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Neurosecretory changes in the hypothalamico-hypophysial system of the rainbow trout (Salmo gairdneri) Carlson, Ian Hedman 1961

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NEUROSECRETORY CHANGES IN THE HYPOTHALAMICO-HYPOPHYSIAL SYSTEM OF THE RAINBOW TROUT (Salmo gairdneri) by IAN HEDMAN CARLSON B. A., The Un i v e r s i t y of B r i t i s h Columbia, 1956 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of ZOOLOGY We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1961 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s m a y b e g r a n t e d b y t h e H e a d o f my D e p a r t m e n t o r b y h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t b e a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f Zoology  T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , V a n c o u v e r &, C a n a d a . D a t e A p r i l 28, 1961 1 A B S T R A C T Variations i n concentration, i f any, of pharmacologically ac t i v e p r i n c i p l e s of the hypothalamico-neurohypophysial system of the rainbow trout Salmo qairdneri during e a r l y periods of transf e r to sea water were investigated. The concentrations of oxytocic and a n t i d i u r e t i c p r i n c i p l e s i n the brains and p i t u i t a r i e s of handled f i s h , f i s h transferred to sea water, and f i s h transferred to fresh water, were measured employing the i s o l a t e d v i r g i n guinea p i g uterus for assaying oxytocic a c t i v i t y , and the ethanol anaesthetized water loaded ra t for assaying a n t i d i u r e t i c a c t i v i t y . Handling the f i s h resulted i n an increase of oxytocic and a n t i d i u r e t i c a c t i v i t y of p i t u i t a r y e x t racts. Transfer of experimental f i s h to a sea water environment re s u l t e d i n a t r a n s i t o r y increase of oxytocic a c t i v i t y of extracts of the p i t u i t a r y and hypothalamus for the f i r s t and second hours with a subsequent return to control l e v e l s . After transfer of the f i s h to a sea water environment the a n t i d i u r e t i c a c t i v i t y of p i t u i t a r y extracts was observed to decrease during the f i r s t and t h i r d hour, with a return to control l e v e l s at the s i x t h hour. This evidence suggests that active p r i n c i p l e s which are known to play active r o l e s i n water balance i n animals higher, phylogenetic-a l l y , than f i s h , are l i b e r a t e d from the hypothalamico-neurohypophysial system of Salmo qairdneri i n response to a hypertonic environment. J. A T A B L E O F C O N T E N T S Page INTRODUCTION 1 MATERIALS AND METHODS V Experimental Conditions 7 Treatment of Tissues 8 Oxytocic Assay 9 A n t i d i u r e t i c Assay 10 RESULTS (Tables: and Figures) 13 TABLE I 13 TABLE II 14 TABLE III 15 TABLE IV ' 16 TABLE V 17 FIGURE 1 18 FIGURE 2 19 FIGURE 3a 20 FIGURE 3b 21 FIGURE 4 22 FIGURE 5 23 FIGURE 6 24 FIGURE 7 25 FIGURE 8 26 RESULTS (Summary) 27 1. Dose Response Relationships I l l (Results Cont'd) Page A. Oxytocic A c t i v i t y 27 B. A n t i d i u r e t i c A c t i v i t y 27 I I . E f f e c t s of Handling 28 I I I . Adaptations to Sea Water 28 A. Oxytocic A c t i v i t y 28 1. P i t u i t a r y 28 2. Brain 28 B. A n t i d i u r e t i c A c t i v i t y 29 1. P i t u i t a r y 29 2. Brain 29 DISCUSSION 30 I. Phylogenetic Relationships of Neurohypophysial Peptides. . 30 A. Chemical Relationships 30 B. Functional Relationships 31 I I . Transfer to Sea Water 32 A. Oxytocic Assays of Extracts 32 B. A n t i d i u r e t i c Assays of Extracts 33 C. Integrated Considerations . . . . . 34 CONCLUSIONS 37 BIBLIOGRAPHY 38 A C K N O W L E D G M E N T I wish to extend to Dr. W. N. Holmes my appreciation of h i s d i r e c t i o n throughout the i n v e s t i g a t i o n and preparation embodied i n t h i s t hesis and for the f i n a n c i a l assistance made possible through a grant to him i n aid of research from the Atomic Energy Commission. I also wish to thank Dr. W. S. Hoarcand Dr. P. A. Larkin for t h e i r suggestions throughout the preparation of t h i s t h e s i s . Further, I wish to extend my gratitude to the F i s h e r i e s Association of B r i t i s h Columbia for the scholarship they awarded to me during the academic year 1960-61. I N T R O D U C T I O N Evidence concerning the presence of active substances i n the neural lobe of mammalian p i t u i t a r i e s has existed f or many years. The work of O l i v e r and Schafer (1895) indicated that intravenous i n j e c t i o n of whole p i t u i t a r y extracts into mammals resul t e d i n a prolonged and intense vasoconstriction. Howell (1898) and Dale (1906) showed that t h i s "pressor" response was associated with the p o s t e r i o r or neural lobe of the p i t u i t a r y gland. However, at the same time Dale also observed that the uterus of the pregnant cat e l i c i t e d powerful contractions i n response to t h i s p o s t e r i o r lobe ex t r a c t . Furthermore milk e j e c t i o n from the mammary glands of l a c t a t i n g cats was observed by Ott and Scott (1910) i n response to i n j e c t e d p o s t e r i o r p i t u i t a r y e x t racts. Another property of p o s t e r i o r p i t u i t a r y extracts was observed by von den Velden (1913) and Formi (1913) while attempting to f i n d some therapeutic value of p o s t e r i o r lobe extracts i n cases of diabetes i n s i p i d u s . These observers found that p o s t e r i o r lobe preparations decreased the urine output i n cases of severe d i u r e s i s . Investigations by Brunn (1921) uncovered yet another a c t i v i t y of the extracts of the mammalian po s t e r i o r p i t u i t a r y , namely, a promotion of water uptake i n amphibia. Further i n v e s t i g a t i o n s by H e l l