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Energy metabolism in carp white muscle Driedzic, William Richard 1975

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ENERGY METABOLISM IN CARP WHITE MUSCLE by WILLIAM RICHARD DRIEDZIC B.Sc. (Hons.), York U n i v e r s i t y , 1970 M.Sc, U n i v e r s i t y of Toronto, 1972 A THESIS SUBMITTED IN THE REQUIREMENTS DOCTOR OF PARTIAL FULFILMENT OF FOR THE DEGREE OF PHILOSOPHY i n the Department of ZOOLOGY We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may b e g r a n t e d b y t h e H e a d o f my D e p a r t m e n t o r b y h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t b e a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f ZOOLOGY T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, C a n a d a D a t e J U L Y 1975. i i ABSTRACT The myotomal muscle of f i s h i s l a r g e l y composed of two tissue types, usually r e f e r r e d to as the red and white f i b e r s . On the basis of histochemi-c a l and biochemical properties i t i s generally accepted that white muscle has a metabolism which i s predominantly anaerobically based, u t i l i z i n g glycogen as i t s f u e l source and that red muscle functions l a r g e l y a e r o b i c a l l y burning fats and/or carbohydrates. In carp (Cyprinus carpio L.) the red f i b e r s are found as a t h i n s u p e r f i c i a l layer below the skin and the white f i b e r s make up the mass of the underlying myotome. Thus, i n this species i t i s possible to r a p i d l y obtain an homogenous sample of white muscle allow-ing the analysis of l a b i l e metabolites. This study investigates the control of g l y c o l y s i s i n white muscle and the source and function of anaerobic NH* production by white muscle. The concentrations of key metabolites were determined i n muscle before exercise and a f t e r maximal a c t i v i t y . During exercise there was an increase i n l e v e l s of glucose-6-phosphate, fructose-6-phosphate and fructose-1-6-di-phosphate which, along with a decrease i n ATP l e v e l s , could account for the increase i n g l u c o l y t i c f l u x by a c t i v a t i o n of phosphofructokinase and pyruvate kinase. I t was found that a f t e r maximal a c t i v i t y the concentration of ATP decreased by about 65%, ADP decreased s l i g h t l y , while AMP remained low and unchanged. Consequently the l e v e l of the free adenylate pool decreased. Simultaneously there was an increase i n the concentration of IMP (inosine monophosphate) and NH^. The increase i n IMP l e v e l and the decrease i n adenylate pool were e s s e n t i a l l y i n 1:1 stoichiometry, a r e s u l t showing that the adenylate pool was decreased by the reaction catalyzed by 5' AMP deaminase. However, the increase i n free Mlt was less than the decrease i n the adenylate i i i pool. The concentration of free amino acids was also determined i n white muscle, before and a f t e r severe environmental hypoxia. During the hypoxic period the t o t a l amount of nitrogen i n the amino a c i d pool increased and there was a tendency f or an increase i n the t o t a l number of free amino acids. On the basis of t h i s study i t i s possible to construct a f a i r l y compre-hensive metabolic scheme for nitrogen metabolism i n carp white muscle during anaerobic work. The energy required for work i s ulti m a t e l y derived from the hydrol y s i s of ATP. When ATP l e v e l s cannot be maintained the content of ADP increases and as t h i s occurs the l e v e l of AMP also increases due to the equilibrium r e l a t i o n s h i p of the adenylates. As the work load on the tissue exceeds i t s aerobic c a p a b i l i t i e s GTP (guanosine triphosphate) l e v e l s drop, 5' AMP deaminase i s activated and the adenylate pool i s decreased. NH^ released from AMP i s subsequently incorporated i n t o the free amino a c i d pool. TABLE OF CONTENTS Abstract Table of Contents L i s t of Tables L i s t of Figures Acknowledgements Chapter I. Introduction Chapter I I . Materials and Methods Chapter I I I . Results Chapter IV. Discussion Chapter V. Concluding Remarks: Red-White Muscle Differences and the Function of the Purine Nucleotide Cycle Chapter VI. L i t e r a t u r e Cited Appendix I. Maintenance of Blood Lactate Levels i n Free Swimming Trout Appendix II Enzyme Nomenclature V LIST OF TABLES Table I. Concentrations of g l y c o l y t i c intermediates i n white muscle of carp under two well defined conditions: r e s t i n g and maximally a c t i v e . Table I I . Concentrations of the adenylates and r e l a t e d meta-b o l i t e s i n white muscle of carp under two well defined conditions: r e s t i n g and maximally active. Table I I I . Energy charge and changes i n concentrations of the adenylate pool and r e l a t e d metabolites i n white muscle of carp under two well defined conditions: r e s t i n g and maximally a c t i v e . Table IV. Concentrations of Krebs cycle intermediates and re l a t e d metabolites i n white muscle of carp under two well defined conditions: r e s t i n g and maximally act i v e . Table V. Concentrations of metabolites and the energy charge value i n white muscle of carp a f t e r various l e v e l s of a c t i v i t y . Table VI. Metabolite concentrations i n carp white muscle before and a f t e r hypoxic s t r e s s . Table VII. Free amino acid concentrations i n carp white muscle before and a f t e r hypoxic s t r e s s . Table VIII. A r t e r i a l and venous blood l a c t a t e concentrations of rainbow trout following exercise to fatigue. Page 30 31 32 33 35 38 39 87 vi LIST OF FIGURES Figure 1. Schematic representation of carp (Cyprinus cafpio) i n cross-section at the l e v e l of the p o s t e r i o r margin of the dorsal f i n . Figure 2. Anaerobic metabolism i n invertebrate f a c u l t a t i v e anaerobes. Figure 3. The percentage aerobic swimming e f f i c i e n c y versus swimming speed of g o l d f i s h . Figure 4. Depletion of the adenylate pool i n white muscle during anaerobic work. Figure 5. Regeneration of the adenylate pool i n white muscle during recovery from anaerobic work. Figure 6. Augmentation of Krebs cycle intermediates during u t i l i z a t i o n of carbohydrate as an energy source. Figure 7. Augmentation of Krebs cycle intermediates during u t i l i z a t i o n of f a t as an energy source. Figure 8. Blood l a c t a t e l e v e l s of i n d i v i d u a l swimming trout at s p e c i f i e d swimming speed and following fatigue. Page 2 10 45 53 54 59 63 84 v i i ACKNOWLEDGEMENTS I would l i k e to thank Dr. P. W. Hochachka for h i s constant i n s p i r a t i o n throughout the course of t h i s study. I would also l i k e to express my thanks to Drs. B i l i n s k i , B r e t t , Hoar, Jones and Randall f o r t h e i r comments and c r i t i c i s m s of the thesi s , to Dr. G. I. Drummond for h i s help i n the early development of the problem, and to Mr. R. Hurst f o r i n s t r u c t i o n i n the extr a c t i o n and analysis of v o l a t i l e acids. I am g r a t e f u l to Dr. I. P. Taylor, Department of Botany, Un i v e r s i t y of B r i t i s h Columbia, for use of both the gas l i q u i d chromatograph and amino analyzer. The su c c i n i c thiokinase and the TH formylase were g i f t s from Dr. W. Bridger, Department of Biochemistry, University of Alb e r t a , and Dr. J. C. Rabinowitz, Department of Biochemistry, University of C a l i f o r n i a , r e s p e c t i v e l y . The study presented i n Appendix I was done i n c o l l a b o r a t i o n with Mr. J. W. Kiceniuk, to whom I am e s p e c i a l l y g r a t e f u l . I would also l i k e to thank Mr. Kiceniuk for c o l l e c t i n g the animals used i n the swimming experiments and f o r exercising some of them. Throughout the study I was the holder of a National Research Council of Canada Graduate Scholarship. F i n a l l y , I wish to thank Cath, Adam and Sean for helping me keep my work i n i t s proper perspective. 1 CHAPTER I. INTRODUCTION 1 a The s k e l e t a l muscle of vertebrates i s composed of a number of character-i s t i c a l l y d i f f e r e n t f i b e r types. At the extreme ends of the spectrum of f i b e r types are those which make up the red and white muscles. In mammals, red, white, and intermediate type f i b e r s usually occur i n complex mixed muscle masses. In some instances, such as the cat soleus, a r e l a t i v e l y pure red muscle may be obtained, but i n mammalian systems, white muscle per se, does not e x i s t as a d i s c r e t e t i s s u e . However, i t i s possible to study white muscle by u t i l i z i n g lower vertebrates. In many f i s h species the white f i b e r s , which constitute 80-95% of the swimming musculature, e x i s t as a d i s c r e t e , e a s i l y separable tissue mass. Where th i s occurs, the red f i b e r s are found as a thin s u p e r f i c i a l layer below the skin forming a thicker t r i a n g l e of muscle at the l e v e l of the l a t e r a l l i n e , with the white f i b e r s making up the mass of the underlying nyotome. The myotomal musculature of carp (Cyprinus carpio L.) i s structured i n this manner (Figure 1). Although there may be gradations i n the s i z e of the f i b e r s (Boddeke jit a l , 1962), a l l of the a v a i l a b l e h i s t o -chemical evidence indicates that white muscle of carp i s an homogenous tissue i n terms of energy generating properties (Ogata and Mori, 1964; Brotchi, 1968). In t h i s species a small number of intermediate type f i b e r s are found, but these occur between the red and white muscle masses. Thus i t i s possible with very l i t t l e d i f f i c u l t y to obtain a r e l a t i v e l y pure preparation of white muscle from carp. I t i s now well established that the red and white muscle of f i s h have markedly d i f f e r e n t metabolic c a p a b i l i t i e s . On the basis of biochemical and h i s t o l o g i c a l properties i t i s generally accepted that white muscle has a metabolism which i s predominantly anaerobic, whereas the red muscle functions l a r g e l y a e r o b i c a l l y . Thus, red muscle i s characterized by a higher content of mitochondria (Buttkus, 1963), myoglobin (Hamoir et a l , 1972), haemoglobin 2 Figure 1. Schematic representation of carp (Cyprinus carpio) i n cross-section at the l e v e l of the posterior margin of the dorsal f i n . Red Muscle White Muscle 3 (Hamoir et a l , 1972), l i p i d (Bone, 1966; L i n e_t a l , 1974), l i p o l y t i c enzymes (George, 1962), Krebs cycle enzymes (Bostrom and Johansson, 1972), and cyto-chrome oxidase (Bostrom and Johansson, 1972) . These properties are r e f l e c t e d by a higher vascular supply to the tissue (Stevens, 1968) and a greater capacity to consume oxygen (Lin et a l , 1974; Wittenberger and Diaciuc, 1965). White muscle, on the other hand, i s poorly vascularized (Stevens, 1968), shows a low oxygen consumption rate (Lin et^ a l , 1974; Wittenberger and Diaciuc, 1965) , and i s biochemically geared for anaerobic metabolism. Consequently, t h i s tissue has a high content of g l y c o l y t i c enzymes (Hamoir e_t a l , 1972) and an extremely active l a c t a t e dehydrogenase designed to channel pyruvate into l a c t a t e (Bostrom and Johansson, 1972). Studies on mammalian systems are i n t o t a l agreement with the above metabolic findings and have been reviewed by Pette and Staudte (1973) and Keul et a l (1972). The control of blood flow to s k e l e t a l muscle i n f i s h i s s t i l l poorly understood. S a t c h e l l (1971) has reviewed this area of l i t e r a t u r e with respect to both exercise and hypoxia. Exercise evokes an o v e r a l l reduction i n peripheral resistance and an increased blood flow through the^vtrunk, region. S a t c h e l l argues that since the white muscle makes up such a large portion of the body musculature, blood flow to t h i s tissue increases during a c t i v i t y . During hypoxia however, peripheral v a s o c o n s t r i c t i o n occurs ( S a t c h e l l , 1971) . It i s w e l l established that during hypoxia blood flow to s k e l e t a l muscle i n mammals i n reduced (Irving, 1964); although not unequivocally established, i t appears that the same mechanism functions i n f i s h . Moreover, i n l i g h t of the poor c i r c u l a t i o n of white muscle under aerobic conditions, i t i s probable that t h i s tissue approximates a closed system during hypoxia. The metabolic c h a r a c t e r i s t i c s of red and white muscle are r e f l e c t e d by 4 a functional difference between these two t i s s u e s . E l e c t r o p h y s i o l o g i c a l studies show that during slow swimming the propulsive force i s derived e n t i r e -l y from the red musculature. At the highest swimming v e l o c i t i e s the white muscle becomes maximally active and together with the red muscle provides the power for locomotion (Bone, 1966; Hudson, 1973) . White muscle was previously thought to be u t i l i z e d only during periods of burst a c t i v i t y (Bone, 1966); however, i t i s now generally accepted, that at l e a s t i n the t e l e o s t , t h i s tissue plays a r o l e over a much wider range of swimming speeds. A l l of the current data i n d i c a t e that at some l e v e l i n the t r a n s i t i o n from low to high swimming speed there i s increasing recruitment of the white f i b e r s . This i s based on: (a) the accumulation of l a c t a t e i n the white muscle of trout (Black ^ t a l , 1962), carp (Johnston and Goldspink, 1973a), c o a l f i s h (Johnston and Goldspink, 1973b), and mackerel (Pritchard et a l , 1971) worked at i n t e r -mediate v e l o c i t i e s ; (b) hypertrophy of white muscle f i b e r s i n c o a l f i s h forced to swim at moderate speeds for extended periods of time (Walker and P u l l , 1973); (c) the repayment of an oxygen debt by salmon during recovery from swimming at a l l elevated speeds (Brett, 1964) and (d) the observation that i n g o l d f i s h the mass of the red muscle f i b e r s alone i s not great enough to meet the o v e r a l l power output of the f i s h (Smit et^ ail, 1971) . During exercise carp white muscle generates a large portion of i t s energy by anaerobic g l y c o l y s i s (Wittenberger and Diaciuc, 1965); thus, during work there i s a decrease i n glycogen with a concomitant increase i n l a c t a t e . The quantitative aspects of this phenomenon, i n numerous species, have recently been reviewed by B i l i n s k i (1974) and need not be repeated here. A l l of the a v a i l a b l e evidence indicates that glycogen i s metabolized i n f i s h by the c l a s s i c a l Embden, Myerhof, Parnas pathway; for each mole of glycogen-derived 5 glucose mobilized, 2 moles of l a c t a t e are formed with a net gain of 3 moles of ATP. Again t h i s area of l i t e r a t u r e has been reviewed i n depth by Hochachka (1969) and Tarr (1972). In many respects the c o n t r o l of g l y c o l y s i s i n f i s h white muscle appears s i m i l a r to other systems studied. Thus, glycogen m o b i l i z a t i o n i s i n i t i a t e d by glycogen phosphorylase, a regulatory enzyme, which i n most tissues i n under a v a r i e t y of controls including hormonal agents I | such as epinepherine and norepinepherine, the adenylates, and free Ca (Drummond, 1971). In f i s h white muscle, Ca appears to be the primary i f not the sole regulator of glycogen phosphorylase since blood flow and hence hormonal signals are minimal (Pocinwong ^ t a l , 1974). The next s i t e of co n t r o l i n the g l y c o l y t i c pathway occurs at the r e a c t i o n catalyzed by phos-phofructokinase (PFK) which catalyzed the conversion of fructose-6-phosphate plus ATP to fructose-l,6-diphosphate plus ADP. The r e a c t i o n catalyzed by PFK represents the f i r s t unique step i n g l y c o l y s i s ; hence, i t i s not s u r p r i s i n g that the enzyme i s p r e c i s e l y regulated by various metabolites i n a manner that controls the rate of g l y c o l y s i s i n accord with the c e l l s ' need f o r energy or g l y c o l y t i c intermediates. In a v a r i e t y of tissues, both substrates and products of the PFK reaction, as w e l l as other f a c t o r s , are a l l o s t e r i c modifiers of the enzyme. Thus, a c t i v a t o r s of PFK include AMP, f r u c t o s e s -phosphate, fructose-1,6-diphosphate, P^, and NH^; whereas ATP, c i t r a t e and creatine phosphate are i n h i b i t o r s (Mansour, 1972) . To complete the a c t i v a t i o n of muscle g l