Function and control of the fish secondary vascular system, a contrast to mammalian lymphatic systems.

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.

A unique mode of tissue oxygenation and the adaptive radiation of teleost fishes.

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.

Endothelium-derived nitric oxide partially mediates salbutamol-induced vasodilatations.

This study examined the ability of salbutamol (selective beta 2-adrenoceptor agonist) to cause endothelium-dependent relaxation in rat aortic rings and depressor response in conscious rats. Salbutamol (0.01-100 microM) concentration dependently relaxed preconstricted aortic rings. The relaxant response was partially attenuated by either mechanical removal of the endothelium or treatment with NG-nitro-L-arginine methyl ester (L-NAME, 100 microM). In conscious rats, either i.v. infused phenylephrine (5 micrograms/kg per min) or i.v. bolus injected L-NAME (12.8 mg/kg), but not the vehicle, caused similar sustained increases in mean arterial pressure (MAP). I.v. infused salbutamol (2-128 micrograms/kg per min, each dose for 5 min) dose dependently decreased MAP in vehicle-treated rats; the depressor responses were potentiated by hypertension induced by phenylephrine. In contrast, the magnitudes of the depressor response to salbutamol in L-NAME-treated rats were less than those in rats pretreated with phenylephrine or the vehicle. I.v. bolus injections of salbutamol (0.25-16 micrograms/kg) also caused dose-dependent and transient decreases in MAP in vehicle-treated rats. The magnitude but not the duration of the depressor response to salbutamol was less in rats treated with L-NAME, compared to those in rats given phenylephrine or the vehicle. These results suggest that endothelium-derived nitric oxide is partially involved in beta 2-adrenoceptor-mediated vasodilatation.

Hepatic mixed function oxygenase activity and glutathione S-transferase activity in mice following ethanol consumption and withdrawal.

The effect of an 8 day liquid diet containing 7% v/v ethanol and the effect of ethanol withdrawal on several drug metabolizing enzyme activities, cytochrome P-450 content and glutathione S-transferase activity (GST) has been studied in male C57/BL mice. After treatment, hepatic microsomal activities toward benzphetamine (BNZ), biphenyl (BPH) and dimethylnitrosamine (DMN) and cytosolic GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB) were determined. Ethanol treatment caused a differential time dependent increase in the metabolism of the 4 xenobiotics. Increased BPH-4-hydroxylase activity correlated most closely with that of the increased concentration of hepatic P-450. That is, both values were increased (5.8-fold) over controls after 8 days of ethanol treatment. Ethanol withdrawal (24 h) resulted in a 46% reduction in the P-450 content and a 26% reduction in BPH-4-hydroxylase activity compared to the elevated values at day 8. By 48 h, the values were no different from controls. DNA-N-demethylase, BNZ-N-demethylase and GST activities all increased after 4 days of ethanol treatment and remained the same at 8 days. However, ethanol withdrawal resulted in differential time dependent changes in the activities towards BNZ, DMN, and CDNB. While DMN-N-demethylase activity returned to control activity within 24 h, BNZ-N-demethylase activity did not change for the first 24 h of withdrawal, but returned to control activity by 48 h. GST activity had not decreased by 48 h of withdrawal. These data suggest that ethanol induces several cytochrome P-450 isozymes that have a time difference in induction by ethanol and reduction following ethanol withdrawal. Furthermore, ethanol induction of GSTs occurs quickly (4 days) and remains elevated at least 48 h after ethanol withdrawal.

Molecular cloning and characterization of a hypoxia-responsive CITED3 cDNA from grass carp.

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.

The effect of feeding and fasting on ammonia toxicity in juvenile rainbow trout, Oncorhynchus mykiss.

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.

Swimming and ammonia toxicity in salmonids: the effect of sub lethal ammonia exposure on the swimming performance of coho salmon and the acute toxicity of ammonia in swimming and resting rainbow trout.

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.

Concentrations of persistent lipophilic compounds in fish are determined by exchange across the gills, not through the food chain.

Uptake of persistent lipophilic toxicants in fish occurs via the food and by transfer across the body surface, notably the gills. Flux rates of most lipid soluble toxicants across the gills is rapid and the animal must eat at very high rates for feeding to have a significant effect on toxicant concentration in the body. The relative rates of uptake via feeding and transfer across the gills are analyzed from a theoretical and experimental standpoint. At the low feeding rates typical of fish, the uptake of toxicants in the food can be ignored when estimating toxicant body concentration.

