The Journal of Experimental Biology 198, 127Ð135 (1995) 127 Printed in Great Britain © The Company of Biologists Limited 1995

INDUCTION OF ORNITHINEÐUREA CYCLE AND METABOLISM AND EXCRETION IN RAINBOW TROUT (ONCORHYNCHUS MYKISS) DURING EARLY LIFE STAGES

PATRICIA A. WRIGHT1,*, ANDREW FELSKIE1 AND PAUL M. ANDERSON2 1Department of Pathology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 and 2Department of Biochemistry and Molecular Biology, School of Medicine, University of Minnesota, Duluth, Minnesota 55812-2487, USA

Accepted 7 September 1994

Summary The ornithineÐurea cycle (OUC) is present in transcarbamylase (OTCase) were first detected in 40-day- elasmobranch fish and many terrestrial vertebrates. old embryos; activities peaked between days 53 and 71 and Recently, a functional OUC has been reported in a few then subsequently decreased. Adult liver activity teleost species, suggesting that all teleost fish have the for GSase was several-fold lower than in whole larval trout for the OUC, but expression is relatively rare. We and OTCase and CPSase III (glutamine- and N- investigated the possibility that the OUC is expressed acetylglutamate-dependent CPSase catalysing the first step during early development in trout as a mechanism for of the OUC) activities were essentially absent in adult liver. detoxifying produced from the catabolism of yolk We conclude that embryonic and larval trout are primarily protein. We followed ammonia and excretion rates, ammoniotelic. Urea is synthesized immediately after tissue ammonia and urea levels and OUC enzyme activities fertilization, but is not excreted until after the embryo is in rainbow trout up to 93 days after fertilization. Both hatched. The results provide evidence for the presence of ammonia and urea tissue concentrations increased several- the OUC in larval rainbow trout, since four of the OUC fold in the first 40 days after fertilization (embryo stage), enzymes are induced just after hatching and the levels of peaking at 1.7mmol N l 1 and 2.5mmol N l 1, respectively. activity are relatively high compared with those in adult Ammonia excretion could be detected in 4-day-old liver tissue. Furthermore, we suggest that all teleosts have embryos, but urea excretion was not initiated until after retained the OUC genes, which are expressed only during hatching (day 45). Urea excretion in larval fish (days 42–93) certain stages of development (embryogenesis), and in a increased several-fold and by day 93 was 14% of total few rare species expression is maintained throughout the nitrogen excretion, as found in adult trout. Glutamine life cycle to cope with unusual environmental conditions synthetase (GSase) and activities were detected in (e.g. alkaline water, air exposure). ‘whole animal’ homogenates just after hatching and the levels of activity increased markedly to day 93. Carbamoyl Key words: ammonia, urea, enzyme activities, embryo, larvae, phosphate synthetase (CPSase) and ornithine carbamoyl phosphate synthetase, liver, trout, Oncorhynchus mykiss.

