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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 U n i v e r s i t y o f 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 a c c e p t t h i s t h e s i s as conforming r e q u i r e d standard  THE  t o the  UNIVERSITY OF BRITISH COLUMBIA  A p r i l , 1961  In the  presenting  requirements  of  British  it  freely  agree for  advanced  I  copying  gain  shall  by or  not  his  of  April  degree  the  and  study.  I  extensive  may  be  granted  representatives. of  copying  this  without  by  of  the  It thesis  is  Columbia,  make  further this  Head  of  thesis my  understood  for  my w r i t t e n  of  University  reference  allowed  1961  at  shall  Zoology  28,  fulfilment  Library  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r &, Canada. Date  p a r t i a l  the  publication be  in  that  for  purposes  or  agree  for  permission  that  Department  an  Columbia,  scholarly  Department  thesis  for  available  that  this  f i n a n c i a l  permission.  1  A B S T R A C T  V a r i a t i o n s i n c o n c e n t r a t i o n , i f any, o f p h a r m a c o l o g i c a l l y a c t i v e p r i n c i p l e s o f the h y p o t h a l a m i c o - n e u r o h y p o p h y s i a l system o f the rainbow t r o u t Salmo q a i r d n e r i d u r i n g e a r l y p e r i o d s o f t r a n s f e r t o sea water were i n v e s t i g a t e d .  The c o n c e n t r a t i o n s o f o x y t o c i c 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 b r a i n s and p i t u i t a r i e s o f handled  fish,  f i s h t r a n s f e r r e d t o sea water, and f i s h t r a n s f e r r e d t o f r e s h water, were measured employing the i s o l a t e d v i r g i n guinea p i g u t e r u s f o r a s s a y i n g o x y t o c i c a c t i v i t y , and the e t h a n o l a n a e s t h e t i z e d water l o a d e d rat  f o r assaying a n t i d i u r e t i c  activity.  H a n d l i n g the f i s h r e s u l t e d i n an i n c r e a s e o f o x y t o c i c and antidiuretic activity of pituitary  extracts.  T r a n s f e r o f e x p e r i m e n t a l f i s h t o a sea water  environment  resulted 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 f o r the f i r s t  subsequent r e t u r n t o c o n t r o l l e v e l s . sea  and second hours w i t h a  A f t e r t r a n s f e r o f the f i s h t o a  water environment the a n t i d i u r e t i c a c t i v i t y o f p i t u i t a r y  was observed to d e c r e a s e d u r i n g the f i r s t  extracts  and t h i r d hour, w i t h a  r e t u r n t o c o n t r o l l e v e l s a t the s i x t h hour. T h i s evidence suggests t h a t a c t i v e p r i n c i p l e s which are known to  p l a y a c t i v e r o l e s i n water balance i n animals h i g h e r , p h y l o g e n e t i c -  a l l y , than f i s h , are l i b e r a t e d from the h y p o t h a l a m i c o - n e u r o h y p o p h y s i a l system o f Salmo q a i r d n e r i i n response t o a h y p e r t o n i c  environment.  J. A  T A B L E  OF  C O N T E N T S  Page 1  INTRODUCTION MATERIALS AND METHODS Experimental  V  Conditions  7  Treatment o f T i s s u e s  8  O x y t o c i c Assay  9  A n t i d i u r e t i c Assay  10  RESULTS ( T a b l e s : and F i g u r e s )  13  TABLE I  13  TABLE I I  14  TABLE I I I  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) 1.  Dose Response R e l a t i o n s h i p s  27  I l l  ( R e s u l t s Cont'd) A. O x y t o c i c  Page Activity  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 o f Handling I I I . Adaptations A. O x y t o c i c  28  t o Sea Water  28  Activity  28  1. P i t u i t a r y  28  2. B r a i n  28  B. A n t i d i u r e t i c  Activity  29  1. P i t u i t a r y  29  2. B r a i n  29  DISCUSSION  30  I. Phylogenetic  R e l a t i o n s h i p s o f Neurohypophysial P e p t i d e s .  .  30  A. Chemical R e l a t i o n s h i p s  30  B. F u n c t i o n a l R e l a t i o n s h i p s  31  I I . T r a n s f e r t o Sea Water A. O x y t o c i c  32  Assays o f E x t r a c t s  B. A n t i d i u r e t i c C. I n t e g r a t e d  32  Assays o f E x t r a c t s  Considerations  33 . . . . .  34  CONCLUSIONS  37  BIBLIOGRAPHY  38  A C K N O W L E D G M E N T  I wish t o extend to Dr. W. N. Holmes my a p p r e c i a t i o n h i s d i r e c t i o n throughout t h e i n v e s t i g a t i o n and p r e p a r a t i o n  of  embodied  i n t h i s t h e s i s and f o r the f i n a n c i a l a s s i s t a n c e made p o s s i b l e through a grant  t o him i n a i d o f r e s e a r c h  from the Atomic Energy  I a l s o wish t o thank Dr. W. S.  Hoarcand  f o r t h e i r s u g g e s t i o n s throughout t h e p r e p a r a t i o n Further,  D r . P. A.  Larkin  of this thesis.  I wish t o extend my g r a t i t u d e t o the F i s h e r i e s  A s s o c i a t i o n o f B r i t i s h Columbia me d u r i n g  Commission.  t h e academic  f o r t h e s c h o l a r s h i p they awarded to  year 1960-61.  I N T R O D U C T I O N  E v i d e n c e c o n c e r n i n g the presence o f a c t i v e substances i n the n e u r a l lobe o f mammalian p i t u i t a r i e s has e x i s t e d f o r many y e a r s .  The  work o f O l i v e r and S c h a f e r (1895) i n d i c a t e d t h a t i n t r a v e n o u s i n j e c t i o n o f whole p i t u i t a r y e x t r a c t s i n t o mammals r e s u l t e d i n a p r o l o n g e d intense vasoconstriction.  Howell  t h i s " p r e s s o r " response was o f the p i t u i t a r y g l a n d .  (1898) and Dale (1906) showed t h a t  a s s o c i a t e d w i t h the p o s t e r i o r o r n e u r a l lobe  However, a t the same time Dale a l s o  t h a t the u t e r u s o f the pregnant  observed  c a t e l i c i t e d powerful c o n t r a c t i o n s i n  response t o t h i s p o s t e r i o r l o b e e x t r a c t . the mammary glands o f l a c t a t i n g c a t s was  Furthermore  o f p o s t e r i o r p i t u i t a r y e x t r a c t s was  milk ejection  from  observed by O t t and S c o t t (1910)  i n response t o 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 r a c t s .  Formi  and  Another p r o p e r t y  observed by von den Velden (1913) and  (1913) w h i l e a t t e m p t i n g t o f i n d some t h e r a p e u t i c v a l u e o f p o s t e r i o r  l o b e e x t r a c t s i n cases o f d i a b e t e s i n s i p i d u s .  These o b s e r v e r s found  that  p o s t e r i o r l o b e p r e p a r a t i o n s decreased the u r i n e output i n cases o f severe diuresis.  I n v e s t i g a t i o n s by Brunn (1921) uncovered y e t another  o f the e x t r a c t s o f the mammalian p o s t e r i o r p i t u i t a r y , namely, a o f water uptake  i n amphibia.  F u r t h e r i n v e s t i g a t i o n s by H e l l e r  activity promotion (1941)  indicated that differences exist i n the amphibian response to preparations from species other than mammalian species.  This suggested the possible  presence of several hormones i n the animal kingdom causing similar responses.  Also, Hogben and de Beer (1925) indicated that i n several  species including f i s h , there was an active p r i n c i p l e i n the posterior p i t u i t a r y that e l i c i t e d a f a l l i n blood pressure i n avian systems. With these i n i t i a l investigations laying the pattern of a c t i v i t i e s of posterior lobe extracts other workers developed methods of separating the active components found i n the posterior p i t u i t a r y . developed a means of separating these f r a c t i o n s . vance oxytocin and vasopressin became commercially  Kamm (1928)  As a r e s u l t of t h i s adavailable.  I t 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 e j e c t i o n i n mammals, blood pressure depression i n birds, and water uptake i n amphibia, while vasopressin was observed to be responsible for  vasoconstriction and the subsequent r i s e i n blood pressure i n birds  and mammals as well as promoting renal tubular water reabsorption i n birds and mammals.  In addition, vasopressin was  shown to stimulate i n v i t r o  uterine contraction i n mammalian systems i f magnesium ions were present i n the supporting medium. Methods of bio-assay have been developed to e x p l o i t these pharmac o l o g i c a l properties of vasopressin and oxytocin.  Assays for oxytocic  a c t i v i t y have been described by Dale and Laidlaw (1912), Kochman (1921), Burn and Dale (1922), Burn (1937), and Fraser (1939) employing the i s o l a t e d guinea pig uterus. using the r a t uterus.  Houlton (1948) also described a similar assay  Methods employing the f a l l i n avian blood  pressure,  c5  as a measure of oxytocic a c t i v i t y , have been described by Paton and Watson (1912),  Gaddum (1928), Coon (1939), Smith (1942), Smith and Vos  (1943), Thompson (1944).  An i n v i t r o assay employing the isolated frog  bladder has been described by Sawyer (i960) who also mentioned a further modification of the r a t 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 i n mammals and the reduction of urine flow i n 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 a l (1946). A n t i d i u r e t i c assays using mice were described by Gibbs (1930) and Burn (1931) modified these techniques for use i n r a t s .  Further modi-  f i c a t i o n s of t h i s method were described by Gilman and Goodman (1937), Ham  and Landis (1942), and J e f f e r s , 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 r e l i a b l e but are r e s t r i c t i v e according to the amount of active material present i n the extract to be assayed.  In the case of small quantities of oxytocin  the guinea pig or r a t uterus have been found to be s u i t a b l e .  While for  small quantities of vasopressin the r a t a n t i d i u r e t i c method of Ingraham and Baratz (1959) or the water uptake assay employing toads (Lauber et a l , 1959) are most u s e f u l .  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 c e l l s and t h e i r contents. The investigations by Herring (1913) on the elasmobranch and teleost neurohypophysial systems indicated the presence of p r i n c i p l e s that evoked pressor, oxytocic, and milk ejection responses i n mammalian subjects.  These observations were l a t e r substantiated by Hogben and  de Beer (1925).  Boyd and Dingwall (1939) demonstrated the presence  of the frog water balance factor i n the neurohypophysial system of the cod.  Heller (1941) substantiated t h i s investigation by showing the  presence of a frog water balance factor i n the neurointermediate lobe of the skate.  Also, H e l l e r indicated the presence of an a n t i d i u r e t i c  p r i n c i p l e i n 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 p r i n c i p l e s from elasmobranchs and teleosts that e l i c i t e d oxytocic and a n t i d i u r e t i c responses i n mammalian systems.  Furthermore,  comparison  with the pharmacological responses of prepared polypeptides by Du Vigneaud (1952 and 1958) indicated that active p r i n c i p l e s existed i n the hypothalami-neurohypophysial systems of elasmobranchs and teleost f i s h similar to oxytocin and vasopressin. However, chromatographic evidence for the existence of vasopressin i n extracts from the neurohypophysial system of elasmobranchs and teleosts i s lacking ( H e l l e r , 1961).  