The hormonal regulation of urea excretion in the Mexican axolotl (Ambystoma mexicanum cope)

GENERAL

AND

COMPARATIVE

ENDOCRINOLOGY

31, 45-52 (1977)

The Hormonal Regulation of Urea Excretion in the Mexican Axolotl (Ambystoma mexicanurn Cope) HARTMUT SCHULTHEISS Zoologisches Institut der Universittit, Lehrstuhl 75 Karlsruhe, Kaiserstrasse 12, West Germany

II,

Accepted August 20, 1976 The neotenic axolotl (Ambystoma mexicanum) is able to produce urea in relatively large amounts; up to 75% of its total waste nitrogen. Well-fed larvae had higher serum urea levels than unfed animals and excreted urea at higher rates. ACTH. corticosterone, cortisol and aldosterone elevated the larval serum urea concentration to double or fourfold that of the controls after 4 injections. Thyroxine elevated urea levels also but only when administered in high doses of 20 to 100 nkflanimal per day. TSH had no significant effect. The amount of urea excreted (biochemical determination) was increased significantly only by aldosterone, cortisol or T* administration, while ACTH- and corticosterone-treated animals excreted about the same amount of urea as controls, though the serum urea levels were much higher. Increased serum urea levels can be explained by reduced excretion rates (14C-urea experiments); this statement does not mean that higher production rates do not occur. Reduced excretion rates may also explain the relatively small amount of urea that was excreted by ACTH and corticosterone-treated animals. A theoretical urea pool (total urea of the body) was calculated from the amount of urea excreted and the urea excretion rates.

ities of the enzymes of the Krebs-Henseleit omithine-urea cycle are also elevated at the beginning of anuran metamorphic climax (Cohen and Brown, 1960, cited by Cohen, 1966; Paik and Cohen, 1960). The same is true for the recovery of 14Cbicarbonate as 14C-urea which also indicates increased urea production (Brown, 1964). Thyroxine (T4) and triiodothyronine (T3), which accelerate metamorphosis, increase enzyme activities as well as urea excretion (Paik and Cohen, 1960; Ashley et al., 1968; Noguchi, 1969). Not only thyroid hormones, but also ACTH and corticosteroids have an effect on urea excretion in anurans. Medda and Frieden (1970) found the T3and T,-influence on urea excretion in Rana cutesbeiunu to be intensified by ACTH. Leist (1970) also found that urea excretion was accelerated during normal metamorphosis of Xenopus laevis by ACTH and corticosteroids.

It has been shown by Huggins et al. (1969) that fresh water teleosts possess all the enzymes of the Krebs-Henseleit ornithine urea cycle so that they are able to form urea from carbon dioxide and ammonia and excrete this as a nitrogeneous end product. Nevertheless, ammonia plays a more dominant part in nitrogen excretion of teleosts than urea. In amphibians one of the most dramatic changes during metamorphosis and adaptation to a terrestrial habitat is the shift from ammonotelism to ureotelism. During the course of metamorphosis there is an increase in urea excretion, whereas ammonia excretion remains fairly constant (Munro, 1939; 1953). Ammonia is a toxic substance which is tolerated in the land-nesting frog (Leptoductylus bufonicus) only up to a concentration of 50 mM, whereas the urea tolerance of these animals is in excess of 400 mM (Shoemaker and McClanahan 1973). Parallel to the increased urea excretion the activ45 Copyright All rights

@ 1977 by Academic Press. Inc. of reproduction in any form reserved.

46

HARTMUT

SCHULTHEISS

Though the axolotl is normally totally aquatic, it is very useful for studies on metamorphosis since this can easily be induced and controlled. In the present studies should be compared the influence of thyroid hormones and corticosteroids on urea levels in the body of neotene Mexican axolotls (Ambystoma mexicanum) and on their urea excretion. The results have already been published in part (Schultheiss, 1973), but additional work was necessary, since neither metamorphosed nor hypophysectomized animals were available, nor was the influence of age and nutrition known at the time of the earlier work. ANIMALS

AND TECHNIQUES

Animals Mexican axolotls, l-2 years old, were used for the experiments. They were bred in our own laboratory in Frankfurt/Main and Karlsruhe (West-Germany) and their weights ranged between 30 and 100 g. Metamorphosed specimens were those animals which were forced to undergo metamorphosis 1.5 years prior to the experiments by adding thyroxine (T,) to the aquarium water (concentration of T4 was 3 x 10-B). Hypophysectomy was performed 4 months prior to the experiments by incising the palate under an operation microscope and removing the pituitary by suction with a small glass pipette. Lightening of the animals’ skins served to indicate a successful operation. Some heads were sectioned for light microscopy after the experiments to ensure that no regeneration of the pars distalis had taken place.

