Seasonal effects of dehydration in air on urea production in the frog Rana pipiens

Camp. Biochem. Physiol., 1974, Vol. 47A, pp. 39 to 50. Pergamon Press. Printed in Great Britain

SEASONAL EFFECTS UREA PRODUCTION

OF DEHYDRATION IN AIR ON IN THE FROG RANA PIPIENS

ARTHUR University of Minnesota,

Department

M. JUNGREIS* of Zoology, Minneapolis,

Minnesota 55455, U.S.A.

(Received 2 March 1973)

Abstract-l.

The concentrations of urea and ammonia in blood or serum and urine of frogs, Ranapipiens, were determined after 6-48 hr of mild dehydration in air. Increased concentrations of urea were observed in blood or serum of frogs dehydrated less than 12.5 hr in the spring-summer, but not in the fallwinter. 2. Ammonia represented only 0.6-3.4 per cent of amino nitrogen excreted in the forms of ammonia and urea. Ammonia was not detected in serum during any season, but was present in blood that was hemolyzed. 3. Administration of Pitressin did not alter the concentration of urea in serum of tap water treated frogs, except at high doses, where it was observed to cause a decrease in the concentration of urea in serum. 4. The increase in serum urea levels was associated with an increase in urea production. The seasonal nature of this response is discussed.

INTRODUCTION IN THE COURSE the activities

of a preliminary

study designed

of liver and kidney

of urea, I found that in response

enzymes

to explore

associated

to mild dehydration

the relationships

with synthesis in hypertonic

between

or production saline (Jungreis,

1971) or in air, frogs, Rana pipiens pipiens, dehydrated in the spring-summer responded differently from those dehydrated in the fall-winter. When frogs were dehydrated in air for up to 12 hr, the concentration of urea in serum increased in the spring-summer, but not the fall-winter. This increase in serum urea represented an increase in the rate of urea production (Jungreis, 1971). The increase in urea production is not mediated by anti-diuretic hormone, and does not represent a shift-in the direction of urea-in the proportion of nitrogen excreted in the forms of ammonia and urea. MATERIALS

AND

METHODS

General procedure Frogs, Rana pipiens pipiem, collected in northern Minnesota, the upper great plains states, or west-central Canada, were obtained from J. R. Schettle Biologicals, Inc. (Stillwater, Minn. 55082). Upon arrival, individual frogs were placed in glass “finger bowls” or in l-pint capacity plastic containers filled to a depth of l-2 cm with pre-aerated tap * Present address: Ohio University,

Department 39

of Zoology, Athens, Ohio 45701.

40

ARTHUR

M.

JUNGREIS

water (changed daily), and kept in continuo~ly darkened constant temperature rooms (20, 22.5 or 25°C) or in the laboratory at room temperature (ZS’C). Animals were not fed after arrival into the laboratory. Prior to each experiment, frogs were manually voided, rinsed with tap water, dried with a paper towel, weighed and placed in either dry containers (experimental) or maintained in water (controls, one frog/container). Upon terminating an experiment, frogs were again manually voided-the urine being collected directly in plastic 1 -ml microcentrifuge tubesdried and re-weighed. Individual frogs were then pithed, and blood collected by heart puncture with heparanized tuberculin syringes. The entire procedure between weighing and collection of blood took 2-4 min. Except for frogs used in some spring-summer experiments, blood was then transferred to conical plastic microcentrifuge tubes, centrifuged at 16,000 g for 2-5 min in a Misco ~i~rocent~fuge, the serum portions decanted, transferred to clean tubes and frozen at - 16°C. For frogs used in some spring-summer experiments, whole blood was not centrifuged, but frozen directly, and hemolyzed blood rather than serum used for analysis. No differences in urea content were observed between whole blood and serum. Measurement

