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The respiration rates of excretory tissues in the cutthroat trout (Salmo clarki clarki) Stott, Gael Harling 1959

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THE RESPIRATION RATES OF EXCRETORY TISSUES IN THE CUTTHROAT TROUT (Salmo c l a r k i c l a r k l ) b y GAEL HARLING STOTT B . S c , 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 , 1958 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n t h e D e p a r t m e n t o f Z o o l o g y We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF B R I T I S H COLUMBIA J u l y , 1959 i ABSTRACT The oxygen consumptions of g i l l and kidney tissues of the cutthroat trout (Salmo clarki clarki) were determined by the direct method of Warburg. The respiration rates of tissues from f i s h ranging from 10 to 100 gm. were examined in relation to body weight. A decline in weight specific oxygen consumption for both t i s -sues was observed. On a log-log plot, the regression coefficient for kidney was -.148 while that for g i l l was -.139. The decline did not support the .73 rule (Brody, 194-5) at the level of tissue respiration. The oxygen consumptions of kidney and g i l l tissues were examined during a 168 hour period after transfer of the f i s h from fresh water to 65$ standard sea water. A sharp i n i t i a l rise in Q02 of kidney tissue was noted during the f i r s t 48 hours after transfer, reaching a maximum at 20 hours. The kidney tissue respiration during the remainder of the experi-mental period remained significantly higher than the parallel control level. The g i l l tissue respiration declined rapidly during the f i r s t 10 hours after transfer and remained s i g n i f i -cantly below the control level during the whole experimental period. The results are discussed in relation to recent observations of Holmes, Chester Jones, Phil l i p s , and Sexton, concerning possible hormonal regulation of salt-electrolyte and water metabolism by vasopressin and adrenocortical steroids in euryhaline species of salmonids.' In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Zoology The University of B r i t i s h Columbia, Vancouver Canada. i i TABLE OF CONTENTS Page INTRODUCTION ( 1) I. Metabolism and Body Size ....( 1) II. The Sites of Osmotic Regulation ( 4) A. Osmoregulation and Environment ( 4) B. Sites of Osmotic Exchange ( 5) 1) Skin... ( 5) 2) Kidney ( 6) 3) G i l l s ( 8) III. Changes Occurring on Transfer to Sea Water....(10) A. Physiological Changes (10) B. Hormonal Changes (11) IV. Scope of Thesis (13) MATERIALS AND METHODS (15) I. Respirometry (15) II. Regression Lines (17) III. Transfer to Sea Water (17) RESULTS AND DISCUSSION (19) I. Tissue Respiration and Body Weight (19) A. Results (19) 1) Kidney (19) 2) G i l l s * (19) B. Discussion (24). i i i I I . Effect of Transfer to Sea Water (27) A. Results.. (27) 1) Kidney (27) 2) G i l l s (28) B. Discussion (34) CONCLUSIONS (38) APPENDIX - EFFECT OF VASOPRESSIN ON OXYGEN CONSUMPTION IN THE KIDNEY (39) I. Materials and Methods (39) A. Effects of Vasopressin in Vitro ...(39) B. Effects of Vasopressin in Vivo .....(39) I I . Results and Discussion (42) BIBLIOGRAPHY (46) iv LIST OF TABLES AND FIGURES Page Table I . . . . ( 2 1 ) Figure 1 (22) Figure 2 (23) Table II (29) Table III (30) Table IV ( 3 D Figure 3 (32) Figure 4 (33) Table V (41) V ACKNOWLEDGMENT I would li k e to thank my research director, Dr. W.N. Holmes, for the help and encouragement he has given me. both in the lab and during the writing of this thesis. I would also l i k e to express my appreciation to the other members of my committee: to Dr. P.A. Larkin for his help with the s t a t i s t i c a l analysis of the results, and to Dr. P. Ford and Dr. CV. Finnegan for their suggestions throughout the year. In addition to the individuals who have given me generous assistance this year, I would also l i k e to thank the National Research Council for the bursary which enabled me to undertake this project. 1 INTRODUCTION As a group, the Salmonoidae have remarkable a b i l i t i e s to withstand wide variations in the tonicity of their environment without undergoing commensurate changes in their body fl u i d s . Indeed, the salmon experiences extreme environmental changes during the natural course of i t s l i f e history. Considerable physiological adaptation with respect to water and electrolyte metabolism must be made for these f i s h to survive. The cutthroat trout (Salmo clarki clarki) in nature may or may not go to sea. The stocks used in this work probably had some "sea run" propensity, and hence displayed a toleration for a f a i r l y wide salinity range. This study involved the examination of some of the physiological changes occurring when the f i s h is transferred abruptly from fresh to sea water. I. Metabolism and Body Size The size of the f i s h available for the study of the physio-logical adaptations associated with the transfer to sea water varied widely during the course of the experiments. Wide variations i n the oxygen uptake of g i l l and kidney tissues from normal f i s h also occurred. Therefore, a knowledge of any weight specific variation between oxygen consumption and total body weight was necessary before valid interpretations of the experi-mental data could be made. There is abundant evidence in the literature to indicate a relationship between body weight and metabolism. Brody (194-5) 2 compared data from mature mammals and found that basal metabol-ism changed regularly from 8 Calories per Kg. for the elephant to 200 Calories per Kg. for the mouse. Kleiber (194-7) carried out a similar comparison of metabolic rates from animals ranging in size from the mouse to the cow. These workers found that the following relationship applied: log M • log b + ot log W (1) or M = bW (2) where M is the metabolism, W, the body weight, the exponent , the slope of the line on a log-log plot, and b, the intercept indicating the value of M when W i s 1. Brody (1945) and Kleiber (1947) concluded that the metabolism of mature homoiotherms tended to vary with the .73-»?5 power of the body weight. Benedict (1938) considered metabolism to be directly proportional to the surface area of the body, i.e. M = bW*^. However, the value of oi for Benedict's data was .73 when certain atypical species were omitted (Brody, 1945). The generalized formula of Brody, giving an exponent of .73? was calculated from the regression of log metabolism vs. log body weight for mature homoiothermic species. With the excep-tion of small metazoans, Zeuthen (1953) found the same inter-specific generalizations applicable to the poikilotherms. He l i s t s oc values of .7 for unicellular organisms, .95 for small metazoans, and .75 for large metazoans. On a log-log plot, he