ABSTRACT In the two decades since the first cloning of the mammalian kidney urea transporter (UT-A), UT genes have been identified in a plethora of organisms, ranging from single-celled bacteria to metazoans. In this review, focusing mainly on vertebrates, we first reiterate the multiple catabolic and anabolic pathways that produce urea, then we reconstruct the phylogenetic history of UTs, and finally we examine the tissue distribution of UTs in selected vertebrate species. Our analysis reveals that from an ancestral UT, three homologues evolved in piscine lineages (UT-A, UT-C and UT-D), followed by a subsequent reduction to a single UT-A in lobe-finned fish and amphibians. A later internal tandem duplication of UT-A occurred in the amniote lineage (UT-A1), followed by a second tandem duplication in mammals to give rise to UT-B. While the expected UT expression is evident in excretory and osmoregulatory tissues in ureotelic taxa, UTs are also expressed ubiquitously in non-ureotelic taxa, and in tissues without a complete ornithine–urea cycle (OUC). We posit that non-OUC production of urea from arginine by arginase, an important pathway to generate ornithine for synthesis of molecules such as polyamines for highly proliferative tissues (e.g. testis, embryos), and neurotransmitters such as glutamate for neural tissues, is an important evolutionary driving force for the expression of UTs in these taxa and tissues.

In teleosts, a branchial metabolon links ammonia excretion to Na + uptake via Rh glycoproteins and other transporters. Ureotelic elasmobranchs are thought to have low branchial ammonia permeability, and little is known about Rh function in this ancient group. We cloned Rh cDNAs ( Rhag, Rhbg and Rhp2 ) and evaluated gill ammonia handling in Squalus acanthias . Control ammonia excretion was <5% of urea-N excretion. Sharks exposed to high environmental ammonia (HEA; 1 mmol −1 NH 4 HCO 3 ) for 48 h exhibited active ammonia uptake against partial pressure and electrochemical gradients for 36 h before net excretion was re-established. Plasma total ammonia rose to seawater levels by 2 h, but dropped significantly below them by 24–48 h. Control Δ P NH 3 (the partial pressure gradient of NH 3 ) across the gills became even more negative (outwardly directed) during HEA. Transepithelial potential increased by 30 mV, negating a parallel rise in the Nernst potential, such that the outwardly directed NH 4 + electrochemical gradient remained unchanged. Urea-N excretion was enhanced by 90% from 12 to 48 h, more than compensating for ammonia-N uptake. Expression of Rhp2 (gills, kidney) and Rhbg (kidney) did not change, but branchial Rhbg and erythrocytic Rhag declined during HEA. mRNA expression of branchial Na + /K + -ATPase ( NKA ) increased at 24 h and that of H + -ATPase decreased at 48 h, while expression of the potential metabolon components Na + /H + exchanger2 ( NHE2 ) and carbonic anhydrase IV ( CA-IV ) remained unchanged. We propose that the gill of this nitrogen-limited predator is poised not only to minimize nitrogen loss by low efflux permeability to urea and ammonia but also to scavenge ammonia-N from the environment during HEA to enhance urea-N synthesis.

SUMMARY Digestion affects nitrogen metabolism in fish, as both exogenous and endogenous proteins and amino acids are catabolized, liberating ammonia in the process. Here we present a model of local detoxification of ammonia by the intestinal tissue of the plainfin midshipman ( Porichthys notatus ) during digestion, resulting in an increase in urea excretion of gastrointestinal origin. Corroborating evidence indicated whole-animal ammonia and urea excretion increased following feeding, and ammonia levels within the lumen of the midshipman intestine increased to high levels (1.8±0.4 μmol N g −1 ). We propose that this ammonia entered the enterocytes and was detoxified to urea via the ornithine-urea cycle (O-UC) enzymes, as evidenced by a 1.5- to 2.9-fold post-prandial increase in glutamine synthetase activity (0.14±0.05 and 0.28±0.02 μmol min −1 g −1 versus 0.41±0.03 μmol min −1 g −1 ) and an 8.7-fold increase in carbamoyl phosphate synthetase III activity (0.3±1.2 versus 2.6±0.4 nmol min −1 g −1 ). Furthermore, digestion increased urea production by isolated gastrointestinal tissue 1.7-fold, supporting our hypothesis that intestinal tissue synthesizes urea in response to feeding. We further propose that the intestinal urea may have been excreted into the intestinal lumen via an apical urea transporter as visualized using immunohistochemistry. A portion of the urea was then excreted to the environment along with the feces, resulting in the observed increase in urea excretion, while another portion may have been used by intestinal ureolytic bacteria. Overall, we propose that P. notatus produces urea within the enterocytes via a functional O-UC, which is then excreted into the intestinal lumen. Our model of intestinal nitrogen metabolism does not appear to be universal as we were unab le to activate the O-UC in the intestine of fed rainbow trout. However, literature values suggest that multiple fish species could follow this model.