of ammonia or urea

The concentration of urea in serum or urine was determined using obrink’s (195.5) modification of the Conway & Byrne (1933) microdiffusion analysis for ammonia. Duplicate S- or lo-,ul samples of serum or urine were incubated with one drop of 25 mgjml aqueous urea solution (Urease-Dunning, obtained from Hynsin, Westcott & Dunning, Inc., Baltimore, Md. 21201) for at least 30 min and the reaction stopped with ca. 0.30 ml of 45% (w/v) potassium carbonate. After incubating the resulting mixture for at least 60 min, ammonia was determined by titrating the absorbing solution [l y0 boric acid in 20% ethanol (pH S*O), containing 0.5% (v/v) of a l/l ( v /v) mixture of methyl red and bromcresol green] back to pH 5.0 with 0.01 N sulfuric acid using a Gilmont Micrometer Buret (0.2 ml capacity). The end point of the titration was determined by eye. Standards of 10 and 20 mM urea were run simultaneously. The accuracy of the method of microdiffusion is 5 per cent for samples whose final concentration is under 2 mM urea, and 1 per cent for samples over 8 mM urea. Me~s~~~ent

of osmotic pressure

The osmotic pressure of undiluted urine was measured by the method of freezing point depression using an Osmette Precision Osmometer. The proportion of urine pressure exerted by urea was determined by assuming that Molar = Osmolal. For example, urea present in urine at a concentration of 20 mM was assumed to have an osmotic pressure of 20 mOsmole. If the measured osmotic pressure of this urine sample was 35 mOsmole, then the percentage of the osmotic pressure exerted by urea was 20/3.5 x 100 per cent = 57 per cent. Characteristics

of frogs associated e&h season

Frogs exhibiting a fall-winter pattern of metabolism are thought to experience a slower rate of growth, to store large quantities of glycogen in liver (Smith, 1950, 1952; Mizell, 1965; Hong et aZ., 1968), to have a higher tolerance to food deprivation (Jungreis & Hooper, 1970) and to have enhanced metabolism of triglycerides (Freeman et al., 1968; Brenner, 1969). Frogs exhibiting a spring-summer pattern of metabolism are said to experience more rapid rates of growth, to store smaller quantities of glycogen in liver (Smith, 1950, 1952; Mizell, 1965; Hong et aZ., 1968), to store larger quantities of fat relative to carbohydrate (Smith, 1950; Mizell, 196.5; Hong et al., 1968; Brenner, 1969), to have a reduced capacity to synthesize glycogen (Courley et al., 1969) and to be less tolerant of food deprivation (Jungreis, 1970).

UREA

PRODUCTION

IN

THE

41

FROG

Responses of spring-summer frogs Zero to forty-eight hr of dehydration in air. One hundred and eighty frogs obtained in early March were placed in a lOO-gal capacity holding tank filled with aerated well water (4°C) for 6 days. During the first 6 days of storage, 52 animals died, whereupon the surviving frogs were transferred to plastic containers partially filled with aerated tap water, and placed in a continuously darkened constant temperature-humidity room (22.5”C, 50% r.h.). Of these 128 frogs, 25 or 19.6 per cent died before completion of the experiment. Following 10 days of pre-treatment, groups of 12-18 frogs were transferred to dry containers for from 6 to 48 hr. Animals were not handled during dehydration. After 6-7, 12, 18, 27 or 48 hr of dehydration in air, 0.3-1.0 ml of blood and 0.2-0.7 ml of urine were collected from individual frogs. Frogs dehydrated for 7 or more hours spontaneously voided their bladders at least once, as evidenced by the presence of 1-3 yellow-green spots on the floor of the container. Frogs used in this experiment were weighed only at the beginning of the experiment. Consequently, weight changes and hematocrit changes were not available for this group of frogs. To provide information on the extent of water loss following initiation of mild dehydration in air, 58 frogs collected in northern Minnesota during July were acclimated in water at 22.5”C for 1 week, and then dehydrated in air (r.h. 50%) for periods up to 7.5 hr. The decline in body weight was then determined. Effects of anti-diuretic hormone (ADH). A reduction in water uptake through the skin necessitates a comparable reduction in urine formation, Urine formation will decline in response to anti-diuretic hormone (Shoemaker, 1965). Consequently, the effect of ADH on the concentration of urea in serum was determined in frogs maintained in tap water. Twentynine frogs collected in northern Minnesota were obtained in late August and acclimated for 7 days (22*5”C, 50% r.h.). Following pre-treatment, frogs were injected intra-abdominally either with two dose levels of Pitressin (Parke-Davis, 20 Pressor Units/cm3 of 0.9% sodium chloride) or with saline (0.7% sodium chloride): Group I, 4 P.U./lOO g. Group II, 20 P.U./lOO g. Group III, 0.10 ml of saline. Animals were then returned to tap water and blood collected after 5 hr. Since Pitressin might not mimic the effects of “natural” amphibian arginine-vasotocin (anti-diuretic hormone), the procedure of Shoemaker (1965) was employed to obtain amphibian ADH. Four toads, Bufo marinus, each weighing cu. 500 g, were acclimated in August at 20°C for 7 days. Following acclimation, toads had their bladders catheterized-to remove the fluid contents-and were placed in 0.25 M sodium chloride (to a depth of 4-6 cm) for 6 days. Following salt dehydration toads were decapitated and blood collected. The presumed increase in titer of ADH in these toads was not verified, since past experience had proved it to be a very reliable procedure. Thirty-two frogs obtained at the same time as, and pre-treated in a comparable fashion to those used in the Pitressin experiments, were divided into four groups and administered intra-abdominally: Group IV, 5 P.U./frog. Group V, B. marinus serum/frog. Group VII,