y c o l y s i s , the a c t i v i t y of PFK i s integrated with the next major control s i t e i n the pathway: pyruvate kinase (PyK). PyK catalyzes the con-version of phosphoenolpyruvate plus ADP to pyruvate plus ATP. An i n t e g r a t i o n of the rate c o n t r o l l i n g enzymes i s achieved i n two ways. F i r s t l y , ADP formed i n the PFK reaction serves as a substrate for PyK, and secondly, at l e a s t i n 6 the lower vertebrates, fructose-1,6-diphosphate functions as a feed forward a c t i v a t o r of PyK (Hochachka and Somero, 1973). The k i n e t i c properties of both PFK and PyK from f i s h muscle have been studied (Freed, 1971; Somero and Hochachka, 1968; Mustafa et a l , 1971); however, the mechanism c o n t r o l l i n g g l y c o l y t i c f l u x i n vivo i n t h i s tissue had not been v e r i f i e d . Thus, i t seemed worthwhile to determine the concentration of metabolites known to regulate t h i s pathway under varying conditions of energy demand. There i s now a s u b s t a n t i a l amount of evidence which suggests that g l y c o l y s i s i s not the only anaerobic metabolic pathway i n white muscle. At l e a s t three studies have shown the m o b i l i z a t i o n of nitrogenous compounds i n f i s h white muscle during exercise. Thus, with exercise to fatigue, the white muscle content of NH^ increases from about 4 to 7 ymoles/gm i n cod (Fraser et a l , 1966) and from about 2.5 to 7 umoles/gm i n T r i a c h i s s c y l l i u m (Suyama et a l , 1960). Kutty (1972) has hypothesized, on the basis of oxygen consumption and NH^ ~ excretion by T i l a p i a mossambica, that at l e a s t a p o r t i o n of the NH^ ~ produced i n the muscle during a c t i v i t y i s of anaerobic o r i g i n . Support for this contention arises from the observations that g o l d f i s h expire increased amounts of NH^ during hypoxia (Dejours et a l , 1968) and that the blood l e v e l of NH^ i n carp increases during hypoxia (Pequin and Serfaty, 1962). I t would be of i n t e r e s t to ascertain i f there are reactions i n white muscle, functional under anaerobic conditions, which could provide a source of NH^ ~. There appear, on the basis of our knowledge of intermediary metabolism, to be only two l i k e l y o r i g i n s of anaerobic NH^: the free adenylate pool and the amino acid pool. I t i s recognized that the enzyme 5' AMP deaminase i s instrumental i n the c o n t r o l l e d release of NHA i n s k e l e t a l muscle (Lowenstein, 1972) . 5* AMP 7 deaminase catalyzes the degradation of AMP to IMP (inosine monophosphate) plus NH^ and occurs i n p a r t i c u l a r l y high t i t r e s i n s k e l e t a l muscle. Further-more, the a c t i v i t y of t h i s enzyme i s at l e a s t 15 times higher i n carp white muscle than i n red muscle or heart ( F i e l d s , personal communication). This reaction was thus considered a l i k e l y candidate for the production of anaerobic MH^. AMP, however, i s i n equilibrium with ATP and ADP by the adenyl-ate kinase reaction: 2 ADP •AMP + ATP Therefore i f 5' AMP deaminase i s responsible for the production of anaerobic NH£ t h i s should be r e f l e c t e d by a decrease i n the e n t i r e adenylate pool. In inse c t f l i g h t muscle, a tissue with no anaerobic capacity, the adenylate pool i n fact remains constant during a c t i v i t y (Gerez and K i r s t e n , 1965) or imposed hypoxia (Ford and Candy, 1972). But i n vertebrate muscle the s i t u a t i o n i s more complex probably due to the varying content of red and white f i b e r s i n d i f f e r e n t mammalian musculatures. In working conditions when oxygen i s not l i m i t i n g the adenylate pool does not decrease i n e i t h e r s k e l e t a l muscle (Edington e_t a l , 1973) or heart (Shafer and Williamson, 1973) , whereas a f t e r an hypoxic stress i t i s found to decrease with a concomitant increase i n IMP plus i t s degradation products (Imai et a l , 1964; Chaudry et a l , 1974; Deuticke and Gerlach, 1966). In f i s h , Jones and Murray (1960) found a 1:1 s t o i c h i o -metric r e l a t i o n s h i p between depletion of adenine nucleotides and accumulation of IMP i n cod muscle a f t e r the animal had been fatigued. In l i g h t of the apparent discrepancies i n t h i s area, a study of adenylate pool a l t e r a t i o n s i n an organism, the carp, possessing a large homogenous white muscle mass was i n i t i a t e d . In a study of anaerobic NliT production, the p o s s i b i l i t y of the fermenta-8 t i o n of amino acids must also be considered. Many invertebrates which are f a c u l t a t i v e anaerobes mobilize both carbohydrates and amino acids anaerobical-l y and th i s apparently provides them with a d d i t i o n a l means of generating ATP over and above c l a s s i c a l g l y c o l y s i s . Figure 2 i s a s i m p l i f i e d representa-t i o n of what i s thought to occur during periods of oxygen deprivation by invertebrate f a c u l t a t i v e anaerobes (Hochachka et a l , 1973). The reaction scheme for carbohydrates i s the same as that which occurs i n vertebrate tissue as f ar as the l e v e l of phosphoenolpyruvate. However, instead of phosphoenol-pyruvate being d i r e c t l y converted to pyruvate i t i s f i r s t carboxylated to form oxaloacetate which i s quickly reduced to malate. Malate apparently has two metabolite fates. One route i n v o l v i n g the re v e r s a l of Krebs cycle reactions leads to the formation of succinate and the concomitant production of ATP. Succinate may accumulate as an end product or be further catabolized to proprionate, a v o l a t i l e acid, with the further production of ATP and C ^ . The other d e s t i n a t i o n of malate i s conversion to pyruvate followed by transamina-t i o n with glutamate to form alanine. Invertebrates have p a r t i c u l a r l y high l e v e l s of free amino acids and i t i s thought that these are i n i t i a l l y transaminated with a-ketoglutarate to form t h e i r respective a-ketoacids. The a-ketoacids formed from amino acids such as leucine and v a l i n e are further catabolized to v o l a t i l e acids again with the production of ATP and CX^. In t e r e s t i n g l y enough, there are data which lend credulence to the hypothesis that such a r e a c t i o n scheme may occur i n f i s h t i s s u e s . The anaero-b i c production of CO 2 by f i s h has been reported several times. Thus, g o l d f i s h i n j e c t e d with l a b e l l e d glucose produce l a b e l l e d CT^ under anoxic conditions (Hochachka, 1961) and g i l l t i s s u e incubated under anaerobic conditions produces C0 9 of metabolic o r i g i n (Ekberg, 1962). This concept of anaerobic 9 production i s supported by numerous reports of r e s p i r a t o r y quotients greater than 1 during both hypoxia and swimming (Kutty, 1968, 1972; Morris, 1967; Mathur, 1967). Furthermore, the anaerobic production of u n i d e n t i f i e d v o l a t i l e acids has been noted on at l e a s t two occasions (Blazka, 1958; Blazka and Kopecky, 1961). Perhaps the observations of the anaerobic production of NH^, CC>2 and v o l a t i l e acids can be explained s i n g u l a r l y , but when a l l of them are considered ±a toto they suggest that something i s unaccounted for i n our current theory of anaerobic metabolism i n f i s h and that the problem should by no means be considered closed. In p a r t i c u l a r , the above unusual observations suggest (1) that l a c t a t e may not be the sole end product of anaerobic glyco-genolysis, and/or (2) glycogen i s not the only anaerobic energy source u t i l i z e d . Although white muscle generates much of i t s energy by anaerobic means, i t also has an aerobic component to i t s metabolism. Moreover, during work t h i s t i s s u e has the capacity to increase i t s oxygen consumption to a small degree (Wittenberger and Diaciuc, 1965). In the mammalian heart and s k e l e t a l muscle the t o t a l amount of Krebs cycle intermediates increases as the work load increases (Shafer and Williamson, 1973; Edington'et'al,' 1973); however, i t had never been ascertained whether or not white muscle per se has the capacity to do so. Consequently, t h i s work also investigates the a b i l i t y of white muscle to augment the s i z e of i t s Krebs cycle pool during increased energy demands. In the present t h e s i s , two types of experiments were c a r r i e d out to examine energy metabolism i n carp white muscle. In one set of experiments, animals were exercised i n order to elucidate anaerobic c o n t r o l mechanisms and Krebs cycle a l t e r a t i o n s . In another study carp were subjected to hypoxic st r e s s and changes i n the free amino acid pool were examined. 10 Figure 2. Anaerobic metabolism i n invertebrate f a c u l t a t i v e anaerobes. Abbreviations not indicated i n text: 1,3 DPG, 1,3 diphos-phoglycerate; 2-KCA, 2-ketoisocaproate; a-KGA, a-ketoglutar-ate; a-KVA, a-ketoisovalerate; OXA, oxaloacetate; PEP, phosphoenolpyruvate; 3 PGA, 3 phosphoglycerate. Modified from Hochachka ^ t a l (1973). NAD+-N A D H -GLYCOGEN \ 3 PGA *-a-KvA 2-KCA Oj-KGA ADP + Pj ATP-maiate • fumarate NADP NADPH Pyruvate |aianine| FP red GTP- succinate ox GDP+Pi succinyl CoA J - C o ASH methylmalonylCoA ADP+P; ATP ADP+ P; . -C02 proprionyl CoA ATP-- C o A S H propnonate ADP+P; ATP / ~ - \ HsobutyrylCoA 1 1  isobutyrate NAD'1" NADH CQ ADP+Pj ATP 7~\ NA D + NADH isovalerylCoA -isovalerate O 11 CHAPTER II MATERIALS AND METHODS l i f t Animals Carp (Cyprinus carpio L.) were seined from l o c a l ponds i n southern B r i t i s h Columbia. F i s h employed i n the swimming experiments were between 12 and 15 cm i n length and weighed between 40 and 60 gm. These animals were maintained at 12 ±1°C i n aerated running water, under a simulated natural photoperiod and fed corn ad l i b . The carp u t i l i z e d i n the hypoxia experiment were about 30 cm i n length and weighed about 1000 gm. These animals were held out of doors i n running aerated water at 12 ±3°C and during the holding period of not longer than three weeks were not fed. Exercise Experiments Fis h were exercised i n a swim tunnel s i m i l a r to that described by Brett (1964). F i s h were introduced into the tunnel and forced to swim at 8.6 cm/sec for 1 hr. Following the introductory phase the f i s h were subjected to 10 min periods of swimming at fi x e d v e l o c i t i e s a f t e r which the v e l o c i t y was rapidly increased. The v e l o c i t y increment was approximately 7 cm/sec. The experiment was terminated either a f t e r successful completion of the 2nd v e l o c i t y l e v e l or when the f i s h was unable to remove i t s e l f from an e l e c t r i f i e d g r i d (15 v AC) at the downstream end of the tunnel. This l a t e r behaviour was defined as fatigue and usually occurred during the 4th speed increment. The water was maintained at 11°C and 100% a i r saturation. C r i t i c a l v e l o c i t y was calculated using the empirical formula of Brett (1964) such that the l a s t v e l o c i t y that the f i s h s u c c e s s f u l l y maintained was added to the v e l o c i t y at which the f i s h fatigued, m u l t i p l i e d by the proportion of the 10 minute period that i t was able to sustain t h i s f i n a l speed. The mean c r i t i c a l v e l o c i t y for the animals of this experiment was 45 cm/sec. F a i l u r e of a f i s h to meet the imposed v e l o c i t y i s due to the l i m i t a t i o n of the oxygen de l i v e r y system and not exhaustion of the white muscle (Jones, 12 1971; Brett, 1964) . A f t e r the experimental period an animal i s s t i l l able to perform burst a c t i v i t y i f forced to do so. Thus, i n the present study there i s no reason to believe that the white muscle was fatigued. Hypoxia Experiments The day before an experiment a f i s h was removed from the holding tank and placed i n a sealed chamber which was just s l i g h t l y l a rger than the animal. Aerated water, about 1 C° higher than the holding temperature, flowed through the chamber at 180 ±10 ml/min. The test chamber was covered with black p l a s t i c i n the evening and uncovered the following morning. The e n t i r e apparatus was immersed i n a water bath which served to maintain the temperature. In the morning, on the day of an experiment, the O2 content of the inflowing water was reduced by nitrogen gas to about 10% a i r saturation as measured by a micro Winkler technique (Kent and H a l l , personal communication). Within 45 minutes the O2 content i n the chamber f e l l to t h i s l e v e l and continued to drop for the duration of the experiment which was terminated a f t e r 4 hours. For co n t r o l f i s h which were kept i n the chamber for the same length of time as the experi-mentals the O2 content never f e l l below 60% of the a i r saturated l e v e l . In an experiment of t h i s type i t i s d i f f i c u l t to quantitate the degree of hypoxic stress to which the animals were exposed. Observations of a q u a l i t a -t i v e nature, however, indicated beyond question that the experimental f i s h were indeed subjected to severe hypoxia. Thus, preliminary studies showed that there was mortality i n animals subjected to experimental conditions for periods any longer than four hours; whereas, the co n t r o l f i s h could survive fo r a second day and probably longer. Furthermore, animals subjected to the reduced oxygen l e v e l demonstrated an extreme hyperventilation and frequently l o s t equilibrium. Neither of these behaviour patterns was observed i n the 13 con t r o l group. During the test period both the co n t r o l and the experimental f i s h demonstrated very l i t t l e a c t i v i t y . Preparation of Tissue f or Biochemical Analysis In the exercise experiments, the f i s h were removed from the holding tank or the swim tunnel and immediately decapitated. In the hypoxia experiment, the animals were removed from the experimental chamber and stunned by a blow to the head. In both studies, a portion of white muscle weighing approximately 1 gm was dissected from immediately below the dorsal f i n , s t a r t i n g at the posterior margin and going a n t e r i o r l y . The tissue sample was then frozen i n l i q u i d nitrogen, within 20 sec a f t e r removal of the f i s h from the water. In the hypoxia experiment, a second sample of ti s s u e , weighing about 30 gm, was also dissected out and frozen i n l i q u i d nitrogen. This tissue sample was used f o r v o l a t i l e acid a n a lysis. Extraction of A l l Metabolites Except V o l a t i l e - Acids The frozen tissue was powdered with mortar and p e s t l e which had been previously cooled and then the sample was placed i n a 40 ml p l a s t i c centrifuge tube into which a Teflon pestle could f i t snugly. The test tube contained an al i q u o t of cold HCIO^ (8% w/v) i n 40% ethanol. The sample was mixed quickly with a glass rod and the amount of HCIO^ was taken up to 3.5 ml/gm ti s s u e . The tissue was homogenized for 2 min at high speed with a V i r t i s (#23) mixer during which time the test tube was maintained i n a dry-ice ethanol bath. The homogenate was spun at 25,000 g (-4°C) f o r 10 min to p r e c i p i t a t e p r o t e i n . The supernatant s o l u t i o n was saved and the p r e c i p i t a t e was resuspended i n the same volume of HCIO^ as previously used. Af t e r a further c e n t r i f u g a t i o n the supernatant solutions were combined and n e u t r a l i z e d to pH 5.5-6.0 with 3 M K 9C0^ containing 0.5 triethanolamine. The p r e c i p i t a t e d KCIO^ was removed by 14 c e n t r i f u g a t i o n and the supernatant s o l u t i o n was stored at -20°C (Williamson and Corkey, 1969). Extraction of V o l a t i l e Acids V o l a t i l e acids were i s o l a t e d by steam d i s t i l l a t i o n (Baker, 1957). The larger piece of frozen ti s s u e was broken into small pieces and placed i n a 500 ml double neck d i s t i l l i n g f l a s k . Ten volumes of H^ O and 2 drops of Antifoam A concentrate (Sigma) (Ackman and Noble, 1973) were added and the pH was adjusted to 2.0-2.5 with 10 N ILjSO^. Between 10 and 11 times the volume of f l u i d o r i g i n a l l y present i n the d i s t i l l i n g v essel was c o l l e c t e d i n 25 ml KOH pH=8. The pH of the d i s t i l l a t e was maintained between 8.0-8.5 with 0.1 N KOH. The d i s t i l l a t e was f l a s h evaporated to dryness and taken up i n 200 y l of acetate free formic a c i d . Enzymatic Analysis of Metabolites A l l of the metabolites, with the exception of the amino acids and the v o l a t i l e acids, were measured enzymatically, Furthermore, each of the enzymat-i c analyses, except the procedures f o r formate and IMP, was based on the absorbance changes of the pyridine nucleotides at 340 my. The change i n amount of the pyridine nucleotides i n ymoles i s calculated from the equation (A b p t i -c a l density.