Hormone

Injections

Four hormone injections were given intramuscularly either once a day (in serum urea experiments) or every other day (urea excretion experiments) to neotenic axolotls. The last injection was given 24 hr before the urea determinations. All hormones were dissolved or suspended in 0.2 ml frog Ringer solution. Control animals were untreated, because preliminary studies proved that Ringer injections had no effect on urea levels or excretion. The following hormones were used at the dosages stated: ACTH: 1 IU Acetropan (Hoechst), Corticosterone: 300 pg 11,21-dihydroxypregnen(4)dion (3,20) (Merck), Cortisol: 300 pg hydrocortison-hemisuccinat(Na) (Hoechst), Aldosterone: 50 pg Aldocorten (Ciba),

TSH: 0.1 IU Thyratrop (Ferring), Thyroxine (T,): 20 or 100 nM t-thyroxine in serum urea experiments, 12.5 nM L-thyroxine in urea excretion experiments.

Biochemical

Determination

of Urea

Urea was determined colorometrically with phenol and hypochlorite as described by Fawcett and Scott (1960); the commercial urea test kit of Boehringer (Mannheim) was used with certain modifications for the sample concentrations. Urea-N was determined by subtracting NH,-N from total waste-N. For NH,-N determination urease in the incubation medium was replaced by distilled water. Standardisation was carried out using solutions of urea and NH&l. Blood samples (50 ~1) were taken from the bulbus arteriosus with heparinized pipettes. Urine (50 ~1) was collected from the cloaka by pressing the animals gently in front of the hindlegs. Aliquotes of aquarium water (200 ~1) were taken after 24 hr of animals’ maintenance in 2 liter of tap water to determine the amount of urea excreted.

Urea Excretion

Rate

Urea excretion rate was determined by measurements of %-urea. Increasing radioactivity in the aquarium water was measured at various times after injection of YJ-urea. This gives an exponential function of the type a = a,(1 - e-9, whereas the internal radioactivity

(1) is described by

(2)

b = b, . cit.

a and b are the radioactivities in the outer and inner medium at the time f respectively, a, is the radioactivity in the aquarium water after total excretion of V-urea; it is identical with b,, the injected radioactivity. Other values of b were given by the difference between injected radioactivity and measured external radioactivity. The logarithm of Eq. (2) gives a straight line, (In b = In b, - At), the slope of which is (3)

The biological half-life (t,,,) of Y-urea was taken graphically from the straight lines of internal radioactivity or by calculation from the formula t

I” -

In 2 (In b, - In b,)l(t,

In 2 - tz)

= 7’

The excretion rate K of urea is -X multiplied 100% when t is given in hours.

(4)

by

UREA EXCRETION K= ln2.

lOO(%). t,iz hr

the animals. Urea nitrogen accounts for a rather small portion (25%) of total waste nitrogen in the blood of 6 month-old animals but much more (75%) in larvae which were 1.5 years old. In metamorphosed axolotls more than 90% of the total waste nitrogen was found to be urea nitrogen but total ureotelism was never found in metamorphosed animals living in water. Feeding of the animals is another very important factor so far as urea content of the blood and urea excretion are concerned. In intact larvae serum urea levels varied from 0.8 to 3.3 mmoles/liter when the food supply varied between insufficient and adequate, whereas ammonia varied only from 1.2 to 2.2 mmoleslliter. The influence of feeding on the amount of urea excreted and the excretion rate in intact larvae, hypophysectomized larvae and metamorphosed animals is shown in Table 1. High nutritional protein supply (bovine heart muscle) resulted in higher urine urea levels and increased urea excretion. In intact and hypophysectomized larvae the excretion rate (K) was doubled, whereas in

(5)

The method was originally used for the measure of THO diffusion across the skin (see Motais et al., 1969; Payan and Maetz, 1971 or Schultheiss et al., 1972).

