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The effects of arginine vasotocin and oxytocin on sodium and water balance in Ambystoma
Comp. Biochem. Physiol., 1965, 17ol. 16, pp. 531 to 546. Pergamon Press Ltd. Printed in Great Britain
THE EFFECTS OF ARGININE VASOTOCIN AND OXYTOCIN ON SODIUM AND WATER BALANCE IN A M B Y S T O M A * R O N A L D H. A L V A R A D O and S H E L D O N R. J O H N S O N Department of Zoology, Oregon State University, Corvallis, Oregon (Received 4 M a y 1965)
Abstract--1. When injected with arginine vasotocin (20-100 mU) both larvae and adults gain weight. The duration of the response is related to dosage. The hormone does not influence the osmotic uptake of water so that the response must result from antidiuresis, a large share of which can be attributed to a reduction in glomerular filtration. 2. Arginine vasotocin stimulates the net uptake of sodium by larvae and adults. In larvae this response lasts approximately 24 hr and is followed by a pronounced net loss of sodium. Adults gain sodium for at least 43 hr. During the initial 24 hr after injection of the hormone into larvae the efflux and the influx are increased above control values. However, the influx is enhanced to a greater extent and the animals gain sodium. After 24 hr the influx returns to normal but the high efflux persists for several hours. This leads to a net loss of sodium. The net uptake of sodium by adults injected with the hormone is the result of a faster influx. 3. Moderate doses (50-100 mU) of oxytocin caused larvae to lose weight and had no effect on the weight of adults on a short-term basis. The hormone had no effect on the osmotic inflow of water in adults or larvae. Presumably the loss of weight in larvae reflects a diuretic condition. Higher doses of oxytocin (500 mU) abolished this response and caused a slight increase in weight. 4. Oxytocin increases the efflux Of sodium from larvae and adults, possibly by inducing diuresis. The hormone had no effect on the influx of sodium in adults or larvae. 5. An effort has been made to interpret the results in terms of their significance in the maintenance of an osmotic and ionic steady state by these animals. INTRODUCTION RECENT interest in the comparative endocrinology of vertebrates has led to intensive investigations on the morphology and function of the hypothalamic-neurohypophysial complex (Green & Maxwell, 1959; Sawyer, 1961a, 1961b, 1963, 1964; Heller, 1963; Scharrer & Scharrer, 1963; Kleeman & Cutler, 1963). A m o n g the functions of this system is the elaboration of hormones which participate in the regulation of hydromineral metabolism. Of the submammalian classes of vertebrates the Amphibia have been most extensively studied. However, within this class most investigations have been limited to the anurans. * Supported by grants from the National Institutes of Health (GM-11817) and the National Science Foundation (G-12471), and by the Oregon State University General Research Fund. 531
532
RONALD H. ALVARADO AND SHELDON R. JOHNSON
Both anurans and urodeles are known to secrete arginine vasotocin (Follett & Heller, 1964). This hormone has been found in all submamma]ian vertebrates which have been examined except for the Elasmobranchii (Heller, 1963). At ]east one other neurohypophysial hormone is found in Amphibia. It is similar to oxytocin in many of its effects but differs in certain pharmaco]ogical properties (Follett & Heller, 1964). In the anuran, Rana esculenta, the hormone has recently been identified as isoleucine-8-oxytocin (Acher et al., 1964). With respect to hydromineral metabolism the responses of amphibians to neurohypophysial hormones may be divided into a water-balance effect and a sodium-balance effect. Comparative studies on several species of anurans and a few species of urodeles have revealed striking species differences in the nature and magnitude of these responses. Brunn (1921) first demonstrated that injection of neurohypophysial extracts into frogs immersed in water leads to an increase in their weight. The response has since been observed in many species of anurans. In this group the hormones act by (1) increasing the permeability of the skin to water (Fuhrman & Ussing, 1951), (2) increasing the permeability of the urinary bladder to water (Ewer, 1952a) and (3) causing renal antidiuresis (Houssay & Potick, 1929). The last response may result from a decrease in glomerular filtration or an increase in tubular reabsorption of water (Sawyer, 1957; Bentley, 1963). For predominantly terrestrial forms the water-balance response has adaptive value and indeed there appears to be a positive correlation between the magnitude of the response and degree of terrestrialism (Steggerda, 1937; Ewer, 1952b). In addition to their effects on water balance in anurans the neurohypophysial hormones also influence sodium metabolism. Active transport of sodium across the isolated frog skin (Fuhrman & Ussing, 1951), the urinary bladder (Ewer, 1952a; Leaf et al., 1958) and the renal tubules (Jard & Morel, 1963) is enhanced by these hormones. Relatively little attention has been devoted to the endocrine control of hydromineral metabolism in urodeles. The hormones secreted by the hypothalamicneurohypophysial complex of urodeles have only recently been characterized (Follett & Heller, 1964). Most investigations on urodeles have involved the injection of high concentrations of hormones of mammalian origin. These studies, and those of Bentley & Heller (1964) using lower doses of arginine vasotocin and oxytocin, have shown that larval and adult urodeles display a water-balance effect (B61ehr~dek & Huxley, 1927; Steggerda, 1937; Dow & Zuckerman, 1939). Sodium balance is also affected by these hormones (Jorgensen et al., 1946; Bentley & Heller, 1964). Neither the water-balance nor the sodium-balance response has been systematically analyzed using purified or synthetic hormones on both larvae and adults of the same species. Such studies may be of interest, not only because they may reveal information about the ontogeny of the responses involved, but also because they afford a direct means of testing the hypothesis that the osmoregulatory aspects of neurohypophysial function have evolved in association with terrestrialism. Finally, studies on urodeles are important from the point of view of the evolution
S O D I U M AND WATER BALANCE I N A M B Y S T O M A
533
of neurohypophysial function, for this group represents the most primitive order in which a water balance effect has been demonstrated. MATERIALS AND METHODS
1. Animals Most of the experiments on larvae and all on adults were performed on Ambystoma tigrinum collected in eastern Washington and shipped by air to Corvallis, Oregon. Some experiments were conducted on larval Ambystoma gracile collected in Corvallis. The larvae of both species were of comparable size. No significant species differences were observed with respect to the parameters under consideration. Larvae were stored in dechlorinated tap water at 8-10°C. Adults were stored in a similar fashion except that they could crawl out of the water at will. None of the animals were fed. For experimental purposes the animals were immersed in artificial pond water (1.3 mM sodium chloride, 0.8 mM calcium chloride, 0.1 mM potassium chloride and 0.2 mM sodium bicarbonate) and equilibrated at 15-17°C for at least 1 week prior to the initiation of an experiment. All experiments were done at this temperature. 2. Anesthetic These animals are sensitive to handling (Jorgensen et al., 1946; Alvarado & Kirschner, 1963). Stress induces diuresis and upsets salt balance. Thus for operations involving extensive handling, such as injections or ligation of the cloaca, the animals were anesthetized by immersion in 0.1% tricaine methane sulfonate (pH adjusted to 7.0). Complete immobilization requires approximately 20 min for larvae, slightly longer for adults. The animals recover within 30 min after removal from the anesthetic. 3. Hormones Synthetic oxytocin (Syntocinon) was obtained from Sandoz Pharmaceuticals. Synthetic arginine vasotocin (AVT) was kindly supplied by Dr. Brian Follett, Washington State University. Doses of AVT are given in pressor units, of oxytocin in rat uterus units. Each hormone was diluted with amphibian Ringer's solution. All injections were made into the peritoneal cavity; the injection volume was 0.1 ml. Both hormone preparations contained chloretone as a preservative and were acidified with acetic acid. Control animals were injected with appropriate solutions of these substances. 4. Water balance Animals were anesthetized, injected with the desired solution and each was immersed in artificial pond water. At specified intervals each animal was weighed (precision + 0.05 g).