Introduction Two major hepatic pathways exist for the production of urea previous studies, on a variety of teleosts, OUC enzyme + in vertebrates. Urea is formed from NH4 (or glutamine) and activities ranged from barely detectable to moderate, while HCO3 in the ornithineÐurea cycle [OUC, six enzymes: substantial uricolytic enzyme activity was present (Cvancara, carbamoyl phosphate synthetase (CPSase), ornithine 1969a; Huggins et al. 1969; Goldstein and Forster, 1965; transcarbamylase (OTCase), argininosuccinate synthetase and Anderson, 1980; Hayashi et al. 1989; Campbell and Anderson, , arginase and the accessory enzyme glutamine synthetase 1991; Cao et al. 1991; Wright, 1993; Wright et al. 1993). (GSase)]. The uricolytic pathway, by which is Moreover, urea constitutes less than 20% of total nitrogen converted to urea, involves three enzymes: uricase, excretion in most freshwater and marine teleosts. It was allantoinase and allantoicase. The OUC is found in marine surprising, therefore, to discover a teleost fish, the tilapia elasmobranch and coelacanth fish but, until recently, was Oreochromis alcalicus grahami, of Lake Magadi, Kenya thought to be absent from the largest group of living (pH10), that excreted all its nitrogenous wastes as urea and had vertebrates, the teleost fish (Brown and Cohen, 1960). In significant OUC enzyme activities (Randall et al. 1989; Wood

*Address for correspondence: Department of Zoology, University of Guelph, Guelph, Ontario, Canada NIG 2W1. 128 P. A. WRIGHT, A. FELSKIE AND P. M. ANDERSON et al. 1989b). Furthermore, studies on the marine toadfish that CPSase activity would be due to a mitochondrial Opsanus beta (Read, 1971; Mommsen and Walsh, 1989; ‘mammalian-type’ CPSase I, i.e. utilized only ammonia as the Walsh et al. 1990) and several freshwater, air-breathing nitrogen-donating and required the presence of N- teleosts from India (e.g. Heteropneustes fossilis, Saha and acetyl-L-glutamate (AGA) for activity (Campbell and Ratha, 1987, 1989) have demonstrated the presence of a Anderson, 1991). In ureosmotic elasmobranchs, however, the functional OUC and enhanced urea excretion rates. It is now CPSase catalysing the first step of the OUC is a mitochondrial clear that the full complement of OUC enzymes is present in CPSase III, which is similar to CPSase I except that it utilizes a few teleost species that are either adapted to unusual the amide group of glutamine in preference to ammonia as the environmental conditions (O. a. grahami) or are periodically nitrogen-donating substrate (Anderson, 1980, 1991). It is air-exposed (O. beta, H. fossilis). Taken together, these likely, therefore, that CPSase III rather than CPSase I fulfils findings suggest that all teleosts have retained the genes for the this role in all fish (Mommsen and Walsh, 1989, 1991; ornithineÐurea cycle, but expression of a functional pathway is Anderson, 1991; Campbell and Anderson, 1991; Cao et al. relatively rare. 1991). When measuring very low levels of CPSase activity, it Why is the OUC functional in a few teleost species, but is also necessary to be sure that the activity measured is not inoperative in the majority of species? It is highly unlikely that due to a CPSase II, which is probably present in most tissues the genes for the OUC would have been conserved during fish (Cao et al. 1991). CPSase II catalyses the first step in the evolution, if they no longer code for functional proteins. The pyrimidine nucleotide pathway; this enzyme utilizes either observation that many teleost species express all or most of the glutamine or ammonia (at high concentrations) as a substrate, OUC enzymes, even if some of the activities are very low, is subject to allosteric inhibition by uridine triphosphate (UTP), suggests (1) that some or all of the OUC enzymes may be does not require AGA for activity and is localised in the cytosol involved in other metabolic pathways, or (2) that the OUC may (Campbell and Anderson, 1991). be functional and expressed at higher levels during the early The present study was initiated to determine whether OUC life stages. In addressing the first possibility, it should be noted enzymes are induced in embryonic and/or larval trout and that arginase, the last enzyme in the OUC, is widely found in whether significant urea formation and/or excretion occurs. teleost tissues. Arginase is thought to be directly