Renal responses to vasoactive hormones in the aglomerular toadfish, Opsanus tau

GENERAL AND COMPARATIVE ENDOCRINOLOGY 43, l-9 (1981)

Renal Responses to Vasoactive Hormones in the Aglomerular Toadfish, Opsanus fau ALBERT Department

of Physiology

and

ZUCKER

AND HIROKO

NISHIMURA’

Biophysics, Universify of Tennessee Memphis, Tennessee 38163

Accepted November

Center

for

the Health

Sciences,

19, 1979

Angiotensin and neurohypophysial hormones have been shown to affect renal and cardiovascular function in glomerular fish. In order to determine if these hormones have any tubular actions, we administered angiotensin, arginine vasotocin (AVT), and isotocin intraarterially into the unanesthetized, chronically cannulated, .aglomerular toadfish Angiotensin (100 ng kg-’ min-I) increased dorsal aortic pressure, but did not increase urine flow or the urinary excretion rate of Na, K, Cl, or Mg. AVT (50 ng kg-’ min-I) increased dorsal aortic blood pressure comparable to the increase after angiotensin. Urine flow and urinary electrolyte excretion increased also in response to AVT, but the effect was highly variable. A bolus injection of isotocin (1000 ng kg-‘) did not elicit a pressor response, but significantl,y increased urine flow, urinary Mg concentration, and the urinary excretion of Mg and Cl. These data suggest that isotocin, and perhaps AVT, may act on teleost renal tubules. AVT caused more variable renal effects than isotocin, presumably due to its cardiovascular actions.

Teleost kidneys produce an angiotensinlike substance upon incubation with homologous plasma (Mizogami et al., 1968), and the histochemical properties of some of their arteriolar cells are similar to mammalian juxtaglomerular cells (Krishnamurthy and Bern, 1969; Oguri et al., 1972). Several studies have tried to elucidate the physiological ‘role of the renin-angiotensin system (RAS) in fish. Plasma renin activity did not increase when teleosts were transferred to hypoosmotic media (Nishimura ef al., 1976; Henderson et al., 1976), but it did increase in response to hemorrhage or hypotension (Nishimura et al., 1979), thus suggesting that the RAS may be important in cardiovascular regulation in teleosts. In glomerular bony fish, pressor doses of angiotensin caused glomerular diuresis and natriuresis, which are largely ascribed to the increase in dorsal aortic pressure (Nishimura and Sawyer, 1976; Sawyer et

al., 1976). However, Henderson et al., (1978) found that angiotensin reduced glomerular filtration rate (GFR) and urine flow in the norepinephrine-infused anesthetized trout (Brown et al., 1978). Arginine vasotocin (AVT), which is chemically similar to mammalian vasopressin, is found in the neurohypophysis of nonmammalian gnathastome vertebrates. In glomerular bony fish, AVT caused a diuresis at pressor doses (Chester-Jones et al., 1969; Henderson and Wales, 1974; Sawyer et al., 1976; Babiker and Rankin, 1978), which in some instances continued after the pressor effect dissipated (Sawyer, 1970). Isotocin, the other teleost neurohypophysial hormone, is vasoactive (Chan and Chester-Jones, 1969), but its renal effects seem to be dependent on dose and salinity accbmation in the eel (ChestesJones et al.; 1969; Babiker and Rankin, 1978). In ,gIomerular vertebrates, diuresis and natriuresis can be caused either by increasing glomerular filtration (in the absence of perfect glomerular-tubular bal-

1 Send reprint requests to Dr. &roko Nishimura, Department of Physiology and Biophysics, University of Tennessee Center for the Health Sciences, Memphis, Tennessee 38163. l

OOi6-6480/81/010001-99$01.00/0

2

ZUCKER AND NISHIMURA

ante) and/or by changing tubular function (transport or permeability). Diuresis and natriuresis produced in glomerular bony fish by pressor doses of angiotensin and AVT were usually accompanied by an increase in GFR. Thus, it is not clear whether these substances also altered tubular function in bony fish. Therefore, we administered angiotensin and neurohypophysial hormones to an aglomerular fish, wherein tubular actions may be more apparent. METHODS Animals

and Maintenance

Toadfish, Opsanus tau, of both sexes, weighing between 190 and 760 g, were purchased from dealers in New York and from Marine Biological Laboratories in Woods Hole, Massachusetts. They were held in 50% seawater (SW) (Instant Ocean, Aquarium Systems, Inc., Ohio) at 15”, fed shrimp weekly (except for the week and a half before surgery), and were allowed to acclimate to laboratory conditions for at least 17 days prior to surgery.