found the same slope for homoiotherms and poikilotherms. Weymouth's (1944) data showed an ot value for crustaceans of 3 .8. However, the intercept for the poikilotherms was lower than that for homoiotherms (Zeuthen, 1953)* Zeuthen (1953) also compared the changes i n metabolism with ontogenetic increases in the size of an organism. He recognized a similar triphasic curve for the oxygen consumption during the development of several poikilotherms. The larval phases showed oC values approaching 1.0 whereas the embryonic and post-larval stages of development showed oC values of approximately .7. As in the case of interspecific metabolism, the intraspecific variation in metabolism with increasing body was not so constant among the poikilotherms as the homoiotherms. (Weymouth, 194-4). Both the intraspecific and interspecific log-log plot of metabolism vs. body weight show a positive regression. If the metabolism i s expressed i n units of metabolism per unit body weight, then a weight specific metabolism i s obtained. When equations (1) and (2) are thus treated, the following equations for weight specific metabolism are obtained: log M - log W = log b * oc log W - log W = log b +• ( OL - 1) log W (3) or M bW^"1^ (4) W The observed decline in weight specific metabolism with the increasing size of the organism has stimulated several workers to investigate the metabolism of individual tissues from animals of differing sizes. Equation (4) can be used to compare Q02 values. However, i t must be assumed, in relating the tissue metabolism per unit weight of tissue to the total body weight 4 by equation (4), that the tissue size and the total body size Increase proportionately. In the case of weight specific metabolism, exponents of -.27 and -.33 correspond to the .73 and the surface rules respectively. Krebs (1950) studied the oxygen consumption of five different tissues from nine mammalian species, finding the Q02 values for homologous tissues lower in the larger animals. He found that the decrease of Q02 values for the kidney cortex was much smaller than the decrease of rate of basal heat production. His results gave an interspecific value for (c£-l) of-.064. Bertalanffy and Pirozynski (1953) did not find any significant regression of kidney QQ2 values with body weight for the rat. Krebs (1950) did not find any tissues which conformed to the classical rules, while Bertalanffy and Pirozynski (1953) found only thymus and dia-phragm conforming ( (ot-1) = -.26). Vernberg (1954) carried out an investigation involving marine teleosts. He examined the weight specific respiration rates of brain, l i v e r , and skeletal muscle for the toadfish (Opsanus tau). and found that only liver showed a negative regression ( (aL-1) ~ -.145). II. The Sites of Osmotic Regulation A. Osmoregulation and Environment In f i s h , osmotic regulation varies according to the envi-ronment. Stenohaline forms are able to tolerate only narrow ranges of salt concentration in the water. In fresh water f i s h , the osmotic concentration of the body fluids (depression of 5 freezing point, A = 0 . 5 7 ° O i s higher than that of their envi-ronment (A=0,) (Black, .1957). Water tends to diffuse into the tissues and salts diffuse from the body to the external environ-ment. The fresh water fish must, therefore, alleviate the tendency towards hydration of body tissues and conserve elec-trolytes. In marine f i s h the opposite problems arise. Since the concentration of the body fluids (A =0.78) is lower than that of sea water (A = 2.0) (Black, 1957), the f i s h tend to lose water across the g i l l s and other body surfaces. They gain water by the ingestion of food and by swallowing sea water, but much of this is lost extra-renally. Smith (1930) found that Ql% of the water swallowed by the eel was absorbed through the gut and 59$ was lost extra-renally, while in the sculpin 63% was ab-sorbed and 31$ lost. Euryhaline fishes are able to tolerate a wider salinity range. On transfer to fresh water, marine forms tend to gain water and lose salts. They adapt by increased urine flow and increased reabsorption of electrolytes. The anadromous and catadromous fishes, such as the salmon and the eel, may u t i l i z e hormonal activity to produce physiological changes enabling them to survive i n their new environments. B. Sites of Osmotic Exchange The main sites of osmotic exchange in the fishes are skin, the kidney, and the g i l l s . 1) The Skin Injury to the skin or removal of the mucus covering.impairs 6 the resistance of the f i s h to the osmotic pressures of their environment. In marine species, injury leads to a water diuresis and an increase in body chloride concentration (Grafflin, 1931). Raffy (194-9) found that Blennius pholis L. became stenohaline i f the mucus was washed off, and the removal of mucus from the elvers of Angullla anguilla led to a rise in the osmotic pressure of the blood and to death (Firly, 1932). Krogh (1937) also found that damage to the skin accelerated the loss of salt by eels held i n fresh water. Pickford (1957) states that the results of Grafflin, Raffy, and F i r l y indicate that the intact skin of teleosts is impermeable to the external medium and that the impermeability depends on the secretion of the mucus glands. The skin of the fresh water teleost has a very low permeability to water. This is emphasized by the find-ing that 1 ml. of water takes 158 days to pass across 1 sq. cm. of goldfish skin, and 5 years to pass across the same area of eel skin (Krogh, 1939, and Keys, 1933). Holmes (1959) also found that the skin of the rainbow trout was only a minor site of sodium transfer. 2) The Kidney Fresh water f i s h have a well developed glomerular kidney, which enables them to excrete water as a hypotonic urine (A = 0.7-0.9) (Black, 1957). The urine flow i s copious, that for the rainbow trout ranging 60-106 ml. per kg. body weight per day (Krogh, 1937). Marshall (1934) found that the catfish produced 300 ml. of urine per kg. per day, and Smith (1932) 7 indicated urine flows of 200-400 ml. per kg. per day for the carp, the goldfish and the eel. In marine fish, glomeruli may be present or absent, but the distal convoluted tubule is normally absent (Edwards, 1935)* The proximal convolution is thus the only indispensible part of the kidney. Marshall and Grafflin (1932) found that water reabsorption takes place in the kidney tubule in glomerular teleosts. Clarke (1934) demonstrated a 78.6$ reabsorption of the glomerular f i l t r a t e by the kidney of the sculpin. Only a small amount of urine i s excreted by marine teleosts, about 3 ml. per Kg. per day (Clarke, 1934). Grafflin (1931) recorded urine flows of 2.5 ml. per kg. per day i n the aglomerular toad-f i s h and less than 4 ml. per kg. per day in the