^Q )(volume of assay mixture)/?, where E = 6.22 cm /ymole, the e x t i n c t i o n c o e f f i c i e n t of the pyridine nucleotides at 340 my. This value represents the number of ymoles of metabolite present per the amount of protein free n e u t r a l i z e d extract added, i f i n the analysis there i s a 1:1 r a t i o between the content of measured metabolites and the oxidation or reduc-t i o n of the pyridine nucleotides. Assays were c a r r i e d out at 37°C on a Unicam SP 1800 dual beam spectophotometer connected to a s t r i p chart recorder. A l l enzymes were purchased from Sigma, St. Louis, Mo. 15 Alanine assay: 4. _L M A T . T J _L u + l a c t a t e dehydrogenase _^ . , „._.+ pyruvate + NADH + H z e l a c t a t e + NAD glutamate-pyruvate alanine + a-ketoglutarate transaminase pyruvate + glutamate Reagents Buffer: 0.5 M t r i s pH 8.1 NADH: 8 mM i n 1% KHC03 (w/v) a-ketoglutarate: 0.1 M i n 0.1 M t r i s pH 7.4 Lactate dehydrogenase: source - beef heart Glutamate-pyruvate transaminase: source - pig heart Procedure LDH i s added to a cuvette containing buffer, NADH (0.17 mM), and neutr a l i z e d p r o t e i n free extract to remove endogenous pyruvate. When the reaction i s complete a-ketoglutarate (0.2 mM) i s added. F i n a l l y glutamate-pyruvate transaminase i s added and the decrease i n O D ^ ^ Q i s recorded. The reaction i s extremely slow, thus i t i s preferable to p l o t an alanine standard curve and int e r p o l a t e to a s c e r t a i n the content of alanine i n the extract. Reaction times - pyruvate, 1 min; alanine, 10 min (Lowry and Passonneau, 1972). 16 Aspartate assay: oxaloacetate + NADH + H + malate dehydrogenase +• malate + NAD + aspartate + a-ketoglutarate glutamate-oxaloacetate transaminase >oxaloacetate + glutamate Reagents Buffer: 50 mM imidazole pH 7 NADH: 8 mM i n 1% KHC0 3 (w/v) a-ketoglutarate: 0.1 M i n 0.1 M t r i s pH 7.4 Malate dehydrogenase: source - p i g heart Glutamate-oxaloacetate transaminase: source - p i g heart . Procedure Malate dehydrogenase i s added to a cuvette containing buffer, NADH (0.17 mM), a-ketoglutarate (0.2 mM), and ne u t r a l i z e d protein free extract. When the reaction i s complete glutamate-oxaloace-tate i s added and the decrease i n 0D-... i s recorded. Reaction 340 time - 10 min (Lowry and Passonneau, 1972). C i t r a t e assay: oxaloacetate + NADH + H + malate dehydrogenase ••malate + NAD c i t r a t e c i t r a t e lyase oxaloacetate + acetate Reagents Buffer: 0.1 M t r i s pH 7.6 NADH: 8 mM i n 1% KHC0 3 (w/v) Zn C l 2 : 1.2 mM i n H 20 Malate dehydrogenase: source - p i g heart C i t r a t e lyase: source - Aerobacter aerogenes 17 Procedure Malate dehydrogenase i s added to a cuvette containing b u f f e r , NADH (0.17 mM), Z n C l 2 (40 pM) and ne u t r a l i z e d p r o t e i n free extract. When the reac t i o n i s complete c i t r a t e lyase i s added and the decrease i n 0°3^Q recorded to determine c i t r a t e content. Reaction time - 3 min (Lowry and Passonneau, 1972). Formate assay: ^ . , , c , . J t e t r a h y d r o f o l i c a c i d formylase formate + ATP + t e t r a h y d r o f o l i c a c i d N (lO)-formyl-tetrahydrofolic a c i d + ADP + P H+ N (lO)-formyl-tetrahydrofolic a c i d •5,10-methenyl-t e t r a h y d r o f o l i c a c i d Reagents Buffer: 1.0 M triethanolamine pH 8.0 Tetrahydrofolic a c i d : 0.01 M pH 7.0 i n 1 M 2-mercaptoethanol ATP: 0.05 M i n 1.0 M triethanolamine MgCl 2: 0.1 M i n H 20 Percho l i c a c i d : 2% (w/v) i n B^O Tetr a h y d r o f o l i c a c i d formylase: source - Clostridium cylindrosporum Pr.66edure Te t r a h y d r o f o l i c a c i d formylase i s added to a centrifuge tube containing buffer, t e t r a h y d r o f o l i c a c i d (0.4 mM), ATP '(1 mM), MgCl 2 (5 mM) and ne u t r a l i z e d protein free extract. A f t e r 2 min at 37°C 1 volume of p e r c h l o r i c acid i s added and the mixture centrifuged to remove pr o t e i n . The dif f e r e n c e i n 0 D3^Q between a blank and a sample i s determined. The ex t i n c t i o n c o e f f i c i e n t 18 2 of 5,10-methenyl-tetrahydrofolic acid at 350 mu i s 24.9 cm / ymole (Rabinowitz and Pr'icer, 1965). Fructose-6-phosphate and fructose-1,6-diphosphate assay: glucose-6-phosphate glucose-6-phosphate + NADP+ dehydrogenase ^ 6-phosphogluconate + NADPH + H + phosphoglucose isoniGir3 . s6 f ructose-6-phosphate • glucose-6-phosphate r- ^ n , ,. , , _ fructose-1,6-diphosphatase - , fructose-1,6-diphosphate - - »- fructose-6-phosphate + P^ Reagents Buffer: 1.0 M t r i s pH 8.8 NADP+: 10 mM i n IL/) MgCl 2: 0.1 M i n H 20 EDTA: 1.2% (w/v) i n H 20 Glucose-6-phosphate dehydrogenase: . source - yeast Phosphoglucose isomerase: source - yeast Fructose-1,6-diphosphatase: source-^arabbit l i v e r Procedure Glucose-6-phosphate dehydrogenase i s added to a cuvette containing buffer, NADP+ (0.33 mM), MgGl 2 (7 mM), EDTA (10 mM), and ne u t r a l i z e d protein free extract. Glucose-6-phosphate dehydrogenase i s added to remove.endogenous glucose-6-phosphate. When the reac t i o n i s complete phosphoglucose isomerase i s added and the increase i n ^340 "*"S r e c o r < ^ e < ^ t o determine the content of fructose-6-phosphate. F i n a l l y fructose-1,6-diphosphatase i s added to determine the 19 l e v e l of fructose-l,6-diphosphate. Reaction times -glucose-6-phosphate, 1 min; fructose-6-phosphate, 5 min; fructose-l,6-diphosphate, 30 min (Racker, 1965). Glucose-6-phosphate and ATP assay: glucose-6-phosphate glucose-6-phosphate + NADP+ dehydrogenase ^ 6-phosphogluconate + NADPH + H + ATP + glucose hexokinase ^ + glucose-6-phosphate Reagents Buffer: 0.05 M triethanolamine pH 7.5 NADP+: 10 mM i n H 20 MgCl 2: 60 mM i n H 20 Glucose: 30 mM i n H 20 Glucose-6-phosphate dehydrogenase: source - yeast Hexokinase: source - yeast Procedure .Glucose-6-phosphate dehydrogenase i s added to a cuvette contain-ing b uffer, NADP+ (0.17 mM), MgCl 2 (1 mM), and ne u t r a l i z e d protein free extract to determine the content of g l u c o s e s -phosphate. When the reac t i o n i s complete glucose (1.0 mM) i s added. F i n a l l y hexokinase i s included and the increase i n O D ^ Q recorded to determine the l e v e l of A l P i Reaction times -glucose-6-phosphate, 1 min; ATP, 8 min (Lamprecht and Trautschold, 1965). 20 a-glycerophosphate assay: a-glycerophosphate + NAD glycerophosphate + dehydrogenase > dihydroxyacetonephosphate + NADH + H Reagents Buffer: glycine-hydrazine pH 9.2 (prepared by Sigma -stock #826-6) NAD+: 10 mM i n H 20 a-glycerophosphate dehydrogenase: source - rabbi t muscle Procedure a-glycerophosphate dehydrogenase i s added to a cuvette contain-ing buffer, NAD+ (0.17 mM), and ne u t r a l i z e d protein free extract and the increase i n 0D o / r i i s recorded. The reac t i o n i s 340 extremely slow, thus i t ,is preferable to p l o t a standard curve and i n t e r p o l a t e to ascer t a i n the content of a-glycerophosphate i n the extract. Reaction time - 10 min (Lowry and Passonneau, 1 9 7 2 ) . Inosine monophosphate assay: hypoxanthine + 20„ + 2 H_0 xanthic oxidase >• u r i c a c i d + 2 H„0 inosine + P. l nucleoside phosphorylase >hypoxanthine + ribose-5-phosphate 5' nucleotidase >inosine + P. Reagents Buffer: 0.05 M KH 2P0 4 pH 7.4 EDTA: 0.1 M i n H o0 21 xanthic oxidase: source - buttermilk nucleoside phosphorylase: source - c a l f spleen 5 1 nucleotidase: source - Crotalus adamanteus venom Procedure The course of the reac t i o n i s followed at 293 mu. Xanthic oxidase i s added to a cuvette containing b u f f e r , EDTA (33 mM), and n e u t r a l i z e d protein free extract. When the re a c t i o n i s complete nucleoside phosphorylase i s added to remove any endo-genous inosine. F i n a l l y 5' nucleotidase i s included to determine the content of IMP. The e x t i n c t i o n c o e f f i c i e n t f o r 2 u r i c acid at 293 my i s 12 cm /umole. Reaction times -hypoxanthine, 1 min; inosine, 1 min; IMP, 20 min (adapted from Coddington, 1965). a-ketoglutarate assay: glutamate a-ketoglutarate + NH* + NADH + H + d e h y d r o g e n a s e , g i u t a m a t e + NAD+ Reagents Buffer: 0.5 M t r i s pH 8.0 NADH: 8 mM i n 1% KHC0 3 (w/v) (NH£) 2S0 4: 35 mM i n H 20 Glutamate dehydrogenase: source - bovine l i v e r Procedure GDH i s added to a cuvette containing b u f f e r , NADH (0.17 mM), (NH^^SO^ (5 mM), and neut r a l i z e d p r o t e i n free extract and the decrease i n OD^Q recorded. Reaction time - 8 min (Bergmeyer and Bernt, 1965).. 22 Pyruvate, ADP and AMP assay: + l a c t a t e dehydrogenase *• l a c t a t e + NAD + pyruvate + NADH + H ADP + phosphoenolpyruvate pyruvate kinase > ATP + pyruvate AMP + ATP adenylate kinase >. 2 ADP Reagents Buffer: 0.05 M triethanolamine pH 7.5 NADH: 8 mM i n 1% KHC03 (w/v) MgCl 2: 120 mM i n H 20 KCl: 750 mM i n H 20 Phosphoenolpyruvate: 15 mM i n 0.05 M triethanolamine pH 7.5 ATP: 6 mM i n 0.05 triethanolamine pH 7.5 Lactate dehydrogenase: source - beef heart Pyruvate kinase: source- rab b i t muscle Adenylate kinase: source - rabbi t muscle LDH i s added to a cuvette containing buffer, NADH (0.17 mM), MgCl 2 (2 mM), KCl (75 mM), and neutralized protein free extract to determine the content of pyruvate. When the reac t i o n i s complete phosphoenolpyruvate (0.25 mM) i s added followed by pyruvate kinase to determine the content of ADP. When the second reaction i s complete ATP (0.1 mM) i s included and the decrease i n O D ^ Q recorded a f t e r the addit i o n of adenylate kinase. For each mole of AMP two moles of NADH are oxidized. Reaction times - pyruvate, 1 min; ADP, 2-4 min; AMP, 6 min (Lowry and Passonneau, 1972). Procedure 23 Succinate assay: + , * T A ™ T _L U + l a c t a t e dehydrogenase . H pyruvate + NADH + H — — 2 »- l a c t a t e + NAD , , , A T V r i pyruvate kinase ^ , A r > T 1 phosphoenolpyruvate + ADP — — • pyruvate + ADP , A . ™ i ^ A succinate thiokinase . . _ . , A T v n . _ succinate + ATP + CoA : — • suc c i n y l CoA + ADP + P i Reagents Buffer: 0.05 M triethanolamine, 10 mM MgSO^, 5 mM EDTA pH 7.4 NADH: 5 mM i n 0.1 M triethanolamine pH 8.2 Phosphoenolpyruvate: 0.1 M i n 0.05 M triethanolamine pH 7.4 ATP: 10 mM i n 0.05 M triethanolamine pH 7.4 CoA, l i t h i u m s a l t : 5 mM i n ^ 0 Lactate dehydrogenase: source - beef heart Pyruvate kinase: source - rabbit muscle Succinate thiokinase: source - E^ . c o l i Procedure LDH i s added to a cuvette containing buffer, NADH (0.17 mM), and neutralized protein free extract to remove, endogenous pyruvate. When the i n i t i a l reaction i s complete phosphoenolpyruvate (1.5 mM) and pyruvate kinase are added to the cuvette to remove endogenous ADP. When the second reaction i s complete, l i t h i u m CoA (0.8 mM) and ATP (0.15 mM) are added. F i n a l l y succinate thiokinase i s added and the decrease i n 0 D o / o i s recorded. Reaction times -340 pyruvate,,1 min; ADP, 3 min; succinate, 30 min (Williamson and Corkey, 1969). Lactate assay: i _L i a A ^ + l a c t a t e dehydrogenase ^ . , T A_ T T . 77+ la c t a t e + NAD p- pyruvate + NADH + H 24 Reagents Buffer: glycine-hydrazine pH 9.2 (prepared, by Sigma-stock #826-6) NAD+: 10 mM i n H 20 Lactate dehydrogenase: source - beef heart Procedure LDH i s added to a cuvette containing buffer, NAD+ (0.33 mM), and neutralized protein free extract and the increase i n O D ^ ^ Q recorded. Reaction time - 45 min (Sigma b u l l e t i n #826). Malate assay: glutamate-oxaloacetate oxaloacetate + glutamate t r a n s a m l n a s e ^ aspartate + a-ketoglutarate T ^ . „,T>+ malate dehydrogenase . _ ^ » T » ^ T T _L T T + malate + NAD 3- ^ • oxaloacetate + NADH + H Reagents Buffer: glycine-hydrazine pH 9.2 (prepared by Sigma-stock #826-6) NAD+: 10 mM i n H 20 Glutamate: 88 mM i n 0.5 M t r i s Glutamate-oxaloacetate transaminase: source - p i g heart Malate dehydrogenase: source - p i g heart Procedure Glutamate-oxaloacetate transaminase i s added to a cuvette.containing buffer, NAD+ (0.17 mM), glutamate (10 mM), and neutralized protein free extract. When the reaction i s complete malate dehydrogenase i s added and the increase i n O D ^ Q i s recorded. Reaction time -60 min (Lowry and Passonneau, 1972) NH^ assay: HTTT + i i ^ T ^ ~KTATVTT . T T + glutamate dehydrogenase . ^  NH, + a-ketoglutarate + NADH + H — - — — • glutamate + NAD+ 25 Reagents Buffer: 0.5 M t r i s pH 8.0 NADH: 8 mM i n 1% KHC03 (w/v) a-ketoglutarate: 0.1 M i n 0.1 M t r i s pH 7.4 Glutamate dehydrogenase: NH^ free, source - bovine l i v e r Procedure GDH i s added to a cuvette containing buffer, NADH (0.17 mM) , a-ketoglutarate (10 mM), and neutralized p r o t e i n free extract and the decrease i n 0D~ / r t recorded. Reaction time - 10 min 340 (Kun and Kearney, 1970). Amino Acid Analysis P r i o r to amino acid analysis the protein free extract, was separated from i n t e r f e r i n g substances by absorption on a column (1 x 15 cm) of Amberlite IR-120 which had been previously.washed with 5% HCl. 2.0 ml of protein free extract adjusted to pH 2-3 with 10 N ^SO^ were applied to the column. The column was washed with 40 ml of d i s t i l l e d water and the amino acids were eluted with 40 ml 2 N NH^OH (Williamson et_ a l , 1967). The eluant was evapor-ated to dryness i n a 1 l i t r e f l a s k taken up i n 5 ml ^ 0 and transferred to a 50 ml f l a s k . The o r i g i n a l 1 l i t r e c o l l e c t i n g f l a s k was washed with 2 ml H^ O which was placed i n the 50 ml f l a s k . The amino acid extract was again evaporated to dryness and stored at -20°C. Immediately p r i o r to analysis the sample was taken up i n c i t r a t e buffer pH 2.2. Amino acids i n 200-500 y l al i q u o t s were separated on a Beckman 120C amino acid analyzer. The ion exchange r e s i n was a sulfonated p o l y s t y r e n e - d i v i n y l benzene copolymer. The operating temperature was 55°C. Lysine, h i s t i d i n e and arginine were separated on a 16 cm column by e l u t i o n with 0.35 N Na c i t r a t e pH 5.25. The a c i d i c amino acids were separated on a 46 cm column by e l u t i o n 26 with 0.2 N Na c i t r a t e pH 3.25. A l l amino acids were detected by ninhydrin reagent. Internal standards, a^-amino-3-guanidinopropionic acid for the basics column and norleucine for the a c i d i c s column, were employed so that a correc-t i o n could be made for the aging of the ninhydrin reagent. The number of moles of each amino acid residue applied was determined by comparison with a standard chromatogram. V o l a t i l e Acid Analysis V o l a t i l e acids were separated by gas l i q u i d chromatography. A Varian Aereograph (#1700) equipped with a flame i o n i z a t i o n detector was used. The columns were 6 f t by 1/8 i n s t a i n l e s s s t e e l packed with Chromsorb 101 80/100 mesh, and were maintained at 130°C. The c a r r i e r gas was N 2 with a flow rate of 56 ml/min. No attempt was made to accurately quantitate the content of v o l a t i l e acids. Calculations Both the amino acid analysis and. the enzymatic analysis provide data i n ymoles of s p e c i f i e d substance per al i q u o t of pro t e i n free n e u t r a l i z e d extract. The a l i q u o t of neutralized p r o t e i n free extract i s converted to the corres-ponding value i n gm fresh tissue by multiplying by (vc)(W) (V + V-,) (V + V. ) c d a h' where, V a t o t a l volume of HC10, added during the extraction V. b amount of water i n the sample of tissue powder V c volume of al i q u o t used for n e u t r a l i z a t i o n V d volume of K^CO^ added to n e u t r a l i z e the above a l i q u o t V c W fresh weight of tissue sample i n grams The water content of carp white muscle was determined to be about 80%. 27 In the r e s u l t s , where appropriate, values are expressed as ± standard error of the mean. Results were analyzed s t a t i s t i c a l l y , by a two sampled t -test f o r data c o l l e c t e d i n the swimming experiment and by the Mann-Whitney U test for data of the hypoxia experiment. In a l l cases a p r o b a b i l i t y of l e s s than 0.05 was considered to be s i g n i f i c a n t . 