SUMMARY Models of branchial transport in teleosts have been reshaped by the recent discovery of Rhesus (Rh) glycoproteins, a family of proteins that facilitate the movement of NH 3 across cell membranes. This study examines the effects of crowding and feeding on ammonia excretion in gulf toadfish ( Opsanus beta ) within the context of Rh glycoproteins and the ammonia-fixing enzyme, glutamine synthetase (GS). Four Rh isoforms (Rhag, Rhbg, Rhcg1 and Rhcg2) were isolated from toadfish. Tissue distributions showed higher levels of mRNA expression in the gills and liver, moderate levels in the intestine and lower levels in the stomach. Crowding significantly lowered branchial Rh expression and ammonia excretion rates in fasted toadfish. A comparison of Rh expression in the digestive tract revealed relatively low levels of Rhcg1 and Rhcg2 in the stomach and high mRNA abundance of Rhbg, Rhcg1 and Rhcg2 in the intestine of fasted, crowded toadfish. We speculate that these trends may reduce secretion and enhance absorption, respectively, to minimize the amount of ammonia that is lost through gastrointestinal routes. By contrast, these patterns of expression were modified in response to an exogenous ammonia load via feeding. Post-prandial ammonia excretion rates were elevated twofold, paralleled by similar increases in branchial Rhcg1 mRNA, gastric Rhcg1 mRNA and mRNA of all intestinal Rh isoforms. These changes were interpreted as an attempt to increase post-prandial ammonia excretion rates into the environment owing to a gradient created by elevated circulating ammonia concentrations and acidification of the digestive tract. Overall, we provide evidence that toadfish modulate both the expression of Rh isoforms and urea synthesis pathways to tightly control and regulate nitrogen excretion.

SUMMARY In their native environment, gulf toadfish excrete equal quantities of ammonia and urea. However, upon exposure to stressful conditions in the laboratory (i.e. crowding, confinement or air exposure), toadfish decrease branchial ammonia excretion and become ureotelic. The objective of this study was to determine the influences of cortisol and ammonia on ammonia excretion relative to expression of Rhesus (Rh) glycoproteins and the ammonia-fixing enzyme, glutamine synthetase (GS). In vivo infusions and/or injections were used to manipulate corticosteroid activity and plasma ammonia concentrations in ureotelic toadfish. Metyrapone treatment to lower circulating cortisol levels resulted in a 3.5-fold elevation of ammonia excretion rates, enhanced mRNA expression of two of the toadfish Rh isoforms (Rhcg1 and Rhcg2), and decreased branchial and hepatic GS activity. Correspondingly, cortisol infusion decreased ammonia excretion 2.5-fold, a change that was accompanied by reduced branchial expression of all toadfish Rh isoforms (Rhag, Rhbg, Rhcg1 and Rhcg2) and a twofold increase in hepatic GS activity. In contrast, maintenance of high circulating ammonia levels by ammonia infusion enhanced ammonia excretion and Rh expression (Rhag, Rhbg and Rhcg2). Toadfish treated with cortisol showed an attenuated response to ammonia infusion with no change in Rh mRNA expression or GS activity. In summary, the evidence suggests that ammonia excretion in toadfish is modulated by cortisol-induced changes in both Rh glycoprotein expression and GS activity.