sculpin pos-sessing glomerular kidneys. Although the urine i s hypotonic ( A = 0.62-0.79), the concentration is very close to that of the blood ( A » 0.72-0.82) (Smith, 1932 and Forster, 1953). There-fore, most of the water must be reabsorbed although Smith attributes the low urine flow in the sculpin to a greatly re-duced glomerular f i l t r a t i o n rate. Studies of the structure and function of the aglomerular kidney of marine fishes have indi-cated that the aglomerular kidney corresponds to the second segment of the proximal convoluted tubule i n glomerular forms (Edwards and Condorelli, 1928, Edwards, 1929, Marshall,.1930 and Grafflin, 1937). The substances to be excreted from the blood are secreted into the kidney tubule accompanied by small amounts of water (Marshall and Grafflin, 1932). Studies involving the 8 isolated kidney of the flounder showed that phosphate bond energy, oxygen, cytochrome oxidase, and dehydrogenases were necessary for phenol red transport (Forster, 194-8, Taggart and Forster, 1950, and Forster and Taggart, 1950). Calcium, magnes-ium, sulfate, and phosphate ions, absorbed through the gut, are excreted actively by the kidney (Smith, 1930, 1932, Forster, 194-8, Forster and Berglund, 1956 and Berglund and Forster, 1958). In euryhaline f i s h entering fresh water, increased glomerular f i l t r a t i o n rate and diuresis are necessary to enable the f i s h to excrete excess water crossing the integument. Clarke (1934-) noticed that the diuresis due to handling of f i s h was caused by increased glomerular f i l t r a t i o n rate with a constant reabsorption of water. Reabsorption of salts by the kidney tubule i s important i n a hypotonic environment. When euryhaline f i s h enter sea water, the increased concentration of body fluids due to water loss automatically reduces the urine loss. 3) The G i l l s Keys (1931a, b), by means of a heart-gill perfusion appara-tus, studied osmoregulation by the g i l l of the eel, Anguilla vulgaris. He found a dilution of the perfusion medium when the external medium was fresh water, due to the inward diffusion of water through the g i l l surface. Krogh (1937) found that fresh water fishes actively absorbed chloride ions from very dilute solutions when the salt content of the body fluids was depleted. These ions were usually accompanied by cations but 9 could be exchanged against bicarbonate. Krogh states that the g i l l s of the goldfish have independent mechanisms for absorbing anions and cations. The anion mechanism absorbs chloride and bromide. The cation mechanism takes up sodium ions, but not potassium. Sexton and Meyer (1955) found that goldfish g i l l s could absorb lithium as well as sodium, but not potassium or cesium. Meyer (1952) and Sexton and Russell (1955) have sug-gested an analogy between the function of g i l l and kidney epithelial tissues. These workers found that mercurial diu-retics inhibited the active sodium uptake and increased the sodium loss from the goldfish g i l l . Marine f i s h , which swallow sea water to prevent dehydra-tion must excrete the excess salts to maintain a low body f l u i d concentration. Smith (1930) noted that osmotic work was necessary for extra-renal excretion of water and electrolytes in a marine environment. He suggested that the g i l l s were the site of this extra-renal regulation. Perfusion experiments by Keys (1931b)involving a hypertonic environment showed that chloride was secreted from the perfusion medium against a large concentration gradient. He pointed out a similarity between the activity of the g i l l of the sea water eel and the normal mammalian kidney. Keys and Wilmer (1932) found secretory c e l l s in the g i l l s of the eel, and indicated a possible correlation between them and the chloride secreting activity of the g i l l s . Copeland (194-8, 1950) studied the histology of the chloride secreting cells of the euryhaline fish, Fundulus heteroclitus. 10 He ascribed the function of chloride exchange to certain columnar acidophilic cells r i c h in mitochondria. The cells have a reversible polarity, secreting chloride in sea water, and absorbing i t from a hypotonic environment. Since an "excretory vesicle" containing chloride was present in sea water and in saline injected f i s h , Copeland (1948) concluded that the activity of the cells was determined by the internal environment. The activity of the cells in fresh water was correlated with a high concentration of alkaline phosphatase (Copeland and Pettengill, 1948). Getman (1950) noticed similar changes in the chloride secreting cells of Anguilla rostrata. Bevelander (1935» 1936) assigned the function of chloride secretion to the whole respiratory epithelium rather than to single ce l l s , although he noticed that the mucus cells, of various stages of secretory activity, could be localized or scattered. Copeland (1950) agrees that the chloride c e l l may be a modified mucus c e l l . III. Changes Occurring on Transfer to Sea Water A. Physiological Changes Busnel (1942) found that exposure of stenohaline and euryhaline fishes to varying s a l i n i t i e s caused changes i n the concentration, the hematocrit, and the pH of the blood. Portier and Duval (1922a, b) and Fontaine, Dellatre, and Callamand (1945) showed that transfer of fresh water fishes to moderate sa l i n i t i e s caused^ an i n i t i a l rise in the blood con-11 centration and the plasma-cell ratio. Busnel (1942) indicated that there is a gradual return to normal. Parry (1958) found that salmonid fishes able to withstand transfer into sea water showed an i n i t i a l rise in blood concentration with a subse-quent f a l l to normal levels. In rainbow trout, Busnel, Drilhon, and Raffy (194-6) found that adult f i s h could with-stand a gradual transfer to high sal i n i t i e s (A= 1.90), but the concentration of their internal milieu increased (from A =.50 to A -1.03). These workers suggested that rain-bow trout were intermediate between the stenohaline (carp) and euryhaline (eel) teleosts in their a b i l i t y to regulate osmotically. B. Hormonal Changes In the mammal, hormonal principles from the neurohypophysis and adrenal cortex are active in water and salt-electrolyte metabolism. Polypeptides have been isolated from the neuro-hypophysis which have vasopressor, antidiuretic, and oxytocic effects. Vasopressin, or antidiuretic hormone, causes increased water reabsorption in the d i s t a l convoluted tubule of the kidney. The amphibian water balance principle from the neuro-hypophysis also causes water retention in the Anura. From the adrenal cortex, the mineralocorticoids, especially aldosterone, effect the retention of sodium by modifying the active reab-sorption from the kidney tubule. These hormones may also govern