534
RONALD H. ALVARADO AND SHELDON R. JOHNSON
5. Osmotic uptake of water Each animal was anesthetized, its urinary bladder was emptied by supra-pubic compression and the cloacal opening was blocked with a purse-string ligature. After injection of the appropriate solution each animal was rinsed, weighed and placed in artificial pond water. Weighings were made at 2 hr intervals for 8 hr. The rate of increase in weight is linear over this period and the rate of uptake of water can be estimated directly. After 8 hr each animal was anesthetized and weighed. The cloaca was unblocked and the urine expelled. Each animal was then reweighed and the mass of urine was obtained by subtraction. A sample of the urine was analyzed for sodium by flame photometry. 6. Glomerular filtration rate The effect of AVT on glomerular filtration was studied by measuring the clearance of Cl~-inulin. The inulin (2.0 t~c) was injected into the peritoneal cavity and the animals were permitted to equilibrate for 24 hr. Each animal was then anesthetized and injected with either 100 mU of AVT or a control solution. After rinsing, each animal was placed in 250 ml of artificial pond water. Samples of the bathing solution were assayed for radioactivity at 1 hr intervals for 10 hr. A serum sample was then obtained and a quantitative fraction assayed for activity. From the total radioactivity in the bath and the serum radioactivity the renal clearance of inulin was estimated. 7. Sodium fluxes The efflux of sodium was measured with Na ~ (Na2~C1). The isotope was diluted in amphibian Ringer's solution and 2-0 t~c was injected into each animal. After 3 days of equilibration each animal was rinsed, anesthetized, treated with the hormone or control solution and immersed in 300 ml of pond water. Samples of the bathing medium were removed at specified intervals and assayed for radioactivity by conventional means (Alvarado & Kirschner, 1963). After 30-50 hr each animal was sacrificed and the specific activity of a serum sample was determined. The efflux of sodium was calculated from the serum specific activity and the rate of appearance of radioactivity in the bath. The net flux was determined by monitoring the concentration of sodium in the bath by flame photometry (precision + 1.0 per cent). The influx was computed from the efflux and the net flux. RESULTS 1. Studies with arginine vasotocin (a) Water balance. No effort was made to establish dose-response relationships for most of the experiments described in this report. However, a pilot study on the effects of various doses of AVT on body weight of larvae showed that doses below 20 mU/animal produce erratic results. For example, a dose of 10 mU/animal produced an increase in weight in only 4 out of 7 animals. The response was small (2-3 per cent increase in weight in 3 hr) and lasted only 3-4 hr. Doses of AVT
SODIUM AND WATER BALANCE IN AMBY3TOMA
53S
above 20 mU/animal produced consistent responses (Fig. 1). The duration of the response is positively correlated with the concentration of the hormone. There is no correlation between dosage and the rate of increase in body weight over the initial 4 hr of the experiments. This suggests that the increase in weight reflects a reduction in water excretion rather than an increase in the osmotic inflow of water, Comparable results were obtained with larval A. gracile. 112
.......*
IOOmU (5)
lOB , s e ~'B
I04 5 0 m U (5) 25rnU (3)
o
IO0
96 Con. 191
92 0
2
I 4
I 6
I 8
I I0
hr
FIG. 1. Effect of A V T o n b o d y w e i g h t in larval A. tigrinum. T h e dose ( m U / a n i m a l )
and the number of observations are indicated at the right. The mean initial weight of the animals was 14"42g + 0-80 (20). Standard errors have been omitted for clarity, however the maximum S.E. observed Was + 2"3. Control larvae lost weight. This response is characteristic of larvae and is less pronounced or absent in adults (Fig. 2). It probably reflects a diuresis induced by stress. Adults also respond to AVT (Fig. 2). Again the dosage is correlated with the duration of the response rather than with the rate of increase in body weight. The adults used in this experiment were about 10 g heavier than the larvae. On a weight basis the dose o f AVT administered to the adults injected with 100mU was 4.2 mU/g. This corresponds with the larvae injected with 50 mU of AVT (dosage 3.7 mU/g). Comparison of the responses elicited at this dosage shows that after 4 hr the rate of increase in weight decreased and, by 6 hr the maximum weight had been reached in larvae. On the other hand, adults continued to gain weight at a
536
RONALD H. ALVARADO AND S H E L D O N R. J O H N S O N
constant rate for 8 hr. T h e experiment was terminated at this time to determim urine volume and composition, so the duration of the response was not established These data indicate that the water-balance effect induced by A V T is more pro. nounced in adults than in larvae. 112
108
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-6
...
104 ,
........
1
s s Je
e pss
.E
Con
i
( 51
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96
I 2
0
I 4
I 6
I 8
hr
FIG. 2. Effect of AVT on body weight in adult A. tigrinum. The dose (mU/animal) and the number of observations are indicated at the right. The mean initial weight of the animals was 25'00 g +_1"60 (10). Standard errors for control animals and those injected with 100 mU are indicated for the 8 hr reading. EFFECT OF A V T ON THE OSMOTIC UPTAKE OF WATER BY LARVAL AND ADULT THE DOSE WAS 1 0 0 m U / A N I M A L . UNLESS OTHERWISE SPECIFICED THE VALUES INDICATED IN THIS AND ALL SUBSEQUENT TABLES REPRESENT MEAN ~ S . E . (?t).
TABLE 1--THE
A. tigrinum.
Initial body wt (g) Stage
Control
AVT
Larva Adult
13-67+ 1.10(10) 34"16+ 2-06(4)
12"54+_0-68(10) 32"06+ 0.44(4)
Rate of increase in wt (% of initial wt/hr) Control 1.28 + 0-09(10) 1.40 + 0"10(4)
AVT 1"21 +_0'06(10) 1-40 + 0-10(4)
(b) Osmotic uptake of water. Table 1 shows that A V T has no effect on the rate of uptake of water in larval or adult A. tigrinum; this has been corroborated by studies on larval A. gracile. T h u s , the hormone does not increase the permeability of the skin to water. This is supported by the observations of Bentley & Heller (1964) on isolated pieces of skin and apparently represents a basic difference between urodeles and anurans. (c) Rate of urine production and glomerular filtration. Table 2 shows that A V T causes a pronounced antidiuresis. T h e mechanisms producing this effect have not
537
SODIUM AND WATER BALANCE IN AMBYSTOMA
been studied in detail; however, a few inulin clearance m e a s u r e m e n t s have been made on larvae. In these forms the major share of the antidiuresis can be ascribed to a reduction in glomerular filtration. T h i s is shown in T a b l e 3. Arginine vasotocin had no significant effect on sodium concentration in the urine of larvae or adults (Table 4). TABLE 2 - - T B E EFFECT OF A V T ON URINE PRODUCTION IN LARVAL AND ADULT A. tigrinum* Initial body wt (g)
Rate of urine production (mg/g hr)
Stage
Control
AVT
Control
AVT
Larva Adult
13-08 + 1"15(9) 34"16 + 2'03(4)
12"30+ 0'67(11) 32"06+ 0'45(4)
10"4 + 2'1(9) 16"2 + 2"0(4)
3"8 + 0"7(11) 4"0 + 0-8(4)
* The dose was 100 mU/animal. TABLE 3 - - T H E EFFECT OF A V T ON INULIN CLEARANCE IN LARVAL A . tigrinum*
Treatment
Control AVT
Body wt (g)
Clearance (ml/g hr)
15"7 + 1"7(5) 16'3 +_2'0(6)
0"025_+0"008(5) 0"004 + 0"002(6)
* Dose 100 mU/animal. TABLE 4
T H E EFEECT OF A V T
ON SODIUM CONCENTRATION IN THE URINE OF LARVAL AND a d u l t A. tigrinum* Sodium concentration (raM/1.)
Stage
Control
AVT
Larva Adult
5.2 + 1.0(10) 7"4 + 1"2(6)
4.6 + 1.4(11) 9"9 _+1-6(5)
* The dose was 100 mU/animal.
(d) Sodium fluxes. T h e effect of A V T on sodium balance in larval .4. tigrinum is expressed in Fig. 3. All values have been corrected to a standard body weight of 10 g and mean values are indicated. Control animals maintained a steady state. Larvae injected with A V T experienced an initial net uptake of sodium which lasted 20-25 hr. T h i s was followed by a net loss of sodium. T h e mean flux values measured over the first 32 hr following treatment are given in T a b l e 5. T h e h o r m o n e increased both the influx and the e m u x ( P < 0.05); however, the influx was enhanced to a greater extent and a net uptake of sodium ensued. Flux measurements showed that the net loss of sodium which begins approximately 25 hr after
RONALD H.
538
ALVARADO AND SHELDON R . JOHNSON
injection results f r o m a r e t u r n of the influx to approximately control values, while the efflux remains high. Eventually the influx and the effiux m u s t converge and the animals r e s u m e a steady state; however, the time course has not been followed. F i g u r e 4 shows that adults also take up net quantities of s o d i u m w h e n injected with A V T . C o m p a r i s o n of Figs. 3 and 4 reveals that there are quantitative
SS
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t
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AVT
.~_ ~
-20
-30
o
I
I
I
I
I
IO
20
30
4o
5o
hr
FIG. 3. Effect of AVT on sodium balance in larval A . tigrinum. The dose was 100 mU/animal. The mean weight of the AVT-treated animals was 15-3 g -+0"8 (6), that of the controls was 16"0 g +_0-8 (6). Vertical lines represent _+1 S.E. T A B L E 5 - - T H E EFFECT OF A V T ON SODIUM FLUXES IN LARVAL AND ADULT A . tigrinum. FLUXES WERE MEASURED OVER 32 HR FOR LARVAE AND 39 HR FOR ADULTS
Stage
Treatment
Body wt (g)
THE
Sodium fluxes (/z-equiv/10 g/hr) Efflux
Influx
Net flux
Larva
Control AVT
14-8+0"7(11) 15"2 + 1-0(12)
1'5+0"1(11) 2.1 +_0"2(12)
1'3_+0-1(11) 2'8 -+0-3(12)
-0-2_+0'2(11) 0'7 -+0.4(12)
Adult
Control AVT
38'4 + 0"7(4) 31"4+1-6(3)
0-7 -+0-1(4) 0"6+0"1(3)
0.9 _+0'1(4) 1"5_+0"1(3)
0"2 + 0.1(4) 0-9+0"1(3)
539
SODIUM AND WATER BALANCE I N . / I M B Y T O M A
differences between larvae and adults, particularly with respect to the duration of the response. In adults the response was pronounced even after 43 hr, when the experiment was terminated. The doses of AVT/unit weight were 6.5 mU/g and 3.2 mU/g for larvae and adults respectively. Thus the sodium-balance effect, as well as the water-balance effect, is more pronounced in adults than in larvae when both are subjected to the same experimental conditions.
50-
=
s]
40-
AVT
E o
s p J
"7"
30
f
s So
+ gao
J
,t /
d
//
Con.
0
-IC
0
l I0
I 20
I 30
I 40
I ,50
hr
FIG. 4. Effect of A V T o n s o d i u m balance in a d u l t A. tigrinum. T h e dose was 100 m U / a n i m a l . T h e m e a n w e i g h t of t h e A V T - t r e a t e d animals was 31"4 g + 2"2 (3), t h a t of t h e controls was 32"4 g + 0"4 (4). Vertical lines indicate + 1 S.E.