0.05 ml B. marinus serum/frog. 0.25 ml of saline.

Group VI,

0.25 ml

Frogs were returned to tap water, and after 6-7 hr, the experiment terminated and blood collected. Responses of fall-winter frogs Zero to forty-eight hr of dehydration in air. Forty-two hibernating frogs, obtained in early November, were acclimated in the laboratory for 12 days at 20°C. The thirty-six surviving animals were sub-divided into four groups, with ten or seven animals being

42

ARTHUR

M.

JUNGREIS

dehydrated in air for 12.5 or 48 hr, respectively, and ten or nine animals, respectively, serving as tap water controls. Eflects of anti-diuretic hormone. Frogs used in these experiments were obtained at the same time as, and acclimated in a similar manner, to those described above. Four groups of eighteen frogs were injected with: Group I, 2 P.U./frog. 0.25 ml of saline.

Group II, 5 P.U./frog.

Group III, 0.10 ml of saline. Group IV,

and returned to tap water for 4, 7 or 14.5 hr, respectively, after which blood was collected from 6 or fewer animals per group. For reasons previously elaborated (see Responses of spring-summer frogs, Effects of anti-diuretic hormone), the effects of serum from dehydrated B. marinus was also investigated. When frogs were injected with Pitressin or saline (Groups I-IV), thirty-six additional frogs were simultaneously administered at two dose levels serum from B. marinus: Group V,

0.10 ml B. ma&us

serum/frog.

Group VI,

0.25 ml B. marinus serum/frog.

RESULTS

Responses of spring-summer frogs to dehydration in air Urea. Urea was determined in blood and urine of spring-summer frogs dehydrated in air or kept in tap water (Table 1). Following 6-7, 12, 18, 27 or 48 hr of treatment, the concentration of urea in blood and urine was significantly elevated relative to tap water controls (P < 0.001). The increased concentration of urea in blood of animals dehydrated for only 6-7 hr is evidence that an increase in urea production has occurred. To verify this, the following calculation was made: At least 80 per cent of the wet weight of R.pipiens is water (Schmid, 1969). Since urea is evenly distributed throughout all body water except that found in the urinary bladder or the kidney (Marshall & Davies, 1914), the quantity of urea in a frog whose bladder is empty can be determined from equation (1):

