Indeed, when P. schlosseri were forced to exercise on land after 24 h of aerial exposure, the alanine level in the muscles increased significantly, with no apparent change in glycogen content. In addition, there was no significant change in the ATP level and energy charge of the muscle. In contrast, when B. boddaerti were exercised on land, glycogen levels in the muscles decreased significantly and lactate levels increased. In addition, muscle energy charge was not maintained and the ATP level decreased significantly. Hence, it was concluded that when P. schlosseri were active on land, they were capable of using certain amino acids as a metabolic fuel, and avoided ammonia toxicity through partial amino acid catabolism. Such a strategy is the most cost-effective way of slowing down internal ammonia build-up without involving energy-expensive ammonia detoxification pathways. Furthermore, an examination of the balance between nitrogenous excretion and accumulation in a 70 g P. schlosseri revealed that degradation of amino acids in general was likely to be suppressed to slow down the build-up of ammonia internally. It is possible that such a strategy may be widely adopted, especially by obligatory air-breathing fishes, to avoid ammonia intoxication during aerial exposure.

Asunto(s)

Alanina/metabolismo , Aminoácidos/metabolismo , Metabolismo Energético , Actividad Motora , Perciformes/metabolismo , Adenosina Trifosfato/metabolismo , Aire , Aminoácidos/sangre , Animales , Ciclo del Ácido Cítrico , Glucógeno/metabolismo , Ácido Láctico/metabolismo , Hígado/metabolismo , Malato Deshidrogenasa/metabolismo , Malatos/metabolismo , Músculos/metabolismo , Oxidación-Reducción , Fotoperiodo , Esfuerzo Físico , Ácido Pirúvico/metabolismo