Camp. Biochem. Physiol. Vol. 91 A, No. 3, pp.

499-508,

1988

Printed in Great Britain

0300-9629188 $3.00+ 0.00 0 1988Pergamon Press plc

IN THE MUDSKIPPER, PERIOPHTHALMUS CANTONENS: CHANGES IN FREE AMINO ACIDS AND RELATED COMPOUNDS IN VARIOUS TISSUES UNDER CONDITIONS OF AMMONIA LOADING, WITH SPECIAL REFERENCE TO ITS HIGH AMMONIA TOLERANCE
KATSUYA IWATA Biological Laboratory, Faculty of Education, Wakayama University, 930 Sakaedani, Wakayama-shi, Wakayama 640, Japan (Received 3 March 1988) Abstract-l. When the fish was kept out of water, the levels of ammonia increased greatly in the liver, skin and muscle, whereas those in the blood and brain increased to a lesser extent. 2. A marked increase in free amino acids (FAA), particularly non-essential ones was found in various tissues during exposure to air; the level of glutamine in the brain of the fish kept out of water for 48 hr increased up to lo-fold that of the fish kept in 20% SW (control). 3. P. canfonensis has a great tolerance to ammonia; it could survive for more than 7 days in 15 mM NH,Cl solution, while the water breathing gobid fishes died within 24 hr at 10 mM NH,Cl. 4. In the fish immersed in 15 mM NH,Cl solution for 3-7 days, changes in ammonia and FAA content in various tissues were essentially the same as those found in the fish kept out of water. 5. By either treatment, urea contents in various tissues were not different from those of the control. 6. Glutamic dehydrogenase and glutamine synthetase levels in the brain of the mudskipper were about lo- and 2-fold higher than those of several water-breathing gobid fishes, respectively. 7. It appears that the mudskipper does not modify the mode of nitrogen metabolism toward ureotelism, but develops the ability to protect the central nervous system against ammonia toxicity in relation to the amphibious mode of life.

NITROGEN

METABOLISM

INTRODUCTION The mudskippers, genera Periophthalmus are unusual among teleost fishes in that they feed on small benthic animals on mudflats out of water and they spend the most-part of their lives on land. It has been known that the levels of ammonia excretion by these fish are

very low during exposure to air (Gordon et al., 1969 and 1978; Morii et al., 1978 and 1979; Iwata et al., 1981). In the previous study (Iwata et al., 1981), it was found that the mudskipper, Periophthalmus cantonensis, kept out of water accumulates a great amount of ammonia as well as free amino acids (FAA) in its tissue, particularly in the muscle, whereas urea increased to a lesser extent. In keeping with this, the activity of glutamic dehydrogenase (GDH) increased in the liver and muscle of the fish kept out of water (Iwata et al., 1981; Iwata and Kakuta, 1983). In the mudskipper, GDH catalysing reductive amination of a-keto-glutarate seems to play a most important role in removing ammonia from the tissues while the fish is subjected to a dehydrated condition. It is known that the aquatic amphibia, Xenopus luevis kept in a condition of water shortage increases the rate of urea production, which is associated with a great increase in the level of carbamoylphosphate synthetase (Balinski et al., 1967; Janssen and

Cohen, 1968). Janssen (1972) demonstrated that the rise of ammonia level in the tissues of the toad placed in 5 mM NH4Cl solution causes a great increase in levels of carbamoylphosphate synthetase and GDH in the liver. This finding suggests that the initial stimulus for urea biosynthesis in the toad is an increase in toxic ammonia in the tissue rather than dehydration stress, although, unfortunately, Janssens results were not confirmed in the study made by Balinsky (1981). In the mudskipper, it is interesting to see whether an increase in ammonia in the tissues stimulates the biosynthesis involving ammonia detoxication as demonstrated in Xenopus Iuevis. Additionally, it is also interesting to know which tissue or organ is mostly responsible for the detoxication while the fish hardly excrete ammonia. In the present study, changes in content of FAA and related compounds in various tissues such as the blood, brain, liver, skin and muscle of the mudskipper kept out of water were compared with those of the fish immersed in NH,Cl solution. Furthermore, the levels of GDH and glutamine synthetase (GS) in the brain, liver and muscle of this fish were compared with those of several water breathing gobid fishes in order to see whether or not the mudskipper has a special ability for the ammonia tolerance or detoxication in relation to its terrestrial mode of life.