T=

U,+O.8

x [U,l x IV,

where T is the quantity of urea present, U, is the quantity excreted per unit time, [ UJ the concentration of urea in serum (PM/g fluid) and W is the body weight (g). Assuming a body weight of 27 g, the quantity of urea present after 67 hr of treatment (data from Table 1) would be [from equation (l)] 0.8 x 4.3 x 27 = 93.7 PM of urea. Since the quantity of excreted urea was not measured, the following approximation was made. Frogs acclimated at 25°C in the spring-summer had a mean rate of urea excretion of 0.21 PM urea/g per hr (Jungreis, 1970). If animals used in this experiment had comparable rates of urea excretion, tap water frogs would have excreted 40.3 PM of urea in a 7-hr period. Therefore, the total quantity of urea associated with tap water controls is 93.7 +40*3 = 134 PM of urea. Different assumptions are needed for dehydrated frogs. If I assume that a 4 per cent decrease in body weight (see Table 2) is attributable exclusively to water loss, then a 25-g frog which started out with 21.6 g of water would have only 20.5 g after 7 hr. Urea present in individual frogs would then be equal to 20.5 x 8.47 or 173.8 PM of urea. An estimate of excreted urea was obtained by assuming

* Standard error of the mean. t Total urinary nitrogen is defined as nitrogen derived from ammonia or urea. 1 Because the experiments being run were designed to end at about the same time on the same day, urine collected from frogs kept in tap water at the beginning of an experiment also represented urine collected at the end of a period of dehydration. Consequently, all tap water treated frogs used as controls for individual experiments were treated as one group.

Ammonia Urea+

No. of frogs Urine urea (mM) No. of frogs Blood ammonia (mM) No. of frogs Urine ammonia (mM) oh Total urinary output

No. of frogs Blood urea (m&l)

Hours of dehydration in air

TABLE ~--THE CONCENTRATIONS OF AMMONIA AND UREAIN BLOOD AND URINE, AND THE PERCENTAGES OF THE URINARY NITROGEN EXCRETED AS AMMONIAOR UREA IN FROGS,R. pipiem, ACCLIMATED AT 22*.5”C, AND DEHYDRATED IN AIR FORUP TO 48 hr IN LATEMARCH

ARTHURM. JUNGREIS

44

TABLE ~--MEAN WEIGHTSANDMEANPERCENTAGE WEIGHTINCREASES IN FROGSACCLIMATED AT 22*5X, .4m DEHYDRATED IN AIR OF 50% R.H. FOR UP ~0 7.5 hr Hours of dehydration in air

Tap

No. of animals Mean wt. (g) Initial Final o/0 Increase

Water

1.75-2.75

3-4

19

9

6

18.9 18.2 - 3.8

19.3 18.8 -2.5

19.6 19.7 +0.6

4.5-5.49 12 20.7 20.0 -3.4

5.5-6.49 5 22.1 21.2 -4.2

6.5-7-S 7 21.7 21.0 -3.3

that the 0.4 ml (average) of urine collected from frogs dehydrated for 7 hr was the minimum voidable volume of urine present in the bladder. This value is in agreement with that predicted from the studies of Hong (1957) and Schmidt-Nielsen & Forster (1954). The concentration of urea in urine was 77.9 PM/ml (data from Table 1). Therefore, the minimal quantity of urea excreted was 0.4 x 77.9 = 31.2 PM, and the total quantity of urea associated with 7 hr dehydrated frogs is 31.2 + 173.8 = 205 PM. This quantity is 71 PM or 53 per cent greater than that present in tap water controls. Thus, the increase in serum urea observed reflects an increase in urea production. Ammonia. Ammonia was determined in blood and urine of frogs dehydrated for up to 48 hr. The quantity of ammonia present in blood approached the minimal quantity measurable by the method of micro-diffusion. This quantity of ammonia is associated with red cell lysis, since freshly drawn blood, if rapidly centrifuged, contains undetectable quantities of ammonia (data not shown). In urine, the concentration of ammonia did not increase prior to 48 hr of dehydration (P> O-10). The absence of a significant increase in urine ammonia when contrasted with the observed increase in urine urea suggests that excreted ammonia originated in the kidney rather than the blood (Jungreis, 1969; Pitts, 1968). Urine urea and its relation to urine osmotic pressure. The osmotic pressure of urine increased in response to dehydration (P-C O*Ol), and reached a maximum after 27 hr. While the proportion of the urine osmotic pressure occupied by urea increased after 6 hr of dehydration, no further increase was noted with length of dehydration (Table 3). Effects of anti-diuretic hormone. Pitressin was administered intra-abdominally to spring-summer frogs at three dose levels: 4, 12 and 20 P.U./lOO g (Table 4). No increase in the concentration of urea in serum was observed (P> 0.10). However, after administration of 12 or 20 P.U./lOO g, a decreased level of urea in serum was noted (P < 0.01, Table 4). While these animals were hydrated in response to anti-diuretic hormone, the increase in water content was insufficient to account for the decrease in urea present (data not shown). That Pitressin entered the circulatory system of these frogs was evidenced by extensive skin darkening within 3 hr