28 CHAPTER III RESULTS 28 a Swimming Experiment Carried. Out, i n Spring 1974 The concentrations of a l l of the g l y c o l y t i c intermediates measured with the exception of pyruvate increased during a c t i v i t y (Table I ) . There was also a tendency f o r a-glycerophosphate, a metabolite associated with the g l y c o l y t i c pathway, to increase. The greatest change occurred i n l a c t a t e l e v e l s which increased by about 10 ymoles/gm. The mass action r a t i o of the phosphofructo-kinase reaction, that i s (ADP)(fructose-l,6-diphosphate)/(ATP)(fructose-6-phosphate), was 1.82 i n the rested f i s h and 2.78 i n the exercised group. These values were displaced from.the equilibrium constant of reaction by about two orders of magnitude (Mahler, and Cordes, 1966), and support the concept of a regulatory r o l e of phosphofructokinase i n t h i s t i s s u e . The r e s u l t s of t h i s study with respect to the adenylate pool are summar-ized i n Tables II and I I I . The content of ATP decreased with a c t i v i t y . When the animals were exercised, ATP concentrations were reduced by about 65%. Levels of ADP also decreased a small but s i g n i f i c a n t amount; however, AMP concentrations remained low and unchanged. Thus i n t h i s complex way, the t o t a l free adenylate pool decreased during the exercise period. Concomitant with t h i s decrease was an increase i n IMP concentration. The increase i n IMP l e v e l and the decrease i n the adenylate pool were e s s e n t i a l l y i n 1:1 s t o i c h i o -metry, a r e s u l t c l e a r l y showing that the adenylate pool was decreased by the conversion of AMP to IMP and NH^. I t i s i n t e r e s t i n g to note that although NH^ concentration increased i n working white muscle, the change i s not as large as for IMP. The energy charge, [ATP] +0.5 [ADP]/[ATP] + [ADP] + [AMP], as defined by Atkinson (1968a), was high i n both groups of animals, 0.89 i n the rested f i s h , and 0.83 i n the exercised group (Table I I I ) . The apparent equilibrium constant of the adenylate kinase reaction was 0.3 i n both the rested and exercised animals. 29 Of the two amino acids measured, aspartate l e v e l s decreased, by a small but s i g n i f i c a n t amount while there was a tendency f or an increase i n the l e v e l of alanine (Table IV). C i t r a t e , malate, a-ketoglutarate and oxaloacetate, compounds associated with the Krebs cycle, did not increase during- a c t i v i t y , and i n f a c t there was a tendency for c i t r a t e to decrease. I t therefore appears that carp white muscle has a l i m i t e d capacity to augment the s i z e of i t s Krebs cycle pool. 30 Table Concentrations of g l y c o l y t i c intermediates i n white muscle of carp under two w e l l defined conditions: r e s t i n g and maximally a c t i v e . Maximally Metabolite Resting a c t i v e Glucose-6-phosphate 0.67 ± 0.05 1.33 ± 0.10* Fructose-6-phosphate 0.11 ± 0.01 0.18 ± 0.03* Fructose-1,6- 0.85 ± 0.08 1.28 ± 0.10* diphosphate Pyruvate 0.11 ± 0.03 0.12 ± 0 . 0 3 Lactate 3.71 + 0.17 12.58 + 1.18* a-glycerophosphate 2.52 + 0.60 4.59 ± 1.30 A l l values are expressed i n micromoles/gm of f r e s h t i s s u e (±S.E.). N = 7. * S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e between groups. 31 Table I I - Concentrations of the adenylates and r e l a t e d metabolites i n white muscle of carp under two w e l l defined conditions: r e s t i n g and maximally a c t i v e . Maximally Metabolite Resting a c t i v e ATP 4.12 ± 0.18 1.87 ± 0.08* ADP 0.97 ± 0.05 0.73 ± 0.18* AMP 0.07 ± 0.02 0.08 ± 0.02 IMP 1.38 ± 0.26 4.01 + 0.16 NHI" 3.00 + 0.30 4.10 ± 0.25* 4 A l l values are expressed i n micromoles/gm of fresh t i s s u e (±S.E.M). N = 7. * S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e between groups. 32 Table I I I . Energy charge and changes i n concentrations of the adenylate pool and r e l a t e d metabolites i n white muscle of carp under two w e l l defined conditions: r e s t i n g and maximally a c t i v e . Adenylate pool IMP NH; Energy charge Resting 5.1 1.38 3.00 0.89 Maximally a c t i v e 2.68 4.01 4.10 0.83 D i f f e r e n c e -2.48 +2.63 • +1.10 -0.06 Adenylate pool, IMP and NH^ are expressed i n micromoles/gm of fresh t i s s u e . N = 7. 33 Table TV. Concentrations of Krebs cycle intermediates and r e l a t e d metabolites i n white muscle of carp under two w e l l defined conditions: r e s t i n g and maximally a c t i v e . Metabolite C i t r a t e Malate Oxaloacetate a-ketoglutarate Aspartate Alanine Resting 0.50 ± 0.06 1.12 ± 0.28 Undetectable <0.10 0.24 + 0.03 2.62 + 0.46 Maximally a c t i v e 0.34 + 0.04 1.16 ± 0.09 Undetectable <0.10 0.14 ± 0.03* 3.12 ± 0.47 A l l values are expressed i n micromoles/gm of f r e s h t i s s u e (±S.E.). N = 7. * S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e between groups. 34 Swimming Experiment Carried Out i n Summer 1974 Results of a second study i n which f i s h were also exercised at an i n t e r -mediate speed are shown i n Table V. Although i t i s now well recognized that white muscle i s a c t i v e at intermediate speeds, these data, however, are d i f f i -c u l t to i n t e r p r e t since the degree of a c t i v i t y of i n d i v i d u a l f i b e r s i s unknown. For t h i s reason the experiment was terminated a f t e r only a few i n d i v i d u a l s were sampled. In l i g h t of the paucity of r e s u l t s a l l of the raw data are presented. Even though the data are l i m i t e d they are i n t e r e s t i n g i n a number of respects. The concentration of la c t a t e i n the c o n t r o l and maximally exercised groups was s i m i l a r to that found i n the spring study. Lactate reached a maximum of about 12 umoles/gm i n both swimming studies. Lactate concentration i n white muscle sampled from f i s h worked at moderate speeds was i n most cases intermediate i n l e v e l r e l a t i v e to that found i n r e s t i n g and maximally a c t i v e muscle. NH^ con-centration increased with a c t i v i t y and i n one i n d i v i d u a l case reached 95 umoles /gm. The l e v e l of NH^ i n the summer sampled f i s h was 5-6 times greater than that i n the spring sampled animals. As expected both ATP and ADP. generally decreased with a c t i v i t y , the lowest l e v e l s occurred i n the maximally exercised group. There was a tendency for AMP to increase i n the most strenuously worked muscle. I t i s i n t e r e s t i n g to note that the energy charge remained high and r e l a t i v e l y constant at a l l three work loads. The l e v e l of IMP was highest i n the maximally exercised group although there was much i n d i v i d u a l v a r i a b i l i t y i n t h i s component. The high content of IMP i n the one rested f i s h i s not unexpected i n l i g h t of the low l e v e l of ATP; however, the low l e v e l of IMP i n two of the f i s h exercised at intermediate speeds remains an enigma. This f i n d i n g may be due to experimental error since i n a l l other cases the l e v e l of the adenylate pool and the concentration of IMP are i n v e r s e l y proportional. Table V. Concentrations of metabolites and the energy charge value i n white muscle,of carp a f t e r various l e v e l s of a c t i v i t y . L e v e l of Animal Metabolite Energy a c t i v i t y number Lactate ATP ADP AMP IMP chargi Rested 1 1.70* q-16.96 5.17 1.01 0.02 0.78 0.92 2 3.26 16.52 2.31 0.72 0.02 2.59 ... 0.87 Intermediate - 3 2.36 25.26 3.99 0.93 0.03 0.38 0.90 4 4.54 23.13 1.98 0.49 0.02 0.52 0.89 5 7.17 19.12 2.77 0.70 0.02 1.24 0.89 Maximally 6 9.90 25.11 1.62 0.67 0.06 3.29 0.83 a c t i v e 7 12.66 95.10 1.07 0.64 0.14 4.06 0.75 *A11 values expressed i n ymoles/gm f r e s h t i s s u e . 36 Hypoxia Experiment The concentration of l a c t a t e i n white muscle of. carp.exposed to severe environmental hypoxia was about 12 ymoles/gm (Table VI). This value was si m i -l a r to that found a f t e r maximal a c t i v i t y by white muscle. The content of l a c t a t e i n the c o n t r o l f i s h of the hypoxia study was almost as high as that of the experimentals. In l i g h t of previous observations (Tables I and V) that the l a c t a t e l e v e l i n white muscle of rested f i s h was about 3 ymoles/gm, i t i s probable that the experimental f i s h , i n the present study, were subjected to some degree of anaerobic s t r e s s . Nevertheless, many p o s i t i v e conclusions may be made from t h i s work. I t has been c l e a r l y shown that the v o l a t i l e acids, acetate, proprionate, butyrate, or valerate (Table VI) were not produced as anaerobic end products i n the white muscle of carp. Accumulation of formate would not be detected with the a n a l y t i c a l techniques employed here; however, using an enzymatic assay i t had been shown that t h i s a c i d was not produced i n white muscle of carp during strenuous exercise. Furthermore, the data show that succinate was not a q u a n t i t a t i v e l y important anaerobic end product i n carp white muscle. The content of free amino acids may be found i n Table VII. Since the c o n t r o l animals were, on the basis of l a c t a t e concentration, subjected to some degree of anaerobic stress, any a l t e r a t i o n i n the free amino acid pool w i l l be minimal and consequently d i f f i c u l t to pick up. Regardless, i t may be said with some confidence that no one amino acid was a q u a n t i t a t i v e l y important anaerobic end product such as alanine i s i n invertebrate animals. No single amino acid was markedly a l t e r e d by the hypoxic conditions; although, there was a tendency for an increase i n the t o t a l free amino acid pool and a l l of the amino acids with the exception.of glycine. The most s i g n i f i c a n t f i n d i n g i n r e l a t i o n to amino acid metabolism was the amount of nitrogen that was 37 locked up i n the free amino acid.pool. This increased by about.6. ymoles/gm (Table VII) and was i n agreement with the tendency f or free NH^ to decrease (Table VI) and the t o t a l free amino acid pool to increase. A lack of 1:1 stoichiometry between the increase i n amino acids and the increase i n nitrogen incorporated into the amino acid pool occurs since h i s t i d i n e , l y s i n e , and arginine contain more than 1 nitrogen atom each. The amount of. nitrogen i n c o r -porated into the free amino acid pool was far i n excess of the decrease i n NH^. When these findings are considered along with the lack of production of v o l a t i l e acids i t may be s a f e l y concluded that there was not an ac t i v e and general amino acid fermentation. Glycine may, however, be an exception to t h i s general r u l e since i t was the only amino acid to decrease during hypoxia. 38 Table VI. Metabolite concentrations i n carp white muscle before and a f t e r hypoxic s t r e s s . Mean Control Range Hypoxic Mean . . Range Lactate NH+ Succinate Acetate Proprionate Butyrate Valerate-9.60* 6.23-13.50 4.32 2.18-8.24 <0.30 <0.2 x 10 -3 12.02 6.90-15.12 3.09 1.90-5.63 <0.30 <0.2 x 10 -3 *A11 values are expressed i n ymoles/gm of fr e s h t i s s u e . N = 4. 39 Table VII. Free amino acid concentration i n carp white muscle before and a f t e r hypoxic s t r e s s . Control Hypoxic Mean Range Mean . Range Asp 0.15* (0.08-0.21) 0.09 (0.07-0.11) Thr 0.47 (0.21-0.70) 0.48 (0.28-0.63) Ser-Gln 0.73 (0.46-1.09) 1.00 (0.76-1.42) Glut 0.22 (0.11-0.31) 0.26 (0.19-0.32) P r o l , 0.17 (0.08-0.23) 0.25 (0.25-0.37) Gly 6.17 (3.69-9.82) 5.42 (4.31-6.94) Ala 0.84 (0.42-1.08) 1.41 (0.80-1.99) Va l 0.29 (0.14-0.48) 0.44 (0.29-0.69) Met 0.08 (0.05-0.12) 0.11 (0.09-0.16) Isoleu 0.26 (0.12-0.43) 0.38 (0.21-0.38) Lec 0.37 (0.23-0.57) 0.55 (0.34-0.83) Tyr 0.06 (0.02-0.11) 0.09 (0.07-0.12) Phe 0.08 (0.04-0.12) 0.33 (0.08-0.73) Lys 1.22 (0.64-2.12) 1.97 (0.63-3.40) Hist 3.71 (2.83-4.66) 4.74 (3.56-6.02) Arg 0.10 (trace-0.16) 0.23 (0.13-0.32) To t a l amino acids 14.93 (13.12-18.18) 17.75 (17.31-18.67) Nitrogen i n amino acid pool 23.74** (21.16-26.24) 29.90** (27.63-31.92) *A11 values are expressed i n ymoles/gm fr e s h t i s s u e . N = 3. ^ ^ S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e between means• 40 CHAPTER IV DISCUSSION 40 a Control of G l y c o l y s i s The s i t u a t i o n with respect.to the c o n t r o l of g l y c o l y s i s i n white muscle seems r e l a t i v e l y s t r a i g h t forward. Concentrations of glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate and l a c t a t e r i s e during the exercise period i n d i c a t i n g that, as expected, the g l y c o l y t i c contribution to energy production i s increased during high work rates. A c t i v a t i o n of the two key regulatory enzymes of g l y c o l y s i s , phosphofructokinase and pyruvate kinase, may be explained on the basis of the known k i n e t i c properties of these enzymes. Thus substrate and product a c t i v a t i o n (Freed, 1971) of phosphofructokinase (by fructose-6-phosphate and fructose-1,6-diphosphate, respectively) with concomitant fructose-1,6-diphosphate feed, forward a c t i v a t i o n of pyruvate kinase, commonly observed i n f i s h muscle pyruvate kinases (Somero and Hochachka," 1968; Mustafa et a l , 1971), could r e a d i l y account for the observed increase i n g l y c o l y t i c r a te. Moreover, d e i n h i b i t i o n of these two enzymes would be expected as a consequence of f a l l i n g l e v e l s of ATP (Freed, 1971; Mustafa et a l , 1971), and of creatine phosphate (Storey and Hochachka, 1974), both processes being f a c i l i t a t e d by fructose-1,6-diphosphate. In these co n t r o l c h a r a c t e r i s t i c s , carp white muscle g l y c o l y s i s appears to be s i m i l a r to other more commonly studied systems. Two inconsistencies with the l i t e r a t u r e , however, deserve mention. F i r s t l y , i t i s evident from the data that the energy charge i s e s s e n t i a l l y i d e n t i c a l at both l e v e l s of muscle metabolism and muscle work. Although i n v i t r o both phosphofructokinase (Shen e_t a l , 1968) and pyruvate kinase (Purich and Fromm, 1973) are stimulated by a decrease i n t h i s parameter, i n vivo i t i s clear that energy charge plays only a modest r o l e i n sustaining the high g l y c o l y t i c rates that support extreme muscle work. Secondly, there i s no evidence whatever that AMP constitutes an uniquely important metabolite 41 s i g n a l to g l y c o l y s i s i n white muscle, as suggested by Newsholme (1972) for heart, because i t s concentration i s s i m i l a r at the widely d i f f e r i n g g l y c o l y t i c rates. In contrast, i f there i s a single adenylate s i g n a l that i s important to a sustained high l e v e l of g l y c o l y s i s i t presumably i s ATP, since i t s o v e r a l l concentration change i s the greatest. However, as s h a l l be argued l a t e r , i n order to take advantage of t h i s metabolic " s i g n a l " the organism must tol e r a t e an o v e r a l l reduction i n the adenylate pool. Lactate and Other P o t e n t i a l End Products I t i s i n t e r e s t i n g to note that i n the three i n d i v i d u a l experiments report-ed here the maximal l e v e l of white muscle l a c t a t e i s c o n s i s t e n t l y about 12 ymoles/gm. However, when carp white muscle was e l e c t r i c a l l y stimulated u n t i l the muscle i t s e l f fatigued, l a c t a t e reached 33 ymoles/gm (Wittenberger and Diaciuc, 1965). The present data may i n d i c a t e an upper l i m i t that l a c t a t e approaches i n vivo for t h i s species. Data which support the concept of a l i m i t to which l a c t a t e i s allowed to normally accumulate have been obtained with rainbow trout. Thus, i n trout strenuously exercised for 5 minutes muscle l a c t a t e increased from 3 to 47 ymoles/gm. Yet i n animals sampled a f t e r 9 and 15 minutes of strenuous exercise, there was no further increase i n muscle la c t a t e above that found i n the animals worked for 5 minutes (Black et a l , 1962) . Stevens and Black (1966) and Hammond and Hickman (1966) also obtained r e s u l t s of a s i m i l a r nature with trout. A l l of the a v a i l a b l e biochemical and h i s t o l o g i c a l evidence suggests that carp white muscle i s p a r t i c u l a r l y w e ll designed for anaerobic metabolism. In f a c t , on the basis of hypoxia studies, one would normally consider carp to be a "good anaerobe". The extreme resistance of t h i s animal to low 0 2 environ-ments was demonstrated by Mazeaud (1973) who induced anoxia i n carp by expos-42 ing them.to a i r for periods up to 2 hours with only infrequent short returns to water. Even at temperatures as high as 10°C the lower l e t h a l l e v e l of dissolved for carp was observed to be about 0.5 mg/1 (-5% a i r saturation) (Downing and Merkins, 1957). Species c l o s e l y r e l a t e d to carp also show an inordinate tolerance to hypoxia. For instance, Basu (1949) found no deaths i n g o l d f i s h held for 9 hours i n water with an 0^ content of only 0.6 mg/1, at 28°C. This p a r t i c u l a r species can i n f a c t survive t o t a l i n t e r r u p t i o n of oxidative phosphorylation by cyanide poisoning (Fry, personal communication). A further extreme case i s demonstrated by Crucian carp which l i v e i n small ponds that become i c e locked, gradually grow anoxic and remain 0^ free for up to 2 months (Blazka, 1958). These findings are very much d i f f e r e n t from those that have been obtained with many other f i s h species. For instance, the salmonids, which are the most a c t i v e l y studied, have been repeatedly shown to succumb between 1 and 2 mg/1 dissolved 0 2 (see Doudoroff and Shumway, 1970, for numerous references). Studies of t h i s nature, of course, do not provide evidence of the anaerobic capacity of white muscle per se. However, i t i s possible to estimate the anaerobic c a p a b i l i t i e s of the swimming musculature with forced exercised experiments. The aerobic e f f i c i e n c y of a working muscle, that i s the energy converted to u s e f u l work/energy a v a i l a b l e from consumed oxygen, i s considered to be about 20-30% ( H i l l , 1950); any values higher than t h i s are thus i n d i c a t i v e of anaero-bic metabolism. The percentage aerobic working e f f i c i e n c y of swimming g o l d f i s h (Figure 3) has been determined by Smit e_t al_ (1971) . At low swimming speeds the g o l d f i s h demonstrates a low aerobic e f f i c i e n c y , as the animal swims fas t e r the percentage e f f i c i e n c y increases, and at v e l o c i t i e s above. 