Heller and Pickering (1961), and Sawyer, Munsick, and  5  van Dyke (1961) have i n d i c a t e d p h a r m a c o l o g i c a l l y and the presence  o f o x y t o c i n and a substance  s y n t h e t i c a l l y prepared  p o l y p e p t i d e o f Du  i n v e s t i g a t i o n s by Sawyer, Munsick, and  chromatographically  s i m i l a r to v a s o t o c i n , the Vigneaud (1958).  van Dyke ( i 9 6 0 and  t h a t v a s o t o c i n i s p r e s e n t i n f i s h , amphibians, and  As  well,  1961)  indicated  birds.  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 o f v a s o p r e s s i n and o x y t o c i n i n f i s h have met  with l i t t l e  success.  There have been no  s u c c e s s f u l 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 h y p o p h y s i a l hormones i n f i s h .  Sawyer (1933) and Dreyer  to neuro-  (1946), have shown  i n c r e a s e d tonus and rythm o f smooth muscle from the stomach and  the  i n t e s t i n e o f f i s h exposed to a medium c o n t a i n i n g p o s t e r i o r lobe  principles.  Bacon (1951  and  1952)  and W i l h e l m i , P i c k f o r d and  Sawyer (1955) have  i n d i c a t e d t h a t i n j e c t i o n s o f o x y t o c i n and/or p o s t e r i o r l o b e p r e p a r a t i o n s caused  e i t h e r maturation  c a r p i o . and  Semotilus  i n pre-spawning Catostomus commersoni,  atromaculatus  Cyprinus  o f the i n d u c t i o n o f the spawning  r e f l e x i n Fundulus h e t e r o c l i t u s . F u r t h e r work by Holmes (1959) has i n d i c a t e d t h a t o x y t o c i n reduces  the r e s p i r a t i o n r a t e o f k i d n e y t i s s u e i n  trout. I n v e s t i g a t i o n s which attempted to a s s o c i a t e the q u a n t i t y o f n e u r o s e c r e t o r y m a t e r i a l p r e s e n t i n the neurohypophysial of  h y d r a t i o n and d e h y d r a t i o n have proved  system w i t h s t u d i e s  to be more s u c c e s s f u l .  by Arvy, F o n t a i n e and Gabe (1954) on Phoxinus phoxinus.  Phoxinus  and A n g u i l l a a n q u i l l a have i n d i c a t e d t h a t immersion o f t h e s e  Studies laevis.  fish in  s t r o n g l y h y p e r t o n i c environments r e s u l t e d i n a d e p l e t i o n o f 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 . Gabe (1954) on C a l l i o n y m u s  F u r t h e r i n v e s t i g a t i o n s by Arvy  and  l y r a and Ammodytes lancepJatus have i n d i c a t e d  t h a t immersion i n h y p e r t o n i c 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 i f 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  MATERIALS  AND  METHODS  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 f a c i l i t i e s 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. outside holding pond.  Sixteen fish were removed from the  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 t h i s i n i t i a l experiment i t was decided that a system of paired experimental and control groups of f i s h be employed for each time period of exposure of the experimental f i s h to 60% sea water. A number of f i s h 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 f i s h to the new surroundings.  At the end of t h i s period f i v e f i s h were removed and  placed i n 60% sea water, whilst f i v e control f i s h from the same group were placed i n i d e n t i c a l containers of fresh water.  Thus for each  experimental period of one hour, three hours, six hours, and twelve hours, f i v e f i s h were placed i n sea water and f i v e f i s h were placed i n fresh water for exactly the same time, hence serving as controls. At the end of the experimental period the f i s h were removed, s a c r i f i c e d by a blow on the head, weighed and decapitated with a subsequent removal of p i t u i t a r i e s and brains.  However, another series  of experiments for confirmatory studies was conducted whereby ten f i s h were employed for experimental and control groups.  The p i t u i t a r i e s  were pooled for each experimental and control group of either f i v e f i s h or ten f i s h .  As well, the hypothalami were pooled for each  group of five or ten f i s h .  The extracts of pooled p i t u i t a r i e s and  hypothalamo were assayed.  Treatment of Tissues P i t u i t a r y and hypothalamic tissues were quickly removed from the decapitated f i s h and placed i n 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 t h i s mixture was  placed i n a b o i l i n g water bath f o r f i v e minutes.  During t h i s period  of time the tissue was macerated with a glass s t 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 o f f and stored at -21°C u n t i l  required for assay. Oxytocic assays were performed with the undiluted supernatant extract.  P r i o r to the assay f o r a n t i d i u r e t i c a c t i v i t y 0.05.ml. of  the supernatant was d i l u t e d to 5 ml. with 0.9% saline f o r p i t u i t a r i e s but not diluted f o r brain tissue extracts.  Inactivation of  neurohypophysial hormones was accomplished by mixing a 1:1 solution of supernatant and 0.05 M. sodium t h i o g l y c o l l a t e at pH7.4.  The  mixture was l e f t standing at room temperature for two hours p r i o r to assay.  Oxytocic Assay The assay f o r oxytocic a c t i v i t y employed the isolated diestrus guinea p i g uterus suspended i n Ringer*s solution as described by Emmens (1950). The composition of the Ringer's solution was as follows:  NaCl KC1 CaCl MgCl2 NaHC03 Dextrose 2  9.0 0.42 0.12 0.0025 0.5 0.5  gm/l II  n II II  n  V i r g i n diestrus guinea pigs of 180-230 grams weight were employed throughout the experiment.  