Surgical

Procedures

Individual toad&h were adapted to a darkened acrylic chamber through which aerated, filtered 50% SW at 15” was passed. Three days later, the animals were anesthetized in bicarbonate-buffered 0.03% tricaine methane sulfonate (Finquel, Ayerst) and maintained on 0.02% of this anesthetic during surgery. Two ventral incisions for ureteral cannulation and a midventral incision for arterial cannulation were made in large toadfish, with a single Y-shaped incision was used for smaller fish. The ureters were cannulated with polyethylene tubing (PE-10) or with expanded polyvinyl chloride catheters (SVE-6; Dural Plastics and Engineering, Australia). A minor branch of the celiac artery was cannulated with PE-10 tubing (the tip floated in celiac artery), and the hepatomesenteric artery was occlusively cannulated with either PE-50 or SVE-6 tubing. Occlusive cannulation or clotting (as noted during autopsy) of the celiac artery resulted in abnormally high rates of urine flow, and such animals were not included in the present studies. Placement of all cannulas and the absence of leaks were verified with the retrograde injection of ink during autopsy. After surgery, toadfish were returned to their chambers, but they were restrained (dorsal side up) by means of ligatures through their pectoral fins and tail in order to prevent free movement. This allowed reduction of the length and thereby the dead space of the urinary catheters. Toadfish did not struggle once they adapted to the restrained state. Animals were allowed at least a complete day to recover from surgery.

Blood and Urine Collection Blood samples (50 ~1) were taken daily (before urine collections began), and hematocrit and plasma electrolytes were determined. Urine was usually collected hourly (or every 2 hr if the rate of urine flow was very slow) in small tared capped tubes; and, due to cannula dead space, the urine produced during one period was usually collected during the following hourly period. For most fish, urine was collected separately from each ureter, and the prorated electrolyte concentra; tions for a given period were determined by computing weighted averages from the individual ureteral excretion data by the following formula: E combined =

V,.E,

+ Vz.E, v,+v,

(1)

where the subscripts refer to the two ureters, V to the rate of urine flow (~1 100 g-l hr-I), and E to the concentration of a specific electrolyte. In a few fish, urine from both kidneys was collected together, and urinary electrolyte concentrations were directly determined.

Experimental

Protocol

I. Control period. The control level for all urinary variables was taken as the mean of the two collection periods (l-2 hr each) preceding infusion or injection. Control blood pressure was the average of values obtained every 30 min during the control periods. 2. Drug infusion. Vasoactive hormones and solvent controls were infused for 30 or 60 min at the rate of 1 ml kg -I hr-’ with an infusion pump (Sage, 335). The experimental interval included the infusion period plus the following hour. This usually corresponded to two urinary collection periods, and the mean of these two values is used as the experimental level. When the rate of urine flow was very slow, the experimental interval only represents a single 2-hr collection. Experimental blood pressure is the mean blood pressure during the infusion period (measured every 5 min) and the following hour (measured every 15 min). 3. Drug injection. Vasoactive hormones were injected intraarterially by means of a microliter syringe. The experimental interval always corresponded to the I-hr urinary collection period following injection. Experimental blood pressure represents the mean of measurements taken every 5 min. 4. Recovery period. The recovery interval is the 2 hr following the experimental interval (two I-hr or one 2.hr urinary collection periods). Blood pressure during the recovery interval is the mean of measurements taken every 30 min. Usually only one experiment was performed per fish per day. However, following an infusion experiment which did not alter urine flow (vehicle control or angiotensin, 10 ng kg-’ min-I), another experiment with a more potent substance was sometimes made.

VASOACTIVE

HORMONES

Angiotensin ([Asn’, VaP] ANG II) was provided by Ciba Pharmaceutical Company; arginine vasotocin (potency, 160 vasopressor unitsimg) and isotocin were kindly provided by Dr. W. H. Sawyer (Columbia University, N.Y). Stock solutions containing angiotensin (100 &ml), AVT (100 kg/ml), and isotocin (10 pg/ml) were stored in solvents; the angiotensin solvent contained 0.002% thimerosal and 0.2% mannitol, while the isotocin and AVT solvent contained 0.05 M acetic acid and 0.5% chlorobutanol. Each stock solution was diluted with 0.9% saline to the proper concentration just before infusion or injection.