499

500
MATERIALS AND METHODS

KATSUYA IWATA

Fish Periophthalmus cantonensis of both sexes, weighing 1.5-2.8 g were collected at the estuary of Shirahama in Wakayama Prefecture, Japan, and they were kept in 20% sea water (SW) for a month before use. Throughout the rearing period, Chironomus larvae were daily supplied as a food. The fish reared in 20% SW were starved for 4 days, and then they were kept out of water for 12, 24 and 48 hr. During the period out of water, five fish were held in a glass container with a tightly fitting cup and wet filter paper on the bottom. The fish which had previously been starved in 20% SW for 6 days were used as control. Other groups of the fish were immersed in 15 mM NH,CI dissolved in 20% SW (pH 7.5) for 3 and 7 days. In all the experimental periods, the fish were held at a temperature of 2&22C. The following water breathing gobid fishes, Tridentiger obscurus, Acanthogobius Jlanimanus (estuary), Bathygobius fuscus, Chasmichthys dolichognathus dolichognathus (tide pool) and Rhinogobius brunneis (fresh water) were collected from each habitat in Wakavama Prefecture in order to compare with the enzyme activities and ammonia tolerance of P. canlonensis. Analysis of amino acids and related compounds

MgCl, (20 wmol), 2-mercaptoethanole (25 umol). sodium L-glutamate (50hmol), hydroxyamine hydrochloride adjusted to pH 7.2 with 2 N NaOH (100 pmol), sodium ATP (IO pmol), creatine phosphate (50 pmol), creatinkinase (0.34.5 EC unit) and-tissue homogenate in a final volume of 1.0 ml. The incubation was carried out at 30C in a centrifuge tube. After incubation, ferric chloride solution (0.37 M FeCl,, 0.67 N HCl and 0.2 M trichloroacetic acid) was added to each reaction mixture and amount of y-glutamylhydroxamate (GHA) formed were measured colormetrically at 535 nm. The enzyme activities in GDH and GS were expressed as tissue activity bmol NADH/min/g wet tissue) and @mol GHA/hr/g wet tissue), respectively.
Statistical analysis

The results were compared Duncans multiple range test.

RESULTS

using Students t-test or

1. Efict of FAA

of keeping and related

the-fish out of water on pool sizes compounds in various tissues

Total blood, brain, liver, skin, lateral muscle and carcass were removed, weighed immediately, and homogenated with cold 2.5% sulfosalicylic acid. Of the tissues examined, total blood, brain and liver of three to five fish from each treatment group were pooled separately. Samples were then centrifuged at 3000 g at 4C for 20 min, and the pellets were washed once with 2.5% sulfosalicylic acid. The original supernatant and the wash were pooled, and stored at -20C until analysed. Amino acids in the pooled supematant were analysed using an automated Hitachi amino acid analyser Model 835. Urea and TMAO (trimethylamine oxide) were determined by the method of Ceriotti and Spandrio (1963) and Bystedt et al. (1959), respectively. Some blood samples were hemolysed with distilled water, and total blood osmolarity was determined by Knauer Semi-micro osmometer (Yanaco-Craft).

Table 1 shows the changes in muscle water content and total blood osmolarity of the fish kept out of water. During the 48 hr out of water, the muscle

water content tended to decrease and the total blood osmolarity increased slightly. Table 2 shows the concentrations of free amino acids (FAA) and related compounds in each tissue of the control fish (20% SW). Among the tissues examined, the lowest level of FAA was found in the blood. The relatively high level of taurine, as found in blood, was mainly due to taurine present in erythrocytes, since separate experiments showed that the concentration of taurine in the red blood cell was about 6-fold higher than that of plasma. In the other tissues, taurine was also present in the highest concentration, ranging from 36% to 60% of the total FAA as listed in Table 2. In the skin, muscle and whole body, glycine and alanine were main constituent of FAA excluding taurine, whereas in the Enzyme preparation and enzyme assays brain and liver, glutamate was present in the highest The brain, liver and lateral muscle of freshly killed fishes concentration. were removed, blotted dry and weighed. These were then As shown in Fig. 1, the levels of ammonia in the homogenized in 10 vol. of ice cold 100 mM imidazole_HCl liver, skin, muscle and whole body increased with the buffer (pH 7.4) with 0.5% triton X-100. The homogenate time course of keeping the fish out of water, which was sonified for 1 min with a sonicator (Ultrasonic Inc.). reached about 15 mM/kg tissue during 48 hr out of An ice bath was used to prevent heating of the homogenate. The sonified homogenate was then centrifuged at 10,OOOg water. On the other hand, in the blood and brain, for 30 min at 4C. The supematant was decanted and used ammonia was kept at a lower level during exposure for glutamic dehydrogenase (GDH) and glutamine synto air. thetase (GS) assays. GDH activities in the homogenates No change was found in the level of urea in the were assayed at 25C in the direction of reductive amination tissues examined. It is noted that urea was uniformly by the same method as reported previously (Iwata et al., distributed in various tissues except for the skin. The 1981). GS activities were assayed using the assay conlevels of taurine and TMAO (not shown in Fig. 1) in dition of Row et al. (1970) with the creatine kinase ATPthe tissues except for the blood and liver of the regenerating system of Vorhaben et al. (1973). The reaction treated fish were not statistically different from those mixture contained imidazole-HCI buffer pH 7.2 (50 pmol),
Table I. Muscle water content and total blood osmolarity of the fish kept in 20% SW (control) and out of water.