Sodium fluxes of adults were measured over a 39 hr period. Table 5 shows that AYT produced a faster influx ( P < 0-05) but had no effect on the efflux.
2. Studies with oxytocin (a) Water balance. Fig. 5 shows the effects of oxytocin on body weight of larval A. tigrinum. Both control and experimental animals lost weight, but the loss was greater in the latter. At 12 hr the difference between animals injected with 100 mU (5.8 mU/g) of oxytocin and control animals is significant (P < 0.05). This experiment
540
RONALD H . ALVARADO AND SHELDON R. JOHNSON
has been repeated using larval A. gracile. Doses of 100 or 200 mU/animal (15 g) produced significant decreases in body weight. However, when the dosage was increased to 500 mU/animal this response was abolished and a slight increase in weight (2 per cent of initial weight in 4 hr) was observed. 104
I00
"~
=
96 ............................ " " ' '- o , , . . . . , , i
92
]I "~"~'t
88
0
I 2
I 4
I 6
I 8
I I0
Con. 14) 50mU 141
IOOrnU(4)
I 12
hr
FIG. 5. The effect of oxytocin on body weight in larval A. tigrinum. The dose (mU/animal) and the number of observations are indicated at the right. The mean initial weight of the animals was 18'18 g + 0"54 (12). Standard errors are indicated for the 12 hr points. Oxytocin (100 m U / a n i m a l - - 3 . 8 mU/g) had no significant effect on weight of adult A. tigrinum over a 12 hr period. Both control and experimental animals remained in a steady state comparable to the control group indicated in Fig. 2. (b) Osmotic uptake of water. T h e injection of 100 m U of oxytocin into larvae (6.3 mU/g) or into adults (3-9 mU/g) had no effect on the rate of osmotic uptake of water. T h e values were essentially the same as those given in Table 1. Neither A V T nor oxytocin has an effect on the permeability of the integument to water. This has been corroborated by studies on larval A. gracile. (c) Rate of urine production. We have been unable to show an effect of oxytocin on the rate of urine production in larval or adult A. tigrinum (Table 6). However, the n u m b e r of observations was small and the rate was measured only over the initial 8 hr after injection. Fig. 5 shows that the loss of weight in larvae occurred between 6 and 12 hr after injection of the hormone, and thus could reflect a delayed diuresis which would not be detected with our methods. Oxytocin had no effect on sodium concentration in the urine of larvae or adults. (d) Sodium fluxes. Larvae injected with 100 m U of oxytocin suffer a net loss of sodium (Fig. 6). Flux measurements revealed that this is the result of a faster efflux
541
S O D I U M AND W A T E R BALANCE I N A M B Y ST O M A
(P< 0"05). The influx was not changed (Table 7). Presumably this reflects an increase in the renal loss of sodium which may be associated with diuresis. Figure 7 shows the effect of oxytocin on sodium balance in adults. During the period of the experiment, control animals did not maintain a steady state but gained TABLE 6--THE
EFFECT OF OXYTOCIN O N THE RATE OF U R I N E P R O D U C T I O N I N LARVAL AND ADULT A .
tigrinum*
Initial body wt (g)
Rate of urine production (mg/g hr)
Stage
Control
Oxytocin
Larva Adult
15"36 + 1"40(3) 26"86 + 1"55(3)
16"00+ 2"95(4) 25"40 + 3"58(4)
Control
Oxytocin
12"7 + 2"1(3) 23-4 + 0-8(3)
13"6 + 1"2(4) 21-0 + 1"8(4)
* The dose was 100 mU/animal.
I01-
L Con. (4)
c o
~o_ u -~ -I0 i
o
% %
-zo-
%
Oxy. (8)
-30 0
I
I
I
I
I
IO
20
30
40
50
hr
FIG. 6. Effect of oxytocin on sodium balance in larval A. tigrinum. The dose was 100 mU/animal. T h e mean weight of the oxytocin-treated animals was 14.9 g + 0.7 (8), that of the controls was 14"6 g _+2"3 (4). Vertical lines indicate +1 S.E.
sodium. The oxytocin-treated adults also gained sodium for 10-12 hr and then began to lose it. At 43 hr the difference between control and oxytocin-treated animals is significant (P< 0.05). It would appear that the effect of oxytocin on sodium balance is more pronounced in larvae than adults and the time course suggests different mechanisms.
542
RONALD H. ALVARADOAND SHELDON R. JOHNSON
Table 7 shows that the efflux of sodium was significantly higher in oxytocintreated adults than in controls (P< 0.05). The fluxes indicated represent mean values measured over the entire 43 hr of the experiment which obscure the details of the pattern indicated in Fig. 7. The difference between the influxes of control and oxytocin-treated adults is not significant. TABLE 7--THE
E F F E C T O F O X Y T O C I N O N S O D I U M FLUXES I N LARVAL A N D A D U L T A . T H E FLUXES W E R E AVERAGED OVER A
tigrinum. *
PERIOD
Sodium fluxes (/~-equiv/10 g hr)
Body wt (g)
Effiux
Influx
N e t flux
Oxytocin
14"5 + 2-3(4) 14'9 + 0'7(8)
2"0 + 0"3(4) 2"7 + 0-2(8)
2'2 + 0"3(4) 2'2 + 0"2(8)
0"2 ± 0'2(4) - 0 " 5 + 0'1 (8)
Control Oxytocin
30.5 + 0"8(8) 28,6 + 1 '7(6)
0"7 + 0"1 (8) 1-3 + 0"1(6)
1'0 _+0"1 (8) 1 '3 + 0"1 (6)
0'3 + 0"2 (8) 0-0 + 0"1 (6)
Stage
Treatment
Larva
Control
Adult
41-HR
* T h e dose was 100 m U / a n i m a l . w
i
20-
g~
-g
Con. (8)
> I0
g® I E
~
--
Oxy
16)
c
-Io
o
I IO
I
I 30
20
I 40
I 50
hr
FIG. 7. Effect of oxytocin on sodium balance in adult A. tigrinum. T h e dose was 100 m U / a n i m a l . T h e m e a n weight of the experimental animals was 28"6 g + 2.2 (6), that of controls was 30"5 g + 0"08 (8). For clarity standard errors are indicated for only the 43 hr points. DISCUSSION
Normal plasma levels of neurohypophysial hormones in amphibia have not been determined. However, concentrations of 10-9-10 -1~' M arginine vasotocin are effective in natriferic, hydroosmotic and antidiuretic tests (Maetz, 1963). These
S O D I U M AND WATER BALANCE I N A M B Y S T O M A
543
concentrations fall within the range of plasma levels of vasopressin found in mammals (Kleeman & Cutler, 1963). Relative to these concentrations the doses administered in the experiments described in this report were massive. There were two reasons for this. First, under the experimental conditions described the sensitivity of the animals to the hormones was much lower than has been reported for some amphibians, including urodeles. For example, Bentley & Heller (1964) reported a significant water balance response in axolotls (36 g) injected with 2.4 x 10-4 mU of AVT. The injection of 5-10 mU of AVT into larval A. tigrinum or A. gracile (15 g) in our experiments produced an effect in only a few instances. The reasons for this discrepancy are unknown. It should be pointed out that most of the animals used in the present study had been subjected to low temperature (8-10°C) for 4-6 weeks prior to the equilibration period. Since season and temperature influence the Brunn effect in anurans (Heller, 1930; Jorgensen, 1950) it is conceivable that this treatment caused a degree of refractoriness. Our animals were anesthetized for injection while those of Bentley and Heller were not. However, separate experiments on A. gracile showed no differences in sensitivity between anesthetized and unanesthetized larvae. The second reason for using high doses was that the sensitivity of some of our methods is limited, so that the duration of the responses under study must be sufficiently long to insure their detection. Doses of AVT above 20 mU/animal consistently caused water retention. There was a direct relationship between the duration of the response and the dosage. The response was more pronounced in adults than larvae. Since the former are partially terrestrial, some adaptive significance may be attached to this difference, however, the physiological mechanisms underlying this difference are unknown. In both larvae and adults the water retention results from antidiuresis. Clearance studies on larvae showed that the antidiuresis can be attributed largely to a decrease in glomerular filtration. This is further supported by the observation that urinary sodium concentration was not influenced by the hormone. This does not exclude the possibility of tubular effects, the significance- of which will be evaluated in future experiments. In anurans the hormone decreases glomerular filtration rate and stimulates tubular reabsorption of sodium and water (Jard & Morel, 1963). The integument of larval and adult urodeles differs from that of most adult anurans in that its permeability to water is not increased by AVT or oxytocin. However, AVT does stimulate the active uptake of sodium across the body surface of both larval and adult urodeles. Oxytocin has no effect. In anurans both AVT and oxytocin are effective (Maetz, 1963). The data from urodeles demonstrate that the AVT-induced natriferic response of the integument may be independent of the hydroosmotic response. Similar observations have been made on the skin of the aquatic anuran, Xenopus (Maetz, 1963). In larvae the etftux as well as the influx of sodium was accelerated by AVT. In adults the efflux of hormone-treated animals equaled that of control animals.