osmoregulation in fishes. The hormones of the neurohypophysis have been isolated in 12 fishes. Heller (1941, 1945) demonstrated the presence of the antidiuretic hormone in the cod pituitary. Fontaine (1956) found strong antidiuretic activity, according to mammalian bioassay techniques, in the pituitary of eels and salmon i n fresh water, and in the pike, a fresh water teleost. Many workers found that f i s h do not respond to the antidiuretic action of either mammalian or f i s h vasopressin by a weight increase (Burgess, Harvey and Marshall, 1933? Boyd and Dingwall, 1939, Fontaine and Raffy, 1950, Callamand, Fontaine, Olivereau, and Raffy, 1951? and Fontaine, 1956). Sexton (1955), however, found that vasopressin caused an increased flow of urine in the goldfish, accompanied by an increased water uptake by the g i l l . These observations may explain the failure of other workers to record a weight increase in fishes injected with vasopressin. Several adrenocortical steroids have,been characterized in f i s h blood. Hatey (1954) studied the corticosteroids i n the blood of the salmon and found the concentration to be highest at the smolt stage. Phi l l i p s , Holmes, and Bondy (1959) characterized C o r t i s o l , cortisone, corticosterone, and aldosterone in the plasma of spawning male salmon. Chester Jones (1956) has outlined the features common to the teleost interrenal organ and the mammalian adrenal cortex. In Astyanax  mexicanus, the interrenal body responded to mammalian co r t i -cotropin (ACTH) as well as to f i s h pituitary extracts, and hypophysectomy of Anguilla anguilla, caused a decrease in the 13 weight of the interrenal which could be restored by ACTH (Rasquin, 1951, Rasquin and Atz, 1952, Fontaine and Hatey, 1953)* He also found that hormones affecting salt-electrolyte metabolism in eutherians (desoxycorticosterone acetate and cortisone) influence the sodium and potassium levels in the blood and muscle i n f i s h (Chester Jones, 1956). IV. Scope of the Thesis The purpose of this thesis is to investigate the changes in the metabolic activity of the g i l l and kidney tissues when cutthroat trout are transferred abruptly from fresh water to a hypertonic medium. The preceding sections show that there is ample evidence for the osmoregulatory function of the g i l l and kidney tissues. From the proved experimental differences in the active excretion of water and electrolytes in fresh water and marine fishes, one might expect variations in the metabolism of these tissues i n the different environments. Oxygen consumption, since tissue oxidation and energy-producing phosphorylation systems are normally linked, i s used as an index of the energy expended by the g i l l s and kidney during osmoregulation. The effects of vasopressin, the posterior pituitary hor-mone active in mammalian water balance, on oxygen consumption was investigated. Holmes (1959) found that vasopressin almost completely blocked renal sodium excretion in saline loaded f i s h . Since vasopressin appears to be active i n fishes, an attempt was made to correlate a possible action of vasopressin 14 in the osmoregulation of fishes, by comparing the metabolic activity of tissues from sea water and vasopressin treated f i s h . 15 MATERIALS AND METHODS I. Respirometry Cutthroat trout, (Salmo cla r k i clarki) which had been rear-ed in the Brit i s h Columbia Game Department Hatchery at Cultus Lake, B.C., were used throughout these experiments. No attempt was made to sex the animals. Prior to use the f i s h were held in running dechlorinated tap water (temperature about 8°C) i n large cement troughs. The oxygen consumption of g i l l and kidney tissue was deter-mined by the direct method of Warburg as outlined by Umbreit, Burris, and Stauffer (1958). The centre well of each flask contained 0.2 ml. of 20$ KOH solution and a wick of Whatman #1 f i l t e r paper. The main compartment contained 2.7 ml» of Krebs' saline solution (1950) modified for trout tissues. Krebs' saline solution was specifically formulated for mammalian tissues. The total osmolar concentration of this solution was varied u n t i l the depression of freezing point and pH were equal to those of cutthroat trout blood ( A = 0.58°C, pH = 7.2). The composition of this modified Krebs' solution was as follows: Substance Concentration Parts NaCl 0.935 103 KC1 1.19 4 KH2P04 2.19 1 MgS04.7H2o 3.97 1 16 Substance Concentration Parts NaHC03 P0 4 Buffer 1.35 3 18 PO^ Buffer: Na2HP04 1.47 4 NaH2P04.H20 1.43 1 No substrates were used i n this medium since previous results had been more variable in the presence of the recommended substrates. The f i s h were k i l l e d by a blow on the head and immediately weighed. After the digestive tract and swim bladder were removed, the dorsal side of the kidney was gently scraped free with curved forceps and the organ removed. The kidney was then blotted to remove blood and l a t e r a l l y sliced freehand with a razor blade. The g i l l s were removed whole and rinsed in ice cold Krebs' solution. The filaments were then cut free of the cartilaginous g i l l bars. Approximately 100 mg. wet weight of tissue were placed in each flask. A l l flasks were gassed with therapeutic oxygen (B.P. and U.S.P. specifications) for 12 minutes and were allowed to equi-librate in the constant temperature bath at 30°C for 15 minutes. The flasks were agitated at the maximum rate and amplitude of shaking. Manometer readings were taken at 10 minute intervals and the total oxygen consumption for the 60 minute period was calculated from a graph of reading vs. time. After the f i n a l 17 reading, the tissues were dried for 48 hours at 101°C to deter-mine the dry weight values. The oxygen consumptions of the tissues were expressed in microlitres ( s mm.3) per mg. dry weight of tissue per hour ( Q c ^ ) * II. Regression Lines i'Og Q02 ^ O T e a c n tissue was plotted against log body weight (in gm.). The ALWAC III-E d i g i t a l computer was used to calculate regression coefficients by the method of least squares. The programme for this s t a t i s t i c a l analysis i s available at the computing centre, U.B.C, Vancouver, B.C. Correlation c o e f f i c i -ents (r) were calculated according to the method outlined by Snedecor (1946). The 95$ confidence limits on mean values of Q02 appropriate to various values of body weight were calculated by the method outlined by Snedecor (1956). The observed regres-sion coefficients were compared to a theoretical regression of -.27 using the procedure of Snedecor (1946). III. Transfer to Sea Water To determine the changes occurring on transfer of the f i s h to a hypertonic medium, the f i s h were pretreated by holding them i n sea water. The sea water was prepared by mixing 3 parts of ocean sea water to 1 part of dechlorinated tap water. This had a salinity of 62-65$ of standard sea water (NaCl = 31.88$), when tested by a conductivity meter, and caused a depression of freezing point, A « l.29°C. During the experiment, the f i s h were transferred from the 18 storage tanks to battery jars containing 4 l i t r e s of sea water for a specified period of time. Two f i s h were placed in each jar, and duplicate determinations were made. Parallel controls were run in jars containing dechlorinated tap water. The experiment was repeated varying the length of time the f i s h were l e f t i n the jars. The water temperature was held constant between 7-8°C by keeping the jars in a trough of running water, and the water i n the jars was aerated by individual bubbling stones connected to a compressed air source. 