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Metabolic regulation in skeletal muscle during exercise : a fish-mammal comparison Dobson, Geoffrey P. 1986

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METABOLIC REGULATION IN SKELETAL MUSCLE DURING EXERCISE: A FISH-MAMMAL COMPARISON GEOFFREY P. DOBSON B.Sc. Monash University, Clayton, V i c , Australia. 1976 M.Sc. Monash University, Clayton, V i c , Australia. 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in The Faculty of Graduate studies, Dept. of Zoology, University of British Columbia, Vancouver, B.C. Canada. We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March, 1986 © Geoffrey Phill ip Dobson, 1986 By 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. Department The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6(3/81) i i A B S T R A C T The aim of the present investigation was to examine the control of anaerobic glycogenolysis in working and fatigued skeletal muscle. The two animals chosen for the study were a teleost fish and the laboratory rat. The rationale behind using a comparative approach to investigate fundamental questions on metabolic control resides in the different abil it ies of each animal to perform exercise, and to their markedly different myofibril lar organization. In the process of defining the hierarchical recruitment of fuel and pathway selection in rainbow trout fast-twitch white skeletal muscle, it was clear that the near-maximal myosin ATPase activity was supported solely by PCr hydrolysis. It was not until the rate and force of contraction decreased that the relative contribution of anaerobic glycogenolysis became Increasingly important. Despite glycogenolysis possessing a lower maximal ATP generating potential than PCr hydrolysis, it has the advantage of being less constrained by time, and is recruited to extend muscle performance, but at submaximal workloads. Demonstration of the same temporal pattern of activation was not attempted for rat skeletal muscle because of complex fiber heterogeneity. The etiology of fatigue after 10 and 30 minutes of burst swimming in trout was due to the near depletion of glycogen In white muscle. Inhibition of anaerobic glycogenolysis was not correlated with l imitations to either the availability of ADP or NAD\ or inhibition of i i i phosphofructokinase (PFK-1). Similarly, the onset of fatigue and inhibition of glycogenolysis in three different skeletal muscles (gastrocnemius, plantaris and soleus) of the rat after 30 min of endurance treadmill running (25 meters/min), was not related to ADP availability, but associated with the near-depletion of muscle glycogen. As the endogenous stores of glycogen became limiting, hexokinase (Hk) appeared to be activated in trout white muscle after 10 min, and in the three rat skeletal muscles after 30 min, Indicating an increase in uptake and phosphorylation of blood-borne glucose. In rats running at a high speed for 2 minutes, g lycogenos i s was maintained through the coordination of glycogen phosphorylase and PFK-1. Muscle performance in these rats was maintained despite large percentage swings in cytosolic redox, the ATP/ADP ratio and phosphorylation potential. A common belief in the literature is that inhibition of glycogenolysis during short-term strenuous exercise is brought about by the pH dependent ATP inhibition of PFK-catalysis. Evidence was provided indicating PFK-1 is operational in skeletal muscle at about pH 6.6 for both the fish and the rat. Fish partially solved the problem of PFK-1 inhibition by lowering ATP, whereas the rat appeared to rely on the synergistic action of a number of positive modulators. A detailed kinetic analysis of purified rabbit muscle PFK-1 revealed that any modulator that increases the ratio of unprotonated to protonated form of the enzyme, could supply the muscle cell with a means of maintaining glycogenolytic flux despite fall ing pH. i v A number of striking differences were apparent between the regulation of glycogenolysis in fish and rat skeletal muscle. The f i rst major difference was the direction of change in cytosolic redox or the NAD+/NADH ratio. In fish white muscle the ratio increased, and the cytosol became more oxidized with exercise, whereas the opposite occurred in rat fast-twitch skeletal muscle. The difference was a consequence of lactate retention in fish white muscle. On the basis of crossover analysis, pyruvate kinase (PK) appeared to be activated in trout white muscle at both fatigue states. However, this was misleading, and also considered a consequence of rising pyruvate and due to lactate retention via the mass action effect at the LDH equilibrium. Obviously, the change in redox and the apparent crossover at PK are linked, and a literature survey revealed that during short-term maximal work, a mammalian skeletal muscle may indeed behave as fish white muscle. The other contrasting feature of this comparative analysis was the demonstration that ATP in trout white muscle can fal l by 80% at exhaustion. No such large percentage reductions in ATP occurred in either of the rat fast-twitch skeletal muscles, or indeed have been reported in skeletal muscle of any other exercising animal. In all cases the total nucleotide pool remained constant. A general conclusion to be drawn from this study is that muscle fatigue should be viewed as a multi-component process in response to l imiting glycogen, and not leveled at any one particular step of the glycogenolytic pathway. V TABLE OF CONTENTS Page*" Abstract (ii) List of Tables : ( vm) List of Figures .' Abbreviations fci-ii) Acknowledgements ( x i v ) Chapter 1. Introduction 1 The contractile cycle 2 Short-term generation of ATP 3 Control of glycogenolysis in skeletal muscle 7 NADVNADH cycles in glycogenolysis 12 Role of ADP, ATP/ADP ratio and phosphorylation potential in regulating glycogenolysis 13 Limits of anaerobic ATP generation 14 Specific aims and study outline 15 Chapter 2 Materials and Methods 20 Section 1. Fish 21 Exercise protocol and muscle dissection 21 Muscle homogenization, extraction and neutralization 23 v i Page* : Glycolytic intermediates 24 Nucleotides and phosphagens 24 Orthophosphate determination 25 Intracellular pH 26 Calculation of free cytosolic ADP 26 Calculation of free cytosolic NADVNADH ratio 27 Crossover theorem: applicability to identify sites of glycogenolysis 28 SECTION 2. Rats 29 Traning and exercise protocol 29 Anaesthesia 30 Muscle dissection 30 Calculation of free cytosolic ADP 31 Analytical methods ; 32 SECTION 3. Phosphofructokinase 32 Assay procedure 33 Data analysis 34 CHAPTER 3: Regulation of anaerobic generating  pathways in rainbow trout fast-twitch white skeletal muscle during exercise. 36 Introduction 37 Results 38 Discussion 42 v i i Page' Summary 48 CHAPTER 4: Regulation of anaerobic glycogenolysis  in three types of rat skeletal muscle during exercise Introduction 58 Results 59 Discussion 63 Summary 74 CHAPTER 5: Phosohofructokinase control in muscle:  nature and reversal of PH dependent ATP inhibition Introduction 91 Results and Discussion 92 Interpretation and physiological significance 96 CHAPTER 6 GENERAL DISCUSSION 108 CHAPTER 7: SUMMARY AND CONCLUSIONS 1 27 REFERENCES:. 133 LIST OF TABLES Page* Table 3.1. Changes in fuel and nucleotide concentrations in trout fast - twitch white muscle following a 10 second sprint 50 Table 3.2. ATP turnover in trout fast -twitch white muscle accompanying high intensity swimming 51 Table 3.3. Changes in fuel, nucleotide concentration, and pH in trout fast -twitch white muscle following a 10 and 30 min burst swim to fatigue and exhaustion 52 Table 3.4. Glycolytic intermediates in trout fast-twitch white skeletal muscle following 10 and 30 minutes of burst swimming to fatigue and exhaustion 53 Table 3.5. Cytosolic free ADP, ATP/ADP ratio, phosphorylation potenital and redox state in trout fast-twitch muscle following 10 and 30 minutes of burst swimming to fatigue and exhaustion 54 Table 4.1. Changes in fuel, nucleotide concentration and pH in rat gastrocnemius skeletal muscle following a 2 min high intensity, and endurance run to fatigue 76 Table 4.2. Changes in fuel, nucleotide concentration and pH in rat plantaris skeletal muscle following a 2 min high intensity, and endurance run to fatigue 77 i x Page* Table 4.3. Changes in fuel and nucleotide concentrations in rat soleus skeletal muscle following a 2 min high intensity, and endurance run to fatigue 78 Table 4.4. Estimates of ATP turnover supported by glycogenolysis in three different rat skeletal muscles during a 2 minute high intensity run 79 Table 4.5. Glycolytic intermediates in rat gastrocnemius skeletal muscle following a 2 minute high intensity run, and 30 minute endurance run to fatigue 80 Table 4.6. Glycolytic intermediates in rat plantaris skeletal muscle following a 2 minute high intensity run, and 30 minute endurance run to fatigue 81 Table 4.7. Glycolytic intermediates in rat soleus skeletal muscle following a 2 minute high intensity run, and 30 minute endurance run to fatigue 82 Table 4.8. Cytosolic free ADP, ATP/ADP ratio, phosphorylation potential and redox state in rat gastrocnemius following a 2 minute high intensity, and 30 minute endurance run to fatigue 83 Table 4.9. Cytosolic free ADP. ATP/ADP ratio, phosphorylation potential and redox state in rat plantaris following a 2 minute high intensity, and 30 minute endurance run to fatigue 84 X Page* Table 4.10. Cytosolic free ADP, ATP/ADP ratio, phosphorlation potential and redox state in rat soleus following a 2 minute high intensity, and 30 minute endurance run to fatigue 85 Tables. 1. Kinetic and regulatory properties of rabbit muscle PFK-1 as a function of pH in the presence of 1.0 mM and 5.0 mM ATP, and 5.0 mli ATP with 10 uM F 2,6-BP at 25°C 99 Table 5.2. Effect of modulator(s) to stabilize rabbit muscle PFK-1 against citrate inhibition 100 LIST OF FIGURES: Page* 3.1. Graphical representation of displacement of individual glycolytic reactions from thermodynamic equilibrium in trout fast -twitch white muscle 55 3.2. Crossover plot of glycolytic intermediates in trout fast -twitch white skeletal muscle after 10 and 30 minutes of burst swimming to fatigue 56 4.1. Graphical representation of displacement of individual glycolytic reactions from thermodynamic equilibrium in rat gastrocnemius skeletal muscle at three different activity states 86 4.2. Crossover plot of glycolytic intermediates in rat gastrocnemius skeletal muscle following a 2 minute high intensity run, and a 30 minute endurance run to fatigue 87 4.3. Crossover plot of glycolytic intermediates in rat plantaris skeletal muscle following a 2 minute high intensity run, and a 30 minute endurance run to fatigue 88 4.4. Crossover plot of glycolytic intermediates in rat soleus skeletal muscle following a 2 minute high intensity run, and a 30 minute endurance run to fatigue 89 - x i i Page* Fig. 5.1. Effect of F 6-P on activity of purified rabbit muscle PFK-1 at various pH values in the presence of 1.0 mli ATP 101 Fig. 5.2. Effect of F 6-P on the activity of purified rabbit muscle PFK-1 at various pH values In the presence of 5.0 mM ATP 102 Fig. 5.3. Effect of F 6-P on the activ ity of purified rabbit muscle PFK-1 at various pH values in the presence of 5.0 mM ATP and 10 uM F 2,6-BP 103 Fig. 5.4. Change in apparent Km or S 0 5 of purified rabbit muscle PFK-1 as a function of pH at 25°C 10A Fig. 5.5. Effect of modulators on the activity of purified rabbit muscle PFK-1 over the physiological pH range. The pH profiles were determined at 25°C in the presence of 0.1 mM F 6-P and 5.0 mM ATP 105 Fig. 5.6. Effect of varying F 2,6-BP and 6 1,6-BP on the activity of purified rabbit muscle PFK-1 at 25°C, pH 7.0, 0.1 mM F 6-P and 5.0 mM ATP 106 x i i i ABBREVIATIONS ABBREVIATION Gly Glu G 1-P G 6-P F 6-P F 1,6-BP DHAP GAP 1,3-DPG 3-PGA 2-PGA PEP Pyr Lact u mol/g wet wt n mol/g wet wt METABOLITE Glycogen Glucose Glucose 1-phosphate Glucose 6-phosphate Fructose 6-phosphate Fructose 1,6-bisphosphate Dihyroxyacetone phosphate Glyceraldehyde 3-phosphate 1,3-diphosphoglycerate 3-phosphoglycerate 2-phosphoglycerate Phosphoenolpyruvate Pyruvate Lactate lO^mole/gram wet weight 10"9mole/gram wet weight xiv ACKNOWLEDGEMENTS I would like to thank my supervisor, Dr. P. W. Hochachka for his support and unbounding enthusiasm throughout the course of the study. I am very grateful for the help and assistance from fellow graduate students in our laboratory, and in particular to Wade 5. Parkhouse. Special thanks go to Dr. A. Belcastro of the Department of Physical Education and Sport Studies, University of Alberta, Edmonton, Canada, for supplying the rats and surgical expertise for the second phase of the study. I would like to thank Drs. E. Yamamoto and H. Abe for teaching the basics of enzymology and H.P.L.C. techniques respectively. Thanks go to the Australian Prime Ministers, Malcolm Fraser and Bob Hawke for their continued help and support in restraining the Victorian Education Department, and Crown Solicitor, from f i l ing a suit against me for an outstanding student loan. Lastly, my largest debt continues to be with my wife who has continually demonstrated patience and diplomatic support. 1 CHAPTER I: GENERAL INTRODUCTION A fundamental goal of the study of tissue metabolism is to elucidate the regulatory mechanisms that adjust metabolic rates to ATP turnover. Skeletal muscle provides an ideal system to investigate such control mechanisms because of its capacity to increase its metabolic rate by many orders of magnitude during rest to work transitions (Cori, 1956). Any systems analysis of metabolic control in skeletal muscle must bear in mind the following: (1) What factors govern the selection of the appropriate fuel at the appropriate time to support a given workload? (2) Which enzymes are important in control of flux, and how are they regulated? (3) How are the various metabolic pathways synchronized with each other in the cel l , and what is the role of compartmentation, and (4) What factors l imit flux and muscle performance? At the outset, the aim of the present thesis dissertation was to cr i t ica l ly re-examine the regulation of anaerobic glycogenolysis in different muscle types of fish and the rat during short-term high intensity exercise, and following endurance exercise to fatigue and exhaustion. In an effort to present a general introduction into the hierarchical control mechanisms involved in the modulation of glycogenolysis, I feel it f i r st appropriate to briefly review relevant aspects of muscle contraction and the role of ATP. 2 THE CONTRACTILE CYCLE Central to our understanding of muscle biochemistry is that each time the myosin 51 head undergoes a pull and release cycle with actin, one molecule of ATP is hydrolysed to ADP, Pi and H + (Perry 1979, Eisenberg and Hil l 1985). Each contraction-relaxation cycle in skeletal muscle is regulated by transient changes in the concentration of free cytosolic Ca + + . According to the steric model of thin filament regulation, Ca"1"1" binds to and el ic i t s a conformational change in troponin, a regulatory protein, which is located at regular intervals (40 nM) along the length of the tropomyosin-actin complex. The C a + + mediated conformational change in troponin leads to a 10 degree rotational shift in the long beadlike tropomyosin from the periphery towards the groove center of the actin double helix. This shift in tropomyosin exposes the actin binding sites to myosin ATPase, and muscle tension is developed as the thick myosin and thin actin filaments slide past each other by several hundreds of nanometers (For a review see Ebashi 1980, Huxley 1985). During relaxation, the active sequestering of free C a + + ( 10 " 5 to 10 " 7 M) by the sarcoplasmic reticulum precludes myosin ATPase activity by altering the steric properties of the troponin-tropomyosin actin complex. In muscle, the myosin ATPase acts as an energy transducer by coupling the chemical reaction of ATP hydrolysis to the generation of force. We can define the power of skeletal muscle as the rate at which the fiber can apply force over a distance , which for the most part 3 depends upon the rate at which ATP can be turned over by the Ca + + activated F-actin myosin ATPase (termed myofibrillar or myosin ATPase). This reaction may be written as : A T P 4 - + HjO —> ADP 3 - + p2 " + H + + work The work term denotes the coupling of ATP hydrolysis to the myofibrillar ATPase. In the myofibril lar compartment, the coupled reaction is displaced by about 7 orders of magnitude from thermodynamic equilibrium. The thermodynamic equilibrium constant for the hydrolysis of ATP is around 2.8 X 10 + 5 M at pH 7.2, 38° C, 1.0 mM Mg + + , ionic strength 0.25 (Veech 1980). SHORT-TERM GENERATION OF ATP Energy metabolism and its regulation is highly dependent on oxygen availability. Aerobic metabolism provides energy at a highly efficient rate from mitochondrial oxidation of the blood borne fuels free fatty acids, glucose and lactate (Hollozy and Booth, 1976, Wahren 1979, Hollozy and Coyle 1984). However, with increasing workload, a point is reached where a disparity between oxygen uti l ization and ATP turnover develops, and ATP generation is largely accomplished by activation of the anaerobic processes of phosphocreatine (PCr) hydrolysis and glycogenolysis (Margaria 4 1972; Hochachka et al., 1983). One consequence of recruiting anaerobic pathways is that muscle work is constrained by time and inversely related to ATP turnover with supra-maximal efforts lasting only a few seconds (Wilkie, 1980; Gollnick , 1982). (1) PHOSPHOCREATINE HYDROLYSIS The f i r s t mechanism to be discussed, PCr hydrolysis, is brought about by the direct transfer of phosphate from phosphocreatine to ADP via the near-equilibrium reaction catalysed by creatine kinase (Lohmann, 1934). PCr 2 " + ADP 3 ' + l-f" ATP 4 - + Cr It is generally well accepted that during maximal ATP turnover in muscle the creatine kinase reaction is driven by the rate at which ADP and H+ ions are released from the myosin SI head during contraction (Kuby et al., 1954; Meyer et al., 1985). On the basis of the high maximal velocity of creatine kinase in skeletal muscle (Kuby et al., 1954), PCr hydrolysis could theoretically support a maximal myofibrillar ATPase activity of 500 u moles ATP/g wet wt /min (Bendall, 1961) for a period of about 16 sec before depletion of PCr. This calculation assumes an endogenous PCr store of 30 u moles/g muscle (Burt et al., 1976, Meyer et al., 1982). During such high flux rates another high activity enzyme, myokinase, may contribute to ATP generation but requires substantially elevated ADP concentrations (Km of 0.33 mM, Noda, 1973) as would be the case when PCr is nearly depleted (Cain and Davies, 1962). Recently, 5 using saturation-transfer 3 1 P NMR, the creatine kinase reaction has been shown to operate close to equilibrium in both frog and cat skeletal muscle (Kushmerick et al., 1980 Gadian et al., 1981). (1 1) ANAEROBIC GLYCOGENOLYSIS The second mechanism of ATP generation in the absence of oxygen in skeletal muscle 1s through the conversion of glycogen to lactate with two substrate phosphorylations of ADP occurring at phosphoglycerate kinase (PGK) and pyruvate kinase (PK). G l u c o s y l u n i t + 3 P f 2 " + 3 A D P 3 " + H + — • 2Lacrate"+ 3 A T P 4 " + 3 H 2 0 The process of anaerobic glycogenolysis generates about one-twelfth of the ATP that can be obtained from the complete oxidation of the glucosyl unit from glycogen to C0 2 and Hp. Why the conversion of glycogen to lactate is preferred over the complete oxidation of either glycogen or exogenous glucose to support high flux rates is thought to be due to the higher ATP yield  per unit time (McGilvery. 