544
RONALD H . ALVARADO AND SHELDON R. JOHNSON
Both results were unexpected because the hormone decreases the renal loss ol sodium. In addition to stimulating the mechanism in the integument responsible for the net accumulation of sodium, the hormone can possibly increase the activity of a parallel exchange diffusion component. This would account for the increase in the isotopically measured effiux. The effects of neurohypophysial hormones on salt and water transfer across the isolated urinary bladder of urodeles have recently been investigated by Bentley & Heller (1964). They were unable to demonstrate any effect of the hormones on the short-circuit current across this epithelium, nor did they observe a hydroosmotic effect. This represents another basic difference between urodeles and anurans. It should be pointed out that their observations were limited to the isolated organ. We have evidence (unpublished observations) that adult A. tigrinum under terrestrial conditions are able to make use of urinary bladder water. Possibly the response is not mediated by hormones in urodeles. Moderate doses of oxytocin (100 mU/animal) caused larvae to lose weight and to suffer a net loss of sodium. Presumably this reflects a diuretic condition. Our techniques were too crude to determine this directly, but the fact that the hormone had no effect on the rate of osmotic uptake of water supports this contention. Furthermore, a diuretic action of this hormone has been reported for anurans (Ewer, 1952b; Uranga & Sawyer, 1960) and for fresh-water fish by Maetz (1963). In fish the response results from an increase in glomerular filtration rate. Nothing is known about the mechanism in urodeles. Curiously, high doses of oxytocin (500 mU/animal) abolished this response and caused a slight increase in body weight in larval A. gracile. This increase in weight was also observed by Heller (1963) and Bentley & Heller (1964) in other urodeles. The mechanism has not been analyzed. The data on the effects of oxytocin on adults are fragmentary. In the few cases studied the hormone (100 mU/animal) had no effect on body weight but did cause the loss of sodium. The time course of this sodium loss differed from that of larvae in that it did not begin until 15-18 hr after injection of the hormone, whereas in larvae the loss was apparent shortly after injection. This suggests that in adults a different, possibly a secondary, response is involved. There is at present insumcient information to permit the formulation of a comprehensive theory of endocrine regulation of hydromineral metabolism in amphibians. However, as a result of the many comparative studies on this problem certain distinct patterns are emerging. The Amphibia evolved from the Choanichthyes and like this group produce two distinct neurohypophysial principles, AVT and a yet unidentified oxytocin-like principle (Follett & Heller, 1964). The transition from fish to amphibian presumably occurred in fresh water where water elimination, not conservation, is the critical problem. It is doubtful that the primitive function of the neurohypophysial hormones is reflected by the typical water balance effect and indeed such an effect is generally absent or reduced in fresh-water forms. The functions of these hormones in fresh-water vertebrates remain the object of conjecture. Jorgensen &
S O D I U M AND WATER BALANCE I N A M B Y S T O M A
545
Larsen (1960) and Sawyer (1961b) have suggested that the primitive function of the hypothalamic-neurohypophysial octapeptides is the regulation of the adenohypophysis which in turn may regulate the release of mineralocorticoids from interrenal tissue. It is also possible that the original role of these hormones was the regulation of reproductive behavior (Wilhelmi et al., 1955). Finally, numerous studies have shown that neurohypophysial hormones play a direct role in the regulation of electrolyte balance (Maetz, 1963). Many of the effects of AVT described in this report can best be interpreted in this context. The level of AVT in the blood might regulate the activity of the sodiumtransporting system in the skin. The time course of the sodium balance response indicates this is a direct effect and is not mediated by other endocrine organs. This is supported by observations on isolated target organs. Arginine vasotocin also causes antidiuresis without changing sodium concentration in the urine. While this reduces the renal excretion of sodium, it also aggravates the water balance problem and thus is of doubtful significance while the animals are in water. However, for terrestrial adults the response assumes considerable significance. Statements about the effects of oxytocin on salt and water metabolism must be qualified because there may be differences between the effects of synthetic oxytocin and the natural "oxytocin-like" principle of urodeles. Our results and others (see Maetz, 1963) suggest that in aquatic forms oxytocin may be a diuretic hormone functioning in the control of the elimination of water by the kidneys. The diuresis entails the loss of some sodium, but if controlled quantities of AVT were released at the same time, this loss could be compensated by increasing the active uptake of sodium across the skin. Furthermore, the AVT would exert a regulatory effect on the kidneys, preventing the possibility of extensive diuresis. By delicately regulating the release of each of the neurohypophysial hormones the animal could integrate the mechanisms involved in maintaining an osmotic and ionic steady state. The factors which trigger and regulate the release of the hormones are not known for lower vertebrates. REFERENCES ACHERR., CHAUVETJ., CHAUVETM. T. & CREPYD. (1964) Phylog6nie des peptides neurohypophysaires: Isolement de la mesotocine (Ileus-ocytocine) de la grenouille, interm6diaire entre la Ser,-Ileus-ocytocine des poissons osseux et l'ocytocine des mammif6res. Biochim. Biophys. Acta 90, 613-615. ALWmADOR. H. & KIRSCHNERL. B. (1963) Osmotic and ionic regulation in Ambystoma tigrinum. Comp. Biochem. Physiol. 10, 55-67. B~LEHRADEKJ. & HUXLEYJ. S. (1927) The effects of pituitrin and of narcosis on waterregulation in larval and metamorphosed Ambystoma..7. Exp. Biol. 5, 89-96. BENTLEYP. J. (1963) Neurohypophysial function in amphibians, reptiles and birds. Syrup. Zool. Soc. Lond. 9, 141-152. BENTLEYP. J. & HELLERH. (1964) The action of neurohypophysial hormones on the water and sodium metabolism of urodele amphibians. J. Physiol. 171, 434-453. BRUNN F. (1921) Beitrag zur Kennmis der wirkung von hypophysenetrakten auf den Wasserhaushilt des Frosches. Z. Ges. Exp. Med. 25, 170-175. Dow D. & ZUCKERMANS. (1939) The effect of vasopressin, sex hormones and adrenal cortical hormone on body water in axolotls..7. Endocrinol. 1, 387-398.
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RONALD H. ALVARADOAND SHELDON R. JOHNSON
EWER R. F. (1952a) The effect of pituitrin on fluid distribution in Bufo regularis Reuss. J. Exp. Biol. 29, 173-177. EWER R. F. (1952b) The effects of posterior pituitary extracts on water balance in Bufo careus and Xenopus laevis, together with some general considerations on anuran water economy. J. Exp. Biol. 29, 429-439. FOLLETT B. K. & HEALER H. (1954) T h e neurohypophysial hormones of lungfish and amphibians. J. Physiol. 172, 92-106. FUHRMAN F. A. & USSING H. H. (1951) A characteristic response of the isolated frog skin potential to neurohypophysial principles and its relation to the transport of sodium and water. J. Cell. Comp. Physiol. 38, 109-130. GREEN J. D. & MAXWELLD. S. (1959) Comparative anatomy of the hypophysis and observations on the mechanism of neurosecretion. In Comparative Endocrinology (Edited by GORBMAN A.), pp. 368-392. Wiley & Sons, New York. HEALER H. (1930) l~ber die Einwirkung von Hypophysenhinterlappenextrakten auf den Wasserhaushalt des Frosches. Arch. Exp. Path. Pharmaeol. 157, 298-322. HEALER H. (1963) Pharmacology and distribution of neurohypophysial hormones. Syrup. Zool. Soe. Lond. 9, 93-106. HoussAY B. A. & POTICK D. (1929) Cited in H. Heller (1950). The comparative physiology of the neurohypophysis. Experientia 6, 368-376. JARD S. & MOREL F. (1963) Actions of vasotocin and some of its analogues on salt and water excretion by the frog. Amer. J. Physiol. 204, 222-226. JORGENSEN C. B. (1950) T h e amphibian water economy, with special regard to the effect of neurohypophysial extracts. Acta Physiol. Scand. 22 (Suppl. 78), 79 pp. JORGENSENC. B. & LARSENL. O. (1960) Comparative aspects of hypothalamic-hypophysial relationships. Ergeb. Biol. 22, 1-29. JORGENSEN C. B., LEVI H. & USSINGH. H. (1946) On the influence of the neurohypophysial principles on the sodium metabolism in the axolotl (Ambystoma mexicanum). Acta Physiol. Scand. 12, 350-371. KLEEMAN C. R. & CUTLER R. E. (1963) The neurohypophysis. Ann. Rev. Physiol. 25, 385-432. LEAr A., ANDERSON J. & PAGE L. B. (1958) Active sodium transport by the isolated toad bladder. J. Gen. Physiol. 41, 657-668. MAa~TZ J. (1963) Physiological aspects of neurohypophysial function in fishes with some reference to the Amphibia. Syrup. Zool. Soc. Lond. 9, 107-140. SAW'~R W. H. (1957) The antidiuretic action of neurohypophysial hormones in Amphibia. In The Neurohypophysis (Edited by HELLER H.), pp. 171-182. Butterworths, London. SAW'~R W. H. (1961a) Comparative physiology and pharmacology of the neurohypophysis. Rec. Prog. Hormone Res. 17, 437-465. SAWYERW. H. (1961b) Neurohypophysial hormones. Pharmacol. Rev. 13, 225-277. SAWYER W. H. (1963) Neurohypophysial secretions and their origin. In Advances in Neuroendocrinology (Edited by NALBANDOVA. V.), pp. 69-80. Univ. of Illinois Press, Urbana. SAWYERW. H. (1964) Vertebrate neurohypophysial principles. Endocrinology 75, 981-990. SCHARRER E. & SCHARRERB. (1963) Neuroendocrinology, 289 pp. Columbia Univ. Press, New York. STEGGEROA F. R. (1937) Comparative study of water metabolism in amphibians injected with pituitrin. Proc. Soc. Exp. Biol. 36, 103-106. URANGA J. & SAWER W. H. (1960) Renal responses of the bullfrog to oxytocin, arginine vasotocin, and frog neurohypophysial extract. Amer. J. Physiol. 198, 1287-1290. WIaHELMI A. E., PICKEORD G. E. & SAWYERW. H. (1955) Initiation of the spawning reflex response in Fundulus by the administration of fish and mammalian neurohypophysial preparations and synthetic oxytocin. Endocrinology 57, 243-252.