19 RESULTS AND DISCUSSION I, Tissue Respiration and Body Weight  A, Results 1) Kidney (Table I and figure 1) The weight specific equation (4) was applied to the QQ2 values obtained for cutthroat trout tissues. The kidney values plotted i n figure 1 were obtained from 83 estimations involving f i s h varying in body weight from 10 to 100 gm. These values were found to have a regression of -.148 with a mean body weight of 23.2 gm. and a mean Qo2 of 6.77. The equation for this line was: log QQ2 = 1.0327 - .148 log W or QQ2 = 10.78 W r.148 The correlation coefficient (r) of .42 was rather low, but with 81 degrees of freedom, the f i t of the line was highly s i g n i f i -cant (p^L.001) (Table I). The weight specific values were used since i t was impossible to remove the entire kidney from the f i s h . 2) G i l l (Table I and figure 2) The equation (4) was again applied to the Q02 values for g i l l tissue of the cutthroat trout. Estimations were made on 143 f i s h varying in weight from 10 to 100 gm. The regression of the values was -.139 with a mean body weight of 26.1 gm. and a mean QQ2 of 5«79« The equation for the line was: 20 log Qo2 = 0.9597 - .1391'log W or Q 0 2 = 9.114 W -•139 The wide variation in values gave an even lower correlation coefficient than the kidney tissue ( r = .32), but with 141 degrees of freedom, the f i t of the slope was significant (p <.001). (Table I). The dotted lines on figures'1 and 2 represent the inter-vals within which 95$ of the mean Q02 values would f a l l for the corresponding body weight. Table I shows the results of the regression lines for g i l l and kidney. TABLE I. REGRESSION OF KIDNEY AND GILL Q02 WITH BODY WEIGHT Tissue N Mean Body Weight i n gm. Mean b 1 S ( l o g y . l o g x ) 3 j>4 P value 5 Kidney 83 23.2 6.77 10.780 -.148 .0112 .42 <.005 > .oo i G i l l 143 26.1 5.79 9.1U -.139 .0100 .32 <.005 > . o o i when body weight = 1 gm. Gradient o f l i n e on a l o g - l o g p l o t S ( l o g y . l o g x) = standard d e v i a t i o n of the regression c o e f f i c i e n t 4 C o r r e l a t i o n c o e f f i c i e n t 5 P value when (oC.-l) compared to t h e o r e t i c a l regression of -.27 (Facing figure 1) FIGURE 1. Regression of kidney Q02 w i t t l body weight of the cutthroat trout. 15 (Facing figure 2) FIGURE 2. Regression of g i l l Q02 with body weight of the cutthroat trout. FIGURE 2 24 B. Discussion Neither the exponent for the kidney nor that for the g i l l s agreed with the surface rule or Brody's (194-5) .73 rule relat-ing body weight and metabolism. The regression coefficients of the 0,02 v a l u e s £ ° r kidney and g i l l (-.148 and -.139) were s t a t i s t i c a l l y compared to a theoretical value of .27 corres-ponding to the .73 rule. Since the comparisons showed a significant difference from the .73 rule for both tissues (kidney, p <.005 >.001, and g i l l , p <£.005 >.001), the obser-vations do not support the .73 rule at the level of tissue respiration. The exponents of the body weight for kidney and g i l l tissues didnot differ significantly (-.148 vs. -.139), sug-gesting that the rate of decline in weight specific oxygen consumption was the same for g i l l and kidney tissue. Bertalanffy and Pirozynski (1953) recalculated Krebs' (1950) data for nine mammalian species and obtained an interspecific (oC-1) value of -.064 for kidney. They did not find any significant regression of kidney G,Q2 values in their own data. Examination of the integration constants (b) showed that the Qo2 values of the kidney and g i l l tissues were on the same level (Table I, .b = 10.78 vs. 9.11). This fact provides further evidence to support the idea that the osmoregulatory work is equally distributed between the g i l l s and kidneys i n teleosts. Holmes (1959) showed that, in the sodium loaded rainbow trout, the excretion of sodium was equally divided 25 between the g i l l s and the kidney. Vernberg (1954) studied the weight specific respiration rates of brain, l i v e r and skeletal muscle from the marine teleost, Opsanus tau. Both brain and muscle showed positive regressions with (ot -1 ) values of .202 and .118 respectively. Liver showed a negative regression of approximately similar value to those obtained for g i l l and kidney of the cutthroat trout ( (oc-l) a - . 1 4 5 ) . Since Vernberg expressed the Q o 2 values in microlitres of oxygen per gm. wet weight of tissue per minute, i t was necessary to evaluate his results in terms of mg. dry weight of tissue per hour to compare the two sets of data. The dry weight values for Vernberg1s data were calculated from the mean percentage water content given in his results. This conversion gave integration constants (b), i.e. 0.02 when W is 1, of .734 for brain, 2.29 for l i v e r , and .110 for muscle. A l l these values were lower than those for excretory tissues i n the fresh water cutthroat trout. As a result of their studies of the oxygen consumptions by the major tissues of the mammalian body, Krebs (1950) and Bertalanffy and Pirozynski (1953) abandoned the theory that the weight specific decline in the total body metabolism was due to an integrated decline in the metabolic rates of the tissues. From the data presented in this thesis and those of Vernberg, there appears to be a parallel decline in the metabolically more active tissues, l i v e r , kidney, and g i l l , i n teleosts. The less active tissues, the brain and skeletal 26 muscle, with lower integration constants, show positive regres-sions. Thus the evidence indicates that the weight specific decline in overall metabolism in teleosts is not due to an integrated decline, and supports the work of Krebs (1950) and Bertalanffy and Pirozynski (1953) for mammals. The correlation coefficients (r) for kidney (.4-2) and for g i l l (.32) are very much lower.than those found by Krebs (1950) and calculated by Bertalanffy and Pirozynski (1953) (r = .846-.969). This inconsistency may have been because the animals were not from highly inbred stock. It could also have been a manifestation in part of the poikilothermic nature of the teleosts. Weymouth, Crimson, Hall, Belding, and Field (1944) reported low correlation coefficients when weight specific oxygen consumption was related to body weight in crustaceans. 