The animals were s a c r i f i c e d by  a blow on the head and the uterus was removed and placed i n a bath of Ringer's solution at room temperature.  A l l f a t t y tissue was removed  from the uterine horns while i n t h i s bath.  One horn of the prepared  uterus was suspended i n the assay bath at 28°C. and allowed to e q u i l i brate i n the oxygen gassed Ringer medium for at l e a s t one-half hour before the assay commenced.  The assay program was similar to that of  Houlton (1948). Standard preparations of P i t o c i n (Parke Davis) were used throughout the experiments.  A stock standard solution of 10 mu.  of oxytocic a c t i v i t y per 0.1 ml. was made and during the assays standards of d i f f e r e n t concentrations were made from t h i s stock solution i n 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. for a l l standards and extracts were recorded.  The uterine responses  From the dose response  curve, the concentration of active p r i n c i p l e s i n the unknown extracts was calculated and expressed i n m i l l i u n i t s of oxytocic a c t i v i t y per p i t u i t a r y or brain per 100 gm. body weight.  An index of per cent of  control a c t i v i t y was also calculated for each pair of experimentals and control f i s h .  A n t i d i u r e t i c Assay The method employed for the assay of a n t i d i u r e t i c a c t i v i t y i n the extracts of the p i t u i t a r i e s and brains was a modification of that described by Baratz and Ingraham (1959).  Female r a t s of 200 gm. body  weight were obtained from the central animal depot several days p r i o r to the assays.  A l l rats were water loaded with 10 ml. of warm tap  water the afternoon p r i o r to assay.  Food was withheld twenty-four  hours before assay, although water was allowed ad libidum. On the morning of assay the r a t to be used was anaesthetized, water loaded  w i t h a volume o f 10% e t h a n o l w a t e r s o l u t i o n e q u i v a l e n t t o 5% o f t h e body w e i g h t .  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 c a n n u l a t e d and a  c a t h e t e r was p l a c e d i n t h e b l a d d e r v i a t h e u r e t h r a i n such a p o s i t i o n t h a t t h e r e would be a syphon e f f e c t .  I f b l e e d i n g o c c u r r e d as a r e s u l t  o f 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 t h r o m b i n was f l u s h e d i n t o t h e b l a d d e r and a l l o w e d t o d r a i n . of the bladder.  No attempt was made t o t i e o f f t h e dead space  D u r i n g t h e assay t h e r a t was r e s t r a i n e d on an e l e -  v a t e d t a b l e , warmed by a r e a d i n g lamp. 5 m l . graduated  The u r i n e was c o l l e c t e d i n a  c y l i n d e r p l a c e d below t h e r a t .  A f t e r s u r g e r y , t h e u r i n e f l o w u s u a l l y i n c r e a s e d and d u r i n g t h i s p e r i o d t h e s p e c i f i c g r a v i t y o f t h e u r i n e was d e t e r m i n e d u s i n g t h e method o f Barbour and H a m i l t o n  (1926).  Once a s t a t e o f c o n s t a n t  u r i n e f l o w and s p e c i f i c g r a v i t y was reached  t h e assay  commenced.  A f o u r - p o i n t p l a n was employed whereby two i n j e c t i o n s o f e x t r a c t s o f p o o l e d p i t u i t a r i e s from e x p e r i m e n t a l  f i s h exposed t o 60% sea  water were compared w i t h two i n j e c t i o n s o f e x t r a c t s from c o n t r o l f i s h o f t h e same time p e r i o d o f exposure t o f r e s h w a t e r . o f p l a n was n e c e s s a r y  This  type  s i n c e t h e r a t o n l y responds r e l i a b l y t o f o u r  injections. A f t e r t h e i n j e c t i o n o f 0.05 m l . o f s u p e r n a t a n t  diluted to  0.5 m l . i n a 0.5 m l . t u b e r c u l i n s y r i n g e , 0.2 m l . o f 0.9% s a l i n e was used t o f l u s h a l l t h e e x t r a c t from t h e cannula i n t o t h e b l o o d stream of the r a t .  D u r i n g t h e s u c c e s s i v e t e n minute p e r i o d s a f t e r  injection  o f t h e 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. initial  Once t h e s p e c i f i c g r a v i t y r e t u r n e d t o t h e  v a l u e , another i n j e c t i o n o f e x t r a c t o r s t a n d a r d was made.  12  This procedure was repeated u n t i l four responses were recorded. S p e c i f i c gravity of the urine was plotted against time and the change i n s p e c i f i c gravity of urine f o r each i n j e c t i o n of extract or standard was determined.  The change i n s p e c i f i c gravity or uterine  contraction caused by the experimental extracts was compared to the change i n s p e c i f i c gravity or uterine contractions caused by the corresponding control extracts and expressed as a percentage using the following formula:  % control activity  a c t i v i t v / p i t u i t a r v / l O O gm. body wt. f i s h i n S. W. a c t i v i t y / p i t u i t a r y / 1 0 0 gm. body wt. f i s h i n F. W.  x  100  In order to calculate per cent control a c t i v i t y from the date of the a n t i d i u r e t i c assays the following method was employed.  The  f i r s t response of the r a t to the experimental extract was compared to the f i r s t response of the r a t to the control extract, and expressed as a percentage.4  % activity  A S D . Gr. x 10 urine S. W. f i s h S p . Gr. x 10"* urine F. W. fish  x  100  A  This c a l c u l a t i o n was repeated for the second response of the r a t to the experimental extract and the second response of the r a t to the control extract.  An average percentage was calculated and presented  i n a tabular form.  iv  T A B L E  I  PITUITARY OXYTOCIC ACTIVITY MgT ABSENT  TREATMENT  NO. FISH  MEAN BODY WT. .. dn .qm; S. E. M.  