involved in Ammonia and urea excretion rates, ammonia and urea tissue urea synthesis via the degradation of dietary arginine levels, and four ornithineÐurea cycle enzyme activities (Cvancara, 1969b; Wright, 1993). The fact that the remaining (CPSase, OCTase, GSase and arginase) were measured in OUC enzymes have relatively low activities probably embryonic and larval rainbow trout (Oncorhynchus mykiss) up discounts a significant involvement in other metabolic to 93 days after fertilization. In addition, an effort was made reactions. The second possibility warrants further attention. to confirm that the CPSase activity detected was due to a Ammonia is formed when yolk protein is metabolised during CPSase III. the endogenous feeding period in embryonic and larval teleosts. The permeability of the egg membrane to nitrogenous wastes is relatively low (Smith, 1947) and it may be valuable Materials and methods to have a mechanism to detoxify excess ammonia during early Animals and experimental protocol development. Moreover, when the larvae hatch, ammonia Fertilized rainbow trout [Oncorhynchus mykiss (Walbaum)] detoxification may continue to be important since metabolism embryos were obtained from Blue Springs Fish Hatchery accelerates, but ammonia may not be efficiently excreted as (Hanover, Ontario, Canada). Eggs were maintained in gill surface area is initially undeveloped (Rombough, 1988). continuous-flow incubating troughs (11–13˚C) for up to 93 Only a few studies have addressed the issue of nitrogen days (water pH 8.0, water hardness 125mg l 1). metabolism and excretion in teleosts during the early life Approximately 2 weeks after hatching (i.e. at day 54), larval stages. Dépêche et al. (1979) demonstrated urea synthesis very fish were fed starter feed (Martin Mills Starter) several times early in embryonic (pre-hatch) development (8 days) from per day. radiolabelled bicarbonate (a substrate for the OUC). Rice and Each experiment (days 4, 8, 16, 30, 37, 40, 45, 50, 53, 58, Stokes (1974) reported that embryonic and larval (post- 64, 71 and 93) was performed on 180 embryos or larval fish hatching) rainbow trout synthesized urea, but that urea was not randomly selected from the incubation trough. Thirty animals excreted to the environment. Two OUC enzymes, OCTase and were placed in each of six chambers containing 50ml of arginase (but not CPSase and argininosuccinate synthetase), aerated water. To measure nitrogen excretion rates, water were detected in larval trout (Rice and Stokes, 1974). Rainbow samples (10ml) were collected at 0h and 4h and immediately trout fingerlings (25–40g) were reported to have the full frozen ( 20ûC) for later determination of urea and ammonia complement of liver OUC enzymes (Chiu et al. 1986); levels. Excretion rates (nmol N g 1 h 1) were calculated as the however, as in the Rice and Stokes (1974) study, the assay difference in concentration (0Ð4h) in nmol N l 1 multiplied by conditions for CPSase activity may not have been appropriate. the volume of the chamber (l) divided by the mass of the tissue The first step in the OUC is catalysed by CPSase I in (g) and the time period (4h) (1nmol l 1 of urea=2nmol N l 1 mammalian ureotelic species. Until recently, OUC capability urea and 1nmol l 1 ammonia=1nmol N l 1 ammonia, where in fish was assessed by measuring enzyme activities, assuming N=nitrogen). At the end of the 4h experiment, animals were Induction of urea cycle enzymes in larval trout 129 blotted and weighed. To determine water content, 10 animals was determined as previously described (Xiong and Anderson, were dried to constant mass in a drying oven at 50ûC and the 1989). Reaction mixtures for arginase contained 0.06moll 1 1 1 difference between wet and dry mass was recorded. The glycine, pH9.7, 4.8mmol l MnCl2, 15.2mmol l remainder of the animals were frozen in liquid N2 (stored at l-[guanido-14C]arginine; the reaction was terminated after 0, 80ûC for up to 2 months) for later analysis of tissue ammonia 30 and 60min (0, 3 and 6min for adult liver) and [14C]urea and urea concentrations and enzyme activities. formed was determined as described previously (Casey and Anderson, 1982; Ruegg and Russell, 1980). Reaction mixtures Water and tissue ammonia and urea analysis for measuring the -glutamyl reaction of GSase Water samples were analyzed for ammonia and urea levels was as described previously (Shankar and Anderson, 1985); - using colorimetric assays described by Verdouw et al. (1978) glutamyl hydroxamate formation was determined after reaction and Rahmatullah and Boyde (1980), respectively. For tissue for 0, 10, 20 and 40min. Reaction mixtures for CPSase 1 1 1 determinations, six subsamples of frozen embryos or larvae contained 10mmol l ATP, 14mmol l MgCl2, 5mmoll (0.25–0.50g) were ground to a fine powder in liquid N2, [14C]bicarbonate, 0.055mol l 1 Hepes, pH7.4, 0.055mol l 1 deproteinized in 1 volume of ice-cold perchloric acid (8%), KCl, 0.5mmol l 1 DTT, 0.25mmol l 1 EDTA and, where and centrifuged at 12000 g for 10min (4ûC). The supernatant indicated, 10mmol l 1 glutamine, 0.5mmol l 1 N-acetyl-L- 1 1 was removed, neutralized with saturated KHCO3 and glutamate (AGA), 100mmol l NH4Cl and/or 2.5mmol l centrifuged again (12000 g for 4min, 4˚C). The final uridine triphosphate (UTP); [14C]carbamoyl phosphate formed supernatant was analyzed for ammonia levels using a Sigma after 60min was determined as previously described diagnostic kit (171-C) and urea levels with Sigma reagents (Anderson et al. 1970). Mitochondrial CPSase III (an OUC (procedure no. 535). Ammonia and urea tissue concentrations enzyme) requires glutamine as a substrate and AGA as a were corrected for tissue water content and expressed as positive effector. Cytosolic CPSase II (involved in pyrimidine mmol N l 1 tissuewater. nucleotide biosynthesis) can use either ammonia or glutamine as a substrate, does not require AGA, and is inhibited by UTP. Preparation of extracts for enzyme analysis To differentiate between CPS II and III, duplicate experiments Tissues were stored at 80ûC for up to 6 weeks. All were performed to determine the effects of substrates and procedures were carried out at 4ûC. Extracts were prepared by effectors on enzyme activity (Table 1). The estimated limits of homogenization of 1.5Ð2g of the whole animal (except for detection for each of the four enzymes assayed, expressed in adult trout, where only liver tissue was used) with 4 volumes terms of molmin 1 g 1 tissue, were 0.0003 (CPSase), 0.01 of extract buffer (0.05moll 1 KCl, 0.05mol l 1 Hepes buffer, (OTCase), 0.08 (GSase) and 0.001 (arginase). pH7.5, 0.5mmol l 1 EDTA, 0.01mol l 1 ATP, 0.015mol l 1 It was not possible to measure argininosuccinate synthetase 1 1 MgCl2, 5mmol l NaHCO3 and 1mmoll DL-dithiothreitol, and lyase activities in this study because the levels of activities DTT), with a PotterÐElvjehem homogenizer followed by brief are typically very low (even in with a very active sonication. ATP, MgCl2 and NaHCO3 were included in the OUC) and insufficient tissue was available. extract buffer for CPSase measurements only, to maximize the stability of the extracted enzyme (Anderson, 1981). The Protein measurement supernatant (5ml) obtained after centrifugation at 12000 g for Protein was determined by the dye-binding method with 10min was passed through a 25ml Sephadex G-25 column Bio-Rad Laboratories reagents as described by Bradford equilibrated with 0.1mol l 1 Hepes, pH7.5, 0.1mol l 1 KCl, (1976), using bovine serum albumin as a standard. 0.5mmol l 1 EDTA and 1mmol l 1 DTT. Fractions containing most of the protein were pooled. Protein concentration was measured before and after the gel filtration chromatography Results step as a measure of the dilution of the extract; calculations of Urea and ammonia excretion rates and tissue levels molmin 1 g 1 tissue (=unitsg 1) were corrected for this Both ammonia and urea concentrations in the embryos dilution. Enzyme activities in each extract were measured increased several-fold in the first 40 days after fertilization, immediately following gel filtration chromatography. peaking at 1.7mm o l Nl 1 and 2.5mm o l Nl 1, respectively (Fig. 1). Following hatching, ammonia levels increased by a further Enzyme assays twofold and then decreased, as did urea concentrations, to OTCase, arginase, GSase and CPSase activities were 1 . 3m m o lNl 1 (ammonia) and 0.4mm o l Nl 1 (urea) by day 93. measured at 26ûC, essentially as previously described (see Ammonia excretion was detected immediately (day 4) in below). Reaction mixtures for OTCase contained 10mmol l 1 embryonic rainbow trout, while urea excretion was not detected ornithine, 5mmoll 1 carbamoyl phosphate, 0.05mol l 1 KCl, until after hatching (Fig. 2). Ammonia excretion increased 0.05mol l 1 Hepes, pH7.4, 0.5mmol l 1 DTT, 0.25mmol l 1 linearly until day 30 and then remained constant until after EDTA and 0.5ml of extract (prepared as above); the reaction hatching. Immediately after hatching (day 44), ammonia was terminated and protein precipitated after 0, 10, 20 and/or excretion rates increased relatively rapidly and were 17-fold 1 40min by addition of 75 l of 2moll HClO4, and citrulline higher than pre-hatch rates by day 93. Until day 57, urea concentration in the supernatant obtained after centrifugation excretion rates remained relatively low but, thereafter, rates 130 P. A. WRIGHT, A. FELSKIE AND P. M. ANDERSON