Water content (%)

Control (20% SW) out of water 24 hr 48 hr Mean + SD. Number of observations 81.4* I.6 (4) 79.7 * I .o (4) 78.8 f 1.6 (5) in parentheses.

Blood osmolarity 287.0 f

(mOsm) (4)

I I .7

312.5 f 23.3 (4) 327.6 f 21.7 (4)

A high ammonia tolerance of the mudskipper

Table 2. Concentrations Blood GlY Ala Ser Asp Glu Gh Pro TY~ CYS Thr Val Ile Leu Met Phe LYS His Arg Total 1.25 0.36 0.28 0.15 0.26 0.22 BLD BLD 0.16 0.15 0.34 0.08 0.17 0.06 0.21 0.19 0.18 0.03 4.09 1.37 I.19 0.05 8.94 1.31 of free amino acids and related compounds in various tissues of the control in parentheses indicate the number of samples. Mean f SD Brain* 2.19 BLD 2.72 0.92 6.83 1.29 BLD BLD 0.58 0.88 0.47 BLD 0.08 BLD 0.77 0.54 0.75 BLD 18.02 5.63 1.20 I .65 23.55 ND Liver (3). I .35 f 0.25 I .72 + 0.37 I.10 f 0.36 0.30 f 0.08 2.27 f I.12 0.64kO.19 BLD BLD 0.44 + 0.07
.T

501
fish. Figures

Skin (9) 2.35 + 0.66 1.46 f 0.34 0.88 f 0.23 0.35 f 0.08 I .56 f 0.39 0.55 kO.15 BLD BLD 0.29 f 0.05 . ... . 0.30 f0.15 0.45 + 0.08 0.05 * 0.07 0.16 k 0.08 0.05 + 0.06 0.22 * 0.34 0.51 * 0.33 0.59 f 0.23 0.10 f 0.08 9.87 f 2.55 7.59 + I .72 0.52 + 0.1 I BLD 20.06 k 3.27 4.99 + I.15

Muscle (9)

Whole body (8)

0.53 + 0.25 0.90*0.19 0.05 ? 0.08 0.17 +0.06 BLD 0.19 * 0.34 0.33 f 0.06 0.33 + 0.06 BLD 10.32 rl_3.1 I 10.96 f 1.66 I .08 k 0.45 0.29iO.16 ll.56?2.72 2.13

3.91 k 0.96 7.83 f 2.22 2.09 5 0.74 I .44 + 0.24 0.62 + 0.17 0.69kO.19 0.23 f 0.03 0.16kO.02 I.48 f 0.23 0.53 + 0.09 0.72 + 0.27 0.78 f0.13 0.11 kO.10 0.13 * 0.09 BLD BLD 0.62 + 0.24 0.11 +0.02 . . ..T... . .. . . .. .. .. . .. . 0.22 + 0.07 0.39 f 0.08 0.35 k 0.06 0.47+0.l8 BLD 0. IO + 0.03 0.12+0.12 0.18+0.05 BLD 0.07 * 0.07 BLD 0.09 * 0.05 0.73 f 0. IO 0.55 k 0.26 0.78 + 0. I9 0.83 f 0.37 BLD 0.06 + 0.05 14.93 + 2.40 5.92+ I.16 I .39 + 0.56 BLD 27.05 f 3.46 I I.61 + 0.66 II.45 & 1.63 3.63 + I .20 1.43 f 0.41 BLD 24.83 & 2.41 8.62 + I.51

