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Recovery from exhaustive exercise in rainbow trout white muscle : a model for studies of the control… Schulte, Patricia Marita 1990

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RECOVERY FROM EXHAUSTIVE EXERCISE IN RAINBOW TROUT WHITE MUSCLE: A MODEL FOR STUDIES OF THE CONTROL OF ENERGY METABOLISM IN VIVO By PATRICIA MARITA SCHULTE B . S c , The U n i v e r s i t y of B r i t i s h Columbia, 1987 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA September, 1990 <£) P a t r i c i a M a r i t a S c h u l t e , 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. The University of British Columbia Vancouver, Canada Department DE-6 (2/88) i i ABSTRACT Recovery from exhaustive exercise i n the white muscle of rainbow trout (Oncorhynchus mvkiss) was used to examine the ro le of the adenylates i n the contro l of energy metabolism and to assess the v a l i d i t y of equ i l ibr ium models of the behaviour of the high energy phosphates. The d i f f i c u l t y of obtaining muscle samples from f i s h makes d e t a i l e d analys i s of the behaviour of the l a b i l e high energy phosphates complex. The use of a new sampling procedure, the in fus ion of a l e t h a l dose of anaesthetic v i a an indwel l ing cannula, minimized t h i s problem. At exhaustion [ATP] and [PCr] were depressed by 75 and 80% respec t ive ly r e l a t i v e to the re s t ing values . [ATP] deplet ion was mirrored by a s to ich iometr ic increase i n [IMP]. During recovery [PCr] returned to the re s t ing l e v e l within 2 hours, but [ATP] recovery was slow and not complete u n t i l 24 hours post exerc i se . In contrast , energy charge and R A T P ( t h e proport ion of the free adenylate pool phosphorylated to ATP) were, i f anything, higher than the re s t ing values by 2 hours post exerc i se . Therefore, [ATP] and energy status can be d i ssoc iated i n t i s sues l i k e f i s h white muscle because of the act ion of the purine nucleot ide c y c l e . At 2 hours post exercise the ca l cu la ted free ADP concentration dropped to less than one tenth the value at res t . As a r e s u l t the [ATP] / [ADP] f r e e r a t i o increased by nearly 6 f o l d . i i i This condition may be required for glycogen resynthesis from la c t a t e i n muscle. Several s i m i l a r equilibrium models of the behaviour of the adenylates and PCr were applied to the f i s h white muscle system. In general, the models well describe the r e l a t i o n s h i p between the high energy phosphates. However, the d e f i n i t i o n of the high energy phosphate pool introduces some complications since this includes the t o t a l [ATP]. Because of the action of AMP deaminase the [ATP] concentration can change without measurable changes i n the energy status, which i s not considered i n any of the models. As long as the extent of IMP formation i s known the models can be applied, but since the formation of IMP may vary from f i s h to f i s h or with exercise regime the models lose much of t h e i r p r e d i c t i v e power. i v TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES v i LIST OF FIGURES v i i i ACKNOWLEDGEMENTS x INTRODUCTION 1 MATERIALS AND METHODS 17 Animals 17 Swimming assessment 17 Surgical procedure 18 Exercise protocol 19 Sampling and muscle dissection 20 Muscle homogenization 21 Biochemical analyses 23 In t r a c e l l u l a r pH 26 Calculations 30 S t a t i s t i c a l analysis 30 Cytosolic redox 31 I n t r a c e l l u l a r free magnesium 32 Model calculations 35 RESULTS 39 General 39 Adenylates 39 Phosphocreatine 42 A r t e r i a l and i n t r a c e l l u l a r pH 42 Carbohydrate metabolism 44 Lactate/pyruvate r a t i o s and redox 45 Plasma lactate and glucose 45 Tissue water 49 In t r a c e l l u l a r free magnesium 49 Model calculations 49 DISCUSSION 59 The resting f i s h 59 ATP overshoot 64 ATP depletion 66 Phosphocreatine and energy metabolism 70 Carbohydrate metabolism 72 In t r a c e l l u l a r pH 78 Free ADP 80 Free AMP 85 RAXP and energy charge 8 6 SUMMARY AND CONCLUSIONS 94 REFERENCES 97 v i LIST OF TABLES Table 1 . Magnesium binding s i t e s and binding constants i n s k e l e t a l muscle 3 4 Table 2 . E q u i l i b r i u m constants used 38 Table 3. A r t e r i a l and i n t r a c e l l u l a r pH estimated by the DMO and homogenate techniques p r i o r to and during recovery fol lowing exhaustive exercise 40 Table 4. Adenylate concentrations i n white muscle p r i o r to and during the recovery from exhaustive exericse i n rainbow trout 4 1 Table 5. Concentrations of phosphocreatine, creat ine and inorganic phosphate i n white muscle 4 3 Table 6. Concentration of l ac ta te , pyruvate, glycogen and glucose i n rainbow trout white muscle 4 6 Table 7 . Lactate/pyruvate r a t i o and c y t o s o l i c redox . . . 4 7 Table 8. Plasma lactate and glucose concentrations . . . . 48 Table 9 . T o t a l t i s sue water content and f l u i d d i s t r i b u t i o n 50 Table 10. Magnesium bound ATP, bound ATP as a f r a c t i o n of the t o t a l creat ine (Fa) and as a f r a c t i o n of the t o t a l adenylates (Ratp); creat ine charge (Fc) and normalized p o t e n t i a l energy pool 55 v i i Table 11. Free ADP concentration ca lcu la ted without reference to ion binding, or using the model equations and several d i f f eren t concentrations of free magnesium 56 Table 12. Free AMP concentration ca lcu la ted at d i f f e r i n g free magnesium concentrations 57 Table 13. Energy charge ca l cu la ted using the t o t a l concentrations of ADP and AMP and according to the model d e f i n i t i o n s 58 v i i i LIST OF FIGURES Figure 1. Model of the concentrations of PCr, ATP, ADP, AMP and P i i n the presence of creatine kinase and adenylate kinase as ATP i s hydrolyzed 6 Figure 2. Calculated equilibrium concentrations of phosphocreatine and adenosine phosphates as high energy phosphate i s discharged 8 Figure 3. Normalized metabolite concentrations as a function of energy content 9 Figure 4. ATP concentration during recovery from exhaustive exercise i n rainbow trout 14 Figure 5. Calculated t o t a l magnesium concentration at rest and at exhaustion for a variety of free magnesium concentrations from 0 to 30 mM 51 Figure 6. 3 Carbon units ([lactate] + 2[glycogen]) in rainbow trout white muscle following exhaustive exercise 74 Figure 7. Results of model calculations applied to rainbow trout white muscle following exhaustive exercise 88 ix Figure 8. The v a r i a t i o n i n creatine charge with the normalized p o t e n t i a l energy pool i n rainbow trout white muscle following exhaustive exercise: the e f f e c t of the purine nucleotide cycle 89 ACKNOWLEDGEMENTS I would l i k e to thank Dr. Peter Hochachka f o r his support and e s p e c i a l l y for providing a stimulating academic environment which i s , i n part, a t t r i b u t a b l e to everyone i n the lab but derives i n large measure from h i s own boundless enthusiasm. A special thanks goes to my friends and colleagues i n the lab, i n p a r t i c u l a r : Chris Moyes, Tim West and Peter Arthur without whose help and suggestions t h i s thesis would not have been possible. I would also l i k e to thank Dr. Dave Randall and his group for allowing me for the use of t h e i r c a p i l l a r y pH electrode and the swim tunnel. Special thanks to Yong Tang for teaching me the homogenate pH technique. Thanks also to Dr. Carefoot for allowing me to use one of his drying ovens. 1 INTRODUCTION C e l l u l a r energy metabolism can be conceptually divided into two d i s t i n c t components, ATP consuming systems and ATP producing systems. One of the fundamental c h a r a c t e r i s t i c s of metabolic organization i s the coupling of energy supply to energy demand. Muscle tissue provides an excellent system for the study of the coupling between these two components because of i t s wide range of energy demand. The metabolic rate of a muscle c e l l can increase many f o l d over the t r a n s i t i o n from rest to work. For example, energy turnover in the frog sartorius muscle increases by nearly 1,000 f o l d in the f i r s t few seconds of tetanic contraction (Krisanda e_t .al., 1988) . The ATP consuming reactions in muscle c e l l s include actomyosin ATPases as well as a variety of ATP-dependent ion pumps. It i s generally considered that ATP demand i s the driving force, or independent variable, i n metabolic coupling. So, as ATP demand increases due to, for example, increased rate of contraction in a muscle, ATP production w i l l also r i s e . However, i t should be noted that cause and effect can be d i f f i c u l t to determine and i t may be that the ATP producing systems of the c e l l are simply responding to the same signal that upregulates the ATP demand. For example, the changes i n [Ca2+] which i n i t i a t e a contraction could also serve to speed up ATP production (Connett, 1990). The control of the ATP consuming reactions i n the muscle c e l l has been extensively studied, but 2 for the purposes of t h i s discussion i t i s s u f f i c i e n t to regard these reactions as a "black box" of the general form: ATP vADP + P± which can be set to a variety of d i f f e r e n t levels of ATP demand. The systems which supply energy can be broken down into three main sub-systems (1) the mitochondrial sub-system , (2) the g l y c o l y t i c sub-system and (3) the phosphate energy sub-system (Connett, 1990) . The mitochondrial and g l y c o l y t i c sub-systems contain the energy producing functions of the c e l l . Both oxidative phosphorylation and g l y c o l y s i s share many of the same control signals. Two main cont r o l l e r s are redox balance and phosphorlyation state, which i s e s s e n t i a l l y some measure of the degree to which the adenylates are phosphorylated to ATP. Erecinska e_t a_l. (1977) have shown that aerobic ATP synthesis responds to changes i n the phosphorylation state of the c e l l . G l ycolysis i s also sensitive to phosphorylation state largely because of the a l l o s t e r i c control of PFK through ATP i n h i b i t i o n and d e i n h i b i t i o n by ADP, AMP and phosphate (Hochachka and Somero, 1984) . These systems act as feed back loops since changes i n the rate of ATP production w i l l a f f e c t phosphorylation state. Mitochondrial redox state i s also involved i n the control of oxidative phosphorylation (Koretsky et a l . , 1984; Katz et a l . , 1988) and cy t o s o l i c redox state i s generally accepted to be an important c o n t r o l l i n g factor of c e l l u l a r metabolism brought about by a network of near equilibrium reactions in 3 which pyridine nucleotides p a r t i c i p a t e as cofactors (Krebs and Veech, 1969) . The interaction of cytosolic.'. and mitochondrial redox i s complex. The role of the phosphate energy sub-system i s of the greatest importance to the present study. This sub-system consists of the energy supply d i r e c t l y available i n the cytosol, that i s , the c y t o s o l i c pools of creatine phosphate and ATP. ATP i s the d i r e c t substrate for most of the energy requiring processes in the c e l l . Phosphocreatine on the other hand i s thought to p a r t i c i p a t e i n only one reaction: ADP + PCr + H* , -1 ATP + creatine catalyzed by the enzyme creatine phosphokinase (CPK). The actual function of the CPK reaction i s controversial. It has been suggested that PCr and creatine may function as a shuttle for the transport of high energy phosphates between compartments of adenylates within muscle c e l l s . The phosphocreatine energy shuttle includes three basic parts, one i n the area of the mitochondrion, one in the area of the myofibril and the actual "shuttle" through the cytosol between the two. According to the theory, at the myofibril PCr i s u t i l i z e d by bound CPK to rephosphorylate the ADP produced by the actomyosin ATPase. The r e s u l t i n g creatine enters the bulk phase of the cytosol. At the mitochondria t h i s creatine i s u t i l i z e d by an isozyme of CPK which i s bound close to the adenylate translocase s i t e . Here, according to the theory, the conditions are favourable for the formation of PCr from t h i s creatine and the ATP produced by 4 oxidative phosphorylation. The PCr then enters the bulk phase of the cytosol and diffuses to the myofibril where the cycle begins again. The basic evidence for the theory i s outlined in Bessman and Savabi (1990) . In apparent contrast, the c l a s s i c theory regarding the function of CPK i s to act as a buffer to [ATP] during temporary mismatching between ATP demand and supply. The e q u i l i b r i a between PCr and the adenylates are such that when net high energy phosphate hydrolysis occurs the PCr pool i s depleted to a greater extent than the ATP pool (McGilvery and Murray, 1974; A l l e n and Orchard, 1987; Connett, 1988) . This analysis treats the contents of the cytosol as e s s e n t i a l l y homogenous and does not require the assumption of compartmentation necessary for the phosphocreatine shuttle. Rather i t assumes that CPK i s i n equilibrium everywhere in the c e l l which appears to be well founded (Meyer et a l . , 1984) . As formulated, these two hypotheses appear to be mutually exclusive. Meyer et. al.. (1984) showed that they are compatible. They showed that the properties of CPK which make i t a temporal buffer of [ATP] also allow i t to function as a s p a t i a l buffer to [ATP]. However, the s p a t i a l buffering function should not be regarded as a shuttle per se since t h i s requires some sort of metabolic compartmentation. Rather, Meyer et a l . (1984) suggest that t h i s s p a t i a l buffering i s an example of f a c i l i t a t e d d i f f u s i o n , equivalent i n p r i n c i p l e to the role of myoglobin i n oxygen transport. Using a t o t a l l y d i f f e r e n t approach Jacobus (1985) reaches a si m i l a r conclusion 5 based on a d i f f u s i o n r e s t r i c t i o n on ADP. These hypotheses are based on viewing energy metabolism as a series of e q u i l i b r i a . A l l of the constituents of the phosphate energy sub-system are i n t e r r e l a t e d v i a the following equations: (1) Cr + MgATP2y- * MgADP" + PCr2" + H+ (2) AMP2" + MgATP 2 =F==^ ADP3" + MgADP" Reaction (1), the CPK reaction was discussed above. Reaction (2) , catalyzed by adenylate kinase (myokinase), AdK, sets the ra t i o s of the various adenine nucleotides. Both CPK and AdK are thought to be at or near equilibrium i n skeletal muscle. The capacity of CPK i n s k e l e t a l muscle i s such that i t should almost always be near equilibrium. Direct measurements by 31P-NMR support t h i s conclusion (Meyer et a l . , 1985). Adenylate kinase i s probably also near equilibrium i n skeletal muscle (McGilvery and Murrray, 1974) since the enzyme capacity i s s i m i l a r to that of CPK i n s k e l e t a l muscle (Connett, 1988) . If these reactions are assumed to be at equilibrium then i t i s possible to model the changes i n the r e l a t i v e concentrations of the adenylates and PCr as "high energy" phosphate i s hydrolyzed. Figure 1 shows the results of such a model (Allen and Orchard, 1987) . It assumes that the following reactions occur: ATP > ADP + Pi PCr + ADP^ ^ ATP + Cr 2ADP^ ATP + AMP changes i n pH were neglected and a l l reactions were assumed to occur at pH = 7.0. However, the AdK and CPK reactions do not 6 Figure 1: Model of concentrations of PCr, ATP, free ADP, and AMP and phosphate i n the presence of creat ine kinase and adenylate kinase as ATP i s hydrolyzed. See A l l e n and Orchard (1987) . Hydrolys i s of ATP was regarded as i r r e v e r s i b l e . Reactions were assumed to occur at pH =7.0 and changes i n pH were ignored. S t a r t i n g concentrations were [ATP]= 7mM; [PCr]= 25mM; [ADP]=[AMP]=[Pi]=0 [ATP][Cr]/[ADP][PCr] = 200 [ADP]2/[ATP][AMP] = 1 7 occur i n vivo as they are shown above. Rather, only the Mg2+ chelates of the adenylates are involved. In order to properly apply these e q u i l i b r i a to experimentally derived metabolite measurements some estimate of both H+ and magnesium binding to the adenylates should be taken into account. This approach was f i r s t outlined by McGilvery and Murray (1974), see figure 2. Connett (1988) further refined t h i s by scaling a l l parameters to the t o t a l creatine pool (PCr+Cr) and considering the effects of changes i n pH and [Mg2+] . Since PCr participates i n only the CPK reaction the t o t a l creatine pool i s e s s e n t i a l l y constant for one tissue over a l l metabolic states. Total [creatine] varies widely from tissue to tissue (Connett, 1988) so scaling a l l parameters in t h i s way allows dir e c t comparison between tis s u e s . As can be seen from figures 1,2 and 3 which outline the behaviour of the adenylates and PCr as phosphate energy i s hydrolyzed according to each model, the careful consideration of ion binding does not make a great deal of difference to t h e i r general predictions. There are c l e a r l y two d i s t i n c t phases in the depletion of the high energy phosphate pool. In the f i r s t phase the adenine nucleotide pool i s protected and only [PCr] decreases with l i t t l e or no change in the adenylates. Connett (1988) terms t h i s the buffering phase. In the second phase, energy i s supplied at the expense of [ATP] and readjustments to the r e l a t i v e proportions of the adenine nucleotides are brought about as a result of the AdK equilibrium. One obvious but often overlooked point that i s made clear by these models i s that 8 24 36 32 28 24 20 16 12 8 4 0 TOTAL HIGH ENERGY PHOSPHATE (mmoles/kg) F i g u r e 2: C a l c u l a t e d e q u i l i b r i u m c o n c e n t r a t i o n s of phosphocreatine and the adenosine phosphates as h i g h energy phosphate i s d i s c h a r g e d . T o t a l Cr = 30 mmoles/kg Free [ATP]+[ADP]+[AMP] = 6 mmoles/kg pH assumed t o be constant at 7.0 f r e e Mg2+ « 0.5mM [ATP] [Cr]/[H +] [ADP]= 1.51xl0 8 M"1 [AMP] [ATP] / [ADP] 2 = 0.364 A f t e r M cGilvery and Murray (1974) 9 1 . 