1975). The higher ATP yield per unit time is l ikely due to localization of glycogen and glycogenolytic enzymes close to the myofibrillar ATPase , and the kinetic structure of the pathway. Glycogenolysis Is a spontaneous reaction in a thermodynamic sense, proceeding with a relatively large decrease in free energy of -57 kcal/mol, pH7.0, 25 °C (Krebs and Kornberg, 1957). Later in 6 1964, Bucher and Russman focused in on the free energy changes of the twelve sequential reactions of the pathway, and compared the thermodynamic equilibrium constant of each reaction determined jn vitro to its respective mass action ratio measured in vivo (concentration ratio of products over reactants). These workers pioneered much of our current understanding on metabolic control by characterizing each enzyme as catalyzing either a near- or non-equilibrium reaction. In general, enzymes that catalyze near-equilibrium or readily reversible reactions (AG°«AG) are present in muscle with activit ies 10-to 100-fold greater than the maximum glycogenolytic flux (Bucher and Russman, 1964; Scrutton and Utter, 1968). Because they catalyze readily reversible reactions these enzymes are considered poor places to control unidirectional flux along a multienzyme pathway. In contrast, those enzymes that catalyze reactions displaced far from thermodynamic equilibrium are generally irreversible and characterized by low maximal activit ies to glycogenolytic flux ratios (Rolleston, 1972). This class of enzymes are important in providing directionality to pathway flux, and serve to integrate flux with the ATP requirements of the cell by being sensitive to a number of external allosteric effectors and covalent modification (Sols 1981). For all practical purposes, the irreversible reactions of glycolysis and glycogenolysis are catalyzed by hexokinase (Hk), glycogen phosphorylase, phosphofructokinase-1 (PFK-1) and PK (Hess, 1962; Williamson, 1965; Scrutton and Utter, 1968). It is not fortuitous therefore that these regulatory enzymes are strategically 7 positioned at the beginning, in the middle and at the end of the pathway, and that all are subject to varying degrees of multimodulation. Before looking at the regulation of the glycolytic and glycogenolytic pathway in more detail, it is worth mentioning that the application of thermodynamic principles provides information about whether a reaction can proceed spontaneously but not about the rate at which it proceeds. Regulation of metabolism therefore is essentially regulation of activity of enzymes and related membrane transport systems (Randle and Tubbs, 1979). CONTROL OF GLYCOGENOLYSIS IN SKELETAL MUSCLE Historically, the early work of Carl F. Cori in 1933 and later in 1956 showing that the rate of accumulation of hexose monophosphates exceeded the rate of lactate formation in tetanically stimulated rat gastrocnemius in situ and isolated frog sartorius muscle, provided the f i r s t unequivocal demonstration of a rate controlling step at the level of PFK-1. Additional studies about 5 years later extended Cori 's observation by implicating glycogen phosphorylase as well (Ozand and Narahara, 1964; Danforth , 1965). This classic series of investigations together with the thermodynamic treatment of Bucher and Russmann (1964) provided the experimental basis for much of our conceptual framework on metabolic control in skeletal muscle. Having briefly discussed the strategic positioning of the 8 various regulatory enzymes of glycogenolysis, the pathway can be subdivided and analysed in four broad sections: (1) Conversion of glycogen to glucose 6-phosphate(G 6-P), (2) Conversion of G 6-P to fructose 1,6-bisphosphate (F 1,6-BP), (3) Conversion of F 1,6-BP to pyruvate, and finally, (4) Conversion of pyruvate to lactate. The following discussion w i l l examine control of glycogenolysis in more detail by reviewing the regulation of three key enzymes; glycogen phosphorylase, PFK-1 and PK. GLYCOGEN PHOSPHORYLASE In skeletal muscle, the rate of glycogen breakdown primarily is under neural and hormonal control (Kavinsky and Meyer, 1977., Cohen 1983). The two best understood mechanisms involve phosphorylation of the inactive b form of glycogen phosphorylase to the active a form by the enzyme, phosphorylase kinase. The f i r s t control mechanism involves neural mediation by C a + + activation. The C a + + binds to calmodulin, a subunit of the inactive phosphorylase kinase, and as all the binding sites become f i l led, the entire molecule shifts to the active conformation leading to the subsequent conversion of glycogen to glucose 1-phosphate in the presence of P, (Boderling and Park, 1974, Cohen 1983). The second mechanism of control is mediated by catacholamine activated cyclic AMP which in turn leads to the phosphorylation of phosphorylase kinase b to a (Cohen 1983). A third mechanism that has been 9 extensively studied in vitro, but not well understood in vivo , involves non-covalent activation of phosphorylase b by either AMP or IMP (Fischer et al., 1971, Griff iths et al., 1976). AMP is regarded the more physiological effector since its apparent activation constant is 50 uM compared to 2 mM for IMP (Rahim et al., 1978). However, the most important of the three mechanisms involved in the activation of glycogen phosphorylase during the init ia l stages of excitation-contraction coupling is the release of C a + + from the sarcoplasmic reticulum. This explains why the activity level of phosphorylase a can rise from 5 to 90% with a short half-time of about 0.7 sec (Helmreich et al., 1965). Since glycogen consists of molecules of different sizes, it is impossible to determine the ratio [Glycogenn_,/Glycogenn] so we write the apparent equilibrium constant as [Gl-P/Pj]. At equilibrium, the ratio is 0.28 at pH 6.8 , 25°C (Fischer et al., 1971). In resting muscle, the mass action ratio may be at least two orders of magnitude lower than the equilibrium constant, assuming that a G 1-P concentration of 0.003 mM and a free P, of 1.0 mM. As glycogen is converted to G 1-P, the G 1-P enters the pathway of glycolysis at the level of G 6-P. This reaction is catalyzed by the near-equilibrium enzyme, phosphoglucomutase (PGM), which requires priming by glucose 1,6-bisphosphate (G 1,6-BP). G 1,6-BP is formed by phosphorylation of G 1-P in the presence of ATP and phosphoglucokinase (Bietner, 1979). Both G 6-P and G 1,6-BP are potent inhibitors of hexokinase and together with P. provide the to most likely means by which the cell selects glycogen over glucose during high muscle work rates (Lueck and Fromm, 1974 ). PHOSPHOFRUCTOK1NASE PFK-1 catalyzes the phosphorylation of fructose 6-phosphate (F 6-P) to F 1,6-BP. This reaction is the f i rst irreversible step of the glycolytic pathway below the G 6-P crossroad. The kinetic behavior of PFK-1 in vitro and in vivo is highly complex (Uyeda, 1979). Using the skeletal muscle enzyme , Passonneau and Lowry (1962) demonstrated that ATP acts as both inhibitor and substrate, that F 6-P acts as both activator and substrate, that both products F 1,6BP and ADP act as activators, that H + ion acts as a potent inhibitor, and that AMP, P(. and ammonia (NH 4 +) all act as activators of PFK-1 below pH 7.2. Furthermore, adding to this long l i s t of effectors, two other metabolites, fructose 2,6-bisphosphate (F 2,6-BP) and G 1,6-BP, have recently been found to be the most potent activators of PFK-1 so far discovered ( Bietner, 1979 ; Hers and VanShaftigen, 1982). The reported inhibition of muscle PFK-1 by PCr has proven to be due to a contaminant of PCr (Fitch et al., 1979). The* question of how PFK-1 operates in working muscle has challenged biochemists for over three decades. At physiological concentrations of F 6-P , it is known that high concentrations of ATP cause a marked inhibition at pH 7.0 (Ui, 1966), a condition that has also been observed at high enzyme concentrations (Bosca et al., 1 1 1985). As the pH decreases, the ATP sensitivity and F 6-P cooperativity increases. This inhibition of PFK-1 is due to the enhanced pH-dependent binding of ATP at an allosteric site (Passoneau and Lowry, 1962; Uyeda, 1979), and can be counteracted by the various positive effectors present in the cell (Trivedi and Danforth, 1966). Bock and Frieden (1976 a,b) have provided evidence to suggest that this interaction arises from F 6-P binding to unprotonated forms of the muscle enzyme, while ATP binds preferentially to the protonated forms at two ionizable groups (Pettigrew and Frieden, 1979). This mechanism of interaction has also been demonstrated with PFK-1 from ascites tumor cells (Sols, 1981). It can be stated with a fair degree of confidence that although PFK-1 as a control point of glycogenolysis is recognized beyond dispute, the mechanism of its action remains unclear. PYRUVATE KINASE PK catalyzes the transfer of the phosphate group from phosphoenolpyruvate (PEP) to ADP with the subsequent formation of pyruvate and ATP. One important regulatory function of this enzyme is to maintain unidirectional flux towards pyruvate formation. In contrast to the liver enzyme, muscle PK typically reveals a hyperbolic saturation curve in response to increasing PEP (Ainsworth and MacFarlane, 1973). On the basis of this response the muscle enzyme has been assumed for a long time less important to flux control than the other non-equilibrium enzymes, glycogen 12 phosphorylase and PFK-1. However, on careful re-examination of the kinetic properties of muscle PK, a pronounced sigmoidicity was apparent at physiological pH when the init ial velocities were determined as a function of either ADP or Mg + + (Phillips and Ainsworth, 1977). Furthermore these studies revealed a very important link between the activity of PFK-1 and PK by demonstrating that the product of the PFK-1 reaction, F 1,6-BP, strongly activates the muscle enzyme. In summary, PK 'turns o f f whenever the ATP concentration is high in the absence of positive effectors, and 'turns on' by feedforward activation through the combined action of PEP and F 1,6-BP availability. Since al l the preceding enzymes of glycolysis up to PFK-1 operate close to thermodynamic equilibrium in skeletal muscle (Veech et al., 1979; Connett, 1985), the activity of PK serves two vital functions: (i) provides directionality to pathway flux, and (ii) regulates the levels of glycolytic Intermediates at each of the respective steps up to PFK-1. NADVNADH CYCLES IN GLYCOGENOLYSIS A knowledge of the redox state of the cytoplasmic free NADVNADH couple is essential for an understanding of the regulation of glycogenolysis in skeletal muscle (Edington, 1970). During high flux rates, the NADH formed at the glyceraldehde 3-phosphate dehydrogenase (GAPDH) step is rapidly re-oxidized further down the pathway by lactate dehydrogenase concomitant 13 with the reduction of pyruvate to lactate (McGilvery, 1983). Since the total amount of nicotinamide adenine dinucleotide present in a cel l is constant at about 0.8 u moles/g wet wt tissue, regulatory mechanisms must exist to maintain the NAD+/NADH ratio within certain l imits if glycogenolysis is to continue (Cleland, 1967). In the rested state, the cytoplasmic NAD+ /NADH ratio in mammalian skeletal muscle was calculated to be about 300 (Edington et al., 1973) . In contrast, this ratio in the mitochondrial compartment is believed to be two orders of magnitude lower (Williamson et al., 1967). ROLE OF ADP. ATP/ADP RATIO AND PH05P0RYLATI0N  POTENTIAL IN REGULATING GLYCOGENOLYSIS The primary signals in muscle that respond to the demands of increasing work are thought to be changes in free C a + + and free cytosolic ADP (Jacobus et al., 1982; Chance et al., 1985). The transient increase in free ADP w i l l occur whenever the rate of ATP hydrolysis slightly exceeds the rate of ATP generation. The rise in free ADP causes a decrease in both the ATP/ADP ratio and phosphorylation potential (ATP/ADP. P4 ratio). A widely held belief is that the arrangement of the ATP generating mechanisms in a cell operate to maintain a high ATP/ADP ratio or phophorylation potential so that the hydrolysis of ATP remains favourable. In the context of this general discussion on coupling anaerobic processes to myosin ATPase, the three parameters (free ADP, ATP/ADP or 14 ATP/ADP. Pj ratios) either directly or indirectly play a cr i t ica l role In regulating the activit ies of the kinase reactions of both PCr hydrolysis and glycogenolysis during high muscle work rates (McGilvery, 1975). That free ADP in the cytosol of muscle has been calculated to be as low as 20 to 40 uM (~5% of the total ADP content in muscle; Veech et al., 1979), further attests to the important catalytic role of this metabolite in control of metabolism . LIMITS OF ANAEROBIC ATP GENERATION Operationally, fatigue can be defined as the inability of an organism or muscle to maintain a pre-determined exercise intensity or myosin ATPase activity (Edwards, 1975; Faulkner, 1983). The term exhaustion may be used synonymously with fatigue but for the purpose of this thesis, emphasis is placed on the term exhaustion to mean the inability of an animal to maintain any exercise intensity with the subsequent loss of postural support and orientation. When a muscle is contracting at maximal speed of shortening, the limited store of PCr must obviously be a prime candidate for the development of fatigue (Vergara et al., 1977)). If the speed of shortening is reduced , the onset of fatigue and exhaustion w i l l be delayed , and in this case may be brought about by glycogen depletion or by a number of interrelated factors that are primarily governed by the intensity and duration of contraction (Hermansen, 15 1981; Gollnick, 1982; Faulkner, 1983). Traditionally, the onset of fatigue in skeletal muscle has been associated with one of the products of ATP hydrolysis, H + ions (Bolitho-Donaldson and Hermansen, 1978; Nassar-Gentina et al., 1978; Sahlin, 1978; Sahlin et al., 1981). The H + ion has a myriad of functions in a muscle cel l , and an increase in its concentration (intracellular pH down to 6.4 ) has been postulated to bring about the onset of fatigue by affecting the kinetic and regulatory properties of PFK-1 (Hollozy et al., 1978; Hermansen, 1981), the C a + + sequestering and releasing properties of the sarcoplasmic reticulum (Nakamura and Schwarts, 1972; F itts et al., 1982), and even the myofibril lar ATPase itself (Portzehl et al., 1969; Fabiato and Fabiato, 1978; Dawson et al., 1980). The neuromuscular junction has also been implicated with a decrease in force development (Bigland-Ritchie et al., 1982, Faulkner, 1983). Superimposed on these potentially l imiting effects of H* ion on the rate of anaerobic glycogenolysis and muscle performance are the effects of concomitant changes in either the ATP/ADP ratio, the phosphorylation potential or the NADVNADH redox couple. SPECIFIC AIMS AND STUDY OUTLINE The central theme of the present thesis is regulation of the anaerobic ATP-generating pathways in vertebrate skeletal muscle during exercise and fatigue, with particular emphasis on glycogenolysis. The animals of choice were a teleost fish and laboratory rat. The rationale behind using two different animal 16 models to investigate the same fundamental questions on metabolic control resides in the different abi l it ies of each to perform exercise, and to their markedly different myofibrillar organization (Ariano et al., 1973; Webb, 1978; Johnston, 1981; Armstrong and Laughlin, 1985). In contrast to the laboratory rat, f ish are highly specialized for fast-start performances (Webb, 1978), a specialization that is predictable on the basis of their myotomal organization. The major bulk of the body musculature of f ish primarily is composed of fast - twitch glycolytic (FG) or white fibers, with red or slow oxidative (SO) appearing as a separate thin triangular strip running longitudinally beneath the lateral line (Johnston, 1977, 1981). In general, the red muscle mass constitutes about 10% of the total body musculature, but this percentage may vary widely depending upon the activity pattern and l i festyle of the species (Greer-Walker and Pull, 1975). Between the red and white muscle layers there is an intermediate zone which is often referred to as fast-oxidative glycolytic or FOG fiber type, after the terminology of Peter et al., 1972 (Johnston, 1981). The myofibril lar organization of higher vertebrates is much more complex and heterogeneous in nature with each skeletal muscle being composed of widely varying percentages of the three basic fiber types (FG, FOG & SO) [Peter et al., 1972]. In the case of the rat, even though 50% of the total musculature of the hindlimb is composed of fast - twitch fibers (FG & FOG), detailed analysis of Its cross-sectional area reveals that muscles with a higher percentage of SO fibers are generally located in the deeper regions, 17 whereas those muscles with a higher percentage of fast - twitch fibers are located at the periphery of the leg (see Armstrong and Laughlin, 1985). The three basic fiber types have been classif ied on the basis of their different structural, biochemical and physiological properties. In general, the FG fibers are typically larger (up to 100A in sarcomere diameter); have a high C a + + /Mg + + activated actomyosin ATP ase; a high anaerobic glycogenolytic and low aerobic capacity; low myoglobin, capillary and mitochondrial density; a high buffering capacity , and easily fatiguable ( Peter et al., 1972; Baldwin et al., 1973; Hollozy and Booth 1976; Johnston 1985). SO fibers on the other hand have a smaller cross-sectional area and display the opposite metabolic profiles as described for FG fibers. Not surprisingly, as discussed above for fish those skeletal muscles with a higher percentage of FG fibers are preferentially recruited for short-term high intensity work, whereas muscles with a higher proportion of SO fibers are recruited during lighter to moderate intensity exercise (Davison et al., 1976., Hollozy and Booth, 1976; Bone et al., 1978, Hollozy and Coyle, 1984). As running speed increases, electromyographic studies on rat hindlimb muscles have shown that there is a progressive shift in activation of motor units from the deep 50 muscles to the more peripheral FG muscles (Armstrong and Laughlin, 1985). The same pattern of activation of muscle types has also been demonstrated In fish swimming at increasing speeds (Bone et al., 1978), except that the SO fibers are located at the periphery. The different spatial 18 organization of muscle types in the rat hindlimb and f ish probably reflect the different weight bearing requirements of quadrapedal terrestrial locomotion verses the near neutral buoyancy of f ish in their aquatic environment. Because of the obvious functional differences between the three basic muscle fiber types, and the different spatial patterns of myofibril lar organization of the two animals, it was conceivable that Identification of f iber-specif ic regulatory mechanisms of glycogenolysis may be more apparent in fish white skeletal muscle than in the comparable fast-twitch skeletal muscles of the rat. The regulatory parameters that w i l l be examined in this study include the free cytosolic ADP concentration, the ATP/ADP ratio, the phosphorylation potential, the redox potential and the interrelated effects of decreasing intracellular pH. Since the NADVNADH and ATP/ADP ratios are functionally linked through the combined GAPDH-PGK-LDH equilibria, the study sets out to establish the relative importance of each parameter in controlling glycogenolysis, and more important, their respective roles in l imiting muscle performance. Moreover, coupling between the anaerobic processes and the myosin ATPase in muscle is coordinated through the ADP or ATP requiring kinases of PCr hydrolysis and glycogenolysis. A commonly held view in the field of exercise biochemistry and physiology is that the onset of fatigue during short-term high Intensity exercise is brought about by the inhibitory effect of pH on PFK-1 (Hollozy et al., 1978; Hermansen, 1981). The present author believes that such statements are not 19 well founded and in addition to examining control of PFK-1 in f ish skeletal muscle at fatigue, and in rat skeletal muscle after two different exercise intensities, a rigorous kinetic study w i l l attempt to show how the enzyme can achieve significant catalytic rates in working muscle despite fall ing pH. The work w i l l be presented in three separate sections. The f i r s t section w i l l consider the regulation of PCr hydrolysis and anaerobic glycogenolysis 1n fast - twitch white muscle of rainbow trout (Salmo gairdneri) after a 10 sec sprint, a 10 min burst swim at approximately 120% V0 2 max to fatigue, and following a 30 min endurance swim to exhaustion. The second section w i l l examine anaerobic glycogenolysis in three different muscle types of the rat after 2 minutes of high intensity treadmill running , and following a 30 min endurance run to fatigue. The third section w i l l deal with a re-examination of the kinetic and regulatory properties of purified rabbit muscle PFK-1 . The fourth and final section with give a general account of the major findings of this interdisciplinary investigation between the fish and rat, and w i l l attempt to summarize the s imi lar it ies and differences between each in regulating, and in the case of fatigue, l imiting glycogenolysis in skeletal muscle accompanying exercise. 