Table 1. Effect of substrates and effectors on CPSase activity Day 64 Adult liver Substrate and/or effector present Experiment 1 Experiment 2 Experiment 1 Experiment 2 None 0.8 0.5 0.9 0.8 Glutamine 1.2 0.8 2.0 1.4 Glutamine + N-acetyl-L-glutamate 2.4 1.7 2.1 1.4 Glutamine + uridine triphosphate 0.3 0.1 0.1 0.2 Ammonia 1.0 0.8 5.8 3.7 Ammonia + N-acetyl-L-glutamate 1.1 0.9 5.9 3.6 Ammonia + uridine triphosphate 0.3 0.2 0.7 0.6

CPSase activity, carbamoyl phosphate synthetase activity, nmolmin−1 g−1 tissue. Duplicate measurements are represented by experiment 1 and 2. increased several-fold. The percentage of nitrogenous wastes The effect of different substrates and effectors on CPSase (ammonia+urea) excreted as urea increased over the 93 day activity in 64-day-old larvae and in adult liver tissue is shown period: days 4Ð40, 0%; days 45Ð64, 7%; and days 71Ð93, 14 %. in Table 1. In the absence of substrate, glutamine or ammonia, Water content remained relatively constant in embryos until there is some activity that may be due to endogenous substrate just before hatching (approximately 65%; Fig. 3). Starting at (including, perhaps, side-chain glutamine amide groups in day 37, there was a linear increase in water content until the protein) in the tissue homogenate. The results clearly show a yolk sac was completely consumed (day 63); water content stimulation of glutamine-dependent CPSase activity by N- remained constant thereafter (approximately 84%; Fig. 3). acetylglutamate (AGA) in larval tissue, indicating the presence of CPSase III activity. This stimulation was not observed in adult Enzyme activities liver and indicates an absence of CPSase III. There is apparently CPSase activity (AGA and glutamine present in assay a mixture of CPSase II and CPSase III in larval tissue (CPSase mixtures=CPSase II+CPSase III) was very low, but could be II activity is inhibited by uridine triphosphate, UTP). Activity in detected in whole animal homogenates beginning at day 40 the presence of ammonia is low in larval tissue but relatively (Fig. 4A). Activity peaked at day 64 and then declined by day high in adult tissue, which is consistent with the presence of 93 to levels comparable to those in adult liver tissue. The ratio CPSase III in larval tissue and its absence in adult liver. of glutamine-dependent CPSase activity with AGA present to The time course of changes in OTCase activity was very that without AGA present is plotted in Fig. 4B. A ratio of more similar to that of total CPSase activity: activity increased after than 1.0 indicates CPS III activity. The ratio peaked at day 53 day 40, peaked at day 71 and then declined before day 93, with and declined gradually to day 93. The ratio in adult liver tissue little or no activity in adult liver (Fig. 4C). GSase activity was was close to 1.0, indicating an absence of CPS III and the low initially (day 16Ð40), but increased rapidly after day 40 presence of CPSase II only. (Fig. 4D). GSase activities in adult liver (units g 1 liver) were

Fig. 1. Ammonia-N (filled circles) and urea-N (open circles) concentrations in whole trout embryos and larvae up to 93 days after fertilization. Solid vertical bars indicate period of hatching and dashed Fig. 2. Ammonia-N (filled circles) and urea-N (open circles) excretion vertical bar indicates when the yolk sac disappeared. Values are rates by trout embryos and larvae up to 93 days after fertilization. means ±1 S.E.M. of six measurements. Other details are as in Fig. 1. Induction of urea cycle enzymes in larval trout 131