Calculation of the Urea Pool In addition to determining “C-urea excretion rates the excreted urea was also determined biochemically as described above. From these determinations the amount of urea excreted per hour per unit mass were derived M (mmoles urea/kg hr). The total urea concentration of the body or the urea pool therefore is the amount of urea excreted (M) divided by the excretion rate (K) multiplied by 100%. P(mMlkg) =

M(mmoles/kg hr) . lOO(%) K(%lhr)

Temperature and Season All experiments were carried out at a water temperature of 20-22” during the period March to May.

RESULTS

It is well known that neotenic axolotls are both ammonotelic and ureotelic, but the ureotelism is increasing with the age of TABLE EFFECTS

47

IN THE AXOLOTL

OF NUTRITION

1 ON UREA

EXCRETIONS

B. Collected data from several experiments

A. Single experiment with 4 animals in each group Treatment

n

Intact larvae 4 fed 4 unfed Hypophysec. larvae fed 4 4 unfed Metamorph. axolotls fed 4 unfed 4

u [mmoles/liter]

M

K

[pmoles/kg hr]

[%/hr]

n

[%/hr]

3.82 k 1.35 1.12 * 0.17

357 2 35 91 ? 110

7.23 2 3.28 4.24 k 0.82

8 53

6.32 + 1.08 3.88 ” 0.20b

2.17 2 0.47 1.70 f 0.82

313 f 106 k

23 9b

7.14 f 1.01 3.11 f 1.03

9 27

7.97 2 1.28 4.52 2 0.33b

696 k 133 208 of: 51b

3.78 f 0.25 3.66 5 0.70

8 32

3.99 k 0.29 3.94 i_ 0.24

22.20 f 4.47 6.47 + 0.8S

K

a Urine urea concentration. U (collected from the cloaca and determined biochemically), amounts of urea excreted, M (biochemically determined in aliquotes of aquarium water), and excretion rates for urea, K (determined from the slope of internal radioactivity after intramuscular injection of’*C-urea). Effects of nutrition: axolotls were either fed until 24 hr before the start of the experiment or unfed for 8 days. Values are means f SEM. b P < 0.001 in Student’s I test between fed and unfed animals.

48

HARTMUT

metamorphosed axolotls after good feeding the excretion rate remained low. Nevertheless the amount of biochemically determined urea (M) excreted was also increased in metamorphosed axolotls as in neotenic ones. To reduce the variability of the results due to feeding differences all the experiments investigating excretion were done with unfed animals (last feeding 8 days before urea determination) which were 1-2 years old. a. Effects of Various Treatments on Serum Urea Levels Hypophysectomy did not change serum urea levels. Metamorphosed axolotls always had higher serum urea levels than neotenic controls. ACTH, cortisol, corticosterone, and aldosterone were very effective in raising urea levels in the blood. After 4 injections of ACTH the urea level was doubled, whereas the steroids were able to increase the concentration fourfold, to the level found in the control animals. TSH, which was given in a dose sufficient to complete metamorphosis within 8 weeks, did not change the urea concentration after 4 days of treatment. T4 increased urea concentration in the blood when administered in rather high doses of 100 r&f per injection but no change was found at a dose of 20 t&I/injection (Fig. 1). b. Effects of Various Treatments on the Amount of Urea Excreted The amount of urea excreted per unit weight and time biochemically determined, was influenced by some of the hormones tested; hypophysectomy had no effect. Metamorphosed animals excreted much more urea than neotenic controls. ACTH, corticosterone and TSH had no significant (P < 0.01) effects on larvae. Only aldosterone, cortisol and T, increased urea excretion (see Table 2). But this increase was not as great as the rise in serum urea levels.