20 CHAPTER 2: MATERIALS AND METHODS 21 MATERIALS AND METHODS. SECTION 1  FISH Rainbow trout (Salmo gairdneri) of both sexes were obtained from the Sun-Valley Trout Farm, Mission. B.C. Canada. Fish were fed ad libitum and maintained in outdoor tanks with a continuous supply of fresh, aerated, dechlorinated tap water. EXERCISE PROTOCOL AND MUSCLE DISSECTION The exercise protocols used for the study included a 10 second supra-maximal sprint, and two prolonged burst swimming events of about 10 and 30 min duration. To avoid confusion, it was necessary to modify the terminology of Hoar and Randall (1978) and define a high intensity burst swim lasting less than 20 sec as a sprint. Two different stocks of fish were used: the f i r s t winter stock (water temperature 4 - 6°C, weighing 60 to 70 g and 18 to 20 cm in length) was used for the sprint protocol; while the second summer stock (water temperature 10° C, weighing 200 to 250 g, and 27 to 30 cm in length) was used for the two prolonged swimming protocols. Fish were exercised in a Brett-type swim tunnel (Brett, 1964). Prior to each experiment, fish were characterized into groups on the basis of steady state swimming performance using stepwise increases in water velocity of 10 min duration (increments of 0.2 body lengths/sec), and continued until a steady state swimming 22 velocity could no longer be maintained (Brett, 1964; Jones and Randall, 1978). This method is useful for selecting f ish of s imilar physiological state and swimming ability. With this exercise procedure, the maximum steady-state swimming velocity achieved prior to fatigue is known as the cr i t ica l velocity or Ucrit (Brett, 1964). At swimming speeds below Ucrit, metabolism has been shown to be predominately aerobic with a small anaerobic component appearing at about 80% Ucrit (Bone et al., 1978). Even though no simple relationship between oxygen uptake and Ucrit has been described for fish, it is reasonable to assume that Ucrit is analogous to the V0 2 max index used to evaluate mammalian exercise performance. A l l fish were transferred by net from black holding boxes to the swim tube and those fish that struggled were discarded. In the case of the sprint protocol, the water velocity was immediately increased to the maximal 90 cm/sec, which corresponded to about 5 body lengths/sec. It was previously determined one week earlier that the time to fatigue was on the average 18 sec. Fish were netted after 10 sec of sprinting, and the white epaxial muscle immediately excised from a site posterior to the dorsal fin. Samples were immediately freeze-clamped in liquid nitrogen (- 200°C) and stored at -70°C until required. The average time between capture of f ish and freeze-clamping muscle was about 6 sec. Those f ish subjected to burst swimming at about 120% Ucrit lasted on average 10 min before fatigue set in and their swimming position could no longer 23 be maintained. The longer burst swimming group were started at the highest maximum water velocity with the speed being continuously decreased as each fish could no longer maintain position in the swim tunnel. After a few minutes, however, the water velocity was increased again to make certain that the anaerobic ATP generating pathways were being recruited at al l times. By continually oscillating the speed control in this way, fish were completely exhausted at both high and low swimming velocities. The procedure was halted when fish could no longer swim. The average time to complete exhaustion was 30 min. White epaxial muscle was dissected out immediately in an identical manner as that described above for sprint fish. MUSCLE HOMOGENIZATION. EXTRACTION AND  NEUTRALIZATION White epaxial muscle was powdered under liquid nitrogen using a pre-cooled mortar and pestle. Tendons and fragments of connective tissue were dissected free and discarded. About 500 mg of powdered tissue was transferred to a pre-cooled pre-weighed vial containing 1.0 ml of ice-cold 0.6 N perchloric acid (PCA) and then accurately re-weighed. A further 1.0 ml PCA was added and the powder homogenized at intermediate to high speed for 15 sec at 0°C using an Ultra-Turrax T18 homogenizer coupled to 10 mm shaft and generator. The homogenization procedure was repeated and the sides of the vial washed down with a further 0.5 ml PCA. The suspension was stirred under low speed and 100 ul was removed in 24 duplicate for glycogen determination. The remaining homogenate was then transferred to two 1.5 ml eppendorf polypropylene test-tubes, and centrifuged for 2 min at 13,000 r.p.m. and 4°C in a microcentrifuge. A known volume (approximately 2.0 ml) of supernatant was removed and immediately transferred to a pre-cooled test-tube containing a pre-determined volume of saturated Tris base [tris(hydroxymethyl)-aminomethane] to bring the pH of the extract to 7.0. The neutralized PCA extract was frozen immediatley in liquid nitrogen and kept at -70°C until required. A l l biochemical analysis was carried out within two to three weeks. The homogenization and neutralization procedure described is rapid and takes about 6 minutes to complete. In a parallel set of validation studies the highly acid-labile phosphocreatine was shown to undergo not more than 5% hydrolysis with this technique. GLYCOLYTIC INTERMEDIATES The total content of all glycolytic intermediates were measured in a Pye-Unicam SP8-100 UV-VIS spectrophotometer at 340 nm using the routine NADH or NAD coupled enzymatic procedures described in Bergmeyer (1983). Each assay was validated with the appropriate standard(s). NUCLEOTIDES AND PHOSPHAGENS The nucleotides ATP,ADP, AMP and IMP, and PCr and creatine (Cr) concentrations were analytically determined by High Performance Liquid Chromatography (HPLC). The procedure was 25 carried out on a Spectrophysics 8000 B HPLC coupled to a Spectroflow 773 Absorbance Detector. The nucleotides were passed through a Brownlee anion-exchange column at a flow rate of 2.0 ml/min and eluted in 30 min using a linear gradient of dihydrogen potassium phosphate (KHj PO A MCB PX1-566-3). The .initial gradient comprised 50 mM KHj P04-HC1 pH 2.31 at 25° and 600 mM KH 2P0 4-HC1, pH 2.63 at 25°C and the nucleotides were detected at 254 nm. The column temperature was 55°C. PCr, Cr and AMP were eluted at the same flow rate of 2 ml/min in 12 min using an isocratic 50 mM KH 2P0 4-HC1 buffer at pH 3.1, and detected at 210 nm. 20 ul of neutralized sample was required for each set of determinations. Following every set of six chromatographic runs, a single 20 ul injection of known standards was passed through the system to check retention times of each metabolite. Internal standards were also used to validate the procedure for muscle analysis. ORTHOPHOSPHATE DETERMINATION Orthophosphate (Pp was determined by the colorometric method of Black and Jones (1983). This method has been subsequently validated with 3 1 P NMR on neutralized muscle extracts from resting and exercising fish (Parkhouse and Dobson, unpublished data). 26 INTRACELLULAR PH Intracellular pH of muscle was estimated by using the homogenate technique described by Costi l l et al., 1982) with the following modifications: Frozen muscle powder ground under liquid nitrogen and Kept at -70°C was homogenized at 0°C in a salt solution (1 : 9 w/v) containing 145 mM KC1, 10 mM NaCl and 5 mM iodoacetate, pH 7.0 at 10°C. The pH of the muscle homogenate was measured at 10°C (body temperature of fish) with a Radiometer Micro-pH electrode coupled to a PHM-71 Acid-Base Analyser. For intracellular pH of rat gastrocnemius and plantaris the identical procedure was used except the measurement temperature was 38°C. CALCULATION OF FREE CYT050LIC ADP Free cytosolic ADP was calculated using the creatine kinase equilibrium as described in detail by Veech et al., (1979). The calculation is based on the assumption that the reaction is maintained near-equilibrium, an assumption that appears to be valid in working muscle (Veech et al., 1979; Kushmerick et al, 1980; Gadian et al., 1981; Shoubridge et al., 1982; Meyer and Kushmeric, 1985). The equilibrium constant for the creatine kinase reaction is 1.66 X 1 0 + 9 M at pH 7.0, free Mg + + 1 mM, ionic strength of 0.25 and temperature 38°C. It should be emphasized that the metabolite concentrations used in the calculation are those determined 27 analytically and include all ionized and complexed species. A l l tissue metabolites are expressed in u moles/g cell water in order to make the metabolite ratios directly comparable to the equilibrium constant measured in vitro. The percentage of tissue weight that is water was determined in a parallel set of studies and found to be 80% , a value that is s imilar to rat skeletal muscle (Veech et al., 1979). The free absolute concentration of ADP in the cytosol of fish white muscle In vivo may differ slightly from the calculated estimates in view of the fact that the apparent thermodynamic equilibrium constant for the creatine kinase reaction used was determined at 38°C. The relative change in free ADP occurring with exercise should however be valid. The equilibrium constant could not be temperature corrected for 10°C because the constant has not been well defined at any temperature other than 38°C, and therefore precludes an accurate calculation of AH 0 (Veech, personal communication). CALCULATING FREE CYTOSOLIC NAPVNAPH RATIO The cytoplasmic NAD+/NADH ratio was calculated using the lactate dehydrogenase equilibrium (Williamson et al., 1967., Veech et al., 1969) according to the folowing expression: NADVNADH = [Pyruvatel/tlactate] X [H +]/Keq where lactate and pyruvate concentrations denote the total 28 measured contents. The equilibrium constant ( Keq) was calculated to be 1.1 x 10" 1 2 IT 1 at 10°C, pH 7.0 and at an Ionic strength of 0.25 assuming a AH° of + 14 kcal/mol. This calculated equilibrium constant is very s imilar to that determined directly at 16°C (Hakala et al., 1956). This method is based on the following two assumptions: (a) that the LDH reaction is maintained near-equilibrium in working muscle, and (b) that the myofibrillar compartment has an ionic strength of 0.25 and free Mg + + of 1.0 mM. CROSSOVER THEOREM: APPLICABILITY TO IDENTIFY  CONTROL SITES OF GLYCOGENOLYSIS The crossover theorem was developed by Chance and Williams (1956) for use in identifying control points of the phosphorylating mitochondrial electron transport chain. Later in the mid-sixties it was applied to glycolysis (see Williamson, 1970). For graphical representation, individual reactions of the glycolytic pathway are listed in order of succession on the abcissa, and the relative changes of their metabolite concentrations (expressed as a percentage of control values) are plotted on the ordinate. The zero line represents the steady state, where no changes in levels of intermediates with time take place. Crossovers between pairs of neighboring metabolites may occur from minus to plus or from plus to minus relative to the zero line. Crossovers indicate sites of interaction or control points along the pathway in response to a change in flux . A detailed account of the theoretical basis for the 29 crossover theorem can be found in Williamson (1970) and Heinrich et al., (1977). MATERIALS AND METHODS: SECTION 2  RATS Adult male Sprague-Dawley rats weighing approximately 300 to 320g were obtained from Acme Biomedical Supplies, Calgary, Canada. Rats were individually housed and maintained in a temperature (25°C) and light (12hr dark/12 hr light cycle) controlled room. They were fed ad libitum on purina rat chow and allowed free access to water. A l l experiments were carried out between 9.00 a.m. and 12.00 noon to avoid any diurnal variation in muscle glycogen (Conlee et al., 1976; Garetto and Armstrong, 1983). TRAINING AND EXERCISE PROTOCOL Rats were trained by running at 30 m/min on a treadmill for 10 min a day 5 days a week for a period of 4 to 8 weeks prior to the experiment. Rats selected for the short-term 2 min high intensity run were familiarized with the protocol for 7 days before the experiment. The 2 min protocol consisted of running at a treadmill speed of 75 m/min for 20 sec and 56 m/min for the remaining 1 min 40sec. These animals were not fatigued after 2 min. The 30 endurance protocol consisted of running at a treadmill speed of 25 m/min until fatigue. Fatigue was characterized by the animal's inability to maintain a running speed of 25m/min. ANAESTHESIA . The exercised animals were anaesthetized with an Intra-perltoneal injection of a 1.5 ml solution comprising 0.5 ml Na pentobarbital (50 mg/ml ) and 1.0 ml curare (lOmg/ml). Curare is a neuromuscular blocking agent and was found to be suitable in preventing muscle twitching following exercise. The response time of the anaesthetic without curare was 60 to 90 sec. Use of this procedure for the control group, however, was not successful and produced noticeable irregular twitching and violent kicking resulting in extremely low PCr values (Dobson, Belcastro and Parkhouse, unpublished data). Despite a longer response time of 3 to 5 min, the preferred technique of anaesthesia for control rats was the use of ether soaked cotton wool placed in a sealed Bell jar. This technique has been widely used for detailed biochemical analysis of muscle in the pre-exercise or "rested ' state (Veech et al., 1979). MUSCLE DISSECTION Following anaesthesia, rats were placed in a supine position and skeletal muscles of the left lower extremity rapidly 31 exteriorized The soleus muscle was the f i r s t to be excised, taking 30 sec, and immediately freeze clamped in liquid nitrogen with pre-cooled aluminum tongs. The plantaris was excised next, taking about 60 sec, followed by the gastrocnemius (80-90 sec). The same procedure was repeated for the right lower leg with respective dissection times of 1 min 50 sec, 2 min 15 sec and 2 min 45 sec. A l l times for individual muscles refer to the time elapsed between the fully anaesthetized state and freeze-clamping in liquid nitrogen. Care was taken not to stretch the muscles during the dissection procedure. The tissues were stored for approximately 2 weeks at -70°C prior to analysis. Before the homogenization procedure, individual soleus muscles were pooled from the left leg of three rats for the 2 min running group, while the plantaris was pooled from the left leg of two rats. The gastrocnemius was not pooled. An identical procedure was adopted for the three muscles of the right leg. No significant biochemical differences were noted between muscles pooled from either the left or right lower extremities. A similar grouping procedure was used for the endurance rats except that muscles were paired on the basis of running time to fatigue. CALCULATION OF FREE CYT050LIC ADP Free cytosolic ADP was calculated using the combined glyceraldehyde 3-phosphate dehydrogenase-phosphoglycerate kinase 32 and lactate dehydrogenase equilibrium expression (GAPDH-PGK) [see Veech et al., 1979]. The same assumptions hold for this method as described for the creatine kinase system. The equilibrium constant for the combined GAPDH-PGK reaction is similar to that described by Veech et al., (1979) as modified by Connett (1985). ANALYTICAL METHODS Al l other analytical methods used were identical to those described in detail in SECTION 1 of the MATERIALS AND METHODS and can be found under their respective headings . MATERIALS AND METHODS: SECTION 3  PH05PH0FRUCT0KINASE Crystalline rabbit muscle PFK-1 (EC 2.7.1.11) was obtained as an ammonium sulphate suspension from Sigma Chemical Company (Type III Lot number: 81F-9590). The specific activity and purity of the enzyme was 110 U/mg protein, which was similar to that used by Bock and Frieden (1976a). One unit of activity is defined as the production of 1.0 u mole of Fructose l,6BP/min at 25°C and pH 8.0. Nucleotides, buffers, and auxiliary enzymes were obtained from Sigma Chemical with the exception of AMP and rabbit muscle a-glycerophosphate dehydrogenase and triose-phosphate isomerase, 33 which were purchased from Boehringer Mannheim. A l l other biochemicals were reagent grade and used as received from the supplier. PFK-1 was dialized at 4°C against two changes of 50 mM Tris-phosphate (pH 8.0 at 4°C), 10 mM 1,4-dithiothreitol, and 1 mM EDTA-Na. After dialysis, the ratio of the absorbance of the enzyme solution at 280 and 260 nm was > 1.65 (Bock and Frieden, 1974). Two batches of the enzyme were used with similar results. ASSAY PROCEDURE Enzyme activity was measured by the addition of an aliquot of the dialized enzyme sample (diluted approximately 1:4 with buffer) to a reaction cuvette, and the rate of formation of F 1,6-BP was monitored at 25°C in a final volume of 1 ml. The assay mixture contained 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-KOH, 0.15 mM NADH, 10 mM MgCl 2, 0.10 mM 1,4-dithiothreitol, 0.01 mM EDTA, 1.0 or 5.0 mM ATP, aldolase (0.8 U), a-glycerophosphate dehydrogenase (1.0 U), and triose-phosphate isomerase (5 U), with various concentrations of F 6-P and pH values (7.67, 7.25, 7.0 and 6.8 at 25°C). The coupling enzymes were either desalted by overnight dialysis or by passing through a Sephadex 6-25 column. The reaction mixture was thermally incubated for 10 min at 25°C, and the reaction initiated by the addition of PFK-1. The absorbance was monitored at 340 nm with a Unicam 5P-1800 dual 34 beam recording spectrophotometer. One unit of PFK activity corresponds to the oxidation of 2 umol NADH, which is equivalent to the production of 1 umol F 1,6-BP per min under the above assay conditions. Initial velocities were linear functions of time and enzyme concentration. DATA ANALYSIS In the presence of positive modulators, init ial velocities were readily obtained from the slopes of recorded optical densities. However, under some circumstances, reaction rates were linear with time for only about 5 to 10 seconds. This was particularly notable in the presence of citrate where the time-dependent loss of activity is mainly due to the conversion of the active tetrameric enzyme to the inactive dimeric form (Lad et al., 1973). It was also evident, but to a lesser degree, at high ATP levels and low pH, when the F 6-P concentrations were 0.0125 mM or lower, again possibly due to the loss of tetrameric enzyme. In such cases, int it ia l velocities were determined by measuring the tangent to the recorded curve and extrapolating back to the time of addition of enzyme. However, in the absence of citrate, at higher F 6-P concentrations and /or in the presence of positive modulators, no such loss of tetramer based enzyme activity was observed, and reaction velocities were linear for periods up to several minutes. Otherwise, wherever possible, this problem was avoided by using high physiological F 6-P concentrations (0.1 mM). 