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Effects of hypophysectomy on the plasma ionic and osmotic balance in rainbow trout, Salmo gairdneri
GENERAL
AND
Effects
COMPARATIVE
49, 240-247 (1983)
ENDOCRINOLOGY
of Hypophysectomy on the Plasma Ionic and Osmotic Balance in Rainbow Trout, Salmo gairdneri BJ~~RN TH. BJ~RNSSON AND TIIU HANSON
Department
of Zoophysiology,
University
of GBteborg,
Box 250 59, S-400 31 Gothenburg,
Sweden
Accepted March 18, 1982 The effects of hypophysectomy on the plasma ionic and osmotic balance in juvenile rainbow trout (S&IO gairdneri) kept in 113 sea water for various periods were studied. Hypophysectomy caused a decrease in the plasma levels of ionized calcium and chloride within a week after the operation. At 2-5 weeks after hypophysectomy, the plasma levels of ionized calcium and chloride were still significantly lower in hypophysectomized fish when compared to sham-operated controls. Hypophysectomy had no effects on the plasma levels of sodium, total calcium, total magnesium, inorganic phosphate, plasma proteins, or plasma osmolality. When 113 sea water adapted (l-4 weeks), hypophysectomized and sham-operated fish were exposed to fresh water for 2 weeks, the plasma levels of ionized calcium, chloride, sodium, and plasma osmolality were significantly lower in hypophysectomized fish when compared to sham-operated fish, while the plasma levels of total calcium, total magnesium, and proteins were higher. No effects were noted on the plasma levels of potassium and inorganic phosphate. The presented data indicate that the pituitary gland plays a significant role in the regulation of plasma calcium and chloride levels in ,S. gairdneri, while its role in the regulation of plasma magnesium and sodium is less clear. The pituitary gland seems to play a minimal role in the plasma regulation of inorganic phosphate and potassium.
Fontaine et al. (1949) were the first to suggest an osmoregulatory role for the teleostean pituitary gland, and to test this hypothesis with the aid of hypophysectomy. Ensuing studies performed on several teleost species, both euryhaline and freshwater (FW) living, have shown that hypophysectomized fish in FW suffer a loss of sodium and chloride from the plasma. These ion changes and the related fall in plasma osmolality are considered to be the main cause of death of hypophysectomized fish in FW (for ref., see Macfarlane, 1974). The length of survival time in FW after hypophysectomy has been suggested to be related to the relative rate of ion loss from plasma as well as the tolerance of the given species to lowered plasma osmolality, sodium and chloride levels (Dharmamba, 1970; Macfarlane, 1974). The limited survival time in FW following hypophysectomy may be increased considerably by placing the fish in a near-isoosmotic envi-
ronment, such as physiological saline or l/3 sea water (SW) (Chidambaram et al., 1972; Oduleye, 1975). It is well established that hypophysectomy does not only affect the sodium/ chloride based osmotic balance of the plasma, but also the levels of the multivalent ions, especially that of calcium in a number of teleost species studied so far (Pang et al., 1973; Chan and Woo, 1978). Owing to the relative inaccessibility of the pituitary gland in salmonid fish, few studies involving hypophysectomy have been carried out on species of this family of teleosts (Donaldson and McBride, 1967; Oduleye, 1975, 1976; Yamazaki, 1976; Komourdjian and Idler, 1977; Komourdjian et al., 1978). However, the introduction of a new technique for hypophysectomizing Salmo gaivdneri (Komourdjian and Idler, 1977), has now made hypophysectomy a practical endocrinological research tool also in salmonid fish. To investigate the effects of hy-
240 0016-6480/83/020240-08$01.50/O Copyright All rights
0 1983 by Academic Press, Inc. of reproduction in any form reserved.
HYPOPHYSECTOMY
pophysectomy on the plasma ionic and osmotic balance in S. gairdnevi a number of experiments, both short-term and longterm, have been carried out. TERI
AND
METHODS
Fish. Juvenile S. gairdireri (40- 100 g bw) were obtained from a local hatchery near Gothenburg (September 1979%March 1980). The fish were kept without food in tanks holding approximately 300 liters of tiltered, recirculating, and aerated FW at a temperature of 10 to 12”, and a photoperiod of 12L:12D. Under these conditions the fish were acclimatized for at least ! week prior to the experiments. Hypoplzysectomy. The fish were hypophysectomized and sham-operated by the buccal approach of Komourdjian and Idler (1977) at different times of the year. The fish were anaesthestized in 2 mgiliter aqueof propoxate (Janssen Pharmaceutics, ium) until all but shallow respiration movements ceased, and this state was kept during the surgery by an aqueous solution (113 SW) of 20 mg tricaine methanesulfonate (MS 222)/1iter, which was pumped over the gills. During recovery from anaesthesia, the fish were kept under observation in a small basin with noncirculating but aerated 113 SW, before being returned to holding tanks, containing noncirculating but aerated 113 SW, at a temperature of 10 to 12” and a photoperiod of 12L:42D. This water was partially changed every 3 - 4 days Expcrimerztal design. IGperiment 1: To examine the short-term effects of hypophysectomy on the plasma ionic and osmotic balance, hypophysectomized and sham-operated