CPSase activity reported here are an order of magnitude lower than can be detected by the more commonly used colorimetric assay procedure, which explains in part why Rice and Stokes (1974) were not able to detect CPSase activity in their study of trout eggs or alevins. The latter study also did not utilize glutamine as a substrate (see Introduction). Chiu et al. (1986) reported unusually high levels of ammonia-dependent CPSase activity (approximately 0.05 molmin 1 g 1 liver) in trout fingerlings; AGA was included in their assay mixtures, but activity with glutamine as a substrate was not measured. Furthermore, they injected fish with [14C]bicarbonate, but were unable to recover labelled arginine, implying that the OUC was not operative. Hence, the presence of a functional OUC in rainbow trout fingerlings remains unproved. Fig. 3. Percentage water content of whole trout embryos and larvae Except for adult liver, enzyme activities were measured up to 93 days after fertilization. Other details are as in Fig. 1. from whole-animal preparations. It is reasonable to assume that the majority of these enzyme activities, expecially CPSase III 18-fold lower than in larval tissue (Fig. 4D). There was a and OTCase, are localized in the liver, which may represent dramatic increase in arginase activity after day 45 and, in only 1Ð2% of the mass of the whole animal. Thus, the levels contrast to GSase and OTCase, the level of activity in adult of these enzymes in developing liver may actually be quite liver was 20-fold higher than in 93-day-old larvae (Fig. 4E). It high. The absolute enzyme activities will also be should be noted that enzyme activities were measured in underestimated in embryo and fry prior to the disappearance whole-animal homogenates and that, prior to day 63, the yolk of the yolk sac (day 63) while, after day 63, the activity values sac was a component of the embryo and fry. After the yolk sac are truly representative of whole-animal tissue. had been consumed (day 63), the absolute enzyme activities We could not detect OUC enzymes prior to day 40, but this were more representative of whole-body levels. may reflect the limit of the sensitivities of the enzyme assays (see Materials and methods) and the need to use whole-animal homogenates. Dépêche et al. (1979) found that the highest rate Discussion of urea synthesis from [14C]bicarbonate occurred in 8-day-old The results presented here indicate that relatively high levels trout embryos. Moreover, we found that urea synthesis in of all four of the OUC enzymes measured out of the six that embryos was initiated a few days after fertilization (Fig. 1). are required for glutamine-dependent urea synthesis, appear at These results imply that the OUC may be functional very early about the time of hatching. Significant OUC enzyme activity in embryonic development, but our assay methods were not in the early life stages is somewhat surprising, since activity in sensitive enough to detect lower levels of activity (see adult trout is very low (Huggins et al. 1969; Wilkie et al. 1993), Materials and methods). Alternatively, uricolysis, the pathway as it is for most teleost species (Huggins et al. 1969; Anderson, by which urea is synthesized from uric acid, may be functional 1980; Cao et al. 1991; Wright, 1993). Our findings may help in early embryos. To our knowledge, uricolytic enzymes to clarify a perplexing question. Why have teleosts apparently (uricase, allantoinase and allantoicase) have not been measured retained the genes for the OUC enzymes when only a few in embryonic or larval trout, although uricolysis is an important species express a functional pathway (Oreochromis alcalicus pathway in adult teleosts (see below). grahami, Randall et al. 1989; Opsanus beta, Mommsen and Urea synthesis in the embryo may function as a mechanism Walsh, 1989; Heteropneustes fossilis, Saha and Ratha, 1987, to detoxify very high ammonia levels in the embryo and early 1989)? We suggest that all teleosts utilize the OUC pathway larva (see Griffith, 1991). The yolk, composed mostly of during embryogenesis, but in the later developmental stages protein and some fat, is the main source of fuel during the the genes are repressed, except in a few rare species. This endogenous feeding period (Smith, 1947; for a review, see hypothesis must be tested in a wide group of teleost species. Kamler, 1992). Although ammonia is excreted during these CPSase activities in larval tissue appear to be due to the early embryonic stages, ammonia concentrations still presence of both CPSase II (required for