35 LIMITATIONS TO RESEARCH When conducting biochemical measurements on skeletal muscle of exercising animals a number of cautionary points should be realized. The f i r s t l imitation is to understand that recovery metabolism may occur between the time of cessation of exercise and freeze clamping the muscle. This 1s not as cr i t ica l for fish muscle because of short sampling times (sec) but may be significant for each of the respective rat skeletal muscles. This may bias the interpretation of the biochemical data in rat skeletal muscle due to complex fiber heterogeneity and longer sampling times. It must be emphasized that concentrations of metabolites represent total contents, and in the case of highly heterogeneous muscles of the rat, represent a relative contribution from all fiber types. This problem is more pronounced in those muscle types that are well perfused with blood e.g. soleus muscle. The problem is less important in the fast-twitch muscles (gastrocnemius and plantaris). However, one should s t i l l be aware that each fast-twitch muscle consists of different percentages of FG and FOG populations. 36 CHAPTER 3: REGULATION OF ANAEROBIC GENERATING  PATHWAYS IN RAINBOW TROUT FAST-TWITCH  WHITE MUSCLE DURING EXERCISE 37 CHAPTER 3. INTRODUCTION It is well established that the temporal activation of phosphocreatine hydrolysis and anaerobic glycogenolysis in working muscle depends to a very large extent on the intensity and duration of contraction (Hohorst et al., 1962; Danforth, 1965). Less clear are the regulatory mechanisms controlling flux, and those fiber specific related mechanisms leading to fatigue (Wenger and Reed, 1976; Karlsson, 1980). Fish provide a particularly useful metabolic system for investigating such mechanisms during high work rates because of their fast-start performance capacity (Webb, 1978), and their myofibril lar organization. As outlined in the introduction, f ish locomotory muscle is composed of two spatially separate muscle masses of different fiber types: white muscle and red (Johnston, 1982). The white muscle mass comprises most of the body musculature with the red muscle appearing as a thin triangular strip running longitudinally beneath the lateral line. In the case of trout, 90% of the body musculature is white and 10% is red (Johnston, 1977, 1981). Thus a major advantage of using fish models to study the biochemistry of exercise is that many of the interpretative problems arising from complex patterns of fiber hetereogenety are minimized. The present study was undertaken to investigate the contribution and control of phosphocreatine hydrolysis and anaerobic glycogenolysis in generating ATP for a short 10 sec sprint, and for two longer (10 and 30 min) swimming protocols to 38 fatigue and exhaustion respectively. RESULTS : FUELS AND ATP TURNOVER (1) PHOSPHOCREAT1NE PCr decreased from 27.03 to 21.36 u mole/g wet wt muscle following a 10 sec sprint (Table 3.1.) Assuming that 1 mole of ATP is produced per mole PCr hydroiyzed, and knowing the duration of each burst during the 10 sec period, the ATP turnover was estimated to be 188 u moles ATP/g wet wt/min (Table 3.2.) This value may be three times higher if we make some reasonable assumptions about the level of PCr in white muscle at the pre-exercise state prior to the swim (see Discussion). In the 10 and 30 min burst swimming fish, PCr in white muscle decreased by about 90%. In both exercise groups the total PCr plus Cr remained constant (Table 3.3.) (2) GLYCOGEN In fish subjected to the 10 sec sprint there was no significant change in white muscle glycogen or lactate (Table 3.1.) In direct contrast, the 10 and 30 min swimming fish util ized 95 and 98% of their endogenous glycogen store (Table 3.3.). A large fraction of the glycogen uti l ized appeared as muscle lactate. Muscle glucose increased 2.5 fold in the 10 min burst swimming fish but no change was observed in white muscle of the 30 min group (Table 3.4.). Assuming that 3 moles of ATP is produced per mole of glucosyl residue from glycogen to lactate, and knowing both the average 39 number of bursts (67± 4 SEM n=6) and the approximate duration of each burst (0.8 sec), the ATP turnover in white muscle during 10 min of burst swimming was estimated to be 78 u moles/g wet wt/min (Table 3.2.). The number of bursts for the 30 min swimming fish was too variable to allow such an estimate of ATP turnover to be made. NUCLEOTIDE AND PHOSPHATE POOL The individual nucleotide concentrations underwent no appreciable change in white muscle following the 10 sec sprint (Table 3.1.). However, major changes occurred during the 10 and 30 min exercise protocols (Table 3.3). One of the most striking was the 54 and 80% fa l l in ATP for the 10 and 30 min trout burst swimming groups respectively. This dramatic decrease in ATP appeared as a stoichiometric increase in IMP, with the total AMP content undergoing a much smaller percentage change (Table 3.3.). In this way, the total nucleotide pool remained essentially constant. The high Pj value for muscle in the pre-exercise state was confirmed by 3 1 P NMR (Parkhouse and Dobson, unpublished data), and was not considered an artifact of the colorometric method used, but more appropriately relate to the fish capture technique (see discussion). The concentration of Pi increased in both groups with exercise (Table 3.3.). 40 THERMODYNAMIC STRUCTURE OF THE GLYCOGENOLYTIC  PATHWAY IN TROUT WHITE MUSCLE The apparent deviation of the calculated mass action ratios from thermodynamic equilibrium in trout white muscle is presented in Fig. 3.1. It is clear that the mass action ratios of those reactions catalyzed by glycogen phosphorylase, Hk, PFK-1 and PK are more than two orders of magnitude displaced from thermodynamic equilibrium. Furthermore, no marked changes were apparent accompanying exercise except at the combined GAPDH-PGK and PGK catalysed steps (Fig. 3.1.). GLYCOLYTIC INTERMEDIATES AND POTENTIAL CONTROL  SITES The glycolytic intermediates in white muscle in the pre- and two exercise states are presented in Table 3.4. In trout burst swimming for 10 min significant changes in the concentrations of glycolytic intermediates were observed, and potential control sites along the pathway were identified at Hk, PFK-1 and PK (Fig. 3.2.). A large crossover also appeared at the combined GAPDH-PGK step (Fig.3.1.). According to this data, in conjuction with the thermodynamic structure of the pathway, it is reasonable to assume that the three most important regulatory enzymes of glycogenolysis in trout white muscle are glycogen phosphorylase, Hk, PFK-1 and perhaps PK. In those fish that underwent 30 min of 41 swimming, the glycolytic intermediates were extensively depleted, and no potential crossovers were apparent except that a greater magnification of change occurred at the combined GAPDH-PGK step (Table 3.4. and Fig. 3.2.). CYTOPLASMIC NAD/NADH RATIO AND MUSCLE PH The cytoplasmic NADVNADH ratios were calculated for the control and for the 10 and 30 min burst swimming trout, and the results obtained are presented in Table 3.5. The NADVNADH ratio increased 2.7 fold from 809 to 2176 in white muscle of the 10 min swimming fish, and increased 2.5 fold for the 30 min group . Intracellular pH of white muscle was 6.93 in the pre-exercise state and decreased to 6.66 and 6.47 for the 10 and 30 minute burst swimming fish respectively (Table 3.3.). CYTOPLASMIC FREE ADP AND PHOSPHORYLATION STATE Free ADP could not be calculated using the combined GAPDH-PGK equilibrium expression because as we have already discussed the reaction was shown to deviate markedly from equilibrium with exercise. Alternatively, free cytoplasmic ADP was calculated using the creatine kinase equilibrium and summarized in Table (3.5.). In the pre-exercise state, free ADP in white muscle was 0.069 u moles/g cell water, which represents about 8% or the total ADP content measured in neutralized PCA extracts (Table 42 3.3.). Free ADP increased about 5 fold during exercise to 0.357 and 0.328 u moles/g cell water for the 10 and 30 min burst swimming trout, which represents 27 and 23% of the total ADP content respectively (Table 3.5.). The phosphorylation potential in white muscle of trout was 4140 in the pre-exercise state and dramatically decreased to 205 and 110 for the 10 and 30 min burst swimming fish. Similarly, the ATP/ADP ratios decreased by over 90% to extremely low values in white muscle accompanying exercise. DISCUSSION COUPLING PCr HYDROLYSIS TO MYOSIN ATPase A fundamental observation of the study on fish white skeletal muscle is the unequivocal demonstration that PCr hydrolysis precludes anaerobic glycogenolysis during near maximal myofibril lar ATP ase activity (Table 3.1.). A conservative estimate of the ATP turnover supported by PCr hydrolysis was 188 u moles ATP/g wet wt muscle/min (Table 3.2.). This value may be as high as 598 u moles/g wet wt/min , since the PCr concentrations in white muscle reported in Table 3.1. are considered serious underestimates of the pre-exercise state prior to the 10 sec sprint. There are several reasons for believing this: First, the pre-exercise values were determined on f ish that had been captured by hand from black holding boxes. One inevitable consequence of this 43 procedure is that struggling results in a rapid loss of PCr, the rate of which has been shown to be proportional to the number of tai l f l ips (Mommsen, Stanely and Dobson, unpublished data). No struggling occurred in the sprint f ish during the transfer by net from the black box to the swim tunnel. Secondly, identical experiments carried out one week earlier on the same fish, but without sacrifice, revealed that the time to fatigue was 18 seconds. That 21 u moles PCr/g wet wt remained in white muscle after 10 sec (Table 3.1.) is entirely consistent with the view that about 40 u moles PCr/g would be present prior to the sprint. Thirdly, based on evidence from 3 1 P NMR, PCr values for a variety of vertebrate FG skeletal muscles 'at rest' are in the range of about 27 to 30 u moles/g wet wt, which represents about 77% of the total PCr and Cr pool (Burt et al., 1976; Meyer et al., 1982; Kushmerick and Meyer, 1985). This important in vivo information from other vertebrate fast -twitch skeletal muscles is consistent with the predicted value of around 40 u moles/g wet wt for trout since the total PCr and Cr pool is a much higher 58 u moles/g wet wt (Table 3.1.). In addition, assuming the free Pf concentration at rest is between 1 and 3 u moles/g wet wt (Kushmerick and Meyer, 1985), the analytically determined 15 u moles P./g muscle in trout added to the PCr value of 21 u moles/g also yields a s imilar estimate of around 40 u moles PCr/g wet wt in white muscle prior to the sprint. 44 COUPLING ANAEROBIC GLYCOGENOLYSIS TO MYOSIN ATPase That anaerobic glycogenolysis cannot pace the near-maximum myofibril lar ATPase in trout white muscle was a significant finding of the present study. The ATP turnover supported by anaerobic glycogenolysis over 10 minutes of burst swimming was estimated to be 78 u moles/g wet wt/min (Table 3.2.). This rate of ATP generation agrees wel l with the value of 90 u moles ATP/g wet wt trout white muscle/min at 10°C calculated on the basis of the maximal enzyme activity of the rate controlling enzyme , PFK-1 (Yamamoto, work in progress). The estimate of 78 u moles ATP/g wet wt/min for trout white muscle is also consistent with the rate of ATP generation from anaerobic glycogenolysis determined for in the isolated frog gastocnemius following intense electrical stimulation (Cori, 1956). BIOCHEMICAL CHARACTERIZATION OF THE  GLYCOGENOLYTIC PATHWAY There is l i t t le doubt that the development of fatigue or exhaustion in trout after 10 and 30 minutes of burst swimming was associated with the depletion of endogenous stores of muscle glycogen. That the onset of fatigue and exhaustion is linked to a near-depletion of muscle glycogen following high intensity 45 prolonged exercise has been reported for a number of vertebrates, including man (Gollnick et al., 1973; Armstrong et al., 1974; Baldwin et al., 1975; Gollnick, 1982). However, despite a s imilar etiology for the development of fatigue and exhaustion for the 10 and 30 min swimming trout respectively, a number of important differences were noted in the steady-state levels of their glycolytic intermediates, and the apparent functional integrity of the glycolytic pathway. Potential control sites along the pathway were identified by crossover analysis at Hk, PFK-1 and PK for the 10 min burst swimming fish (Fig. 3.2.). Because glycogen was largely depleted no crossover appeared at this locus. Even though anaerobic uti l ization of blood-borne glucose cannot support the same myofibril lar ATPase activity as glycogen because of the low catalytic potential of Hk (Johnston, 1977), the apparent crossover at Hk indicates increased uptake and phosphorylation of glucose at this time. The possible role of glucose util ization w i l l be discussed in the General Discussion Chapter 6. Facil itation of Hk activity may have occurred in response to the decrease in G 6-P (Table 3.4.), or in response to more subtle changes in the potent regulator, G 1,6-BP (Bietner, 1979). Explaining the apparent crossover at PFK-1 is more challenging. The finding that an increase in F 6-P and a decrease in F 1,6-BP concentration occurred after 10 min indicates that F 6-P is being delivered faster than PFK-1 can catalyze its conversion to products. Often fatigue has been related to the pH dependent ATP inhibition of 46 PFK-1, but this does not appear to be the case for trout white muscle. The 54% decrease in ATP would be expected to offset the inhibitory effect of pH, particularly in the presence of a number of positive modulators, P j f AMP and NH 4* (Table 3.3.). This proposal is also supported by the slight increase in the mass action ratio favouring F 1,6-BP formation. In direct contrast to the 10 min burst swimming fish, no crossover points were detected at either Hk or PFK-1 for the 30 min group because of the large decreases in glycolytic Intermediates in white muscle (Fig.3.2. and Table 3.3.). In fact, the sum of all of the glycolytic intermediates from G 6-P to pyruvate decreased by a dramatic 80% compared to the 25% decrease for the 10 min burst swimming fish. This near total depletion of glycolytic intermediates was accompanied by a 80% fal l in ATP, a 20 fold reduction in the ATP/ADP ratio, a 38 fold reduction in the cytoplasmic phosphorylation potential and a 2.5 fold increase in the NADVNADH ratio (Table 3.5.). Obviously, in the absence of the appropriate compensatory mechanisms, these major metabolic perturbations at exhaustion must have a profound effect on the kinase reactions of PCr hydrolysis and glycogenolysis. Interestingly, the dramatic fa l l in ATP was stoichiometrically matched with a rise in IMP (Table 3.3.). That IMP accumulates in trout white muscle following exercise provides direct evidence for the activation of AMP deaminase, and operation of the purine nucleotide cycle in this species (Lowenstein, 1972). The has also 47 been shown for carp white muscle (Driedzic and Hochachka, 1976). One interesting feature common to both fatigue states was the marked displacement of the combined GAPDH-PGK reaction from thermodynamic equilibrium (Figs. 3.1. and 3.2.). Because the combined GAPDH-PGK reaction is unique among the glycolytic steps by interacting with both the phosphorylation potential and redox couple (Scopes, 1973), reasons for the apparent shift from near-equilibrium with exercise are complex. Notwithstanding, one contributing factor appears to be the effect of change in the ATP/ADP ratio on the PGK equilibrium since the mass action ratio undergoes a major 20 and 41 fold reduction favoring 1,3-diphosphoglycerate formation for both the 10 and 30 minute swimming groups respectively (Fig. 3.1). Rovetto et al., (1975) proposed that inhibition of glycolysis in the ischemic myocardium was due to the effect of H + ion and l imiting supplies of NAD + on GAPDH. This explanation does not apply for trout white muscle at the two activity states since the NADVNADH increased, not decreased (Table 3.5.). The 2 to 3-fold increase in the free cytosolic NADVNADH ratio means that NAD + was being reduced by the GAPDH reaction at a slower rate than NADH was oxidized by LDH. The most likely explanation for this effect may be inhibition of PGK by the low ATP/ADP ratio which may have have an uncoupling effect on the GAPDH and LDH reactions, and in this way effect redox balance. Interestingly, the lactate/pryuvate ratio did not change significantly with exercise which means that the change in redox was largely affected by increasing H* ion (Table 3.5.). 