0 0 0 .2 0 .4 0 . 6 0 .8 1 .0 N O R M A L I Z E D P O T E N T I A L E N E R G Y P O O L ( P C r + 2 A T P + A D P ) F i g u r e 3 : N o r m a l i z e d m e t a b o l i t e c o n c e n t r a t i o n s as a f u n c t i o n o f " e n e r g y c o n t e n t " , t o t a l a d e n y l a t e s = [ATP]+[ADP]+[AMP] t o t a l c r e a t i n e = [ P C r ] + [ C r ] c r e a t i n e c h a r g e (Fc) = [ P C r ] / t o t a l c r e a t i n e e n e r g y c h a r g e (EC)= fATP 1+0.5 fADP1 t o t a l a d e n y l a t e s R a t p = [ A T P ] / t o t a l a d e n y l a t e s R a d p = [ A D P ] / t o t a l a d e n y l a t e s Ramp = [ A M P ] / t o t a l a d e n y l a t e s A l l v a l u e s were c o m p u t e d u s i n g [Mg 2 + ] =lmM p H = 7 .0 t o t a l a d e n y l a t e s / t o t a l c r e a t i n e = 0 . 2 A f t e r C o n n e t t (1988) 10 large changes i n [PJ can arise without substantial changes in [ATP] (Allen and Orchard, 1987) . Note that i f [PCr] f a l l s by 20% [ADP] f r e e w i l l increase by 200% and [PJ by 160% without any change i n [ATP]. This aspect of the CPK reaction probably plays an important physiological role i n the muscle c e l l . Phosphate i s a known activator of both glycogenolysis, through action on phosphorylase (Morgan and Parmeggiani, 1964) and gl y c o l y s i s through the activation of PFK (Hochachka, 1980) and at high level s i s known to be an i n h i b i t o r of actomyosin ATPase (Cooke and Pate, 1990). As mentioned before, Connett's analysis (figure 3) also takes into account the effects of changing [Mg2+] and pH. Changing pH al t e r s the extent of the f i r s t , or buffering, phase of high energy phosphate depletion. A l k a l i n i z a t i o n of the cytosol results i n less e f f e c t i v e buffering (ie: more rapid depletion of [ATP]) while a c i d i f i c a t i o n (as i s seen i n working sk e l e t a l muscle) leads to an increase in the range of buffering. Changes i n [Mg2+] have similar effects with high [Mg2+] decreasing the extent of the buffering phase. When energy charge or RAXP ( [ATP ] / ( [ATP ] + [ADP ] free+ [AMP ] f r e e) values are high (>0.5) a factor of two change in [Mg2+] results in a less than 10% change in the value of the energy charge or RATP. These effects are greater when energy charge i s low. As a result changes i n [Mg2+] are probably only important under conditions of extreme energy depletion when [ATP] and hence energy charge and RflTP start to f a l l . Since ATP i s a major i n t r a c e l l u l a r magnesium chelator, 11 large changes i n [Mg 2 +] f r e e would be expected at that time. The mathematical and chemical precision of Connett's approach allow him to make several i n t e r e s t i n g statements regarding the phosphate energy sub-system of the c e l l . Among the most s t a r t l i n g of these i s that the f r a c t i o n of the adenine nucleotide pool i n the form of ATP and, by analogy, i n the form of AMP amd IMP i s simply a function of the "creatine charge" (the f r a c t i o n a l phosphorylation of creatine •( [PCr] / [Cr] T) ) , the pH, [Mg2+] , and [K+] . It i s d i f f i c u l t to adequately test these models i n vivo in mammalian sk e l e t a l muscle systems since even at fatigue [ATP] seldom f a l l s by less than 20% (Wilkie, 1981) . Therefore only events that are analogous to the buffering phase have been examined i n any d e t a i l . In f i s h white muscle, however, i t has been shown that [ATP] may be depleted by up to 90% following exhaustive exercise (Dobson and Hochachka, 1987; Mommsen and Hochachka, 1988). In addition rainbow trout are known to subsequently recover over a period of about 24h (Mommsen and Hochachka, 1988; M i l l i g a n and Wood, 1986) . This recovery could provide an ideal system i n which to examine models such as that of Connett (1988) since the system starts out with low potential energy ([PCr]+2[ATP]+[ADP]) gradually returning to a state of high pot e n t i a l energy over a time course s u f f i c i e n t l y slow to make sampling for conventional biochemical analyses f e a s i b l e . In f i s h white muscle there i s an additional series of reactions involving the adenylates which must be considered. 12 These reactions are c o l l e c t i v e l y c a l l e d the purine nucleotide cycle (Lowenstein, 1972). (1) AMP2- :> IMP2- + NH3 (2) IMP2- + aspartate 1" + GTP4" > adenylosuccinate + GDP3-+ Pi (3) adenylosuccinate • > fumarate + AMP2 Reaction (1) catalyzed by AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) i s thought to be the only enzyme of the cycle activated during exercise i n many muscle types (Flanagan et a l . , 1986), including trout white muscle (Mommsen et a l . , 1988). Reactions (2), catalyzed by adenylosuccinate synthase (IMP: L-aspartate ligase (GDP), EC 6.3.4.4) and (3), catalyzed by adenylosuccinate lyase (adenylosuccinate AMP lyase EC 4.3.2.2), are thought to be active only during recovery. The action of AMP deaminase i s probably not of significance during the buffering phase of phosphate energy depletion since at rest [AMP]f r e e i s on the order of 15nM. The Km of the AMP deaminase reaction for AMP i s about 0.4mM (Smiley and Suelter, 1967), very much greater than the i n vivo concentrations. [AMP]f increases to approximately 3 uM as PCr i s depleted to about 10% of i t s i n i t i a l l e v e l (assuming an equilibrium constant of 1 for AdK at pH 7.0 and [MgATP] of 8mM) (Krisanda et a l . , 1990) s t i l l s ubstantially lower than the Km. During the second phase of high energy phosphate depletion the adenylate kinase reaction becomes important and [AMP] ris e s rapidly (figures 1,2 and 3) . It i s during t h i s phase that the adenine nucleotide pool w i l l be depleted by the actions of AMP deaminase. The muscle mass of f i s h provides an excellent system for 13 investigating muscle energy metabolism for a number of reasons. Most important i s the organization of the myotomal mass of f i s h . Fish muscle i s s p a t i a l l y separated into groups by f i b r e type (Johnston, 1981) . In the trout the myotome i s d i f f e r e n t i a t e d into two main f i b r e types, white and red, on the basis of histochemical staining for the aerobic enzymes and m y o f i b r i l l a r ATPase. The red, slow twitch, muscle mass i s found primarily i n a narrow longitudinal s t r i p running beneath the l a t e r a l l i n e while the white, fast twitch, muscle comprises the bulk of the muscle mass (Johnston, 1981) . The white muscle mass i n trout does include a small percentage of red type f i b r e s , but t h i s i s n e g l i g i b l e compared to the highly heterogenous nature of most mammalian muscles. There have been several studies which investigated the nature of the recovery period following exhaustive exercise (Milligan and Wood, 1986; Dobson and Hochachka, 1987; Mommsen and Hochachka, 1988, Pearson et a l . , 1990). However, no one study has measured a l l the parameters needed to assess energy metabolism i n the context of the models of the c y t o s o l i c phosphate energy sub-system discussed e a r l i e r . In fact, the time course and nature of the changes i n the concentrations of PCr and the adenylates vary substantially from experiment to experiment (see figure 4). Two of the three studies outlined i n these figures report an actual increase i n [ATP] r e l a t i v e to rest at some point during recovery. This behaviour i s not consistent with the metabolic models discussed e a r l i e r since 14 Figure 4. ATP concentration during recovery from exhuastive exercise i n rainbow trout. The ATP overshoot. A l l concentrations were converted to umoles/g wet weight using data available i n the o r i g i n a l papers. restexh 2h 4h 24h restexh 2h 4h rwtexh 2h 4h recovery time 16 they suggest that there may have been some synthesis of new adenylate skeletons to enhance the ATP pool. Adenylate synthesis can occur v i a either a de novo or salvage pathway, with phosphoribosyl pyrophosphosphate (PRPP) as a common intermediate. Possible sources of substrate for the salvage pathway could include both adenylate breakdown products such as adenosine, and any breakdown products of RNA since the nucleotides are f r e e l y interconvertible. However, hypothesizing that the [ATP] overshoot seen during recovery i s due to ATP synthesis by either pathway presents a paradox. Both of these anabolic pathways are energetically expensive and, i n fact, are activated by high energy charge (Kunjara et a l . , 1987). How the c e l l can engage i n these processes at a time when i t s energy reserves (including ATP and hence energy charge) are c r i t i c a l l y depleted i s an open question. The aim of the present study i s two-fold: f i r s t , to characterize the metabolic events occurring i n f i s h white muscle during recovery from exhaustive exercise, with p a r t i c u l a r reference to the high energy phosphate compounds and t h e i r breakdown products, in order to further investigate the existence and possible causes of previously observed [ATP] overshoots; second, to test a number of equilibrium models, i n p a r t i c u l a r that of Connett (1988). The f i s h system i s ideal for t h i s purpose since i t i s one of the few vertebrate systems i n which a profound [ATP] depletion followed by a f u l l recovery i s seen, which may allow the models to be tested to t h e i r l i m i t s . 17 MATERIALS AND METHODS  Animals Rainbow trout (Oncorhynchus mykiss) of both sexes (mean weight 560 ± 8 9g (SEM) ) were obtained from West Creek Trout Ponds, Aldergrove, B r i t i s h Columbia. Fish were held outdoors i n one 2m diameter c i r c u l a r tank supplied with flow-through dechlorinated Vancouver tap water between 8 and 12°C. Fish were fed by hand d a i l y to s a t i a t i o n with Oncor P a c i f i c Salmon Feed (Moore - Clarke). Swimming Assessment In the evening in d i v i d u a l f i s h were transfered to a Brett type swim tunnel (1964) and the next day U c r l t was determined according to the procedure outlined i n (Beamish, 1978). Three uncannulated and one cannulated f i s h of t h i s stock were used. Uc r i t was calculated according to the following formula: Ucrit= Ui + ( t i / t i i x Uii) where Ui = the highest v e l o c i t y maintained for the prescribed time. Uii= v e l o c i t y increment (lOcm/s) t i = time the f i s h swam at the "fatigue" v e l o c i t y (min) t i i = prescribed time period at each v e l o c i t y (10 min) 18 This v e l o c i t y was then corrected for the presence of the f i s h i n the flume as follows: Uc = U s(l + A i / A i i ) where Uc = corrected v e l o c i t y Us = ve l o c i t y i n absence of f i s h A i = cross sectional area of f i s h (/0.5d/0.5w) Aii= cross sectional area of the tunnel Uc was found to be 2.8 6 ± 0.03 body lengths/second (mean ± SEM) for the uncannulated f i s h and 2.82 body lengths per second for the cannulated f i s h . It i s important to know U c r l t for any stock of f i s h i f you wish to study metabolism i n white muscle because white muscle in f i s h i s poorly recruited at low swimming speeds (below Ucrit) (Johnston, 1981) while higher speeds res u l t i n the recruitment of white f i b r e s and the onset of fatigue. Surgical Procedure Trout (starved for one day) were anaesthetized in a buffered (NaHC03) tricanemethanesulphonate (MS-222) solution at a concentration of 1:6,000 g/L and then transferred to the operating table where they were maintained under anaesthetic by use of forced v e n t i l a t i o n of a buffered MS-222 solution at a concentration of 1:16,000 c h i l l e d to approximately 15 °C and bubbled with oxygen. Dorsal a o r t i c cannulae were implanted using a modification of the technique of Sovio e_t a_l. (1972) . In b r i e f , two stitches were put i n place i n the roof of the mouth and a heat f l a r e d sleeve (PE 200) run through the front of the roof of the mouth and out i n the area of the nares. Sharpened guitar wire was f i t t e d inside a piece of polyethylene tubing (PE 50) about 25 cm long and then used to pierce the dorsal aorta s u p e r f i c i a l l y at the l e v e l of the f i r s t or second g i l l arch. The wire was then removed and the cannula advanced (up to about 5cm). The cannula was then run out through the implanted sleeve and fi x e d i n place on the roof of the mouth using the sutures. Fish were allowed to recover from surgery for at least 48 hours p r i o r to the beginning of experimentation during which time they were not fed. P r i o r to experimentation, cannulated f i s h were held i n an opaque black plexiglass box divided into six compartments each just s u f f i c i e n t l y large to hold the f i s h . The box was supplied with flow through dechlorinated Vancouver tap water which was between 8 and 12 °C throughout the duration of the experiment. Exercise Protocol F i s h were transferred by net to a Brett-type swim tunnel and allowed to f a m i l i a r i z e themselves with the tunnel for 15 to 20 minutes with the water flowing at low speed. Flow was then increased to the maximum the f i s h could attain (generally about 130% Ucrlt) . Any f i s h which could not atta i n at least 120% of the U c r i t for the stock were discarded. The speed was held constant u n t i l the f i s h could no longer maintain t h i s v e l o c i t y (about 3 minutes) at which point the speed was decreased by about 30 percent. This speed was held for about 5 minutes and then gradually increased and the cycle begun again. The flow speed was continually o s c i l l a t e d in t h i s way as the maximum attainable speed gradually decreased u n t i l the f i s h could no longer maintain i t s pos i t i o n in the swim tunnel even at the slowest speeds. At t h i s point the f i s h no longer responded to being grasped by the investigator. When exhausted, the f i s h i s unresponsive but capable of v e n t i l a t i o n and i s f l a c c i d , not s t i f f . In only one case out of over a hundred f i s h used i n t h i s and other experiments was a f i s h driven into rigor at exhaustion. The entire exercise procedure took approximately 25-30 minutes to complete. Fish were either k i l l e d immediately following exercise (at exhaustion) or transferred back to the holding box. Fish were then randomly assigned to a treatment group and k i l l e d after 2, 4, 8, or 24 hours of recovery as required. Resting f