tish were exposed to 113 SW for 1,3, or 7 days. Experinzenr 2: To examine the long-term effects of hypophysectomy, hypophysectomized and shamoperated fish were exposed to 113 SW for 2-5 weeks. &perinzent 3: Preliminary experiments showed that when hypophysectomized fish were exposed to FW direct!y after the operation the mortality was high. However. when the fish were acclimatized to 113 SW for at least 1 week after the operation, and then exposed to FW the hypophysectomized fish survived for at least 5 weeks (Bjornsson et al., 1981). Therefore, to examine the long-term effects of hypophysectomy in FW, hypophysectomized and sham-operated fish were acciimatized to 113 SW for at least 1 week after the operation. and then exposed to FW for 2 weeks. Due to the difficult nature of the operation, the period to obtain sufficient number of operated fish for the various experiments varied. Thus, the postoperative period for some of the experimental groups is given as 2-4 weeks. Blood samplbzg md plasma analysis. At the termination of an experiment, the fish was stunned by a
OF
RAINBOW
TROUT
241
blow on the head, and 0.5% 1.0 ml of blood was drawn from the caudal vessels using a l-ml syringe with a 0.4 x Z-mm needle. Plasma was obtained by centr&tgation, and stored at -20” until analysis. After the fish had been sacrificed, the completeness of the operation was accertained by visual inspec.:ior: under fight microscope. Supposedly hypophyseceomized fish, which proved to have pituitary remnants were excluded from the experiment, as were shamoperated fish with damaged pituitary glands. Plasma sodium and potassium concentrations were determined by flame emission spectroscopy (Turner Model 510 spectrometer) using an internal lithium standard. and plasma chloride concentration was determined by amperometric titration (Radiometer CM 10 chloride titrator). Plasma calcium ion concentration was determined using ion-exchanger membrane eiectrodes (Radiometer F2112Ca selectrodes), while total plasma calcium and magnesium concentrations were determined by atomic absorption spectroscopy (Perkin-Elmer 303 atomic absorption spectrophotometer). Plasma osmolaiity was determined by vapour pressure osmometry (Wescor 5100 B vapour pressarre osmometer). Inorganic phosphate and protein concentrations in plasma were measured by the analytical procedures of Dryer ef ai. (i9S7) and Henry et al. (1957). respectively, using a Carl Zeiss speetrophotometer. Minteramd~sis. The FW and SW used in this study was analyzed using the same analytical procedures as described for plasma analysis (FW: Chloride, 1.2 ,&VI: sodium. 2.2 m.M; potassium, 0.2 mM, total caicimm, 0.5 m&f: tot21 magnesium, 0.5 phate,
i-7-S
are shown in Table 1. fooilowing ~y~o~~ysectomy, nificant
decrease
there is a sig-
in the plasma
level
of
6
5.3 +0.4 0.285
0.529
4.2 eo.3
3.9 kO.5
4.3 +0.6
3.8 +0.3
4.2 20.5
K+ (mM)
157.7 k1.9
161.8 k2.0
155.0 k4.8
152.0 t2.6
160.3 22.4
158.8” -+1.7
Na+ @Ml
TABLE
1
0.660
2.35 io.13
2.36 -to.07
2.26 kO.05
2.29 kO.20
2.16 iO.08
2.33 -co.07
CaTO (mM)
CONSTITUENTS
0.001*
0.78 i-O.08
1.10 kO.07
0.82 kO.10
0.96 20.05
0.56 kO.07
0.85 to.03
CaZ+ (mM)
IN RAINBOW
0.165
1.67 kO.09
1.56 kO.08
1.65 kO.09
1.54 10.11
1.54 io.07
1.46 kO.02
Mg,” bM)
TROUT
0.575
2.31 20.18
2.24 +0.22
1.99 -co.10
2.29 20.14
2.54 io.09
2.24 to.10
(mM)
Pi”
KEPT
IN
0.660
2.84 20.20
3.26 -co.43
4.00 20.35
2.66 20.17
1.90 ‘-co.30
3.18 +0.37
Protein (g%‘o)
l/3 SW
sham-operated controls, collectively tested (P < 0.017).
0.0143
133.3 k1.5
143.2 -r-1.3
137.5 21.5
140.0 kO.9
141.5 kl.2
140.5 kO.7
Cl@Ml
ON PLASMA
u Number of animals in each group. b Total calcium. c Total magnesium. d Inorganic phosphate. e All data are expressed as means i SE. * Hypophysectomized groups statistically different from corresponding
P=
Hypophysectomized
6
Sham-operated
7
4
Hypophysectomized
4
3
Sham-operated
4
4
1
n”
OF HYPOPHYSECTOMY
Hypophysectomized
Sham-operated
Treatment
EFFECTS
Time after treatment (days)
SHORT-TERM
0.078
301.9 21.8
311.3 k2.7
300.9 t3.0
301.4 11.3
305.6 k5.7
302.9 22.5
Osmolality (mOsm/kg)
HYPOPHYSECTOMY
ionized calcium (P = 0.001) and chloride (P = 0.014). Hypophysectomy had no effects on the plasma levels of sodium, potassium, total calcium, total magnesium, inorganic phosphate, proteins, or plasma osmolality (P b 0.017). Long-term ef,ects of hypophysectomy. The long-term effects of hypophysectomy on fish kept in 113 SW after the operation are shown in Table 2. At 2-5 weeks after ophysectomy, there is a marked decrease in plasma levels of ionized calcium (P = 0.0001) and chloride (Z’ = 0.003), while no effects are seen on plasma levels of sodium, potassium, total calcium, total magnesium, inorganic phosphate, proteins, or plasma osmolahty (P 2 0.017). The long-term effects of hypophysectomy on fish kept in 113 SW (1-4 weeks) r the operation, and then exposed to for 2 weeks are shown in Table 3. HI3 SW adapted (l-4 weeks) hysectomized and sham-operated fish were exposed to FW for 2 weeks, there is a marked decrease in plasma levels of ionized calcium (P < O.O0006), chloride (P < lasma osmolality (P < ition, there is a decrease in Bevel (P = 0.009). On the other hand, there is a marked increase in total plasma levels of calcium (P < 006), magnesium (P < 0.00006), and proteins (P = 0.0001). There is no effect on plasma levels of potassium or inorganic phosphate (P 2 0.017).