SUMMARY The gulf toadfish ( Opsanus beta ) is a facultatively ureotelic fish that excretes primarily urea under conditions of crowding or confinement. To examine the relationship between ammonia production, urea production and the ornithine–urea cycle (O–UC) enzyme activity and mRNA expression,we subjected toadfish to two-day and seven-day crowding regimes. Plasma cortisol levels were measured and liver tissue was assayed for ammonia and urea concentrations. Liver glutamine synthetase (GS), carbamoyl phosphate synthetase III (CPS), ornithine carbamoyl transferase (OCT) and arginase (ARG)activities were also measured. Quantitative PCR was utilized to determine liver GS, CPS, OCT, ARG, argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) mRNA expression. Hepatic ammonia concentrations decreased with increased duration of crowding whereas liver urea and circulating cortisol levels increased. An elevation in enzyme activity with increased duration of crowding was observed for all four O-UC enzymes examined. By contrast, mRNA expression was variable for the O–UC enzymes and only CPS and ASS had mRNA expression levels that were elevated in crowded fish. These results suggest that the activities of O–UC enzymes are better predictors for urea production than O–UC enzyme mRNA expression levels.

SUMMARY Gulf toadfish, Opsanus beta , are one among a group of unusual teleosts that excrete urea as their predominant nitrogen end product in response to stressful conditions. Under conditions of crowding or confinement,fasted toadfish excrete the majority of their nitrogen waste in large pulses of urea (>90% of total nitrogen) lasting up to 3 h. An earlier study demonstrated that cortisol has an inhibitory influence on urea pulse size. The present study tested the hypothesis that cortisol mediates changes in urea pulse size in ureotelic toadfish through the glucocorticoid receptor (GR) and not the mineralocorticoid receptor (MR). In vivo pharmacological investigations were used to manipulate the corticosteroid system in crowded toadfish, including experimentally lowering plasma cortisol levels by the injection of metyrapone, blocking cortisol receptors through exposure to either RU-486 (GR antagonist) and spironolactone (MR antagonist), or through exogenous infusion of the tetrapod mineralocorticoid aldosterone (tetrapod MR agonist). The data demonstrate that lowering the activity of cortisol, either by inhibiting its synthesis or by blocking its receptor, resulted in a two- to threefold increase in pulse size with no accompanying change in pulse frequency. Treatment with spironolactone elicited a minor (∼1.5-fold)reduction in pulse size, as did aldosterone treatment, suggesting that the anti-mineralocorticoid spironolactone has an agonistic effect in a piscine system. In summary, the evidence suggests that urea transport mechanisms in pulsing toadfish are upregulated in response to low cortisol, mediated primarily by GRs, and to a lesser extent MRs.

SUMMARY The diversity of mechanisms of ammonia and urea excretion by the gills and other epithelia of aquatic organisms, especially fish and crustaceans, has been studied for decades. Although the decades-old dogma of `aquatic species excrete ammonia' still explains nitrogenous waste excretion for many species,it is clear that there are many mechanistic variations on this theme. Even within species that are ammonoteles, the process is not purely `passive',often relying on the energizing effects of proton and sodium–potassium ATPases. Within the ammonoteles, Rh (Rhesus) proteins are beginning to emerge as vital ammonia conduits. Many fishes are also known to be capable of substantial synthesis and excretion of urea as a nitrogenous waste. In such species, members of the UT family of urea transporters have been identified as important players in urea transport across the gills. This review attempts to draw together recent information to update the mechanisms of ammonia and urea transport by the gills of aquatic species. Furthermore, we point out several potentially fruitful avenues for further research.