e r (1941) indicated that differences exist in the amphibian response to preparations from species other than mammalian species. This suggested the possible presence of several hormones in the animal kingdom causing similar responses. Also, Hogben and de Beer (1925) indicated that in several species including fi s h , there was an active principle in the posterior pituitary that e l i c i t e d a f a l l in blood pressure in avian systems. With these i n i t i a l investigations laying the pattern of ac t i v i t i e s of posterior lobe extracts other workers developed methods of separating the active components found in the posterior pituitary. Kamm (1928) developed a means of separating these fractions. As a result of this ad-vance oxytocin and vasopressin became commercially available. It was now possible to categorize the observed responses with respect to the hormone responsible. Oxytocin was observed to be responsible for uterine contraction and milk ejection in mammals, blood pressure depression in birds, and water uptake in amphibia, while vasopressin was observed to be responsible for vasoconstriction and the subsequent rise in blood pressure in birds and mammals as well as promoting renal tubular water reabsorption i n birds and mammals. In addition, vasopressin was shown to stimulate in vitro uterine contraction in mammalian systems i f magnesium ions were present in the supporting medium. Methods of bio-assay have been developed to exploit these pharma-cological properties of vasopressin and oxytocin. Assays for oxytocic activity have been described by Dale and Laidlaw (1912), Kochman (1921), Burn and Dale (1922), Burn (1937), and Fraser (1939) employing the isolated guinea pig uterus. Houlton (1948) also described a similar assay using the rat uterus. Methods employing the f a l l in avian blood pressure, c5 as a measure of oxytocic activity, have been described by Paton and Watson (1912), Gaddum (1928), Coon (1939), Smith (1942), Smith and Vos (1943), Thompson (1944). An in vitro assay employing the isolated frog bladder has been described by Sawyer (i960) who also mentioned a further modification of the rat uterus technique employing the hen oviduct. A seldom used assay method, that of milk ejection, has been described by Cross and Harris (1952) and by van Dyke, Adamsons and Engle (1955). Assays for vasopressin involve two responses, namely, the elevation of blood pressure in mammals and the reduction of urine flow in hydrated animals. Pressor assays have been described by Dale and Laidlaw (1912), Hogben (1924), Kamm (1928), and Stewart (1949), employing dogs and other methods employing rats have been described by Landgrebe et al (1946). Antidiuretic assays using mice were described by Gibbs (1930) and Burn (1931) modified these techniques for use in rats. Further modi-fications of this method were described by Gilman and Goodman (1937), Ham and Landis (1942), and Jeffers, Livezy and Austin (1942), Sawyer (1958), and Ingraham (1959). Lauber, Kautz, Eversole (1959) describe a very sensitive method for vasopressin assay employing the toad Bufo marinus. Many of the current bio-assay methods are sensitive and reliable but are restrictive according to the amount of active material present in the extract to be assayed. In the case of small quantities of oxytocin the guinea pig or rat uterus have been found to be suitable. While for small quantities of vasopressin the rat antidiuretic method of Ingraham and Baratz (1959) or the water uptake assay employing toads (Lauber et a l , 1959) are most useful. Another method for determining the presence of neurohypophysial hormones involves histochemical techniques. The use of Gomori*s staining techniques has been reviewed by Scharrer (1945). Other techniques have been described by Scharrer (1954 a and b), Bargman (1949), Halmi (1952), Dawson (1953), and Gabe (1953). In general, the histochemical methods described are useful for determining the presence of most neurosecretory cells and their contents. The investigations by Herring (1913) on the elasmobranch and teleost neurohypophysial systems indicated the presence of principles that evoked pressor, oxytocic, and milk ejection responses in mammalian subjects. These observations were later substantiated by Hogben and de Beer (1925). Boyd and Dingwall (1939) demonstrated the presence of the frog water balance factor in the neurohypophysial system of the cod. Heller (1941) substantiated this investigation by showing the presence of a frog water balance factor in the neurointermediate lobe of the skate. Also, Heller indicated the presence of an antidiuretic principle in the neurohypophysial system of both the skate and the cod. Later investigations by Sawyer, Munsick, and van Dyke (1961) and Heller and Pickering (1961) indicated beyond any doubt the presence of principles from elasmobranchs and teleosts that e l i c i t e d oxytocic and antidiuretic responses in mammalian systems. Furthermore, comparison with the pharmacological responses of prepared polypeptides by Du Vigneaud (1952 and 1958) indicated that active principles existed in the hypothalami-neurohypophysial systems of elasmobranchs and teleost fish similar to oxytocin and vasopressin. However, chromatographic evidence for the existence of vasopressin in extracts from the neurohypophysial system of elasmobranchs and teleosts i s lacking (Heller, 1961). Heller and Pickering (1961), and Sawyer, Munsick, and 5 van Dyke (1961) have indicated pharmacologically and chromatographically the presence of oxytocin and a substance s i m i l a r to vasotocin, the s y n t h e t i c a l l y prepared polypeptide of Du Vigneaud (1958). As well, i n v e s t i g a t i o n s by Sawyer, Munsick, and van Dyke (i960 and 1961) indicated that vasotocin i s present i n f i s h , amphibians, and b i r d s . E f f o r t s to demonstrate the p h y s i o l o g i c a l a c t i v i t y of vasopressin and oxytocin i n f i s h have met with l i t t l e success. There have been no successful i n v e s t i g a t i o n s showing an a n t i d i u r e t i c response to neuro-hypophysial hormones i n f i s h . Sawyer (1933) and Dreyer (1946), have shown increased tonus and rythm of smooth muscle from the stomach and the i n t e s t i n e of f i s h exposed to a medium containing p o s t e r i o r lobe p r i n c i p l e s . Bacon (1951 and 1952) and Wilhelmi, Pickford and Sawyer (1955) have ind i c a t e d that i n j e c t i o n s of oxytocin and/or po s t e r i o r lobe preparations caused e i t h e r maturation i n pre-spawning Catostomus commersoni, Cyprinus  carpio. and Semotilus atromaculatus of the induction of the spawning r e f l e x i n Fundulus h e t e r o c l i t u s . Further work by Holmes (1959) has indicated that oxytocin reduces the r e s p i r a t i o n rate of kidney ti s s u e i n t r o u t . Investigations which attempted to associate the quantity of neurosecretory material present i n the neurohypophysial system with studies of hydration and dehydration have proved to be more succe s s f u l . Studies by Arvy, Fontaine and Gabe (1954) on Phoxinus phoxinus. Phoxinus l a e v i s . and Anguilla a n q u i l l a have indicated that immersion of these f i s h i n strongly hypertonic environments r e s u l t e d i n a depletion of Gamori p o s i t i v e p i t u i t a r y neurosecretory m a t e r i a l . Further i n v e s t i g a t i o n s by Arvy and Gabe (1954) on Callionymus l y r a and Ammodytes lancepJatus have indicated that immersion i n hypertonic sea water caused a depletion of neurosecretory material in the nucleus preopticus and the neurohypophysis. Upon returning these fish to normal sea water the neurosecretion was almost replenished within one hour. In addition, i f these fish were placed in 25% sea water an accumulation of neurosecretory material was evidenced. If for the time being we discard the objections against correlating the Gomori positive neurosecretory material and pharmacologically active principles in the neurohypophysis we may have sufficient evidence for a liberation of these principles in response to environmental changes in tonicity. Houston (1958) and Vickers (i960) have indicated that upon transfer to sea water the serum electrolyte levels in Rainbow trout Salmo qairdneri rise markedly. If we consider the classical experiment of Verny (1947) whereby increased tonicity of the blood resulted in a release of the antidiuretic material we may have sufficient evidence to presume a similar releasing factor may exist in fish. Homer Smith (1953) postulated that an antidiuretic activity was associated with the low urine output of marine teleosts when compared to the high urine output of fresh water teleosts. Studies by McBean and Holmes (1961) have indicated that a markedly reduced inulin excretion is evidenced by trout adapted to sea water when compared to fresh water controls. Fontaine (1956) speculated that oxytocin may act upon the afferent arteriole and/or venule of the glomerulus causing vasoconstriction, and hence a reduced glomerular filtration. If we consider the evidence to date, concerning the roles of neurohypophysial hormones in teleosts during adaptation to a sea water environment, we find very little information that is truly enlightening. The present work is an attempt to quantify the changes if any, that occur in the concentrations of active neurohypophysial principles in the trout, Salmo qairdneri, during the early period of adaptation to a sea water environment. 7 M A T E R I A L S A N D M E T H O D S Experimental Conditions Hatchery raised trout, Salmo qairdneri. were used throughout these experiments. The fish were maintained in an outside holding pond supplied with running dechlorinated water at an average o temperature of 8 C. Experimental fish were removed from this holding pond to inside facilities which consisted of rectangular cement troughs supplied with running dechlorinated tap water of an average o temperature of 8 C. The plan of the experiment required a number of fish to be placed in 60% sea water for various time periods. Upon completion of exposure to the sea water environment the fish were removed, weighed, and decapitated. The pituitary and hypothalamic region of each brain was removed. Changes in concentration of hormonal material as a result of handling were investigated. Sixteen fish were removed from the outside holding pond. Six of these animals were weighed and decapitated, whilst five fish were held in the hatchery for one hour in fresh water before sacrifice, the other five being placed in 60% sea water for one hour. On the basis of this i n i t i a l experiment i t was decided that a system of paired experimental and control groups of fish be employed for each time period of exposure of the experimental fish to 60% sea water. A number of fish were removed from the outside holding pond and transferred to the inside holding f a c i l i t i e s and a period of twenty-four hours was allowed for acclimation of the fish to the new surroundings. At the end of this period five fish were removed and placed in 60% sea water, whilst five control fish from the same group were placed i n identical containers of fresh water. Thus for each experimental period of one hour, three hours, six hours, and twelve hours, five fish were placed in sea water and five fish were placed in fresh water for exactly the same time, hence serving as controls. At the end of the experimental period the fish were removed, sacrificed by a blow on the head, weighed and decapitated with a subsequent removal of pituitaries and brains. However, another series