MU/PIT/lOO gm. BODY WT. av. (range) 6.9 (6.5-7.4)  non handled  6  221 gm. ±18.6  handled  5  200 gm. ±16.9  9.3 (9.2-9.4)  1 hr. 60% S. W.  5  182 gm. ±15.0  14.6 (14.2-15.1)  1 hr. F. W.  5  200 gm. ±16.9  9.3 (9.2-9.4)  3 hr. 60% S. w.  5  170 *24.3  9.3 (9.1-9.6)  3 hr. F. W.  5  161 *21.1  9.6 (8.4-10.9)  % CONTROL 74.6 100  157 100 98.5 100 97.4  6 hr. 60% S. w.  5  196 *12.7  7.8 (6.7-8.9)  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 i n gm. * S p . G r . x 10 S. E . M. 1st 2nd 221 77 73 4  TREATMENT  NO. FISH  non handled  6  % CONTROL 66.1  U8.6 handled c o n t r o l  5  200 ±16.9  117  110  1 h r . 6 0 % S. W.  5  182 tl5.8  40  32  1 h r . F. W.  5  200 ±16.9  117  110  170 ±24.3  10  45  100  65  100  44  102.2  3 h r . 6 0 % S. W.  5  3 h r . F. W.  5  161 + 21.1  6 h r . 6 0 % S. W.  5  196 ±12.7  73  5  194 *14.4  67  46  5  189 ±15.0  115  62  192 * 9.5  143  6 h r F. W.  12 h r . 6 0 % S. W.  12 h r . F. W.  5  55  100  31.6  100  39.5  100  96.5  100  T A B L E  I I I  DIENCEPHALIC OXYTOCIC ACTIVITY Mg ABSENT 7  non handled  6  221 ±18.6  MU/lOO gm BODY WT. av. (range) 8.3 (7.1-9.6)  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  TREATMENT  NO. FISH  MEAN BODY WT. i n qm. S. E. M.  % CONTROL 72  ll  T A B L E  I V  PITUITARY OXYTOCIC ACTIVITY Mg PRESENT  TREATMENT  NO. FISH  MEAN BODY WT. i n gm. S. E . M.  MU/lOO gm. MU/PIT . BODY WT. av. range) 10.2 10.9 9.6-10.8)  % CONTROL  control  30  93.6 i 5.0  1 h r . 6 0 % S. W.  10  92.7 ±7.4  7.8 7.0- 8.6  8.5  78.7  2 h r . 6 0 % S. W.  10  91.0 ±2.9  6.2 5.7-6.7  7.5  69.4  3 h r . 6 0 % S. W.  10  89.0 ±2.5  6.5 6.1- 6.9  7.4  68.8  4 h r . 6 0 % S. W.  10  117.4 ±13.8  8.4 8.0-8.8  7.2  67.6  6 h r . 6 0 % S. W.  10  91.6 ±3.9  7.6 7.3-7.9  8.3  76.9  8 h r . 6 0 % S. W.  10  87.8 ±6.5  7.6 7.0-8.2  8.7  80.6  10 h r . 6 0 % S. W.  10  85.8 ±2.5  7.5 7.2- 7.8  8.8  81.0  12 h r . 6 0 % S. W.  10  90.9 ±4.7  8.1 7.5-8.6  8.9  82.6  24 h r . 6 0 % S. W.  10  68.2 ±3.5  7.4 7.0-7.8  10.8  100  100  1  T A B L E  TREATMENT  NO. FISH  V  MEAN BODY WT. i n qm. S. E . M.  control  30  93.6 £5.0  MU/BRAIN av. (range) 15.7 (14.8-16.6)  1 h r . 6 0 % S. W.  10  92.7 ±7.4  17.7 (16.0-19.4)  113  2 h r . 6 0 % S. W.  10  91.0 ±2.9  37.8 (30.8-34.8)  240  w.  10  89.0 ± 2.5  4 h r . 6 0 % S. w.  10  117.4 ±13.8  26.1 (22.4-29.7)  166  w.  10  90.9 4.7  23.7 (20.4-27.0)  151  68.2 ^3.5  17.4 (15.1-19.7)  111  3 h r . 6 0 % S.  12 h r . 6 0 % S.  i 24 h r . 6 0 % S.  w.  10  28.6 (25.3-31.9)  % CONTROL  100  183  (Facing figure l ) FIGURE 1 .  Dose response curves from two v i r g i n d i e s t r u s guinea p i g u t e r u s p r e p a r a t i o n s .  (Facing figure FIGURE 2.  2)  T y p i c a l 4 p o i n t A. D. H. assay employing v a s o p r e s s i n (Parke D a v i s ) as s t a n d a r d .  Q  SPECIFIC O O O o  GRAVITY  o o  o o  o o  O  o  O  OF O O CD O  URINE  O O  1  0 . 0 5 mu. VASOPRESSIN  0.10 mu. VASOPRESSIN •n  m  a  c _ m z ho  0 . 0 5 mu. VASOPRESSIN  I  O c  3)  CO  0.10 mu. VASOPRESSIN  CM  _r  O ro O  (Facing FIGURE 3 a .  f i g u r e 3a)  Comparison o f p i t u i t a r y e x t r a c t s from handled f i s h and non-handled f i s h w i t h r e s p e c t to o x y t o c i c and antidiuretic activities.  mu. ro  of  OXYTOCIN  b  b  CD  CT)  b  b  ro O O  o b  ~i  <  o  c33  CD Z  —1  PO  GUI  =  FISH  HANDLED  O  NON-HANDLED  FISH  z m  —  Z H  -  >  CO GO  AY  HANDLED  —  FISH  > —  o c H  X  -<  o o o  >  O —I  < H  •<  m 33  cGO  NON-HANDLED  FISH  i—i  8 m CO 0)  HAN D L E D  FISH  o c  > m  —i  JO 33 NON-HANDLED  TJ  FISH  o  CO CO > -<  HANDLED  NON-HANDLED  ro O  o A  C\7  FISH  FISH  en O  SPECIFIC  co  o  GRAVITY  ro  o o  o 10  e  3)  O >  m  m o  o  O  33 3>  —i  > o  (Facing FIGURE 3b.  figure  3b)  Comparison o f o x y t o c i c and a n t i d i u r e t i c a c t i v i t y of experimental fish pituitary extracts.  ACTIVITY/PITUITARY/IOO gm % CONTROL  BODY WT. OF  S.W.  FISH  ACTIVITY=  X IOO  ACTIVITY/PITUITARY/IOO gm  BODY WT. OF  F.W.  FISH  I 50 > r-  u < _1  o cr  100  o u 50  _L  0  4 HOURS  5  6 IN  60%  FIGURE 3b  7 SEA  8 WATER  _|_ 9  10  11  12  (Facing FIGURE 4.  figure  4)  T y p i c a l 4 p o i n t A. D. H. assay o f p i t u i t a r y e x t r a c t s o f handled and non-handled f i s h .  SPECIFIC O O O O  O O ro O  o o -o o  GRAVITY o o at o  OF  URINE  o o  GO  O  ro O  O  o  0 . 0 2 5 ml. NON-HANDLED  EXTRACT  0 . 0 2 5 ml. HANDLED EXTRACT  8  2 m ro  m  0 . 0 2 5 ml. HANDLED EXTRACT  -  x o c  0.025 ml. NON-HANDLED  CO  w -  EXTRACT  (Facing figure 5) FIGURE 5.  Typical 4 point A. D. H. assay of p i t u i t a r y extract of paired experimental and control f i s h .  1.0120 -  1.0100 -  1.0080 -  I. 0060  1.0040 -  1.0020 -  1.0000 TIME  IN  HOURS  FIGURE 5  (Facing figure FIGURE 6.  6)  E f f e c t of addition of Mg"""in Ringer bath indicating depressed a c t i v i t y due to depletion of the a n t i d i u r e t i c principle.  ACTIVITY/PITUITARY/IOO gm. BODY WT. OF % CONTROL  S.W.  FISH  ACTIVITY =  X IOO  ACTIVITY/PITUITARY/IOO  gm. BODY WT. OF  F.W.  FISH  200  >  OXYTOCIC  ACTIVITY  (PLUS  OXYTOCIC  ACTIVITY  (MINUS Mg.)  ANTIDIUERETIC  ACTIVITY  6 HOURS  Mg.)  8 IN  60%  FIGURE 6  SEA  OL\-  10 WATER  12  (Facing figure 7) FIGURE 7. Oxytocic a c t i v i t y i n brain extracts of experimental f i s h .  OXYTOCIC ACTIVITY/BRAIN/IOO gm. % CONTROL  BODY WT.  S.W.  ACTIVITY^  *  OXYTOCIC ACTIVITY/BRAIN/IOO gm.  X 6 HOURS  8 IN  60%  FIGURE 7  BODY WT. F.W.  10 SEAWATER  12  100  (Facing figure  FIGURE 8.  