48 Lastly, another observation that was common to both activity states was the crossover indicated at the PK locus. However this apparent crossover can be explained as a consequence of the LDH equilibrium. This w i l l be considered in more detail in the General Discussion Chapter 6. The main findings of this chapter can be summarized  as follows: (i) During supra-maximal work rates, the myosin ATPase was shown to be supported solely by PCr hydrolysis. The ATP turnover was estimated to be between 188 and 598 u moles/g wet wt muscle/min (ii) Fatigue after 10 min was related to the near-depletion of glycogen in white muscle. The glycolytic pathway appeared functional at this time with control sites being identified at Hk and PFK-1. PFK-1 did not appear to be inhibited by low muscle pH (pH 6.66) and was not considered causal to the onset of fatigue. The preferential fuel at this time was exogenous glucose. ( i i i ) Total exhaustion after 30 min of burst swimming was similarly related to glycogen depletion, but differed from the 10 min by showing a dramatic 80% reduction in the sum of glycolytic intermediates from G 6-P to pyruvate. No control sites were Identified along the pathway. (iv) The ATP concentration dramatically decreased in white muscle by 54 and 80% following the 10 and 30 min burst swims to 49 fatigue and exhaustion respectively. The total nucleotide pool remained constant through the operation of the purine nucleotide cycle. (v) In both fatigue states, the free cytosolic NAD+/NADH ratio and ADP increased. These regulatory parameters were considered not to be l imiting to glycogenolytic flux. (vi) The free cytosolic ATP/ADP ratio and phosphorylation potential decreased by over 70 to 80%. This was due to the increase in free ADP and the decrease in ATP. Associated with these changes was a marked displacement of the PGK, and the combined GAPDH-PGK reactions from thermodynamic equilibrium. (vii) The apparent crossover at PK was misleading and considered to be a consequence of the LDH equilibrium. TABLE 3.1. CHANGES IN FUEL AND NUCLEOTIDE CONCENTRATIONS IN TROUT FAST-TWITCH WHITE MUSCLE FOLLOWING A 10 SECOND SPRINT PHOSPHOCREATINE GLYCOGEN NUCLEOTIDE AND PHOSPHATE POOL A c t i v i t y PCr Cr Total Glycogen Lactate ATP ADP AMP IMP To t a l P i NH£ Pre-exercise (4) 27.03 31.89 58.92 20.40 1.79 +1.29 +0.92 +2.09 +0.92 +0.43 4.99 0.745 0.045 0.145 5.925 15.2-2 0.943 +.26 +.081 +.009 +.024 +.425 +0.56 +.162 10 Sec Sprint (6) 21.36 38.62 59.98 22.12 2.20 +0.76 +0.58 +0.73 +3.72 +0.37 (S) (S) (NS) (NS) (NS) 4.15 0.656 0.025 0.116 4.947 19.90 0.806 +.85 +.034 +.003 +.029 +.377 +1.76 +.124 (NS) (NS) (NS) (NS) (NS) (S) (NS) Metabolite concentrations (u moles/gm wet wt muscle) are given as the mean+SEM with the number of f i s h sampled i n parentheses. S t a t i s t i c a l s i g n i f i c a n c e (P) between the sp r i n t and c o n t r o l group was evaluated using the two-tailed Student's t - t e s t . P<0.05 (S) S i g n i f i c a n t (NS) Not s i g n i f i c a n t . TABLE 3.2. ATP TURNOVER IN TROUT FAST-TWITCH WHITE MUSCLE ACCOMPANYING HIGH INTENSITY SWIMMING ESTIMATED TIME TO MAJOR FUEL ATP TURNOVER E V E N T % V0 2 MAX FATIGUE UTILIZED u moles/g wet wt/min 10 sec Sprint 150 18 + 1.2 PH0SPH0CREATINE 188 - 598 (6) 10 min Burst 120 10+2.1 GLYCOGEN 78 (6) ATP turnover was calculated assuming 1 mole of ATP produced per mole of PCr hydrolysed, and 3 moles ATP produced per mole of glucosyl residues from glycogen. The number of f i s h sampled are i n parentheses. Estimates of percentage maximal oxygen consumption were calculated on the basis of a c r i t i c a l v e l o c i t y around 3 (see Materials and Methods). TABLE 3.3 CHANGES IN FUEL, NUCLEOTIDE CONCENTRATION AND pH IN TROUT FAST-TWITCH WHITE MUSCLE FOLLOWING A 10 AND 30 MINUTE BURST SWIM TO FATIGUE AND EXHAUSTION PH0SPH0CREATINE GLYCOGEN A c t i v i t y PCr Cr Total Glycogen Lactate Pre-exercise 19.83 31.50 51.33 22.40 5.76 (6) +0.92 +0.87 +1.37 +1.68 + .49 10 Min Burst 1.56 49.62 51.18 1.33 35.88 (6) + .59 +0.48 +2.29 + .61 +1.30 (S) (S) (NS) (S) (S) 30 Minute 1.51 51.39 52.90 0.38 33.39 Endurance + .85 +1.76 +1.35 + .08 +1.58 (6) (S) (S) (NS) (S) (S) NUCLEOTIDE AND PHOSPHATE POOL pH ATP ADP AMP IMP Total P i NH£ 7.334 0.738 0.063 0.304 8.439 25.72 1.030 6.93 + .291 + .034 + .017 + .080 + .355 +1.93 + .140 + .02 3.350 1.072 0.271 4.087 8.773 46.75 3.581 6.66 + .913 + .044 + .072 + .895 + .245 +2.81 + .508 + .02 (S) (S) . (S) (S) (NS) (S) (S) (S) 1.346 1.123 0.244 6.074 8.704 45.23 5.517 6.48 + .138 + .068 + .032 + .464 + .626 +1.83 + .319 + .04 (S) (S) (S) (S) (NS) (S) (S) (S) Metabolite concentrations (u moles/g wet wt muscle) are given as the mean+SEM with the number of f i s h sampL in parentheses. S t a t i s t i c a l s i g n i f i c a n c e (P) between the exercise and control f i s h was evaluated using the two-tailed Student's t - t e s t . P<0.05 (S) Sig n i f i c a n t (NS) Not s i g n i f i c a n t . 53 TABLE 3.4. GLYCOLYTIC INTERMEDIATES IN TROUT FAST-TWITCH WHITE MUSCLE FOLLOWING 10 AND 30 MINUTES OF BURST SWIMMING TO FATIGUE AND : EXHAUSTION Metabolite Pre-exercise 10 Min 30 Min Glucose 0.800+.060 2.101+.151 (S) 0.890+.110 (NS) Glucose 1-phosphate 0.175+.039 0.076+.009 (S) 0.025+.006 (S) Glucose 6-phosphate 0.455+.077 0.279+.118 (NS) 0.036+.014 (S) Fructose 6-phosphate 0.073+.009 0.157+.011 (S) 0.025+.019 (S) Fructose 1,6-bisphosphate 2.074+.251 1.145+.043 (S) 0.074+.010 (S) Dihydroxyacetone phosphate 1.001+.009 0.559+.073 (S) 0.330+.030 (S) Glyceraldehyde 3-phosphate 0.061+.021 0.100+.021 (NS) 0.118+.021 (NS) 1,3-diphosphoglycerate 0.097+.021 0.157+.029 (NS) 0.127+.018 (NS) 3-phosphoglycerate 0.661+.096 0.597+.137 (NS) 0.052+.013 (S) 2-phosphoglycerate 0.062+.007 0.048+.007 (NS) 0.023+.002 (S) Phosphoenolpyruvate 0.062+.007 0.052+.007 (NS) 0.011+.002 (S) Pyruvate 0.043+.012 0.397+.008 (S) 0.194+.037 (S) Lactate 5.76 + .49 35.9 + 1.3 (S) 33.4 + 1.6 (S) Metabolite concentrations (u moles/g wet wt muscle) are given as the mean+ SEM (n = 6). S t a t i s t i c a l s i g n i f i c a n c e (P) between the exercise and control group was evaluated using the two-tailed Student's t - t e s t . P<0.05 (S) S i g n i f i c a n t (NS) Not s i g n i f i c a n t . 54 TABLE 3.5. CYTOSOLIC FREE ADP, ATP/ADP RATIO, PHOSPHORYLATION POTENTIAL AND REDOX STATE IN TROUT FAST-TWITCH WHITE MUSCLE FOLLOWING 10 AND 30 MINUTES OF BURST SWIMMING TO FATIGUE AND EXHAUSTION Parameter Pre-exercise 10 Min 30 Min Free ADP 0.069+. 001 0.357+. ,069 (S) 0.328+. 070 (S) % Total ADP 7.7+ .41 26.5+ 4.7 (S) 22.6+ 3.8 (S) ATP/ADP 133+ 7.0 12+ 4. • (S) 5.0+ 1 . (S) ATP/ADP P i 4140+ 489 205+ 80 (S) 110+ 18. (S) NAD+/NADH 809+ 208 2176+ 464 (S) 2053+ 507 (S) Lactate/Pyruvate 192+ 53 129+ 41 (S) 164+ 27 (S) The cytoplasmic free ADP concentration was calculated from the measured t o t a l t i s s u e contents of the creatine kinase equilibrium (see Materials and Methods). A l l concentrations are expressed as u moles/g c e l l H 20 (n = 6). The ATP/ADP r a t i o and phosphorylation p o t e n t i a l (ATP/ADP Pj_) were calculated using free ADP. S t a t i s t i c a l s i g n i f i c a n c e (P) between each exercise group and control f i s h was evaluated using the two-tailed Student's t - t e s t . P<0.05 (S) Sign-i f i c a n t (NS) Not s i g n i f i c a n t . 5 5 F i g . 3.1. Graphical representation of displacement of i n d i v i d u a l g l y c o l y t i c reactions from thermodynamic equilibrium i n trout f a s t - t w i t c h white s k e l e t a l muscle. Thermodynamic equilibrium constants were determined at pH 7.0 and unit a c t i v i t y of water at 25°C. Unless otherwise s p e c i f i e d equilibrium constants are from Burton (1957). (a) Noltmann (1972) (b) Bohme et a l . , (1975) (c) Connett (1985) (d) Veech et a l . , (1979). M.A.R. i s the mass action r a t i o . 55a Phos PGM PFK TPI PGK Eno cr oo <D C M * 6 o o ir> to Hk PGI Aid GAP-PGK PGM LDH 1 v > f " > f > f \ r > f > f | > t 1 45. -° i f ) i f ) o b o C O 6 o 9 O X CO r-C M O i — X o I f ) o o C O C O 6 o C O PK >< CO CD C M < - 8 1 CD o c r 5 +6 0 t 10Min BURST SWIM TROUT W. MUSCLE - 8 1 + 6 T 30 Min ENDURANCE SWIM 56 F i g . 3.2. Crossover plot of g l y c o l y t i c intermediates i n trout f a s t - t w i t c h white s k e l e t a l muscle a f t e r 10 and 30 minutes of burst swimming to fatigue. Changes i n g l y c o l y t i c intermediates are expressed as a percentage of pre-exercise values. 56a G1-P F6-P DHAP 1.3DPG 2PGA PYR Gly G6-P F1.6P GAP 3PGA PEP LACT 57 CHAPTER 4: REGULATION OF ANAEROBIC  GLYCOGENOLYSIS IN THREE TYPES OF RAT SKELETAL MUSCLE DURING EXERCISE 58 CHAPTER 4: INTRODUCTION Having examined some aspects of regulation of anaerobic glycogenolysis in trout fast -twitch white muscle during both short-and long-term high intensity swimming to fatigue and exhaustion, an obvious extension was to investigate whether s imilar metabolic changes occurred in fast - twitch skeletal muscles of the rat during a s imilar set of exercise protocols. To a f i r s t approximation, the two muscle types most s imilar to trout white muscle in terms of biochemical and physiological properties are the two ankle extensors, the gastrocnemius and plantaris. The gastrocnemius is a mixed fast-twitch muscle comprising 58% FG, 38% FOG & 4% SO fiber types, while the plantaris is comprised of 53% FOG, 41% FG & 6% SO fibers (Ariano et al., 1973). A third muscle, the soleus, a SO fiber type, was selected to highlight any fiber specific mechanisms in the control of glycogenolysis that may occur during exercise and fatigue. Despite a great number of studies on the different biochemical strategies of the various muscle types of the rat to different running intensities and duration (Baldwin et al., 1973., Terjung et al., 1974; Baldwin et al., 1975; Fitts et al., 1982), no single study has focused in particular on the control of glycogenolysis at these different work intensities. The aim of this study was to investigate metabolic regulation of glycogenolysis in 3 different muscle types of the rat during a 2 min high intensity run, and following an endurance run to fatigue. 59 RESULTS GLYCOGEN. PHOSPHOCREATINE AND ATP TURNOVER Glycogen underwent approximately a 50% reduction in concentration in all three types of skeletal muscles following the 2 min high intensity run, and a dramatic 90 to 95% depletion following the endurance run to fatigue (Tables 4.1, 4.2 & 4.3). Assuming that 3 moles of ATP is produced per mole of glucosyl from glycogen to lactate, the ATP turnover was estimated for each muscle type and the results obtained presented in Table 4.4. The concentration of PCr decreased by 50% in the gastrocnemius and plantaris, and by 30% in the soleus accompanying the 2 min run. Further percentage reductions in PCr were observed following the endurance run to fatigue with a 60 to 65% fal l in the gastrocnemius and plantaris, and a modest 25% fa l l in the soleus (Tables 4.1, 4.2. & 4.3.). The concentration of P{ increased significantly in the gastrocnemius and plantaris during both exercise protocols, but not in the soleus. As expected, the sum of both PCr & Pj, and PCr & Cr concentrations for each muscle type remained constant with exercise. NUCLEOTIDES The concentration of ATP in the gastrocnemius and plantaris decreased only by 10 to 30%, with no significant change occurring in the soleus accompanying both running protocols. The total ADP 60 content in each muscle s imilarly did not change appreciably with exercise. AMP changed only slightly in all three muscle types with exercise. Because of the variable levels of IMP in each muscle at the pre-exercise state, larger percentage increases were observed in the gastrocnemius than in plantaris after the 2 min run. Greater increases were apparent following the endurance run with a 30 fold increase in IMP in the gastrocnemius and a 5 fold increase in the plantaris. No significant change in IMP concentration occurred in the soleus with exercise. The total adenine nucleotide pool remained constant in al l three muscles. NH 4 + concentration underwent a 2 to 4 fold increase in the soleus and gastrocnemius muscles respectively after the 2 min high intensity run but no significant change occurred in the plantaris. Endurance running to fatigue was associated with higher NH 4 + levels in the gastrocnemius (a 7 fold increase) and plantaris (a 2 fold increase), but not in the soleus (Tables 4.1., 4.2. & 4.3). THERMODYNAMIC STRUCTURE OF THE GLYCOGENOLYTIC  PATHWAY IN RAT SKELETAL MUSCLE Graphical representation of individual reactions of glycolysis showing their relative displacement from thermodynamic equilibrium in rat gastrocnemius is shown in Fig.4.1. The mass action ratios of those reactions catalyzed by HK and PFK-1 are displaced by over five orders of magnitude from thermodynamic equilibrium, while those catalyzed by glycogen phosphorylase and 6! PK are displaced by about two orders of magnitude. This thermodynamic structure of glycolysis did not appreciably change with exercise (Fig. 41.). Similar thermodynamic profiles were observed for the plantaris and soleus muscles, and have not been included. GLYCOLYTIC INTERMEDIATES AND POTENTIAL CONTROL  SITES The glycolytic intermediates of rat gastrocnemius, plantaris and soleus muscles at the three activity states are presented in Tables 4.5., 4.6. & 4.7. During the 2 min high intensity, and endurance run to fatigue, major changes were observed in many of the glycolytic intermediates and potential control sites were identified at glycogen phosphorylase and PFK-1 in al l three muscle skeletal muscles following the 2 min run, but only at HK following the endurance run to fatigue (Figs. 4.2., 4.3. 4.4). CYTOPLASMIC NAD/NADH RATIO AND MUSCLE PH The cytoplasmic NADVNADH ratio for rat gastrocnemius and plantaris is presented in Tables 4.8. &4.9. The NADVNADH ratio decreased by about 50 to 70% in both muscles accompanying the 2 min high intensity run. This pattern of change was not significantly altered following the endurance run to fatigue. That is, despite the plantaris becoming more reduced and stat ist ical ly significant relative to the control state, when compared with the 62 gastrocnemius at either of the two exercise states significance was lost due to the large standard errors (P < 0.05) [Table 4.9.]. The decrease in the NADVNADH ratio was accompanied by a five fold increase in the lactate/pyruvate ratio in both gastrocnemius and plantaris muscles following each of the exercise protocols. A smaller 2-fold increase in the lactate/pyruvate ratio was observed in the soleus after the 2 min run, and a 3 to 4- fold increase after the endurance run (Table 4.10.) Intracellular pH decreased from 6.92 to about 6.6 in both the gastrocnemius and plantaris skeletal muscles following the 2 min high intensity run, and to about 6.7 following the endurance run to fatigue (Tables 4.1., 4.2. & 4.3). Intracellular pH was not measured in the soleus. CYTOPLASMIC FREE ADP AND PHOSPHORYLATION STATE Free cytoplasmic ADP was calculated using the GAPDH-PGK-LDH equilibrium. In the pre-exercise state, free ADP was 0.060, 0.041 and 0.037 u moles/g cell water for the gastrocnemius, plantaris and soleus muscles respectively (Tables 4.8., 4.9. & 4.10.) These values represent about 5, 4 and 4% of the total ADP content measured (Tables 4.1., 4.2. & 4.3.). During the 2 min high intensity run, free cytoplasmic ADP increased about 3 fold in each of the three muscles, and between 4 to 6-fold following the endurance run to fatigue. In the latter group, these concentrations represent 29, 14 and 25% of the total ADP content measured. Free 63 ADP was not estimated using the CPK equilibrium because intracellular pH was not measured in the soleus. The cytoplasmic ATP/ADP ratios , and phosphorylation potentials were calculated using the free ADP from the combined GAPDH-PGK expression. The values obtained are summarized in Tables (4.8., 4.9. & 4.10.). In the pre-exercise state, the ATP/ADP ratio was 137, 175 and 127 for the gastrocnemius, plantaris and soleus muscles respectively. In al l muscles, the ATP/ADP ratio decreased by about 70% following the 2 min high intensity run, and by over 80% following the endurance run. Similarly, the phosporylation potential (ATP/ADP P}) dramatically decreased in each muscle following exercise but larger percentage drops occurred in the gastrocnemius and plantaris (Tables 4.8., 4.9. & 4.10.). DISCUSSION COUPLING GLYCOGENOLYSIS TO MYOSIN ATPase Estimates of the ATP turnover supported by glycogenolysis in rat gastrocnemius, plantaris and soleus following 2 min of high intensity running were calculated to be 30, 28, and 16 u moles/g wet wt muscle/min respectively (Table 4.4.). These values are conservative estimates and assume that the generation of ATP by glycogenolysis was totally anaerobic. If however there was a small but significant aerobic component to glycogen breakdown, the 64 estimates for each respective muscle would be considerably higher. This difference between the anaerobic and aerobic catabolism of glycogen is due to the higher ATP yield on a molar basis accompanying complete oxidation (McGilvery, 1983). If we make the assumption that 10% of the glycogen util ized was completely oxidized , the ATP generated per unit time would be 64, 61 and 34 u moles ATP/g wet wt/min for the gastrocnemius, plantaris and soleus respectively (Table 4.4.). This range of ATP turnovers calculated for the different rat skeletal muscles during short-term high intensity running is well within the theoretical maximum values predicted on the basis of in vitro maximal velocities of PFK-1. The maximum velocity of PFK-1 for rat plantaris is about 100 u moles F 1,6BP formed /gm wet wt muscle/min (unit) at 30°C, and about 23 units for the soleus (Baldwin et al., 1982). Because of s imilar percentages of fast-twitch fibers, the PFK-1 activity for the gastrocnemius was assumed s imilar to plantaris . After making the appropriate temperature correction assuming a Q 1 0 of 1.8, the maximum rate of ATP generation from glycogenolysis would be around 492 u moles ATP/g/min for plantaris and gastrocnemius, and around 113 u moles ATP/g/min for the soleus at 38°C. 65 VALIDITY OF USING ELECTRICAL STIMULATION STUDIES  IN INTERPRETING DIFFERENT MUSCLE RECRUITMENT  PATTERNS DURING EXERCISE Using the perfused rat hindquarter preparation, Spriet et al., (1985) demonstrated s imilar percentage decreases in glycogen content from the gastrocnemius and plantaris following 5 min of high frequency stimulation, but found no significant change occurring in the soleus. The soleus has long been considered an important postural muscle in the rat (see Armstrong and Laughlin, 1985), but the contrasting findings of the present study, and those of others (Gardiner et al., 1982) demonstrate unequivocally that the soleus is recruited to a very large extent during high speed locomotion. The marked discrepency between these results and those obtained using the isolated rat hindlimb preparation is believed due to the different muscle recruitment patterns and running gaits accompanying quadrapedal terrestrial locomotion. A point of emphasis is that care should be taken when extrapolating biochemical and physiological information from in situ electrical stimulation studies to whole animal exercise performance. REGULATION OF GLYCOGENOLYSIS DURING 2 MIN OF HIGH  INTENSITY RUNNING Using crossover analysis to identify the potential control sites along the pathway, it was apparent that glycogen phosphorylase and 66 PFK-1 are pivotal in coordinating glycogenolytic flux with the ATP requirements of the myofibril lar ATPase (Figs. 4.2., 4.3., & 4.4). One way that these enzymes achieve such a high degree of control over fuel selection is via the branchpoint regulation of G 6-P (Lueck and Fromm, 1974). That the concentration of G 6-P significantly increased in gastrocnemius, plantaris and soleus during the 2 min run (Tables 4.5., 4.6. & 4.7.), attests to the importance of this key metabolite to inhibit Hk, and select glycogen as principal fuel. This increase in G 6-P has also been confirmed in single rat skeletal muscle fibers accompanying electical stimulation (Hintz et al., 1982). Furthermore, accumulation of glucose in al l three muscles without an apparent crossover during the 2 min run also indicates inhibition of Hk at this time (Figs 4.2., 4.3. & 4.4. and Tables 4.5., 4.6. & 4.7.). Notwithstanding, an increase in G 1,6-BP may also be important in regulating the activity of Hk (Bietner, 1979). PFK-1 not only regulates the concentration of G 6-P, but on the basis of crossover analysis showing that F 6-P rises and F 1,6-BP decreases in al l three muscles accompanying an increase in glycogenolytic flux, suggests that indeed this is a major rate controlling step of the pathway. According to the original formulation of the crossover theorem of Chance and Williams (1956), an increase in substrate concentration and a decrease in product at one site along a multi-enzyme pathway indicates inhibition at this locus. However, this theorem as discussed in the Materials and Methods, Section 1, was originally proposed for the sequence of carriers in the mitochondrial electron transport chain 67 where the total concentration of each substrate-product pair is conserved at al l times. This property