SCHULTHEISS

SERU.4 UREA 13

I2

11

6

FIG. 1. Hormonal influences on serum urea levels in the Mexican axolotl. Hypophysectomy was performed 4 months, and metamorphosis was induced 1.5 years, prior to the experiments. Hormone injections (into intact larvae) were made on 4 successive days until 24 hr before the start of the experiment. Each column gives the mean of 8 determinations + SEM. (Differences between control groups may be the result of unequal feeding.)

c. Effects of Various Treatments on the Half-Life, Excretion Rate and Pool of Urea Figure 2 shows how internal radioactivity decreases with time after injection of 14Curea. Radioactivity is represented on a logarithmic scale so that a straight line is derived. With the help of the dotted lines the half-lives of 14C-urea in the body can

UREA EXCRETION

EFFECT

TABLE 2 TSH, T4, HYPOPHYSECTOMY, AND METAMORPHOSIS UREA (M), THE i4C-U~~~ EXCRETION RATE (K), AND THE CALCULATED UREA POOL (W

OF ACTH, CORTICOSTEROIDS, THE AMOUNT OF EXCRETED

M

P

[%/hr]

f test

73.3 + 6.2 66.7 + 6.3 298.0 t 47.8

ns P < 0.001

4.69 2 0.49 3.73 +- 0.46 4.65 r 0.73

ns ns

1.68 2 0.19 2.05 f 0.35 8.32 lr. 1.77

P < 0.001

8 8

140.4 2 9.9 163.0 2 12.7

IlS

3.55 r 0.17 1.74 f 0.21

P < 0.001

4.03 f 0.36 10.38 r 1.32

P < 0.001

8 8

123.6 ? 13.2 141.0 + 19.6

ns

3.48 -c 0.16 3.44 -c 0.29

tlS

3.58 2 0.39 4.62 + 0.69

ns

P < 0.001

5.15 z!z0.79 20.70 f 3.37

P < 0.001

n

Control Hypophysect. Metamorph.

8 8 8

Control ACTH Control TSH

Control T.4 Aldosterone Cortisol

K

ON

t test

Treatment

Control Corticosterone

49

IN THE AXOLOTL

8 8

bmolesikg

hr]

132.7 5 14.3 137.0 2 6.6 122.0 221.0 174.0 228.0

f c 2 +

3.0 17.3 10.2 14.1

IlS

2.87 2 0.33 0.74 2 0.08

[mmolesfkg]

2.71 f 0.55

P < 0.005 2.08 f 0.15 P co.005 1.03 2 0.04 P < 0.001 1.02 * 0.06

ns P < 0.01

P < 0.01

a Four hormone injections were given on every other day. Determinations injection. Values are the means of n determinations * SEM.

5.16 11.05 17.15 22.25

k f f 2

1.12 1.57 1.62 1.02

t test ns

P < 0.025 P < 0.001 P < 0.001

were started 24 hr after the last

FIG. 2. Hormonal influences on the excretion velocity of ‘*C-urea. Four hormone injections (into intact larvae) were given on alternate days. The internal radioactivity is plotted on a log scale against time. The biological half-lives of urea in the body are indicated by the dotted lines. The number of animals tested is given in parentheses. For statistics see excretion rates (K) in Table 2. Hypophysectomized and metamorphosed animals were those referred to in Fig. 1.