6 lengths/sec the working e f f i c i e n c y i s greater than can be accounted for by aerobic means alone. 43 At the highest speeds the g o l d f i s h can a t t a i n the working e f f i c i e n c y reaches almost. 100%.. Thus, i t may be said with some certitude that much of the energy required for intense swimming by the g o l d f i s h i s generated by anaerobic means and i n the extreme case approaches 80% of the energy output of the animal. In a comparable study employing rainbow trout, Webb (1971) has shown that the o v e r a l l contribution of anaerobic metabolism to intense swimming* i s n e g l i g i b l e . Yet i n trout, exercised strenuously for only, a few minutes, the concentration of muscle l a c t a t e increases to 40-50 ymoles/gm from a r e s t i n g value of about 3 ymoles/gm (Black et_ a l , 1962; Stevens and Black, 1966; Hammond and Hickman, 1966). Even i n mammalian muscle, the content of la c t a t e normally reaches 35 ymoles/gm during a c t i v i t y (Edington jit a l , 1972). Since carp white muscle i s apparently capable of performing high l e v e l s of anaerobic work one would pr e d i c t that i t would also produce, high.amounts of l a c t a t e . Yet, despite the anaerobic c a p a b i l i t i e s of carp white muscle, l a c t a t e accumulation i n t h i s tissue i s low by vertebrate standards. The phenomenon of a lack of p r o p o r t i o n a l i t y between the amount of energy which must be generated anaerobically and the accumulation of l a c t a t e has been observed for other species. For instance, Blazka and Kopecky (1961) claimed that a f t e r 4 hours of anoxia the Crucian carp accumulated only 0.5 ymoles of lactate/gm. During hypoxic excursions (ending i n anoxia) bullheads accumulated only about 0.7 ymoles lactate/gm of muscle; by comparison l a c t a t e l e v e l s increased by about 15 and 30 ymoles/gm i n rainbow and brown trout, respective-l y , although the hypoxic stress i n the l a t t e r two cases was much less severe (Burton and Spehar, 1971). C l e a r l y , white muscle of some species has a high anaerobic c a p a b i l i t y but i t does not accumulate an extraordinary amount of l a c t a t e . I t i s known that l a c t a t e i s not excreted during anaerobic work 44 (Prosser et. a l , 1957) and i n l i g h t " o f p e r i p h e r a l v a s o c o n s t r i c t i o n during hypoxia ( S a t c h e l l , 1971) i t i s u n l i k e l y that there could be an e f f e c t i v e de-p o s i t i o n i n other tissues such as the l i v e r . These findings therefore suggest that something i s yet unanswered about the manner i n which carp white muscle deals with low oxygen a v a i l a b i l i t y . The data of the present study show that carp white muscle is" not the s i t e of metabolic, pathways which have evolved i n f a c u l t a t i v e anaerobes. The produc-tio n of v o l a t i l e acids anaerobically by trout (Blazka, 1958) and Crucian carp (Blazka and Kopecky, 1961) had been suggested; however, t h i s f i n d i n g was r e -futed for muscle and l i v e r by Burton and Spehar (1971) who considered Blazka's f i n d i n g to be an a r t i f a c t . This study confirms the work of Burton and Spehar (1971) at l e a s t f o r muscle; however at the present time the formation of these products i n tissues other than muscle and l i v e r cannot be ruled out. The f a i l u r e to f i n d v o l a t i l e acids argues strongly against the p o s s i b i l i t y of an ac t i v e amino acid fermentation, since during anaerobic work i n invertebrates i t i s believed that v o l a t i l e end products are derived from the catabolism of amino acids (Hochachka e_t a l , 1973). The p o s s i b i l i t y of an a c t i v e amino acid fermentation i s further negated by the observation that during hypoxia there i s a tendency for the free amino acid pool to increase not decrease. Further-more, there i s not a reorganization of the amino acid pool which could ind i c a t e a p r e f e r e n t i a l u t i l i z a t i o n of some amino acids. Glycine may be an exception to t h i s r u l e since there i s a tendency for t h i s amino acid to decrease during hypoxia. I t i s known though that f i s h have an a c t i v e glycine anabolism under both aerobic and hypoxic conditions (Demael-Suard et a l , 1974) and that under some circumstances the concentration of free glycine i n carp white muscle may be as high as 30 ymoles/gm (Creach, 1966). C e r t a i n l y the r o l e of glycine 45 Figure 3. The percentage aerobic swimming e f f i c i e n c y versus swimming speed of g o l d f i s h . Aerobic e f f i c i e n c y i s defined as energy required to develop power/energy a v a i l a b l e from consumed oxygen. The graph i s pl o t t e d from the data of Smit e_t a l (1971). 46 metabolism should be the subject of future.work. This study also shows that succinate i s not a s i g n i f i c a n t anaerobic end product i n f i s h white muscle whereas i n the oyster heart even a f t e r a short anoxic period i t reaches 5 ymoles /gm ( C o l l i c u t , personal communication). In the invertebrates, succinate i s formed anaerobically by a r e v e r s a l of the Krebs cy c l e . One would expect t h i s to occur only under conditions of complete anoxia; t h i s was not the case i n the present study, and i t i s doubtful whether t h i s ever occurs as a normal course of events i n the carp. In conclusion, i t would be of extreme i n t e r e s t to a s c e r t a i n i f under anaerobic conditions glycogen i s q u a n t i t a t i v e l y converted to l a c t a t e i n carp white muscle. Stevens and Black (1966) have provided s t a t i s t i c a l evidence that l a c t a t e i s the sole end product of anaerobic glycogen catabolism i t trout; however, s i m i l a r data do not e x i s t f or any other species. NHI" Levels The r e s u l t s of the swimming experiment c a r r i e d out i n spring 1974 show quite conclusively that the adenylate pool i s a source of anaerobic NH^ pro-duction i n carp white muscle. In that particular.experiment, the increase i n NH^ content i n muscle during a c t i v i t y was l e s s than the adenylate pool decrease or IMP increase. One explanation for the lack of 1:1 stoichiometry between + + IMP and NH^ increase i s that some NH^ i s being released into the blood. This would provide an explanation for the observed phenomenon of anaerobic NH^ production by swimming f i s h (Kutty, 1972). However, on the basis of the hypoxia study, there appears to be another fate of NH^ released from the free adenylate pool. This aspect s h a l l be discussed under amino a c i d metabol-ism. The NH^ content of carp white muscle sampled i n the exercise experiment 47 c a r r i e d out i n summer. 1974 i s most i n t r i g u i n g . . The values are. extremely high and i n f a c t appear to be the highest ever reported i n the l i t e r a t u r e f o r skele-t a l muscle. I t i s well known that carp can mobilize t h e i r muscle proteins to serve as an energy source (Creach and Serfaty, 1974) and t h i s i s probably what the animals i n the present study are doing. This concept i s supported by the observation that NH^ content increases quite markedly during a c t i v i t y . The data fur t h e r suggest that carp muscle has the capacity to t o t a l l y u t i l i z e some amino acids d i r e c t l y as an energy source i n s i t u and that p r i o r conversion to carbohydrates i n other tissues i s not necessary. I t i s not clear why the animals were mobilizing t h e i r p r o t e i n stores i n the present study. One p o s s i -b i l i t y i s that even though the f i s h were fed on a d a i l y basis, the supplied di e t may have been inadequate. Otherwise these f i s h may.undergo a seasonal switch i n f u e l source as i s thought to occur i n salmonids during s m o l t i f i c a -t i o n (Saunders, personal communication). C l e a r l y t h i s problem warrants further consideration. Adenylate Pool Size The stoichiometric r e l a t i o n s h i p between adenylate pool depletion and IMP accumulation c l e a r l y shows, that the adenylate pool i s reduced by the reaction catalyzed by 5' AMP deaminase. The regulatory nature of t h i s enzyme from carp white muscle has been well characterized ( F i e l d s , personal communication; Purzycha-Preis and Zydowo, 1969). The enzyme i s activated by ADP (K about 0.5 mM) and potently i n h i b i t e d by GTP (guanosine triphosphate) (K^ about 50 uM). From the present study the enzyme appears to be c o n t r o l l e d l a r g e l y by the removal of GTP i n h i b i t i o n . GTP l e v e l s are i n i t i a l l y low i n f i s h muscle (Jones and Murray, 1960; Gras et_ a l , 1967) and as demands for high energy phosphates increase during a c t i v i t y GTP l e v e l s must f a l l , for GTP i s only formed i n 48 e s s e n t i a l l y two ways, f i r s t l y by transphosphorylation with ATP and secondly by the Krebs cycle reaction catalyzed by succinate.thiokinase. When ATP. l e v e l s are reduced the rate of GTP production by the former r e a c t i o n must also be reduced. Furthermore, as energy demands are placed on white muscle, g l y c o l y s i s i s activated far more than Krebs cycle a c t i v i t y (Wittenberger and.Diaciuc, 1965), consequently the proportion of triphosphorylated nucleotides that GTP represents must decrease. In f a c t , Jones and Murray (1960) were unable to detect GTP i n muscle of fatigued cod. Be that as i t may, the question s t i l l remains of the p h y s i o l o g i c a l s i g n i f i -cance of the reduced adenylate pool during high muscle work rat e s . One simple explanation may be that 5' AMP deaminase functions i n concert with the adeny-l a t e kinase reaction to maximize ATP production by a mass ac t i o n e f f e c t . However, there may be a more important thermodynamic explanation.for the ob-servation. Thus, as ATP l e v e l s drop during muscle work the r a t i o change of [ADP][P_^]/[ATP] could d r a s t i c a l l y reduce the free energy of ATP hydrolysis according to the following r e l a t i o n s h i p : [ADP][P.] AG = AG° + RT In — [ATP] During muscle work, con t r o l of t h i s r a t i o may become in c r e a s i n g l y d i f f i c u l t since not only i s there a change i n the ADP/ATP r a t i o , there also occurs an increase i n P^ concentrations (Hammond and Hickman, 1966) . These considera-tions emphasize that i n the absence of external c o n t r o l l i n g mechanisms large drops i n ATP concentrations could not be tolerated because they would occur concomitantly with increasing ADP l e v e l s of comparable magnitude, a. s i t u a t i o n that i s c l e a r l y prevented. In t h i s connection the regulation of. energy charge may be l e s s c r i t i c a l to g l y c o l y t i c control, than to. the maintenance of a s u i t -49 able r e l a t i o n s h i p between. ADP and ATP l e v e l s . That r e l a t i o n s h i p could be adjusted by the concerted action of adenylate kinase and AMP deaminase, the AMP formed from the adenylate kinase reaction being removed by AMP deaminase i n order to minimize ADP accumulation. Amino Acid Metabolism The r e s u l t s of the hypoxia study show that during anaerobic metabolism nitrogen i s incorporated into the free amino acid pool. The source of t h i s nitrogen i s most l i k e l y free NH^ l i b e r a t e d from the adenylate pool. The only known mechanisms for f i x i n g free NH^ i n muscle are by the reactions catalyzed by glutamate dehydrogenase and glutamine synthetase. The l a t t e r enzyme catalyzes the formation of glutamine from glutamate and NH^ ~; however, since the concentration of glutamine i s quite low i n f i s h muscle t h i s can be ruled out as a major nitrogenous sink. I t thus seems l i k e l y , that glutamate dehydro-genase, which has been shown to occur i n f i s h muscle (McBean et a l , 1966), functions to f i x free NH^ into the amino acid pool by the following reaction: NH^ ~ + a-ketoglutarate + NADH •glutamate + NAD+ Carp white muscle has a very high capacity to u t i l i z e amino acids for energetic purposes under aerobic conditions (Creach and Serfaty, 1974). Thus, given an acti v e aerobic catabolism of amino acids there must be at any given time a pool of p a r t i a l l y oxidized products. A wide spectrum of glutamate transaminase a c t i v i t y has been demonstrated i n f i s h muscle (Siebert e_t a l , 1964) and i t i s probable that the small increase i n a number of.amino acids during anaerobic metabolism i s due to transamination of pre- e x i s t i n g a-ketoacids with glutamate. The glutamate dehydrogenase reaction could not only serve to maintain low NH^ le v e l s during anaerobic metabolism but may also confer an energetic advantage to the tissue since i t would provide an a d d i t i o n a l method of ox i d i z i n g NADH. 50 Proposed Scheme of Nitrogen Metabolism On the basis of the hypoxia and the swimming experiments i t i s possible to construct a f a i r l y comprehensive metabolic scheme for nitrogen metabolism during anaerobic work i n carp white muscle (Figure 4). The energy required fo r work i s ul t i m a t e l y derived from the hydrolysis of ATP to ADP and P^ (reaction a ). When ATP l e v e l s cannot be maintained by the energy production pathways the content of ADP increases, and as the ADP l e v e l increases ATP and AMP are formed by the adenylate kinase reaction simply because of a mass action e f f e c t (reaction b). As the work load on. the tissue exceeds i t s aerobic capa-b i l i t i e s GTP l e v e l s drop (reaction c and d), AMP deaminase i s activated (reac-t i o n e) and the adenylate pool i s decreased. This i s possible since AMP,, the substrate of the AMP deaminase reaction, i s made a v a i l a b l e by adenylate kinase. NH^ released from AMP i s fixed into glutamate by glutamate dehydrogenase (reaction f) and i s subsequently transferred to a v a r i e t y of a-keto acids to form amino acids (reaction g). The question then remains as to how the adenylate pool i s replenished i n the recovery period following fatigue. Lowenstein (1972) has proposed that the 5' AMP deaminase reaction i s one step i n a reaction span that i s termed the purine nucleotide cycle (Figure 5). According to Lowenstein, IMP further reacts with GTP and aspartate to form adenylosuccinate. The adenylosuccinate i n turn i s converted to AMP plus fumarate. I t has been shown i n homogenates of mammalian s k e l e t a l muscle that the cycle functions i n concert with g l y