SUMMARY After a meal, dogfish exhibit a metabolic alkalosis in the bloodstream and a marked excretion of basic equivalents across the gills to the external seawater. We used the H + , K + -ATPase pump inhibitor omeprazole to determine whether these post-prandial alkaline tide events were linked to secretion of H + (accompanied by Cl – ) in the stomach. Sharks were fitted with indwelling stomach tubes for pretreatment with omeprazole (five doses of 5 mg omeprazole per kilogram over 48 h) or comparable volumes of vehicle (saline containing 2% DMSO) and for sampling of gastric chyme. Fish were then fed an involuntary meal by means of the stomach tube consisting of minced flatfish muscle (2% of body mass) suspended in saline (4% of body mass total volume). Omeprazole pre-treatment delayed the post-prandial acidification of the gastric chyme, slowed the rise in Cl – concentration of the chyme and altered the patterns of other ions, indicating inhibition of H + and accompanying Cl – secretion. Omeprazole also greatly attenuated the rise in arterial pH and bicarbonate concentrations and reduced the net excretion of basic equivalents to the water by 56% over 48 h. Arterial blood CO 2 pressure ( P a CO 2 ) and plasma ions were not substantially altered. These results indicate that elevated gastric H + secretion (as HCl) in the digestive process is the major cause of the systemic metabolic alkalosis and the accompanying rise in base excretion across the gills that constitute the alkaline tide in the dogfish.

SUMMARY The goal of the present study was to investigate the role of circulating cortisol and urea in the transcriptional regulation of branchial glutamine synthetase (GS), which incorporates NH 3 into glutamate to form glutamine, and the toadfish urea transporter, tUT, which is involved in urea excretion across the gill of the gulf toadfish. GS (of which there are two isoforms, LGS and GGS) and tUT mRNA expression and activity were measured in toadfish exposed to treatments that would induce variable stress responses. In addition, the role of circulating urea in tUT regulation was investigated by infusing toadfish with urea alone or in combination with intraperitoneal injection of RU486, a corticosteroid type II receptor antagonist. There was a 4.8-fold upregulation in the mRNA expression of the gill-specific GS isoform(GGS) in response to cortisol infusion and a similar upregulation in the more ubiquitous isoform (LGS). Furthermore, there was a significant 1.9-fold and 3.3-fold upregulation in the mRNA expression of the toadfish urea transporter,tUT, in response to stress through crowding or exogenous cortisol loading through infusion, respectively. In addition, tUT was found to have a urea-sensitive component to transcriptional regulation that was independent of circulating cortisol concentrations. However, the changes measured in mRNA expression of GGS, LGS and tUT did not correspond with changes in protein activity. To determine the cell type(s) involved in glutamine production and urea excretion, we attempted to localize GGS, LGS and tUT using in situ hybridization. This study is the first to show that GGS and tUT expression appear to occur in gill mitochondria-rich cells of toadfish,suggesting that these cells play a combined glutamine production and urea excretion role, which may have implications for predator avoidance.

SUMMARY In order to study the physiological consequences of voluntary feeding in the gastrointestinal tract of a ureotelic marine elasmobranch, dogfish (fasted for 96 h) were sampled at various times up to 360 h after consuming a 5–6% ration of teleost fish (hake) under natural feeding conditions. Digestion and absorption were completed between 120 and 360 h post-feeding. The tissue masses of different segments of the gastrointestinal tract increased and decreased markedly as the chyme moved through, mainly because of fluid engorgement rather than hyperplasia. In fasted dogfish, the cardiac and pyloric stomachs contained only small volumes of highly acidic fluid (pH 1.77±1.12, 2.05±0.08) similar in composition to seawater. Feeding resulted in gastric pHs of 3.20±0.31 and 3.95±0.40 at 6 h, followed by slow declines through 60 h. An alkaline tide in the blood also occurred at 6 h. In the face of large changing masses of highly acidic chyme in the stomachs, the pH (6.50±0.10), ionic composition and volume of chyme in the intestine (spiral valve) were precisely regulated from 6 to 60 h post-feeding at very different values from those in the stomachs, and intestinal HCO – 3 remained low (5.12±0.83 mmol l –1 ). The colon was usually empty and its pH constant at 7.20±0.16 at all times. Despite the ingestion of strongly hypo-osmotic teleost tissue, the osmolality of the chyme remained in equilibrium with that of the blood plasma in all segments at all times after feeding. Much of the osmotic equilibration was because of the secretion of urea into the chyme,particularly in the intestine. After feeding, gastric fluid concentrations of Na + and Mg 2+ declined, K + and Ca 2+ increased, whereas Cl – exhibited little change, indicating that additional drinking of seawater was minimal. Na + ,K + , water and especially Cl – were absorbed in the intestine, whereas Mg 2+ and Ca 2+ were largely excluded. Our results illustrate the complex integration of digestive and ionoregulatory function in the elasmobranch digestive tract, and marked differences from the teleost pattern.