UREAPRODUCTION IN THE FROG

45

TABLE ~-THE OSMOTICPRESSUREOF URINEANDTHE PERCENTAGE OF THE OSMOTICPRESSURE REPREEENTEDBYUREA OBSBRVEDTOINCREASl3INSPRING-SUMMERFROGS, R.$~i@ts, ACCLIMATED AT 22*5"c, AND SUBJECTED TO DEHYDRATION IN AIR OF 50% R.H. FOR UP TO 48hr Length of time dehydrated in air (hr) 0 (tap water) 6 18 27 48

No. of frogs

(7r urea) 100% (total r)

21 5 1 2 2

39.8 79.4 53.3 66.7 41.2

f 3.36* its6.42 f 6.61 + 26.5

v urine (mOsmole) 66.0 106.8 94.6 210.5 232.1

f 18*7* + 20.6 _+15.9 f 32.5

* Standard error of the mean. TABLE ~-THE EFFECTS OF PITRBESIN AND SERUM FROM DEHYDRATED THE CONCENTRATION OF UREA IN SERUM OF SPRING-SUMMER FROGS, AT 20°C (SEE TEXT)

TOADS, B. marinus, ON R. pa@?rS, ACCLIMATED

No. A Treatment Pitressin 4 P.U./lOO g 20 P.U./lOO g Saline control 0.25 ml/frog

Days of acclimation

of frogs

Time (hr)

Serum urea (mW

Skin darkening

7 7

8 8

4 6

159+1.49* 7.32 + 0.77

7

10

5

13.1 f. 2.12

8 8

10 8

6 7

7.42 f 0.36 17.2 + 0.81

0.15 ml/frog

8

9

6

15.6kO.67

-

Saline control 0.15 ml/frog

8

10

7

9.52 + 0.43

-

B Pitressin 12 P.U./lOO g 0.05 ml/frog

B. marinus serum

+++++ +++++ -

+++++ -

* Standard error of the mean.

after administration of this drug. When serum from dehydrated toads, B. marinus, was administered, a significant increase in the concentration of urea in serum was observed (Table 4, P-c 0.01). Responses of fall-winter frogs to dehydration in air

Fall-winter frogs were dehydrated in air following the identical procedures No increase in the concentration of serum urea was used in the spring-summer. observed after 12.5 hr of dehydration (P> O-lo), although a significant increase

46

ARTHUR M. JUNGRBIS

was

observed after 48 hr (P-C 0.001) (Table 5). When Pitressin was administered, no increase in serum urea was observed (P> O-10) (Table 6). After 14.5 hr of treatment, frogs administered 5 P.U./animal ( = 20 P.U./lOO g) had significantly reduced quantities of urea in serum (PC O*OOl),in agreement with the response of spring-summer animals. However, spring-summer frogs responded after only 6 hr, while fall-winter frogs had not responded even after 7 hr. It is noteworthy that frogs exhibiting a 9 per cent increase in body weight did not exhibit an elevated level of urea in serum. When serum from dehydrated B. ma&us was administered to fall-winter frogs, no increase in the concentration of serum urea was observed (2’~ O-10) (Table 6), a result which contrasts markedly with the response of frogs during the spring-summer. TABLE S-THE CONCENTRATION OF UREA IN SERUM OF FALL-WINTER FROGS, R. pipiens, ACCLIMATED AT 20”C, AND DEHYDRATEDIN AIR-WAS NOT OBSERVEDTO INCREASEFOLLOWING 12.5 hr OF DEHYDRATION IN AIR, ALTHOUGH A SIGNIFICANT INCREASEIN THE CONCENTRATION OF UREA OBSERVEDFOLLOWING 48 hr OF DEHYDRATION IN AIR