NH,
Urea GABA Tau TMAO

BLD = below the limit of detection. ND = not determined. Pooled samples.

of the control. The level of taurine in the blood decreased in the fish kept out of water for 48 hr, whereas that in the liver tended to increase in the fish exposed to air for 24-48 hr. In the tissues examined, non-essential amino acids (NEA) such as glutamate, glutamine, alanine and glycine increased with the time course of keeping the fish out of water (Fig. 2). Possibly, some of glutamine had been hydrolysed to yield glutamate and ammonia during the extraction and quantitating procedures. The sum of these amino acids (Glu + Gln) are thus given in Fig. 2. In the blood, there was a trend to increase in Glu + Gln in the fish kept out of water. In the brain of the fish kept out of water for 48 hr, glutamate and glutamine levels were about twice and IO-fold those of the control, respectively. It is noted that alanine also increased in the brain of the treated fish kept for 2448 hr. Among the essential amino acids (EA) in the brain, histidine increased slightly in the fish treated for 24-48 hr. A similar trend was also found in the liver, although glutamine as well as glutamate in the liver increased less markedly. In the muscle and whole body of the fish kept out of water, the NEA such as alanine, Glu + Gln, glycine and probably serine increased greatly, although a relative increase in glycine was low. The EA such as lysine and histidine increased slightly in the muscle and whole body of the fish treated for 24-48 hr. Similar trends were also observed in the skin, although the levels of glycine and the EA in its tissue of the fish kept out of water for 48 hr were not significantly different from those of the control. Figure 3 shows the distribution of ammonia-N and total NEA-N within each tissue of the fish body. These values were obtained by multiplying the concentrations of these compounds in each tissue expressed as nitrogen (pg atoms/g each tissue) by the

proportion of each tissue weight with respect to total body weight (Table 3). On a percentage distribution basis, 60-70% of ammonia and NEA within the whole body were found to be contained in the muscle; 8-15% of those were occupied by the skin, and only a small fraction in the liver and brain. Thus, a major site of storage of ammonia and FAA during exposure to air was the muscle tissue. In the present study, the rates of ammonia excretion by the fish kept out of water for 24 and 48 hr were 3.0 and 7.2 pmole/g fish, respectively, which were about one-fifth of the fish kept in 20% SW (control). Adding each rate to the total increment of ammonia-N and NEA-N (ANH, + ANEA) in the fish body during each period out of water gives the predicted rate of total ammonia production during exposure to air. This rate during 24 hr out of water (15 pmole/g fish) was about the same as the rate of ammonia excretion of the control fish, but that for 48 hr out of water (23 pmole/g fish) decreased to about 75% of the control. On the other hand, the increments of ammonia-N (ANH,-N) and NEA-N (ANEA-N) within the fish body during 24 hr out of water comprised 45 and 35% of the total ammonia production, respectively, while those values for 48 hr out of water were 41 and 27%, respectively. 2. Efect of keeping thejish in NH&I solution on pool sizes of FAA and related cornpow& in various tissues When the fish was immersed in 15 mM NH&l solution, neither the water content in muscle nor the total blood osmolarity differed from that of the control fish. As in the fish kept out of water, the levels of ammonia in the blood and brain of the fish immersed in 15 mM NH,CI for 3 and 7 days were much lower than those of other tissues which were about the same as ambient concentration (Fig. 4).

502

KATSUYA IWATA
Micro-mole / g tissue I Taurine

Ammonia

I 0

urea

10

20

Brain

0"' of
water

12 hr

Fig. 1. Changes in ammonia, urea and taurine content in various tissues of the mudskipper kept out of water. Each range indicates the standard deviation.

difference was not found in the levels of taurine and urea between the treated and the control fish. Changes in the individual amino acid content in various tissues of the fish immersed in NH&l were essentially the same as those of the fish kept out of water (Fig. 5). However, the level of Glu + Gln in the brain of the fish in NH&l for 7 days was lower than that found in the fish kept out of water for 48 hr; the levels of alanine in the blood, muscle and whole body increased greater than those of the fish out of water. As shown in Fig. 3, in the fish immersed in NH&l solution, a major site of ammonia and FAA accumulation was the muscle, as in the fish exposed to air.
Any significant

liver examined in the present study was plotted against the level of ammonia in the blood (Fig. 6), a highly significant correlation (t = 0.86-0.87, n = 6) was found between them (P < 0.05). It should be noted that the response of the brain against a slight increase of ammonia in the blood was much greater than that of the liver. A highly significant correlation (r = 0.78, P < 0.01) was found in the concentration of NEA in the muscle as plotted against the level of ammonia in itself (Fig. 7).
4. Comparison of tissue GDH and GS levels among various gobid fishes