SUMMARY Because urea is bioenergetically expensive to synthesize, few aquatic teleostean (bony) fish make or excrete much urea beyond early development and excrete the majority of nitrogenous waste as the readily diffusible ammonia. The gulf toadfish is one of a few adult teleostean fish that excretes predominately urea. Most studies of chemosensing by fish predators have focused on amino acids as odorants, but we tested the chemo-attractiveness of both urea and ammonia. We report that characteristic `prey-attack' behaviors by a key toadfish predator, gray snapper, were elicited by low ammonia concentrations (<100 nmol N l -1 ) and similar urea concentrations blunted the ammonia-induced component of attacks. Thus, urea functions as a cloaking molecule, explaining why toadfish co-excrete urea with ammonia. Furthermore, ammonia waste is an important chemical attractant for piscine predators.

SUMMARY In order to investigate the metabolic poise of the elasmobranch rectal gland, we conducted two lines of experimentation. First, we examined the effects of feeding on plasma metabolites and enzyme activities from several metabolic pathways in several tissues of the dogfish shark, Squalus acanthias, after starvation and at 6, 20, 30 and 48 h post-feeding. We found a rapid and sustained ten-fold decrease in plasma β-hydroxybutyrate at 6 h and beyond compared with starved dogfish, suggesting an upregulation in the use of this substrate, a decrease in production, or both. Plasma acetoacetate levels remain unchanged, whereas there was a slight and transient decrease in plasma glucose levels at 6 h. Several enzymes showed a large increase in activity post-feeding, including β-hydroxybutyrate dehydrogenase in rectal gland and liver, and in rectal gland, isocitrate dehydrogenase, citrate synthase, lactate dehydrogenase, aspartate amino transferase, alanine amino transferase, glutamine synthetase and Na + /K + ATPase. Also notable in these enzyme measurements was the overall high level of activity in the rectal gland in general. For example, activity of the Krebs' TCA cycle enzyme citrate synthase (over 30 U g -1 ) was similar to activities in muscle from other species of highly active fish. Surprisingly, lactate dehydrogenase activity in the gland was also high (over 150 U g -1 ), suggesting either an ability to produce lactate anaerobically or use lactate as an aerobic fuel. Given these interesting observations, in the second aspect of the study we examined the ability of several metabolic substrates (alone and in combination) to support chloride secretion by the rectal gland. Among the substrates tested at physiological concentrations (glucose, β-hydroxybutyrate, lactate,alanine, acetoacetate, and glutamate), only glucose could consistently maintain a viable preparation. Whereas β-hydroxybutyrate could enhance gland activity when presented in combination with glucose, surprisingly it could not sustain chloride secretion when used as a lone substrate. Our results are discussed in the context of the in vivo role of the gland and mechanisms of possible upregulation of enzyme activities.

SUMMARY We investigated the consequences of feeding for acid–base balance,nitrogen excretion, blood metabolites and osmoregulation in the Pacific spiny dogfish. Sharks that had been starved for 7 days were surgically fitted with indwelling stomach tubes for gastric feeding and blood catheters for repetitive blood sampling and were confined in chambers, allowing measurement of ammonia-N and urea-N fluxes. The experimental meal infused via the stomach tube consisted of flatfish muscle (2% of body mass) suspended in saline (4% of body mass total volume). Control animals received only saline(4% of body mass). Feeding resulted in a marked rise in both arterial and venous pH and HCO 3 – concentrations at 3–9 h after the meal, with attenuation by 17 h. Venous Ṗ O2 also fell. As there were negligible changes in Ṗ CO2 ,the response was interpreted as an alkaline tide without respiratory compensation, associated with elevated gastric acid secretion. Urea-N excretion, which comprised >90% of the total, was unaffected, while ammonia-N excretion was very slightly elevated, amounting to <3% of the total-N in the meal over 45 h. Plasma ammonia-N rose slightly. Plasma urea-N,TMAO-N and glucose concentrations remained unchanged, while free amino acid and β-hydroxybutyrate levels exhibited modest declines. Plasma osmolality was persistently elevated after the meal relative to controls, partially explained by a significant rise in plasma Cl – . This marked post-prandial conservation of nitrogen is interpreted as reflecting the needs for urea synthesis for osmoregulation and protein growth in animals that are severely N-limited due to their sporadic and opportunistic feeding lifestyle in nature.