Arginine

Blood pressure was measured with a strain gauge pressure transducer (Hewlett-Packard, 1280C) and recorded continuously (Hewlett-Packard, 7782A); sodium and potassium were measured with a flame photometer (Instrumentation Laboratory, 143), chloride by coulometric titration (Buchler, Cotlov titrator), and magnesium with an atomic absorption spectrophotometer (Perkin-Elmer, 290). Unless otherwise specified, a paired t test was used to determine if the values during the experimental and recovery periods were significantly different from the control levels.

RESULTS Angiotensin

Infusion of the angiotensin vehicle (6% angiotensin solvent) did not alter dorsal aortic pressure or any of the measured renal variables (Tables 1 and 2). Although the angiotensin (100 ng kg -I min -‘) infusion increased dorsal aortic pressure, it did not TABLE VEHICLES

AND HORMONES

(1Values are mean i 1 SE. *P < 0.05. **p < 0.01.

1

ON BLOOD

PRESSURE

(mm Hg) IN THE TOADFIS@

Control

Experiment

Recovery

7 9

19.4 -c 1.0 18.0 2 1.1

20.2 + 1.2 22.2 ir 1.7**

19.3 i 1.1 16.9 F 1.1”

4 7

18.5 t 1.0 17.5 t 1.8

16.5 + 0.4 21.3 + 2.5**

17.5 i 0.9 17.5 s 2.4

4

19.7 k 1.4

19.1 + 1.3

18.7 -+ 1.5

N

Angiotensin vehicle Angiotensin (100 ng kg-’ mini) AVT vehicle AVT (50 ng kg-’ min-*) Isotocin (1000 ng kg-‘)

Vasotocin

Infusion of the AVT vehicle (3% AVT solvent) did not alter blood pressure or renal function in the toadfish (Tables 1 and 2). AVT (50 ng kg-l min-l) infused for 1 hr caused a significant increase in bIood pressure during the experimental interval (Table 1). Although AVT’s maximum pressor effect during infusion was smalier than that of angiotensin, following the termination of infusion the pressor effect of AVT remained longer than that of angiotensin (Pig. 1). Following AVT, urine flow increase seven out of nine aminals; approximately 60% as a group mean (Table 2). The variance of urine flow also increased (F, test, P < 0.05; Sokal and Rohlf, 1973). Nowever, the increase in urine flow was not statistically significant when determined by a paired t test or Wilcoxon’s signed rank test (P > 0.1). Similarly, urine flow and variability increased following a bolus injection of AVT (500 ng kg-l; n = 4). AVT also increased the mean urinary excretion of

Analysis

OF VARIOUS

3

significantly change any of the measured urinary variables (Tables 1 and 2). Similarly, no renal effect was noted when angiotensin was infused at a lower dose (10 ng kg-l min-‘; n = 4) or injected as a bolus (500 ng kg-‘; 12 = 3) except a slight but significant elevation in urinary K excretion after 10 ng kg-l min-l.

Drugs

EFFECT

IN TOADFISH

(meq

liter-‘)

100 g-’

UN,V (peq

UK

liter-‘)

(pl 100 g-’

u ml (meq

V

THE

hr-I)

hr-‘)

EFFECT

2.02 2 0.29 1.97 2 0.23 2.14 f 0.24

16.9 16.8 13.2

C E R

f f i

t 2.00 2 1.89 r 1.89

1.22 * 0.30 1.26 c 0.30 1.29 ? 0.30

103.0 101.0 110.4

11.56 11.93 10.87

C E R

C E R

C’ E R

AVT,

ISOTOCIN,

Angiotensin vehicle (N = 8, n = 6)*

OF ANGIOTENSIN,

1.91 !C 0.27 2.04 ‘- 0.45 2.01 f 0.43

2.65 2.64 2.86

1.41 1.44 1.32

1.12 + 0.36 1.04 k 0.29 1.14 c 0.29

14.72 15.35 14.87 98.6 94.2 99.7

? 3.46 jl 2.66 c 2.46

FUNCTION

-c 0.35 ‘: 0.40 f 0.49

-c 0.45 k 0.47 k 0.35

zi 14.4 -c 15.4 ? 18.5

k 4.40 t 4.29 2 3.83

Angiotensin 100 ng kg-’ min-’ (N = 8, n = 6)b

2 ON URINARY

+ 16.1 + 17.5 t 16.5

80.7 81.6 81.4

14.01 12.91 14.63

AVT vehicle (N = 8, n = 5)”