c o l y s i s (Tornheim and Lowenstein, 1974). The theory,'however, predicts only a transient increase i n IMP with general maintenance of the adenylate pool. C l e a r l y the cycle per se does not operate during a c t i v i t y i n carp white muscle since there i s an accumulation of IMP. Moreover, the cycle i s not simply operating at a new steady state ( i . e . at 51 a l t e r e d l e v e l s of the adenylates and IMP) since during anaerobic metabolism i n t h i s t i s s u e there i s no a l t e r a t i o n i n the free amino.acid pool. I f the cycle were to become a c t i v e only during recovery of the adenylate pool a f t e r exercise, the discrepancy between these r e s u l t s and those of Tornheim and Lowenstein (1974) would be apparent rather than r e a l . Since the enzymes of the purine nucleotide cycle apparently are present i n white muscle ( F i e l d s , personal communication), i t i s probable that they supply a pathway for the regeneration of the adenylate pool from IMP during recovery following anaerobic work. Thus, i n white muscle the reaction pathway shown i n Figure 5 i s a " c y c l e " only i n a formal sense, because the two arms of the cycle are f u n c t i o n a l l y separated i n time. One arm, catalyzed by AMP deam-inase, i s formally a c a t a b o l i c pathway leading to AMP h y d r o l y s i s ; the other arm i s formally an anabolic pathway leading.to AMP formation during recovery. The co n t r o l properties of AMP deaminase as well as adenylosuccinate synthetase, which catalyzes the formation of adenylosuccinate, are e n t i r e l y consistent with t h i s model. Thus, during white muscle work, AMP deaminase would be deinhibited due to dropping concentrations of GTP, and at the same time, GDP concentrations are presumably increased.. GDP i s a potent i n h i b i t o r of adenylosuccinate syn-thetase (Muirhead and Bishop, 1974) and t h i s e f f e c t , coupled with reduced a v a i l a b i l i t y of one of i t s substrates (GTP), r e a d i l y explains how t h i s arm of the purine nucleotide cycle i n white muscle i s held at a reduced rate at the same time as AMP deaminase i s being strongly d e i n h i b i t e d . The f i n a l aspect to the picture i s the r o l e of amino acids.during the recovery period. The n i t r o -gen that i s stored i n the amino a c i d pool during anaerobic: work can be r e i n c o r -porated into the adenylate pool by a ser i e s of transaminations. In'this r e -spect the fumarate produced by the purine nucleotide cycle would play an 52 i n t e g r a l r o l e i n providing oxaloacetate for the formation.of aspartate with a v a r i e t y of' amino acids. This metabolic scheme, makes good b i o l o g i c a l sense, however i t remains to be tested. Krebs Cycle Pool Size There i s no increase i n four of the Krebs cycle intermediates even when white muscle i s maximally a c t i v e . I t i s thus apparent that t h i s tissue has very l i t t l e capacity i f any to augment.the s i z e of i t s Krebs cycle pool i n concert with increased energy demands. This i s very much d i f f e r e n t from mammalian heart. (Shaferrand Williamson, 1973) or s k e l e t a l muscle (Edington et^ a l , 1973) i n which the concentration of Krebs cycle intermediates may be highly elevated during strenuous work. In heart burning glucose augmentation of Krebs cycle intermediates i s f u l l y accounted for by aspartate depletion. Aspartate i s transaminated with a-ketoglutarate to form oxaloacetate and g l u t a -mate. The glutamate then undergoes a second transamination with pyruvate to form alanine and regenerate a-ketoglutarate. Thus aspartate carbon appears as Krebs cycle intermediates and aspartate nitrogen appears as alanine (Shafer and Williamson, 1973). The same phenomenon may occur to a l i m i t e d extent i n working white muscle of f i s h since glycogen i s the f u e l source. This accounts for the small but s i g n i f i c a n t decrease i n aspartate and the tendency for alanine to increase (see Chapter V for an extended discussion of t h i s p o i n t ) . 53 Figure 4. Depletion of the adenylate pool i n white muscle during anaerobic work. 53a (a) (b) ATP 2 ADP-->ADP + P, -> ATP + AMP (d) GTP + ADP-suc c i n y l CoA-GDP ->GDP + ATP •> succinate GTP (e) AMP- ->IMP + NH; (f) NH^ + NADH + a-ketoglutarate (g) glutamate a-ketoacid -> glutamate + NAD -> a-ketoglutarate amino a c i d 54 Figure 5. Regeneration of the adenylate pool i n white muscle during recovery from anaerobic work. Abbreviations not indicated i n text: ASP, aspartate; a-KGA, a-ketoglutarate; OXA, oxaloacetate. f u m a r a t e nucleotide a d e n y l o s u c c i n a t e cycle GDP+P: GTP ASP a-KGA a m i n o a c i d m a l a t e OXA g l u t a m a t e a - k e t o a c i d 55 CHAPTER V CONCLUDING REMARKS: RED-WHITE MUSCLE DIFFERENCES AND THE FUNCTION OF THE PURINE NUCLEOTIDE CYCLE 55 a The s k e l e t a l muscle of vertebrates i s composed of c h a r a c t e r i s t i c a l l y d i f f e r e n t t i s s u e types. At the extreme ends of the spectrum are the f i b e r s which make up the red and white muscles. These two f i b e r types may be d i s -tinguished by numerous c r i t e r i a such as the content of mitochondria, myoglobin, haemoglobin and oxidative enzymes, blood flow and consumption rate (see Chapter 1). It i s generally accepted that red muscle functions l a r g e l y a e r o b i c a l l y u t i l i z i n g f a t s or . carbohydrates as i t s f u e l source whereas white muscle has an extraordinary anaerobic component to i t s metabolism based upon glycogen u t i l i z a t i o n . In t h i s respect the mammalian heart i s very s i m i l a r to red muscle (Keul et a l , 1972). Meaningful studies at the metabolite l e v e l of mammalian red or white muscle are e s s e n t i a l l y impossible since the two f i b e r types e x i s t i n mixed bundles; however, i n many f i s h species red and white muscle occur as d i s c r e t e e a s i l y separable t i s s u e masses. In the present study I have taken advantage of the unique d i s t r i b u t i o n of muscle f i b e r s i n f i s h to elucidate the c o n t r o l of energy metabolism i n white muscle alone. Furthermore, i n considering the r e s u l t s within the framework of red-white muscle differences i t has been possible to i n t e r p r e t some heretofore i n e x p l i c a b l e . f i n d i n g s i n the l i t e r a t u r e . Regulation of the Adenylate Pool Size During strenuous a c t i v i t y by white muscle, when anaerobic metabolism i s highly ac t i v a t e d , there i s an o v e r a l l reduction i n the adenylate pool content. This occurs because ATP concentration cannot be maintained over a c e r t a i n minimum. However, when heart or red muscle i s forced to work there i s no decrease i n the t o t a l l e v e l of adenylates since ATP content i s maintained once a new steady state i s attained (Neely et a l , 1972; Gerez and K i r s t e n , 5 6 1965). But when either of these two tissues i s subjected to hypoxia there i s a decrease i n the adenylate pool (Imai et a l , 1964; Deuticke and Gerlach, 1966; Neely et a l , 1973; Chaudry et a l , 1974).just as occurs i n white muscle. In s k e l e t a l muscle t h i s i s accomplished by a c t i v a t i o n of 5' AMP deaminase and the decrease i n the adenylates i s i n .1:1 stoichiometry with e i t h e r IMP increase or the sum of IMP and i t s degradation products (Imai et a l , 1964; Deuticke and Gerlach, 1966). I t i s proposed that following recovery from anaerobic work the adenylate pool i s restored by a r e a c t i o n span known as the purine nucleotide cycle (see Chapter W„ Figure 5l)l.I, 2). In heart, however, the adenylate pool i s reduced by 5' nucleotidase which catalyzes the conversion of AMP to adenosine plus ribose-5-phosphate (Rubio et_ a l , 1973). Regardless, i n a l l three t i s s u e s the phenomenon i s the same: thus, when the anaerobic component pf metabolism i s activated r e l a t i v e to the aerobic component there occurs a reductioh'iri the s i z e of the adenylate pool. But heart contains the enzymes of the purine nucleotide cycle (Lowenstein, 1972; Muirhead and Bishop, 1974) and moreover red muscle i s r a r e l y subjected to hypoxia i n the normal course of events. The question then arises, as to what other functions the purine nucleotide cycle has i n the c e l l . A clue to the question comes from a recent study by Winder et al,(1974) i n which i t i s shown that adenylosuccinate, the f i n a l enzyme i n the purine nucleotide cycle, i s t i g h t l y correlated with Krebs cycle a c t i v i t y and not g l y c o l y s i s . However, before pursuing t h i s r e l a t i o n s h i p further i t i s necessary to describe a generally unappreciated aspect of Krebs cycle function. Strategies of Krebs Cycle A c t i v a t i o n It i s i n t u i t i v e l y obvious that i n the t r a n s i t i o n fromaa r e s t i n g to a working state involving an increased rate of oxygen consumption there must be 5'7 a concomitant a c t i v a t i o n of the Krebs cy c l e . I t i s important to r e a l i z e that the Krebs cycle can be activated i n but two fundamental ways: (a) by increasing the turnover rate or the rate of "spinning", with no change i n pool s i z e of intermediates, or (b) by increasing the steady state l e v e l of Krebs cycle intermediates as w e l l as increasing "spinning" r a t e . In the  f i r s t instance, there i s no change i n the maximum c a t a l y t i c p o t e n t i a l of the  cycle; i n the second, there i s an increase i n c a t a l y t i c p o t e n t i a l of the  cycle that i s proportional to the augmentation of cycle intermediates. Change i n Krebs cycle "spinning" rate i s apparently achieved through t i g h t meta-b o l i t e r egulation of key cycle enzymes. This aspect of Krebs cycle control i s now f a i r l y w e l l described (Atkinson, 1968b; LaNoue and Williamson, 1971; LaNoue et a l , 1972) and w i l l not be emphasized here. S u f f i c e to i n d i c a t e , however, that i n any given metabolic state, merely increasing acetylCoA a v a i l a b i l i t y can increase the "spinning" r tate of the Krebs cycle at the i n i t i a l l y low, basal l e v e l s of cycle intermediates. The main reason why t h i s mechanism M i t s e l f i s usually.inadequate i s a shortage of oxaloacetate. Basal l e v e l s of oxaloacetate are very low, on t h i s there i s widespread agree-ment. Actual values are d i f f i c u l t to estimate, because of compartmentation and of i n s t a b i l i t y during,extraction. The best a v a i l a b l e estimates, however, ind i c a t e that oxaloacetate concentrations may be ,as low as 1S5 uM (Shafer and Williamson, 1973; Garland and Randle, 1964; Williamson, 1965), and t h i s low oxaloacetate a v a i l a b i l i t y severely l i m i t s the rate at which Krebs cycle spinning can be increased. In f a c t , at any given metabolic .state, i t i s a r e l a t i v e u n a v a i l a b i l i t y of oxaloacetate that probably sets the basal rate of Krebs cycle spinning (LaNoue and Williamson, 1971; LaNoue et a l , 1972) and therefore, i t i s generally agreed, large Krebs cycle a c t i v a t i o n requires 58 augmenting the cycle intermediates. How i s t h i s augmentation achieved? Krebs Cycle Augmentation During U t i l i z a t i o n of Carbohydrate Mechanisms for augmenting the Krebs cycle intermediates are dependent upon whether carbohydrate or f a t i s being u t i l i z e d as the f u e l source. Let us f i r s t examine what occurs during the catabolism of carbohydrate as t h i s process has been more f u l l y studied. When glucose-derived pyruvate i s being shuttled into the mitochondria to form acetylCoA, two problems temporarily a r i s e : f i r s t l y , there i s a requirement for more oxaloacetate to handle the newly formed acetylCoA, and secondly, a redox imbalance i s created i n the g l y c o l y t i c path. Both problems (the requirement for oxaloacetate and the redox imbalance) are solved by aspartate transaminase catalyzed m o b i l i z a t i o n of aspartate. This s i t u a t i o n i s . p a r t i c u l a r l y w e ll documented i n r a t heart burning glucose (Shafer and Williamson, 1973), where the augmentation of Krebs cycle i n t e r -mediates i s f u l l y accounted for by aspartate depletion. During the t r a n s i -t i o n or a c t i v a t i o n period,.aspartate-derived oxaloacetate i s reduced to malate.in the c y t o s o l , a process that accounts for a large f r a c t i o n of the required NAD for sustaining g l y c o l y s i s (the other f r a c t i o n coming from a-glycerophosphate and l a c t a t e dehydrogenases). The malate then moves into the mitochondria where i t i s reconverted to oxaloacetate, f o r sparking c i t r a t e synthase (Figure 6\)'; l ) c Two points to emphasize here are (a) that  aspartate-carbon appears as Krebs cycle intermediates, and (b) that aspartate  nitrogen appears as alanine because the aspartate transaminase r e a c t i o n i s coupled to alanine transaminase through the cosubstrates glutamate and a-ketoglutarate. The l a t t e r two are tumbled between aspartate and alanine transaminase i n t h i s s i t u a t i o n and t o t a l . a l a n i n e accumulation equals augmenta-5 9 Figure 6. Augmentation of the Krebs cycle intermediates during u t i l i z a t i o n of carbohydrate as an energy source. 59a glucose alanine cc-ketoglutarate aspartate pyruvate t acetyl C o A glutamate oxaloacetate 60 t i o n of Krebs cycle intermediates. P r e c i s e l y the same mechanism occurs i n the a c t i v a t i o n of metabolism, i n red muscle (Ruderman and Berger, 1974) and to a l i m i t e d extent i n white muscle. Under these ;g§ndit'ionTs pyruvate derived from glucose i s the unquestionable amino acceptor (Ruderman and Berger, 1974). Thus, by t h i s simple process, the Krebs cycle i s set at a new and higher c a t a l y t i c p o t e n t i a l f or sustaining a prolonged work load. The l i m i t e d capacity of white muscle to augment the s i z e of the Krebs cycle pool i s r e f l e c t e d by the content of aspartate which i n t h i s t i s s u e i s only about 0.25 ymoles/gm. In heart, however, aspartate l e v e l s are much higher as i s the capacity to increase oxaloacetate content (Neely et_ a l , 1972) . The aspartate content of red muscle, per se, i s not known but i t has been shown that during a c t i v i t y Krebs cycle intermediates i n t h i s t i s s u e may increase to l e v e l s even higher than those found i n heart (Edington et a l , 1973) . In working white muscle there i s a decrease i n the adenylate pool with a concomitant increase i n free NH^ ". But i n working heart or red muscle, there are no measurable changes i n the t o t a l adenylate pool, although t r a n s i t o r y changes i n ATP, ADP, and. AMP concentrations can occur (Neely et a l , 1972; + Shafer and Williamson, 1973). As f a r as the data currently i n d i c a t e , no NH^ i s produced by working heart (Shafer and Williamson, 1973) or red muscle (Gerez and K i r s t o n , 1965); the only form of nitrogenous "waste" product that accumulates i s alanine and i t i s the only s i g n i f i c a n t form of nitrogen c a r r i e r being released from muscle during aerobic glucose metabolism. Glutamine, an equally important nitrogen c a r r i e r under some conditions, i s not released from heart or red muscle burning glucose unless the system i s supplied with exogenous amino acids (Odessey et a l , 1974). (In the l a t t e r case, glutamine 61 i s formed by mechanisms discussed below.) G l y c o l y t i c I n h i b i t i o n During Fat and Amino Acid Catabolism If a,glucose-perfused heart i s transferred to acetate as the chief exo-genous carbon source, glucose u t i l i z a t i o n drops to nearly zero (Randle et a l , 1970), and the same appears true during metabolism of p a l m i t i c acid (Randle et a l , 1964) and of keto acids formed from v a l i n e , i s o l e u c i n e , and leucine (Johnson and Connelly, 1972). These are c r u c i a l observations for they indi c a t e that when fa t s or amino acids are oxidized, carbohydrate (the only major anaerobic f u e l ) i s being "spared" by feedback i n h i b i t o r y loops from mitochondrial metabolism to key steps i n g l y c o l y s i s . Although further d e t a i l s concerning these i n h i b i t o r y i n t e r a c t i o n s undoubtedly w i l l be elucidated, i t i s already known that creatine phosphate, ATP, and c i t r a t e are a l l p o t e n t i