50

HARTMUT

SCHULTHEISS

easily be determined. Urea has the shortest Within 30 days in Munro’s experiments half-life in the bodies of intact and urea excretion was enhanced from about 80 hypophysectomized larval axolotls, as well pmoles urea/kg fresh weight per hour to 240 as in untreated metamorphosed animals. gmoles/kg hr (own recalculation from MunDifferences between larval and meta- ro’s data). These values are in the range of morphosed axolotls were found only in the present experiments in which 4 injecwell-fed animals as discussed above. The tions of T4 increased urea excretion from mean half-life of urea in controls (n = 53) 120 to 220 pmoles/kg hr. During anuran was 20 hr; hypophysectomized (n = 27) metamorphic climax values of excreted and metamorphosed (n = 32) animals ex- urea are much higher than in the urodele creted somewhat faster with t,,, = 18 and 19 axolotl even if metamorphosis is induced in hr, respectively, though the difference was the latter by high doses of T4. Rana curesnot statistically significant. TSH did not beiana reached 583 pmoles/kg hr (Stage change the half-life, whereas T4 prolonged XXIV) in Brown’s experiments (Brown er it by more than 50% (34 hr). ACTH and al., 1959) and in Rana pipiens it became corticosteroids extended the half-life much 1340 pmoles/kg hr (own recalculation) in more than T4 so that it was 45 hr with experiments of Ashley et al. (1968) after 2 ACTH treatment, and 68 and 69 hr with days’ immersion in a lop6 M T3 solution at aldosterone and cortisol, respectively. The 30”. The increasing excretion of urea in most effective hormone was corticosterone Rana species after TX-induced metamorwhich slowed down the rate of excretion phosis is strongly correlated with tail reducgiving a half-life of 102 hr. Increasing half- tion (Medda and Frieden, 1970). Tail reduclives means decreasing excretion rates (see tion means that large quantities of proteins animals and techniques). From the excre- are hydrolized and amino acids are deaminated in the liver. This leads to the suggestiotrrate (K) for 14C-urea and the excreted amount of urea (M) the urea pool (P) can be tion that urea production and excretion are mainly reflections of the catabolic procalculated which should represent the total climax. As urea in the body per unit weight. Table 2 cesses during metamorphic urodeles do not resorb the whole tail, ungives the values of M, K, and P from different experiments. For each experiment new like anurans, the catabolic processes are not so pronounced. Thus it is understandcontrol animals were taken to avoid differences due to unequal feeding or unknown able that in the Mexican axolotl the factors. amounts of urea excreted are never as high Metamorphosed animals had an urea as in anurans. It is not unlikely that thyroid pool which was about four times larger than hormones act via the amino acid pool, in the controls and hypophysectomized lar- which is increased by catabolism, rather vae. ACTH and corticosteroids increased than directly on urea production. Noguchi the pool by between 100 and 400% that of (1969) assumed that this was the case when the controls, while TSH was ineffective. he found that not only T, but also acThe calculated theoretical urea pool was tinomycin D (which increases catabolism), doubled by T4. and several amino acids, activated liver arginase. Furthermore, Dolphin and Frieden DISCUSSION (1955) found the same effect when hyTotal nitrogen excretion is increased drocortisone or epinephrine were adminismarkedly when metamorphosis is induced tered. Medda and Frieden (1970) found in the Mexican axolotl. The increase is due urea excretion to be enhanced by ACTH to a rise in urea-N excretion while NH,-N over and above the stimulation produced remains relatively constant (Munro, 1953). by TB. A small increase in urea excretion by

UREA

EXCRETION

ACTH was found in the Mexican axolotl also, though aldosterone and cortisol were more effective and raised the amount of urea excreted by 43 and 87% above that of the controls. The effects of ACTH and carticosteroids on urea excretion could also reflect their action on catabolic processes. On the other hand corticosterone did not enhance the amount of urea excreted; this may be due to its action in stimulating synthesis of liver proteins (Schultheiss, 1973) by which catabolism may have been compensated. It may also be, however, that the amount of urea excreted was not increased up to the time of determination because of a reduction in the rate of excretion. The 14C-urea method has shown that corticosteroids increase serum urea levels and urea pools by reducing the excretion rates. Although the excretion rates were markedly reduced, the amounts of urea excreted were much higher in aldosterone, cortisol, and T,-treated axolotls than in untreated larvae. Corticosteroids have more pronounced effects on the urea excretion rate than does T4, thus they result in higher serum urea levels and larger urea pools. Since TSH has no effect on the accumulation of urea, and T4 is effective only in very high doses, the possibility must be considered that the interrenal gland may play an important part in the renal handling of urea during metamorphosis. Recently it has been shown that in the toad Bufo marinus increasing salinities result in parallel elevation of both urea concentrations and corticosteroid levels (Garland and Henderson, 1975). From these and other data it is not unlikely that corticosteroid production is enhanced in amphibians whenever water shortage occurs and urea excretion is reduced by the animals to reduce water loss. These suggestions must be considered in the light of enzyme activation of the ornithine-urea cycle by corticosteroids (Radke and Hanke, 1976) and the fact that

IN

THE

51

AXOLOTL

accelerated urea synthesis, demonstrated by 14C0, incorporation into liver urea, resuits from ACTH and T4 treatment of neotenic axolotls (Schultheiss, unpublished data). ACKNOWLEDGMENT I am indebted to Professor Dr. W. Hanke who encouraged me to do these experiments and read the manuscript critically.