of experiments for confirmatory studies was conducted whereby ten fish were employed for experimental and control groups. The pituitaries were pooled for each experimental and control group of either five fish or ten f i s h . As well, the hypothalami were pooled for each group of five or ten fi s h . The extracts of pooled pituitaries and hypothalamo were assayed. Treatment of Tissues Pituitary and hypothalamic tissues were quickly removed from the decapitated fish and placed in acetone at -21°C. Upon extraction the acetone was drained from the test tubes and the tissues were dried by evaporation with a stream of compressed a i r . One ml. of 0.9% saline was added to the tissue to be extracted and this mixture was placed in a boiling water bath for five minutes. During this period of time the tissue was macerated with a glass st i r r i n g rod. After heating, the mixture was centrifuged at 3,000 r.p.m. and 10°C for ten minutes. The supernatant was drawn off and stored at -21°C until required for assay. Oxytocic assays were performed with the undiluted supernatant extract. Prior to the assay for antidiuretic activity 0.05.ml. of the supernatant was diluted to 5 ml. with 0.9% saline for pituitaries but not diluted for brain tissue extracts. Inactivation of neurohypophysial hormones was accomplished by mixing a 1:1 solution of supernatant and 0.05 M. sodium thioglycollate at pH7.4. The mixture was l e f t standing at room temperature for two hours prior to assay. Oxytocic Assay The assay for oxytocic activity employed the isolated diestrus guinea pig uterus suspended in Ringer*s solution as described by Emmens (1950). The composition of the Ringer's solution was as follows: NaCl 9.0 KC1 0.42 CaCl 2 0.12 MgCl2 0.0025 NaHC03 0.5 Dextrose 0.5 Virgin diestrus guinea pigs of 180-230 grams weight were employed throughout the experiment. The animals were sacrificed by a blow on the head and the uterus was removed and placed in a bath of Ringer's solution at room temperature. A l l fatty tissue was removed gm/l II n II II n from the uterine horns while in this bath. One horn of the prepared uterus was suspended in the assay bath at 28°C. and allowed to eq u i l i -brate in the oxygen gassed Ringer medium for at least one-half hour before the assay commenced. The assay program was similar to that of Houlton (1948). Standard preparations of Pitocin (Parke Davis) were used throughout the experiments. A stock standard solution of 10 mu. of oxytocic activity per 0.1 ml. was made and during the assays standards of different concentrations were made from this stock solution in order that a dose response curve could be obtained for each assay. The extracts of unknown concentration were assayed on single volumes of 0.05 ml. of the supernatant extract solution. The uterine responses for a l l standards and extracts were recorded. From the dose response curve, the concentration of active principles i n the unknown extracts was calculated and expressed in milliunits of oxytocic activity per pituitary or brain per 100 gm. body weight. An index of per cent of control activity was also calculated for each pair of experimentals and control f i s h . Antidiuretic Assay The method employed for the assay of antidiuretic activity in the extracts of the pituitaries and brains was a modification of that described by Baratz and Ingraham (1959). Female rats of 200 gm. body weight were obtained from the central animal depot several days prior to the assays. A l l rats were water loaded with 10 ml. of warm tap water the afternoon prior to assay. Food was withheld twenty-four hours before assay, although water was allowed ad libidum. On the morning of assay the rat to be used was anaesthetized, water loaded w i t h a volume of 10% ethanol water s o l u t i o n e q u i v a l e n t to 5% of the body weight. The r i g h t e x t e r n a l j u g u l a r v e i n was cannulated and a catheter was placed i n the bladder v i a the ure t h r a i n such a p o s i t i o n t h a t there would be a syphon e f f e c t . I f bleeding occurred as a r e s u l t of c a t h e t e r i z a t i o n a s o l u t i o n o f thrombin was f l u s h e d i n t o the bladder and allowed to d r a i n . No attempt was made to t i e o f f the dead space of the bladder. During the assay the r a t was r e s t r a i n e d on an e l e -vated t a b l e , warmed by a reading lamp. The u r i n e was c o l l e c t e d i n a 5 ml. graduated c y l i n d e r placed below the r a t . A f t e r surgery, the u r i n e flow u s u a l l y increased and during t h i s p e r i o d the s p e c i f i c g r a v i t y of the u r i n e was determined us i n g the method of Barbour and Hamilton (1926). Once a s t a t e of constant u r i n e flow and s p e c i f i c g r a v i t y was reached the assay commenced. A f o u r - p o i n t plan was employed whereby two i n j e c t i o n s of e x t r a c t s of pooled p i t u i t a r i e s from experimental f i s h exposed to 60% sea water were compared with two i n j e c t i o n s of e x t r a c t s from c o n t r o l f i s h of the same time p e r i o d of exposure to f r e s h water. This type of plan was necessary s i n c e the r a t only responds r e l i a b l y to four i n j e c t i o n s . A f t e r the i n j e c t i o n of 0.05 ml. of supernatant d i l u t e d to 0.5 ml. i n a 0.5 ml. t u b e r c u l i n s y r i n g e , 0.2 ml. of 0.9% s a l i n e was used to f l u s h a l l the e x t r a c t from the cannula i n t o the blood stream of the r a t . During the successive ten minute periods a f t e r i n j e c t i o n o f the e x t r a c t , u r i n e samples were c o l l e c t e d and s p e c i f i c g r a v i t y measurements were made. Once the s p e c i f i c g r a v i t y returned to the i n i t i a l value, another i n j e c t i o n of e x t r a c