SUMMARY The marine gulf toadfish ( Opsanus beta ) is an unusual teleost fish as it is able to switch between ammoniotelism and ureotelism in response to a variety of laboratory conditions. The present study integrates field work conducted in Biscayne and Florida Bays, USA with laboratory studies to examine ureotelism during the early life history stages of O. beta . Adult toadfish voluntarily nested in artificial shelters placed amongst seagrass beds and were found to be predominantly ureotelic under natural conditions as the internal shelter water had mean urea and ammonia concentrations( N =51) of 14.2±1.6 μmol N l –1 and 8.9±0.9 μmol N l –1 , respectively. Toadfish successfully spawned in shelters, providing eggs, larvae and juvenile toadfish for laboratory study. In the lab, juvenile toadfish were also ureotelic and urea was excreted in pulsatile events that accounted for 62.0±5.9% of total urea-N excreted. Excretion rates of urea-N and ammonia-N were 1.018±0.084 μmol N h –1 g –1 and 0.235±0.095 μmol N h –1 g –1 ,respectively. Field-collected eggs, larvae and juveniles expressed significant levels of the ornithine–urea cycle enzymes carbamoyl-phosphate synthetase III, ornithine transcarbamylase and arginase and the accessory enzyme glutamine synthetase, all of which increased in activity as toadfish developed through early life stages. In juveniles, the ammonia 96-h LC 50 value was 875 μmol N l –1 and there was a 3-fold increase in ornithine transcarbamylase activity in the 1000 μmol N l –1 NH 4 Cl treatment. The results are discussed in the context of the causal factor(s) for ureotelism in toadfish. Furthermore,the results of this study suggest it is unlikely that the adaptive significance of ureotelism in toadfish is a means to prevent fouling nests with ammonia and in turn poisoning offspring; however, additional study is warranted.

SUMMARY When injected arterially, serotonin (5-hydroxytryptamine; 5-HT) has been shown to elicit naturally sized urea pulse events in the gulf toadfish, Opsanus beta . The goal of the present study was to determine which 5-HT receptor(s) was involved in mediating this serotonergic stimulation of the pulsatile excretion mechanism. Toadfish were surgically implanted with caudal arterial catheters and intraperitoneal catheters and injected with either 8-OH-DPAT (1 μmol kg –1 ), a selective 5-HT 1A receptor agonist, α-methyl-5-HT (1 μmol kg –1 ), a 5-HT 2 receptor agonist, or ketanserin, a 5-HT 2 receptor antagonist (0.01, 0.1, 1 and 10 μmol kg –1 ) plus α-methyl-5-HT. 8-OH-DPAT injection did not mediate an increase in urea excretion, ruling out the involvement of 5-HT 1A receptors in pulsatile excretion. However, within 5 min,α-methyl-5-HT injection caused an increase in the excretion of urea in>95% ( N =27) of the fish injected, with an average pulse size of 652±102 μmol N kg –1 ( N =26). Withα-methyl-5-HT injection there was no corresponding increase in ammonia or [ 3 H]PEG 4000 permeability. Urea pulses elicited byα-methyl-5-HT were inhibited in a dose-dependent fashion by the 5-HT 2 receptor antagonist ketanserin, which at low doses caused a significant inhibition of pulse size and at higher doses significantly inhibited the occurrence of pulsatile excretion altogether. However, neither 8-OH-DPAT nor α-methyl 5-HT injection had an effect on plasma cortisol or plasma urea concentrations. These findings suggest the involvement of a 5-HT 2 -like receptor in the regulation of pulsatile urea excretion.