8)  A hypothetical phylogenetic d i s t r i b u t i o n o f known a c t i v e p o s t e r i o r lobe p r i n c i p l e s . (From Sawyer e t a l 1959)  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 )  PIG  Lysine  MAMMALS Lactation  vasopressin  CysTyr P h e G l u ( N H  appears  ^  2  ) A s p ( N H ) C y S Pro A r g • G l y ( N H )  Arginine  2  2  vasopressin  R E P T I L E S 8 BIRDS AMPHIBIANS Ant/diuresis  appears  Neural  appears  lobe  BONY  C y s T y r l l e u G l u ( N H ) A s p ( N H ) C y S P r o - L e U • Gly ( N H )  FISH  2  ELASMOBRANCHS UNKNOWN  2  Oxytocin  2  ^  PEPTIDE  AGNATHA VERTEBRATES INVERTEBRATES  Cys-Tyr-I l e u - G l u ( N H ) A s p ( N H ) C y S P r o - A r g 2  ^  \  Arginine  2  vasotocin  -^ASCIDIANS-  y^7?  ACTIVITY  A R T H R O P O D S - 7>  FIGURE 8  ACTIVITY  Gly ( N H ) 2  R E S U L T S  I.  Dose Response Relationships A.  Oxytocic A c t i v i t y Throughout the assays of f i s h extracts for oxytocic a c t i v i t y ,  known doses of a standard solution of P i t o c i n (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 i n figure 1.  Since the responses  f i t t e d the l i n e closely no further s t a t i s t i c a l treatment of t h i s data was f e l t to be necessary.  B.  Antidiuretic Activity A plot of a t y p i c a l four-point assay for a n t i d i u r e t i c a c t i v i t y  employing a standard solution of P i t r e s s i n (Parke Davis) i s shown i n figure 2.  This assay of standard vasopressin served only as an  indicator of dose response r e l a t i o n s h i p s .  Other t y p i c a l responses to  extracts of f i s h p i t u i t a r i e s are shown i n figures 3, 4, and 5.  In these  cases comparative differences i n s p e c i f i c gravity between treatments indicated the r e l a t i v e amounts of a n t i d i u r e t i c p r i n c i p l e present i n the extract assayed.  II.  E f f e c t s of Handling Handled f i s h evidenced  a greater amount of oxytocic and a n t i d i u r e t i c  a c t i v i t y i n t h e i r p i t u i t a r i e s when compared to the non-handled f i s h (Tables I and I I , figures 3a arid 4).  S i m i l a r l y , the brains of handled  f i s h had a greater amount of oxytocic a c t i v i t y than did the brains of non-handled f i s h (Table I I I ) . III.  Adaptation to Sea Water A.  Oxytocic A c t i v i t y 1.  Pituitary Assays for oxytocic a c t i v i t y of f i s h p i t u i t a r y extracts  i n a Ringer solution without Mg**ions indicated an i n i t i a l r i s e i n oxytocic a c t i v i t y per p i t u i t a r y for the f i r s t hour a f t e r 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 p r i n c i p l e s on the oxytocic response.  Treatment of p i t u i t a r y extracts with 0.05M sodium t h i o -  g l y c o l l a t e indicated that only known p i t u i t a r y polypeptides were present, since complete i n a e t i v a t i o n of the oxytocic response occurred.  2.  Brain Assays for oxytocic a c t i v i t y with Mg ion present i n  the Ringer's solution indicated the presence of an o x y t o c i c - l i k e substance that dramatically increased i n concentration during the f i r s t two hours after transfer to sea water (Table V, figure 7).  Assays  without Mg^ion i n the Ringer's solution indicated a s l i g h t increase  i n oxytocic-like a c t i v i t y i n the brains of the one-hour experimental f i s h with a subsequent return to control l e v e l s at three hours (Table III and figure 7).  Treatment of these extracts with 0.05M  sodium t h i o g l y c o l l a t e f a i l e d to reduce the a c t i v i t y . B.  Antidiuretic Activity 1.  Pituitary A n t i d i u r e t i c assays using the change i n urine s p e c i f i c  gravity of the water loaded rat indicated  a very marked depletion of  a n t i d i u r e t i c a c t i v i t y of the p i t u i t a r i e s during the three-hour period after exposure to a sea water environment (Table I I , figure 3b, 5, and  6).  Figure 3b indicated  relationship of t h i s decrease with the  increased p i t u i t a r y oxytocic a c t i v i t y for experimental f i s h . An example of the magnitude of t h i s response after three hours i n sea water i s shown i n figure 5.  This depletion of a n t i d i u r e t i c a c t i v i t y was  s i m i l a r l y r e f l e c t e d i n the uterine response to oxytocic material when the assay was conducted i n the presence of Mg^ion (figure 6). This p i t u i t a r y a n t i d i u r e t i c material from both experimental and control f i s h was completely inactivated  by treatment of the extracts  with  0.05M sodium t h i o g l y c o l l a t e . 2.  Brain A n t i d i u r e t i c a c t i v i t y of the extracts  and  of experimental  control f i s h brains was extremely small and rapidly  while conducting the assay. these extracts  inactivated  As a r e s u l t of the unsuitable nature of  t h i s portion of the investigation was discontinued.  Dl  1.  S C U S S I O N  Phvlogenetic Relationships of Neurohypophysial Peptides A.  Chemical Relationships The recent observations by H e l l e r and Pickering (1961) and  Sawyer, Munsick and van Dyke (1961) which have indicated the existence of argenine vasotocin i n the neurohypophysis of elasmobranchs, teleosts, amphibians, r e p t i l e s , and birds has tended to modify our thinking concerning neurohypophysial  hormones i n non-mammalian vertebrates. Previous  consideration of t h i s topic involved oxytocin and vasopressin linked i n a 1:1 r a t i o .  Furthermore the pharmacologic and chromatographic evidence  presented by these workers has shown the absence of vasopressin, both i n the argenine and lysine forms i n the hypothalamo-neurohypophysial system of the lower vertebrates.  