does not hold for the glycolytic pathway, particularly during high flux rates where control is leveled at multiple sites located at strategic points along the pathway. Thus a substrate-product pair at any one locus along the glycolytic pathway may vary in concentration at any time depending on the activity state of muscle. From this study It is clear that inhibition is not occurring at the PFK-1 locus, and that activation in this case is characterized by F 6-P rising in parallel with flux . This proposal supports the early work of Bucher and Russman (1964), later to be confirmed by Wilson et al., (1967) and Edington et al., (1973). An important question to consider is how does F 6-P increase during high flux rates? The most obvious reason is that glycogen phosphorylase is regulated to deliver F 6-P faster than PFK-1 can convert it to product. This makes a great deal of sense because an increase in F 6-P not only activates but more importantly stabilizes PFK-1 against pH-dependent ATP inhibition (Passoneau and Lowry, 1962; Mansour, 1972). The finding that F 6-P increases is also supported on theoretical grounds in light of glycogen phosphorylase having a greater maximal activity compared to PFK-1 (Harris et al., 1976; Baldwin et al., 1982). Thus faci l i tat ion of PFK-1 activity is probably brought about by feedforward activation by F 6-P, together with the complex interaction between the long l i st of positive modulators (Sols, 1981; Bosca et al., 1985). Superimposed on this basic system of metabolic control by 68 glycogen phosphorylase and PFK-1, are the ADP-requiring enzymes of PGK and PK. In order that glycogenolysis and myosin ATPase are tightly coupled in skeletal muscle during high work rates, both the activit ies of PGK and PK must be paced with the rate of product formation of ADP from ATP hydrolysis. The activity of PGK may further be controlled and coordinated by the non-equilibrium PK enzyme through regulation of metabolite levels which would have an impact further back along the pathway via the series of near-equilibrium catalyzed reactions. During the 2 min high intensity run the free cytosolic ADP concentration increased about 3 fold in each of the rat skeletal muscles (Tables 4.5., 4.6. & 4.7.). An important consequence of free ADP rising is that both the cytosolic ATP/ADP ratio and phosphorylation potential decrease. Following the 2 min run, the ATP/ADP ratio decreased by about 70 to 80% in the three muscles. Similar percentage decreases were seen in the phosphorylation potential. These ratios are important not only in the regulation of mitochondrial phosphorylation (Slater, 1976), but provide a direct index between ATP hydrolysis and the ATP generating mechanisms of glycogenolysis in the cytosol. This study demonstrates that glycogenolytic flux in skeletal muscle can be sustained in the face of wide variations of both the ATP/ADP ratio and phosphorylation potential. However, even though the rats were not fatigued after 2 minutes, al l indications on the basis of these low potentials suggest that fatigue was fast approaching. This prediction was borne out in a parallel set of validation studies carried out on 69 individuals from the same general stock runnning the same treadmill protocol. This experiment showed that a marked reduction in performance or fatigue occurred between 2 min 15 and 2 min 30 sec. Accompanying the fa l l in ATP in the gastrocnemius and plantaris, was a stoichiometric rise in IMP and NH 4 +. During exercise this irreversible reaction in skeletal muscle is catalyzed by the allosteric enzyme, AMP deaminase (Lowenstein, 1972). The maximal activity of AMP deaminase in rat skeletal muscle is about 200 u mol/g wet wt/min at pH 7.0 and 38°C (Goodman and Lowenstein, 1977). The reaction is believed to be activated by the dual effect of ADP and AMP availability via the myokinase equilibrium, and by the increase in H* ions generated from the hydrolysis of ATP at the myosin S 1 head (Sahlin, 1978; Dudley and Terjung, 1985). That no significant change occurred in the total AMP content with exercise in either the gastrocnemius and plantaris was not surprising, since like ADP, a large fraction of the total AMP is bound, and it is the free concentration that is catalytically important (Veech et al., 1979). Using the free ADP estimates shown in Table 4.8. & 4.9.., the concentration of AMP can be calculated using the myokinase equilibrium according to the following expression: AMP F R E E = [ADP]2/ATP. Keq , where the Keq is 1.05 at 38°C, 1 mM free Mg + + at an ionic strength of 0.25 (see Dudley and Terjung, 1985). Using this method, the free AMP increased from 0.42 to 4.0 n mol/g cell water in the gastrocnemius, and from 0.22 to 2.6 n mol/g cell water in the plantaris accompanying the 2 min 70 run. The 10 fold increase in the concentration of free AMP following short-term high intensity running, together with the increase in H + ion (a pH of about 6.62, Tables 4.1. & 4.2.) attests to the importance of both these factors in activating AMP deaminase at this time. If the myokinase reaction is indeed in equilibrium, as was assumed in the above calculations, then the freely available AMP accounts for only 0.5 and 1.4% of the total content of AMP in plantaris and gastrocnemius respectively at the pre-exercise state. These values increase to 6 and 14% of the total AMP respectively following the 2 min run. Thus it appears that virtually all the AMP in the muscle cell is compartmentalized away and not available to either the myokinase or AMP deaminase. However, unlike the well known binding of ADP to actin, there is no readily identifiable structural protein to which AMP binds in skeletal muscle. In recovery, many studies have shown that the adenine nucleotides in rat skeletal muscle are replenished via reamination of IMP through the action of adenolsuccinate sythetase and adenylsuccinate lyase (Aragon and Lowenstein, 1980). These two reactions together with the AMP deaminase reaction constitute the purine nucleotide cycle (Lowenstein, 1972). Although several functions have been attributed to the purine nucleotide cycle, recent evidence has shown that in rat skeletal muscle the complete cycle does not operate during moderate to intense contractile activity (Meyer and Terjung, 1979, 1980). Many studies have viewed the metabolic importance of the cycle's operation in muscle as (i) maintenance of a high ATP/ADP ratio, (ii) maintenance of 71 glycogenolytic flux through modulation of PFK-1 by NH 4 + ion, and ( i i i ) 'spark' the Krebs cycle by delivering fumarate during steady-state work or in recovery (Lowenstein, 1972, Meyer and Terjung, 1979, Dudley and Terjung, 1985). While not doubting the importance of these functions, the present author's interpretation for the primary function of the purine nucleotide cycle in fast -twitch skeletal muscle is to provide a sink for the loss of adenine nucleotide that occurs when the muscle fa i l s to balance ATP supply to ATP usage during high rates of muscle contraction. It performs this v ital function by forming a relatively metabolically inert compound, IMP. It is interesting that in heart muscle where a tight coupling between ATP supply and demand is maintained at al l times despite large fluctuations in workload, the purine nucleotide cycle has no functional importance (Taetgmeyer, 1985). Thus the role of the purine nucleotide cycle in fast - twitch skeletal muscle to control glycogenolysis or to provide fumarate to the Krebs cycle should be viewed as secondary consequences of the primary function. In direct contrast to these changes described for rat fast -twitch skeletal muscle, the ATP remained constant in the soleus (Table 4.3.). This has also been confirmed by electrical stimulation studies of Meyer and Terjung (1979) who showed that on intense stimulation of an isolated rat soleus even after the blood supply had been severely restricted, ATP fal ls only marginally with very l i t t le change in IMP. It appears therefore that unlike fast -twitch muscles, the soleus has a number of protective 72 mechanisms like the myocardium that ensure the ATP concentration is defended despite high workloads. As discussed in the general introduction and Chapter 3, control of glycogenolysis is also mediated by the cytosolic redox state. During the 2 min high intensity run, the NADVNADH ratio decreased in both the gastrocnemius and plantaris (Tables 4.8. & 4.9.). This decrease was accompanied by an increase in the lactate/pyruvate ratio. The disproportionate increase in the lactate/pyruvate ratio compared to the concomitant decrease in redox potential is due to the effect of increasing H + ion on the LDH equilibrium. Similar conclusions have been forwarded for rat skeletal muscle (Aragon and Lowenstein, 1980) and for human muscle following short-term high intensity exercise (Sahlin et al., 1976). CONTROL OF THE GLYCOGENOLYTIC PATHWAY AFTER  ENDURANCE RUNNING TO FATIGUE It was clear from the present study that the etiology of fatigue after 30 min of endurance running was s imilar to the 30 min endurance swim of the trout and linked to the near total depletion of muscle glycogen. The concentration of glycogen decreased by 94, 92 and 88% in the gastrocnemius, plantaris and soleus muscles respectively (Tables 4.1., 4.2. & 4.3.). That low glycogen and high lactate content was measured in all three muscles further demonstrates the large dependence of each muscle 73 type to this exercise intensity, as indeed was the case for the 2 min run. On the basis of crossover analysis no control points were located at glycogen phosphorylase and PFK-1 in the 3 skeletal muscles examined (Figs. 4.2., & 4.3.), with the possible exception of the soleus (Fig. 4.4.). An apparent crossover may have occurred at the PFK-1 locus in soleus but the small percentage changes in F 6-P and F 1,6-BP relative to control levels make 1t diff icult to draw any f irm conclusions. One obvious control site however was apparent at the Hk locus in al l three muscle types. Enhanced uptake and phosphorylation of glucose through faci l itat ion of Hk may be linked to either (i) Glycogen replenishment through the 3 step conversion from G 6-P to G 1-P, G 1-P to uridine diphosphate glucose, and finally the transfer of glucosyl units into the residues of the glycogen complex, or (ii) catabolism of glucose to pyruvate which itself can either be converted to lactate or undergo complete oxidation to C0 2 and H 20.. Accompanying the large reductions in the levels of glycolytic intermediates in the fast-twitch rat skeletal muscles, and to a lesser extent in the soleus, were large increases in free ADP (Tables 4.8., 4.9. & 4.10.). On the basis of these findings it was clear that fatigue was not related to ADP availability. However, the dramatic fa l l in both the ATP/ADP ratio and phosphorylation potential must obviously have had a significant effect on the control of glycogenolysis. The redox changes accompanying fatigue were also dramatic, with the muscle becoming more reduced. In 74 other words, NAD* may have become limiting to the GAPDH reaction and glycolysis at this time. The main findings of this chapter can be summarized  as follows: (i) During 2 min of high intensity, and endurance running to fatigue, the 50% decrease in muscle glycogen and high lactate levels in the fast-and s low-twitch fibers attests to the importance of recruiting anaerobic glycogenolysis to support myosin ATPase. Unlike trout white muscle, the rapid efflux of lactate from rat skeletal muscle during exercise (Hermansen, 1981) makes it more diff icult to assess the exact percentage contribution of anaerobic glycogenolysis to muscle work. (ii) Coordination of glycogenolytic flux during the 2 min run was achieved through the hierarchical regulation of glycogen phosphorylase and PFK-1. In this way, F 6-P was shown to increase with flux. As a point of emphasis, PFK-1 was totally operational in the fast-twitch muscles despite a muscle pH of 6.6. ( i i i ) Maintenance of glycogenolytic flux and running performance over the 2 min period was accompanied by a 3 fold increase in free cytosolic ADP, a 70 to 80% fal l in the ATP/ADP ratio and phosphorylation 'potential, and a 50% decrease in the NADVNADH ratio. Unlike fish white muscle the lactate/pyruvate ratios significantly increased. A slight fa l l in ATP occurred in both fast - twitch muscles and was matched with a stiochiometric rise in 75 IMP. No change in ATP occurred in the soleus despite its large anaerobic contribution to muscle work. (iv) The etiology of fatigue following the endurance run was the near-depletion of muscle glycogen in each of the three muscle types. Crossover analysis revealed faci l itat ion of Hk at this time with the concomitant switch in fuel from glycogen to exogenous glucose. The concentration of ATP continued to fa l l while free ADP continued to rise leading to further reductions in the ATP/ADP ratio and phosphorylation potentials. The NADVNADH ratio was low in gastrocnemius and plantaris but not stat ist ical ly different from the 2 min run. .TABLE 4.1. CHANGES IN FUEL., NUCLEOTIDE CONCENTRATION AND pH IN RAT GASTROCNEMIUS SKELETAL MUSCLE FOLLOWING A 2 MINUTE HIGH INTENSITY, AND ENDURANCE RUN TO FATIGUE PHOSPHOCREATINE GLYCOGEN NUCLEOTIDE AND PHOSPHATE POOL pH A c t i v i t y Pre-exercise (5) 2 Min Run (5) PCr Cr Total Glycogen Lactate ATP ADP AMP IMP Tot a l NH, 17.71 28.96 46.67 +.42 +.17 +.34 9.83 39.02 48.85 +.65 +.81 +.55 (S) (S) (NS) 38.56 2.58 +2.19 +.57 18.39 13.26 +1.50 +1.60 (S) (S) 6.570 0.915 0.031 0.046 7.56 14.10 0.231 +.077 +.036 +.005 +.007 +.05 +.28 +.057 5.301 0.864 0.029 0.654 6.85 +.318 +.026 +.004 +.196 +.34 (S) (NS) (NS) (S) (NS) 19.47 1.000 +0.92 +.253 (S) (S) 6.92 + .04 6.63 + .04 (S) 30 Minute 6.37 42.66 49.03 2.26 9.56 4.710 0.892 0.035 1.368 7.01 26.35 1.680 6.71 Endurance +.98 +1.3 +.99 +.19 +2.35 +.453 +.033 +.004 +.308 +.21 +1.21 +.121 +.04 (6) (S) (S) (NS) (S) (S) (S) (NS) (NS) (S) (NS) (S) (S) (S) Metabolite concentrations (u moles/g wet wt muscle) are given as the mean+SEM with the number of rats sampled i n parenetheses. S t a t i s t i c a l s i g n i f i c a n c e (P) between each exercise and con t r o l group was evaluated using the two-tailed Student's t - t e s t . P<0.05 (S) S i g n i f i c a n t (NS) Not s i g n i f i c a n t . TABLE 4.2. CHANGES IN FUEL, NUCLEOTIDE CONCENTRATION AND pH IN RAT PLANTARIS SKELETAL MUSCLE FOLLOWING A 2 MINUTE HIGH INTENSITY, AND ENDURANCE RUN TO FATIGUE A c t i v i t y PHOSPHOCREATINE GLYCOGEN NUCLEOTIDE AND PHOSPHATE POOL pH Pre-exercise (6) PCr Cr Total Glycogen Lactate 15.50 25.74 41.24 +1.49 +1.03 +2.13 32.90 3.22 +1.19 +.28 ATP ADP AMP IMP To t a l 5.731 0.871 0.045 0.211 6.86 +.15 +.056 +.004 +.058 +.25 P i NHJ 14.39 0.700 6.92 +1.37 +.250 +.07 2 Min Run (6) 8.39 35.24 43.63 +.99 +1.50 +1.00 (S) (S) (NS) 13.99 11.40 5.280 0.953 0.052 0.580 6.87 +1.21 +1.94 +.230 +.079 +.008 +.186 +.06 (S) (S) (NS) (NS) (NS) (NS) (NS) 17.91 0.744 6.62 +1.39 +.151 +.03 (S) (NS) (S) 30 Minute 5.67 38.57 44.24 2.53 8.70 4.404 1.060 0.081 1.165 6.71 23.63 1.596 6.68 Endurance +.90 +1.51 +1.41 +.15 +1.4 +.301 +.046 +.017 +.079 +.26 +0.54 +.127 +.07 (5) (S) (S) (NS) (S) (S) (S) (S) (NS) (S) (NS) (S) (S) (S) Metabolite concentrations (u moles/g wet wt muscle) are given as the mean+SEM with the number of rat s sampled i n parentheses. S t a t i s t i c a l s i g n i f i c a n c e (P) between each exercise and c o n t r o l group was evaluated using the two-tailed Student's t - t e s t . P<0.05 (S) S i g n i f i c a n t (NS) Not s i g n i f i c a n t . TABLE 4.3. CHANGES IN FUEL AND NUCLEOTIDE CONCENTRATIONS IN RAT SOLEUS SKELETAL MUSCLE FOLLOWING A 2 MINUTE HIGH INTENSITY, AND ENDURANCE RUN TO FATIGUE PHOSPHOCREATINE GLYCOGEN NUCLEOTIDE AND PHOSPHATE POOL A c t i v i t y PCr Cr Total Glycogen Lactate ATP ADP AMP IMP To t a l P i NH£ Pre-exercise (5) 9.29 22.11 31.40 24.70 3.84 +.93 +4.36 +1.85 +1.47 +.80 3.770 0.815 0.069 0.164 4.82 12.51 0.714 +.167 +.043 +.010 +.109 +.186 +1.06 +.070 2 Min Run (6) 6.59 25.94 32.53 +.75 +1.65 +1.58 (S) (NS) (NS) 14.48 7.26 3.940 0.847 0.076 0.115 +.85 +.51 +.157 +.029 +.018 +.024 (S) (S) (NS) (NS) (NS) (NS) 4.98 12.52 1.474 +.167 +0.47 +.385 (NS) (NS) (S) 30 Minute 6.87 24.86 31.73 3.01 7.73 3.753 0.764 0.075 0.151 4.74 13.39 1.539 Endurance +.24 +1.27+1.20 +.38 +.41 +.169 +.040 +.022 +.074 +.163 +0.99 +.131 (5) (S) (NS) (NS) (S) (S) (NS) (NS) (NS) (NS) ( NS) (NS) (S) Metabolite concentrations (u moles/g wet wt muscle) are given as the mean+SEM with the number of rats sampled i n parentheses. S t a t i s t i c a l s i g n i f i c a n c e (P) between each exercise and con t r o l group was evaluated using the two-tailed Student's t - t e s t . P<0.05 (S) S i g n i f i c a n t (NS) Not s i g n i f i c a n n t . TABLE 4.4. ESTIMATES OF ATP TURNOVER SUPPORTED BY GLYCOGENOLYSIS IN THREE DIFFERENT RAT SKELETAL MUSCLES DURING A 2 MINUTE HIGH INTENSITY RUN ATP TURNOVER, u moles/g wet wt/min MUSCLE TYPE 100% Conversion to Lactate 90% Conversion to Lactate 10% Complete Oxidation GASTROCNEMIUS 30 64 PLANTARIS 28 61 SOLEUS 1 6 34 ATP turnover was calculated assuming that 3 moles of ATP are produced per"mole glucosyl unit from glycogen to la c t a t e , and that 37 moles of ATP are formed per mole of glucosyl unit from glycogen to CO2 and H2O. For d e t a i l s see Discussion. 80 TABLE 4.5. GLYCOLYTIC INTERMEDIATES IN RAT GASTROCNEMIUS SKELETAL MUSCLE FOLLOWING A 2 MINUTE HIGH INTENSITY, AND 30 MINUTE ENDURANCE RUN TO FATIGUE Metabolite Pre-exercise 2 Min 30 Min -(5) (5) (6) Glucose 0.467+.052 1.080+.050 (S) 0.938+.146 (S) Glucose 1-phosphate 0.044+.009 0.079+.013 (S) 0.029+.003 (NS) Glucose 6-phosphate 0.784+.171 1.870+.250 (S) 0.458+.052 (NS) Fructose 6-phosphate 0.137+.073 0.367+.062 (S) 0.095+.011 (NS) Fructose 1,6-bisphosphate 0.348+.031 0.111+.021 (S) 0.049+.010 (S) Dihydroxyacetone phosphate 0.062+.005 0.048+.006 (S) 0.028+.003 (S) Glyceraldehyde 3-phosphate 0.028+.005 0.017+.002 (NS) 0.016+.001 (S) 1,3-diphosphoglycerate 0.030+.006 0.024+.001 (NS) 0.037+.011 (NS) 3-phosphoglycerate 0.182+.014 0.130+.011 (S) 0.120+.006 (S) 2-phosphoglycerate 0.013+.001 0.011+.004 (NS) 0.016+.004 (NS) Phosphoenolpyruvate 0.048+.004 0.029+.007 (S) 0.029+.005 (S) Pyruvate 0.089+.009 0.091+.009 (NS) 0.063+.006 (S) Lactate 2.58+.57 13.26+1.60 (S) 9.56+2.40 (S) Metabolite concentrations (u moles/g wet wt muscle) are given as the mean+ SEM with the number of rats sampled i n parentheses. S t a t i s t i c a l s i g n i f i c a n c e (P) between each exercise and control group was evaluated by using the two-t a i l e d Student's t - t e s t . P <0.05 (S) S i g n i f i c a n t (NS) Not s i g n i f i c a n t . 81 TABLE 4.6. GLYCOLYTIC INTERMEDIATES IN RAT PLANTARIS SKELETAL MUSCLE FOLLOWING A 2 MINUTE HIGH INTENSITY,AND 30 MINUTE ENDURANCE RUN TO FATIGUE Metabolite Pre-exercise 2 Min 30 Min (6) (6) (5) Glucose 0.460+.022 0.688+.147 (S) 0.649+.010 (S) Glucose 1-phosphate 0.065+.008 0.079+.007 (NS) 0.038+.001 (S) Glucose 6-phosphate 0.525+.038 0.678+.047 (S) 0.249+.029 (S) Fructose 6-phosphate 0.211+.019 0.300+.024 (S) 0.092+.009 (S) Fructose 1,6-bisphosphate 0.296+.033 0.186+.021 (S) 0.063+.006 (S) Dihydroxyacetone phosphate 0.074+.003 0.058+.004 (S) 0.028+.001 (S) Glyceraldehyde 3-phosphate 0.037+.006 0.023+.004 (NS) 0.026+.003 (NS) 1,3-diphosphoglycerate 0.029+.006 0.023+.003 (NS) 0.023+.004 (NS) 3-phosphoglycerate 0.203+.011 0.166+.006 (S) 0.139+.007 (S) 2-phosphoglycerate 0.028+.004 0.014+.004 (S) 0.005 (S) Phosphoenolpyruvate 0.063+.002 0.049+.003 (S) 0.043+.003 (S) Pyruvate 0.133+.014 0.112+.009 (NS) 0.076+.006 (S) Lactate 3.22+.28 11.40+1.90 (S) 8.70+1.40 (S) Metabolite concentrations (u moles/g wet wt muscle) are given as the mean+ SEM with the number of rat s sampled i n parentheses. S t a t i s t i c a l s i g n i f i c a n c e (P) between each exercise and control group was evaluated using the two-t a i l e d Student's t - t e s t . P<0.05 (S) S i g n i f i c a n t (NS) Not s i g n i f i c a n t . 