pyrimidine nucleotide accumulate to 1.7mmol N l 1 in the egg. Tissue ammonia biosynthesis) and CPSase III (required for urea synthesis). The levels are even higher in newly hatched trout (3.0mmolN l 1 apparent absence of CPSase III in adult liver (no stimulation at day 44; Fig. 1), despite an acceleration in the rate of of glutamine-dependent CPSase activity by AGA) is consistent ammonia excretion (Fig. 2). As a comparison, in resting adult with the absence of OTCase activity and, therefore, OUC- trout, plasma ammonia concentrations are between 0.05 and dependent urea synthesis. The levels of CPSase II activity in 0.50mmol l 1 (depending on the feeding history, among other adult liver and those estimated for larval and embryonic tissue factors), whereas intracellular levels are approximately are comparable to those reported in liver extracts of other fish 2 mmol l 1 (Wright and Wood, 1988). Ammonia is species (Anderson, 1980; Cao et al. 1991). The levels of compartmentalized in adult tissues according to pH gradients 132 P. A. WRIGHT, A. FELSKIE AND P. M. ANDERSON

Fig. 4. Activities and ratios of activities of ornithineÐurea cycle enzymes in whole-animal homogenates up to 93 days after fertilization and in adult liver tissue (units= molmin 1). Values are means ±1 S.E.M., except N=1 for day 45 (see below). (A) Carbamoyl phosphate synthetase (CPSase) activity was measured in the presence of glutamine and AGA and therefore represents both CPSase II (AGA-independent) and CPSase III (AGA-dependent) activities (N=2Ð4). (B) The ratio of CPSase activity measured in the presence of glutamine and AGA to that measured in the presence of glutamine alone. A ratio of 1.0 or greater indicates CPSase II and III activity, whereas a ratio approximately equal to 1.0 indicates only CPS II activity. See Materials and methods for details. (N=2Ð4). (C) OTCase activity (N=2Ð7). (D) GSase activity (N=2Ð7). (E) Arginase activity (N=2Ð7). and/or electrical potential gradients across cell membranes concentrations as low as 0.15mmol l 1 can have severe effects (Wright and Wood, 1988; Wright et al. 1988; Wood et al. on the survival of rainbow trout eggs (Solbé and Shurben, 1989a). It is probable, therefore, that ammonia is also 1989). Clearly, excessive accumulation of ammonia can be compartmentalized in the embryo and larvae. Nevertheless, extremely toxic to salmonids in the early life stages, just as it urea synthesis during early development may be essential to is to adult fish (Lumsden et al. 1993). prevent the toxic accumulation of ammonia, while nitrogen The elevation of tissue ammonia concentrations just after stored as urea is far less harmful at equivalent concentrations. hatching may be associated with the exhaustive muscular Indeed, when recently hatched trout were exposed to activity required for the larvae to escape from the egg capsule. conditions that favour ammonia retention (0.2mmol l 1 In exercising muscle, ammonia is produced anaerobically from ammonia or pH9.0 water for 4h), urea excretion increased the breakdown of adenylates (Mommsen and Hochachka, sixfold (P. A. Wright and M. Land, unpublished data). It has 1989). Exercising fish have much higher plasma ammonia also been demonstrated that external water ammonia levels than fish at rest (Wright and Wood, 1988; Wright et al. Induction of urea cycle enzymes in larval trout 133

1988). The observation that tissue ammonia levels remain teleost fish is uricolysis and/or arginolysis, as most species lack elevated for about 10 days after hatching (days 45Ð55), a functional OUC (Goldstein and Forster, 1965; Huggins et al. indicates that ammonia excretion is limited by the gills, which 1969; Wright et al. 1993; Wright, 1993). CPSase III was not are initially underdeveloped (Rombough, 1988). Urea levels detected and OTCase was close to the limit of detection in adult increase in parallel with ammonia levels (Fig. 1), suggesting liver tissue in the present study, indicating the absence of a that ammonia detoxification plays an important role in the complete OUC. Although the genes for the OUC enzymes are developing larvae. obviously present in rainbow trout, expression of CPSase III Urea accumulation in the embryo prior to hatching may and OTCase was suppressed in adults. It appears that signify a role for urea in osmoregulation. In marine suppression of CPSase III and OTCase was initiated