t or standard was made. 12 This procedure was repeated until four responses were recorded. Specific gravity of the urine was plotted against time and the change in specific gravity of urine for each injection of extract or standard was determined. The change in specific gravity or uterine contraction caused by the experimental extracts was compared to the change in specific gravity or uterine contractions caused by the corresponding control extracts and expressed as a percentage using the following formula: % control activitv/pituitarv/lOO gm. body wt. fi s h in S. W. x 100 activity activity/pituitary / 100 gm. body wt. fish in F. W. In order to calculate per cent control activity from the date of the antidiuretic assays the following method was employed. The f i r s t response of the rat to the experimental extract was compared to the f i r s t response of the rat to the control extract, and expressed as a percentage.-4 % activity A S D . Gr. x 10 urine S. W. fish x 100 A S p . Gr. x 10"* urine F. W. fish This calculation was repeated for the second response of the rat to the experimental extract and the second response of the rat to the control extract. An average percentage was calculated and presented in a tabular form. iv T A B L E I PITUITARY OXYTOCIC ACTIVITY MgT ABSENT MEAN BODY WT. MU/PIT/lOO gm. TREATMENT NO. FISH .. dn .qm; BODY WT. % CONTROL non handled 6 S. E. M. 221 gm. ±18.6 av. (range) 6.9 ( 6 . 5 - 7 . 4 ) 74 .6 handled 5 200 gm. ±16.9 9 . 3 ( 9 . 2 - 9 . 4 ) 100 1 hr. 60% S. W. 5 182 gm. ±15.0 14 .6 ( 1 4 . 2 - 1 5 . 1 ) 157 1 hr. F. W. 5 200 gm. ±16.9 9 . 3 ( 9 . 2 - 9 . 4 ) 100 3 hr. 60% S. w. 5 170 *24 .3 9 . 3 ( 9 . 1 - 9 . 6 ) 98 .5 3 hr. F. W. 5 161 *21.1 9 . 6 ( 8 . 4 - 1 0 . 9 ) 100 6 hr. 60% S. w. 5 196 *12.7 7 .8 ( 6 . 7 - 8 . 9 ) 97 .4 6 hr. F. W. 5 194 214 .4 8 .0 ( 8 . 0 - 8 . 0 ) 100 12 hr. 60% S. w. 5 189 ±15.0 8 . 0 ( 7 . 8 - 8 . 2 ) r-88.7 12 hr. F. W. 5 192 ±9.5 9 .0 ( 8 . 5 - 9 . 6 ) 100 T A B L E I I PITUITARY ANTIDIURETIC ACTIVITY MEAN BODY WT. URINE 4 TREATMENT NO. FISH i n gm. *Sp. Gr. x 10 % CONTROL S. E. M. 1st 2nd non handled 6 221 77 73 66.1 U8.6 handled control 5 200 117 110 100 ±16.9 1 hr. 60% S. W. 5 182 40 32 31.6 tl5.8 1 hr. F. W. 5 200 117 110 100 ±16.9 3 hr. 60% S. W. 5 170 10 45 39.5 ±24.3 3 hr. F. W. 5 161 100 65 100 + 21.1 6 hr. 60% S. W. 5 196 73 44 102.2 ±12.7 6 hr F. W. 5 194 67 46 100 * 1 4 . 4 12 hr. 60% S. W. 5 189 115 62 96.5 ±15.0 12 hr. F. W. 5 192 143 55 100 * 9.5 T A B L E I I I DIENCEPHALIC OXYTOCIC ACTIVITY Mg7ABSENT MEAN BODY WT. MU/lOO gm TREATMENT NO. FISH in qm. BODY WT. % CONTROL non handled 6 S. E. M. 221 ±18.6 av. (range) 8.3 (7.1-9.6) 72 handled cont. 5 200 ±16.9 11.5 (10.6-12.4) 100 1 hr. 60% S. W. 5 182 ±15.8 13.5 (12.9-14.1) 117 1 hr. F. W. 5 200 *16.9 11.5 (10.6-12.4) 100 3 hr. 60% S. W. 5 170 *24.3 13.3 (12.5-14.1) 102 3 hr. F. W. 5 161 ±12.1 13.0 (12.8-13.2) 100 6 hr. 60% S. W. 5 196 ±12.7 13.8 (11.8-15.9) 104 6 hr. F. W. 5 194 ±14.4 13.2 (12.6-13.9) 100 12 hr. 60% S. W. 5 189 *15.0 12.4 (11.2-13.6) 96 12 hr. F. W. 5 192 ±9.5 12.9 (12.9-12.9) 100 l l T A B L E I V PITUITARY OXYTOCIC ACTIVITY Mg PRESENT MEAN BODY WT. MU/lOO gm. TREATMENT NO. FISH i n gm. MU/PIT . BODY WT. S. E. M. av. control 30 1 hr. 60% S. W. 10 2 hr. 60% S. W. 10 3 hr. 60% S. W. 10 4 hr. 60% S. W. 10 6 hr. 60% S. W. 10 8 hr. 60% S. W. 10 10 hr. 60% S. W. 10 12 hr. 60% S. W. 10 24 hr. 60% S. W. 10 93.6 i 5.0 92.7 ±7.4 91.0 ±2.9 89.0 ±2.5 117.4 ±13.8 91.6 ±3.9 87.8 ±6.5 85.8 ±2.5 90.9 ±4.7 68.2 ±3.5 range) 10.2 9.6-10.8) 7.8 7.0- 8.6 6.2 5.7-6.7 6.5 6.1- 6.9 8.4 8.0-8.8 7.6 7.3-7.9 7.6 7.0-8.2 7.5 7.2- 7.8 8.1 7.5-8.6 7.4 7.0-7.8 10.9 8.5 7.5 7.4 7.2 8.3 8.7 8.8 8.9 10.8 % CONTROL 100 78.7 69.4 68.8 67.6 76.9 80.6 81.0 82.6 100 1 T A B L E V TREATMENT NO. FISH MEAN BODY WT. i n qm. MU/BRAIN % CONTROL control 30 S. E. M. 93.6 £5 . 0 av. (range) 15.7 (14.8-16.6) 100 1 hr. 60% S. W. 10 92.7 ±7.4 17.7 (16.0-19.4) 113 2 hr. 60% S. W. 10 91.0 ±2.9 37.8 (30.8-34.8) 240 3 hr. 60% S. w. 10 89.0 ± 2.5 28.6 (25.3-31.9) 183 4 hr. 60% S. w. 10 117.4 ±13.8 26.1 (22.4-29.7) 166 12 hr. 60% S. w. 10 90.9 i 4.7 23.7 (20.4-27.0) 151 24 hr. 60% S. w. 10 68.2 ^3.5 17.4 (15.1-19.7) 111 (Facing figure l ) FIGURE 1 . Dose response curves from two v i r g i n d i e s trus guinea p i g uterus preparations. (Facing figure 2) FIGURE 2. Typical 4 point A. D. H. assay employing vasopressin (Parke Davis) as standard. Q S P E C I F I C G R A V I T Y OF URINE O O O o o o O o o o o o O O O CD O O O O ro O 1 0 . 0 5 mu. VASOPRESSIN 0.10 mu. VASOPRESSIN •n m a c _ m z ho I O c 3) CO 0 .05 mu. VASOPRESSIN CM 0.10 mu. VASOPRESSIN _ r (Facing figure 3a) FIGURE 3a. Comparison of p i t u i t a r y extracts from handled f i s h and non-handled f i s h with respect to oxytocic and a n t i d i u r e t i c a c t i v i t i e s . mu. of O X Y T O C I N ro b b CT) b CD b o b ~i ro O O H A N D L E D F I S H N O N - H A N D L E D F I SH H A N D L E D F I S H < o = X -< O CD —1 c Z o 33 o PO GUI o — z Z m H > > - O > —I CO — GO o < AY c H — H •< m 33 c GO N O N - H A N D L E D F I S H i—i 8 m CO 0) HAN D L E D F I S H N O N - H A N D L E D F I SH H A N D L E D F I S H o c JO T J o CO CO > -< > —i m 33 O > O m o 33 3> e 3) m — i o > o N O N - H A N D L E D F ISH ro O o en O co o o o ro o A S P E C I F I C GRAV ITY 10 C\7 (Facing figure 3b) FIGURE 3b. Comparison of oxytocic and a n t i -d i u r e t i c a c t i v i t y of experimental f i s h p i t u i t a r y extracts. ACTIVITY/PITUITARY/IOO gm BODY WT. OF S.W. FISH % CONTROL ACTIVITY= X IOO ACTIVITY/PITUITARY/IOO gm BODY WT. OF F.W. FISH > r-u < _1 o cr o u I 50 100 50 _ L _|_ 0 4 5 6 HOURS IN 6 0 % FIGURE 3b 7 8 9 SEA WATER 10 11 12 (Facing figure 4) FIGURE 4. Typical 4 point A. D. H. assay of p i t u i t a r y extracts of handled and non-handled f i s h . S P E C I F I C G R A V I T Y OF U R I N E O O O O O O ro O o o -o o o o at o o o GO O O o ro O 0 . 