The presented data suggest that the pituitary gland may piay a significant role in the calcium regulation in S. gairdneri. Even when byp~physectomized fish are kept in hypercalcemic environment (113 SW), ionized plasma calcium level shows a rapid (within 1 week) and long-lasting (2-5 weeks) decrease. This effect, which is augmented when the fish is exposed to a hyp~ca~cem~c environment (FW), is not reflected in changes in total plasma calcium
OF
RAINBOW’
TROUT
is observation is the currently held opinion bound and ionized calcium a simple equilibrium (se contrast to total plas studies have been carri
243
not in line with that proteinin plasma are in calcium, few out on ion,ized
calcium level that were not acc~~~a~~ed by changes in total plasma calcium in intact Gasterosteus ncdeatus. These findings and the results presented here demonstrate clearly the necessity of of free or ionized plasm the total plasma level o studying calcium regu%
to plasma proteins, as taneous increase in to plasma protein level. The results present
in fish. It remains to be inther the observed Increase in ca~c~~rn~b~~d~~g is nonspecific or due to specific ca~c~~rn-b~~d~~g proteins I The appearance of specific ca~c~~~-b~~~i~~ peplasma level fo 1978). The nature of the hypercalce the pituitary gland of teleost fis be elucidated. There is growi t this factor may be ~~~~act~~
Recently
it has been shown
was suggested that the failure to demonstrate hypocalcemic ffect of exogenous calcitonin in intact fis might be due to an
” Number of animals in each group. * Total calcium. c Total magnesium. d Inorganic phosphate. e All data are expressed as means ? SE. * Hypophysectomized groups statistically
P=
Hypophysectomized
different
6
9
5
Sham-operated
8
18
9
7
5
2-4
from
0.022
152.3 k1.1
155.3 kO.8
150.9 k1.5
153.5 21.4
142.1 kO.9
143.4” %l.O
Na+ (n-M
K+
0.070
2.3 -co.2
2.4 kO.2
3.3 20.1
4.0 kO.3
3.1 LO.2
3.5 20.3
(mM)
0.097
2.24 kO.08
2.37 kO.08
2.59 kO.07
2.78 20.05
2.16 20.10
2.22 kO.10
0.0001*
1.46 kO.05
1.54 r0.02
0.76 20.07
0.93 to.04
1.27 to.06
1.41 LO.10
CaZ+ (mM)
collectively
0.059
0.83 kO.09
0.83 kO.04
0.85 -co.03
0.89 kO.02
0.83 20.02
0.047
2.13 20.14
2.36 io.10
0.072
2.08 kO.20
2.37 co.10
2.53 kO.15
3.16 kO.13
4.58 kO.39
4.13 io.ll
k%)
Protein
IN l/3 SW
(P < 0.017).
1.86 kO.05
2.39 kO.14
2.50 rO.ll
2.47 kO.08
(mM)
Pid
KEPT
tested
TROUT
0.77 r0.02
IN RAINBOW
controls,
2 CONSTITUENTS
sham-operated
0.003*
136.3 %1.2
137.1 kO.5
129.8 -cl.8
134.5 20.8
134.1 t1.1
138.6 21.4
(mM)
Cl-
TABLE ON PLASMA
corresponding
OF HYPOPHYSECTOMY
Hypophysectomized
Sham-operated
Hypophysectomized
Sham-operated
Treatment
EFFECTS
Time after treatment (weeks)
LONG-TERM
0.019
304.6 k2.6
302.6 a1.3
294.1 k1.6
302.3 21.9
306.6 %2.0
319.6 k5.4
Osmolality (mOsm/kg)
EFFECIS
Time after treatment
HYPOPHYSECI‘OMY
2-4
113 SW
FW
weeks
+ 2 weeks
1 week 113 SW + 2 weeks FW
or
” Number of animals in each group. ’ Total calcium. e ‘F&al magnesium. ri Inorganic phosphate. c All data are expressed as means r SE. * Hypophysectomized groups statistically
P= P<
Hypophysectomized
Sham-operated
Hypophysectomized
Sham-operated
Treatment
LONG-TERM
different
-
16
from
corresponding
0.066
0.009”
co.17
FO.04 3.30
2.50
2.81 LO.07
2.57 to.07
-to.06
collectively
tested
O.OOOQ6* ~.___
+0.02
0.63 -to.01 0.79
0.76 kO.02
0.69 to.02
(mM)
Mg.,”
Pi”
AND
3.86
2.47
0.0001”
-co.17
(P < 0.017).
0.039
CO.09
kO.16 4.50
7.43 kO.85
1.92 -co.05 kO.08 2.36
4.46 kO.18
k%)
Protein
THEN
2.07 20.05
(mM)
IN 113 SW
1.48
KEPT
io.04 0.92
0.76 to.04
0.97 kO.02
(mM)
Ca?+
FIRSI
o.ooo&?”
controls.