TABLE AND VEHICLES

2.61 2.57 2.43

1.08 1.58 1.04

92.7 85.7 84.3

13.30 21.28 14.41

? 0.38 ‘- 0.37 2 0.34

2 0.18 k 0.36 t 0.20

k 16.9 i 12.0 t 14.4

” 2.18 2 6.89 f 3.40

AVT 50 ng kg-’ min-* (N = 9, n = 7)b

IN THE AGLOMERULAR

+ 0.27 c 0.29 2 0.25

_t 16.2 + 16.2 2 15.5

+ 2.03 2 2.40* -t 1.94

2.25 2 0.54 1.97 k 0.37 1.78 _’ 0.29

0.99 1.10 0.96

86.9 79.1 75.4

10.63 13.48 12.18

Isotocin 1000 ng kg-l (N = 7, n = 7)*

TOADFISH”

100 g-l

U,IV (yeq

1OU g-l

hr-I)

hr-*)

hr-*)

c

E R

rtr 10.2 f 12.9 -c 14.0

c 0.008 r 0.006 -’ 0.008

1.16 1.62 1.66

125.4 129.0 134.8

n is the sample

-+ 0.70 -t- 0.64 ir 0.58

+- 26.6 f 30.4 i- 27.2

1.97 k 0.50 1.86 F 0.47 2.17 ” 0.48

150.7 141.1 143.2

0.027 0.025 0.028

VKV, UC,, and UcIV;

1.03 2 0.19 I.09 -c 0.20 0.97 4 0,2I

c

R

104.4 118.1 99.1

c E

I 18.8 + 37.7 -+ 21.7

1.57 F- 0.33 1.55 r 0.29 1.34 k 0.23

C E R

+- 9.5 2 9.4 2 9.0

* 0.003 f 0.003 rt 0.003

133.0 130.0 126.7

OA21 0.022 0.022

C l-3 R

E R

IL Values are mean + SE. ’ N is the sample size fQr V, UN~, UN,V, UK, ’ C, control; E, experimental; R, recovery. *P < 0.05.

UMJJ (keq

(meq

liter-‘)

liter-‘)

UC1 (meq

V MC4

100 g-’

UKV

(peq

k 15.2 _’ 14.6 c 16.4

size for

U,,

and

U,,V.

2.44 3.60 2.90

143.2 128.6 147.0

1.87 3.39 2.23

136.1 143.1 142.3

i 13.2 zk 12.8 r 11.6 z!z 0.68 i: 0.65 k 0.60

0.034 0.065 0.037

CL 0.017 f 0.017 +- 0.014

1.60 ‘-c 0.52 1.40 2 0.38 1.62 ” 0.52

96.6 94.4 93.8

1.97 2.05 1.98

119.2 120.4 120.3

0.042 0.043 0.043

+ 0.78 i 1.58 _t 1.14

k 34.6 r 23.6 t 28.6

f 0.39 * 1.41 rt 0.70

? 10.3 k 8.8* 2 9.8

a 0.008 t 0.030 t 0.013

1.38 2.02 1.82

143.8 156.0 155.0

0.70 0.99 0.80

127.7 134.4 136.2

0.021 0.025 0.021

iz 0.32 r 0.50* t 0.46

-c 29.6 -c 29.8* t 28.6

iz 0.26 f 0.38* -c 0.30

k 12.3 t 14.4 + 12.4

-+ 0.004 t 0.005 rt 0.003

ZUCKER

AND

NISHIMURA

AVT (50ng/kg z

7.5 -

w zi [L

5.0 -

2

per mid

2.5 ()-2,5 _ I 0

i TIME

2 (Hours)

3

I 4

FIG. 1. Time course of dorsal aortic blood pressure response to 60-min infusions of angiotensin and AVT in the toadfish. Values expressed as mean (of 2- 10 animals) increase from control level 2 1 SE. Angiotensin control blood pressure: 19.0 f 0.5 mm Hg, n = 9; AVT control blood pressure: 19.1 + 0.6 mm Hg, it = 10.