0 2 5 ml. N O N - H A N D L E D E X T R A C T 0 . 0 2 5 ml. HANDLED EXTRACT 2 m 8 m ro -0 .025 ml. H A N D L E D E X T R A C T x o c CO 0 .025 m l . N O N - H A N D L E D E X T R A C T w -(Facing figure 5) FIGURE 5. Typical 4 point A. D. H. assay of pituitary extract of paired experi-mental and control fish. 1.0120 -1.0100 -1.0080 -I. 0060 1.0040 -1.0020 -1.0000 TIME IN HOURS FIGURE 5 (Facing figure 6) FIGURE 6. Effect of addition of Mg"""in Ringer bath indicating depressed activity due to depletion of the antidiuretic principle. ACTIVITY/PITUITARY/IOO gm. BODY WT. OF S.W. FISH % CONTROL ACTIVITY = X IOO ACTIVITY/PITUITARY/IOO gm. BODY WT. OF F.W. FISH 2 0 0 > OXYTOCIC ACTIVITY ( P L U S Mg.) O X Y T O C I C ACTIVITY (MINUS Mg.) O -A N T I D I U E R E T I C A C T I V I T Y L\-6 8 10 HOURS IN 6 0 % S E A WATER FIGURE 6 12 (Facing figure 7) FIGURE 7. Oxytocic activity in brain extracts of experimental fi s h . OXYTOCIC ACTIVITY/BRAIN/IOO gm. BODY WT. S.W. % CONTROL ACTIVITY^ * 100 OXYTOCIC ACTIVITY/BRAIN/IOO gm. BODY WT. F.W. X 6 8 10 HOURS IN 6 0 % S E A W A T E R FIGURE 7 12 (Facing figure 8) FIGURE 8. A hypothetical phylogenetic d i s t r i b u t i o n of known active p o s t e r i o r lobe p r i n c i p l e s . (From Sawyer et a l 1959) P I G C y s T y r - P h e G l u ( N H ) Asp (NH J - C y S P r o - l _ V S G I y ( N H ) Lysine vasopressin M A M M A L S Lactation appears R E P T I L E S 8 BIRDS A M P H I B I A N S Ant/diuresis appears Neural lobe appears BONY FISH E L A S M O B R A N C H S UNKNOWN PEPTIDE CysTyr P h e G l u ( N H 2 ) A s p ( N H 2 ) C y S Pro A r g • Gly ( N H 2 ) ^ Arginine vasopressin C y s T y r l l e u G l u ( N H 2 ) A s p ( N H 2 ) C y S P r o - Le U • Gly (NH 2 ) Oxytocin ^ AGNATHA V E R T E B R A T E S I N V E R T E B R A T E S Cys-Tyr-I leu - G l u ( N H 2 ) A s p ( N H 2 ) C y S P r o - A r g Gly ( N H 2 ) ^ Arginine vasotocin \ - ^ A S C I D I A N S - ACTIVITY y^7? ARTHROPODS - 7> ACTIVITY FIGURE 8 R E S U L T S I. Dose Response Relationships A. Oxytocic Activity Throughout the assays of fish extracts for oxytocic activity, known doses of a standard solution of Pitocin (Parke Davis) were employed for reference purposes. Dose response curves were plotted for each uterine preparation. Representative dose response curves for two uterine preparations are shown in figure 1. Since the responses fi t t e d the line closely no further s t a t i s t i c a l treatment of this data was f e l t to be necessary. B. Antidiuretic Activity A plot of a typical four-point assay for antidiuretic activity employing a standard solution of Pitressin (Parke Davis) i s shown in figure 2. This assay of standard vasopressin served only as an indicator of dose response relationships. Other typical responses to extracts of fish pituitaries are shown in figures 3, 4, and 5. In these cases comparative differences in specific gravity between treatments indicated the relative amounts of antidiuretic principle present in the extract assayed. I I . Effects of Handling Handled fish evidenced a greater amount of oxytocic and antidiuretic activity i n their pituitaries when compared to the non-handled fish (Tables I and II, figures 3a arid 4). Similarly, the brains of handled fish had a greater amount of oxytocic activity than did the brains of non-handled fish (Table III). III. Adaptation to Sea Water A. Oxytocic Activity 1. Pituitary Assays for oxytocic activity of fish pituitary extracts in a Ringer solution without Mg**ions indicated an i n i t i a l rise in oxytocic activity per pituitary for the f i r s t hour after transfer to sea water with a return to control levels for the remainder of the experimental period (Table I, figures 3b, and 6). Addition of Mg**" ions modified the response of the uterus (Table V and figure 6) which suggested the influence of other active principles on the oxytocic response. Treatment of pituitary extracts with 0.05M sodium thio-glycollate indicated that only known pituitary polypeptides were present, since complete inaetivation of the oxytocic response occurred. 2. Brain Assays for oxytocic activity with Mg ion present in the Ringer's solution indicated the presence of an oxytocic-like substance that dramatically increased in concentration during the f i r s t two hours after transfer to sea water (Table V, figure 7). Assays without Mg^ion in the Ringer's solution indicated a slight increase i n oxytocic-like activity in the brains of the one-hour experimental fis h with a subsequent return to control levels at three hours (Table III and figure 7). Treatment of these extracts with 0.05M sodium thioglycollate failed to reduce the activity. B. Antidiuretic Activity 1. Pituitary Antidiuretic assays using the change in urine specific gravity of the water loaded rat indicated a very marked depletion of antidiuretic activity of the pituitaries during the three-hour period after exposure to a sea water environment (Table II, figure 3b, 5, and 6). Figure 3b indicated relationship of this decrease with the increased pituitary oxytocic activity for experimental f i s h . An example of the magnitude of this response after three hours in sea water i s shown in figure 5. This depletion of antidiuretic activity was similarly reflected in the uterine response to oxytocic material when the assay was conducted i n the presence of Mg^ion (figure 6). This pituitary antidiuretic material from both experimental and control fish was completely inactivated by treatment of the extracts with 0.05M sodium thioglycollate. 