Serum urea (mM) -~No. of frogs Air dehydration Tap water

12.5 hr 13.0 + l%* 9.57 + 1.27

10 10

No. of frogs

48 hr

7 9

32.5 f 2.60* 6.95 f 1.11

* Standard error of the mean.

DISCUSSION

In response to short-term dehydration in hyper-osmotic sodium chloride, spring-summer but not fall-winter frogs, R. p. pipiens, had increased concentrations of urea in serum and increased rates of urea production (Jungreis, 1971). When frogs were subjected to mild dehydration in air, comparable seasonal patterns of response were observed. The increase in urea associated with airdehydrated frogs is not associated with a decrease in ammonia excretion with concomitant enhancement of urea excretion, since ammonia represents only a small fraction of the total nitrogen excreted in the forms of ammonia and urea. Nor is the increase designed to conserve water, since frogs in the fall-winter fail to respond to dehydrating conditions with an increase in urea production. Why then does this increase

in urea production

that the observed

increase

an effect by Pitressin following

administration

also supports of massive

be caused by other pituitary by skin darkening.

occur ? The

seasonal nature of the response

is not associated

hormone

with water

this conclusion. quantities

The

of Pitressin

contaminants

is evidence

loss, per se. The decrease (12-20

absence

in serum

P.U./lOO

in the preparation,

of

urea

g) may

as evidenced

~-THE

l

Standard error of the mean.

12.8 t 2.00 11.4f1.02

6 6

9.93 f o-93 10.6 f 0.77

5 5

3 6

11*2t 1.17 12.4& 1.53

13.3 + 0,85 7.02 & 1.35

+3.0

+4.6 +9-o

-

+++++ ++-I-++

-

2 E:

5! 1 g

!

6 6

11-l + 1.25 12.2 + 0.96

13.6 f 4.07

B. murinusserum 0.10 ml/frog 0.25 ml/frog

6 6

5

10.5 & 1.02 12.2 * 1.21

10.7 * 1.59

-

6 6

6

0

E 3

10.0 f 1.66

15.5 rt 1.91*

Skin darkening

6

5

14.5 hr

0.25 ml/frog Pitressin 2 P.U./frog 5 P.U./frog

11*4* 1*00*

N

%

6

7 hr

8

11.0 j: 1*18*

N

OF

6

4 hr

OF UREA IN SERUM

7 hr weight change (%)

THE CONCENTRATION

0.10 ml/frog

N

Serum urea (mM)

EFFECTS OF PITRSSSIN AND SERUM FROM DEHYDRATED TOADS, B. marinus,~~ FALL-WINTER FROGS, R.&&m (SEETSXT)

Saline control

Treatment

TABLE

48

ARTHUR

M.