Oxytocin f i r s t appears at the l e v e l of the bony  fishes and continues to be present throughout the animal kingdon.  A  summary of the phylogenetic r e l a t i o n s h i p s of these polypeptide hormones and t h e i r structures has been presented  by Sawyer et a l (1959) (figure 8).  In the l i g h t of-these considerations, therefore, we must consider, when dealing with active p r i n c i p l e s from t e l e o s t sources that we are dealing with oxytocin and vasotocin.  Thus we must i n t e r p r e t the present  observations i n 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 i n mammalian systems i t e l i c i t s a c t i v i t y similar to both oxytocin and vasopressin when assayed i n 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 i n a l l assays except those involving uterine preparations without Mg  .  The presence of  oxytocin d i d not appreciably a l t e r the response of the mammalian, avian, r e p t i l i a n , and amphibian assay systems to vasotocin.  I t would appear,  then, that vasotocin i s a functional intermediate between vasopressin and oxytocin and the existence of t h i s functional intermediate has only been demonstrated i n non-mammalian vertebrates. Hi H e r and Pickering (1961) gave a more complete summary of the r e l a t i v e responses of mammalian, avian, and amphibian systems to vasotocin, oxytocin, and argenine and lysine vasopressins.  I t would appear from t h i s  summary that vasotocin i s i n fact responsible f o r the difference i n responses observed employing the frog water uptake assay described by H e l l e r (1941).  Further, avian response such as the hen oviduct assay  indicated that vasotocin i s much more active than oxytocin.  Maetz et a l  (1959) had proposed the presence of N a t r i f e r i n , a new polypeptide i n the neurohypophysis of animals below the mammals. was approximately  present  This preparation  ten times as active as the same measurable quantity of  VJ  oxytocin, using a mammalian assay to standardize the a c t i v i t y , i n promoting an increased sodium flux across i n v i t r o amphibian skin preparations. This evidence tends to suggest that vasotocin i s more active i n lower forms, excepting f i s h , since there have been no reported e f f e c t s of vasotocin on water and e l e c t r o l y t e balance i n f i s h . II.  Transfer to Sea Water A.  Oxytocic Assays of Extracts I f we consider the summary of Sawyer, Munsick, and van Dyke (1959),  p a r t i c u l a r l y the response:of  the r a t uterus with and without Mg ion i n  solution, we see that the a c t i v i t y of vasotocin i s markedly increased i n the presence of Mg^ion.  The present i n v e s t i g a t i o n has employed a similar  technique, varying the concentration of Mgf* ion, to demonstrate the v a r i a b i l i t y i n composition us, we can now  of extracts.  Considering the evidence  i n t e r p r e t the r e s u l t s indicated i n figure 6.  before  In the case  where no Mcj" ion i s i n solution the uterus responds only to oxytocin. 1  On  the other hand, i n the presence of Mg"* ion the uterus responded to both oxytocin and vasotocin.  Thus, any decrease i n vasotocin below control  l e v e l s would be indicated as, i n fact, was the case. has been shown to inactivate a l l known neurohypophysial Munsick, and van Dyke (i960).  Sodium t h i o g l y c o l l a t e hormones (Sawyer,  The p i t u i t a r y oxytocic responses were  t o t a l l y eliminated when our extracts were s i m i l a r l y treated.  Thus the  uterine responses observed from p i t u i t a r y extracts are t o t a l l y due to only either oxytocin or vasotocin.  On the other hand, the brain extracts  contained a considerable amount of oxytocic a c t i v i t y that was inactivated by sodium t h i o g l y c o l l a t e .  not  This suggested the presence of some  unknown substance that fluctuates upon, handling of f i s h and upon placement  i n hypertonic environments. neurointermediate  The presence of a similar substance i n the  lobe of elasmobranchs was reported by Sawyer, Munsick  and van Dyke (1961).  However, since r e l a t i v e l y large amounts of tissue  were employed for obtaining extracts i n the present work there exists a p o s s i b i l i t y of i n t e r f e r i n g substances such as 5 hydroxy-tryptamine being present. Considering a l l the evidence concerning the presence and absence i n non-mammalian vertebrates and the physiological properties, both oxytocic and a n t i d i u r e t i c , of vasotocin, i t would appear that the present observations were at least i n part due to t h i s polypeptide. trout to 60% sea water evidenced  Transfer of  a decline i n oxytocic a c t i v i t y only i n  assays that employed magnesium ions i n the Ringer s o l u t i o n .  I f we  accept  the presence of vasotocin, then t h i s decline i n a c t i v i t y after transfer to sea water can be explained only by a depletion of vasotocin i n the p i t u i t a r i e s of the experimental B.  fish.  A n t i d i u r e t i c Assays of Extracts Sawyer, Munsick, and van Dyke (1961), and H e l l e r and Pickering  (1961) showed that vasotocin caused a n t i d i u r e s i s i n water loaded, anaesthetized r a t s .  ethanol  Furthermore, these investigations have shown the  presence of vasotocin i n the p i t u i t a r y of trout.  Thus, i f we consider the  change i n a n t i d i u r e t i c a c t i v i t y upon transfer of trout to sea water as indicated by figures 3b, 5, and 6, we see there was of an active substance, perhaps vasotocin.  i n fact a depletion  Inactivation with sodium  t h i o g l y c o l l a t e indicated the absence of substances other than oxytocin, vasotocin, and the vasopressins.  