27 II. Effect of Transfer to Sea Water A. Results The mortality rate after transfer of cutthroat trout from fresh water to sea water was dependent on the concentration of the sea water. After transfer to 100$ standard sea water, from a sample of 10 f i s h , 5 were dead within 22 hours and a l l were dead by 30 hours. The f i s h survived for 96 hours in 75$ standard sea water but were i n poor condition at the end of this period. A l l the f i s h survived and were in good condition 168 hours after transfer into 65$ standard sea water. For this reason, 65$ standard sea water was used in a l l these experiments. The body weight of the f i s h used in this experiment was within a 15 gnu range. Since the regression of QQ2 and body weight on a log-log plot was slight, and the scatter of the Q02 values for a single weight was large, the uncorrected values for QQ2 were used i n plotting the graphs. 1) Kidney (Tables II and IV, and figure 3) Control f i s h kept in 4 l i t r e s of fresh water during the experimental period showed a slight i n i t i a l rise in kidney QQ2 value followed by an apparent f a l l , between 48 and 168 hours, below the zero hour control level. This variation did not differ significantly from the zero hour oxygen consumption (p>.l). The experimental animals, transferred into 65$ standard 28 sea water, showed an i n i t i a l rise i n kidney Q Q 2 values during the f i r s t 4-8 hours. This rise was maximal at 20 hours and highly significant when compared to the parallel controls (p^.001). From 48 hours onwards, there was a progressive decline in the Q Q 2 values which levelled off slightly higher than the parallel controls. The combined values for the 48-168 hour samples showed that the mean Q Q 2 value for the experimental f i s h was significantly higher than the mean value for the controls (p<C.001). 2) G i l l s (Tables III and IV, and figure 4) During the experimental period of 168 hours there was no significant change in the oxygen consumption of g i l l tissue from control f i s h kept in 4 l i t r e s of fresh water. G i l l tissue from animals exposed to sea water showed a significant decline during the f i r s t 10 hours. The combined values for the 48-168 hour samples showed that the mean Qo2 value for the experi-mental f i s h was significantly lower that the mean value for the controls (p 4.001). A l l curves on figures 3 and 4 were drawn freehand to the best f i t of the points. The f i r s t point on each figure corresponds to the zero hour control f i s h . TABLE I I . Changes i n the oxygen consumption of kidney t i s s u e s a f t e r t r a n s f e r of f i s h to sea water. Time a f t e r Transfer (HP.) SEA WATER .FRESH WATER P V a l u e 2 Mean Body-Weight (Gm.) No. o f F i s h \ Mean Body Weight (Gm.) No. of F i s h Q°a 0 14.02 + 1.54 1 4 7.211 + 0.288 14.02 + 1.54 4 7.211 + 0.288 5 14.02 + 0.69 4 7.988 + 0.739 14-78 + 0.37 4 9.242 + 0.500 <.2 > .1 10 22.00 + 0.95 4 10.356 + 0.283 — -15 21.95 + 0.64 4 10.996 + 0.580 — - • 20 21.37 + 3.55 3 11.840 ± 0.453 — • - -— 24 13.88 + 1.57 4 10.086 + 0.500 14.05 + 0.70 4 6.922 + 0.620 ^.01 >.005 30 12.92 + 0.88 4 7.411 ± 0.760 -48 13.84 + O.64 8 8.193 +0.600 14.5° 1 0-87 4 5.422 + 0.303 .^005 >.001 72 13.20 + 1.62 4 8.007 + 0.231 15.12 + 0.69 4 7.165 i 0.803 ^.4 7 .2 96 17.90 + 1.68 4 7.860 +0.289 16.22 + 2.36 4 7.586 + 0.445 >.5 120 19.02 +5.57 2 8.579 + 1.556 13.45 i 2.10 4 5.963 +. 0.504 C2 7 .1 144 10.95 + 1.02 4 6.143 + 0.283 12.50 + 1.08 4 6.713 ± 0.590 <.5 7 .4 168 15.50 + 1.76 4 7.902 + 0.737 12.35 i 1.32 4 6.715 + 0.600 <.2 7 .4 Standard e r r o r of mean value P value compared to corresponding freshwater controls TABLE I I I . Changes i n the oxygen consumption o f g i l l t i s s u e s a f t e r t r a n s f e r of f i s h to sea water. Time a f t e r Transfer (Hr.) SEA WATER FRESH WATER P V a l u e 2 Mean Body-Weight (Gm.) No. of F i s h Q °2 Mean Body Weight (Gm.) No. of F i s h Q °2 0 14.02 + 1.541 4 6.A10 + 0.144 3A.02 + 1.54 A 6.410 + 0.144 5 14.02 + 0.69 4 5.4U + 0.300 14.78 + 0.37 4 6.776 + 0.236 C025 >.oio 10 22.00 + 0.95 4 4.846 + 0.100 - — 15 21.95 + 0.64 4 5.582 + O.462 - — 20 21.37 + 3.55 3 5.790 + 0.095 - — 24 13.88 + 1.57 A 7.552 + 0.215 14.05 + 0.70 A 6.060 + 0.276 ^.010 ?.005 30 12.92 + 0.88 A 4.487 + 0.202 - — 48 12.76 +0.99 3 5.177 + 0.537 14.50 + 0.87 4 5.314 ± 0.266 >.5 72 13.20 + 1.62 4 5.457 + 0.253 15.12 + 0.69 4 6.396 + 0.305 < . l > .05 96 17.90 +1.68 4 5.001 + 0.366 15.53 + 1.05 3 6.159 + 0.217 <.050 ^ .025 120 19.02 +5.57 2 4.320 + 0.344 13.45 + 2.10 A 6.183 + 6.340 ^.01 > .005 1U 10.95 + 1.02 4 5.294 + 0.231 12.50 + 1.08 A 7.609 + 0.556 ^.01 > .005 168 15.50 + 1.76 A 5.687 + 0.331 12.35 + 1.32 4 5.826 + 0.359 >.A0 Standard e r r o r on mean value P value compared to corresponding freshwater c o n t r o l s TABLE IV Comparison of combined values (48-I68 hr.) f o r g i l l and kidney t i s s u e r e s p i r a t i o n o f f i s h i n sea water and f r e s h water. Tissue SEA WATER FRESH WATER P V a l u e 2 Mean Q n  U 2 N Mean Q °2 N G i l l 5.237 *• 0.425 1 21 6.254 + 0.510 23 < 0.001 Kidney 7.856 + 0.825 26 6.593 + 0.707 24 < 0.001 x Standard er r o r o f mean value 2 P value o f combined values compared corresponding combined c o n t r o l values over the same period (Pacing figure 3) FIGURE 3. Effect of transfer of cutthroat trout to seawater on the kidney QQ2 values. * SEA WATER TIME AFTER TRANSFER (HR.) FIGURE 3 ( f a c i n g f i g u r e 4) FIGURE 4. E f f e c t o f t r a n s f e r o f c u t t h r o a t t r o u t t o s e a w a t e r on t h e g i l l Q02 v a l u e s . TIME AFTER TRANSFER (HR.) FIGURE 4 34 B. Discussion There are three possible sites of osmotic regulation i n fishes: the kidney tubule, the g i l l epithelium, and the skin. The well developed glomerular kidney possessed by fresh water teleosts.enables them to excrete a copious dilute urine. Urine flows vary from 60-400 ml, per kg. body weight per day (Smith, 1932, Marshall, 1934, and Krogh, 1937). In contrast to these high rates of diuresis, the marine teleosts have a relatively scant urine flow of less than 4 ml. per kg. per day (Grafflin, 1931). Although Smith (1955) attributes the low urine flow in the sculpin to a greatly reduced glomerular f i l t r a t i o n rate, Clarke (1934) reported a 78.6$ tubular reab-sorption of glomerular f i l t r a t e in the sculpin. There is no evidence at present to indicate that the anadromous species, such as the salmon, or euryhaline species, such as the trout, undergo such