2. Brain Antidiuretic activity of the extracts of experimental and control fish brains was extremely small and rapidly inactivated while conducting the assay. As a result of the unsuitable nature of these extracts this portion of the investigation was discontinued. D l S C U S S I O N 1. Phvlogenetic Relationships of Neurohypophysial Peptides A. Chemical Relationships The recent observations by Heller and Pickering (1961) and Sawyer, Munsick and van Dyke (1961) which have indicated the existence of argenine vasotocin in the neurohypophysis of elasmobranchs, teleosts, amphibians, reptiles, and birds has tended to modify our thinking concerning neurohypophysial hormones in non-mammalian vertebrates. Previous consideration of this topic involved oxytocin and vasopressin linked in a 1:1 ratio. Furthermore the pharmacologic and chromatographic evidence presented by these workers has shown the absence of vasopressin, both in the argenine and lysine forms in the hypothalamo-neurohypophysial system of the lower vertebrates. Oxytocin f i r s t appears at the level of the bony fishes and continues to be present throughout the animal kingdon. A summary of the phylogenetic relationships of these polypeptide hormones and their structures has been presented by Sawyer et al (1959) (figure 8). In the light of-these considerations, therefore, we must consider, when dealing with active principles from teleost sources that we are dealing with oxytocin and vasotocin. Thus we must interpret the present observations in terms of oxytocin and vasotocin rather than the mammalian analogues oxytocin and vasopressin. B. Functional Relationships Unwittingly, investigators have observed the responses of mammalian, avian, and amphibian systems to vasotocin for many years. Although vasotocin i s not present in mammalian systems i t e l i c i t s activity similar to both oxytocin and vasopressin when assayed in mammalian systems. Sawyer, Munsick, and van Dyke (1959) compared neurohypophysial extracts from avian sources with synthetic vasotocin and a mixture of vasotocin and oxytocin and found that vasotocin was very active in a l l assays except those involving uterine preparations without Mg . The presence of oxytocin did not appreciably alter the response of the mammalian, avian, reptilian, and amphibian assay systems to vasotocin. It would appear, then, that vasotocin i s a functional intermediate between vasopressin and oxytocin and the existence of this functional intermediate has only been demonstrated in non-mammalian vertebrates. Hi Her and Pickering (1961) gave a more complete summary of the relative responses of mammalian, avian, and amphibian systems to vasotocin, oxytocin, and argenine and lysine vasopressins. It would appear from this summary that vasotocin i s in fact responsible for the difference in res-ponses observed employing the frog water uptake assay described by Heller (1941). Further, avian response such as the hen oviduct assay indicated that vasotocin i s much more active than oxytocin. Maetz et al (1959) had proposed the presence of Natriferin, a new polypeptide present in the neurohypophysis of animals below the mammals. This preparation was approximately ten times as active as the same measurable quantity of VJ oxytocin, using a mammalian assay to standardize the activity, in promoting an increased sodium flux across in vitro amphibian skin preparations. This evidence tends to suggest that vasotocin i s more active in lower forms, excepting fi s h , since there have been no reported effects of vasotocin on water and electrolyte balance in fi s h . II. Transfer to Sea Water A. Oxytocic Assays of Extracts If we consider the summary of Sawyer, Munsick, and van Dyke (1959), particularly the response:of the rat uterus with and without Mg ion i n solution, we see that the activity of vasotocin is markedly increased i n the presence of Mg^ion. The present investigation has employed a similar technique, varying the concentration of Mgf* ion, to demonstrate the var i a b i l i t y in composition of extracts. Considering the evidence before us, we can now interpret the results indicated in figure 6. In the case where no Mcj1" ion i s in solution the uterus responds only to oxytocin. On the other hand, in the presence of Mg"* ion the uterus responded to both oxytocin and vasotocin. Thus, any decrease in vasotocin below control levels would be indicated as, in fact, was the case. Sodium thioglycollate has been shown to inactivate a l l known neurohypophysial hormones (Sawyer, Munsick, and van Dyke (i960). The pituitary oxytocic responses were totally eliminated when our extracts were similarly treated. Thus the uterine responses observed from pituitary extracts are total l y due to only either oxytocin or vasotocin. On the other hand, the brain extracts contained a considerable amount of oxytocic activity that was not inactivated by sodium thioglycollate. This suggested the presence of some unknown substance that fluctuates upon, handling of fish and upon placement in hypertonic environments. The presence of a similar substance in the neurointermediate lobe of elasmobranchs was reported by Sawyer, Munsick and van Dyke (1961). However, since relatively large amounts of tissue were employed for obtaining extracts in the present work there exists a possi b i l i t y of interfering substances such as 5 hydroxy-tryptamine being present. Considering a l l the evidence concerning the presence and absence in non-mammalian vertebrates and the physiological properties, both oxytocic and antidiuretic, of vasotocin, i t would appear that the present observations were at least in part due to this polypeptide. Transfer of trout to 60% sea water evidenced a decline in oxytocic activity only in assays that employed magnesium ions in the Ringer solution. If we accept the presence of vasotocin, then this decline in activity after transfer to sea water can be explained only by a depletion of vasotocin in the pituitaries of the