Na, K, Cl, and Mg by 50- lOO%, but only the increase in Cl concentration was statistically significant. Isotocin Isotocin (1000 ng kg-l) did not alter blood pressure (Table 1). However, it caused a significant increase in urine flow, urinary Mg concentration, and the urinary excretion of Cl and Mg (Table 2). DISCUSSION

In aglomerular fish, urine is thought to be formed by the osmotic movement of fluid secondary to divalent ion secretion into the tubules. The tubular fluid is then modified by solute (and fluid) reabsorption in more distal segments of the nephron (Hickman and Trump, 1969). Urine flow during control periods for the toadfish used in this study averaged at 12.8~1 100 g-l h-l, which is similar to that reported by Lahlou et al. (1969) and Howe and Gutknecht (1978) in the same species. Urine flow rate measured hourly fluctuated approximately 20% despite careful positioning of the urinary catheters (as confirmed during autopsy). Although the

tubules may indeed function intermittently, another possible explanation is that, since the kidney is attached to the body wall musculature, contraction of the body wall (during swimming or moving) might cause expulsion of urine. Although this much variation in urine flow is normal for intact fish, we use a mean period of 2-4 hr as the control level to minimize the influence of possible short-term intermittency. Nevertheless, small changes in urine flow during the experimental interval may not have been detected in the unanesthetized preparation. Angiotensin We could not find any effects of moderate or high doses of angiotensin on urine flow or urinary concentration or excretion of Na, Cl, and Mg. Churchill et al. (1979) reported that the infusion of angiotensin (mean dose, 68 ng kg-’ min-‘) caused diuresis and natriuresis in the unanesthetized aglomerular goosefish. However, since their fish were placed with the ventral side up during the experiment, renal hemodynamics may have been different from those in our preparation.

VASOACTIVE

HORMONES

IN

TOADFlSH

7

water eels, Chester-Jones et al. (1969) showed that isotocin was diuretic, while Babiker and Rankin (1978) found that isototin could be both diuretic and antidinretic. However, only a diuretic response was found in seawater-adapted eels (Babiker and Rankin, 1978). In the aglomerular toadfish, the bolus injection of isotocin increased urine flow, AVT urinary Mg concentration, and the urinary Although the AVT vehicle contained a excretion of Cl and Mg. Lahlou et al. (1969) small amount (0.015%) of a vasodilator also reported (preliminary results) that an (chlorobutanol), the vehicle alone did not inject,ion of isotocin (1000 ng kg+) insignificantly alter dorsal aortic blood pres- creased urine flow by 50% in toadfish. sure or any urinary variable. Although in our study urine flow never increased 50%, a consistent diuresis was obInfusion of AVT into toadfish increased urine flow by 60% as a mean. However, the served. These results suggest that the presresponse in individual animals was vari- ence of the glomerulus is not a prerequiable, ranging from a slight decrease to a site for the renal action of isotocin in three-fold increase. This variability may in- teleosts. Therefore, although the dose used dicate that, in addition to its effect on the is not physiological, it indicates that renal kidney, another action of AVT, most likely tubular isotocin receptors may exist. The its cardiovascular effects, influences renal fact that isotocin increased both Mg and Cl function. excretion in the toadfish may indicate that Sawyer (1970) found in the African the secretion of Mg is related to the movelungfish that when AVT was injected ment of Cl into the aglomerular tubules. abruptly, it had a greater renal effect than Isotocin may affect urine flow by increasing when it was infused, even if the infused the movement of these ions, and therefore dose was several times larger. However, in the ‘osmotic flow of water into the tubules. Recently, Babiker and Rankin (f939) rethe aglomerular toadfish, we could not detect a different renal response between the ported that neither AVT nor isotocin adbolus injection and the infusion of AVT. ministered into the renal portal system of Lahlou et al. (1969) reported that AVT in- the isolated perfused angler-fish (aglomerular) kidney at doses as high as 500 ng kg-’ jections at doses as high as 1000 ng kg-l caused a maximal increase of 30% in urine had an effect on urine flaw, composition, or flow in the toadfish. electrolyte excretion, However, since they used a high venous (renal portal) perfusion Isotocin pressure (27-30 cm H,O), the discrepancy Isotocin has been shown to decrease dor- between their results and our resuhs may sal aortic blood pressure in eels (Chan and be ascribed to a different renal hemodyChester-Jones, 1969). However, as re- namic status between. the two preparations. ported by Lahlou et al. (1969), we did not To conclude, in the toadfish, there is no find any effect of isotocin on dorsal aortic relation between the pressor and the renal blood pressure in the toad&h. Contradiceffects of vasoactive hormones. Since tory results have been reported on the ef- toadfish do not have glomeruli, it is unlikely fect of isotocin on urine flow in freshwater that vasoactive substandes cause a pressure glomerular teleosts. Maetz et al. (1964) did diuresis by increasing renal arterial b$.ilronot find any effect in the goldfish. In freshstatic pressure or by increasing e&rent Likewise, preliminary results show that although norepinephrine (250 ng kg-l; n = 3) increased blood pressure in the toadfish, it did not affect any urinary variable. This result is of interest in contrast to the antidiuresis caused by this drug in the eel, presumably by preglomerular constriction (Nishimura and Sawyer, 1976).