3. Relationship
content

between

tissue FAA and ammonia

When the levelof Glu + Gln either in the brain or

Figure 8 shows GDH and GS activities in the brain, liver and muscle as compared with those of several water breathing gobid fishes from different habitats. In the mudskipper, GDH and GS activities

high ammonia tolerance of the mudskipper

Micro-mole / g tissue

503

10

20

30

40

Blood

Liver

Fig. 2. Changes in free amino acid content in various tissues of the mudskipper kept out of water. Each piled bar indicates that glycine (horizontal striped bar), the sum of glutamate and glutamine (shaded bar), alanine (solid bar), serine (vertical striped bar), the other non-essential amino acids (open bar) and total essential amino acids (cross striped bar). Each range indicates the standard deviation.

either in the fish kept out of water or immersed in NH&l solution were not significantly different from those of the control. Both the enzyme levels in the mudskipper as shown in Fig. 8 are the means of various treatments. The brain GDH level of the

5. Comparison of ammonia tolerance between the mudskipper and water breathing gobid jishes

mudskipper was about IO-fold higher than that of the water breathing gobid fishes; little difference in GDH activities was found in the liver among them. The muscle GDH level of the mudskipper was higher than that of any other species examined (P < 0.01) although the level was low as compared with that of the brain and liver. There were also similar trends in GS levels with respect to the highest enzyme activities in the brain and muscle of the mudskipper (P < 0.01). The liver enzyme differed little.

Table 4 shows the survival time of various gobid fishes in the ammonium chloride solution. At 10 mM NH&I dissolved in 20% SW, the water breathing gobid fishes all died within 24 hr. In contrast, at 15-20 mM NH& no mudskipper died, and the behavior was apparently normal during the experimental period (one week).

DISCUSSION

The present results are essentially the same as our earlier results (Iwata et al., 1981) with respect to the following items: (a) the mudskipper accumulates a

504
0

KATSUYA IWATA 5 10 15 20
Table 3. The tissue weight expressed as a percentage of body weight of the mudskipper Tissue MWCle Skin Liver Brain Skin Others Mean ? SD. Percentage 47.8 12.1 1.6 0.6 f + + k 2.6 2.6 0.5 0. I

Control Out of 24 hr

water 48 hr
NH4Cl (7 days)

Control Out of 24 hr water 48 hr

N&Cl (7 days) HUsCle Skin Others

Fig. 3. Ammonia and total non-essential amino acid (NEA) content in the whole body of the various treatments expressed as nitrogen (pg atoms/g fish) and the distribution of those content within each tissue. large amount of ammonia in the body during the period out of water, (b) in keeping with this, this fish has a high ammonia tolerance, (c) FAA, but not urea play a central role in ammonia detoxication. However, in the present study, GDH activities in the

tissues of the fish kept either out of water or in NH&l solution were not different from those of the control. In the previous study, GDH levels in the fish exposed to air for 72 hr increased significantly. The reason for this discrepancy is not clear at present, although in the present study, no attempt was made to separate a mitochondrial fraction from the tissues as in the earlier study. It is well known that ammonia interferes with oxidative metabolism in the mammalian tissues, and especially its toxicity profoundly affects brain metabolism (Campbell, 1973). In the present study, alanine increased markedly in the tissues which retained a high concentration of ammonia, indicating that ammonia may stimulate glycolitic pathway in those tissues. In the mudskipper kept out of water and in NH&l solution, the level of glutamine as well as glutamate

Ammonia

0
I

5
I I

15
I II

I 012
I

IJl-eFl
I II

Taurirle

10
I

20
I

30

Whole

Fig. 4. Changes in ammonia, urea and taurine content in various tissues of the mudskipper immersed in 15 mM NH,CI.