i s h were kept in the holding box for at least 48 hours p r i o r to sampling. Sampling and Muscle Dissection Resting f i s h are notoriously d i f f i c u l t to sample. Any 21 struggling results in a rapid depletion of the high energy phosphates, p a r t i c u l a r l y PCr (Dobson and Hochachka, 1987) . To reduce t h i s problem f i s h were anaethetized p r i o r to sampling. At the s p e c i f i e d sampling time 2mL of blood was withdrawn and placed on i c e . This volume was replaced with a 65mg/mL solution of sodium pentobarbitol (Somnotol). This dose rapidly k i l l s the f i s h with minimal struggling. As soon as v e n t i l a t i o n ceased (30 seconds to 3 minutes) the f i s h was removed from the water and a 1 cm thick steak was s l i c e d from the area immediately behind the dorsal f i n using a double-bladed hatchet (Faupel et a l . , 1972). The steak was rapidly freeze-clamped i n l i q u i d nitrogen with pre-cooled aluminium tongs. The steak was kept i n l i q u i d nitrogen u n t i l deproteinized. Muscle Homoqenization, Extraction and Neutralization A section of epaxial white muscle was removed from the steak under l i q u i d nitrogen and quickly transferred to a precooled mortar. It i s c r i t i c a l to maintain the tissue sample at low temperature to avoid changes i n the concentrations of the high energy phosphates. To t h i s end the mortar was kept in a styrofoam cooler which was f i l l e d with l i q u i d nitrogen at a l l times such that the mortar was nearly submerged. The mortar was s i m i l a r l y nearly f i l l e d with l i q u i d nitrogen. In t h i s way the sample was submerged i n l i q u i d nitrogen throughout the 22 procedure. The muscle was then coarsely ground with the mortar and pestle and any pieces of skin and bone removed. The l i q u i d nitrogen was then replenished i f necessary and the remaining muscle was ground to a very fine powder. Approximately l g of the powder was then transferred to a pre-weighed c h i l l e d test tube containing lmL of ice cold 7% perchloric acid (PCA) which was quickly re-weighed, once any remaining l i q u i d nitrogen had boiled o f f . Then 3mL of cold PCA was added and the mixture was homogenized using an U l t r a turrax tissue grinder. The tissue was ground for 15 seconds three times separated by one minute to allow the t i p to cool and prevent excessive heating of the homogenate. Throughout homogenization the tube was held i n a slurry of s a l t water and ice at a temperature of between -5 and -10 °C. Two 200 uL aliquots of the homogenate were removed and stored at -80 °C for l a t e r determination of glycogen. The remainder was centrifuged at 12,000g for 9 minutes at a temperature between 0 and 2 °C. A known volume of the supernatant was removed and 500 uL of a saturated solution of TRIZMA base was added as a buffer. This mixture was then t i t r a t e d with 10M KOH u n t i l a pH of between 7.0 and 7.2 was reached i n order to precipitate the KC104. This mixture was again centrifuged i n the cold at 12,000g for 3 minutes and the pH of the supernatant checked and adjusted with 10M KOH or HCl as necessary. The neutralized extract was stored at -80 °C u n t i l assayed. Extraction e f f i c i e n c y of t h i s method for the l a b i l e high energy phosphates was assessed using a variety of methods. In the f i r s t v a l i d a t i o n study the tissue homogenate was simulated by using a solution of bovine serum albumin (BSA lmg/mL) containing an aliquot of concentrated PCr standard solution. This mixture was then extracted and neutralized as above and percentage recovery assessed. Recovery was 91.77 + 6.36 % (mean ± SEM n=3) . PCr recovery was also assessed by adding a known amount of PCr standard to a muscle homogenate in PCA. Recovery was 100.84 ± 5.73% (n=3) . This procedure was repeated with ATP supplementation. Recovery was 103.3 ± 4.84% <n=4) . Biochemical Analyses Chromatography: High performance l i q u i d chromatography (HPLC) was used to measure ATP, ADP, AMP and IMP. The procedure was ca r r i e d out using an LKB 2152 HPLC co n t r o l l e r and 2150 titanium pump coupled to a 2220 recording integrator. The separation was performed on an Aquapore AX-300 7 um weak anion exchanger (Brownlee labs). The separation was si m i l a r to that of Harmsen et_ aT. (1982) and a modification of that used by Parkhouse et a_l. (1987) . To b r i e f l y outline, e l u t i o n was i s o c r a t i c for the f i r s t 5 minutes of the run using 60 mM KH2P04 (pH 3.2) followed by a l i n e a r gradient from 60mM KH2P04 (pH 3.2) to 750 mM KH2P04 (pH 3.5) 24 o v e r 10 m i n u t e s . T h i s c o n c e n t r a t i o n a n d pH was t h e n m a i n t a i n e d f o r 12 m i n u t e s . The c o l u m n was r e - e q u i l i b r a t e d f o r 6 m i n u t e s w i t h s t a r t i n g b u f f e r b e f o r e t h e n e x t r u n . Column t e m p e r a t u r e was 55°C a n d f l o w r a t e was 2mL/minute t h r o u g h o u t . R a p i d e l u t i o n o f ATP i s f a v o u r e d by h i g h [KH 2P0 4] a n d h i g h pH w h i l e l o w pH and l o w [ K H 2 P 0 J a r e r e q u i r e d f o r a d e q u a t e s e p a r a t i o n o f AMP and IMP. The c o n d i t i o n s s e l e c t e d were t h e r e s u l t o f a c o m p r o m i s e b e t w e e n t h e s e f a c t o r s a n d t h e t i m e r e q u i r e d f o r r e e q u i l i b r a t i o n t o t h e s t a r t i n g b u f f e r w h i c h i n c r e a s e s w i t h t h e d i f f e r e n c e b e t w e e n t h e two s o l u t i o n s . D e t e c t i o n was by a b s o r b a n c e o f u l t r a v i o l e t l i g h t (254nm) u s i n g a BIO-RAD f l o w t h r o u g h UV m o n i t o r . S t a n d a r d c u r v e s were c o n s t r u c t e d f o r a l l m e t a b o l i t e s o v e r t h e r e l e v a n t c o n c e n t r a t i o n s by p r e p a r i n g a m i x e d s o l u t i o n i n 60mM KH 2P0 4. t h e s t a n d a r d c u r v e s were l i n e a r t h r o u g h o u t t h e r a n g e r e q u i r e d ( r 2 = 0 . 9 9 ) . 20uL o f n e u t r a l i z e d e x t r a c t was u s e d f o r HPLC d e t e r m i n a t i o n i n a l l c a s e s s i n c e t h i s amount p r o v i d e d e a s i l y v i s u a l i z e d on s c a l e p e a k s f o r a l l m e t a b o l i t e s u n d e r a l l e x e r c i s e a n d r e c o v e r y c o n d i t i o n s . The c o e f f i c i e n t o f v a r i a t i o n b e t w e e n d u p l i c a t e s was a l w a y s l e s s t h a n 5 p e r c e n t . C o m m e r c i a l p r e p a r a t i o n s o f KH 2P0 4 s a l t s c a n c o n t a i n i m p u r i t i e s w h i c h a b s o r b i n t h e UV r a n g e , t h u s i n c r e a s i n g t h e b a s e l i n e a b s o r b a n c e o f t h e b u f f e r s o l u t i o n a n d r e d u c i n g t h e s e n s i t i v i t y o f d e c t e c t i o n o f t h e compounds o f i n t e r e s t . HPLC g r a d e , l o w a b s o r b a n c e s a l t s a r e a v a i l a b l e , b u t a r e e x t r e m e l y e x p e n s i v e . I u t i l i z e d a m o d i f i c a t i o n o f t h e t e c h n i q u e o f R e i s s e t a l . (1984) t o p r e - p u r i f y a n a l y t i c a l r e a g e n t g r a d e KH 2P0 4 t o 25 the required quality. A IM stock solution was passed through a column (Econo column, BIO RAD laboratories 5cm diameter X 30cm) packed with an anion exhchange resin (AG1 X8, chloride form), a cation exchanger (chelex 100, sodium form) and activated charcoal (14-60 mesh). Chelex has high s e l e c t i v i t y for divalent cations, p a r t i c u l a r l y copper, iron and heavy metals. It i s used to remove the trace metal contamination present in the a n a l y t i c a l grade s a l t . The activated charcoal served to remove any aromatic contaminants which may leach from the resins. The stock solution of phosphate was kept at 4 °C and constantly r e c i r c u l a t e d through the column by means of a p e r s i t a l t i c pump. Immediately p r i o r to use the stock buffer was diluted, brought to the appropriate pH and vacuum f i l t e r e d (0.22 um). With t h i s technique the baseline disturbance caused by the impurities present i n the high concentration buffer was reduced by 74%. Spectrophotometry: A l l determinations were performed on a Perkin-Elmer Lambda 2 UV/visible spectrophotometer. Lactate, pyruvate, phosphocreatine and glucose were measured using routine NAD/NADH linked assays, e s s e n t i a l l y as i n Bergemeyer (1974) . Muscle glycogen was measured by digesting neutralized homogenate with amyloglucosidase. Total glucose (liberated from glycogen plus the free glucose pool) was then measured as before and the contribution of free glucose subtracted out (Bergmeyer, 1974). 26 Inorganic phosphate was determined c o l o r i m e t r i c a l l y using the technique of Black and Jones (1983). A l l assays were performed in duplicate. If the c o e f f i c i e n t of v a r i a t i o n between these duplicates was greater than 6% a t h i r d run was performed and the o u t l i e r discarded. Because of observed wide v a r i a t i o n i n the case of the phosphate assay, t r i p l i c a t e s were routinely performed and i f the c o e f f i c i e n t of v a r i a t i o n between these was greater than 6% a fourth was run and the o u t l i e r discarded. A l l assays were validated with appropriate standards. I n t r a c e l l u l a r pH Mean i n t r a c e l l u l a r pH (pHJ was determined using two methods: the d i s t r i b u t i o n of the weak acid 5,5 dimethyloxazolidine 2,4 dione (DMO) and the tissue homogenate technique of Portner et. a_l. (in press) . DMO d i s t r i b u t i o n : Use of the weak organic acid DMO as an In v i t r o marker for pHi has been widespread since i t s introduction by Waddell and Butler (1959) . This technique has, more recently, been employed in vivo i n the rainbow trout to estimate pH± i n a variety of tissues following exhaustive exercise (Milligan and Wood, 1986) . These authors consider t h i s technique capable of r e f l e c t i n g pH± changes i n trout white muscle on the scale of 15 minutes (Milligan and Wood, 1985) . 27 Approximately 12 hours p r i o r to sampling trout were injected through the dorsal aort i c cannula with 10 uCi 3H-mannitol and 2.5 uCi 14C-DMO (New England Nuclear) i n Cortland's f i s h physiological saline to a t o t a l volume of 500 uL. The pH of the a r t e r i a l blood sample collected just p r i o r to somnotol i n j e c t i o n was measured immediately using a Radiometer microelectrode (type E5021) maintained at 10°C v i a a r e c i r c u l a t i n g water bath and linked to a Radiometer 2 6 pH meter. White muscle leve l s of 3H and 1 4C r a d i o a c t i v i t y were measured i n lmL of the PCA extract used for metabolite assays using Amersham ACSII aqueous f l u o r . This method has the disadvantage that the PCA extraction included an at least 6 f o l d d i l u t i o n of the sample necessitating counting a large volume of the li m i t e d extract. However, t h i s method yiel d s low quench colorless samples which i s i t s main advantage over NCS digestion which yields highly coloured samples. This colour may be corrected i n two ways. F i r s t , the samples can be counted d i r e c t l y a f t e r being neutralized with 60 uL of g l a c i a l acetic acid, so long as the appropriate quench curve i s generated to adequately correct for the colour. This approach, however, can introduce gross inaccuracies, especially with respect to the estimation of dpms for low energy beta emitters l i k e t r i t i u m . It i s also possible to decolorize the samples with benzoyl peroxide as suggested by the manufacturer. However, i n t h i s case high chemical quench i s induced and again estimates of dpm for t r i t i a t e d samples can be compromised. To decrease 28 chemiluminescence a l l samples were stored i n the dark least overnight, or u n t i l chemiluminescence was reduced to an acceptable l e v e l , and then counted on an LKB 1214 rackbeta l i q u i d s c i n t i l l a t i o n counter using dual label quench correction. Total tissue water was determined by drying a l-2g sample of tissue taken from an area adjacent to the main sampling s i t e to constant weight i n a drying oven at 75 °C. Tissue e x t r a c e l l u l a r f l u i d volume (ECFV, mL/g) was calculated according to the equation: ECFV = Tissue [3H] mannitol (dpm/g) Plasma [3H] mannitol (dpm/g)/ plasma water (mL/g) I n t r a c e l l u l a r f l u i d volume was then determined as: ICFV = t o t a l tissue water - ECFV Tissue pHi was calculated according to the equation: pHi = pK D M 0 + log( [DMO], . (iO<e H e -e k D M O > + 1) -1) [DMO] e where [DMO]e and [DMO], represent e x t r a c e l l u l a r and i n t r a c e l l u l a r DMO concentration respectively and are calculated as: [DMO]e = plasma 1 4C DMO/ plasma water (dpm/mL) (dpm/mL) [DMO] i - (tissue 1 4C DMO - (ECFV.DMOJ / ICFV) pK D M 0 was taken from Malan et. a l . (1976) . Some caution must be exercised with respect to the use of mannitol as an e x t r a c e l l u l a r space marker. Recent studies have shown that mannitol i s slowly taken up and metabolized in various tissues y i e l d i n g increasing estimates of ECFV 29 (decreasing pEL) over time. However, t h i s e f f e c t has been shown to be minor i n trout white muscle over the time course employed i n t h i s study (Chris Wood, pers. comm.) Measurement of Homogenate pH The estimation of i n t r a c e l l u l a r pH by the measurement of the pH of a tissue homogenate has been frequently used for human muscle biopsies (see for example, C o s t i l l et a l . , 1982) . Direct measurement of pH has a number of advantages over the use of DMO. It eliminates the assumption that blood pH i s respresentative of the pH of the i n t e r s t i t i a l f l u i d and the dependance on the accurate measurement of t h i s pH. It can, i n p r i n c i p l e , r e g i s t e r very rapid pH transients without the problems of p a r t i a l e q u i l i b r a t i o n which may complicate the DMO technique. However, i t i s d i f f i c u l t to be assured that the measured pH bears any relationship to the actual i n t r a c e l l u l a r pH. Recently Portner et a_l. (in press) have developed a new "homogenate technique" which they have applied to the measurement of muscle i n t r a c e l l u l a r pH in, among other animals, rainbow trout. The method i s very simple. In b r i e f , an approximately 200mg sample of muscle, ground under l i q u i d nitrogen to a fine powder, was added to an Eppendorf tube which was then quickly f i l l e d with a solution of 160mM KF and ImM NTA ( n i t r i l o t r i a c e t i c acid, disodium salt) . The mixture was s t i r r e d b r i e f l y with a needle and capped. The insoluble f r a c t i o n was 30 spun down (3-5 seconds in a microcentrifuge at room temperature). Then aliquots of the supernatant were taken for repeated measurement of pH i n a c a p i l l a r y pH electrode thermostatted to 10°C. Variation i n pH between repl i c a t e s from the supernatant was usually below +0.005pH units and from the same muscle powder below ±0.02 pH units. Calculations S t a t i s t i c a l Analysis A l l data are reported as means + standard error of the mean (SEM). Metabolite differences between rest and/or the various recovery times were assessed using a one way ANOVA followed by Tukey's HSD on those parameters which showed a s i g n i f i c a n t F r a t i o . In a l l cases B a r t l e t t ' s test for homogeneity of variances was used to v e r i f y that the assumptions of the above tests were not v i o l a t e d . When the assumptions of homogeneity of variance and normality were not met simple logarithmic transformation of the data proved s u f f i c i e n t to remove the problem. In a l l cases p=0.05 was taken to be the l i m i t of signi f i c a n c e . A l l of the above tests were performed on a microcomputer using the SYSTAT s t a t i s t i c a l package (SYSTAT i n c . ) . In the case of pH, although the data i s reported in thi s way for ease of comprehension and interpretation, a l l s t a t i s t i c a l analyses were performed using calculated [H +]. It does, i n fact, make l i t t l e difference when the pH range being 31 considered i s t h i s small (see B o u t i l l i e r et al.., 1980 for f u l l a n a l y s i s ) . The comparison of the two techniques for estimating i n t r a c e l l u l a r pH poses p a r t i c u l a r problems because the DMO d i s t r i b u t i o n technique i s known to produce pH estimates with greater variance than the homogenate technique (Y. Tang pers. comm.). If t h i s i s the case with the data from t h i s study then parametric techniques of analysis are inappropriate. However, since i n most cases a pair of data points (1 DMO and 1 homogenate) was taken for each f i s h , a number of quite powerful non parametric tests are available. The sign test, signed rank test and Wilcoxon signed rank test were a l l performed to compare these data. Cytosolic Redox: The c y t o s o l i c NAD+/NADH r a t i o was calculated using the lactate dehydrogenase equilibrium (Williamson et .al., 1967; Veech et. al.., 1969) according to the following expression: NADVNADH = ( [pyruvate] x [H+] )/( [lactate] xKeq) where lactate and pyruvate concentrations denote the t o t a l measured contents. The equilibrium constant (Keq) was calculated to be l . l x l O - 1 2 M"1 at 10°C, pH 7.0 at an ioni c strength of 0.25, assuming a H° of +14 kcal/mol (Hakala et .al., 1956) . The use of t h i s method requires the assumption that the LDH reaction i s maintained at near equilibrium i n working muscle. 