8

ZUCKER

AND

resistance. The results from the present study did not reveal any tubular action of angiotensin in the toadfish. This supports the contention that the diuresis and natriuresis caused by angiotensin in glomerular bony fish resulted primarily from increased glomerular filtration coupled with poor glomerular tubular balance (Nishimura and Sawyer, 1976). The modest increase in urine flow and excretion of Mg and Cl following isotocin administration suggests that isotocin acts on a tubular transport mechanism. AVT may interact with the same mechanism as isotocin, but it appears that its other actions, perhaps cardiovascular, make the response highly variable. ACKNOWLEDGMENTS We are pleased to thank Dr. W. Sawyer for determining the pressor potency of AVT, Dr. Paul Churchill for allowing us to examine a preprint, and Charles Faust and Olivette Rhodes for technical assistance. The work reported in this study was supported by Grant PCM 75-20645 from the National Science Foundation and Grant AM-17824 from the National Institutes of Arthritis, Metabolism, and Digestive Diseases. Part of this study was presented at the December 1977 Annual Meeting of the American Society of Zoologists, Toronto. H. Nishimura is an Established Investigator of the American Heart Association.

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NISHIMURA anguilla L.): Effects of the caudal neurosecretory system, corpuscles of Stannius, neurohypophysial peptides and vasoactive substances. J. Endocrinol. 43, 21-3 1. Churchill, P. C., Mqlvin, R. L., Churchill, M. C., and McDonald, F. D. (1979). Renal function in Lophius americanus: Effects of angiotensin II. Amer. J. Physiol. 236, R297-R301, or Amer. J. guilla

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5, R297-R301. Henderson, 1. W., Brown, J. A., Oliver, J. A., and Haywood, G. P. (1978). Hormones and single nephron function in fishes. In “Comparative Endocrinology” (P. J. Gaillard and H. H. Boer, eds.), pp. 217-222. Elsevier/North-Holland Biomedical Press, Amsterdam. Henderson, I. W., Jotisankasa, V., Mosley, W., and Oguri, M. (1976). Endocrine and environmentd influences upon plasma cortisol concentrations and plasma renin activity of the eel, Anguilla anguitla L. J. Endocrinol. 70, 81-95. Henderson, I. W., and Wales, N. A. M. (1974). Renal diuresis and antidiuresis after injections of arginine vasotocin in the freshwater eel (AnguiNu anguilla L.). .I. Endocrinol. 61, 487-500. Hickman, C. P., and Trump, B. F. (1969). The kidney. In “Fish Physiology” (W. S. Hoar and D. J. Randall, eds.), Vol. 1, pp. 91-239. Academic Press, New York. Howe, D., and Gutknecht, J. (1978). Role of urinary bladder in osmoregulation in marine teleost, Opsanus tau. Amer. J. Physiol. 235, R48-R54; or Amer. J. Physiol.: Regulatory Phpsiol. 4, R48-R54.

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VASOACTIVE

HORMONES

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Oguri, M., Ogawa, M., and Sokabe, H. (1972). Juxtaglomerular cells in aglomerular teleosts. Bull. Japan. Sot. Sci. Fish. 38, 195-200. Sawyer, W. H. (1970). Vasopressor, diuretic, and na-

IN

TOADFISH

9

triuretic responses by lungfish to arginine vasototin. Amer. J. Physiol. 218, 1789-1794. Sawyer, W. H., Blair-West, J. R., Simpson, P. A., and Sawyer, M. K. (1976). Renal responses of Australian lungfish to vasotocin, angiotensin II, and NaCl infusion. Amer. J. Physiol. 231, 593 -602.

Sokal, R., and Rohlf, F. J. (1973). “Introduction to Biostatistics.” Freeman, San Francisco. Vander, A. J. (1975). “Renal Physiology.” McGrawHill, New York.