SUMMARY We characterized the expression of the nitrogen metabolism enzyme glutamine synthetase [GSase; L-glutamate: ammonia ligase (ADP-forming), E.C. 6.3.1.2] in tissues of the gulf toadfish Opsanus beta subjected to unconfined(ammonotelic) and confined (ureotelic) conditions. Enzymological results demonstrate that mass-specific GSase activities rank in the order of brain> liver > stomach ≈ kidney > intestine > gill > heart/spleen> muscle. When tissue mass is used to calculate a glutamine synthetic potential, the liver has the greatest, followed by muscle > stomach and intestine, with minor contributions from the remaining tissues. Additionally,during confinement stress, GSase activity increases significantly only in liver (fivefold) and muscle (twofold), tissues that previously showed significant expression of the other enzymes of urea synthesis. Western analyses of samples on SDS gels demonstrated that GSase-specific protein content reflected enzyme activity, and all tissues except muscle had a single,similarly sized GSase subunit of 49.4 kDa; muscle showed staining of two bands of 36.8 and 98.9 kDa, which may possibly result from another gene product or post-translational modification. RT-PCR and RACE-PCR revealed the presence of a second GSase cDNA from gill tissue that shares only 73% nucleotide and amino acid sequence similarity with the GSase cDNA previously cloned from liver, and that lacks a mitochondrial leader-targeting sequence. RT-PCR and restriction digestion experiments demonstrated that mRNA from the original `liver' GSase is expressed in all tissues examined (liver, gill, stomach, intestine, kidney, brain and muscle),whereas the new `gill' form shows expression primarily in the gill. Gill GSase activity shows apparently exclusive expression in the soluble compartment,while other tissues expressing the `liver' form show both cytoplasmic and mitochondrial activities. Phylogenetic analysis of a number of GSases demonstrates that the toadfish gill GSase has a greater affinity for a clade that includes the Xenopus GSase genes and one of two Fugu GSase genes, than it has for a clade containing the toadfish liver GSase and other described teleost GSase genes. The results are discussed in the context of a prior hypothesis on an ammonia-trapping mechanism in the gill of the toadfish.

SUMMARY A recent proposal to store anthropogenic carbon dioxide in the deep ocean is assessed here with regard to the impacts on deep-living fauna. The stability of the deep-sea has allowed the evolution of species ill-equipped to withstand rapid environmental changes. Low metabolic rates of most deep-sea species are correlated with low capacities for pH buffering and low concentrations of ion-transport proteins. Changes in seawater carbon dioxide partial pressure ( P CO 2 ) may thus lead to large cellular P CO 2 and pH changes. Oxygen transport proteins of deep-sea animals are also highly sensitive to changes in pH. Acidosis leads to metabolic suppression, reduced protein synthesis,respiratory stress, reduced metabolic scope and, ultimately, death. Deep-sea CO 2 injection as a means of controlling atmospheric CO 2 levels should be assessed with careful consideration of potential biological impacts. In order to properly evaluate the risks within a relevant timeframe,a much more aggressive approach to research is warranted.

SUMMARY Female squirrelfish (Holocentridae) accumulate higher concentrations of hepatic zinc than any other known organism. In the liver cells, up to 70% of zinc is bound to metallothionein (MT), which is expressed at extremely high levels. These attributes are related to reproduction in ways that have not been fully characterized. In the present study, we have demonstrated that female-specific zinc and MT accumulation and distribution are strongly correlated to the onset of sexual maturity in Holocentrus adsenscionis . Sexual maturation not only resulted in increased concentrations of zinc in the liver and plasma, but also increased levels of hepatic MT mRNA. Furthermore, mature female squirrelfish exhibited greater proportions of MT protein in the nuclear liver cell fractions. To characterize the physiology further, we have examined the influence of the female sex hormone 17 β-estradiol (E 2 ). E 2 was not sufficient to elicit an increase in hepatic zinc concentrations or MT mRNA levels. E 2 administration did, however, result in increased levels of MT in the nuclear fraction as well as overall hepatic MT protein. E 2 also increased concentrations of zinc in the plasma. The changes in zinc concentration in the bloodstream followed the same time course as vitellogenin(VTG) transport from the liver. However, the high ratio of molar concentrations of zinc to VTG in the bloodstream suggest that VTG may not be the primary vehicle for hepato-ovarian zinc transport in squirrelfish.