32 I n t r a c e l l u l a r free Magnesium: I n t r a c e l l u l a r free magnesium [Mg2+] i s an important factor in the understanding of reactions i n the cytosol which involve ATP and the other phosphorylated intermediates. However, the value of t h i s parameter i s uncertain. A variety of techniques have been used to estimate the fr e e l y d i f f u s i b l e pool of [Mg2+] including microelectrodes (Hess et al.., 1982) , dyes (Baylor et al.,1982) and NMR (Kirkels et a l . , 1989). Values for mammalian ske l e t a l muscle f a l l into two ranges: approximately ImM amd 3-5mM (Connett, 1985). Connett (1985) has applied a binding s i t e approach to the estimation of [Mg2+] i n the dog g r a c i l i s muscle. Skeletal muscle contains a number of proteins and other compounds which are known to bind magnesium and for which the magnesium binding constants are known. If the concentrations of these compounds and the t o t a l magnesium i s known for a tissue then the magnesium bound to the substances can be calculated. Assuming that these are the major chelators of magnesium then the free magnesium can be calculated by subtraction. As a result of t h i s assumption the [Mg 2 +] f calculated in t h i s way represents a l i k e l y maximum for [Mg 2 +] f . The major proteins known to bind magnesium along with t h e i r concentrations i n sk e l e t a l muscle and t h e i r binding constants are l i s t e d i n table 1. In addition ATP and PCr are major contributors to magnesium binding the appropriate magnesium binding constants are l i s t e d in table 2. 33 Binding i s estimated as: [Mg 2 +] x = [Mg 2 +] f{l+ S [X] T.K X M V (B + K X M g[Mg 2 +] f where X = the concentration of the compound of interest (protein or metabolite) B = 1 for proteins B = 1 + KJJ [H +] f for phosphorylated compounds subcripts: T = t o t a l concentration f = free concentration KMg = [MgX]/[Mg2+] [X] [MgX] = concentration of magnesium bound form of X Total magnesium was calculated for values of [Mg2+] between 0 and 20mM. These were then compared to the t o t a l magnesium concentration of rainbow trout white muscle which has been measured as 38.6 + 1.7 mEq/L at rest and 37.0 ± 0.4 mEq/L immediately following exhaustive exercise, or approximately 19mM (Parkhouse, 1987) . Since t h i s approach yields only an estimate of [Mg 2 +] f the effe c t s of reasonable variations i n [Mg 2 +] f on the model calculations were assessed. 34 Table 1 : Magnesium binding s i t e s and binding constants in skeletal muscle after Connett (1985) Compound Myosin Troponin Parvalbumin+ Calmodulin Concentration in muscle (mM) 0.32 0.32 0 .14 Binding constant (M"1) 3xl0 5 5xl0 4 lxlO 4 35 Model Calculations: The scaled creatine kinase model (Connett, 1988) was applied to the data from t h i s study. 1) ion binding: The binding of ions was handled as with the ca l c u l a t i o n of i n t r a c e l l u l a r [Mg2+] . So the binding of any phosphorylated compound with the ions [ H + ] , [Mg2+] , and [ K + ] can be described as: K = [XYn"m] / [Xm+] [Yn_] where X refers to the cation binding for example Mg2+, H + , K + and Y refers to the anionic phosphate compound (ATP, ADP, PCr et c . ) . The t o t a l concentration of any of these anions can be written as the sum of the free i o n i c and the bound forms: [Y] = [YH .B, where = 1 + £K£ . x m + and the subscript i refers to the p a r t i c u l a r Y: c =PCr, t =ATP, d =ADP, m =AMP, p =P± . The enzyme e q u i l i b r i a and the ionic binding can then be combined into two variables: RCPK = B T . K ^ J p / ( B D . B C K A ' T | [ H + ] . K C P K ^ A d k = Bt • . K A Q | . K A D K / ( K A T P . B D 2 ) 2) Calculation of free c y t o s o l i c ADP and AMP: The variables defined above can be used i n conjunction with the d e f i n i t i o n of the e q u i l i b r i a to calculate the [ADP] and [AMP] fr e e l y available i n the cytosol: 36 [AMP] = [ADP]2 . RAdK / [ATP] [ADP] = [ATP] . [Cr] .R^ / [PCr] Since a large f r a c t i o n of both ADP and AMP i s known to be bound to, for example, protein i n vivo i t i s important to know [ADP]£ and [AMP]f in order to calculate variables l i k e energy charge and to accurately assess the energy status of the c e l l . 3) D e f i n i t i o n of intermediate pools: Connett (1988) defines a number of pools of intermediates which serve to describe the phosphate energy subsystem of the c e l l . A l l concentration terms used i n these d e f i n i t i o n s refer to the free c y t o s o l i c pools involved in the equilibrium reactions as set out i n the introduction. ; The t o t a l creatine pool [Cr] T = [PCr] + [Cr] i s constant for a tissue over a l l metabolic states and i s thus used to scale a l l other pools to obtain dimensionless equations. The t o t a l free adenine nucleotide pool, [ATP] + [ADP] + [AMP], ([Ad] T) i s the reactive adenine nucleotide pool in the cytosol. Since [Ad] T may or may not be constant i t i s necessary to define the variables RAXP = [AXP]/[Ad] T . The phosphate bond or potential energy pool: Pe = [PCr] + 2[ATP] + [ADP] r e f l e c t s the pool of high energy phosphate bonds available in the t i s s u e . 4) D e f i n i t i o n of dimensionless concentrations: Since [Cr] T i s constant for any one tissue a l l other 37 concentrations can be scaled to [Cr] T to produce a number of dimensionless concentrations: Fc = [PCr]/[Cr] T F a = [Ad] T/[Cr] T F P e = Pe / [ C r ] T F P 1 = [P i l / [ C r ] T The adenylate energy charge (Atkinson, 1977) can also be calculated using the free concentrations of ADP and AMP. EC = 0.5 ((2 [ATP] + [ADP])/[Ad] T ) Connett also derives equations to show that RATP , F a , and E.C. can be calculated i f F c, R^ and RAdK are known. These equations were applied to the data from the present study and the results compared to the RATP, Fa, and EC calculated using the d e f i n i t i o n s shown above. 5) Equilibrium constants: In order to properly apply Connett's model to the data from the present study, values of the equilibrium constants appropriate' to in vivo conditions must be used. There i s a certain amount of va r i a t i o n i n the values of the equilibrium constants reported in the l i t e r a t u r e . I have used the values selected by Connett since these values were determined under conditions i n which a l l binding was accounted for. The values were corrected to conditions of ionic strength equivalent to that in vivo (u=0.17-0.2) and wherever possible to a temperature of 10°C using the Van't Hoff equation: In (Kz/KJ = (- /JH/RT) ( (1/T2)-(1/'T1) (T in °K) The equilibrium constants used are l i s t e d i n table 2. T a b l e Z Equilibrium Constants used. A l l values are corrected to i o n i c strength u = 0.17-0.2 and 10°C except where noted. constant 2 6.69 mM"1 K A M T P 1.13xl0 7 M"1 K&„ 550.69 M"1 KA*„ 5.84 M"1 1534 M"1 K A * P 6.10xl0 6 M"1 K J ^ H 83.99 M"1 K*P 2.92 M"1 K & 2 0 M"1 K P " C r 3.31x10* M"1 O 4 6.05 M'1 Kp 4, 8 . 91x106 M"1 1.7 6 M"1 K $ P 36.66 M"J K * , P 0.99 M"1 K C P K 4 . 86xl0 9 M"1 K A d K 8 .1 AH (Kcal/mole) 3.3 0 .087 1.5 6.0 3.3 0.522 1 . 0 6..0 * 5.59 -11.59 6 .14 3.24 6.0 -2.4 * at 25°C ** at 38°C 39 RESULTS ; General The homogenate technique of Portner et a l . (in press) gave estimates of i n t r a c e l l u l a r pH which were not s t a t i s t i c a l l y distinguishable from the estimates yielded by the DMO d i s t r i b u t i o n technique (see table 3) . However, the v a r i a t i o n associated with the exercise group means taken from the DMO estimates were s t a t i s t i c a l l y d i f f e r e n t from the v a r i a t i o n within groups derived from homogenate measurements. Since the homogenate technique appeared to y i e l d greater precision a l l subsequent calculations with and discussions of i n t r a c e l l u l a r pH w i l l be based on estimates of i n t r a c e l l u l a r pH derived from measurements of the pH i n the homogenates. Adenylates Changes in the concentrations of the adenylates and IMP are outlined i n table 4. Immediately following exhaustive exercise [ATP] f e l l to 24% of the resting value while [IMP] increased nearly 10-fold. Neither [AMP] nor [ADP] changed s i g n i f i c a n t l y . Since TAN also did not change, the decrease i n [ATP] can be accounted for by the increase i n [IMP] and no adenine nucleotides were lo s t from the c e l l as a result of the exercise protocol. During the f i r s t two hours of recovery there was a nearly 40 Table 3 : A r t e r i a l pH and i n t r a c e l l u l a r pH estimated by the DMO and homogenate techniques p r i o r to and during recovery following exhaustive exercise. pHA: a r t e r i a l pH; pH^: i n t r a c e l l u l a r pH (mean ± SEM). PHA pHi (Homog) p ^ (DMO) rest 7.77±.01 7.27+.03 7 .14±.01 exh 7.20±.07** 6.66+.03" 6.78±.ll 2h 7.36±.07* 6.57+.03** 6.52±.13 4h 7.31±.07* 6.57±.02** 6.49±.24 8h 7.63+.17 6.89+.11** 7.01+.19 24h 7.77±.05 7.21+.02 7.22+.04 n = 5 rest; 7 exh.; 5 2h; 5 4h; 4 8h; 6 24h. s i g n i f i c a n t l y d i f f e r e n t from corresponding resting value at p<0.001 s i g n i f i c a n t l y d i f f e r e n t from corresponding resting value at p<0.005 41 Tab le 4 : Adeny la te c o n c e n t r a t i o n s i n white muscle p r i o r t o and d u r i n g recovery from e x h a u s t i v e e x e r c i s e i n rainbow t r o u t , (umoles/g wet we ight ; Dean 1 SEM). TAN - t o t a l adenine n u c l e o t i d e s - [ATPJ+[ADP]+[AMP]+[IMP) [ATP] [ADP) [AMP ] [IMP] [TAN] r e s t 7 .5210.49 0.76810.069 0.07010.013 0.5410.19 8.9010. 2 e x h a u s t i o n 1 .8210 .19" 0.65410.065 0.062+0.016 5 .2810 .47" 7.8210. 60 2h 3 . 7210 .56" 0.61410.068 0.05710.016 2 . 9 8 * 0 . 5 1 " 7 . 3 7 i 0 . 52 4h 3 . 5 7 1 0 . 5 2 " 0.602*0.045 0.04910.021 2 .7710 .45" 6 . 7 8 i 0 . 26 8h 3 . 4510 .55" 0.49210.022 0.06510.015 2.77+0.45" 6.7810. 26 24h 7.0210.62 0.70910.049 0.05310.011 0.2810.07 8 . O 6 1 O . 63 s i g n i f i c a n t l y d i f f e r e n t from cor respond ing r e s t i n g va lue at p<0.001 n - 5 at r e s t , 2h, 4h; 4 at 8h; 7 at e x h . ; 6 at 24h two-fold increase i n [ATP] followed by a long plateau phase during which there was l i t t l e change. However, even at 8h there was no s t a t i s t i c a l l y s i g n i f i c a n t difference from the [ATP] at exhaustion. By 24h [ATP] had f u l l y recovered. Throughout recovery the changes in [ATP] were d i r e c t l y r e f l e c t e d by the changes i n [IMP]. No s i g n i f i c a n t changes were observed in [ADP] or [AMP] and thus [TAN] was conserved at a l l points during recovery.. Phosphocreatine Changes i n the concentrations of the creatine containing compounds and inorganic phosphates are outlined i n table 5. At exhaustion [PCr] decreased to less than 2,0% of the resting value, while [Cr] increased more than 2 f o l d . As was the case with the adenylates, t o t a l creatine remained unchanged, with the decrease i n [PCr] being accounted for by the increase i n [Cr]. The concentration of inorganic phosphate increased i n p a r a l l e l with [Cr] as a result of the hydrolysis of PCr. In contrast to [ATP], [PCr] i s f u l l y recovered by 2h post exercise (see tables 4 and 5) and does not change s i g n i f i c a n t l y thereafter. As before [Cr] and [P±] r e f l e c t e d these changes. A r t e r i a l and I n t r a c e l l u l a r pH Changes i n pHA and pRL are shown i n table 3. A r t e r i a l pH dropped by nearly 0.6 of a pH unit and i n t r a c e l l u l a r pH by very s l i g h t l y more at exhaustion. A r t e r i a l pH showed a gradual 43 T a b l e 5 : C o n c e n t r a t i o n s o f p h o s p h o c r e a t i n e , c r e a t i n e and i n o r g a n i c phosphate i n white muscle p r i o r t o and d u r i n g t h e r e c o v e r y from e x h a u s t i v e e x e r c i s e i n rainbow t r o u t (umoles/g wet weight; mean + SEM). [PCr] [Cr] t o t a l Cr (n) r e s t 22.62±2.69 19. ,36±2 .30 28.58±4. 96 41.98+2.21 (5) exh 4.26±1.14" 40. .7 6+0 .68** 50.12+2. 66* 45.03+1.23 (7) 2h 25.37+3.26 19. .82+2 .10 30.28+3. 82 45.19+2.22 (5) 4h 20.44+3.49 21. ,04+0 .96 28.83+3. 61 41.47+2.69 (5) 8h 21.97+3.76 20. ,80±2 .76 28.40±3. 38 39.87±0.62 (4) 24h 26.23+1.01 15. .74±2 .13 19.82±2. 95 41.97+1.26 (6) s i g n i f i c a n t l y d i f f e r e n t from c o r r e s p o n d i n g r e s t i n g v a l u e at p<0.005 s i g n i f i c a n t l y d i f f e r e n t from c o r r e s p o n d i n g r e s t i n g v a l u e at p<0.001 44 recovery over the 24h time course and by 8h post exercise was not s i g n i f i c a n t l y d i f f e r e n t from the resting value. I n t r a c e l l u l a r pH, on the other hand, remained low for at least 4h post exercise (in fact showing a s l i g h t non-significant decline over the f i r s t two hours) . However, by 8h post exercise pHA had increased s i g n i f i c a n t l y over that of the exhausted state and by 24h post exercise was not s i g n i f i c a n t l y d i f f e r e n t from the resting value. Carbohydrate Metabolism Changes i n the concentrations of lactate, pyruvate, glycogen and glucose i n white muscle are shown in table 6. [Lactate] increased more than 10-fold at exhaustion and t h i s increase could be accounted for by the decrease in glycogen content, although the large variations i n resting glycogen and the fact that the sampling was terminal and not s e r i a l makes a very rigorous analysis of the conservation of carbon units within white muscle impossible. Recovery of [lactate] to resting l e v e l s was r e l a t i v e l y slow (approximately 2 umoles/g/h) and not s t a t i s t i c a l l y s i g n i f i c a n t u n t i l 8 hours when i t had declined by 50%. By 24h [lactate] had returned to resting l e v e l s . There was no appreciable gylcogen replenishment during the f i r s t two hours of recovery, but by 4h [glycogen] was not s t a t i s t i c a l l y s i g n i f i c a n t l y d i f f e r e n t from either [glycogen] at rest or at exhaustion. At 24h [glycogen] was s t a t i s t i c a l l y s i g n i f i c a n t l y d i f f e r e n t from the value at exhaustion. Because of the large v a r i a t i o n i n [glycogen] from f i s h to f i s h i t i s d i f f i c u l t to p r e c i s e l y describe the time course of i t s recovery. Total tissue glucose increased nearly f i v e f o l d at exhaustion and, i n d i r e c t contrast to both [lactate] and [glycogen], remained elevated throughout the recovery period. Pyruvate concentration increased at exhaustion to more than twice the resting value and remained elevated over the f i r s t two hours of recovery af t e r which point i t rapidly decreased, recovering e n t i r e l y by 8h post exercise. Lactate/pyruvate r a t i o s and redox The lactate/pyruvate r a t i o increased at exhaustion and over 8h of recovery such that by 4h post exercise i t was s i g n i f i c a n t l y d i f f e r e n t from the resting value (table 7). But by 24h i t was f u l l y recovered. The variation between f i s h i n calculated NADVNADH r a t i o (redox) was so large that there was no s i g n i f i c a n t change from the resting values at exhaustion or during recovery. Plasma Lactate and Glucose Plasma [lactate] increased by nearly 20-fold at exhaustion (table 8) and remained elevated through 8h of recovery but by 24h post exercise i t was not s i g n i f i c a n t l y d i f f e r e n t from the resting value. Plasma [glucose] increased s i g n f i c a n t l y late i n recovery and was s t i l l elevated at 24h post exercise (table 8). 