CmM)
CaTD
TROUT
0.00006* -.-
sham-operated
0.00006”:
k2.7
io.4
al.1
134.9
5.0 kO.7 101.0
114.8 +2.5
3.3 kO.3 to.3 4.3
131.9 +0.9
3.6 50.4
(mM)
21.3 144.6
147.1
145.7 +1.4
19 19
149.5’ +0.8
K+
CmM)
Nat
3 IN RAINBOW
Cl-
TABLE CONS-SIYUENTS
(mA4)
11
II [’
ON PLASMA
Exposure
0.00006”
il.8
52.0 293.0
302.1
289.9 k1.7
300.6 t2.0
Osmolality (mOsm/kg)
1.0 FW
-
2
2 0
z? e
%
2 4
::
246
BJijRNSSON
AND HANSSON
increased prolactin secretion, which will obscure the calcitonin effect. Therefore, it may be suggested that hypophysectomy unmasks the effect of calcitonin and/or other endogenous hypocalcemic principles, thus resulting in the hypocalcemia observed. However, the presence of hypercalcemic principles other than prolactin in the teleost pituitary gland cannot be excluded. Thus, Parsons et al. (1978) have described the existence of a hypercalcemic principle (“hypercalcin”) related to parathyroid hormone, in the pituitary gland of Gadus rnorhua and Anguilla anguilla. Although the plasma level of ionized magnesium was not measured in the present study, the increase in total plasma magnesium in hypophysectomized fish exposed to FW suggests that the regulation of plasma magnesium and calcium may be similar. These results are in accordance with observations on hypophysectomized Fundulus heteroclitus (Srivastava and Pickford, 1972) and A. japonica (Chan and Woo, 1978), which show that hypophysectomy affects the total plasma levels of calcium and magnesium in a similar way. Prolactin is considered to be necessary for the survival of many teleost species in FW (Utida et al., 1971) and is capable of elevating plasma chloride levels of intact as well as hypophysectomized fish (Olivereau and Lemoine, 1973; Gallis et al., 1979). Our results indicate that the pituitary gland may play a significant role in the plasma chloride regulation in S. gairdneri. However, it remains to be investigated whether the pituitary gland factor responsible for the maintenance of normal plasma chloride levels in S. gairdneri is prolactin. The slight effect of hypophysectomy on plasma sodium levels in S. gairdneri observed in this study confirms the findings by Komourdjian and Idler (1977). Thus, the pituitary gland seems to play a less important role in the plasma sodium regulation in this species than it does in most other teleost species studied so far.
It is noteworthy that the decrease in plasma osmolality in hypophysectomized fish exposed to FW is less than expected from the changes in measured ion levels. Furthermore, the concomitant increase in plasma protein levels seems insufficient to account for this discrepancy. However, other organic plasma constituents may play a role in the osmoregulatory process in S. gairdneri, as has been demonstrated for F. heteroclitus (Pickford et al., 1969). In conclusion, the presented data indicate that the pituitary gland plays a significant role in the regulation of plasma calcium and chloride levels in S. gairdneri, while its role in the regulation of plasma magnesium and sodium is less clear. The pituitary gland seems to play a minimal role in the plasma regulation of inorganic phosphate and potassium in S. gairdneri. ACKNOWLEDGMENTS This work was supported by grants from the Swedish Natural Science Research Council, the Magnus Bergwall Foundation, and the Adlerbertska Research Foundation.
REFERENCES Bjornsson, B. Th., Haux, C., and Hansson, T. (1981). Effects of hypophysectomy on the regulation of plasma levels of sodium and chloride in rainbow trout in fresh water. In “Exogenous and Endogenous Influences on Metabolic and Neural Control,” pp. 201-202. Pergamon Press, Oxford. Chan, D. K. O., and Woo, N. Y. S. (1978). Effect of hypophysectomy on the chemical composition and intermediary metabolism of the Japanese eel, Anguilla
japonica.
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169- 178. Chidambaram, S., Meyer, R. K., and Hasler, A. D. (1972). Effects of hypophysectomy, pituitary autografts, prolactin, temperature and salinity of the medium on survival and natremia in the bullhead. Ictalurus melas. Comp. Biochem. Physiol. 43A, 443-457. Dacke, C. G. (1979). “Calcium Regulation in SubMammalian Vertebrates,” p. 11. Academic Press, London/New York. Dharmamba, M. (1970). Studies of the effects of hypophysectomy and prolactin on plasma osmolarity and plasma sodium in Tilapia mossambica. Gen. Camp. Endocrinol. 14, 256-269.
HYPOPHYSECTOMY Donaldson, E. M., and McBride, J. R. (1967). The effects of hypophysectomy in the rainbow trout Sdn70 gairdnerii (Rich.) with special reference to the pituitary-interrenal axis. Gen. Camp. En&crinol.
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Dryer, R. L.. Tammes, A. R., and Routh, J. I. (1957). The determination of phosphorus and phosphatase with N-phenyl-p-phenylenediamine. J. Biol.
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Komourdjian, M. P., Burton, M. P., and Idler, D. R. (1978). Growth of rainbow trout, Salnto gairdneri, after hypophysectomy and somatotropin therapy. Gen. Camp. EFzdocrinol. 34, 158-162. Lebmann, E. L. ( 1975). “Nonparametrics: Statistical Methods Based on Ranks,” pp. 132-141. Holder-i-Day, San Francisco. Macfarlane. N. A. A. (1974). Effects of hypophysectomy on osmoregulation in the euryhaline flounder, Platichrhvs flesus (L.), in sea water and in fresh waler. Comp. Biochem. Physiol. 47A, 201-217.
Oduleye, S. 0. (1975). The effect of hypophysectomy and prolactin therapy on water balance of the brown
trout
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357-366. Oduleye, S. 0. (1976). The effects of hypophysectomy. prolactin therapy and environmental calcium on freshwater survival and salinity tolerance in the brown trout Salmo trutta L. .I. Fish Biol. 9, 463 -470. Olivereau, M., and Lemoine, A.-M. (1973). Action de
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Pang, P. K. T., Schreibman, M. P., and Griffith. R. W. (1973). Pituitary regulation of serum calcium levels in the killifish, F~cn&!las heterocli?ris L. Gen.
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Siegel, S. (1956). “Nonparametric Statistics for the Behavioral Sciences,” pp. 116- 137. McGraw-Hill Kogakusha, Tokyo. Srivastava, A. K., and Pickford, G. E. (1972). Effects of hypophysectomy on the blood serum of male killifish, F~ndulus heteociitus, in salt water. C~Z. Camp. Endocrinol. 19, 290-303. IJtida, S., Hatai. S., Hirano, T., and Kamemoto, F. 1. (1971). Effect of prolactin on survival and plasma sodium levels in hypophysectomized medaka Ory:ius
iatiprs.
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566-573. Wendelaar Bonga, S. E. (1980). Effect of synthetic salmon calcitonin and low ambient calcium on plasma calcium. ultimobranchial cells. Stannius bodies, and prollactin cells in the teleost Gnsterosteus
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9% 108. Wendelaar Bonga, S. E. (1981). Effect of synthetis salmon calcitonin on protein-bound and free plasma calcium in the teleost Gasfrrosieus acuieatus.