by day 93 elasmobranchs and the coelacanth, urea is retained in relatively (Fig. 4B,C); however, further studies are necessary to confirm high concentrations (approximately 800mmol N l 1) to this observation. Arginase and GSase are, however, expressed balance the osmotic pressure of sea water. In a freshwater in adult liver tissue. Both enzymes are involved in other non- environment, however, it is hard to imagine why an OUC metabolic pathways. Arginase is ubiquitous in fish accumulation of osmolytes within the organism would be tissues where it converts dietary arginine to urea (see necessary. There is also no evidence that urea plays a role in Introduction). GSase catalyses the conversion of glutamate and osmoregulation in adult teleost fish, either marine or freshwater ammonia to glutamine and is important in a number of tissues species (Wood, 1993). for ammonia detoxification and glutamine synthesis (Ritter et It has previously been reported that embryonic and larval al. 1987; Chamberlin et al. 1991). GSase is also essential in trout did not excrete urea, only ammonia (Smith, 1947; Rice the teleost liver to supply glutamine for the synthesis of and Stokes, 1974). Using very sensitive water assay methods, which, in turn, are converted to urea via uricolysis. The we have presented new evidence that urea is excreted just after regulation of OUC enzyme expression or suppression in teleost the embryos hatch (day 45). Once larval fish have escaped from fish is not understood. The cloning of the OUC enzyme, the egg capsule, urea can be excreted across the gills. CPSase III, from fish tissue (J. Hong, W. L. Salo, and P. M. Immediately post-hatching (day 45), urea excretion was very Anderson, in preparation) and the availability of a specific low (Fig. 2), although the urea tissue concentration was CPSase III probe for measuring expression of mRNA will be relatively high (Fig. 1). At the end of the experiments (day 93), helpful in addressing this question. urea excretion had increased considerably (Fig. 2), but the urea In summary, this study presents evidence for the induction tissue concentration was reduced compared with that in 45- of the OUC enzymes, including CPSase III, in larval rainbow day-old embryos (Fig. 1). This large increase in the urea trout. Urea synthesis occurred in embryos almost immediately diffusing capacity in larval fish probably relates to the rapid after fertilization, but urea excretion to the environment was expansion of the gill surface area that occurs in the first half not initiated until after the embryos had hatched. Ammonia of the alevin stage (endogenously feeding larvae, age 42Ð53 levels increased during embryonic development, despite days) (Rombough, 1988). excretion of ammonia to the environment. The observation that The observation that urea is excreted only after the embryos urea tissue levels parallel the increases in ammonia tissue hatch is not surprising. The embryo is surrounded by an concentrations provides evidence that urea synthesis is acellular layer called the chorion or egg capsule. The important in the prevention of ammonia toxicity during the permeability of the chorion is relatively low because it early life stages in trout. functions to protect the developing embryo from the external environment (Cottelli et al. 1988). Oxygen, and The authors wish to thank Joseph Korte for excellent ammonia permeate many biological membranes relatively technical assistance with the enzyme measurements. We are easily and also diffuse across the chorion (Rombough, 1988). also indebted to Dr Hugh Ferguson for providing us with Both anions (Cl ) and cations (H+, K+, Ca2+) can also expertise and facilities for rearing the trout eggs. Funding for permeate the chorion (Peterson and Martin-Robichaud, 1987). this project was provided by the Natural Sciences and In recent years, the universal permeability of urea has been Engineering Research Council to P.A.W. and the National questioned, as urea transport in some tissues has been shown Science Foundation (Grant no. DCB-9105797) to P.M.A. to be dependent on a specific carrier protein (mammalian kidney tubules, Chou and Knepper, 1989: mammalian erythrocytes, Brahm, 1983; toad bladder, Levine et al. 1973). References Our results imply that the chorion lacks a specific urea ANDERSON, P. M. (1980). 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