THE EFFECTS OF ARGININE VASOTOCIN AND OXYTOCIN ON SODIUM AND WATER BALANCE IN A M B Y S T O M A * R O N A L D H. A L V A R A D O and S H E L D O N R. J O H N S O N Department of Zoology, Oregon State University, Corvallis, Oregon (Received 4 M a y 1965)
Abstract--1. When injected with arginine vasotocin (20-100 mU) both larvae and adults gain weight. The duration of the response is related to dosage. The hormone does not influence the osmotic uptake of water so that the response must result from antidiuresis, a large share of which can be attributed to a reduction in glomerular filtration. 2. Arginine vasotocin stimulates the net uptake of sodium by larvae and adults. In larvae this response lasts approximately 24 hr and is followed by a pronounced net loss of sodium. Adults gain sodium for at least 43 hr. During the initial 24 hr after injection of the hormone into larvae the efflux and the influx are increased above control values. However, the influx is enhanced to a greater extent and the animals gain sodium. After 24 hr the influx returns to normal but the high efflux persists for several hours. This leads to a net loss of sodium. The net uptake of sodium by adults injected with the hormone is the result of a faster influx. 3. Moderate doses (50-100 mU) of oxytocin caused larvae to lose weight and had no effect on the weight of adults on a short-term basis. The hormone had no effect on the osmotic inflow of water in adults or larvae. Presumably the loss of weight in larvae reflects a diuretic condition. Higher doses of oxytocin (500 mU) abolished this response and caused a slight increase in weight. 4. Oxytocin increases the efflux Of sodium from larvae and adults, possibly by inducing diuresis. The hormone had no effect on the influx of sodium in adults or larvae. 5. An effort has been made to interpret the results in terms of their significance in the maintenance of an osmotic and ionic steady state by these animals. INTRODUCTION RECENT interest in the comparative endocrinology of vertebrates has led to intensive investigations on the morphology and function of the hypothalamic-neurohypophysial complex (Green & Maxwell, 1959; Sawyer, 1961a, 1961b, 1963, 1964; Heller, 1963; Scharrer & Scharrer, 1963; Kleeman & Cutler, 1963). A m o n g the functions of this system is the elaboration of hormones which participate in the regulation of hydromineral metabolism. Of the submammalian classes of vertebrates the Amphibia have been most extensively studied. However, within this class most investigations have been limited to the anurans. * Supported by grants from the National Institutes of Health (GM-11817) and the National Science Foundation (G-12471), and by the Oregon State University General Research Fund. 531
532
RONALD H. ALVARADO AND SHELDON R. JOHNSON
Both anurans and urodeles are known to secrete arginine vasotocin (Follett & Heller, 1964). This hormone has been found in all submamma]ian vertebrates which have been examined except for the Elasmobranchii (Heller, 1963). At ]east one other neurohypophysial hormone is found in Amphibia. It is similar to oxytocin in many of its effects but differs in certain pharmaco]ogical properties (Follett & Heller, 1964). In the anuran, Rana esculenta, the hormone has recently been identified as isoleucine-8-oxytocin (Acher et al., 1964). With respect to hydromineral metabolism the responses of amphibians to neurohypophysial hormones may be divided into a water-balance effect and a sodium-balance effect. Comparative studies on several species of anurans and a few species of urodeles have revealed striking species differences in the nature and magnitude of these responses. Brunn (1921) first demonstrated that injection of neurohypophysial extracts into frogs immersed in water leads to an increase in their weight. The response has since been observed in many species of anurans. In this group the hormones act by (1) increasing the permeability of the skin to water (Fuhrman & Ussing, 1951), (2) increasing the permeability of the urinary bladder to water (Ewer, 1952a) and (3) causing renal antidiuresis (Houssay & Potick, 1929). The last response may result from a decrease in glomerular filtration or an increase in tubular reabsorption of water (Sawyer, 1957; Bentley, 1963). For predominantly terrestrial forms the water-balance response has adaptive value and indeed there appears to be a positive correlation between the magnitude of the response and degree of terrestrialism (Steggerda, 1937; Ewer, 1952b). In addition to their effects on water balance in anurans the neurohypophysial hormones also influence sodium metabolism. Active transport of sodium across the isolated frog skin (Fuhrman & Ussing, 1951), the urinary bladder (Ewer, 1952a; Leaf et al., 1958) and the renal tubules (Jard & Morel, 1963) is enhanced by these hormones. Relatively little attention has been devoted to the endocrine control of hydromineral metabolism in urodeles. The hormones secreted by the hypothalamicneurohypophysial complex of urodeles have only recently been characterized (Follett & Heller, 1964). Most investigations on urodeles have involved the injection of high concentrations of hormones of mammalian origin. These studies, and those of Bentley & Heller (1964) using lower doses of arginine vasotocin and oxytocin, have shown that larval and adult urodeles display a water-balance effect (B61ehr~dek & Huxley, 1927; Steggerda, 1937; Dow & Zuckerman, 1939). Sodium balance is also affected by these hormones (Jorgensen et al., 1946; Bentley & Heller, 1964). Neither the water-balance nor the sodium-balance response has been systematically analyzed using purified or synthetic hormones on both larvae and adults of the same species. Such studies may be of interest, not only because they may reveal information about the ontogeny of the responses involved, but also because they afford a direct means of testing the hypothesis that the osmoregulatory aspects of neurohypophysial function have evolved in association with terrestrialism. Finally, studies on urodeles are important from the point of view of the evolution
S O D I U M AND WATER BALANCE I N A M B Y S T O M A
533
of neurohypophysial function, for this group represents the most primitive order in which a water balance effect has been demonstrated. MATERIALS AND METHODS
1. Animals Most of the experiments on larvae and all on adults were performed on Ambystoma tigrinum collected in eastern Washington and shipped by air to Corvallis, Oregon. Some experiments were conducted on larval Ambystoma gracile collected in Corvallis. The larvae of both species were of comparable size. No significant species differences were observed with respect to the parameters under consideration. Larvae were stored in dechlorinated tap water at 8-10°C. Adults were stored in a similar fashion except that they could crawl out of the water at will. None of the animals were fed. For experimental purposes the animals were immersed in artificial pond water (1.3 mM sodium chloride, 0.8 mM calcium chloride, 0.1 mM potassium chloride and 0.2 mM sodium bicarbonate) and equilibrated at 15-17°C for at least 1 week prior to the initiation of an experiment. All experiments were done at this temperature. 2. Anesthetic These animals are sensitive to handling (Jorgensen et al., 1946; Alvarado & Kirschner, 1963). Stress induces diuresis and upsets salt balance. Thus for operations involving extensive handling, such as injections or ligation of the cloaca, the animals were anesthetized by immersion in 0.1% tricaine methane sulfonate (pH adjusted to 7.0). Complete immobilization requires approximately 20 min for larvae, slightly longer for adults. The animals recover within 30 min after removal from the anesthetic. 3. Hormones Synthetic oxytocin (Syntocinon) was obtained from Sandoz Pharmaceuticals. Synthetic arginine vasotocin (AVT) was kindly supplied by Dr. Brian Follett, Washington State University. Doses of AVT are given in pressor units, of oxytocin in rat uterus units. Each hormone was diluted with amphibian Ringer's solution. All injections were made into the peritoneal cavity; the injection volume was 0.1 ml. Both hormone preparations contained chloretone as a preservative and were acidified with acetic acid. Control animals were injected with appropriate solutions of these substances. 4. Water balance Animals were anesthetized, injected with the desired solution and each was immersed in artificial pond water. At specified intervals each animal was weighed (precision + 0.05 g).