JUNGREXS

The increase in urea production does not result from de nova synthesis of urea cycle enzymes. Boer&e (1973) failed to find a significant increase in liver or kidney arginase (P> O-05) of R, pipiens in response to dehydration in air. However, arginase is not rate limiting in the synthesis of urea (Brown & Cohen, 1959). While McBean & Goldstein (1967) could measure an increase in the specific activities of the two rate-limiting enzymes in Xenopzrs 2aevis liver: carbamate kinase and ornithine-carbamate transferase, following dehydration in O-25 M sodium chloride, the increases could not account for the enhanced rate of urea synthesis in aivo. A major difference between spring-summer and fall-winter frogs appears to be the relative availability of mobilizable glucose. Spring-summer frogs are reported to have reduced intracellular pools of glucose (Hong et al., 1968), a reduced capacity to produce or store glycogen (Gourley et al., 1969), as well as decreased quantities of glycogen in liver (Smith, 1952; Mizell, 1965 ; Hong et al., 1968; Brenner, 1969). Metabolism of amino acids appears to be of greater importance to frogs in the spring-summer than the fall-winter in maintaining the concentration of glucose in serum (Jungreis, 1970; Jungreis & Hooper, 1970). These seasonal differences are due, in part, to changes in corticosteroid regulation (Varute, 1969; Jungreis et al., 1970) and pituitary function (Sekiguchi, 1968; Kemenade, 1969). As a metabolic stress, dehydration in air should cause an increase in the rate of glucose uptake and utilization. The absence of adequate ~rbohydrate reserves during the spring-summer should cause these animals, in response to dehydration stress, to have enhanced rates of gluconeogenesis. An increase in urea production would result from an increased rate of amino acid metabolism. I had proposed that the increase in urea production was mediated by glucoco~icoids (Jungreis, 1971). However, following dehydration in hyperosmotic saline, a decrease in corticosterone titer was observed (Jungreis et al., 1970), a response opposite of that ,predicted. Glucocorticoid involvement cannot be ruled out entirely for several reasons. Aldosterone, a mineralocorticoid, also has glucocorticoid activity (Middler et aZ., 1969), and the ratio of aldosterone to corticosterone synthesis in amphibia is very high (Crabbe, 1963; Johnston et al., 1967; Jungreis et aZ., 1970). Further experimentation is required to settle this matter. Ammonia was present in hemolyzed R. pip&m blood (Table 1, and S&mid, 1968), but not freshly drawn blood. No ammonia could be detected in freshly drawn blood if it was immediately centrifuged, whereas a delay of as little as 15 min prior to centrifugation could result in the appearance of measurable quantities of ammonia (Jungreis, unpublished). The proportions of amino nitrogen excreted as ammonia or urea were determined in frogs dehydrated in air for up to 48 hr. The percentage of amino nitrogen excreted as ammonia ranged from O-6 to 3.4 per cent, a fraction so small that ammonia can play only a minor role in the elimination of excretory nitrogen. The quantity of ammonia present in urine was related to the pH of blood, and is associated with conservation of the alkaline reserve of blood (Jungreis, 1969).