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Biol.

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Brown, G. W., Jr. (1964). The metabolism of amphibia. In “Physiology of the Amphibia” (J. A. Moore, ed.), pp. l-98. Academic Press, New York. Brown, G. W., Jr., Brown, W. R., and Cohen, P. P. (1959). Comparative biochemistry of urea synthesis. II. Levels of urea cycle enzymes in metamorphosing Rann catesbeiana tadpoles. J. Biol. Chem. 234, 1775-1780. Cohen, P. P. (1966). Biochemical aspects of metamorphosis: Transition from ammonotelism to ureotelism. Harvey Lectures Ser. 60, 119-164. Dolphin, J. L., and Frieden, E. (1955). Biochemistry of amphibian metamorphosis. II. Arginase activity. J. Biol. Chem. 217, 735-744. Fawcett, J. K., and Scott, J. E. (1960). A rapid and precise method for the determination of urea. J. Clin. Purhol. 13, 156-159. Garland, H. O., and Henderson, J. W. (1975). Influence of environmental salinity on renal and adrenocortical function in the toad Bufo marinus. Gen. Comp. Endocrinol. 27, 136-143. Huggins, A. K., Skutsch, G., and Baldwin, E. (1969). Ornithine-urea cycle enzymes in teleostean fish. Comp.

Biochem.

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Leist, K.-H. (1970). Die Bedeutung des Adenohypophysen-Interrenal-Systems fir den Osmomineralhaushalt des Krallenfrosches (Xenopus luetis Daudin) im Verlauf der Metamorphose. Zoo/. fb. Physiot. 75, 375-401. Medda, A. K.. and Frieden, E. (1970). Effect of prolactin, growth hormone and ACTH on the urea excretion of bullfrog tadpoles during normal and induced metamorphosis. Endocrinology 87, 356-365. Motais, R., Isaia, J., Rankin, J. C., and Maetz, J. (1969). Adaptive changes of the water permeability of the teleost gill epithelium in relation to external salinity. J. Exp. Eiol. 51, 529-546. Munro, A. F. (1939). Nitrogen excretion and arginase

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activity during amphibian development. Biochem. J. 33, 2. Munro, A. F. (1953). The ammonia and urea excretion of different species of amphibia during their development and metamorphosis. Biochem. J. 54, 29-36. Noguchi, T. (1969). Regulatory mechanism of hepatic arginase activity of anuran tadpoles during metamorphosis. J. Fat. Sci. Univ. Tokyo, Sect. IV 11, 555-577. Paik, W., and Cohen, P. P. (1960). Biochemical studies on amphibian metamorphosis. I. The effect of thyroxine on protein synthesis in the tadpole. J. Gen. Physiol. 43, 683-696. Payan, P., and Maetz, J. (1971). Balance hydrique chez les Elasmobranches: Argument en faveur d’un controle endocrinien. Gen. Comp. Endocrinoi. 16, 535-554.

SCHULTHEISS Radke, P., and Hanke, W. (1976). The influence of ACTH and corticosteroids on the activity of enzymes, correlated with urea production in ranidae. Gen. Comp. Endocrinol. 29, 286-287. Schultheiss, H. (1973). The influence of ACTH and corticosteroids on the nitrogen metabolism during the metamorphosis of the Mexican axolotl (Ambystoma mexicanum Cope). Zooi. Jb. Physiol. 77, 199-227. Schultheiss, H., Hanke, W., and Maetz, J. (1972). Hormonal regulation of the skin diffusional permeability to water during development and metamorphosis of Xenopus laevis Daudin. Gen. Comp. Endocrinol. 18, 400-404. Shoemaker, V. H., and McClanahan, L. L. (1973). Nitrogen excretion in the larvae of the landnesting frog (Leptodactylus bufonicus). Camp. Biochem. Physiol. 44A, 1149-I 156.