46 Table 6 : Concentration of lactate, pyruvate, glycogen and glucose in rainbow trout white muscle prior to and during the recovery from exhaustive exercise, (mean 1 SEM) . (n) [Lactate] [pyruvate] [glycogen] [glucose] rest (5) 3.92±0.94 0.11310.060 33.9013.63 0.5410.22 exh. (7) 41.7212.63" 0.28910.044" 10.4815.47* 2.48+0.48 2h (5) 36.65±4.62" 0.30510.062' 9.4313.52* 2.2610.34 4h (5) 34.91±1.94" 0.16610.047 15.9014.79 2.3910.18 8h (4) 20.6512.81" 0.06810.020 19.0613.84 2.9610.36 24h (6) 4.85+1.29 0.06910.022 25.8114.08 3.9810.95 UNITS: umoles/g wet weight, except glycogen: umoles glucosyl units/g wet weight. * significantly different from corresponding resting value at p<0.05 ** significantly different from corresponding resting value at p<0.001 Table 7 (n) rest (6) exh. (7) 2h 4h 8h 24h (5) (5) (4) (5) Lactate/pyruvate r a t i o and c y t o s o l i c redox p r i o r to and during the recovery from exhaustive exercise i n rainbow trout white muscle (mean ± SEM). [la c t a t e ] / [pyruvate] 55.06±15.72 159.55±19.53 227.421135.84 360.76±161.83* 359.06±86.55* 104.75144.56 redox 1361.901435.55 1404.411144.06 1772.591474.99 1171.581332.60 336.29134.97 845.461173.91 * s i g n i f i c a n t l y d i f f e r e n t from corresponding resting value at p<0.05 redox = NADVNADH ([pyruvate] / [lactate]) x ([H+]/Keq) Table 8 : Plasma lac ta te and glucose concentrations (mM) during recovery from exhaustive exercise i n rainbow trout (0. mvkiss) . Mean ± SEM. time glucose lac ta te n rest 1 0 . 4 9 ± 2 .22 0 . 6 9 ± 0 . 2 7 5 exh. 14 . 0 3 ± 0 .69 1 3 . 0 5 ± 2 . 6 4 7 2h 1 3 . 8 5 ± 1 .43 1 2 . 0 4 ± 1 . 4 3 5 4h 18.2914 .11 1 7 . 1 2 ± 2 . 8 5 5 8h 1 9 . 4 8 ± 5 .81 1 2 . 2 2 ± 8 . 5 8 4 24h 1 9 . 4 7 ± 2 .21 1 . 1 1 ± 0 . 3 8 6 49 Tissue Water Changes i n tissue water and f l u i d d i s t r i b u t i o n are shown in table 9. No changes i n any of these parameters r e l a t i v e to the resting state were observed at any point during recovery. I n t r a c e l l u l a r Free Magnesium Total magnesium at a variey of [Mg 2 +] f from 0 to 20 mM i s shown i n figures 5 and 6, calculated with the appropriate [ATP], PCr, and pH for rest and exhaustion. These two points would be expected to represent the extremes of [Mg 2 +] f since ATP i s the major magnesium chelator and the greatest change i n [ATP] occurs between rest and exhaustion. From t h i s figure i t i s clear that at rest [Mg2+] i s approximately lOmM while at exhaustion i t could be as high as 15mM. Therefore a l l model calculations w i l l be performed with [Mg2+]f=10mM and a variety of [Mg 2 +] f from 1 to 20mM w i l l be calculated to assess the s e n s i t i v i t y of these calculations to changes i n free magnesium. Model Calculations In general the effects of changing [Mg2+] were small. The eff e c t s of changing [Mg 2 +] f on Energy charge free AMP and free ADP are shown in tables 11, 12 and 13. A l l other parameters were affected by less than 1% by changes i n [Mg 2 +] f over the range 5 -20 mM (data not shown). Changes in the calculated parameters with exercise and Table 9: Total tissue water content and f l u i d d i s t r i b u t i o n i n rainbow trout white muscle p r i o r to and during recovery from exhaustive exercise. mL/kg wet weight (mean ± SEM). Total tissue ECFV ICFV water rest 0.767±.007 0.046±.008 0.720±.007 exhaustion 0.768±.006 0.034±.002 0.7281.004 2h 0.763±.014 0.046±.008 0.722±.014 4h 0.7771.005 0.0511.004 0.730±.010 8h 0.7841.005 0.0751.016 0.7001.012 24h 0.7791.006 0.0771.015 0.7031.012 no s i g n i f i c a n t differences were detected. 51 f igure 5: Calculated t o t a l magnesium concentration at rest and at exhaustion for a v a r i e t y free magnesium concentrations from 0 to 30 mM based on an estimate of magnesium b ind ing . Total t i s sue magnesium for rainbow trout white muscle from Parkhouse et al., 1987. 52 free m a g n e s i u m (mM) 53 recovery are shown in tables 10 - 13. As was the case with [ATP] , F a declined to about 20% of the resting value at exhaustion and slowly recovered so that by 24h post exercise F a was not s i g n i f i c a n t l y d i f f e r e n t from the re s t i n g value. In contrast, RATP decreased only s l i g h t l y at exhaustion and by 2h post exercise was elevated over the resting value (table 10). As expected, changes i n F c p a r a l l e l e d the changes in [PCr], declining to approximately 16% of the resting value at exhaustion. By 2h post exercise F c had f u l l y recovered. Potential energy (F P e ) declined to approximately 20% of the resting value at exhaustion but by 2h post exercise i t had recovered to nearly 80% of the value at rest (table 10). This recovery was followed by a long plateau phase, as was the case with F a. By 24h post exercise F P e was not di f f e r e n t from the resting value. [ADP]f did not change s i g n i f i c a n t l y with exercise, but by 2h of recovery i t had declined by an order of magnitude (table 11). [ADP]f gradually increased over the remainder of the recovery period so that by 24h post exercise i t was not s i g n i f i c a n t l y d i f f e r e n t from the resting value. Free ADP calculated without reference to ion binding was similar i n magnitude to free ADP calculated assuming [Mg 2 +] f = lOmM. The discrepancy between the values was greatest during the recovery period. [AMP]f increased s i g n i f i c a n t l y at exhaustion but by 2h post exercise was almost two orders of magnitude less than the 54 resting value (table 12). As was the case with [ADP]f, [AMP]f gradually increased -over the course of the recovery period but by 24h post exercise was s t i l l less than half of the resting value. Since i t i s d i f f i c u l t to accurately estimate the i n t r a c e l l u l a r [Mg 2 +] f the actual changes i n free AMP are d i f f i c u l t to assess since [AMP]f i s greatly affected by changes in [Mg 2 +] f (table 12) . Energy charge (EC) estimated using the model calculations decreased s l i g h t l y but s i g n i f i c a n t l y at exhaustion but had recovered by 2h post exercise. In contrast, EC calculated without taking into account that large fractions of the ADP and AMP pool are bound i n vivo decreased s i g n i f i c a n t l y at exhaustion and remained depressed u n t i l 24h post exercise (see table 13). 55 Table 10 : Magnesium bound ATP, bound ATP as a f r a c t i o n of the t o t a l c r e a t i n e (Fa) and as a f r a c t i o n of the t o t a l adenylates (Ratp), creatine charge (Fc) and normalized p o t e n t i a l energy pool c a l c u l a t e d according to Connett (1988) at [Mg2*] =0.10mM. (umoles/g wet weight, mean ± SEM) time MgATP Fa Ratp Fc Fpe re s t 7.50±.49 .1984±.0145 .9972 + .0006 .4926±.0525 .8893±.0562 exh 1.81±.19 .0418±.0048 .9872±.0039 .0776±.0194 .1610±.0181 2h 3.71 + .55 .0907+.0124 .9995±.0001 .5114±.0547 .6929+.0606 4h 3.55±.52 .0949±.0140 .9992±.0003 .4324±.0630 .6222±.0597 8h 3.42±.56 .0980+.0179 . 9989+. 0003 .4966±.0950 . 6927±.108O 24h 7.00±.61 .1855±.0128 .99841.0003 .5881+.0452 .9591±.0340 n = 5 r e s t ; 7 exh.; 5 2h; 5 4h; 4 8h; 6 24h. 56 Table 11:Free ADP c o n c e n t r a t i o n c a l c u l a t e d without r e f e r e n c e t o i o n b i n d i n g or us ing the model (Connett 1988) equa t ions and s e v e r a l d i f f e r e n t c o n c e n t r a t i o n s o f free magnesium. Mean t S E M . t ime r e s t e x h . 2h 4h 8h 24b Tree ADP c a l c u l a t e d without r e f e r e n c e t o i o n b i n d i n g Keq - 4 .8x10' 0.02681.0054 0.02651.0078 0.00281.0008 0.003B1.0015 0.00601.0024 0.01551.0034 Free ADP c a l c u l a t e d a c c o r d i n g t o Connett (1988) 0.01491.0031 0.01171.0034 O .OOl l t . 0003 0.00161.0006 0.00291.0003 0.00851.0019 f r e e Magnesium (mM) 5 0.02091.0043 0.01871.0055 0.00181.0004 0.00261.0010 0.00421.0019 0.01201.0026 10 0.02061.0038 0.01901.0056 0.00191.0005 0.00271.0011 0.00421.0019 0.01191.0026 20 0.01831.0037 0.01731.0051 0.00171.0004 0.00251.0010 0.00381.0017 0.01061.0025 n at r e s t - 5 ; e x h . - 7 ; 2h -5 ; 4h -5 ; Bh-4; 24h-6. 57 Table 12 : Free AMP concentration c a l c u l a t e d at d i f f e r i n g free magnesium concentrations, (umoles/g wet weight; mean ± SEM) free [AMP] time magnesium concentration lOmM 15mM 20mM res t 0.0016±.0005 0.0016±.O005 exh. 0.00571.0024 0.0058±.0024 0.0033±.0014 2h 0.00003+.00001 0.00002±. 00001 '. 0.00004±. 00003 4h 0.00007±.00005 0.00007+.00005 0.00008±.00006 24h 0.00055±.0002 0.00055±.0005 n = 5 r e s t ; 7 exh.; 5 2h; 5 4h; 4 8h; 6 24h. 58 Table 13 : Energy charge c a l c u l a t e d using the t o t a l concentrations of ADP and AMP and according to the model d e f i n i t i o n s (Connett, 1988) at [Mg 2*]free = lOmM and 15mM. (mean ± SEM) time rest exh. 2h 4h 8h 24h Energy Charge ( t o t a l ADP and AMP) 0.9455+.0044 0.8434±.0116 0.9124±.0124 0.9138±.O080 0.9190±.0083 0.9474±.0016 lOmM 0.9998±.0001 0. 9969±.001'4 0.9999±.0000 0.9999±.0000 0.9999±.0000 0.9999±.0000 Energy Charge Magnesium Concentration 15mM 0.9998±.0001 0.9969+.0014 0.9999±.0000 l.OOOOt.OOOO 0.9999±.0000 0.99991.0000 n = rest 5; exh. 7; 2h 5; 4h 5; 8h 4; 24h 6. 59 DISCUSSION The Resting Fish The d i f f i c u l t y i n obtaining representative samples from resting f i s h i s shown by the large v a r i a t i o n i n the reported l i t e r a t u r e values for a variety of metabolites. ATP and, i n p a r t i c u l a r , phosphocreatine are well known to be very l a b i l e . Dobson and Hochachka (1987) showed that PCr could decrease as much as 70% aft e r as few as 3 t a i l flaps during sampling. Similarly, ATP was shown to decrease by nearly 30% . The sampling protocol used i n the present experiment largely avoided these problems so that [ATP], [PCr] and glycogen were as high or higher than previous reports. Literature values for resting [PCr] range from 13.05 umoles/g tissue (Mommsen and Hochachcka, 1988) to approximately 25.6 umoles/g tissue (Milligan and Wood, 1986) . However, caution must be exercised in comparing these results because t o t a l creatine concentration may vary between stocks of trout or with body s i z e . [PCr] i t s e l f may not be e a s i l y compared, but the use of the normalized parameter the creatine charge, [PCr]/[PCr]+[Cr], avoids t h i s problem. For example, Dobson et a l . (1987) report a resting [PCr] of 27.03 umoles/g tissue, which i s higher than that measured i n t h i s study (see table II), but [Cr], at 31.89 umoles/g, was also elevated. So creatine 60 charge was 0.459 versus 0.539 in the present study. Connett (1988) reports creatine charges ranging from 0.35 to 0.84 in a variety of sk e l e t a l muscles of a number of mammals from rats to humans. Unfortunately very few studies of exercise i n f i s h have reported [Cr] so i n many cases t h i s comparison cannot be made. Even i n the present study i t seems l i k e l y that a certain amount of PCr hydrolysis occurred during freezing. However, i t i s impossible to estimate the extent of t h i s hydrolysis or to "back calculate" to the true resting animal. Inorganic phosphate concentration i s p a r t i c u l a r l y high in the resting trout r e l a t i v e to measurements made for muscles of other animals however, i t i s very similar to previous estimates of [PA] i n resting f i s h (for example, 25.72+1.93 umoles/g tissue, Dobson et a l . (1987)) . This method has been validated for trout white muscle by Dobson (1987) and Parkhouse (1986) using 3 1P NMR and i t appears to y i e l d accurate estimates of the P± present. The high P, observed i s l i k e l y an a r t i f a c t of the sampling or extraction process because in. vivo 31P NMR of white muscle i n both the resting carp and goldfish has shown P± to be almost undetectable (Van den T h i l l a r t et a l . , 1989). This suggests that the true resting P± i s somewhere around 1 umole/g ti s s u e . The question then i s : what i s the source of the phosphate measured i n the tissue extract? The f i r s t and most l i k e l y source of phosphate i s the hydrolysis of PCr. However, the measured resting [PJ i s nearly 1.5 times the resting [Cr] (see table 5). So, while PCr 61 hydrolysis during freezing may be a major source of the phosphate t h i s cannot be the only phosphate release occurring. Hydrolysis of ATP i s probably not a major contributor to the high resting phosphate since even i f a l l of the ADP, AMP and IMP measured was i n fact the result of ATP hydrolysis during sampling the most phosphate that could possibly be accounted for i s roughly 2 umoles/g t i s s u e . One large possible source of free phosphate i s c e l l u l a r proteins. In contrast to phosphate release by ATP or PCr hydrolysis, t h i s release from proteins would l i k e l y be a result of the extraction protocol rather than sampling time. The perchloric acid extraction method used to p r e c i p i t a t e the proteins i n the sample may be s u f f i c i e n t l y harsh to s t r i p phosphate groups from a wide variety of proteins. Since many c e l l u l a r proteins are known to be phosphorlyated t h i s i s a p o t e n t i a l l y large source of free phosphate. Even though the measured [PJ i s l i k e l y to be f a l s e l y elevated the changes i n [Pi] from rest with exercise and recovery should s t i l l be v a l i d since a l l the samples were handled i n the same way. As was the case with [PCr], there are large variations i n resting [ATP] reported in the l i t e r a t u r e ranging from about 4 umoles/g tissue (Milligan and Wood, 1986) to 7.33 umoles/g tissue (Dobson e_t a_l., 1987) . Again, these variations may be attributable to either variations in the t o t a l adenine nucleotides (TAN= ATP+ADP+AMP+IMP) or to differences i n sampling technique. Since TAN i s seldom measured distinguishing between these alternatives i s often impossible. TAN seems to be closely 62 regulated within a stock of f i s h since, i n the present study the c o e f f i c i e n t of variation for resting TAN was less than 7%. But inte r stock variations can be large. For example, i n a single study by Dobson et al_. (1987) one stock of f i s h had a resting TAN =4.99 umoles/g tissue while another had TAN =8.44 umole/g ti s s u e . This difference may be due to size since the f i r s t stock of f i s h were small, 60-70g, and the second much larger, 200-250g. However, t h i s i s not a consistent trend since Mommsen and Hochachka (1988) report a resting TAN=8.09 umole/g tissue for a stock of small f i s h , 30-55g. These differences serve to underline the importance of measuring factors l i k e t o t a l creatine and TAN since concentrations of ATP or PCr alone may be misleading. Reported resting [lactate] i s p a r t i c u l a r l y variable ranging as high as approximately 12 umoles/g tissue (Milligan and Wood, 1986) . Pearson et. al.. (1990) have reported the lowest resting [lactate] to date, approximately 1.5 umole/g tissue, by using o r a l l y administered diazepam. Sodium pentobarbitol has been previously observed to cause a g l y c o l y t i c activation and increase [lactate] in rat l i v e r (Seitz et. al.., 1973). So i t i s possible that the observed resting [lactate], 3.92 umoles/g tissue i s s l i g h t l y elevated over the true resting value. The large variations i n reported resting glycogen, as low as 8.41 umoles/g tissue (Mommsen and Hochachka, 1988), may be a result of differences i n feeding history rather than sampling protocol. In preliminary experiments resting white muscle [glycogen] was measured on a di f f e r e n t batch of the same stock of f i s h used i n t h i s study, but which had not been feeding properly for several months. The mean resting glycogen for these f i s h was 15.06 +1.45 umole glucosyl units"/g tissue (n=3) , s i g n i f i c a n t l y lower (p=0.05) than