82 TABLE 4.7. GLYCOLYTIC INTERMEDIATES IN RAT SOLEUS SKELETAL MUSCLE FOLLOWING A 2 MINUTE HIGH INTENSITY,AND 30 MINUTE ENDURANCE RUN TO FATIGUE Metabolite Pre-exercise 2 Min 30 Min (5) (6) (5) Glucose 0.971+.089 1.46+.06 (S) 1.71+.19 (NS) Glucose 1-phosphate 0.014+.. 005 0.018+.004 (NS) 0.023+.003 (NS) Glucose 6-phosphate 0.431+.071 0.695+.045 (S) 0.229+.026 (S) Fructose 6-phosphate 0.080+.017 0.158+.016(S) 0.097+.017 (NS) Fructose 1,6-bisphosphate 0.156+.024 0.132+.012 (NS) 0.058+.005 (S) Dihydroxyacetone phosphate 0.056+.003 0.043+.006(NS) 0.036+.003 (S) Glyceraldehyde 3-phosphate 0.036+.006 0.031+.001 (NS) 0.032+.003 (NS) 1,3-diphosphoglycerate 0.055+.007 0.026+.005 (S) 0.020+.008 (S) 3-phosphoglycerate 0.165+.010 0.164+.007 (NS) 0.188+.014 (NS) 2-phosphoglycerate 0.034+.006 0.028+.009 (NS) 0.020+.005 (NS) Phosphoenolpyruvate 0.065+.016 0.060+.004 (NS) 0.036+.005 (NS) Pyruvate 0.128+.012 0.117+.009 (NS) 0.081+.009 (S) Lactate 3.84+.80 7.26+.51 (S) 7.73+.41 (S) Metabolite concentrations (u moles/g wet wt muscle) are given as the mean+_ SEM with the number of rats sampled i n parentheses. S t a t i s t i c a l s i g n i f i c a n c e (P) between each exercise and control group was evaluated using the two-t a i l e d Student's t - t e s t . P 0.05 < (S) S i g n i f i c a n t (NS) Not s i g n i f i c a n t 83 TABLE 4.8. CYTOSOLIC FREE ADP, ATP/ADP RATIO, PHOSPHORYLATION POTENTIAL AND REDOX STATE IN RAT GASTROCNEMIUS FOLLOWING A 2 MINUTE HIGH INTENSITY,AND 30 MINUTE ENDURANCE RUN TO FATIGUE Parameter Pre-exercise 2 Min (5) 30 Min (6) Free ADP 0.060+.014 0.166+.022 (S) 0.260+.077 (S) % Total ADP 5.2+1.2 19.2+1.9 (S) 29.1+7.1 (S) ATP/ADP 137 +29 40 +6.6 (S) 23 +6.2 (S) ATP/ADP P i 7773 +1851 1643+408 (S) 698 +238 (S) NAD+/NADH 488 +144 154+19 (S) 165 +46 (S) Lactate/Pyruvate 30 +5.8 154+28 (S) 157 +42 (S) The cytoplasmic free ADP concentration was calculated from the measured t o t a l contents of the combined GAPDH-PGK-LDH equilibrium (see Materials and Methods). A l l concentrations are expressed as u moles/g c e l l H2O. The ATP/ADP r a t i o and phosphorylation p o t e n t i a l (ATP/ADP P-^ ) were calculated using free ADP. S t a t i s t i c a l s i g n i f i c a n c e (P) between each exercise and control group was evaluated using the two-tailed Student's t - t e s t . P<0.05 (S) S i g n i f i c a n t . 84 TABLE 4.9. CYTOSOLIC FREE ADP, ATP/ADP RATIO, PHOSPORYLATION POTENTIAL AND REDOX STATE IN RAT PLANTARIS FOLLOWING A 2 MINUTE HIGH INTENSITY, AND 30 MINUTE ENDURANCE RUN TO FATIGUE Parameter Free ADP % Total ADP ATP/ADP ATP/ADP P i NAD+/NADH Lactate/Pyruvate Pre-exercise (5) 0.041+.005 4.34+0.60 175+21 9729 +1443 532 +108 22 + 4 2 Min (6) 0.135+.026 (S) 13.43+3.05 (S) 49+9 (S) 2183+678 (S) 251+52 (S) 111+26 (S) 30 Min (5) 0.160+.028 (S) 13.95+2.68 (S) 34+11 (S) 1165+326 (S) 167+22 (S) 116+16 (S) The cytoplasmic free ADP concentration was calculated from the measured t o t a l contents of the combined GAPDH-PGK-LDH equilibrium (see Material and Methods). A l l concentrations are expressed as u moles/g c e l l H2O. The ATP/ADP r a t i o and phosphorylation p o t e n t i a l (ATP/ADP Pi) were calculated using free ADP. S t a t i s t i c a l s i g n i f i c a n c e (P) between each exercise and control group was evaluated using the two-tailed Student's t - t e s t . P<0.05 (S) S i g n i f i c a n t . 85 TABLE 4.10. CYTOSOLIC FREE ADP, ATP/ADP RATIO, PHOSPHORYLATION POTENTIAL AND REDOX STATE IN RAT SOLEUS FOLLOWING A 2 MINUTE HIGH INTENSITY, AND 30 MINUTE ENDURANCE RUN TO FATIGUE Parameter Pre-exercise (5) 2 Min (6) 30 Min (5) Free ADP 0.037+.006 % Total ADP 3.7+0.7 ATP/ADP 127+23 ATP/ADP P± 8121+1707 NAD+/NADH Lactate/Pyruvate 2 9 + 4 0.127+.026 (S) 12.0+2.4 (S) 3 9 + 8 (S) 2472+506 (S) 6 5 + 9 (S) 0.244+.068 (S) 24.7+5.9 (S) 1 9 + 4 (S) 1149+254 (S) 102+15 (S) The cytoplasmic free ADP concentration was calculated from the measured t o t a l contents of the combined GAPDH-PGK-LDH equilibrium (see Materials and Methods). A l l concentrations are expressed as u moles/g c e l l H2O. The ATP/ADP r a t i o and phosphorylation p o t e n t i a l (ATP/ADP P- l) were calculated using free ADP. S t a t i s t i c a l s i g n i f i c a n c e (P) between each exercise and con t r o l group was evaluated using the two-tailed Student's t - t e s t . P<0.05 (S) S i g n i f i c a n t . 86 F i g . 4.1. Graphical representation of displacement of i n d i v i d u a l g l y c o l y t i c reactions from thermodynamic equilibrium i n rat gastrocnemius s k e l e t a l muscle at three d i f f e r e n t a c t i v i t y states. Thermodynamic equilibrium constants were determined at pH 7.0 and unit a c t i v i t y of water at 25°C or 38°C (*). Unless otherwise s p e c i f i e d values for equilibrium constants were obtained from Burton (1957). (a) Fischer et a l . , (1971) (b) Noltmann, (1972) (c) Bohme et a l . , (1975)* (d) Connett, (1985)* (e) Veech et a l . , (1979)* M.A.R. i s the mass action r a t i o . 86a Phos *PGM PFK TPI PGM PK Hk PGI Aid GAPPGK Eno LDH 1 f > t f > f \f S <9 S ^ s o O" CO g S - o ' 0 X X § § g § g 5 « g s 9 | 87 F i g . 4.2. Crossover plot of g l y c o l y t i c intermediates i n rat gastrocnemius s k e l e t a l muscle following a 2 minute high i n t e n s i t y run, and a 30 minute endurance run to f a t i g u e . Changes i n g l y c o l y t i c intermediates are expressed as a percentage of pre-exercise values. ( O ) 2 Min run. ( • ) 30 Min run. 88 F i g . A.3. Crossover plot of g l y c o l y t i c intermediates i n rat p l a n t a r i s s k e l e t a l muscle following a 2 minute high i n t e n s i t y run, and a 30 minute endurance run to fa t i g u e . Changes i n g l y c o l y t i c intermediates are expressed as a percentage of pre-exercise values. ( O ) 2 Min run. ( • ) 30 Min run. 89 F i g . A.A. Crossover plot of g l y c o l y t i c intermediates i n rat soleus s k e l e t a l muscle following a 2 minute high i n t e n s i t y run, and a 30 minute endurance run to fatigue. Changes i n g l y c o l y t i c intermediates are expressed as a percentage of pre-exercise values. ( O ) 2 Min run. ( • ) 30 Min run. o [ I I I 1 I I I I 1 Gly Glu G6-P F1.6BP GAP 3 PGA PEP LACT G1-P F6-P DHAP 1.3DPG 2 PGA PYR 90 CHAPTER 5: PHOSPHQFRUCTOKINA5E CONTROL IN  MUSCLE: NATURE AND REVERSAL OF DH-DEPENDENT ATP INHIBITION 91 CHAPTER 5: PFK-1 CONTROL IN MUSCLE: NATURE AND  REVERSAL OF pH-PEPENDENT ATP INHIBITION INTRODUCTION For at least two decades it has been known that PFK-1 is highly sensitive to H + ions (Ui, 1966; Hofmann, 1976). The basis for this effect is the ATP-induced inhibition of PFK-1 catalysis ( Lardy and Parks, 1956). Although ATP is a substrate for the reaction (binds to a high-affinity catalytic site), at higher physiological concentrations of about 5 mM, it also is inhibitory [binds to a low-aff inity allosteric site(s)], and this inhibition is more pronounced at a pH below 7.2 (Lardy and Parks, 1956; Uyeda, 1979). According to Frieden et al., (1976), low pH increases the ratio of protonated to unprotonated ionization groups at the ATP binding site(s), which faci l i tates ATP binding and inhibits PFK-1. From these types of in vitro kinetic studies on purified PFK-1 and from studies on cel l - free muscle homogenates (Wu and Davis, 1981), the view has generally prevailed that glycolytic function is very pH sensitive over the physiological range because of the extreme pH sensitivity of PFK-1 (Hermansen, 1981; Connett et al., 1984). This rationalization is clearly an oversimplification in light of the study of Meyer et al., (1982a) on cat biceps showing that in the init ia l stages of recovery from fatigue (pH of 6.4), reactivation of glycolysis and muscle work occurred before the pH returned to its control value of around 7.1. The conclusion to be drawn from 92 these types of studies, and from the work presented in Chapter 3 & 4 on fish and rat skeletal muscle, is that low pH does not necessarily l imit glycolysis, and by implication means that mechanisms must exist in muscle to reverse or preclude inhibition of PFK-1 during exercise. Moreover, there are extreme cases in the literature where muscle pH has been reported to fa l l to 5.9 before there is any curtailment of either muscle work or glycolysis (Bailey and Seymour, 1983). The aim of this study was to re-investigate the kinetic and regulatory properties of purified rabbit muscle PFK-1 in an attempt to clar ify how this enzyme can achieve significant catalytic rates in vivo despite fall ing pH. RESULTS AND DISCUSSION INTERACTING PH. ATP AND FRUCTOSE 2.6-BP EFFECTS The effects of F 6-P on the activity of rabbit muscle PFK-1 in the presence of either 1.0 or 5.0 mM ATP, or 5 mM ATP plus 10 uM F 2,6-BP at various pH values are shown in Figs. 5.1., 5.2. & 5.3. The kinetic and regulatory properties determined under these conditions are summarized in Table 5.1. and Fig. 5.4. As the pH is lowered from 7.67 to 6.8 at 25°C, the apparent Km or S Q 5 for F 6-P increases fourfold at 1 mM ATP with a 5 to 10% change in Vmax (Fig. 5.1; Table 5.1.). Over this pH range the nature of F 6-P binding to PFK-1 displays typical Michaelis Menten-type kinetics with 93 corresponding Hil l coefficients of around 1.0 (Table 5.1.). However, at the higher physiological concentrations of ATP, the effect of pH on PFK-1 catalysis is markedly different (Fig. 5.2.), that is, decreasing the pH from 7.67 to 6.8 at 25°C causes a dramatic decrease in the affinity for F 6-P and a shift from hyperbolic to increasingly sigmoidal reaction kinetics. Although the estimates of S Q g values are admittedly subject to greater error at low pH, the data indicate a 50-fold increase in the 5 0 5 value at pH 6.8 compared with pH 7.67 (Table 5.1.). In contrast, the Vmax under these conditons is s imilar to that at 1.0 mM ATP, with only modest changes occurring as a function of pH. An increased regulatory behavior of rabbit muscle PFK-1 at low pH and high ATP is commonly observed for PFK-1 isolated from a variety of other tissues and animal species (Uyeda, 1979; Sols, 1981). The pH-dependent inhibiton of PFK-1 catalysis at 5.0 mM ATP, however can be nearly completely abolished in the presence of 10 uM F 2,6-BP with an accompanying dramatic leftward shift in the F 6-P saturation curve, and lower Hil l coefficient (Fig. 5.3.). In other words, in the presence of 10 uM F 2,6-BP and inhibitory concentration of ATP, the rabbit muscle enzyme behaves as if the ATP concentration was low at any given pH. Conversely, at high ATP concentration and at a low pH of 6.8, PFK-1 behaves in the presence of 10 uM F 2,6-BP as if the pH had been increased to about 7.15, or a 0.35 pH unit shift at any given F 2,6-BP concentration (ca Figs. 5.2. & 5.3). A point of emphasis is that the catalytic response of PFK-1 to F 2,6-BP is most pronounced at inhibitory concentrations of ATP 94 and low pH. That F 2,6-BP exerts l i t t l e or no effect at pH values above 7.4 or low ATP is consistent with the view that its role is to offset the pH-dependent ATP inhibition of PFK-1 catalysis. MULT I MODULATORY EFFECTS The previous experiments established the conditions of interaction between three modulators of PFK-1: FT ions, ATP and F 2,6-BP. Since PFK-1 is influenced by numerous other metabolites and ions ( Passoneau and Lowry, 1962; Mansour, 1972; Bloxham and Lardy, 1973; Sols, 1981), it was of interest to assess the relative importance of some of these to release PFK-1 from ATP inhibition over a s imilar pH range. It is clear from Fig. 5.5. that at physiological concentration of F 6-P (0.1 mM), the most potent single de-inhibitor (or activator) of rabbit muscle PFK-1 is F 2,6-BP. The second most powerful activator is G 1,6-BP (50 uM) which is about 60% as effective as F 2,6-BP. AMP (0.5 mM), already known to reverse ATP inhibition at low pH in frog muscle (Trivedi and Danforth, 1966), along with NH 4 + ion (5.0 mM) or P{ (20 mM) each contribute about 40% the activation potential of F 2,6-BP. When combined, however, G 1,6-BP and AMP together or AMP, NH 4 + and Ps together act in synergism and account for about 100% and 80% of the effectiveness of F 2,6-BP, respectively (Fig. 5.5.). That F 2,6-BP is the most powerful activator of PFK-1 over the physiological pH range is in complete agreement with the recent study of Uyeda et al., (1981). 95 Interestingly, one important development that emerged from the present study was that the apparent Ka of F 2,6-BP was about 100-fold lower than that for G 1,6-BP at pH 7.0; i.e. the apparent K a of rabbit muscle PFK-1 for F 2,6-BP is 30 nM compared to 4 uM for G 1,6 BP with respective Hi l l coefficients of 1.1 and 2.1 (Fig. 5.6.). Citrate is another important modulator of PFK-1 catalysis (Kemp and Foe, 1983). Its inhibitory action is synergistic with ATP, and the mechanism of inhibition is believed to involve a depolymerization of the active tetrameric enzyme into inactive dinners (Lad et al., 1973; Goldhammer and Paradies, 1979). The inactive dinners have been termed 2HE 2 by Bock and Frieden (1976 a,b). Because the effect of citrate is clearly dependent on pH and ATP concentration, it was of interest to examine the nature and extent of citrate inhibition in the presence of the positive modulators under physiological conditions. At 5.0 mM ATP and 0.1 mM F 6-P, citrate (0.5 mM) exerts a strong inhibitory effect (50% loss of activity at pH 7.0) when either AMP (0.5 mM), NH 4 + (5.0 mM), or Pj (20 mM) is present, compared with the 25% loss of activity in the presence of F 2,6-BP (10 uM) or G 1,6-BP (50 uM) (Table 5.2.). However, several combinations, F 2,6-BP and AMP or AMP, Pj and NH 4 + together, stabil ize PFK-1 against citrate inhibition (< 5% loss of activity). This stabilization of PFK-1 against citrate inhibition by F 2,6-BP, G 1,6-BP, and the other postive modulators adds to the growing l ist of regulatory functions for these effectors under near physiological conditions. 96 INTERPRETATION AND PHYSIOLOGICAL SIGNIFICANCE These new kinetic data impact on our understanding of the mechanisms underlying the regulatory behavior of PFK in vertebrate skeletal muscle. With regard to the mechanism of interaction between H + ions, ATP and PFK-1 catalysis, an important point to emphasize is that at inhibitory physiological concentrations of ATP (5.0 mM), the ATP dependence of the reaction is most pronounced over the pH range between 6.8 and 7.0. This observation strongly suggests that binding of ATP to the site(s) of regulation on PFK-1 depends on the protonation of imidazole groups which have a pK of about 6.9 at 25°C. This interpretation agrees well with the conclusion of Bock and Frieden (1976 a,b) and Frieden et al., (1976). These workers further proposed that PFK-1 exists in at least two forms, termed E 4 and HE4. According to their kinetic model, ATP binds preferentially to HE4, the protonated tetramer; whereas F 6-P, F 1,6-BP and AMP preferentially bind to E4, the unprotonated tetramer. Thus, the effect of change in pH is to alter the ratio of unprotonated/protonated tetrameric forms, with the binding of ATP being favoured at low pH. As a f i r s t approximation, it is assumed that adding F 2,6-BP to the reaction cuvette leads to a drop in the apparent pK of the ATP binding site(s) to about 6.6 or even lower. In terms of the ratio of unprotonated to protonated forms of the enzyme, this effect is equivalent to increasing the pH of the medium by about 0.35 pH units (Figs 5.2. & 5.3.). This shift in 97 equilibrium favoring EA formation greatly faci l itates F 6-P binding while at the same time reducing the allosterlc binding of ATP (Frieden et al., 1976), and thereby reducing or reversing the effect of ATP inhibition at low pH. Notwithstanding, a s imilar interpretation adequately explains the action of G 1,6-BP to reverse the ATP inhibiton of PFK catalysis. The effect of citrate is rather more complex. The data presented demonstrate that, in the presence of either F 2,6-BP or G 1,6-BP or a combination of F 2,6-BP with AMP or AMP, Pi and NH 4 + together, PFK-1 catalysis is partially or fully stabilized against citrate inhibition. This finding suggests that the presence of positive modulators not only increases the ratio of unprotonated to protonated forms of the enzyme but stabilizes the E 4 form by protecting it against citrate inhibition, which may involve a change in the polymerization state (Lad et al., 1973, Kemp and Foe, 1979). In conclusion, the strong inhibitory effect of physiological concentrations of ATP on PFK-1 catalysis, in metabolic terms, must be considered to be one of the most fundamental kinetic and regulatory properties of the muscle enzyme. This study has highlighted several mechanisms of modulation that could allow PFK-1 to function in a physiological pH range that has long been considered to be inhibitory. In effect, any modulator that increases the ratio of unprotonated to protonated forms of the tetrameric enzyme could supply the muscle cell with a means of controlling the pH dependent ATP inhibition of PFK catalysis. Aside from the controlling effect of H + per se. the modulators thought to be most 98 effective in shifting the equilibrium towards the E^form in muscle include F 2,6-BP, G 1,6-BP, F 1,6-BP, AMP, NH 4 + and ? v One of the most striking aspects of PFK-1 regulation in white skeletal muscle of f ish highlighted in the present study was the decrease in the ATP concentration which in effect minimizes the need for multimodulation by the traditional set of positive effectors. The relevance of these and other metabolic interactions in trout and rat skeletal muscle during exercise w i l l be discussed in the General Discussion, Chapter 6. TABLE .5.1. Kinetic and regulatory properties of rabbit muscle phosphofructokinase as a function of pH in the presence of 1.0 mM and 5.0 mM ATP, and 5.0 mM ATP with 10 uM fructose 2,6-P2 at 25°C S . or K fructose 6-P (uM) H i l l coeff ic ient, n o.5 m ' pH 7.67 7.25 7.0 6.8 7.67 7.25 7.0 6.8 1.0 mM ATP 50 71 100 218 1.0 1.0 1.0 1.0 ^ 5.0 mM ATP 72 133 707 4500 1.5 1.85 2.0 5.0 mM ATP + 59 63 86 138 2.6 2.6 1.5 1.0 10 uM F-2,6-P 2 The values presented were calculated from the data shown in Figs. 1, 2 and 3 and as described in Materials and Methods. 100 TABLE 5.2. Effect of modulatorCs) to stabi l ize rabbit muscle phosphofructoklnase against c i trate inhibit ion ~cc .. r \ Percentage decrease of act iv i ty txrector\si_ after addition of 0.5 mM c i trate AMP (0.5 mM) 52 P (20 mM) 53 NH* (5 mM) 53 F-2,6-P 2 C10 uM) 25 G-l,6-P 2 (50 uM) 27 F-2,6-P 2 + AMP less than 5.0 AMP, P^ and NH* less than 5.0 Assay conditions: 50 mM Hepes-KOH, pH 7.0 at 25°C and physiological concentrations of ATP (5.0 mM) and F-6-P (0.1 mM) 101 F i g . 5.1. E f f e c t of fructose 6-phosphate on a c t i v i t y of p u r i f i e d rabbit muscle phosphofructokinase-1 at various pH values i n the presence of 1.0 mM ATP. Assay conditions are described i n Materials and Methods: Section 3. Fructose 6 - P (mM) 102 F i g . 5.2. E f f e c t of fructose 6-phosphate on a c t i v i t y of p u r i f i e d rabbit muscle phosphofructokinase at various pH values i n the presence of 5.0 mM ATP at 25°C. Assay conditions are described i n the Materials and Methods: Section 3. 103 F i g . 