534
RONALD H. ALVARADO AND SHELDON R. JOHNSON
5. Osmotic uptake of water Each animal was anesthetized, its urinary bladder was emptied by supra-pubic compression and the cloacal opening was blocked with a purse-string ligature. After injection of the appropriate solution each animal was rinsed, weighed and placed in artificial pond water. Weighings were made at 2 hr intervals for 8 hr. The rate of increase in weight is linear over this period and the rate of uptake of water can be estimated directly. After 8 hr each animal was anesthetized and weighed. The cloaca was unblocked and the urine expelled. Each animal was then reweighed and the mass of urine was obtained by subtraction. A sample of the urine was analyzed for sodium by flame photometry. 6. Glomerular filtration rate The effect of AVT on glomerular filtration was studied by measuring the clearance of Cl~-inulin. The inulin (2.0 t~c) was injected into the peritoneal cavity and the animals were permitted to equilibrate for 24 hr. Each animal was then anesthetized and injected with either 100 mU of AVT or a control solution. After rinsing, each animal was placed in 250 ml of artificial pond water. Samples of the bathing solution were assayed for radioactivity at 1 hr intervals for 10 hr. A serum sample was then obtained and a quantitative fraction assayed for activity. From the total radioactivity in the bath and the serum radioactivity the renal clearance of inulin was estimated. 7. Sodium fluxes The efflux of sodium was measured with Na ~ (Na2~C1). The isotope was diluted in amphibian Ringer's solution and 2-0 t~c was injected into each animal. After 3 days of equilibration each animal was rinsed, anesthetized, treated with the hormone or control solution and immersed in 300 ml of pond water. Samples of the bathing medium were removed at specified intervals and assayed for radioactivity by conventional means (Alvarado & Kirschner, 1963). After 30-50 hr each animal was sacrificed and the specific activity of a serum sample was determined. The efflux of sodium was calculated from the serum specific activity and the rate of appearance of radioactivity in the bath. The net flux was determined by monitoring the concentration of sodium in the bath by flame photometry (precision + 1.0 per cent). The influx was computed from the efflux and the net flux. RESULTS 1. Studies with arginine vasotocin (a) Water balance. No effort was made to establish dose-response relationships for most of the experiments described in this report. However, a pilot study on the effects of various doses of AVT on body weight of larvae showed that doses below 20 mU/animal produce erratic results. For example, a dose of 10 mU/animal produced an increase in weight in only 4 out of 7 animals. The response was small (2-3 per cent increase in weight in 3 hr) and lasted only 3-4 hr. Doses of AVT
SODIUM AND WATER BALANCE IN AMBY3TOMA
53S
above 20 mU/animal produced consistent responses (Fig. 1). The duration of the response is positively correlated with the concentration of the hormone. There is no correlation between dosage and the rate of increase in body weight over the initial 4 hr of the experiments. This suggests that the increase in weight reflects a reduction in water excretion rather than an increase in the osmotic inflow of water, Comparable results were obtained with larval A. gracile. 112
.......*
IOOmU (5)
lOB , s e ~'B
I04 5 0 m U (5) 25rnU (3)
o
IO0
96 Con. 191
92 0
2
I 4
I 6
I 8
I I0
hr
FIG. 1. Effect of A V T o n b o d y w e i g h t in larval A. tigrinum. T h e dose ( m U / a n i m a l )
and the number of observations are indicated at the right. The mean initial weight of the animals was 14"42g + 0-80 (20). Standard errors have been omitted for clarity, however the maximum S.E. observed Was + 2"3. Control larvae lost weight. This response is characteristic of larvae and is less pronounced or absent in adults (Fig. 2). It probably reflects a diuresis induced by stress. Adults also respond to AVT (Fig. 2). Again the dosage is correlated with the duration of the response rather than with the rate of increase in body weight. The adults used in this experiment were about 10 g heavier than the larvae. On a weight basis the dose o f AVT administered to the adults injected with 100mU was 4.2 mU/g. This corresponds with the larvae injected with 50 mU of AVT (dosage 3.7 mU/g). Comparison of the responses elicited at this dosage shows that after 4 hr the rate of increase in weight decreased and, by 6 hr the maximum weight had been reached in larvae. On the other hand, adults continued to gain weight at a
536
RONALD H. ALVARADO AND S H E L D O N R. J O H N S O N
constant rate for 8 hr. T h e experiment was terminated at this time to determim urine volume and composition, so the duration of the response was not established These data indicate that the water-balance effect induced by A V T is more pro. nounced in adults than in larvae. 112
108
IOOmU (3) 4 0 m U (2) .. ......
-6
...
104 ,
........
1
s s Je
e pss
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i
( 51
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96
I 2
0
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FIG. 2. Effect of AVT on body weight in adult A. tigrinum. The dose (mU/animal) and the number of observations are indicated at the right. The mean initial weight of the animals was 25'00 g +_1"60 (10). Standard errors for control animals and those injected with 100 mU are indicated for the 8 hr reading. EFFECT OF A V T ON THE OSMOTIC UPTAKE OF WATER BY LARVAL AND ADULT THE DOSE WAS 1 0 0 m U / A N I M A L . UNLESS OTHERWISE SPECIFICED THE VALUES INDICATED IN THIS AND ALL SUBSEQUENT TABLES REPRESENT MEAN ~ S . E . (?t).
TABLE 1--THE
A. tigrinum.
Initial body wt (g) Stage
Control
AVT
Larva Adult
13-67+ 1.10(10) 34"16+ 2-06(4)
12"54+_0-68(10) 32"06+ 0.44(4)
Rate of increase in wt (% of initial wt/hr) Control 1.28 + 0-09(10) 1.40 + 0"10(4)
AVT 1"21 +_0'06(10) 1-40 + 0-10(4)
(b) Osmotic uptake of water. Table 1 shows that A V T has no effect on the rate of uptake of water in larval or adult A. tigrinum; this has been corroborated by studies on larval A. gracile. T h u s , the hormone does not increase the permeability of the skin to water. This is supported by the observations of Bentley & Heller (1964) on isolated pieces of skin and apparently represents a basic difference between urodeles and anurans. (c) Rate of urine production and glomerular filtration. Table 2 shows that A V T causes a pronounced antidiuresis. T h e mechanisms producing this effect have not
537
SODIUM AND WATER BALANCE IN AMBYSTOMA
been studied in detail; however, a few inulin clearance m e a s u r e m e n t s have been made on larvae. In these forms the major share of the antidiuresis can be ascribed to a reduction in glomerular filtration. T h i s is shown in T a b l e 3. Arginine vasotocin had no significant effect on sodium concentration in the urine of larvae or adults (Table 4). TABLE 2 - - T B E EFFECT OF A V T ON URINE PRODUCTION IN LARVAL AND ADULT A. tigrinum* Initial body wt (g)
Rate of urine production (mg/g hr)
Stage
Control
AVT
Control
AVT
Larva Adult
13-08 + 1"15(9) 34"16 + 2'03(4)
12"30+ 0'67(11) 32"06+ 0'45(4)
10"4 + 2'1(9) 16"2 + 2"0(4)
3"8 + 0"7(11) 4"0 + 0-8(4)
* The dose was 100 mU/animal. TABLE 3 - - T H E EFFECT OF A V T ON INULIN CLEARANCE IN LARVAL A . tigrinum*
Treatment
Control AVT
Body wt (g)
Clearance (ml/g hr)
15"7 + 1"7(5) 16'3 +_2'0(6)
0"025_+0"008(5) 0"004 + 0"002(6)
* Dose 100 mU/animal. TABLE 4
T H E EFEECT OF A V T
ON SODIUM CONCENTRATION IN THE URINE OF LARVAL AND a d u l t A. tigrinum* Sodium concentration (raM/1.)
Stage
Control
AVT
Larva Adult
5.2 + 1.0(10) 7"4 + 1"2(6)
4.6 + 1.4(11) 9"9 _+1-6(5)
* The dose was 100 mU/animal.
(d) Sodium fluxes. T h e effect of A V T on sodium balance in larval .4. tigrinum is expressed in Fig. 3. All values have been corrected to a standard body weight of 10 g and mean values are indicated. Control animals maintained a steady state. Larvae injected with A V T experienced an initial net uptake of sodium which lasted 20-25 hr. T h i s was followed by a net loss of sodium. T h e mean flux values measured over the first 32 hr following treatment are given in T a b l e 5. T h e h o r m o n e increased both the influx and the e m u x ( P < 0.05); however, the influx was enhanced to a greater extent and a net uptake of sodium ensued. Flux measurements showed that the net loss of sodium which begins approximately 25 hr after
RONALD H.
538
ALVARADO AND SHELDON R . JOHNSON
injection results f r o m a r e t u r n of the influx to approximately control values, while the efflux remains high. Eventually the influx and the effiux m u s t converge and the animals r e s u m e a steady state; however, the time course has not been followed. F i g u r e 4 shows that adults also take up net quantities of s o d i u m w h e n injected with A V T . C o m p a r i s o n of Figs. 3 and 4 reveals that there are quantitative
SS
/
20 "6
~
I
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t\
s
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s
E
/1 ""
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t
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AVT
.~_ ~
-20
-30
o
I
I
I
I
I
IO
20
30
4o
5o
hr
FIG. 3. Effect of AVT on sodium balance in larval A . tigrinum. The dose was 100 mU/animal. The mean weight of the AVT-treated animals was 15-3 g -+0"8 (6), that of the controls was 16"0 g +_0-8 (6). Vertical lines represent _+1 S.E. T A B L E 5 - - T H E EFFECT OF A V T ON SODIUM FLUXES IN LARVAL AND ADULT A . tigrinum. FLUXES WERE MEASURED OVER 32 HR FOR LARVAE AND 39 HR FOR ADULTS
Stage
Treatment
Body wt (g)
THE
Sodium fluxes (/z-equiv/10 g/hr) Efflux
Influx
Net flux
Larva
Control AVT
14-8+0"7(11) 15"2 + 1-0(12)
1'5+0"1(11) 2.1 +_0"2(12)
1'3_+0-1(11) 2'8 -+0-3(12)
-0-2_+0'2(11) 0'7 -+0.4(12)
Adult
Control AVT
38'4 + 0"7(4) 31"4+1-6(3)
0-7 -+0-1(4) 0"6+0"1(3)
0.9 _+0'1(4) 1"5_+0"1(3)
0"2 + 0.1(4) 0-9+0"1(3)
539
SODIUM AND WATER BALANCE I N . / I M B Y T O M A
differences between larvae and adults, particularly with respect to the duration of the response. In adults the response was pronounced even after 43 hr, when the experiment was terminated. The doses of AVT/unit weight were 6.5 mU/g and 3.2 mU/g for larvae and adults respectively. Thus the sodium-balance effect, as well as the water-balance effect, is more pronounced in adults than in larvae when both are subjected to the same experimental conditions.
50-
=
s]
40-
AVT
E o
s p J
"7"
30
f
s So
+ gao
J
,t /
d
//
Con.
0
-IC
0
l I0
I 20
I 30
I 40
I ,50
hr
FIG. 4. Effect of A V T o n s o d i u m balance in a d u l t A. tigrinum. T h e dose was 100 m U / a n i m a l . T h e m e a n w e i g h t of t h e A V T - t r e a t e d animals was 31"4 g + 2"2 (3), t h a t of t h e controls was 32"4 g + 0"4 (4). Vertical lines indicate + 1 S.E.
Sodium fluxes of adults were measured over a 39 hr period. Table 5 shows that AYT produced a faster influx ( P < 0-05) but had no effect on the efflux.