the resting glycogen from f i s h of the same stock which had been feeding (see table 6). The f i s h used i n t h i s experiment have the highest resting glycogen yet reported for rainbow trout. It i s d i f f i c u l t to say what set of metabolite concentrations are t r u l y representative of a "resting" trout since d i f f e r e n t stocks may i n fact show considerable v a r i a t i o n . However, i t i s clear that the f i s h i n the present study are as close, or closer, to a "resting" state than any that have been studied previously. However, 31P NMR studies of the carp and gold f i s h suggest that some changes, p a r t i c u l a r l y i n [PCr], Px and pH may have occurred. Using 31P NMR, Van den T h i l l a r t et a l . (198 9) showed that these parameters are remarkably sensitive i n f i s h muscle. Even something so routine as MS-222 anaesthetia can cause a depression in PCr and a concommitant increase in phosphate and decrease i n pH± (to below 7.0 in some cases) . These changes were not reversed as long as the animal was kept anaesthetized. It would be useful to perform a similar experiment on rainbow trout i n order to compare metabolite concentrations obtained from freeze clamped tissues with those co l l e c t e d yin vivo' with 31P NMR. 64 ATP overshoot In contrast to previous studies of recovery following exhaustive exercise no overshoot i n [ATP] was observed during recovery. The reason for t h i s discrepancy most l i k e l y l i e s i n the values for the resting f i s h . Resting [ATP] i n t h i s study was nearly double that reported by both Dobson and Hochachka (1987) and M i l l i g a n and Wood (1986) . In addition, i t was not s i g n i f i c a n t l y d i f f e r e n t (p=0.05) from the highest [ATP] observed during the "overshoot" i n either of the previous studies (figure 4) . This strongly suggests that the resting [ATP] may be depressed. Since IMP, and hence TAN, was not measured i n either of these studies, one cannot eliminate the p o s s i b i l i t y that the low resting [ATP] i s simply a result of low TAN rather than the result of stress related hydrolysis of ATP with a concommitant increase i n IMP; however, i f t h i s i s the case then the high [ATP] observed during the overshoot must be a result of de novo synthesis or salvage. This i s t h e o r e t i c a l l y possible, but unlikely because of the high energy cost. Synthesis of ATP from IMP v i a the purine nucleotide cycle requires 1 GTP while the formation of ATP de novo requires 5 ATP and 1 GTP. ATP can be formed from adenosine and phosphoribosyl pyrophosphate i n a reaction that' i s termed the salvage pathway of adenylate synthesis which i s a much less costly a l t e r n a t i v e . However, PRPP i s present i n muscle c e l l s at very low concentrations (between 1 and 10 nmoles/g tissue, Hisata, 1975; Ipata et a l . , 1987) and the formation of PRPP requires 2 ATP. The alternative 65 hypothesis, that measured resting [ATP] was depressed due to some sampling a r t i f a c t has already been shown to occur i n f i s h muscle (see above) and i s thus far more l i k e l y to be the explanation for the observed "overshoot". In t h i s case i t i s merely necessary to explain why stress related hydrolysis of ATP i s less l i k e l y in samples from a trout at 12 or 24h after exhaustive exercise than at "rest". Even 24h after exhaustive exercise the f i s h in the present study were markedly less responsive to stimulus than the "resting" f i s h . It was possible to handle the post exercise f i s h without any struggling while in the case of the resting f i s h even the sl i g h t e s t disturbance could cause agitation. In t h i s case i t seems l i k e l y that the high [ATP] reported by both Dobson and Hochachka (1987) and M i l l i g a n and Wood (1986) during recovery r e l a t i v e to t h e i r respective resting values are the result of the refractory condition of the f i s h during recovery combined with the great d i f f i c u l t y i n obtaining samples from resting f i s h . In Dobson and Hochachka (1987) resting creatine charge was low at only 0.388 as opposed to 0.539 in the present study. This suggests that both the low [ATP] and [PCr] may be a result of a certain amount of struggling and stress related hydrolysis during sampling. This i s an inte r e s t i n g observation since one might expect PCr hydrolysis to be more extensive before [ATP] began to decrease. The a c t i v i t y of AMP deaminase i n t h i s tissue i s high so i t may be possible to see a decline i n [ATP] in p a r a l l e l with the decline i n [PCr] . In fact, t h i s phenomenon was 66 observed but not discussed by Dobson and Hochachka (1987) . The data of M i l l i g a n and Wood (1986) are more d i f f i c u l t to explain since resting [PCr] i s r e l a t i v e l y high at approximately 25 umoles/g wet weight. The f i r s t p o s s i b i l i t y i s that t o t a l creatine was unusually high i n these f i s h , perhaps as a result of d i e t . This would cause a si t u a t i o n i n which although [PCr] was high at rest creatine charge would be low. However, since t o t a l creatine was not measured t h i s hypothesis can neither be accepted nor rejected. F a i l i n g t h i s i s should be noted that a sit u a t i o n i n which [ATP] i s low and [PCr] (or creatine charge) i s high i s not phy s i o l o g i c a l l y impossible. In fact, t h i s i s preci s e l y the sit u a t i o n of the f i s h in the present experiment at 2, 4 and even 8h post exercise. It may be that the "resting" f i s h used by M i l l i g a n and Wood (1986) were i n a sim i l a r physiological state. This would also help to explain the unusually low glycogen and high lactate observed i n the resting f i s h i n t h e i r study. ATP depletion and Purine Nucleotide Cycling As has been shown i n previous studies (Mommsen and Hochachka, 1988; Dobson et al_., 1987; Dredzic and Hochachka, 1976) when ATP i n f i s h white muscle i s depleted with exercise there i s a stoichiometric accumulation of IMP (see table 4) . This stoichiometry i s maintained throughout the recovery period which suggests that the AMP 5' nucleotidase route of adenylate depletion (to adenosine, and hence inosine and hypoxanthine) i s 67 not of importance in trout white muscle. In addition, Mommsen and Hochachka (1988) have suggested that the two arms of the purine nucleotide cycle are temporally separated so that AMP deamination occurs only during exercise and IMP reamination occurs only during recovery. The results of t h i s study support t h i s conclusion. Large decreases i n [ATP] are often considered to be deleterious. In mammalian systems f a l l i n g [ATP] i s thought to be a sign of impending c e l l death (Nayler, 1983) . However, there may be a number of benefits to purine nucleotide cycl i n g with exercise which may outweigh possible disadvantages caused by large changes i n [ATP]. For example, i t has been suggested that AMP removal from the tissue pool could serve to draw the adenylate kinase reaction i n the ATP producing di r e c t i o n , and second, that the accompanying increase i n ammonium ions could regulate g l y c o l y t i c flux at the l e v e l of PFK (see Van Waarde, 1988 for review). Mommsen and Hochachka have suggested that the ammmonia formed by AMP deamination may contribute to H+ ion buffering. This could be important under conditions l i k e anaerobic exercise i n which there i s a large production of protons. One important function of the purine nucleotide cycle may be the conservation of adenylate backbones. Unlike the purine bases, IMP i s not permeable to the c e l l membrane thus preventing loss of adenlyate backbones to the blood and reducing the cost of adenylate repletion (see previous section). Another function of the purine nucleotide cycle may be to 68 maintain adenylate energy charge (Flanagen e_t a l . , 1986) . Energy charge i s only s l i g h t l y perturbed during exercise and recovery i n spite of the very low [ATP] (see table 11). It i s probably more accurate to think of parameters l i k e RATP being important in maintaining c e l l v i a b i l i l t y than [ATP] per se. In mammalian red muscle tissues which have r e l a t i v e l y low a c t i v i t i e s of purine nucleotide cycle enzymes decreases i n [ATP] result i n increases of [ADP] and [AMP] and a resultant decrease in RATP. While i n white type muscles the action of AMP deaminase prevents the accumulation of AMP and ADP and causes RAXP (and energy charge) to remain high. Thus [ATP] may not be important i n the maintainance of v i a b i l i t y in t h i s type of c e l l . AMP deamination to IMP i s c l e a r l y a rapid process, able to keep pace with the demands of exercise. However, IMP reamination i s protracted, requiring up to 24h to complete. The reason for t h i s sluggishness may be related to the a c t i v i t y of adenylosuccinate synthetase and adenylosuccinate lyase, although these enzymes have never been measured i n f i s h muscle. Alt e r n a t i v e l y , the slow recovery may be a result of the limited a v a i l a b i l i t y of both GTP and aspartate. Aspartate concentrations in f i s h muscle following exhaustive exercise are low (0.055 + 0.008 umoles/g tissue, Mommsen and Hochachka, 1988), far below the Km of adenlyosuccinate synthetase, 0.4mM. [GTP] has seldom been measured i n f i s h but has been reported to be very low in the muscle of fatigued f i s h (Dredzic and Hochachcka, 1976). Differences i n , for example, aspartate a v a i l a b i l i t y could help 69 to explain some puzzling differences i n the time course of [ATP] recovery i n trout between studies. The f i s h of M i l l i g a n and Wood (1986), Mommsen and Hochachka (1988), and the present study a l l require around 24h of recovery before [ATP] returns to the resti n g l e v e l . However, Dobson and Hochachka (1987), using f i s h of similar size to those used by Mommsen and Hochachka (1988) showed f u l l recovery of [ATP] within 2 hours (figure 4). It i s possible that t h i s discrepancy may be due simply to the speed of purine nucleotide c y c l i n g (ie: the a v a i l b i l i t y of aspartate or GTP) . This p o s s i b l i t y requires further investigation. It i s often stated that mammalian skeletal muscle [ATP] never f a l l s below about 75% of resting values even i n the advanced stages of muscle fatigue (see for example, Wilkie, 1981) . However, t h i s i s probably an over generalization even for mammalian skel e t a l muscle and i s c l e a r l y untrue for trout white muscle since [ATP] decreases to less than 25% of the resting value at exhaustion (see table I ) . The situ a t i o n may be sim i l a r i n the white skeletal muscle of mammals as well. Meyer and Terjung (1980) report a greater than 50% decrease i n [ATP] with intense stimulation In s i t u of the s u p e r f i c i a l white section of the gastrocnemius i n the rat . In red type muscles ATP content i s maintained near normal even during intense stimulation which produces rapid fatigue. The response of [ATP] to exercise may represent a basic difference between red and white type skeletal muscles. It i s possible that reductions i n [ATP] are seldom observed i n s k e l e t a l muscle i n mammals because of the d i f f i c u l t y 70 i n obtaining an unmixed muscle sample as a res u l t of the highly heterogenous nature of most mammalian muscles. This has not been a problem with studies on trout because of the clear separation of f i b r e types. Phosphocreatine and Energy Metabolism The quick recovery in phosphocreatine when [ATP] i s s t i l l s u bstantially below the resting l e v e l i s one-of the fundamental observations of t h i s study. This phenomenon has never been previously observed and, in fact, Dobson and Hochachka (1987) stated that "ATP replenishment must be completed before PCr concentrations can be returned to pre-exercise values" . This i s c l e a r l y not the case, at least with respect to the present study. Although [ATP] recovers very slowly, by 2h post exercise energy charge has largely recovered even when calculated on the basis of t o t a l metabolite pools (see table 13) . This was not the case with the results of Dobson and Hochachka (1987) where energy charge (calculated on the basis of t o t a l metabolite pools) did not recover u n t i l [ATP] recovery was complete. As discussed in the introduction, a number of d i f f e r e n t models of energy metabolism suggest that [PCr] (or creatine charge) i s related to energy charge and RATP (the proportion of the adenylates phosphorylated to ATP). In tissues in which the t o t a l adenylate pool (ATP+ADP+AMP) remains more or less constant (ie: l i t t l e IMP or adenosine i s formed) energy charge and [ATP] track each other. However, in tissues i n which the size of the 71 adenylate pool changes, such as f i s h white muscle, energy charge and [ATP] can be dissociated as i n the present study. It i s therefore more accurate to state that [PCr] cannot return to resting levels u n t i l RATP or EC have returned to normal. It cannot be overemphasized that the usefulness of [ATP] as an index of metabolic state i s probably limited, e s p e c i a l l y in tissues l i k e fast twitch s k e l e t a l muscle i n which the size of the adenylate pool varies so greatly. The reason why energy charge and hence phosphocreatine recovers so quickly in these f i s h r e l a t i v e to the f i s h used i n other experiments i s more d i f f i c u l t to assess. F i r s t i t should be noted that i t i s impossible to calculate the true EC using the free concentrations of ADP and AMP because no previous study of recovery from exhaustive i n f i s h has measured the components necessary to calculate the free ADP or AMP. However, even EC calculated using the t o t a l concentrations recovers much more quickly i n t h i s study than i n previous experiments. The reason for t h i s discrepancy may have to do with the high resting glycogen of t h i s stock of f i s h . In Mommsen and Hochachka (1988) and Dobson and Hochachka (1987) energy charge at exhaustion was 0.398 and 0.511 respectively (based on the t o t a l concentrations of ADP and AMP), much lower than in the present study (see table 13). In both other experiments the exhausted f i s h had substantially less than 1 umole glucosyl units/g wet weight i n the muscle and i t i s suggested that exhaustion was a result of fuel depletion. However, in the present study glycogen levels at exhaustion were, i n fact, higher than the resting glycogens of both Dobson and Hochachka (1987) and Mommsen and Hochachka (1988) . This suggests that either the f i s h i n the present study were not f u l l y exhausted or that factors other than fuel depletion were the cause of the exhaustion. There i s a large body of evidence from mamalian studies which suggests that the b u i l d up of metabolic end products (lactate, Pi and pH in particular) may be causal factors i n muscle fatigue. For example, M i l l e r e_t a_l. (1988) showed that Pi and [H+] are associated with muscle fatigue i n vivo i n human beings. In an elegant series of experiments on skinned muscle fi b r e s i t has been shown that the application of phosphate can prevent muscle contraction and cause fatigue (see Cooke and Pate 1990 for review). It i s possible that the f i s h i n the present study became "exhausted" and could no longer swim because of end product accumulation rather than lack of f u e l . In t h i s case i t would not be surprising to see less change in energy charge. And t h i s difference i n energy depletion could account for the d i f f e r e n t rate of recovery in energy charge and hence [PCr]. Carbohydrate Metabolism Two main questions dominate current interest in carbohydrate metabolism during recovery from severe exercise i n f i s h white muscle: what i s the fate of lactate? and, what i s the source of the glycogen resynthesized? In p r i n c i p l e there are a number of possible fates of lactate: 1) e f f l u x from the tissue 73 (followed either by oxidation or by recoversion to glucose and/or glycogen in the l i v e r ) , 2) conversion to pyruvate and oxidation i n s i t u , 3) conversion to pyruvate and then alanine (which i s then metabolised along with the amino acid pool), and 4) reconversion to glycogen i n s i t u . As can be seen from the above, the possible sources of glycogen are intimately involved with the possible fates of lactate. F i r s t , glycogen could be formed from lactate in s i t u . Second, glycogen could be formed via Cori c y c l i n g . In the Cori cycle, lactate formed in the muscle i s car r i e d i n the blood to the l i v e r where i t i s reconverted to glucose. This glucose i s then car r i e d back to the muscle where i t can be converted to glycogen. Cori c y c l i n g has been thought to be necessary to muscle because of the apparent absence of some of the enzymes needed to convert pyruvate to glycogen i n that t i s s u e . However, there i s also a great deal of evidence to suggest that Cori cy c l i n g i s not necessary, at least for some sk e l e t a l muscles. Opie and Newsholme (1967) and Crabtree e_t a l . (1972) demonstrated the presence of both malic enzyme and PEPCK i n rodent and frog skeletal muscles. McLane and Holloszy (1979) demonstrated rapid u t i l i z a t i o n of lactate to form glycogen in both fast twitch red and fast twitch white muscle i n the perfused hind limb of the rat. Slow oxidative f i b r e s , on the other hand, did not u t i l i z e lactate to form glycogen. M i l l i g a n and McDonald (1988) have suggested that Cori cyc l i n g plays only a small role i n glycogen resynthesis i n coho salmon white muscle following exhaustive exercise since greater 74 Figure 6: 3 carbon units ([lactate] +2[glycogen]) in rainbow trout white muscle following exhaustive exercise (umoles/g wet weight). 