.A high ammonia

tolerance

of the mudskipper

505

Micro-mole ! g tissue

Blood

Liver

Skin

Whole

Fig. 5. Changes

in free amino acid content in various tissues of the mudskipper immersed NH,CI. The symbol in each piled bar is the same as in Fig. 2.

in 15 mM

al

25

, w VI .3 * : 0) 3 0 E x.
ci
4

l
20

15

0 0
A A

/
10

c-3 +

$
/

5-

, rl

0
Ammonia Fig. 6. Changes in concentration of the sum of glutamate and glutamine in the brain (circles) and liver (triangles) as a function of the level of blood ammonia @mole/g tissue). Ammonia

10 (rmole/g

15

20 tissue)

Fig. 7. Changes of total non-essential centration in the muscle as a function tissue.

amino acid conof ammonia in its

506 GDH

KATSUYA

IWATA

GS

Fig. 8. Glutamic dehydrogenase, GDH bmole/min/g tissue) and glutamine synthetase, GS (nmole/hr/g tissue) levels between the mudskipper and water breathing gobid tissues. E (estuary), T (tide pool) and F (fresh water) show each habitat of the gobid fishes examined. Each range indicates the standard deviation. Values in parentheses indicate the

number of samples in each tissue. markedly increased in response to the rise of blood ammonia level (Fig. 6). A similar glutamine concentration in the brain has been observed in goldfish kept in water containing ammonia (Levi et al., 1974). An extremely high GDH activity was present in the mudskipper brain. In contrast, very low GDH activities have been observed in the brain of other fishes including several water breathing gobid fishes (present study), Carassius auratus cuvieri (Iwata, unpublished data) and channel catfish (Wilson, 1973). The level of glutamine synthetase in the mudskipper brain is about the same as in goldfish (Waarde and Kesbeke, 1982) and eel brain (Lund and Goldstein, 1969), which are higher than in the brain of MyoxoTable 4. Comparison of survival time in ammonium tion between the mudskipper and water breathing Ambient ammonia cont.* (NH&Cl mM)
P. cwttonensis T. obscurus

chloride solugobid fishes Duration of survival >7 <4 <24 <2 <24 days days hr days hr

1969), channel catfish (Wilson and Fowlkes, 1975), rainbow trout (Walton and Cowey, 1977) and the water breathing gobid fishes examined in the present study. Smart (1978) has demonstrated that the acute toxic action of ammonia to fish is primarily associated with an impairment of cerebral functions as observed in mammals. Therefore, the highest GDH and glutamine synthetase activities in the mudskipper brain seem to serve a protection against ammonia penetrating into the brain by a rapid formation of glutamate and glutamine. This specialized ability appears to play a most important role in allowing this fish to tolerate a high ammonia loading. Comparing the changes in FAA in the tissues of the fish kept out of water with those of the fish immersed in NH&I solution, it is interesting to note that qualitatively similar amino acids such as glutamate, alanine, glutamine, glycine and probably serine increase in various tissues of the fish of both the treatments. This fact suggests that the increase in these amino acid syntheses is triggered by ammonia rather than by dehydration stress. The levels of FAA, particularly glutamate and glutamine, in the brain of the fish kept out of water were found to be higher than those in the fish immersed in NH&l solution. In addition to the fish in NH&l solution having a lower blood ammonia level, the lower FAA level as found in the brain of the fish in NH&l may be attributed to a lower blood osmolarity as compared to the fish kept out of water, since the levels of FAA in the brain of skates is known to change markedly with changing in blood osmolarity (Forster et al., 1978). On the other hand, a higher level of alanine as found in the tissues of the fish immersed in NH&l solution may be associated with an impairment of gas exchange caused by submersion in the solution with high ammonia concentration. It is very interesting to note that ammonia in the blood of the treated fish kept at a lower level in spite of a large amount of ammonia accumulated in the other tissues such as the liver and muscle. Similar results have been observed in goldfish immersed in water containing ammonia (Levi et al., 1974) and Xenopus Zeavis kept in a moist peat (Unsworth and Crook, 1967). In addition, it is known that after ammonia administration, the concentration of ammonia in the erythrocytes of carp is always higher than in the plasma (Ogata and Murai, 1987). These results indicate that ammonia retained in the tissues or cells hardly diffuse into the blood, especially into the plasma. As shown in Fig. 3, a major site of ammonia accumulation is the muscle tissue, and its large storing capacity for ammonia seems to contribute largely to the lowering of the circulating blood ammonia level while the fish is suffering from ammonia loading. Under the circumstances of the fish exposed to air, the skin may play some role in accumulating ammonia as well as in eliminating tissues (Morii et al., 1978). ammonia through its

cephalus scorpius (Lund and Goldstein,

R. brunneus

15-20 mM (pH 7.5) 7 mM (pH 7.3) IOmM 6 mM (pH 7.3) IOmM in 20% sea water.