a l i n h i b i t o r s of both phosphofructokinase and pyruvate kinase (Storey and Hochachka, 1974), and these s i n g l y or i n combination are thought to dampen g l y c o l y s i s during a c t i v e f a t oxidation. In a d d i t i o n , the a-keto acids formed from v a l i n e , i s o l e u c i n e , and leucine are believed to i n h i b i t glucose u t i l i z a -t i o n by competing with pyruvate for pyruvate dehydrogenase (Johnson and Connelly, 1972). Whatever the mechanism for the i n h i b i t o r y i n t e r a c t i o n between mitochondrial metabolism and g l y c o l y s i s , the important point to bear i n mind i s that when f a t i s . u t i l i z e d , glucose becomes l a r g e l y unavailable as a source of pyruvate. Thus, Krebs cycle priming by aspartate transamination to oxaloacetate, coupled to pyruvate transamination to alanine, as described above (Figure 6:)Y 1 ) , i s a reaction span that may quickly run out of a key substrate (pyruvate) under conditions,favouring f a t metabolism. Not s u r p r i s -i n g l y , the amount of alanine formed i n the heart under these conditions does not account for the augmentation of Krebs cycle intermediates (Randle et a l , 1970). Whereas 0^ uptake increases by nearly 10-fold, alanine release from s k e l e t a l muscle increases by l e s s than 2-fold ( F e l i g and Wahren, 1971). Sometimes aspartate i s depleted, but i t s depletion does not account for newly formed Krebs cycle intermeddiates (Randle et_ a l , 1970). At other times, aspartate i n f a c t may.be accumulated during t r a n s i t i o n to a c t i v e f a t oxidation (Neely et_ a l , 1972). What, then, i s the source of Krebs cycle intermediates during a c t i v a t i o n . o f f a t t y a c i d oxidation? Krebs Cycle Augmentation During M o b i l i z a t i o n of Fat A key i n s i g h t into the above question comes from a consideration of the i n t e r a c t i o n between fat and amino a c i d catabolism. In contrast to the i n -h i b i t o r y e f f e c t s on g l y c o l y s i s brought about by f a t t y acid or amino acid catabolism,tthe i n t e r a c t i o n s between f a t t y a c i d and amino acid catabolism  appear to be of a s y n e r g i s t i c nature. Thus, the oxidation of amino acids, p a r t i c u l a r l y v a l i n e , i s o l e u c i n e , and leucine, i s potently enhanced by f a t t y acids such as octanoate (Buse et a l , 1972). During periods of t r a n s i t i o n from low to high rates of f a t t y acid oxidation, the catabolism of v a l i n e and i s o l e u c i n e feeds carbon into the Krebs cycle at the l e v e l of succinylCoA, while the carbon of leucine feeds into the Krebs cycle as acetylCoA. It appears that ilt these amino acids, not aspartate, that prime the Krebs  cycle during, a c t i v a t i o n of. f a t metabolism. The f i r s t two (valine and i s o l e u -cine) increase the c a t a l y t i c p o t e n t i a l of the Krebs cycle by increasing the a v a i l a b i l i t y of oxaloacetate and generally augmenting the pool s i z e of cycle intermediates, while leucine, along with f a t t y acids, increases the amount of oxidizable 2-carbon substrate (acetylCoA), and thus leads d i r e c t l y to an increase i n Krebs cycle turnover rate (Figure 7>)'i :2)~v This model explains (a) why leucine perfusion of mammalian muscle leads to a 5-fold drop i n 63 Figure 7. Augmentation of the Krebs cycle intermediates during u t i l i z a t i o n of f a t as an energy source. GTP GDP+.Pi i valine isoleucine glutamate oxal NHA • glutamate rATP K-ADP+P; ,* -ketoglutarate— f /aspartate l /oxaloacetate glutamine r cycle j aloacetate 1 fumarate <* -keto acids "malate cytosol I CoA derivatives ^malate ^  tumarate „ oxaloacetate succinate ' KREBS * citrate \VCYV. I - 5 ^ succinyl CoA • isocitrate acetyl CoA mitochondrion c - ketoglutarate fatty acids leucine 64 v a l i n e released into the efferent c i r c u l a t i o n by that muscle (Ruderman and Berger, 1974), and (b) why exercise leads to a measurable uptake of v a l i n e and i s o l e u c i n e ( F e l i g and Wahren, 1971). Whereas the carbon of v a l i n e , i s o l e u c i n e , and leucine enters the Krebs cycle, the nitrogen appears f i r s t i n aspartate, but ultimately i n glutamine and to a l e s s e r extent i n alanine. Unlike muscle burning glucose (which releases alanine as the nitrogen c a r r i e r ) , muscle burning f a t and/or amino acids, releases both glutamine and alanine, and of the two, glutamine appears the predominant form of "waste" nitrogen, removing from muscle 2-4 times as much nitrogen/mole of amino a c i d as does alanine. If muscle i s perfused with leucine.or v a l i n e , glutamine, as well as smaller amounts of alanine, are again released i n t o the efferent flow (Ruderman and Berger, 1974). These data, therefore, are consistent with the following reaction scheme (see also Figure §yy: 1):, In t h i s view, glutamate and a-ketoglutarate. tumble between aspartate trans-aminase and transaminases for v a l i n e , i s o l e u c i n e , and leucine (Figure 7)': 65 As a means for regenerating a-ketoglutarate required for m o b i l i z i n g these amino acids, aspartate transaminase i s favoured over alanine transaminase because of l i m i t i n g a v a i l a b i l i t y of pyruvate; however, any alanine which i s released from f a t burning muscle i s formed by alanine transaminase (Ruderman and Berger, 1974), the pyruvate for the r e a c t i o n presumably a r i s i n g from a r e s i d u a l g l y c o l y t i c a c t i v i t y that can i n f a c t be increased with exogenous glucose (Odessey et a l , 1974). The Aspartate-Oxaloacetate Cycle Accordingly, c y t o s o l i c aspartate transaminase function during a c t i v a t i o n of f a t catabolism i s favoured i n the d i r e c t i o n of aspartate production; j u s t the opposite, of course, occurs at t h i s enzyme locus during aerobic.glucose catabolism. The aspartate formed t h i s way may accumulate to a new steady state l e v e l (Neely et a l , 1972) or i t may be depleted (Randle- et a l , 1970), but for a f u l l appreciation of i t s fate and function we must f i r s t inquire as to the o r i g i n of glutamine. On t h i s question, recent evidence ( H i l l s , 1972; Ruderman and Berger, 1974) strongly indicates that glutamine formation i n muscle burning various amino acids (including v a l i n e and leucine) i s catalyzed by glutamine synthetase: glutamate + ATP ( + NH^ • glutamine + ADP + P C l e a r l y , a source.of NH^ i s required for t h i s r e a c t i o n . .As glutamate dehydro-genase i s not present i n heart or red muscle, i t i s widely accepted that the primary pathway for the c o n t r o l l e d release of amino nitrogen i n muscle t i s s u e i s the purine nucleotide cycle (Lowenstein, 1972). Therefore, i t i s proposed that i n red muscle or heart, the r e a c t i o n steps of the purine nucleotide cycle function as_ jl cycle during m o b i l i z a t i o n of amino acids, p a r t i c u l a r l y v a l i n e and i s o l e u c i n e . The aspartate i n i t i a t i n g the 66 purine nucleotide cycle i n f a c t can be regenerated from the fumarate formed by i t , since fumarate can be converted to oxaloacetate. This scheme, termed the aspartate-oxaloacetate c y c l e , provides a source of oxaloacetate for the aspartate transaminase and i n e f f e c t i s a c y c l i c , c a t a l y t i c mechanism ( i n i t i a t e d by aspartate and reforming aspartate), that primes (a) the flow of  nitrogen through the purine nucleotide cycle to glutamine, and (b) the flow  of carbon from iso l e u c i n e and v a l i n e into the pool of Krebs cycle intermedi- ates. For t h i s reason, I predict that during muscle a c t i v a t i o n , depletion of v a l i n e and i s o l e u c i n e should be inversely proportional to glutamine formation and release.from muscle. For t h i s reason, too, the augmentation of Krebs cycle intermediates should be i n v e r s e l y p roportional to v a l i n e and i s o l e u c i n e depletion. In t h i s view, whether or not aspartate i s depleted, accumulated, or unchanged during t r a n s i t i o n to a c t i v e f a t oxidation appears to depend upon the a v a i l a b i l i t y of i s o l e u c i n e and v a l i n e . C l e a r l y some of the fumarate formed by the aspartate-oxaloacetate cycle could feed i n t o , <and be retained within, the pool of Krebs cycle intermediates; that amount should appear as an aspartate depletion. If i s o l e u c i n e and v a l i n e reserves are adequate, however, aspartate may a c t u a l l y accumulate. In a balanced s i t u a t i o n , c l e a r l y aspartate would neither be depleted nor accumulated. A l l three a l t e r n a t i v e s i n f a c t have been observed (Neely e t a l , 1972; Randle et a l , 1970), though never previously explained. Thus, i t appears that when red muscle and/or heart burn f a t , a c t i v a t i o n of the Krebs cycle requires the simultaneous m o b i l i z a t i o n of amino acids, p a r t i c u l a r l y v a l i n e and i s o l e u c i n e . At least during the a c t i v a t i o n period, the "cogs".of the Krebs cycle appear to mesh with those of the aspartate-67 oxaloacetate cycle which primes the flow of amino acid carbon into the Krebs cycle and the flow of amino nitrogen through the purine nucleotide cycle to glutamine (Figure 7). Several advantages accrue from t h i s kind of metabolic organization. F i r s t l y , the only major anaerobic source of energy (carbohyd-rate) i s maximally "spared" during aerobic metabolism. Secondly, i f one compares the energy yielded during v a l i n e (or isoleucine) priming versus that gained by the aspartate priming mechanism, about 10 times as much u t i l i z a b l e energy ( i n the form of ATP equivalents) i s obtained (Krebs, 1964). T h i r d l y , t h i s mechanism allows amino acids to be used i n muscle both f o r priming the Krebs cycle as well as d i r e c t l y f o r energy production without muscle r e q u i r i n g a urea-cycle for handling "waste" nitrogen. F i n a l l y , f o r reasons that are s t i l l unclear, but probably involving a greater absolute augmentation of cycle intermediates, the maximum degree of Krebs cycle a c t i v a t i o n obtainable appears higher during f a t oxidation than i t i s during glucose oxidation (Neely e_t a l , 1972). In conclusion, i t i s i n t e r e s t i n g to note that heart muscle i s capable of burning a v a r i e t y of substrates, f a t t y acids being preferred i n vivo (Neely and Morgan, 1974); red muscle f i b e r s appear to be rather s i m i l a r to the heart i n t h i s respect, whereas, as we have seen, white muscle displays a strong and unique dependence upon carbohydrate. Summary Much of the data i n r e l a t i o n to energy metabolism i n mammalian s k e l e t a l muscle i s d i f f i c u l t to i n t e r p r e t due to the heterogeneity of t h i s t i s s u e . In the present study t h i s problem has been circumvented by the u t i l i z a t i o n of a species of f i s h i n which red and white muscle e x i s t as d i s c r e t e e a s i l y separable tissue masses. During, t r a n s i t i o n to work i n white muscle, as w e l l as during hypoxic stress i n red muscle and heart, there occurs a reduction i n the t o t a l content of the adenylate pool. In s k e l e t a l muscle t h i s i s accom-68 plished. by a c t i v a t i o n of 5' AMP deaminase,, the f i r s t enzyme i n a. r e a c t i o n span known as the purine nucleotide c y c l e . Following recovery from anaerobic work the second arm of the purine nucleotide cycle i s turned on, serving to regenerate AMP and hence the adenylate pool. However, the main function of the purine nucleotide cycle i n red muscle and heart i s r e l a t e d to aerobic metabolism. When muscle undergoes a t r a n s i t i o n from a r e s t i n g to a working state, there i s an increase i n the si z e of the Krebs cycle pool. The simplest method for t h i s augmentation presents i t s e l f when carbohydrates are u t i l i z e d as a f u e l source; under these conditions, aspartate conversion to oxaloacetate, catalyzed by aspartate transaminase, serves to spark the Krebs c y c l e . Because aspartate transaminase Is coupled to alanine transaminase, the increase i n Krebs cycle pool si z e i s q u a n t i t a t i v e l y accounted for by alanine accumulation. When muscle burns f a t , however, t h i s augmentation mechanism i s reduced i n importance due to i n h i b i t i o n of g l y c o l y s i s and to reduced a v a i l a b i l i t y of pyruvate. Under these conditions, the catabolism of v a l i n e and .Isoleucine i s favoured and these appear to be the predominant sources of carbon for augment-ing Krebs cycle pool size.. Although the carbon of these branched chain amino acids appears as Krebs cycle intermediates, the nitrogen appears f i r s t i n aspartate, but then i s released as NH^ by the purine nucleotide c y c l e ; the NH^ then serves as substrate for glutamine synthetase, i n which circumstance glutamine becomes the primary means for removal of amino nitrogen from muscle. Thus, when muscle burns f a t the purine nucleotide cycle functions to channel amino acids i n t o the Krebs c y c l e . 69 CHAPTER VI LITERATURE CITED 69 a Ackman, R. G. and Noble, D. 1973. Steam d i s t i l l a t i o n : a simple technique for recovery of petroleum hydrocarbons from tainted f i s h . J . F i s h . Res. Bd. Canada' 30: 711-714. Atkinson, D. E. 1968a. The energy charge of the adenylate pool as a regulatory parameter. 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L., Baldwin, K. M. and Holloszy, J . 0. 1974. E f f e c t of exercise on AMP deaminase and adenylosuccinate i n rat s k e l e t a l muscle. Am. J. Phy s i o l . 227: 1411-1414. Wittenberger, C. and Diaciuc, I. V. 1965. E f f o r t metabolism of l a t e r a l muscles i n carp. J . F i s h . Res. Bd. Canada 22: 1397-1406. 79 APPENDIX I BLOOD LACTATE LEVELS IN FREE SWIMMING TROUT BEFORE AND AFTER STRENUOUS EXERCISE RESULTING IN FATIGUE 79 a INTRODUCTION The myotomal musculature of f i s h i s l a r g e l y composed of two systems commonly re f e r r e d to as the red and white f i b e r s . The two f i b e r types may be distinguished i n numerous ways including haemoglobin, myoglobin and mitochon-d r i a l content, vascular supply and enzymatic properties. On the basis of these c h a r a c t e r i s t i c s i t i s generally accepted that red muscle has a metabolism that i s a e r o b i c a l l y based burning f a t s and/or carbohydrates whereas white muscle functions l a r g e l y anaerobically u t i l i z i n g glycogen with the concomitant production of l a c t a t e . During slow swimming the propulsive force i s derived e n t i r e l y from the red musculature. But at the highest swimming v e l o c i t i e s the white muscle becomes maximally a c t i v e and together with the red muscle supplies the power for locomotion. At some point i n the t r a n s i t i o n from low to high swimming speeds, there i s recruitment of the white f i b e r s (see Intro-duciton to thesis f o r references). I t i s well recognized that some f i s h can maintain swimming, speeds j u s t below t h e i r c r i t i c a l v e l o c i t y f o r extended periods of time (Brett, 1964). During excursions of t h i s nature new steady state l e v e l s must be attained and i n conditions involving white muscle a c t i v i t y l a c t a t e cannot be allowed to accumulate i n that tissue and hence must be eliminated. Numerous workers have found that blood from rainbow trout sampled by cardiac puncture on restrained f i s h a f t e r periods of moderate a c t i v i t y contained elevated l e v e l s of l a c t a t e (Black, 1957; Black et a l , 1960; Black et a l , 1966; M i l l e r et a l , 1959). In t h i s study the l a c t a t e content of blood taken s e r i a l l y by indwelling catheters from i n d i v i d u a l rainbow trout (Salmo gairdneri) during swimming at well defined v e l o c i t i e s i s recorded. I t i s reported that under the present conditions, there i s no marked increase i n the concentration of blood l a c t a t e during the 80 swimming period at any time p r i o r to fatigue; however, l a c t a t e concentration r i s e s r a p i d l y following fatigue. The f a t e of l a c t a t e produced i n the white muscle of f i s h remains an open question. In mammalian systems which have been better studied i t i s known that 80-90% of the blood l a c t a t e i s oxidized to C0 2 and water (Drury and Wick, 1956); the s i t e of oxidation being heart (Keul et a l , 1972), s k e l e t a l muscle ( J o r f e l d t , 1970), l i v e r (Rowell et a l , 1972) and kidney (Levy, 1962). In r e s t i n g condi-tions approximately 15% of the la c t a t e i s converted to glucose (Reichard et_ a l , 1963) i n the l i v e r (Rowell et a l , 1966); however, during a c t i v i t y t h i s process ( i . e . the C o r i cycle) may be q u a n t i t a t i v e l y more important (Keul et a l , 1972). Black, on the basis of h i s studies with f i s h , was forced to conclude that i n these animals the Co r i cycle i s of l i t t l e importance (Black et^ a l , 1966). Moreover, following anaerobic stress glycogen does not return to prestress l e v e l s even a f t e r 24 hours (Black et^ a l , 1962; Heath and Pr i t c h a r d , 1965), therefore i t i s probable that i n f i s h , as with mammals, most of the blood l a c t a t e i s oxidized to C0 2. B i l i n s k i and Jonas (1972) have shown that the capacity to channel l a c t a t e through the c i t r i c a c i d cycle per un i t weight of tissue decreases i n the following order: g i l l , kidney, red muscle, l i v e r , heart, white muscle. An attempt has been made to quantitate the i n vivo s i g n i f i c a n c e of g i l l as a s i t e of la c t a t e u t i l i z a t i o n by sampling blood before and a f t e r i t s passage through t h i s t i s s u e . The r e s u l t s suggest that l a c t a t e i s removed from the blood by the g i l l s during the recovery period following strenuous a c t i v i t y . 