experimental fi s h . B. Antidiuretic Assays of Extracts Sawyer, Munsick, and van Dyke (1961), and Heller and Pickering (1961) showed that vasotocin caused antidiuresis i n water loaded, ethanol anaesthetized rats. Furthermore, these investigations have shown the presence of vasotocin i n the pituitary of trout. Thus, i f we consider the change in antidiuretic activity upon transfer of trout to sea water as indicated by figures 3b, 5, and 6, we see there was in fact a depletion of an active substance, perhaps vasotocin. Inactivation with sodium thioglycollate indicated the absence of substances other than oxytocin, vasotocin, and the vasopressins. Again, since vasopressin i s absent in lower vertebrates, the observed response was probably due to vasotocin, since Berdi and Cerlette (1956) have shown oxytocin to be a diuretic agent, this would eliminate any probable antidiuretic effect due to oxytocin. If we now consider the brain extracts, and their high oxytocic-like activity, in the light of the work of Berdi and Cerlette (1956), we may conjecture that the interference from this unknown source perhaps in the direction of diuresis, was so great as to totally suppress the antidiuretic activity of the vasotocin present. Further, Knoble (1957), pointed out that thioglycollate inaetivation of active principles present in concentrated extracts, as i s the case of brain extracts, i s unsatis-factory. This may indicate that there was in fact a relatively high concentration of oxytocic-like material present in the hypothalamic region of the brain under the present experimental conditions. C. Integrated Considerations The present evidence indicates that vasotocin may be implicated in the transfer of fish to a hypertonic medium. We have observed a depletion of a pharmacologically active substance in the pituitary of Salmo gairdneri upon transfer to sea water. Arvy, Fontaine and Gabe (1954) and Arvy and Gabe (1954) have indicated the depletion of a Gomori positive stainable material from the hypothalamo-neurohypophysial system in fish upon transfer to a hypertonic environment. Our evidence tends to further substantiate these authors* observations. Russel, Rennels and Drager (1955) studied the variations of oxytocin and Gomori positive materials i n the hypothalamus and pituitary of the rat. Three treatments were carried out; electroshocking, dehydration, and adrenalectomy. The oxytocic content did not vary on shocking, f e l l to 20% of normal levels on drinking 2.5% saline for ten days, while after adrenalectomy the oxytocic content doubled. On the other hand, the Gomori positive material failed to change during shocking, was depleted during saline treatment, but did not change after adrenalectomy. This evidence would suggest that Gomori positive staining i s not always a reliable index of the oxytocic level in tissues. The effects of handling were considered to be a problem in i t s e l f . For the purpose of this investigation i t was f e l t that an experimental design had to be adopted so that control groups and experi-mental groups were handled equally thus eliminating any unwanted handling effects. The biological significance of the increase of active principles in the posterior lobe as a result of handling remains in the realm of speculation. Since vasotocin i s involved, i t would appear dangerous to state that vasotocin may have a similar property to vasopressin, causing the release of ACTH in fish as does vasopressin in mammalian forms (Sawyer, Munsick, and van Dyke, 1959). Further consideration was given to the fluctuation in levels of active principles of the pituitary and the hypothalamus. It was f e l t that a depletion of the pituitary contents was indicative of a secretion of the hormones into the circulatory system. Further, an increase in the hypothalamic region of the brain was an indication of increased synthesis. After a depletion of pituitary contents a replenishment to control levels was considered to be evidence of a higher rate of production of active principles in the hypothalamic region than was being secreted. It would appear from this, that fluctuations in pituitary concentrations of posterior lobe hormones were an indication of a mobilization of these hormones. Only by measuring circulatory levels of active principles would i t be possible to state the rate of secretion, the amount secreted and the duration of time of secretion. This information was considered to be virtu a l l y impossible to obtain when consideration was given to the ava i l a b i l i t y of assay techniques that were sufficiently sensitive for measuring circulating levels of active posterior lobe principles of fish exposed to a hypertonic environment. C O N C L U S I O N S 1 . F i s h that were exposed to excessive handling evidenced a greater amount of a n t i d i u r e t i c and oxytocic a c t i v i t y per p i t u i t a r y than non-handled f i s h . 2. Transfer of f i s h to sea water resulted i n a reduction of a n t i d i u r e t i c a c t i v i t y of p i t u i t a r y extracts during the f i r s t and t h i r d hour a f t e r t r a n s f e r with a return to control l e v e l s at the si x t h hour. 3. Increased p i t u i t a r y oxytocic a c t i v i t y was evidenced i n the p i t u i t a r y extracts of f i s h transferred to sea water for one hour, while con t r o l l e v e l s were maintained for the remainder of experimental periods. 4. Oxytocic a c t i v i t y of brain extracts was increased upon transf e r of f i s h to sea water. 5 . Addition yof Mg**-ions to the Ringer bath res u l t e d i n an enhanced oxytocic response i n d i c a t i n g the presence of active p r i n c i p l e s other than oxytocin, i n both the brain and p i t u i t a r y extracts. 6. 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