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Wendelaar Bonga, S. E., and Greven, J. A. A. (i978). The relationship between prolactin cell activity, environmental calcium, and plasma calcium in the teleost Gasterosteus aculeatus. Gbservations on stanniectomized 6sh. Gen. C’onry. Eudocrinol. 36, 90- 101. Yamazaki, F. (1976). Application of hormones in fisn cuirure. j. Fish. Res. Bd. Canad. 33, 948-958.
AND
Effects
COMPARATIVE
49, 240-247 (1983)
ENDOCRINOLOGY
of Hypophysectomy on the Plasma Ionic and Osmotic Balance in Rainbow Trout, Salmo gairdneri BJ~~RN TH. BJ~RNSSON AND TIIU HANSON
Department
of Zoophysiology,
University
of GBteborg,
Box 250 59, S-400 31 Gothenburg,
Sweden
Accepted March 18, 1982 The effects of hypophysectomy on the plasma ionic and osmotic balance in juvenile rainbow trout (S&IO gairdneri) kept in 113 sea water for various periods were studied. Hypophysectomy caused a decrease in the plasma levels of ionized calcium and chloride within a week after the operation. At 2-5 weeks after hypophysectomy, the plasma levels of ionized calcium and chloride were still significantly lower in hypophysectomized fish when compared to sham-operated controls. Hypophysectomy had no effects on the plasma levels of sodium, total calcium, total magnesium, inorganic phosphate, plasma proteins, or plasma osmolality. When 113 sea water adapted (l-4 weeks), hypophysectomized and sham-operated fish were exposed to fresh water for 2 weeks, the plasma levels of ionized calcium, chloride, sodium, and plasma osmolality were significantly lower in hypophysectomized fish when compared to sham-operated fish, while the plasma levels of total calcium, total magnesium, and proteins were higher. No effects were noted on the plasma levels of potassium and inorganic phosphate. The presented data indicate that the pituitary gland plays a significant role in the regulation of plasma calcium and chloride levels in ,S. gairdneri, while its role in the regulation of plasma magnesium and sodium is less clear. The pituitary gland seems to play a minimal role in the plasma regulation of inorganic phosphate and potassium.
Fontaine et al. (1949) were the first to suggest an osmoregulatory role for the teleostean pituitary gland, and to test this hypothesis with the aid of hypophysectomy. Ensuing studies performed on several teleost species, both euryhaline and freshwater (FW) living, have shown that hypophysectomized fish in FW suffer a loss of sodium and chloride from the plasma. These ion changes and the related fall in plasma osmolality are considered to be the main cause of death of hypophysectomized fish in FW (for ref., see Macfarlane, 1974). The length of survival time in FW after hypophysectomy has been suggested to be related to the relative rate of ion loss from plasma as well as the tolerance of the given species to lowered plasma osmolality, sodium and chloride levels (Dharmamba, 1970; Macfarlane, 1974). The limited survival time in FW following hypophysectomy may be increased considerably by placing the fish in a near-isoosmotic envi-
ronment, such as physiological saline or l/3 sea water (SW) (Chidambaram et al., 1972; Oduleye, 1975). It is well established that hypophysectomy does not only affect the sodium/ chloride based osmotic balance of the plasma, but also the levels of the multivalent ions, especially that of calcium in a number of teleost species studied so far (Pang et al., 1973; Chan and Woo, 1978). Owing to the relative inaccessibility of the pituitary gland in salmonid fish, few studies involving hypophysectomy have been carried out on species of this family of teleosts (Donaldson and McBride, 1967; Oduleye, 1975, 1976; Yamazaki, 1976; Komourdjian and Idler, 1977; Komourdjian et al., 1978). However, the introduction of a new technique for hypophysectomizing Salmo gaivdneri (Komourdjian and Idler, 1977), has now made hypophysectomy a practical endocrinological research tool also in salmonid fish. To investigate the effects of hy-
240 0016-6480/83/020240-08$01.50/O Copyright All rights
0 1983 by Academic Press, Inc. of reproduction in any form reserved.