2. Studies with oxytocin (a) Water balance. Fig. 5 shows the effects of oxytocin on body weight of larval A. tigrinum. Both control and experimental animals lost weight, but the loss was greater in the latter. At 12 hr the difference between animals injected with 100 mU (5.8 mU/g) of oxytocin and control animals is significant (P < 0.05). This experiment
540
RONALD H . ALVARADO AND SHELDON R. JOHNSON
has been repeated using larval A. gracile. Doses of 100 or 200 mU/animal (15 g) produced significant decreases in body weight. However, when the dosage was increased to 500 mU/animal this response was abolished and a slight increase in weight (2 per cent of initial weight in 4 hr) was observed. 104
I00
"~
=
96 ............................ " " ' '- o , , . . . . , , i
92
]I "~"~'t
88
0
I 2
I 4
I 6
I 8
I I0
Con. 14) 50mU 141
IOOrnU(4)
I 12
hr
FIG. 5. The effect of oxytocin on body weight in larval A. tigrinum. The dose (mU/animal) and the number of observations are indicated at the right. The mean initial weight of the animals was 18'18 g + 0"54 (12). Standard errors are indicated for the 12 hr points. Oxytocin (100 m U / a n i m a l - - 3 . 8 mU/g) had no significant effect on weight of adult A. tigrinum over a 12 hr period. Both control and experimental animals remained in a steady state comparable to the control group indicated in Fig. 2. (b) Osmotic uptake of water. T h e injection of 100 m U of oxytocin into larvae (6.3 mU/g) or into adults (3-9 mU/g) had no effect on the rate of osmotic uptake of water. T h e values were essentially the same as those given in Table 1. Neither A V T nor oxytocin has an effect on the permeability of the integument to water. This has been corroborated by studies on larval A. gracile. (c) Rate of urine production. We have been unable to show an effect of oxytocin on the rate of urine production in larval or adult A. tigrinum (Table 6). However, the n u m b e r of observations was small and the rate was measured only over the initial 8 hr after injection. Fig. 5 shows that the loss of weight in larvae occurred between 6 and 12 hr after injection of the hormone, and thus could reflect a delayed diuresis which would not be detected with our methods. Oxytocin had no effect on sodium concentration in the urine of larvae or adults. (d) Sodium fluxes. Larvae injected with 100 m U of oxytocin suffer a net loss of sodium (Fig. 6). Flux measurements revealed that this is the result of a faster efflux
541
S O D I U M AND W A T E R BALANCE I N A M B Y ST O M A
(P< 0"05). The influx was not changed (Table 7). Presumably this reflects an increase in the renal loss of sodium which may be associated with diuresis. Figure 7 shows the effect of oxytocin on sodium balance in adults. During the period of the experiment, control animals did not maintain a steady state but gained TABLE 6--THE
EFFECT OF OXYTOCIN O N THE RATE OF U R I N E P R O D U C T I O N I N LARVAL AND ADULT A .
tigrinum*
Initial body wt (g)
Rate of urine production (mg/g hr)
Stage
Control
Oxytocin
Larva Adult
15"36 + 1"40(3) 26"86 + 1"55(3)
16"00+ 2"95(4) 25"40 + 3"58(4)
Control
Oxytocin
12"7 + 2"1(3) 23-4 + 0-8(3)
13"6 + 1"2(4) 21-0 + 1"8(4)
* The dose was 100 mU/animal.
I01-
L Con. (4)
c o
~o_ u -~ -I0 i
o
% %
-zo-
%
Oxy. (8)
-30 0
I
I
I
I
I
IO
20
30
40
50
hr
FIG. 6. Effect of oxytocin on sodium balance in larval A. tigrinum. The dose was 100 mU/animal. T h e mean weight of the oxytocin-treated animals was 14.9 g + 0.7 (8), that of the controls was 14"6 g _+2"3 (4). Vertical lines indicate +1 S.E.
sodium. The oxytocin-treated adults also gained sodium for 10-12 hr and then began to lose it. At 43 hr the difference between control and oxytocin-treated animals is significant (P< 0.05). It would appear that the effect of oxytocin on sodium balance is more pronounced in larvae than adults and the time course suggests different mechanisms.
542
RONALD H. ALVARADOAND SHELDON R. JOHNSON
Table 7 shows that the efflux of sodium was significantly higher in oxytocintreated adults than in controls (P< 0.05). The fluxes indicated represent mean values measured over the entire 43 hr of the experiment which obscure the details of the pattern indicated in Fig. 7. The difference between the influxes of control and oxytocin-treated adults is not significant. TABLE 7--THE
E F F E C T O F O X Y T O C I N O N S O D I U M FLUXES I N LARVAL A N D A D U L T A . T H E FLUXES W E R E AVERAGED OVER A
tigrinum. *
PERIOD
Sodium fluxes (/~-equiv/10 g hr)
Body wt (g)
Effiux
Influx
N e t flux
Oxytocin
14"5 + 2-3(4) 14'9 + 0'7(8)
2"0 + 0"3(4) 2"7 + 0-2(8)
2'2 + 0"3(4) 2'2 + 0"2(8)
0"2 ± 0'2(4) - 0 " 5 + 0'1 (8)
Control Oxytocin
30.5 + 0"8(8) 28,6 + 1 '7(6)
0"7 + 0"1 (8) 1-3 + 0"1(6)
1'0 _+0"1 (8) 1 '3 + 0"1 (6)
0'3 + 0"2 (8) 0-0 + 0"1 (6)
Stage
Treatment
Larva
Control
Adult
41-HR
* T h e dose was 100 m U / a n i m a l . w
i
20-
g~
-g
Con. (8)
> I0
g® I E
~
--
Oxy
16)
c
-Io
o
I IO
I
I 30
20
I 40
I 50
hr
FIG. 7. Effect of oxytocin on sodium balance in adult A. tigrinum. T h e dose was 100 m U / a n i m a l . T h e m e a n weight of the experimental animals was 28"6 g + 2.2 (6), that of controls was 30"5 g + 0"08 (8). For clarity standard errors are indicated for only the 43 hr points. DISCUSSION
Normal plasma levels of neurohypophysial hormones in amphibia have not been determined. However, concentrations of 10-9-10 -1~' M arginine vasotocin are effective in natriferic, hydroosmotic and antidiuretic tests (Maetz, 1963). These
S O D I U M AND WATER BALANCE I N A M B Y S T O M A
543
concentrations fall within the range of plasma levels of vasopressin found in mammals (Kleeman & Cutler, 1963). Relative to these concentrations the doses administered in the experiments described in this report were massive. There were two reasons for this. First, under the experimental conditions described the sensitivity of the animals to the hormones was much lower than has been reported for some amphibians, including urodeles. For example, Bentley & Heller (1964) reported a significant water balance response in axolotls (36 g) injected with 2.4 x 10-4 mU of AVT. The injection of 5-10 mU of AVT into larval A. tigrinum or A. gracile (15 g) in our experiments produced an effect in only a few instances. The reasons for this discrepancy are unknown. It should be pointed out that most of the animals used in the present study had been subjected to low temperature (8-10°C) for 4-6 weeks prior to the equilibration period. Since season and temperature influence the Brunn effect in anurans (Heller, 1930; Jorgensen, 1950) it is conceivable that this treatment caused a degree of refractoriness. Our animals were anesthetized for injection while those of Bentley and Heller were not. However, separate experiments on A. gracile showed no differences in sensitivity between anesthetized and unanesthetized larvae. The second reason for using high doses was that the sensitivity of some of our methods is limited, so that the duration of the responses under study must be sufficiently long to insure their detection. Doses of AVT above 20 mU/animal consistently caused water retention. There was a direct relationship between the duration of the response and the dosage. The response was more pronounced in adults than larvae. Since the former are partially terrestrial, some adaptive significance may be attached to this difference, however, the physiological mechanisms underlying this difference are unknown. In both larvae and adults the water retention results from antidiuresis. Clearance studies on larvae showed that the antidiuresis can be attributed largely to a decrease in glomerular filtration. This is further supported by the observation that urinary sodium concentration was not influenced by the hormone. This does not exclude the possibility of tubular effects, the significance- of which will be evaluated in future experiments. In anurans the hormone decreases glomerular filtration rate and stimulates tubular reabsorption of sodium and water (Jard & Morel, 1963). The integument of larval and adult urodeles differs from that of most adult anurans in that its permeability to water is not increased by AVT or oxytocin. However, AVT does stimulate the active uptake of sodium across the body surface of both larval and adult urodeles. Oxytocin has no effect. In anurans both AVT and oxytocin are effective (Maetz, 1963). The data from urodeles demonstrate that the AVT-induced natriferic response of the integument may be independent of the hydroosmotic response. Similar observations have been made on the skin of the aquatic anuran, Xenopus (Maetz, 1963). In larvae the etftux as well as the influx of sodium was accelerated by AVT. In adults the efflux of hormone-treated animals equaled that of control animals.