Again, since vasopressin i s absent i n  lower vertebrates, the observed response was probably due to vasotocin,  since Berdi and Cerlette (1956) have shown oxytocin to be a d i u r e t i c agent, t h i s would eliminate any probable a n t i d i u r e t i c e f f e c t due to oxytocin. I f we now  consider the brain extracts, and their high oxytocic-  l i k e a c t i v i t y , i n the l i g h t of the work of Berdi and Cerlette (1956), we may  conjecture that the interference from t h i s unknown source perhaps i n  the d i r e c t i o n of d i u r e s i s , was  so great as to t o t a l l y suppress the  a n t i d i u r e t i c a c t i v i t y of the vasotocin present.  Further, Knoble (1957),  pointed out that t h i o g l y c o l l a t e i n a e t i v a t i o n of active p r i n c i p l e s present i n concentrated factory.  extracts, as i s the case of brain extracts, i s unsatis-  This may  indicate that there was  i n fact a r e l a t i v e l y high  concentration of o x y t o c i c - l i k e material present  i n the hypothalamic region  of the brain under the present experimental conditions. C.  Integrated  Considerations  The present evidence indicates that vasotocin may i n the transfer of f i s h to a hypertonic medium. depletion of a pharmacologically  be implicated  We have observed a  active substance i n the p i t u i t a r y 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 p o s i t i v e stainable material from the hypothalamo-neurohypophysial system i n f i s h upon transfer to a hypertonic  environment.  substantiate these authors* observations.  Our evidence tends to further Russel, Rennels and Drager (1955)  studied the variations of oxytocin and Gomori p o s i t i v e materials i n the hypothalamus and p i t u i t a r y of the r a t . electroshocking, dehydration,  Three treatments were carried out;  and adrenalectomy.  The oxytocic content did  not vary on shocking, f e l l to 20% of normal l e v e l s on drinking 2.5% saline for ten days, while a f t e r adrenalectomy the oxytocic content doubled.  On  the other hand, the Gomori p o s i t i v e material f a i l e d to change during shocking, was depleted during saline treatment, but did not change a f t e r adrenalectomy.  This evidence would suggest that Gomori p o s i t i v e staining  i s not always a r e l i a b l e index of the oxytocic l e v e l i n t i s s u e s . The e f f e c t s of handling were considered to be a problem i n itself.  For the purpose of this i n v e s t i g a t i o n i t was  experimental  f e l t that an  design had to be adopted so that control groups and experi-  mental groups were handled equally thus eliminating any unwanted handling effects.  The b i o l o g i c a l significance of the increase of active p r i n c i p l e s  i n the posterior lobe as a r e s u l t of handling remains i n the realm of speculation.  Since vasotocin i s involved, i t would appear dangerous to  state that vasotocin may  have a s i m i l a r property to vasopressin,  causing  the release of ACTH i n f i s h as does vasopressin i n mammalian forms (Sawyer, Munsick, and van Dyke, 1959). Further consideration was  given to the f l u c t u a t i o n i n levels of  active p r i n c i p l e s of the p i t u i t a r y and the hypothalamus. that a depletion of the p i t u i t a r y contents was the hormones into the c i r c u l a t o r y system. hypothalamic region of the brain was  I t was  felt  i n d i c a t i v e of a secretion of  Further, an increase i n the  an i n d i c a t i o n of increased synthesis.  After a depletion of p i t u i t a r y contents a replenishment to control l e v e l s was  considered to be evidence of a higher rate of production of active  p r i n c i p l e s i n the hypothalamic region than was  being secreted.  appear from t h i s , that fluctuations i n p i t u i t a r y concentrations  I t would of  posterior lobe hormones were an i n d i c a t i o n of a mobilization of these hormones.  Only by measuring c i r c u l a t o r y levels of active p r i n c i p l e s 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  v i r t u a l l y impossible to obtain when consideration was given to the a v a i l a b i l i t y of assay techniques that were s u f f i c i e n t l y sensitive for measuring c i r c u l a t i n g levels of active posterior lobe p r i n c i p l e s of f i s h exposed to a hypertonic environment.  C O N C L U S I O N S  1.  F i s h t h a t were exposed t o e x c e s s i v e h a n d l i n g evidenced a g r e a t e r  amount o f a n t i d i u r e t i c and o x y t o c i c handled  a c t i v i t y p e r p i t u i t a r y than non-  fish.  2.  Transfer  o f f i s h t o sea water r e s u l t e d  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 hour a f t e r t r a n s f e r w i t h a r e t u r n 3.  p i t u i t a r y extracts control  of fish transferred  l e v e l s a t the s i x t h hour.  a c t i v i t y was evidenced i n the t o sea water f o r one hour, w h i l e  l e v e l s were m a i n t a i n e d f o r the remainder o f e x p e r i m e n t a l  4. of  d u r i n g the f i r s t and t h i r d  to control  Increased p i t u i t a r y oxytocic  i n a reduction of  Oxytocic a c t i v i t y of brain extracts  was i n c r e a s e d  periods.  upon t r a n s f e r  f i s h t o sea water.  5. oxytocic  A d d i t i o n yof Mg** ions t o the R i n g e r bath r e s u l t e d -  i n an enhanced  response i n d i c a t i n g the presence o f a c t i v e p r i n c i p l e s o t h e r than  oxytocin,  i n both the b r a i n and p i t u i t a r y e x t r a c t s .  6.  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