a drastic reduction of glomerular f i l t r a t i o n rate when they are transferred to a hypertonic medium. Smith (1932) states that marine teleosts drink large amounts of sea water to offset the water lost by diffusion through the g i l l s . It is assumed that a similar phenomenon occurs on transfer of euryhaline species to sea water. The absorption of both salts and water from the gut and the loss of water by diffusion through the g i l l s would cause a rise in the tissue f l u i d concentration reflected by a rise in the blood concentration (Portier and Duval, 1922 a,b, Busnel, 1942, and Fontaine, Dellatre, and Callamand, 1945). In mammals, the 35 kidney controls the loss of water through the antidiuretic effect of increased active tubular reabsorption of water. The rapid rise i n trout kidney QQ 2 upon transfer to sea water indicates an increased energy turnover which may be associated with increased tubular reabsorption of water and/or electro-lytes. Immediately upon transfer, the f i s h tends to dehydrate. Parry (1958) found that salmonid fishes transferred to sea water showed an i n i t i a l rise i n blood concentration with a subsequent f a l l to normal levels. The f i s h must, therefore, conserve body water. The i n i t i a l rise i n QQ 2 could thus be associated with some antidiuretic process. Physiological doses of vasopressin administered intra-peritoneally to the cutthroat trout are capable of increasing the QQ 2 values of kidney tissue by an increment equal to that observed upon transfer to sea water (Holmes, unpublished, and Appendix). Adrenocortical steroids are capable of increasing the oxygen consumption also, but not to the extent of vaso-pressin (Holmes, unpublished). Previous work on this subject (Holmes, 1959, Chester Jones, Phillips and Holmes, 1959) indicated that adrenocortical steroids were effective in enhancing the reabsorption of sodium by the kidney of the rainbow trout. *' One would not expect this to occur in a f i s h situated in a hypertonic environment. However, the increased reabsorption of water may be accompanied by increased reab-sorption of salts. The demonstration of appreciable amounts of Cortisol, cortisone, corticosterone, and aldosterone in 36 Sockeye salmon blood (Phillips, Holmes, and Bondy, 1959) sug-gests that the synthetic pathways of adrenocortical steroids are at least present among the Salmonoidea. The shape of the experimental curve in figure 3 may pos-sibly be explained as follows: the i n i t i a l rise and peak values for the QQ 2 values may be due to regulatory effects of vasopressin and adrenocortical steroids, while the maintained higher level may be due to a persistent effect of adreno-cortical steroids. In mammals, vasopressin has been shown to have a short term action, while the adrenocortical steroids i may have more lasting effects, as in the case of stress. The later stages of the adaptation of euryhaline fishes to sea water may include anatomical, as well as functional changes in the kidney. Ford (1958) has shown that salmon raised i n sea water have fewer glomeruli than those raised i n fresh water. Black (1957) has also pointed out a gradation i n the functional activity of glomeruli in marine teleosts. Holmes (1959) found that saline loaded rainbow trout exhibit an enhanced net output of sodium in the presence of adrenocortical steroids. Although these steroids caused the classical effect, increased reabsorption of salts, in the kidney, they increased the extra-renal output. The extra-renal increment was greater than the increased tubular reab-sorption. Inhibition of the extra-renal re-uptake of sodium from the environment seemed to be a major factor in enhancing the net output. Sexton (1955) reported a similar inhibition 37 of sodium re-uptake i n the presence of desoxycorticosterone acetate in the goldfish. The observed decline in g i l l Q02 values is consistent with an inhibitory mechanism. The shape of the experimental curve i n figure 4 may be explained by an inhibited re-uptake of salts by the g i l l epithelium, which thus expends less energy in sea water than in fresh water. The inhibition may be controlled by adreno-cortical steroids. Anatomical or histological changes may also follow the hormonally induced functional changes in the g i l l s , the number of chloride secreting cells in Fundulus  heteroclitus varying in fresh water and sea water (Copeland, 1948, 1950). 38 CONCLUSIONS The weight specific oxygen consumption of g i l l and kidney tissue for the cutthroat trout (S,almo cla r k i clarki) decreased with increasing body weight. The following relationships were found: Kidney: Q02 - 10.78 W " ' I 4 8 G i l l : Qo2 = 9.114- W -»139 The decline of the oxygen consumption did not support Brody*s .73 rule or the surface rule at the level of tissue respiration. On transfer of the cutthroat trout from fresh water to sea water, changes occurred in the oxygen consumption of the excretory tissues. A sharp i n i t i a l rise in the Q02 of kidney tissue was noted during the f i r s t 48 hours after transfer, reaching a maximum at 20 hours. The tissue respiration during the remainder of the experi-mental period remained significantly higher than the parallel control level. The g i l l tissue respiration declined rapidly during the f i r s t 10 hours after transfer and remained significantly below the control level during the whole experimental period. Hormonal regulation of salt-electrolyte and water metabolism by vasopressin arid adrenocortical steroids may be correlated with these changes in energy output i n euryhaline species of salmonids. 39 APPENDIX - EFFECT OF VASOPRESSIN ON OXYGEN CONSUMPTION IN THE KIDNEY In an attempt to correlate antidiuretic processes and the transfer of the cutthroat trout to sea water, the effects of vasopressin, administered in vitro and in vivo^ on the kidney oxygen consumption values was investigated. I. Materials and Methods A. Effects of Vasopressin in Vitro To test the effects of vasopressin in vitro, 5 milliunits (mU) of vasopressin (Parke-Davis pitressin) were added to the experimental flasks. This was accomplished by making up a solution of modified Krebs' medium containing 2.0 mU of vaso-pressin per ml. The experimental flasks contained 2.5 ml* of the vasopressin f o r t i f i e d medium, while the control flasks contained 2.5 ml. of Krebs' medium in the main compartment. Substrates, 0.2 ml. of .11 disodium succinate, .1 M disodium fumarate, or .1 M trisodium citrate, were added to the main compartment. Substrates containing .1 M disodium succinate plus .1 M disodium malonate, and .1 M disodium fumarate plus .1 M disodium malonate were used to test the effects of malon-ate inhibition. The respirometry was carried out as before. B. Effects of Vasopressin in Vivo To determine the effects of vasopressin In vivo, the f i s h were taken from the storage tanks and. injected intraperitoneal-40 l y with a dose of 10 mU of vasopressin. This was administered as 0.1 ml. of vasopressin solution containing 100 mU per ml. The f i s h were held in dechlorinated tap water for 5 hours u n t i l sacrificed. Two sets of flasks were prepared: one as outlined before with 2.7 ml. of modified Krebs' medium i n the main compartment, and the other differing by containing only 2.5 ml. of modified Krebs1 medium and 0.2 ml. of substrate. The substrates were the same as those used before, and the respirometry was carried out as explained earlier. 