HYPOPHYSECTOMY
pophysectomy on the plasma ionic and osmotic balance in S. gairdnevi a number of experiments, both short-term and longterm, have been carried out. TERI
AND
METHODS
Fish. Juvenile S. gairdireri (40- 100 g bw) were obtained from a local hatchery near Gothenburg (September 1979%March 1980). The fish were kept without food in tanks holding approximately 300 liters of tiltered, recirculating, and aerated FW at a temperature of 10 to 12”, and a photoperiod of 12L:12D. Under these conditions the fish were acclimatized for at least ! week prior to the experiments. Hypoplzysectomy. The fish were hypophysectomized and sham-operated by the buccal approach of Komourdjian and Idler (1977) at different times of the year. The fish were anaesthestized in 2 mgiliter aqueof propoxate (Janssen Pharmaceutics, ium) until all but shallow respiration movements ceased, and this state was kept during the surgery by an aqueous solution (113 SW) of 20 mg tricaine methanesulfonate (MS 222)/1iter, which was pumped over the gills. During recovery from anaesthesia, the fish were kept under observation in a small basin with noncirculating but aerated 113 SW, before being returned to holding tanks, containing noncirculating but aerated 113 SW, at a temperature of 10 to 12” and a photoperiod of 12L:42D. This water was partially changed every 3 - 4 days Expcrimerztal design. IGperiment 1: To examine the short-term effects of hypophysectomy on the plasma ionic and osmotic balance, hypophysectomized and sham-operated tish were exposed to 113 SW for 1,3, or 7 days. Experinzenr 2: To examine the long-term effects of hypophysectomy, hypophysectomized and shamoperated fish were exposed to 113 SW for 2-5 weeks. &perinzent 3: Preliminary experiments showed that when hypophysectomized fish were exposed to FW direct!y after the operation the mortality was high. However. when the fish were acclimatized to 113 SW for at least 1 week after the operation, and then exposed to FW the hypophysectomized fish survived for at least 5 weeks (Bjornsson et al., 1981). Therefore, to examine the long-term effects of hypophysectomy in FW, hypophysectomized and sham-operated fish were acciimatized to 113 SW for at least 1 week after the operation. and then exposed to FW for 2 weeks. Due to the difficult nature of the operation, the period to obtain sufficient number of operated fish for the various experiments varied. Thus, the postoperative period for some of the experimental groups is given as 2-4 weeks. Blood samplbzg md plasma analysis. At the termination of an experiment, the fish was stunned by a
OF
RAINBOW
TROUT
241
blow on the head, and 0.5% 1.0 ml of blood was drawn from the caudal vessels using a l-ml syringe with a 0.4 x Z-mm needle. Plasma was obtained by centr&tgation, and stored at -20” until analysis. After the fish had been sacrificed, the completeness of the operation was accertained by visual inspec.:ior: under fight microscope. Supposedly hypophyseceomized fish, which proved to have pituitary remnants were excluded from the experiment, as were shamoperated fish with damaged pituitary glands. Plasma sodium and potassium concentrations were determined by flame emission spectroscopy (Turner Model 510 spectrometer) using an internal lithium standard. and plasma chloride concentration was determined by amperometric titration (Radiometer CM 10 chloride titrator). Plasma calcium ion concentration was determined using ion-exchanger membrane eiectrodes (Radiometer F2112Ca selectrodes), while total plasma calcium and magnesium concentrations were determined by atomic absorption spectroscopy (Perkin-Elmer 303 atomic absorption spectrophotometer). Plasma osmolaiity was determined by vapour pressure osmometry (Wescor 5100 B vapour pressarre osmometer). Inorganic phosphate and protein concentrations in plasma were measured by the analytical procedures of Dryer ef ai. (i9S7) and Henry et al. (1957). respectively, using a Carl Zeiss speetrophotometer. Minteramd~sis. The FW and SW used in this study was analyzed using the same analytical procedures as described for plasma analysis (FW: Chloride, 1.2 ,&VI: sodium. 2.2 m.M; potassium, 0.2 mM, total caicimm, 0.5 m&f: tot21 magnesium, 0.5 phate,
i-7-S
are shown in Table 1. fooilowing ~y~o~~ysectomy, nificant
decrease
there is a sig-
in the plasma
level
of
6
5.3 +0.4 0.285
0.529
4.2 eo.3
3.9 kO.5
4.3 +0.6
3.8 +0.3
4.2 20.5
K+ (mM)
157.7 k1.9
161.8 k2.0
155.0 k4.8
152.0 t2.6
160.3 22.4
158.8” -+1.7
Na+ @Ml
TABLE
1
0.660
2.35 io.13
2.36 -to.07
2.26 kO.05
2.29 kO.20
2.16 iO.08
2.33 -co.07
CaTO (mM)
CONSTITUENTS
0.001*
0.78 i-O.08
1.10 kO.07
0.82 kO.10
0.96 20.05
0.56 kO.07
0.85 to.03
CaZ+ (mM)
IN RAINBOW
0.165
1.67 kO.09
1.56 kO.08
1.65 kO.09
1.54 10.11
1.54 io.07
1.46 kO.02
Mg,” bM)
TROUT
0.575
2.31 20.18
2.24 +0.22
1.99 -co.10
2.29 20.14
2.54 io.09
2.24 to.10
(mM)
Pi”
KEPT
IN
0.660
2.84 20.20
3.26 -co.43
4.00 20.35
2.66 20.17
1.90 ‘-co.30
3.18 +0.37
Protein (g%‘o)
l/3 SW
sham-operated controls, collectively tested (P < 0.017).
0.0143
133.3 k1.5
143.2 -r-1.3
137.5 21.5
140.0 kO.9
141.5 kl.2
140.5 kO.7
Cl@Ml
ON PLASMA
u Number of animals in each group. b Total calcium. c Total magnesium. d Inorganic phosphate. e All data are expressed as means i SE. * Hypophysectomized groups statistically different from corresponding
P=
Hypophysectomized
6
Sham-operated
7
4
Hypophysectomized
4
3
Sham-operated
4
4
1
n”
OF HYPOPHYSECTOMY
Hypophysectomized
Sham-operated
Treatment
EFFECTS
Time after treatment (days)
SHORT-TERM
0.078
301.9 21.8
311.3 k2.7
300.9 t3.0
301.4 11.3
305.6 k5.7
302.9 22.5
Osmolality (mOsm/kg)
HYPOPHYSECTOMY
ionized calcium (P = 0.001) and chloride (P = 0.014). Hypophysectomy had no effects on the plasma levels of sodium, potassium, total calcium, total magnesium, inorganic phosphate, proteins, or plasma osmolality (P b 0.017). Long-term ef,ects of hypophysectomy. The long-term effects of hypophysectomy on fish kept in 113 SW after the operation are shown in Table 2. At 2-5 weeks after ophysectomy, there is a marked decrease in plasma levels of ionized calcium (P = 0.0001) and chloride (Z’ = 0.003), while no effects are seen on plasma levels of sodium, potassium, total calcium, total magnesium, inorganic phosphate, proteins, or plasma osmolahty (P 2 0.017). The long-term effects of hypophysectomy on fish kept in 113 SW (1-4 weeks) r the operation, and then exposed to for 2 weeks are shown in Table 3. HI3 SW adapted (l-4 weeks) hysectomized and sham-operated fish were exposed to FW for 2 weeks, there is a marked decrease in plasma levels of ionized calcium (P < O.O0006), chloride (P < lasma osmolality (P < ition, there is a decrease in Bevel (P = 0.009). On the other hand, there is a marked increase in total plasma levels of calcium (P < 006), magnesium (P < 0.00006), and proteins (P = 0.0001). There is no effect on plasma levels of potassium or inorganic phosphate (P 2 0.017).