20 rest exh 2h 4h 8h 24h r e c o v e r y t ime 76 than 80% of the t o t a l blood a c t i v i t y after the i n j e c t i o n of [ 1 4C]-lactate was recovered as lactate. In a p a r a l l e l study we have confirmed t h i s suggestion by showing that glucose uptake into trout white muscle can account for no more than 10% of the glycogen repletion observed following exhaustive exercise (West et a l . i n preparation). On t h i s basis I would suggest that the majority of the lactate formed during exhaustive exercise (see table 6) i s retained i n the tissue and reconverted to glycogen. However, examination of figure 7 suggests that there i s a small, non s i g n i f i c a n t decrease i n the t o t a l 3 carbon units (glycogen +2 lactate) from rest to the f u l l y recovered (24h) f i s h . This d e f i c i t combined with the p o s s i b i l i t y that up to 10% of the glycogen reformed could have come from glucose (see above) suggests that there has been at least some loss of lactate from the t i s s u e . The two most l i k e l y fates of t h i s lactate are e f f l u x to the blood or oxidation i n s i t u . It i s impossible to d i s t i n g u i s h between these options on the basis of the data from t h i s study. However, in a series of p a r a l l e l experiments we have found white muscle mitochondria to d i f f e r substantially from red muscle mitochondria with respect to pyruvate oxidation (Moyes et a l . i n preparation) . For example, during the early phases of recovery there are large increases i n [pyruvate] (see table 7) and white muscle mitochondria have been shown to be extremely sensitive to [pyruvate] over t h i s range. M i l l i g a n and Wood (1986) suggested that during the early phases of recovery in trout the majority of the lactate i s 77 oxidized i n s i t u because during t h i s time there i s no increase i n glycogen and the decrease i n [lactate] which i s seen cannot be accounted for based on the increase i n [lactate] i n the blood. This also appears to be the case with the trout used i n the present study. During the f i r s t two hours of recovery there was no apparent synthesis of glycogen but [lactate] decreased by approximately 5 umoles/g muscle. Roughly 60% of the body mass of a trout i s white muscle (Stevens, 1968) so for a 1kg trout t h i s would be 600g of white muscle and a whole body lactate load of about 3,000 umoles. Trout whole body e x t r a c e l l u l a r f l u i d volume has been measured as about 240 mL/kg (Milligan and Wood, 1986b) . If we assume a l l of the lactate appeared i n the ECF t h i s would y i e l d an increase i n ECF [lactate] i n the hypothetical 1kg trout of roughly 12.5 mM. During t h i s time there was no increase in plasma lactate (table 8). But the blood lactate pool i s not s t a t i c and lactate i s continually lost from the pool, either oxidized or converted to glucose and/or glycogen i n the l i v e r so even though there was no measureable increase i n plasma lactate there was l i k e l y at least some ef f l u x of lactate into the blood. The rate of lactate turnover i s known to be approximately 12 umol/min/kg i n the coho salmon following exhaustive exercise (Milligan and McDonald, 1988) which would correspond to a loss of roughly 6mM lactate from the ECF during the relevant time period (2h). This could account for perhaps half of the decrease i n tissue [lactate] . It i s , however, d i f f i c u l t to make firm conclusions based on calculations of t h i s 78 sort because of the questionable nature of some of the assumptions. For example, i t i s unlikely that a l l of the white muscle i s equally active during exercise or builds up lactate to the same extent. On the other hand i f blood lactate and ECF lactate are not the same then i t might be more appropriate to use blood volume rather than ECFV i n the c a l c u l a t i o n above. If the blood volume of a 1kg trout i s approximately 150mL then the decrease i n tissue [lactate] would be equivalent to an increase in blood [lactate] of about 20mM c l e a r l y much greater than that actually observed. In t h i s case the amount of lactate oxidation r e l a t i v e to the amount of efflux into the blood would greatly increase. While fine scale conclusions about the fate of lactate during the early phases of recovery are impossible, i t i s clear that these data support the contention of M i l l i g a n and Wood (1986a) that the majority of the decrease in white muscle [lactate] i s l i k e l y due to i n s i t u oxidation during the f i r s t two hours of recovery i n which there i s no glycogen synthesis. Because of the f a i r l y large v a r i a t i o n from f i s h to f i s h i n both [glycogen] and [lactate] i t i s d i f f i c u l t to make r e a l l y firm conclusions about the fate of lactate and the o r i g i n of glycogen during recovery. An experimental design i n which s e r i a l samples were taken from the same f i s h would be an i d e a l way to investigate t h i s problem further. I n t r a c e l l u l a r pH The large v a r i a t i o n i n i n t r a c e l l u l a r pH associated with the 79 DMO estimates (see table 3) i s e a s i l y understood since the DMO derived pH i s a calculated parameter involving 7 separate measurements each of which i s subject to a wide variety of non d i r e c t i o n a l errors. In addition, the DMO technique may be influenced by marker e q u i l i b r a t i o n time and extraction method as well as a variety of other factors. The homogenate method on the other hand i s a d i r e c t measurement of pH, although how the measured pH i s related to the true i n t r a c e l l u l a r pH i s a matter open to question. The o r i g i n a l technique (Portner et al.. i n press) includes a complex ca l c u l a t i o n to take into account protons contributed by the e x t r a c e l l u l a r compartment which was neglected here. However, because of the small volume and low buffering capacity of the ECF i n trout white muscle the contribution of t h i s compartment to the measured pH i s negligable, e s s e n t i a l l y outside the l i m i t s of resolution of the pH meter. That anaerobic exercise i s associated with a decrease i n pH i s well known and the magnitude of the pH decrease i n the present study (table 3) i s s i m i l a r to that observed by M i l l i g a n and Wood (1986) following an exhaustive exercise bout i n rainbow trout. But the actual source of t h i s pH decrease i s both complex and controversial. Mommsen and Hochachka (1988) show that the r e l a t i v e contribution of H+ from g l y c o l y s i s and ATP hydrolysis varies with the pH and also with [Mg2+] . The fate of these protons i s equally unclear. Heisler (1984) suggests that the majority of the protons are transferred to the water 80 following severe exercise. In contrast, i t has been found that over 90% of the protons are retained i n the white muscle after exhaustive exercise i n trout (Y. Tang, unpublished). It i s possible that glycogen repletion i s the primary sink for protons during recovery. Immediately following exhaustion there i s a s l i g h t , non s i g n i f i c a n t drop i n the pH. While t h i s may simply be an a r t i f a c t of the small sample size, i t should be noted that during t h i s time the complete recovery of phosphocreatine occurs. In t h i s d i r e c t i o n the CPK reaction produces a proton (at and above pH 7.0 exactly 1 proton i s released per Cr phosphorylated (Lawson and Veech, 1979)). A similar decline i n i n t r a c e l l u l a r pH was observed by Van den T h i l l a r t e_t al_. (1989) during recovery from anoxia i n carp and gold f i s h using 31P-NMR. In t h i s case the pH decline was also coincident with the resynthesis of PCr. Free ADP Recent evidence suggests that free [ADP] in the cytosol ([ADP]f) i s at i n much lower concentrations than those that are measured enzymatically. Estimates of resting [ADP]f generally f a l l i n the range of 1-10 % of the measured ADP (less than 0.01 umol/g (Jacobus et a l . , 1982; Shoubridge et a l . , 1984; C h a l l i s et a l . , 1989) to approximately 0.07 umol/g (Dawson e t a l . , 1977; Wilkie, 1981; Meyer et a l . , 1985)) . In the present study resting free ADP ranges from less than 3% of the measured t o t a l ADP up to about 7% (see tables 4 and 11) depending upon the technique 81 used for the calculation, well within the range of previously computed values. Free ADP when calculated without reference to ion binding i s about 1.5 f o l d greater than free ADP calculated according to the model equations at [Mg 2 +] f = lOmM (see table 11) . Considering the uncertainty of the value of some of the equilibrium constants used t h i s discrepancy i s remarkably small. The Keq used to calculate the [ADP] f without using the model equations was determined at excess [Mg 2 +] f (Eldar and Degani, 1989) . If a Keq determined at ImM [Mg2+] (Lawson and Veech, 1979), corrected to 10°C, i s used then [ADP]f = 0.054 umol/g tissue at rest, very much higher than that derived from the model calcu l a t i o n s . This v a r i a t i o n i n the measured values of the equilibrium constants introduces a degree of uncertainty into the estimates of absolute concentrations of free ADP and AMP. However, the r e l a t i v e changes should be r e l i a b l e . It i s l i k e l y that the calculated "resting" [ADP]f i s f a l s e l y elevated as a result of PCr hydrolysis on freezing. NMR studies on mammalian white s k e l e t a l muscles suggest that roughly 85% of t o t a l creatine content i s present as PCr at rest (Meyer et. al.., 1985, Shoubridge et a l . , 1984). NMR measurements in the carp also show that resting pHA in f i s h white muscle i s approximately 7.4 . If these conditions are assumed to be the case at rest i n the trout then free ADP can be calculated using the model equations. In t h i s case [ADP]f would be about 0.002 umol/g tissue, similar to the value calculated for the 2h f i s h (table 11). So i t i s l i k e l y that the estimated "resting" free 82 ADP i s somewhat elevated r e l a t i v e to the true resting value because of PCr hydrolysis during freezing. This may explain why [ADP]f did not increase s i g n i f i c a n t l y from rest to exhaustion as expected. This also may have been a problem with the samples taken during recovery. For example, considering just the two hour point: i f the measured [PCr] i s "correct" (ie, no PCr hydrolysis occurred during freezing), then [ADP]f i s similar to the estimated "resting" [ADP]f and recovery o f " t h i s parameter i s complete, although ATP/ADP r a t i o would be disturbed. A l t e r n a t i v e l y , i f the measured PCr i s low because of PCr hydrolysis on freezing then the "correct" [ADP]f at t h i s point would be even lower than that reported i n the table. It seems reasonable to assume that similar amounts of ATP hydrolysis occurred i n samples from resting and recovering f i s h since a l l samples were treated i n the same way. If so, the trends observed should be s i m i l a r although the absolute concentrations would be proportionally lower. [ADP]f may have important physiological significance since at these extremely low levels there i s a strong dependence of mitochondrial oxygen uptake on [ADP]. Bishop and Atkinson (1984) showed that at [ADP] equivalent to that calculated for the resting trout white muscle, oxygen uptake was less than 10% of the maximal rate i n v i t r o . S i m i l a r l y at [ATP]/[ADP] ratios greater than 200, as i s the case in the trout at rest, respiratory rate i s less than 10% of the state three rates. This 83 suggests that trout white muscle mitochondria are far from t h e i r potential maximum oxygen uptake at rest. It should be noted that the experiments of Bishop and Atkinson (1984) were performed on mitochondria from rat hearts and as such may d i f f e r from trout white muscle mitochondria; however, no data are available for the behaviour of non mammalian or white muscle mitochondria at such low level s of ADP. More int e r e s t i n g l y , [ADP]f actually declines r e l a t i v e to the resting l e v e l at 2h post exercise (table 11) and as a consequence the [ATP]/[ADP]f r a t i o increases enormously to over 2, 000 i n spite of the decrease i n [ATP]. Under these circumstances the data of Bishop and Atkinson suggest that i f anything the oxygen uptake by the white muscle mitochondria would be less than that seen at rest. During recovery from exhaustive exercise energy demand should be increased over the basal l e v e l , so how i s t h i s demand met? It i s possible that t h i s additional demand may be met g l y c o l y t i c a l l y but t h i s i s unlikely since trout white muscle i s probably a c t i v e l y gluconeogenic during recovery from exhaustive exercise as was discussed e a r l i e r . What i s more l i k e l y i s that factors other than [ADP]f are af f e c t i n g mitochondrial metabolism. There are many possible candidates for factors which can affec t mitochondrial metabolism. For example changes i n redox state are known to be a co n t r o l l e r of mitochondrial metabolism i n mammalian hearts (Koretsky et a l . , 1989). Connett (1987) suggests that mitochondrial and cy t o s o l i c redox are linked. If 84 t h i s i s the case i t seems unlikely that redox plays an important role since c y t o s o l i c redox did not change s i g n i f i c a n t l y over the course of exercise and recovery (table 11) . However, the rela t i o n s h i p between mitochondrial and c y t o s o l i c redox i s s t i l l unclear so i t i s impossible to rule out a role for mitochondrial redox. Changes i n pH are well known to affect maximal oxygen uptake i n i s o l a t e d mitochondria. In i s o l a t e d red muscle mitochondria from the carp a decrease i n pH from 7.3 to 6.5 stimulated state 3 oxygen uptake by nearly two f o l d (Moyes et a l . , 1988) . Increases i n phosphate concentration are also known to stimulate oxygen uptake by i s o l a t e d mitochondria (Bishop and Atkinson, 1984) . Although there was no increase i n t o t a l phosphate from rest to 2h post exercise (table 5) free phosphate i s very l i k e l y to have increased. [ATP] declined by roughly 4mM from rest to 2h post exercise, associated with an equivalent increase in IMP. This would i n p r i n c i p l e release 2 moles of phosphate per mole of ATP, or an increase of up to 8 mM phosphate from rest to 2h post exercise. If, as discussed previously, resting free [phosphate] i s less than ImM t h i s i s a p o t e n t i a l l y large signal to increase oxygen uptake over basal l e v e l s . The question then i s why maintain [ATP]/[ADP] rat i o s at such high levels during recovery. One p o s s i b l i t y relates to the fact that the tissue i s gluconeogenic at that time. High [ATP]/[ADP] rat i o s could be required to maintain flux through the pyruvate kinase back reaction. Although reversible, pyruvate 85 kinase normally catalyzes the reation i n the g l y c o l y t i c d i r e c t i o n ; reversal requires high [ATP] and [pyruvate] and very low [ADP] and [PEP] . The fact that t h i s may also l i m i t oxidative metabolism may not be of importance. Free AMP The potential depression of the [PCr] measurements due to PCr hydrolysis may also have resulted in the overestimation the calculated concentration of AMP; but whatever the free [AMP], at rest i t i s c l e a r l y very low. The Km of AMP deaminase for AMP i s on the order of 0. 