544
RONALD H . ALVARADO AND SHELDON R. JOHNSON
Both results were unexpected because the hormone decreases the renal loss ol sodium. In addition to stimulating the mechanism in the integument responsible for the net accumulation of sodium, the hormone can possibly increase the activity of a parallel exchange diffusion component. This would account for the increase in the isotopically measured effiux. The effects of neurohypophysial hormones on salt and water transfer across the isolated urinary bladder of urodeles have recently been investigated by Bentley & Heller (1964). They were unable to demonstrate any effect of the hormones on the short-circuit current across this epithelium, nor did they observe a hydroosmotic effect. This represents another basic difference between urodeles and anurans. It should be pointed out that their observations were limited to the isolated organ. We have evidence (unpublished observations) that adult A. tigrinum under terrestrial conditions are able to make use of urinary bladder water. Possibly the response is not mediated by hormones in urodeles. Moderate doses of oxytocin (100 mU/animal) caused larvae to lose weight and to suffer a net loss of sodium. Presumably this reflects a diuretic condition. Our techniques were too crude to determine this directly, but the fact that the hormone had no effect on the rate of osmotic uptake of water supports this contention. Furthermore, a diuretic action of this hormone has been reported for anurans (Ewer, 1952b; Uranga & Sawyer, 1960) and for fresh-water fish by Maetz (1963). In fish the response results from an increase in glomerular filtration rate. Nothing is known about the mechanism in urodeles. Curiously, high doses of oxytocin (500 mU/animal) abolished this response and caused a slight increase in body weight in larval A. gracile. This increase in weight was also observed by Heller (1963) and Bentley & Heller (1964) in other urodeles. The mechanism has not been analyzed. The data on the effects of oxytocin on adults are fragmentary. In the few cases studied the hormone (100 mU/animal) had no effect on body weight but did cause the loss of sodium. The time course of this sodium loss differed from that of larvae in that it did not begin until 15-18 hr after injection of the hormone, whereas in larvae the loss was apparent shortly after injection. This suggests that in adults a different, possibly a secondary, response is involved. There is at present insumcient information to permit the formulation of a comprehensive theory of endocrine regulation of hydromineral metabolism in amphibians. However, as a result of the many comparative studies on this problem certain distinct patterns are emerging. The Amphibia evolved from the Choanichthyes and like this group produce two distinct neurohypophysial principles, AVT and a yet unidentified oxytocin-like principle (Follett & Heller, 1964). The transition from fish to amphibian presumably occurred in fresh water where water elimination, not conservation, is the critical problem. It is doubtful that the primitive function of the neurohypophysial hormones is reflected by the typical water balance effect and indeed such an effect is generally absent or reduced in fresh-water forms. The functions of these hormones in fresh-water vertebrates remain the object of conjecture. Jorgensen &
S O D I U M AND WATER BALANCE I N A M B Y S T O M A
545
Larsen (1960) and Sawyer (1961b) have suggested that the primitive function of the hypothalamic-neurohypophysial octapeptides is the regulation of the adenohypophysis which in turn may regulate the release of mineralocorticoids from interrenal tissue. It is also possible that the original role of these hormones was the regulation of reproductive behavior (Wilhelmi et al., 1955). Finally, numerous studies have shown that neurohypophysial hormones play a direct role in the regulation of electrolyte balance (Maetz, 1963). Many of the effects of AVT described in this report can best be interpreted in this context. The level of AVT in the blood might regulate the activity of the sodiumtransporting system in the skin. The time course of the sodium balance response indicates this is a direct effect and is not mediated by other endocrine organs. This is supported by observations on isolated target organs. Arginine vasotocin also causes antidiuresis without changing sodium concentration in the urine. While this reduces the renal excretion of sodium, it also aggravates the water balance problem and thus is of doubtful significance while the animals are in water. However, for terrestrial adults the response assumes considerable significance. Statements about the effects of oxytocin on salt and water metabolism must be qualified because there may be differences between the effects of synthetic oxytocin and the natural "oxytocin-like" principle of urodeles. Our results and others (see Maetz, 1963) suggest that in aquatic forms oxytocin may be a diuretic hormone functioning in the control of the elimination of water by the kidneys. The diuresis entails the loss of some sodium, but if controlled quantities of AVT were released at the same time, this loss could be compensated by increasing the active uptake of sodium across the skin. Furthermore, the AVT would exert a regulatory effect on the kidneys, preventing the possibility of extensive diuresis. By delicately regulating the release of each of the neurohypophysial hormones the animal could integrate the mechanisms involved in maintaining an osmotic and ionic steady state. The factors which trigger and regulate the release of the hormones are not known for lower vertebrates. REFERENCES ACHERR., CHAUVETJ., CHAUVETM. T. & CREPYD. (1964) Phylog6nie des peptides neurohypophysaires: Isolement de la mesotocine (Ileus-ocytocine) de la grenouille, interm6diaire entre la Ser,-Ileus-ocytocine des poissons osseux et l'ocytocine des mammif6res. Biochim. Biophys. Acta 90, 613-615. ALWmADOR. H. & KIRSCHNERL. B. (1963) Osmotic and ionic regulation in Ambystoma tigrinum. Comp. Biochem. Physiol. 10, 55-67. B~LEHRADEKJ. & HUXLEYJ. S. (1927) The effects of pituitrin and of narcosis on waterregulation in larval and metamorphosed Ambystoma..7. Exp. Biol. 5, 89-96. BENTLEYP. J. (1963) Neurohypophysial function in amphibians, reptiles and birds. Syrup. Zool. Soc. Lond. 9, 141-152. BENTLEYP. J. & HELLERH. (1964) The action of neurohypophysial hormones on the water and sodium metabolism of urodele amphibians. J. Physiol. 171, 434-453. BRUNN F. (1921) Beitrag zur Kennmis der wirkung von hypophysenetrakten auf den Wasserhaushilt des Frosches. Z. Ges. Exp. Med. 25, 170-175. Dow D. & ZUCKERMANS. (1939) The effect of vasopressin, sex hormones and adrenal cortical hormone on body water in axolotls..7. Endocrinol. 1, 387-398.
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RONALD H. ALVARADOAND SHELDON R. JOHNSON
EWER R. F. (1952a) The effect of pituitrin on fluid distribution in Bufo regularis Reuss. J. Exp. Biol. 29, 173-177. EWER R. F. (1952b) The effects of posterior pituitary extracts on water balance in Bufo careus and Xenopus laevis, together with some general considerations on anuran water economy. J. Exp. Biol. 29, 429-439. FOLLETT B. K. & HEALER H. (1954) T h e neurohypophysial hormones of lungfish and amphibians. J. Physiol. 172, 92-106. FUHRMAN F. A. & USSING H. H. (1951) A characteristic response of the isolated frog skin potential to neurohypophysial principles and its relation to the transport of sodium and water. J. Cell. Comp. Physiol. 38, 109-130. GREEN J. D. & MAXWELLD. S. (1959) Comparative anatomy of the hypophysis and observations on the mechanism of neurosecretion. In Comparative Endocrinology (Edited by GORBMAN A.), pp. 368-392. Wiley & Sons, New York. HEALER H. (1930) l~ber die Einwirkung von Hypophysenhinterlappenextrakten auf den Wasserhaushalt des Frosches. Arch. Exp. Path. Pharmaeol. 157, 298-322. HEALER H. (1963) Pharmacology and distribution of neurohypophysial hormones. Syrup. Zool. Soe. Lond. 9, 93-106. HoussAY B. A. & POTICK D. (1929) Cited in H. Heller (1950). The comparative physiology of the neurohypophysis. Experientia 6, 368-376. JARD S. & MOREL F. (1963) Actions of vasotocin and some of its analogues on salt and water excretion by the frog. Amer. J. Physiol. 204, 222-226. JORGENSEN C. B. (1950) T h e amphibian water economy, with special regard to the effect of neurohypophysial extracts. Acta Physiol. Scand. 22 (Suppl. 78), 79 pp. JORGENSENC. B. & LARSENL. O. (1960) Comparative aspects of hypothalamic-hypophysial relationships. Ergeb. Biol. 22, 1-29. JORGENSEN C. B., LEVI H. & USSINGH. H. (1946) On the influence of the neurohypophysial principles on the sodium metabolism in the axolotl (Ambystoma mexicanum). Acta Physiol. Scand. 12, 350-371. KLEEMAN C. R. & CUTLER R. E. (1963) The neurohypophysis. Ann. Rev. Physiol. 25, 385-432. LEAr A., ANDERSON J. & PAGE L. B. (1958) Active sodium transport by the isolated toad bladder. J. Gen. Physiol. 41, 657-668. MAa~TZ J. (1963) Physiological aspects of neurohypophysial function in fishes with some reference to the Amphibia. Syrup. Zool. Soc. Lond. 9, 107-140. SAW'~R W. H. (1957) The antidiuretic action of neurohypophysial hormones in Amphibia. In The Neurohypophysis (Edited by HELLER H.), pp. 171-182. Butterworths, London. SAW'~R W. H. (1961a) Comparative physiology and pharmacology of the neurohypophysis. Rec. Prog. Hormone Res. 17, 437-465. SAWYERW. H. (1961b) Neurohypophysial hormones. Pharmacol. Rev. 13, 225-277. SAWYER W. H. (1963) Neurohypophysial secretions and their origin. In Advances in Neuroendocrinology (Edited by NALBANDOVA. V.), pp. 69-80. Univ. of Illinois Press, Urbana. SAWYERW. H. (1964) Vertebrate neurohypophysial principles. Endocrinology 75, 981-990. SCHARRER E. & SCHARRERB. (1963) Neuroendocrinology, 289 pp. Columbia Univ. Press, New York. STEGGEROA F. R. (1937) Comparative study of water metabolism in amphibians injected with pituitrin. Proc. Soc. Exp. Biol. 36, 103-106. URANGA J. & SAWER W. H. (1960) Renal responses of the bullfrog to oxytocin, arginine vasotocin, and frog neurohypophysial extract. Amer. J. Physiol. 198, 1287-1290. WIaHELMI A. E., PICKEORD G. E. & SAWYERW. H. (1955) Initiation of the spawning reflex response in Fundulus by the administration of fish and mammalian neurohypophysial preparations and synthetic oxytocin. Endocrinology 57, 243-252.