The presented data suggest that the pituitary gland may piay a significant role in the calcium regulation in S. gairdneri. Even when byp~physectomized fish are kept in hypercalcemic environment (113 SW), ionized plasma calcium level shows a rapid (within 1 week) and long-lasting (2-5 weeks) decrease. This effect, which is augmented when the fish is exposed to a hyp~ca~cem~c environment (FW), is not reflected in changes in total plasma calcium
OF
RAINBOW’
TROUT
is observation is the currently held opinion bound and ionized calcium a simple equilibrium (se contrast to total plas studies have been carri
243
not in line with that proteinin plasma are in calcium, few out on ion,ized
calcium level that were not acc~~~a~~ed by changes in total plasma calcium in intact Gasterosteus ncdeatus. These findings and the results presented here demonstrate clearly the necessity of of free or ionized plasm the total plasma level o studying calcium regu%
to plasma proteins, as taneous increase in to plasma protein level. The results present
in fish. It remains to be inther the observed Increase in ca~c~~rn~b~~d~~g is nonspecific or due to specific ca~c~~rn-b~~d~~g proteins I The appearance of specific ca~c~~~-b~~~i~~ peplasma level fo 1978). The nature of the hypercalce the pituitary gland of teleost fis be elucidated. There is growi t this factor may be ~~~~act~~
Recently
it has been shown
was suggested that the failure to demonstrate hypocalcemic ffect of exogenous calcitonin in intact fis might be due to an
” Number of animals in each group. * Total calcium. c Total magnesium. d Inorganic phosphate. e All data are expressed as means ? SE. * Hypophysectomized groups statistically
P=
Hypophysectomized
different
6
9
5
Sham-operated
8
18
9
7
5
2-4
from
0.022
152.3 k1.1
155.3 kO.8
150.9 k1.5
153.5 21.4
142.1 kO.9
143.4” %l.O
Na+ (n-M
K+
0.070
2.3 -co.2
2.4 kO.2
3.3 20.1
4.0 kO.3
3.1 LO.2
3.5 20.3
(mM)
0.097
2.24 kO.08
2.37 kO.08
2.59 kO.07
2.78 20.05
2.16 20.10
2.22 kO.10
0.0001*
1.46 kO.05
1.54 r0.02
0.76 20.07
0.93 to.04
1.27 to.06
1.41 LO.10
CaZ+ (mM)
collectively
0.059
0.83 kO.09
0.83 kO.04
0.85 -co.03
0.89 kO.02
0.83 20.02
0.047
2.13 20.14
2.36 io.10
0.072
2.08 kO.20
2.37 co.10
2.53 kO.15
3.16 kO.13
4.58 kO.39
4.13 io.ll
k%)
Protein
IN l/3 SW
(P < 0.017).
1.86 kO.05
2.39 kO.14
2.50 rO.ll
2.47 kO.08
(mM)
Pid
KEPT
tested
TROUT
0.77 r0.02
IN RAINBOW
controls,
2 CONSTITUENTS
sham-operated
0.003*
136.3 %1.2
137.1 kO.5
129.8 -cl.8
134.5 20.8
134.1 t1.1
138.6 21.4
(mM)
Cl-
TABLE ON PLASMA
corresponding
OF HYPOPHYSECTOMY
Hypophysectomized
Sham-operated
Hypophysectomized
Sham-operated
Treatment
EFFECTS
Time after treatment (weeks)
LONG-TERM
0.019
304.6 k2.6
302.6 a1.3
294.1 k1.6
302.3 21.9
306.6 %2.0
319.6 k5.4
Osmolality (mOsm/kg)
EFFECIS
Time after treatment
HYPOPHYSECI‘OMY
2-4
113 SW
FW
weeks
+ 2 weeks
1 week 113 SW + 2 weeks FW
or
” Number of animals in each group. ’ Total calcium. e ‘F&al magnesium. ri Inorganic phosphate. c All data are expressed as means r SE. * Hypophysectomized groups statistically
P= P<
Hypophysectomized
Sham-operated
Hypophysectomized
Sham-operated
Treatment
LONG-TERM
different
-
16
from
corresponding
0.066
0.009”
co.17
FO.04 3.30
2.50
2.81 LO.07
2.57 to.07
-to.06
collectively
tested
O.OOOQ6* ~.___
+0.02
0.63 -to.01 0.79
0.76 kO.02
0.69 to.02
(mM)
Mg.,”
Pi”
AND
3.86
2.47
0.0001”
-co.17
(P < 0.017).
0.039
CO.09
kO.16 4.50
7.43 kO.85
1.92 -co.05 kO.08 2.36
4.46 kO.18
k%)
Protein
THEN
2.07 20.05
(mM)
IN 113 SW
1.48
KEPT
io.04 0.92
0.76 to.04
0.97 kO.02
(mM)
Ca?+
FIRSI
o.ooo&?”
controls.