4mM for the enzyme from rabbit muscle (Smiley and Suelter, 1967) several orders of magnitude higher than even the greatest [AMP]f calculated for the f i s h muscle: on the order of 6 uM. The a c t i v i t y of AMP deaminase i n rainbow trout white muscle i s approximately 40 umoles/minute/g (Fijisawa and Yoshino, 1987) . If t h i s enzyme was at V m a x t h i s would be s u f f i c i e n t capacity to produce about 1 mmole/g muscle over the 30 minute exercise bout used i n t h i s experiment. However, [AMP]f i s far below the Km for the enzyme. If, as a f i r s t approximation, we assume Michaelis Menten kinietics, then at the observed [AMP]f at exhaustion there would be the capacity to produce about 9 umoles IMP/g muscle over the 30 minute protocol, close to the observed increase in [IMP] of about 5 umoles/g. This c a l c u l a t i o n makes the assumption that [AMP]f i s constant at the highest l e v e l recorded throughout the exercise bout and that IMP production i s constant, neither of which i s l i k e l y to be the 86 case. However, i t i s clear that the capacity of AMP deaminase i s s u f f i c i e n t to account for the observed IMP production at physiological levels of [AMP ] f. On the other hand at the [AMP ] f calculated for the 2h post exercise group (table 12) at most 0.042 umoles IMP/g tissue could be formed over the same t h i r t y minute period, less than 0.5% of the capacity at exhaustion. This i l l u s t r a t e s the point that AMP deaminase function i s exquisitely sensitive to changes i n [AMP]f. RaTP and Energy Charge Although both RATP and EC decline s i g n i f i c a n t l y at exhaustion the actual difference between groups was extremely small and recovery was complete by 2h post exercise. When EC i s calculated without taking account the fact that the majority of ADP and AMP are bound in vivo these differences are magnified and recovery appears slower (table 13) . This raises the question of the general relevance of indices l i k e energy charge to the c e l l . The i n i t i a l formulation of the energy charge concept by Atkinson (1977) dealt with the t o t a l pools of ADP and AMP. Since then i t has become widely recognized that the majority of ADP and AMP are bound to proteins i n vivo and thus only the free forms should be considered in calculations of energy charge. Calculating energy charge from the t o t a l concentrations of ADP and AMP can have the eff e c t of magnifying changes i n the energy charge as can be seen with the results from t h i s study. When rigorously calculated i t seems that deviations i n energy charge 87 are very small even under conditions of recovery from exhaustive exercise. The energy demands of the c e l l must vary a great deal under these conditions and i t seems reasonable to expect a putative control parameter to vary as well. Energy charge seems to be a far from ideal control signal since., at least i n t h i s tissue, i t does not seem to undergo measurable changes over the physiological operating range, rather i t seems to be quite closely conserved. It may be that energy charge or similar calculated ratios may serve as indices of c e l l v i a b i l i t y but th e i r relevance to c e l l u l a r metabolism remains open to question. The discrepancy between the energy charge calculated with the t o t a l and free pools of ADP and AMP underscores the importance of dealing with only the reactive pools of metabolites when assessing the energy status of the c e l l . Since t h i s approach requires an estimate of i n t r a c e l l u l a r pH very few of the studies on exercise metabolism i n f i s h white muscle can be rigorously analyzed with respect to energy metabolism. The action of AMP deaminase i s responsible for the maintenance both RATP and EC close to resting levels at a l l times. If [ATP] had declined by 75% without the concomitant production of IMP or adenosine the concentrations ADP and AMP would have increased many f o l d causing RAXP and EC to f a l l and the c e l l to enter the depleting phase of high energy phosphate metabolism (figure 3) . Because of the action of AMP deaminase both RATP and EC are preserved and the c e l l stays in the buffering phase. Connett (1988) states that "because AMP i s the 88 Figure 7: Results of the model calculations (Connett, 1988) applied to rainbow trout white muscle following exhaustive exercise. Fc = creatine charge = [PCr]/total creatine Raxp = [AXP]/([ATP] + [ADP]f+[AMP]f EC = energy charge 89 6 O rest 0 . 0 0 .2 0 . 4 0 .6 0 . 8 1.0 1.2 Normofized poterttio! energy pool (Fpe) F i g u r e 8. The v a r i a t i o n i n c r e a t i n e charge w i t h the n ormalized p o t e n t i a l energy p o o l i n rainbow t r o u t white muscle f o l l o w i n g e x h a u s t i v e e x e r c i s e : the e f f e c t of the p u r i n e n u c l e o t i d e c y c l e . Fc = c r e a t i n e charge = [ P C r ] / t o t a l c r e a t i n e Fpe = [PCr] + 2[ATP] + [ADP] 90 substrate for the deamination reactions that deplete the adenine nucleotide pool i t i s only during t h i s [depleting] phase that the adenine nucleotide pool w i l l be depleted". This i s not the case i n trout white muscle since even at exhaustion RATP and EC do not decline substantially while there i s c l e a r l y a great deal of IMP formed. At most the f i s h have reached the t r a n s i t i o n between the depleting and buffering phases. The model (figure 3) also predicts how F c should change with changing potential energy as high energy phosphates are depleted. It i s possible to apply the same analysis to the results from the present study (figure 7 ). It seems clear from t h i s figure that at a l l times during the exercise and recovery period the muscle was in the buffering phase of high energy phosphate depletion. Since the c e l l remains i n the buffering phase i t becomes much more d i f f i c u l t to test the derived equations with which Connett estimates RATP and EC simply on the basis of F c (and pH and ion concentration) . Agreement seems to be f a i r l y "close but since the range observed i s so small i t i s impossible to come to any firm conclusion. If the data on the relationship between F c arid F P e are examined i n another way i t becomes apparent that there are some discrepancies between the predicted and the observed behaviour. Figure 8 again shows the relationship beween F c and F P e, however, each group has been analyzed separately. From t h i s i t i s clear that the data f a l l into two separate groups: one including the 24h recovery group and the resting f i s h and a second 91 containing the 2,4 and 8h recovery groups. The reason for this difference l i e s in the d e f i n i t i o n of potential energy: Pe = PCr + 2ATP + ADP [ADP] i s very low at a l l times r e l a t i v e to [PCr] and [ATP] so i t can be neglected. [PCr] i s roughly constant, between 20 and 25 umoles/g wet weight, in a l l groups except the exhausted f i s h and so [ATP] i s the major factor influencing Pe i n these groups. The reason for the discrepancy between these two groupings i s therefore " h i s t o r i c a l " . It l i e s in the fact that the 2,4 and 8h post exercise groups have undergone exhaustive exercise which decreased the size of the t o t a l adenylate pool (ATP+ADP+AMP) . This combined with the slow rate of purine nucleotide cycl i n g results i n F a d i f f e r i n g between the two groups. Since Connett's o r i g i n a l model was computed for a constant F a = 0.2 i t i s hardly surprising that a discrepancy appears. What remains to be seen i s whether t h i s constitutes a basic flaw i n the model. F P e i s used in t h i s context almost as an index of work done or umoles of phosphate energy hydrolyzed. As long as the t o t a l adenylate pool remains constant t h i s approximation i s v a l i d and F c w i l l be l i n e a r l y related to F P e. This w i l l c e r t a i n l y be the case i n red muscles where the a c t i v i t y of AMP deaminase i s lower. In white muscles, however, there may be s u f f i c i e n t AMP deaminase to cause a certain loss of adenylates even when [PCr] i s high. And c e r t a i n l y during recovery the s i t u a t i o n becomes complex beause of the variable rate of purine nucleotide cycl i n g so that i t may be impossible 92 to describe energy metabolism i n t h i s way using a single curve. As a result one of the predictions of the model, that energy state can be assessed simply by knowing the creatine charge and the pH, cannot hold for white muscles since the action of AMP deaminase must also be taken into account. There i s an additional complication to be addressed i f the model i s to adequately represent events in. vivo and that i s the e f f e c t of pH. It has already been shown that the e f f e c t of decreasing the pH i s to increase the extent of the buffering phase (see introduction). The effect of decreasing the pH on, for example, [AMP] i s to oppose the increases caused by the decreasing [PCr] . So the changes in [AMP] with exercise w i l l depend upon the r e l a t i v e rates of change of [PCr] and pH. For example i f i n i t i a l l y [PCr] declined with l i t t l e change i n pH there could be a very large increase in the concentration of free AMP. If pH then began to decline with l i t t l e change i n the [PCr] free AMP concentration would start to decline. The effects of differences i n the time course of decreases i n [PCr] and pH with exercise would be seen mostly i n the free concentrations of ADP and AMP. This could have extremely important consequences i n white muscle such as that of the trout. For example, i f [AMP]f was even t r a n s i e n t l y elevated into the range of the for AMP deaminase (which can be accomplished under t h i s model by dropping [PCr] by about 70% without changing pH i n f i s h muscle) a great deal of IMP could be formed. Since f i s h white muscle i s g l y c o l y t i c a l l y very active, [PCr] would quickly start 93 to r i s e again and pH to f a l l but by t h i s point the adenylate pool could have been depleted by the formation of IMP. This would cause complications for the application of the model in white muscles since the adenylate pool size could vary unpredictably. What must be examined i s the relationship between changes i n pH and PCr and the adenylates" i n muscle during exercise. U n t i l t h i s i s done i t becomes very d i f f i c u l t to accurately model energy metabolism p a r t i c u l a r l y i n white type muscles. 94 SUMMARY AND CONCLUSIONS The observation that trout white muscle c e l l s are never out of the "buffering zone" of the models of high energy phosphate depletion, even immediatly after exhaustive exercise, highlights an important c h a r a c t e r i s t i c of these models of c e l l u l a r energy state: that they are best applied to transient events. The adenylates and phosphocreatine behave in the way outlined in figures 1 through 3 over a very short time scale such that one i s , i n princple, unlikely to be able to measure the situ a t i o n described for the "depleting" phase of high energy phosphate depletion under normal physiological conditions i n a healthy muscle c e l l . Any perturbations i n the energy state of the c e l l w i l l be quickly compensated for and the c e l l returned to the buffering zone. For example, when energy demand increases [PCr] tr a n s i e n t l y decreases and even before the c e l l enters the "depleting" phase, [ADP] r i s e s dramatically (figure 1) . Under aerobic conditions t h i s would serve to increase mitochondrial oxygen consumption to meet the new demand and as a result, restore the [PCr]/[total Cr] r a t i o . In tissues with high anaerobic capacity, l i k e f i s h white muscle, the situ a t i o n i s more complex . With an increase i n [ADP], oxygen consumption or g l y c o l y t i c rate also increase as before. As the c e l l approaches the depleting phase of high energy phosphate metabolism, increasing concentrations of AMP activate AMP deaminase. This p u l l s the adenylate kinase reaction i n the ATP forming di r e c t i o n and quickly returns the c e l l to the buffering zone 95 simultaneously reducing the adenylate pool size (ATP+ADP+AMP) by the formation of IMP. So even though the energy state of the c e l l i s returned to normal, [ATP] i s depressed. By viewing the system i n t h i s way i t i s easy to see how [ATP] i n f i s h white muscle could be depressed during sampling even though [PCr]/[total Cr] r a t i o i s r e l a t i v e l y high. The action of AMP deaminase i s also resposible for deviation from the behaviour predicted by the models (figure 8). The observation that [PCr] recovers before [ATP] i s a result of the slow action of the reaminating arm of the purine nucleotide cycle. This results i n a d i s s o c i a t i o n of ATP concentration from energy status which i s not predicted by the models of high energy phophate metabolism. One of the fundamental differences between white and red type muscles may l i e i n t h i s d i s s o c i a t i o n of [ATP] from the energy charge or RATP. This c h a r a c t e r i s t i c of white muscle has an important impact on the ov e r a l l metabolism of the f i s h during recovery from exhaustive exercise. According to the calculations, free ADP i s in a range that could be l i m i t i n g to mitochondrial oxidative metabolism. At rest the mitochondria may be working at less than 10% of t h e i r potential maximum rate. Although not measured, i t i s reasonable to assume that with exercise t h e i r rate of oxygen consumption would rapidly increase as [ADP]f increases, or the ATP/ADP r a t i o decreases, but the energy demands are such that maximal capacity i s i n s u f f i c i e n t and the tissue must rely on anaerobic sources of energy. From exhaustion to 2h post exercise 96 energy status (as r e f l e c t e d by [PCr]/total [Cr] ratio) i s quickly restored. This, i n combination with the low pH drives down the free ADP concentrations to below resting l e v e l s . Low [ADP]f or high [ATP]/[ADP]f r a t i o s severely l i m i t the rate of mitochondrial oxidative phosphorylation, although t h i s effect may be complicated by e f f e c t s of pH, free phosphate and redox (mitochondrial or c y t o s o l i c ) . The high [ATP]/[ADP]f r a t i o s set up conditions which are favourable for glycogen synthesis from lactate. This suggests that there may be a trade off invovled in which rate of glycogen synthesis i s optimized r e l a t i v e to the rate of oxidative phosphorylation and vice versa. The retention of hydrogen ions which i s c h a r a c t e r i s t i c of trout white muscle may be necessary to set the required [ATP]/[ADP] r a t i o required for gluconeogenesis. [ATP] replenishment per se must be thought of as an e n t i r e l y seperate phenomenon with no direct bearing on energy metabolism or carbohydrate metabolism. Energy metabolism and carbohydrate metabolism i n f i s h white muscle following exhaustive exercise must be regarded as an integrated system responding to a number of c o n t r o l l i n g factors including free ADP, inorganic phosphate concentration and pH. Without considering a l l of these parameters i t i s impossible to accurately assess the behaviour of these systems. 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