Ionoregulatory changes in the gill epithelia of coho salmon during seawater acclimation.

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.

Five tropical air-breathing fishes, six different strategies to defend against ammonia toxicity on land.

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.

Air breathing and ammonia excretion in the giant mudskipper, Periophthalmodon schlosseri.

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.

Alkaline environmental pH has no effect on ammonia excretion in the mudskipper Periophthalmodon schlosseri but inhibits ammonia excretion in the related species Boleophthalmus boddaerti.

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.

Ammonia toxicity in fish.

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.

An in vivo study of common carp (Cyprinus carpio L.) liver during prolonged hypoxia.

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.

Tribute to R. G. Boutilier: acid-base transfer across fish gills.

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.

Adenosine/nitric oxide crosstalk in the branchial circulation of Squalus acanthias and Anguilla anguilla.

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.

Cardio-respiratory modeling in fishes and the consequences of the evolution of airbreathing.

The microcirculation of the respiratory organ of water and air breathing vertebrates is similar and can be described as sheet flow. The gross morphologies of the systems, however, are very different and reflect the properties of the medium. The fish heart has a single ventricle that forces blood first through the gills and then through the body. The pressure in the gills is higher than in the systemic circulation, the reverse of the situation seen in mammals. The gill epithelium is thicker than that in the lung and is involved in ionic and acid-base functions carried out in the kidney of mammals. Gills stick together in air. Therefore, fish breathe air using some other structure, such as the gut or mouth, the swimbladder, or the skin. The gills are retained for carbon dioxide excretion and ion and acid-base regulation. This results in a separation of oxygen uptake and carbon dioxide excretion. The gills are often modified in air-breathing fish such that venous blood flows to well developed gills for carbon dioxide and acid excretion, whereas oxygenated blood flow bypasses the gills. This is the beginning of a separation of flows in the heart which is more highly developed in amphibians and reptiles and complete in mammals. The loss of gills requires transfer of ionic and acid base regulation processes to the skin in amphibia and to the kidney in reptiles and mammals, allowing a completely terrestrial existence. The organization of the venous system is influenced by the degree of support offered by the medium.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of water pH on swimming performance in rainbow trout (Salmo gairdneri, Richardson).

A Brett-type respirometer was used to measure the effect of water pH on swimming performance of rainbow trout (Salmo gairdneri). Variations in water pH between 6 and 9 had no measurable effect on maximum aerobic swimming speed. At water pH 4, 5, and 10, however, the critical velocity was only 55, 67, and 61% respectively of that recorded for fish in water of pH 7. Exposure to acid conditions increased coughing and breathing frequency. Acid exposure resulted in a decrease whereas alkaline exposure resulted in an increase in both whole blood and red blood cell pH. Blood gas and acid-base characteristics showed little change during swimming at ∼2.0 BL/second, but exhaustive swimming resulted in a marked and immediate drop in blood pH in fish in acid, alkaline and neutral water. The blood acid-base status was restored to resting levels after exercise in neutral and alkaline water, but the acidosis was maintained following exercise in acid water. Fatigue occurred earlier and blood lactate levels increased to a higher level in fish swum to exhaustion in acid or alkaline water, compared with fish in neutral water.

The effect of sub-lethal ammonia exposure on fed and unfed rainbow trout: the role of glutamine in regulation of ammonia.

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.

Ammonia distribution and excretion in fish.

This paper reviews the literature concerning ammonia production, storage and excretion in fish. Ammonia is the end product of protein catabolism and is stored in the body of fish in high concentrations relative to basal excretion rates. Ammonia, if allowed to accumulate, is toxic and is converted to less toxic compounds or excreted. Like other weak acids and bases, ammonia is distributed between tissue compartments in relation to transmembrane pH gradients. NH3 is generally equilibrated between compartments but NH4 (+) is distributed according to pH. Ammonia is eliminated from the blood upon passage through the gills. The mechanisms of branchial ammonia excretion vary between different species of fish and different environments, and primarily involves NH3 passive diffusion and NH4 (+)/Na(+) exchange. Water chemistry near the gill surface may also be important to ammonia excretion, but a more accurate measurement of the NH3 gradient across the gill epithelium is required before a more detailed analysis of NH3 and NH4 (+) excretion can be made.