l NH,Cl

was dissolved

Waarde and Kesbeke (1982) have reported that in goldfish, glutamine synthetase activity is detectable even in the muscle, and a formation of glutamine in the muscle contributes considerably to the total

A high ammonia

tolerance

of the mudskipper

507

production in the fish body. In the muscle of the mudskipper, glutamine synthetase activity was also detectable, and glutamine synthetase as well as GDH levels both were found to be higher than those in the water breathing gobid fishes and in goldfish as reported by Waarde (1981) and Waarde and Kesbeke (1982). Moreover, in an earlier paper (Iwata and Kakuta, 1983), it has been found that the properties of GDH in the muscle of the mudskipper are somewhat different from those of a water breathing gobid fish with respect to the apparent K,,, value for ammonia and ADPGTP interaction, which are well suited to its amphibious mode of life. GOT and GPT activities in the muscle of this fish were found to be also high, particularly GOT activity was about twice as much as that found in the liver (Iwata and Kakuta, unpublished data). In the fish kept either out of water or in NH&l solution, the NEA such as glutamate, alanine, glycine and glutamine increased greatly in the muscle, and those located in the muscle occupied about 70% of the total NEA in the fish body (Fig. 3). This fact together with higher activities of the enzymes in the muscle as described above strongly suggests that most FAA accumulated in the muscle is produced by itself, but little is delivered from other tissues such as the liver. Thus, the muscle of the mudskipper appears to play an important role not only in accumulating a large amount of ammonia, but also in removing ammonia from the fish body when the fish hardly excrete any ammonia. In conclusion, the mudskipper seems to solve the problem of how to deal with toxic ammonia accumulated during its terrestrial life as follows: this fish kept in a condition of ammonia loading allows the cellular compartment, particularly muscle cells, to accumulate ammonia and to partly convert ammonia into amino acids, while the blood ammonia is regulated at a lower level, and ammonia penetrating into the brain is detoxified by a powerful GDH-glutamine synthetase system in its tissue. Therefore, it appears that the mudskipper does not modify the mode of nitrogen metabolism toward ureotelism, although Gordon et al. (1978) have suggested that this fish has a potential for the transition to ureotelism while it is out of water. Jungreis (1976) has stated that in amphibia kept in a dehydrated condition, the increase in urea synthesis is a secondary response to the higher levels of amino acids present in the blood and tissues following dehydration stress. According to the Jungreiss context, it may thus be proposed that the increase in amino acid synthesis triggered by ammonia loading, as found in the mudskipper, is a fundamental reaction independent of differences in the mode of nitrogen metabolism.
Acknowledgements-1 would like to thank Dr M. Sakaguchi, Research Institute of Food Science, Kyoto University, for valuable advice and for critically reviewing the manuscript.

REFERENCES Balinsky J. B., Choritz E. L., Coe C. G. L. and Van der Schans G. S. (1967) Amino acid metabolism and urea