CmM)
CaTD
TROUT
0.00006* -.-
sham-operated
0.00006”:
k2.7
io.4
al.1
134.9
5.0 kO.7 101.0
114.8 +2.5
3.3 kO.3 to.3 4.3
131.9 +0.9
3.6 50.4
(mM)
21.3 144.6
147.1
145.7 +1.4
19 19
149.5’ +0.8
K+
CmM)
Nat
3 IN RAINBOW
Cl-
TABLE CONS-SIYUENTS
(mA4)
11
II [’
ON PLASMA
Exposure
0.00006”
il.8
52.0 293.0
302.1
289.9 k1.7
300.6 t2.0
Osmolality (mOsm/kg)
1.0 FW
-
2
2 0
z? e
%
2 4
::
246
BJijRNSSON
AND HANSSON
increased prolactin secretion, which will obscure the calcitonin effect. Therefore, it may be suggested that hypophysectomy unmasks the effect of calcitonin and/or other endogenous hypocalcemic principles, thus resulting in the hypocalcemia observed. However, the presence of hypercalcemic principles other than prolactin in the teleost pituitary gland cannot be excluded. Thus, Parsons et al. (1978) have described the existence of a hypercalcemic principle (“hypercalcin”) related to parathyroid hormone, in the pituitary gland of Gadus rnorhua and Anguilla anguilla. Although the plasma level of ionized magnesium was not measured in the present study, the increase in total plasma magnesium in hypophysectomized fish exposed to FW suggests that the regulation of plasma magnesium and calcium may be similar. These results are in accordance with observations on hypophysectomized Fundulus heteroclitus (Srivastava and Pickford, 1972) and A. japonica (Chan and Woo, 1978), which show that hypophysectomy affects the total plasma levels of calcium and magnesium in a similar way. Prolactin is considered to be necessary for the survival of many teleost species in FW (Utida et al., 1971) and is capable of elevating plasma chloride levels of intact as well as hypophysectomized fish (Olivereau and Lemoine, 1973; Gallis et al., 1979). Our results indicate that the pituitary gland may play a significant role in the plasma chloride regulation in S. gairdneri. However, it remains to be investigated whether the pituitary gland factor responsible for the maintenance of normal plasma chloride levels in S. gairdneri is prolactin. The slight effect of hypophysectomy on plasma sodium levels in S. gairdneri observed in this study confirms the findings by Komourdjian and Idler (1977). Thus, the pituitary gland seems to play a less important role in the plasma sodium regulation in this species than it does in most other teleost species studied so far.
It is noteworthy that the decrease in plasma osmolality in hypophysectomized fish exposed to FW is less than expected from the changes in measured ion levels. Furthermore, the concomitant increase in plasma protein levels seems insufficient to account for this discrepancy. However, other organic plasma constituents may play a role in the osmoregulatory process in S. gairdneri, as has been demonstrated for F. heteroclitus (Pickford et al., 1969). In conclusion, the presented data indicate that the pituitary gland plays a significant role in the regulation of plasma calcium and chloride levels in S. gairdneri, while its role in the regulation of plasma magnesium and sodium is less clear. The pituitary gland seems to play a minimal role in the plasma regulation of inorganic phosphate and potassium in S. gairdneri. ACKNOWLEDGMENTS This work was supported by grants from the Swedish Natural Science Research Council, the Magnus Bergwall Foundation, and the Adlerbertska Research Foundation.
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japonica.
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Endocrinol.
35,
169- 178. Chidambaram, S., Meyer, R. K., and Hasler, A. D. (1972). Effects of hypophysectomy, pituitary autografts, prolactin, temperature and salinity of the medium on survival and natremia in the bullhead. Ictalurus melas. Comp. Biochem. Physiol. 43A, 443-457. Dacke, C. G. (1979). “Calcium Regulation in SubMammalian Vertebrates,” p. 11. Academic Press, London/New York. Dharmamba, M. (1970). Studies of the effects of hypophysectomy and prolactin on plasma osmolarity and plasma sodium in Tilapia mossambica. Gen. Camp. Endocrinol. 14, 256-269.
HYPOPHYSECTOMY Donaldson, E. M., and McBride, J. R. (1967). The effects of hypophysectomy in the rainbow trout Sdn70 gairdnerii (Rich.) with special reference to the pituitary-interrenal axis. Gen. Camp. En&crinol.
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Dryer, R. L.. Tammes, A. R., and Routh, J. I. (1957). The determination of phosphorus and phosphatase with N-phenyl-p-phenylenediamine. J. Biol.
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Komourdjian, M. P., Burton, M. P., and Idler, D. R. (1978). Growth of rainbow trout, Salnto gairdneri, after hypophysectomy and somatotropin therapy. Gen. Camp. EFzdocrinol. 34, 158-162. Lebmann, E. L. ( 1975). “Nonparametrics: Statistical Methods Based on Ranks,” pp. 132-141. Holder-i-Day, San Francisco. Macfarlane. N. A. A. (1974). Effects of hypophysectomy on osmoregulation in the euryhaline flounder, Platichrhvs flesus (L.), in sea water and in fresh waler. Comp. Biochem. Physiol. 47A, 201-217.
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SaEmo
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‘247
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Pang, P. K. T., Schreibman, M. P., and Griffith. R. W. (1973). Pituitary regulation of serum calcium levels in the killifish, F~cn&!las heterocli?ris L. Gen.
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Siegel, S. (1956). “Nonparametric Statistics for the Behavioral Sciences,” pp. 116- 137. McGraw-Hill Kogakusha, Tokyo. Srivastava, A. K., and Pickford, G. E. (1972). Effects of hypophysectomy on the blood serum of male killifish, F~ndulus heteociitus, in salt water. C~Z. Camp. Endocrinol. 19, 290-303. IJtida, S., Hatai. S., Hirano, T., and Kamemoto, F. 1. (1971). Effect of prolactin on survival and plasma sodium levels in hypophysectomized medaka Ory:ius
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566-573. Wendelaar Bonga, S. E. (1980). Effect of synthetic salmon calcitonin and low ambient calcium on plasma calcium. ultimobranchial cells. Stannius bodies, and prollactin cells in the teleost Gnsterosteus
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Wendelaar Bonga, S. E., and Greven, J. A. A. (i978). The relationship between prolactin cell activity, environmental calcium, and plasma calcium in the teleost Gasterosteus aculeatus. Gbservations on stanniectomized 6sh. Gen. C’onry. Eudocrinol. 36, 90- 101. Yamazaki, F. (1976). Application of hormones in fisn cuirure. j. Fish. Res. Bd. Canad. 33, 948-958.