41 TABLE V. THE EFFECT OF SUBSTRATE ON THE UTILIZATION BY ISOLATED KIDNEY TISSUES OF VASOPRESSIN IN VITRO AND IN VIVO Substrate Treatment N Mean Q02 Corrected mean Q02 % increase over controls * None Controls 30 9.236 9.236 0 Vasopressin VP.A 3 4 10.126 9.255 0.2 (Controls) 4 3 10.104 VP.B 5 26 9-832 9.832 6.4 (Controls) 30 9.236 Succinate Succinate 12 11.666 11.856 28.4 * (Controls) 11 9.087 Succinate + VP.A 8 15.190 11.917 29.0 (Succinate) 8 15.112 Succinate + VP.B 12 12.400 11.965 29.5 * VP.B 11 10.188 Fumarate Fumarate 4 8.888 12.152 20.6 (Controls) 4 7.377 Fumarate + VP.A 4 10.525 15.169 6 4 . 3 (Fumarate) 4 7.726 Fumarate + VP.B 4 11.953 12.170 3 1 . 7 (VP.B) 4 9.655 C i t r a t e Citrate 4 10.659 9.820 6.3 (Controls) 4 10.024 Citrate + VP.A 3 13.348 9.149 -0.9 (Citrate) 4 13.255 Citrate + VP.B 3 9.580 10.351 11.9 (VP.B) 3 9.098 Succinate S + M 4 I I . 4 0 9 11.159 20.8 + Malonate (Controls) 4 9.442 S + M * VP.A 4 n . 3 6 9 11.673 26.4 (S + M) 3 10.868 S + M + VP.B 4 9.327 9 . 8 4 6 6.6 (VP.B) 4 9.312 Fumarate F + M 4 11.169 10.246 10.9 + Malonate (Controls) 4 9.956 F + M + VP.A 3 10.930 10.213 10.6 (F + M) 3 IO .966 F + M + VP.B 4 9.570 9.833 6 . 4 (VP.B) 4 9.568 Controls f o r the various samples corrected to value f o r combined controls 2 Controls = 100$ 3 VP.A - Vasopressin i n v i t r o , added to f l a s k s 4 Bracketed values act as p a r a l l e l controls f o r preceding samples 5 VP.B - Vasopressin i n vivo, injected into f i s h » P value comparing value and bracketed control s i g n i f i c a n t at the 5% l e v e l 42 II. Results and Discussion Most of the results shown i n Table V were not significant-l y different from the controls. This may have been due to the small sample size. The experiments could not be repeated because of lack of fish, since a disease of the trout at the Cultus Lake Hatchery necessitated destroying the stock of cutthroat trout. Thus, inferences only could be made concern-ing the results. These inferences were: 1) Both succinate and fumarate enhanced the oxygen consumption of control tissue. Citrate was not active as a substrate. 2) Fumarate, only, was active as a substrate i n the presence of vasopressin in vitro, succinate acting at the same levels in the absence and presence of vasopressin. 3) Both succinate and fumarate were inhibited by malonate to the same degree in the control experiments. 4) In the presence of vasopressin in vitro, fumarate was inhibited to a greater degree than succinate by the addi-tion of malonate. The oxygen consumption levels for the vasopressin i n vivo experiments were not markedly different from the control values, indicating that either the dosage of vasopressin was too low to cause an effect or the time between injection and and sacrifice of the f i s h was not optimum. Other experiments (Holmes, unpublished) indicated that injection of vasopressin 4-3 significantly raised the level of kidney Qo2« Substrates from the tricarboxylic acid cycle i n catalytic amounts have been found to stimulate cellular metabolism (Krebs and Johnson, 1937)? although work on bacterial whole c e l l preparations indicates that the a b i l i t y of the c e l l to u t i l i z e the substrates depends on the permeability of the c e l l wall to the substrate. As a rule, certain forms of bacteria cannot u t i l i z e citrate as a substrate u n t i l they have manufactured permease enzymes which enable the substrate to enter the c e l l (Barrett, Larsen, and Kallio, 1953). This fact seemed to hold true also i n the cutthroat kidney slices where fumarate and succinate were active and the larger citrate molecule was not. Sexton and Russell (1955) noted an increased uptake of oxygen by the filaments of the gold-f i s h g i l l in a succinate f o r t i f i e d medium, citing this as proof of the presence of succinic dehydrogenase in the g i l l s of the goldfish. The results i n this thesis showed that succinate (28.4$) and fumarate (20.6$) increased the oxygen consumption of kidney tissues, and both were inhibited by malonate to the same degree (succinate depressed to 20.8$ and fumarate to 10.9$). This might indicate succinic dehydro-genase activity. Vasopressin, added to excretory tissues i n vitro, has been found to enhance their activity. Bentley (1958) found that vasopressin, i n the presence df a substrate, increased water transport across the wall of the isolated urinary bladder 44 of the toad. After testing the effects of several metabolic inhibitors, he concluded that oxidative metabolism u t i l i z i n g phosphate bond energy acted i n water transfer. Vasopressin, added in vitro to trout kidney slices did not enhance the QO2 significantly without substrate (0.2$). It caused a much greater enhancement in the presence of fumarate (64.3$) than in the presence of succinate (29.0$) as a substrate. Also, malonate caused a much greater depression of the Q02 of the kidney slices with vasopressin i n vitro, when fumarate was a substrate (10.6$) than when succinate was the substrate (26.4$). Thus i t might appear that vasopressin acts at an enzymatic level, activating fumarate as a substrate. The succinic dehydrogenase enzyme may be involved since malonate is active in inhibiting the fumarate as a substrate. However, i f succinic dehydrogenase were involved and fumarate was inhibited to a greater degree than succinate, one would suspect that the tricarboxylic acid cycle was reversed by vasopressin. This i s not so since energy i s released only by the forward reactions of the cycle. The relatively small malonate inhibition of succinate as compared to fumarate i n the presence of vasopressin I n vitro might suggest the involve-ment of the actual fumarate molecule as the target for vaso-pressin. The results in this thesis do, however, agree with Bentley's (1958) finding that vasopressin can act in vitro i n the presence of a substrate. 45 To summarize: vasopressin is active in vitro in the presence of a substrate with trout kidney slices. The nature of the substrate may indicate the action of vasopressin at an enzymatic level. 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