synthesis in naturally aestivating Xenopus laeuis. Comp. Biochem. Physiol. 22, 59-68. Balinsky J. B. (1981) Adaptation of nitrogen metabolism to hyperosmotic environment in amphibia. J. exp. Zoo/. 215, 335-350. Bysted J., Swenne L. and Aas H. W. (1959) Determination -of TMAO in fish muscle. J. Sci. F. Agric. 10, 301-304. Campbell J. W. (1973) Nitrogen excretion. In Comoaratioe Animal Physiology (Edited by Presser C. L.), 3rd edn, pp. 279-316. Saunders, Philadelphia. Ceriotti G. and Spandrio L. (1963) A spectrophotometric method for determination of urea. Clinic. Chimicu. Acra 8, 295-299. Forster R. P., Hannafin J. A. and Goldstein L. (1978) Osmoregulatory role of amino acids in brain of the elasmobranch, Raja erinacea. Camp. Biochem. Physiol. &IA, 25-30. Gordon M. S., Boetius I., Evans D. H., McCarthy R. and Ogleby L. C. (1969) Aspects of the physiology of terrestrial life in amphibious fishes-I. The mudskipper, Periophrhalmus sorbinus. J. exp. Biol. 50, 141-149. Gordon M. S., Ng W. W. S. and Yip A. Y. W. (1978) Aspects of the physiology of terrestrial life in amphibious fishes-III. The Chinese mudskipper, Periophthalmus cantonensis. J. exp. Biol. 72, 57-75. Iwata K., Kakuta I., Ikeda M., Kimoto S. and Wada N. (1981) Nitrogen metabolism in the mudskipper, Periophrhalmus cantonensis: A role of free amino acids in hetoxication of ammonia produced during its terrestrial life. Comn. Biochem. Phvsiol. 68A. 589-596. Iwata K. and Kakuta I. (i983) A comparison of catalytic properties of glutamate dehydrogenase from liver and muscle between amphibious Periophthalmus canronensis and water-breathing gobid fishes Tridentiger obscurus obscurus. Bull Jap. Sot. Sri. Fish. 49, 1903-1908. Janssen P. A. and Cohen P. P. (1968) Biosynthesis of urea in the estivating african lugfishes and in Xenopus laeuis under conditions in water shortage. Camp. Biochem. Physiol. 24, 887-898. Janssen P. A. (1972) The influence of ammonia on the transition of ureotelism in Xenopus laevis. J. exp. Zool. 182, 357-366. Jungreis A. M. (1976) Partition of excretory nitrogen in amphibia. Coma. Biochem. Phvsiol. 53A. 133-141. Levi G., Morisi 6.. Coletti A. and Catanzarb R. (1974) Free amino acids in fish brain: Normal levels and changes upon exposure to high ammonia concentrations in uivo, and upon incubation of brain slices. Camp. Biochem. Physiol. 49A, 623-636. Lund P. and Goldstein L. (1969) Glutamine synthetase in tissues of lower vertebrates. Camp. Biochem. Physiol. 31, 205-210. Morii H., Nishikata K. and Tamura 0. (1978) Nitrogen excretion of mudskipper fish Periophthalmus canronensis and Boleophthalmus pecrinirosrris in water and on land. Camp. B&hem. Physiol. 6QA, 189-193. Morii H., Nishikata K. and Tamura 0. f1979) Ammonia and urea excretion from mudskipper fishes Periophrhalmus cantonensis and Boleophthalmus peclinirostris transferred from land to water. Camp. Biochem. Physiol. 63A, 23-28. Ogata H. and Murai T. (1987) Effects of ammonium chloride administration on ammonia and free amino acids levels in erythrocytes and plasma of carp. Nippon Suisan Gukkaishi 53, 1257-1260. Row W. B., Ronzio R. A. and Wellner V. P. (1970) Glutamine synthetase (sheep brain). In Method in Enzymology XVII (Edited by Tabor H. and Tabor C. W.). pp..90&9IO. Academic Press, New York. Smart G. R. (1978) Investigation of the toxic mechanisms of ammonia to fish gas exchange in rainbow trout (Sulmo gairdneri) exposed to acutely lethal concentrations. _ -. . -. . -- ^- .^. J. rish liiol. 12, YJ-IW.
II

508

KATSUYAIWATA

Unsworth B. R. and Crook E. M. (1967) The effect of water shortage of the nitrogen metabolism of Xenopus laeois.
Comp. Biochem. Physiol. 23, 831-845.

of amidase and amide synthetase

in goldfish tissues.

Comp. Biochem. Physiol. 71B, W-603.

Vorhaben J. E., Wong L. and Campbell J. W. (1973) Assay for glutamine synthetase activity. Biochem. J. 135,
893-896.

Walton M. J. and Cowey C. B. (1977) Aspects of ammoniagenesis in rainbow trout, Sulmo gairdneri. Comp.
Biochem. Physiol. 57B, 143-149.

Waarde A. Van (1981) Nitrogen metabolism in goldfish, Curassius aurafus (L.): Activities of transamination reactions, purine nucleotide cycle and glutamate dehydrogenase in goldfish tissues. Comp. Biochem. Physiol. 68B, 407413. Waarde A. Van and Kesbeke F. (1982) Nitrogen metabolism in goldfish, Carassius auratus (L.): Activities

Wilson R. P. (1973) Nitrogen metabolism in channel catfish, lcralurus puncfafus--I. Tissue distribution of asparate and alanine transferases and glutamate dehydrogenase. Comp. Biochem. Physiol. 46B, 617-624. Wilson R. P. and Fowlkes P. L. (1976) Activity of glutamine synthetase in channel catfish tissues determined by an improved tissue assay method. Comp. Biochem. Physiol. 54B, 365-368.