UREAPRODUCTION IN THE FROG

49

REFERENCES BOERNKEW. E. (1973) Adaptations of amphibian arginase-I. Response to dehydration. Comp. Biochem. Physiol. 44B, 647-655. BRENNERF. J. (1969) The role of temperature and fat deposition in hibernation and reproduction in two species of frogs. Herpetologica 25, 105-113. BROWNG. W. & COHENP. P. (1959) Comparative biochemistry of urea synthesis. J. biol. Chem. 235. 1769-l 774. CONWAYE. J.‘& BYRNE A. (1933) An absorption apparatus for the micro-determination of certain volatile substances-I. Micro-determination of ammonia. Biochem. J. 27, 419-429. CRABBE J. (1963) Nature and metabolism of adrenocortical secretion in the toad Bufo marinus. Gen. & compar. Endocr. 3, 692-693. FREEMANS. E., SATCHELLD. G., CHANGC. S. & GAY W. S. (1968) The effect of high K+ solutions and fatty acid substrates on metabolic pathways in toad heart. Comp. Biochem. Physiol. 26, 31-44. GOURLEYD. R. H., SUH T. K. & BRUNTONL. L. (1969) Seasonal differences and the effect of insulin on pyruvate uptake oxidation and synthesis to glycogen by frog skeletal muscle Comp. Biochem. Physiol. 29, 509-524. HONG S. K. (1957) Effects of pituitrin and cold on water exchange in frogs. Am. J. Physiol. 188, 439-442. HONG S. K., PARK C. S., PARK Y. & KIM J. K. (1968) S easonal changes of anti-diuretic hormone action on sodium transport across frog skin. Am.J. Physiol. 215, 439-443. JOHNSTONC. I., DAVISJ. O., WRIGHT F. S. & HOWARDSS. S. (1967) Effects of renin and ACTH on adrenal steroid secretion in the American bullfrog. Am. J. Physiol. 213, 393-399. JUNGREISA. M. (1969) Seasonal effects of dehydration on ammonia and urea production in the frog, Rana pipiens. Am. Zool. 9, 205A. JUNGREISA. M. (1970) The effects of long-term starvation and temperature acclimation on glucose regulation and nitrogen catabolism in the frog Rana pipiens-II. Summer animals. Comp. Biochem. Physiol. 32, 433-444. JUNGREISA. M. (1971) Seasonal effects of hyper-osmotic sodium chloride on urea production in the frog, Ranapipiens. ‘J. exp. Zool. 178, 403-414. JUNGREISA. M. & HOOPERA. B. (1970) The effects of long-term starvation and temperature acclimation on glucose regulation and nitrogen catabolism in the frog Rana pips&s-I. Winter animals. Comp. Biochem. Physiol. 32, 417-432. JUNGREISA. M., HUIBREGTSEW. H. & UNGAR F. (1970) Corticosteroid identification and corticosterone concentration in serum of Rana pipiens during dehydration in winter and summer. Con@. Biochem. Physiol. 34, 683-689. KEMENADEJ. A. M. (1969) The effects of metopirone and aldactone on the pars distalis of the pituitary, the interrenal tissue, and the interstitial tissue of the testis in the common frog, Rana temporaria. Z. Zellforsch. mikrosk. Anat. 96, 466-477. MCBEAN R. L. & GOLDSTEINL. (1967) Ornithine urea cycle activity in Xenopus laewis, adaptation to saline. Science, Wash. 57, 931-932. MARSHALLE. K. & DAVISD. M. (1914) Urea, its distribution in and elimination from the body. J. biol. Chem. 18, 53-80. MIDDLER S. A., KLEEMANC. R., EDWARDSE. & BRODY D. (1969) Effect of adenohypophysectomy on salt and water metabolism in the toad Bufo marinus with studies on hormone replacement. Gen. &? compar. Endocr. 12, 290-304. MIZELL S. (1965) Seasonal changes in energy reserves in the common frog, Rana pipiens. J, cell camp. Physiol. 66, 251-258. OBRINK K. J. (1955) A modified Conway unit for micro-diffusion analysis. Biochem. J. 59, 3446.

ARTHURM. JUNGREIS

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PITTS R. F. (1968) Physiology of the Kidney and Other Body Fluids. 2nd Ed. Yearbook Medical Publishing Co., Chicago. SCHMIDW. D. (1968) Natural variation in nitrogen excretion of amphibians from different habitats. Ecology 49, 180-185. SCHMID W. D. (1969) Physiological specializations of amphibians to habitats of varying aridity. In Physiological Systems in Semi-arid Environments (Edited by HOFF C. C. & RIEDESELM. L.), pp. 135-142. University of Mexico Press. SCHMIDT-NIELSEN B. & FORSTER R. P. (1954) The effect of dehydration and low temperature on renal function in the bullfrog. J. cell. &f camp. Physiol. 44, 233-246. SEKICUCHIT. (1968) Independency of the adrenocortical cell from phyophyseal control as elucidated in hypophysectomized bull frogs. Endocrinology Jap. 15, 70-81. SHOEMAKER V. H. (1965) The stimulus for the water-balance response to dehydration in toads. Comp. Biochem. Physiol. 15, 81-88. SMITH C. L. (1950) Seasonal changes in blood sugar, fat body and liver glycogen, and gonad in the common frog Rana temporaria. J. exp. Biol. 26, 412-419. SMITH C. L. (1952) Environmental temperature and the glycogen content of the frog’s liver (Rana temporaria). Nature, Lond. 170, 74-75. VARUTEA. T. (1969) Seasonal variations in summer cells of adrenal of male frogs Rana tigrina. Ind. J. exp. Biol. 7, 270-273. Key

Word Index-Dehydration;

urea production;

frog excretion; Rana pipiens.