81 MATERIALS AND METHODS Animals Female trout (Salmo gairdneri) (40-53 cm) were purchased from a commercial supplier (Trout Lodge, Ephrato, Wash., U. S. A.) and transported:to U. B. C. by tank truck, where they were held i n large c y l i n d r i c a l , tanks (8000 l i t r e s ) . Constant inflow of fresh dechlorinated water (6-7°C) was maintained at a l l times. F i s h used for experiments were p h y s i c a l l y trained f or periods of at le a s t two weeks p r i o r to use. Training was accomplished by t r a n s f e r r i n g the f i s h to 2000 l i t r e c i r c u l a r tanks i n which the water was kept i n motion by water j e t s driven by pumps. The water v e l o c i t y varied from nearly 0 at the centre of the tank to 35-40 cm/sec at the circumference. The f i s h tended to swim constantly i n the high water v e l o c i t y zone. A l l f i s h were fed Clark's Trout P e l l e t s s i x times weekly. Cannulation Techniques Dorsal a o r t i c cannulation was accomplished by the method of Smith and B e l l (1964) (MS222 anaesthesia) using a 50 cm length of PE60 tubing terminated at the proximal end with a 12 mm section of 21a Huber point needle. Ventral cannulation was accomplished using a cannula s i m i l a r to the dorsal one except that the needle was 2 cm long and bent at 60° 6 mm from the t i p . . This cannula was inserted into the v e n t r a l aorta through the tongue at the l e v e l of the t h i r d g i l l arch. The cannula was sutured to the tongue and extended out of the mouth. A f t e r p a r t i a l recovery on the operating table (constant r e s p i r a -tory frequency) the f i s h was transferred to a water tunnel (Brett j 1964), of 2 126.5 cm c r o s s - s e c t i o n a l area and 35 l i t r e volume, to recover for a minimum of 18 hrs at a water v e l o c i t y of 9 cm/sec. 82 Experimental Design The f i s h were exercised i n a seri e s of 60 min stepwise increasing v e l o c i t y increments, u n t i l fatigue occurred. Each v e l o c i t y increment was about 0.25 lengths/sec. Blood samples, p r i o r to fatigue, from four individual' f i s h , were taken at min 60 of the test period. Fatigue was defined as the i n a b i l i t y of the f i s h to remove i t s e l f from the e l e c t r i f i e d g r i d (10-20 v AC) at the down-stream end of the tunnel. The water was maintained at 6-7°C and 100% a i r saturation. C r i t i c a l v e l o c i t y was calculated using the empirical formula of Brett (1964) such that the l a s t v e l o c i t y that the f i s h s u c c e s s f u l l y maintained was added to the v e l o c i t y at which the f i s h fatigued m u l t i p l i e d by the propor-t i o n of the 60 minute period that i t was able to sustain t h i s f i n a l speed. The mean c r i t i c a l v e l o c i t y f o r the animals of t h i s experiment was 1.6 lengths/ sec. A n a l y t i c a l Techniques Blood samples (0.3-1.0 ml) were taken from the ends of the cannulae with a 1 ml Hamilton syringe. In some cases both a r t e r i a l and venous samples were taken from the same animal. When t h i s occurred the venous sample was obtained 1 minute before the a r t e r i a l sample. Upon removal of the blood i t was immedi-ate l y d i l u t e d 1.0:3.5 v/v with cold 8% HCIO^. The sample was centrifuged to remove pr o t e i n and the supernatant was neutralized with 3 M K^CO^ containing 0.5 M triethanolamine. KCIO^. was removed by c e n t r i f u g a t i o n and an a l i q u o t of the supernatant was analyzed for l a c t a t e enzymatically. Assays were c a r r i e d out on a Unicam SP 1800 dual beam spectophotometer connected to a s t r i p chart recorder. A r t e r i a l and venous blood l a c t a t e content were compared with the Wilcoxon test f o r paired observations and a p r o b a b i l i t y of l e s s than 0.05 was considered to be s i g n i f i c a n t . 83 RESULTS AND DISCUSSION There i s no change i n l a c t a t e concentration between a r t e r i a l . a n d venous blood when samples are taken.prior to fatigue. In order to avoid duplicate samples from the same animal,, only a r t e r i a l blood l a c t a t e l e v e l s of i n d i v i d u a l swimming trout before and immediately a f t e r fatigue are shown i n Figure 8. C l e a r l y , there i s no increase i n blood l a c t a t e at any sustained swimming speed, even though, i n many cases, the exercise l e v e l approaches the c r i t i c a l v e l o c i t y and i s maintained at that l e v e l for 60 minutes. On the basis of heart rate and v e n t i l a t i o n frequency, following a c t i v i t y , on the animals of t h i s experi-ment (Kiceniuk, personal communication) and other evidence c i t e d i n Chapter I, i t appears that there must be white muscle involvement at l e a s t at the highest sustainable v e l o c i t i e s . Webb (1971) was of the opinion that i n trout, the white muscle comes into play at about.80% of the c r i t i c a l v e l o c i t y , yet there i s no increase i n blood l a c t a t e at speeds up to 93% of the c r i t i c a l v e l o c i t y . Since, i n the present study blood was sampled.only a f t e r a steady state l e v e l had been attained i t must be concluded that the rate of elimination of l a c t a t e from white muscle i s equal to the rate of u t i l i z a t i o n elsewhere. I t i s thought that blood flow to the l i v e r i s reduced during exercise ( S a t c h e l l , 1971), thus i t i s u n l i k e l y that t h i s tissue i s a major s i t e of l a c t a t e deposition. As indicated above, the g i l l s are not a major s i t e of l a c t a t e u t i l i z a t i o n during the exercise period. I t i s possible that l a c t a t e produced i n the white muscle i s further oxidized i n the red muscle as Wittenberger and Diaciuc (1965) have suggested occurs i n the carp. I t would be of i n t e r e s t to a s c e r t a i n i f there are differences between trout and carp i n t h e i r capacity to eliminate l a c t a t e produced i n white muscle. Following fatigue there i s a rapid increase i n blood l a c t a t e concen-84 Figure 8. Blood l a c t a t e l e v e l s of i n d i v i d u a l swimming trout at s p e c i f i e d swimming speed and following f a t i g u e . M u l t i p l e points at a given percentage c r i t i c a l v e l o c i t y are representative of repeat runs on d i f f e r e n t days. A l l blood samples taken from do r s a l aorta except one represented by which i s from v e n t r a l aorta. The curve i s a regression l i n e drawn through a l l points p r i o r to fatigue. 84a 85 t r a t i o n to about 2.5 umoles/ml (Figure 8; Table V I I I ) . This represents a 4-to 5-fold increase over the l e v e l at the highest sustained speed. Elevated l e v e l s of blood l a c t a t e immediately following strenuous exercise have been repeatedly shown (Black et a l , 1966; Stevens and Black, 1966; Hammond and Hickman, 1966). Furthermore, although the data over the recovery period are l i m i t e d they f i t the general pattern often described and discussed, of a rapid increase i n blood l a c t a t e concentration, immediately following anaerobic work, reaching a maximum i n 2-4 hours and then slowly returning to normal (Black e_t a l , 1962; Black et a l , 1966; Hammond and Hickman, 1966). The f a c t that blood l a c t a t e increases to such a degree following a c t i v i t y i ndicates that although l a c t a t e i s eliminated from white muscle during a c t i v i t y a large amount i s also retained. The question remains as to how much la c t a t e i s eliminated r e l a t i v e to how much i s allowed to accumulate during sustained swimming. The post fatigue l e v e l s of blood l a c t a t e reported here are i n agreement with the findings of others i n absolute value and i n the manner i n which content increases and then decreases. However, i t had been previously claimed that blood l a c t a t e l e v e l s i n trout exercised at moderate speeds are 2-3 times higher than i n unexercised f i s h (Black, 1957; Black et a l , 1966; M i l l e r et a l , 1959). I t i s quite clear from Figure 8 that blood l a c t a t e does not increase markedly even at high sustained v e l o c i t i e s . The d i s s i m i l a r i t y between the present data and the data of others may be due to methodological procedures. In a l l previous studies blood was obtained by cardiac puncture whereas i n the present work blood was sampled by indwelling catheters. The two techniques appear to y i e l d s i m i l a r r e s u l t s when blood i s sampled a f t e r the exercise period; however, blood taken from f i s h p r i o r to fatigue by cardiac puncture contains elevated l e v e l s of l a c t a t e . I t i s possible that i n manipulating the 86 f i s h f o r cardiac puncture.there.is an.increased cardiac, output which flushes an increased amount of l a c t a t e out of the white muscle. Another unaccounted for parameter i n sampling at moderate swimming speeds i s the a c t i v i t y of the animal per se during the sampling period. Thus, blood l a c t a t e increases following cessation of exercise i n v o l v i n g white muscle a c t i v i t y but does not increase when swimming i s allowed to continue. Randall (personal communica-tion) has suggested that following cessation of a c t i v i t y there may be a l o c a l i z e d hyperaemia i n the white musculature which causes a massive f l u s h i n g out of l a c t a t e . This could explain not only the findings at intermediate v e l o c i t i e s but also the general phenomenon of a rapid increase i n blood l a c -tate concentration following strenuous exercise. Whatever the answer may be, i t i s c l e a r that t h i s problem warrants r e s o l u t i o n . Table VIII l i s t s the animals in. which both a r t e r i a l and venous blood samples were taken following fatigue. Of the eleven, measurements made, eight showed a negative arterial-venous difference which on the average i s about 0.4 ymoles/ml. I t therefore appears that i n the recovery period following strenuous exercise there i s a net uptake of l a c t a t e from the blood during i t s passage through the g i l l s . A 1000 gm .trout has about 50 ml of blood and a c i r c u l a t i o n time of approximately 2 min (Randall,.1970). Thus the g i l l s of a trout t h i s s i z e could take up on the average about 10 umoles 'lactate/min. A f t e r entering the g i l l l a c t a t e must either be excreted or further metabolized. During exercise f i s h excrete a minimal amount of l a c t a t e (Karuppannan, 1972), but there i s no reason to believe that following exercise t h i s l e v e l increases. I t i s probable that any l a c t a t e taken up by the g i l l s i s reconverted to pyruvate, which may be further u t i l i z e d i n a v a r i e t y of ways. G i l l has the capacity to oxidize l a c t a t e t o t a l l y to C0„ and water ( B i l i n s k i and Jonas, 1972) 87 Table VIII... A r t e r i a l and venous blood l a c t a t e concentrations of rainbow trout following exercise to f a t i g u e . F i s h Time following Venous A r t e r i a l A-V fat i g u e (min) (ventral aorta) (dorsal aorta) d i f f e r e n c e 1 1080 1.1* 1.0 -0.1 2 120 10.3 9.8 -0.5 3 1 3.3 2.8 -0.5 30 . 5.4 5.6 +0.2 60 8.4 7.1 -1.3 660 1.5 1.8 +0.3 4 1 1.8 0.6 -1.2 60 3.1 3.0 -0.1 90 4.1 4.9 +0.8 120 6.4 5.6 -0.8 135 6.8 5.3 -1.5 Mean -0.4 *Lactate concentration i n umoles/ml blood. 88 and the i n vivo data of Rao (1968) indi c a t e that the oxygen consumption of g i l l of a 1000 gm trout i s great enough to completely oxidize 10 umoles lactate/min. Furthermore, although studies on the intermediary metabolism of the f i s h g i l l are notably lacking, i t i s known that the crustacean g i l l has an extremely high gluconeogenic capacity (Thabrew e_t al_, 1971) . I f the f i s h g i l l i s at a l l s i m i l a r to the analagous crustacean t i s s u e , a 1000 gm trout could e a s i l y d i r e c t a large f r a c t i o n of the l a c t a t e i n t o glucose. In t h i s respect i t i s i n t e r e s t i n g to note that f i s h g i l l has p a r t i c u l a r l y high glycogen deposits which l i e i n close proximity to an abundant mitochondrial system (Conte, 1969). I t may be that glycogen serves as the metabolic f u e l for t h i s t i s s u e . I t i s also possible that there i s a fu n c t i o n a l e lectron shuttle system between white muscle and g i l l ; such that l a c t a t e formed i n muscle.is simply converted to pyruvate i n the g i l l and subsequently returns to the muscle. In t h i s case the g i l l would be functioning to oxidize NADH produced from the la c t a t e dehydrogenase r e a c t i o n . During a bout of strenuous a c t i v i t y a 1000 gm trout may accumulate 30,000 umoles of l a c t a t e i n i t s white muscle (Black e_t al, 1962; Stevens and Black, 1966; Hammond and Hickman, 1966). Since i t then takes from 12-24 hours for blood l a c t a t e to return to normal i t i s possible that the g i l l plays a heretofore unrecognized r o l e i n the metabol-ism of t h i s metabolite during the recovery period. 89 SUMMARY Rainbow trout,(Salmo gairdneri.) were exercised i n a serie s of 60 minute stepwise increasing v e l o c i t y increments. There i s no increase i n blood l a c t a t e concentration at any time during the exercise period; even though, some of the animals were exercised at 93% of t h e i r c r i t i c a l v e l o c i t y on a sustained basis. The data ind i c a t e that under these conditions the rate of production of l a c t a t e by white muscle i s equal to i t s rate of u t i l i z a t i o n elsewhere. Immediately following fatigue blood l a c t a t e l e v e l r a p i d l y increases. During the recovery period there appears to be a net uptake of l a c t a t e by the g i l l s . 90 APPENDIX II ENZYME NOMENCLATURE 90 a adenylate kinase (E.C. 2.7.4.3) adenylosuccinase (E.C. 4.3.2.2) adenylosuccinate synthetase (E.C. 6.3.4.4) alanine transaminase (E.C. 2.6.1.2) AMP deaminase (E.C. 3.5.4.6) aspartate transaminase (E.C. 2.6.1.1) c i t r a t e lyase (E.C. 4.1.3.8) c i t r a t e synthase (E.C. 4.1.3.7) fructose-1,6-diphosphatase (E.C. 3.1.3.11) glutamate dehydrogenase (E.C. 1.4.1.2) glutamate-oxaloacetate transaminase (E.C. 2.6.1.1) glutamate-pyruvate transaminase (E.C. 2.6.1.2) glutamine synthetase (E.C. 6.3.1.2) glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49) a-glycerophosphate dehydrogenase (E.C. 1.1.1.8) glycogen phosphorylase (E.C. 2.4.1.1) hexokinase (E.C. 2.7.1.2) l a c t a t e dehydrogenase (E.C. 1.1.1.27) malate dehydrogenase (E.C. 1.1.1.38) nucleoside phosphorylase (E.C. 3.2.2.1) 5' nucleotidase (E.C. 3.1.3.5) phosphofructokinase (E.C. 2.7.1.11) phosphoglucoisomerase (E.C. 5.3.1.9) pyruvate dehydrogenase (E.C. 1.2.4.1) pyruvate kinase (E.C. 2.7.1.40) 91 suc c i n i c thiokinase (E.C. 6.2.1.5) t e t r a h y d r o f o l i c acid formylase (E.C. 3.5.1.10) xanthic oxidase (E.C. 1.2.3.2) 

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