5.3. E f f e c t of fructose 6-phosphate on a c t i v i t y of p u r i f i e d rabbit muscle phosphofructokinase at various pH values i n the presence of 5.0 mM ATP with 10 uM fructose 2 bisphosphate at 25°C. Assay conditions are described i n Materials and Methods: Section 3. Fructose 6 - P (mM) 10.4. F i g . 5.4. Change i n apparent K M or S Q ^ of p u r i f i e d r a b b i t muscle phosphofructokinase for fructose 6-phosphate as a function of pH at 25°C. Data plotted from fructose 6-phosphate saturation curves shown i n F i g s . 5.1., 5.2. & 5.3. 3.8 105 F i g . 5.5. E f f e c t of modulators on the a c t i v i t y of p u r i f i e d rabbit muscle PFK-1 over the p h y s i o l o g i c a l pH range. The pH p r o f i l e s were determined at 25°C i n the presence of 0.1 mM F 6-P and 5.0 mM ATP ( • ), 5.0 mM ATP with ei t h e r 0.5 mM AMP, 20 mM P ± or 5.0 mM NH£ ( © ); 5.0 mM ATP with 50 uM glucose 1,6-bisphosphate ( A ); 5.0 mM ATP with 0.5 mM AMP, 20 mM P ± and 5.0 mM NH4+ ( O ), 5.0 mM ATP with 10 uM Fructose 2,6-bisphosphate or 50 uM glucose 1,6-bisphosphate and 0.5 mM AMP ( 8 ). Assay conditions described i n Materials and Methods: Section 3. 106 F i g . 5.6. E f f e c t of varying fructose 2,6-bisphosphate and glucose 1,6-bisphosphate on a c t i v i t y of p u r i f i e d rabbit muscle PFK-1 at 25°C at p h y s i o l o g i c a l concentrations of F 6-P (0.1 mM), ATP (5.0 mM) i n 50 mM HEPES-KOH buffer, pH 7.0 at 25°C. Assay conditions are described i n Materials and Methods: Section 3. 25 c E i O CO CD 2 0 15 o E =* 10 OHh Fructose 2,6-P 2 F2,6-F2» GI.6-R KQ 30 nM 4JUM n .10 2.10 j i i M i n i J I J I • • l I I I I Glucose l ,6-P 2 J i i i i 1 1 1 1 o 03 J I 1_J 10 10' I0 ; 10' Fructose 2 ,6 -P 2 or Glucose l , 6 - P 2 (nM) 107 CHAPTER 6: GENERAL DISCUSSION 106 CHAPTER 6: GENERAL DISCUSSION FUELS AND SHORT-TERM GENERATION OF ATP The near-maximum ATP turnover in trout white skeletal muscle was estimated to be between 188 and 598 u moles/g wet wt/min (Table 3.4). This range was in close agreement with the in vitro maximum velocity of myofibril lar ATPase determined for rabbit muscle (Bendall, 1961). PCr hydrolysis was the principal pathway uti l ized at this time with l i t t le or no contribution from anaerobic glycogenolysis. That PCr hydrolysis precludes anaerobic glycogenolysis in trout muscle (Table 3.1.) supports the earlier work on isolated frog skeletal muscle during tetanic stimulation (Danforth, 1965). While not doubting that the same temporal pattern of fuel and pathway activation exists in mammalian skeletal muscle during maximal work rates, published data dealing with this question are rare owing to methodological problems of sampling mammalian skeletal muscle and complex fiber heterogeneity (Karlsson, 1980). The general conclusion to be drawn from these types of studies is that when the myosin ATPase is activated maximally, creatine kinase outcompetes the kinase reactions of glycogenoysis for the ADP being released at the myosin 51 head. That creatine kinase has preferential access to the products of ATP hydrolysis during maximal work rates may relate to the different kinetic properties of the muscle enzyme (Vmax, Km for ADP), and to its juxtaposition with myosin ATPase (Bessman and Carpenter, 109 1985). It is not until the rate and force of contraction is lowered that the relative contribution of anaerobic glycogenolysis to support the myofibril lar ATPase becomes more important. This general pattern of preferential fuel uti l ization in skeletal muscle during sub-maximal high intensity exercise was shown for trout swimming at approximately 120% V0 2 max for 10 minutes (Chapter 3) , and for the rat treadmill running at high speed for 2 minutes (Chapter 4). In the case of trout white skeletal muscle, the ATP turnover supported by anaerobic glycogenolysis was estimated to be 78 u moles/g wet wt/min (Table 3.2.), while that determined for both rat gastrocnemius and plantaris muscles was estimated to be 64 and 61 u moles/g wet wt/min respectively(Table 4.4.). The assumption used for these latter estimates for the rat was that 10% of the glycogen util ized was completely oxidized (see Chapter 4) . The ATP turnover estimated for the soleus was lower than that for each of the fast-twitch muscles (Table 4.4.), but since this muscle is predominately comprised of s low-twitch fibers, the aerobic component to glycogen util ization may have been higher than the 10% assumed, and therefore minimizing the differences between the three muscle types examined. Nonetheless one f irm conclusion that can be made from this study on the rat was that both fast- and s low-twitch muscles contributed heavily to anaerobic generation of ATP during short-term high intensity running (Tables 4.1., 4.2. & 4.3.). The advantage of recruiting 1 10 anaerobic over aerobic metabolic pathways is that these systems can sustain higher myosin ATPase activit ies over shorter periods of time. Differences in the anaerobic potential of skeletal muscle among different species of the animal kingdom must obviously reflect the differences in locomotory needs associated with complex predator-prey interactions occurring in their natural environment. GLYCOGENOLYSIS: THE PATHWAY The thermodynamic structure of the glycogenolytic pathway in trout fast-twitch white skeletal muscle, and in the three skeletal muscle types of the rat showed no major differences with exercise and appeared highly conservative (Figs 3.1 & 4.1.), except at the combined GAPDH-PGK and the PGK equilibria in trout white muscle (Fig. 3.1.). This information, together with the published kinetic properties for each of the glycolytic enzymes provides strong support for the proposal that glycogen phosphorylase, Hk, PFK-1 and perhaps PK are the most likely candidates for regulating and coordinating glycogenolytic flux in muscle of both species. Aldolase, however, displayed a mass action ratio quite different from its equilibrium constant in all of the rat skeletal muscles, but to a lesser extent in fish white muscle. This apparent difference can be rationalized in terms of aldolase reacting to a specific beta anomeric or acyclic form of F 1,6-BP which represents a small fraction of the total F 1,6 BP content measured in muscle (Midelfort 111 et al .,1976). In view of Its high catalytic potential, and preferential binding to specific forms of F 1,6-BP, aldolase is generally believed to operate near-equilibrium in mammalian muscle (Reynolds et al., 1971; Connett, 1985 ) . CONTROL OF GLYCOGENOLYSIS DURING SHORT-TERM  HIGH-INTENSITY EXERCISE AND FATIGUE Even though the exercise protocol for the 10 min burst swimming trout was not directly comparable to the high intensity run for the rat, a number of interesting s imi lar it ies and differences in the control of glycogenolysis emerged. On the basis of crossover analysis, control sites were identified at Hk and PFK-1 in f ish white (Fig 3.2.), and glycogen phosphorylase and PFK-1 in al l three muscle types of the rat (Figs. 4.2, 4.3. & 4.4.). As discussed in Chapter 3, the apparent crossover at Hk in fish white muscle was obviously linked to the near-depletion of endogenous glycogen. According to the conventional view, the concomitant decrease in G 6-P concentration has a strong de-inhibitory effect on Hk activity with the subsequent increase in uptake and phosphorylation of blood borne glucose (Leuck and Fromm, 1974). Available evidence suggests that this de-inhibition of Hk is brought about by the direct binding of G 6-P to an allosteric or regulatory site on the enzyme (Collowick, 1973). 1 12 Indeed, if the apparent K; of Hk for G 6-P in fish muscle was in the low mM range, then the decrease in G 6-P from 0.455 to 0.279 u moles/g wet wt/ min accompanying 10 minutes of swimming, would indeed confirm its importance in controlling glucose phosphorylation. The role of glucose phosphorylation in trout white muscle following exercise is uncertain but may either assist to replenish glycogen stores or alternatively be fluxed through the glycolytic pathway to provide ATP for recovery metabolism. It should be pointed out that glycogen replenishment in trout white muscle is a slow process and can take up to 24 hrs after strenuous exercise (Black et al., 1962). In contrast to white muscle of trout, the endogenous stores of glycogen in the three rat skeletal muscles following the 2 min high intensity run did not become limiting (Tables 4.1. 4.2., & 4.3.). This has also been reported for humans following short-term high intensity running to fatigue (Gollnick et al., 1973; Gollnick, 1982). This study indicated that glycogenolytic flux in rat skeletal muscle was maintained and coordinated by the action of glycogen phosphorylase and PFK-1. That no crossover occurred at Hk is indicative of the importance of both these enzymes to regulate G 6-P and suppress glucose phosphorylation at this time. A striking analogy between trout and rat skeletal muscle was the finding that F 6-P increased and F 1,6-BP decreased following high intensity exercise. That an apparent crossover was evident in the direction described above in trout white muscle, suggested 113 that PFK-1 was not causal to the fatigue process. Substantiation of this claim also comes from the presence of a number of positive modulators at this time (AMP, Pi & NH 4 +), whose synerg ist ic action, together with an increased F 6-P concentration, would be expected to defend PFK-1 activity in trout white muscle despite fall ing pH. Moreover, the 55% decrease in ATP concentration itself would be expected to play a major role in minimizing the pH-dependent ATP inhibition of PFK-1 catalysis (Table 3.3.). The situation regarding PFK-1 regulation In rat skeletal muscle was less complicated. The parallel increase in F 6-P with glycogenolytic flux in muscle confirms the earlier observations of Bucher and Russman (1963), Wilson et al., (1967) and Edington et al., (1973). As outlined in detail in Chapter 4, the relative accumulation of reactants and depletion of products at the PFK-1 locus, means that delivery of F 6-P at pH 6.6 was occurring at a faster rate than the enzyme could catalyze its conversion to F 1,6-BP. This proposal was consistent with the maximum catalytic capacities of both glycogen phosphorylase and PFK-1 (Harris et al., 1976; Baldwin et al., 1982). During the 2 min high intensity run, PFK-1 in rat muscle may be considered rate l imiting to pathway flux. On the basis of the work presented on the regulation of rabbit muscle PFK-1 described in Chapter 5, the statement that PFK-1 is completely inhibited at low pH can no longer be made with confidence. FT ions should not be thought of as inhibiting PFK-1 cer se. but more appropriately, should be viewed as a specific 1 14 mechanism of control, since pH-dependent inhibition depends upon the non-catalytic role of ATP binding. That ATP binding can be dramatically modified by the presence of positive modulators either singly or in combination was clearly demonstrated in Fig. 5.5 . It was shown that the action of positive modulators is to offset the pH-dependent ATP inhibition of PFK-1 in such a way as to broaden the pH profile into the physiological range that is often reported to be inhibitory to the enzyme. The most potent activators of PFK-1 are the recently discovered F 2,6-BP (Hers and VanShaftigen, 1982), and G 1,6-BP (Hofer and Pette, 1968; Bietner, 1979), but as yet their precise role in skeletal muscle during exercise remains to be clearly established. Notwithstanding, because both modulators have been shown to increase in several working muscle preparations in a way that is consistent with their apparent K for activation (Fig 5.6; Hofer and Pette, 1968; Hue et al., 1982; Storey, 1983), it may be assumed that these regulators play an important role in activating or maintaining glycogenolytic flux in working muscle. PYRUVATE KINASE Another intriguing aspect of the present comparative analysis of glycogenolytic control in skeletal muscle was the occurrence of an apparent crossover at PK in trout fast-twitch white muscle but not in the rat (Figs. 3.2. and 4.2., 4.3. & 4.4.). In contrast to working ! 15 mammalian skeletal muscle, trout retains lactate in white muscle for periods up to 24 hours after strenuous exercise (Black et al., 1962). The phenomenon of lactate retention in fish white muscle is an unresolved question but may be due to an inadequate circulation during high intensity exercise. However, this proposal does not explain why it takes up to 24 hr for trout white muscle to reach pre-exercise lactate levels since blood flow has been shown to increase following strenuous exercise (Neumann et al., 1983). A more plausible explanation of lactate retention in trout white muscle during recovery relates to differences in the properties of the membrane to lactate transport. As a consequence of the near-equilibrium reaction catalyzed by LDH, the high lactate levels reported in this study for trout white muscle accompanying short-term exercise (Table 3.3.) must be counterbalanced by high pyruvate levels if near-equilibrium is to be maintained. This is particularly important since the other reactants and products of the LDH reaction, the redox potential and H +ion concentration, undergo smaller percentage increases (Table 3.5.). Thus in this way, increasing pyruvate leads to an apparent but misleading crossover at pyruvate kinase in trout white muscle. An extreme case among the fishes has been reported for skipjack tuna where lactate in white muscle may reach levels as high as 80-100 u moles/g wet wt with a concomitant rise in pyruvate to about 1.0 u mole/g wet wt muscle (Guppy et al., 1979). 1 16 NAD/NADH CYCLES In order that glycogenolytic flux is maintained in working skeletal muscle during exercise an adequate supply of NAD + is required. As outlined in the introduction, this is accomplished during short-term high intensity exercise by the high activit ies of GAPDH and LDH. One of the most challenging aspects of this study was to provide an explanation for the contrasting cytosolic redox potentials in skeletal muscle of the fish and the rat accompanying short-term high intensity exercise (Tables 3.5., 4.8., 4.9. & 4.10.). The redox state in the cytosol of trout fast-twitch white muscle became more oxidized with exercise, while the cytosol in rat fast -twitch skeletal muscles became more reduced. Since the NADVNADH ratio is coupled to both the ATP/ADP and ATP/ADP. P, ratios through the combined GAPDH-PGK-LDH equilibria (see Veech et al., 1979), the high NADVNADH ratio in trout white muscle should be associated with a high ATP/ADP ratio if al l the enzymes remain at near equilibrium and tightly coupled. As we see from Table 3.5., this prediction was not borne out, and the increased NADVNADH ratio in trout white muscle may be linked to an apparent uncoupling between GAPDH-PGK and LDH. This was supported by the large deviation of the mass action ratio from the thermodynamic equilibrium constant at this time (Fig. 3.1.). In addition, since the lactate/pyuvate ratio did not significantly change with exercise, increase in the NADVNADH ratio in trout white skeletal muscle 1 17 primarily was due to the increase in H + ion (See Chapter 3). In direct contrast, the thermodynamic integrity of the glycolytic pathway in rat skeletal muscle remained unchanged with exercise, with each of the respective dehydrogenases catalyzing near-equilbrium reactions (Fig. 4.1.). That is, the decrease in the cytosolic NADVNADH ratio occurred concomitantly with a decrease in the ATP/ADP ratio and phosphorylation potential in the gastrocnemius and plantaris decreased following the 2 min high intensity run. The redox changes reported in this study for rat skeletal muscle support the previous electrical stimulation studies on the rat hindlimb preparation of Aragon and Lowenstein (1980), and human skeletal muscle following strenuous exercise (Sahlin et al., 1976). Similarly, these studies demonstrated that the increased reduction of the cytosol was also accompanied by a rise in the lactate/pyruvate ratio (see Tables 4.8. & 4.9.). Of particular relevance to this paradox between redox changes in skeletal muscle of the fish and rat are the early fluorometric studies of Jobsis and Duffield (1967) and Jobsis and Stainsby (1967) carried out on skeletal muscle of the frog, toad and dog. These workers found that in response to electrical stimulation (about 5 muscle twitches/sec) NADH in all muscles consistently went oxidized. Even though this change in redox is identical to that described for trout white muscle, the results according to Jobsis and coworkers are not directly applicable since they claim that the fluorescent signal detected was from mitochondrial NADH and not from the cytosol. The exact basis for this discriminating effect 118 between these two compartments in muscle however has never been fully clarified. A few years later, Edington et al., (1973) examined the changes in the free cytosolic redox state in a rat hindlimb preparation following 30 sec of intense electrical stimulation. The interesting aspect of this study was the finding that accompanying electrical stimulation, the cytosolic redox potential became more oxidized. The difference between this preparation and the perfused rat hindilimb preparation of Aragon and Lowenstein (1980) was that the former was devoid of an adequate blood supply and behaved as an isolated system. Thus the rat hindlimb muscle in the study of Edington et al., (1973) behaved as fish white skeletal muscle. The 6 fold increase in the NADVNADH ratio reported was due to the retention of lactate in muscle and subsequent increase in pyruvate associated with the LDH equilibrium. The increase in pyruvate would also lead to an apparent crossover at the PKJ locus, as discussed in the previous section for fish. It seems probable that like trout skeletal muscle, this phenomenon may be either a consequence of membrane limitations to lactate transport or an inadequate supply of blood necessary to remove lactate as it is formed during high work rates. Similarly, a 10 fold increase in pyruvate and high lactate levels have been reported in cat gastrocnemius after 30 seconds of tetanus (Wilson et al., 1967). The NADVNADH ratio in this case increased from 575 ± 8.5 (n = 2) to 748 ± 26 SEM (n = 3), and was accompanied by a 2 fold increase in the lactate/pyruvate ratio. These calculations assume that the intramuscular pH fel l from 7.0 to 6.6. A recent study on human I 19 muscle metabolism also confirms that the muscle cytosol can become oxidized in vivo during maximal treadmill exercise (Cheetham et al., 1985). On the basis of the lactate/ pyruvate ratios reported, and assuming the pH of the cytosol decreased from 7.0 to 6.6, the free NADVNADH ratio was calculated to increase from 524 to 830 following the sprint. The conclusion to be drawn from the work presented in this thesis on fish and rat, as well as from the above in vitro and in vivo electrical stimulation and human performance studies, is that it appears that the redox state of muscle depends primarily upon the rate of which lactate is retained or effluxed from skeletal muscle. Thus, in tetanically stimulated or maximally working vertebrate muscle, pyruvate concentrations rise in response to the increase in lactate via the LDH equilibrium, and the cytosol init ial ly becomes more oxidized. The increase In pyruvate may be transient and primarily depends on the rate at which lactate is effluxed from the cytosolic compartment of muscle into the blood. That the cytosol may become more oxidized in fish and mammalian skeletal muscle at the onset of heavy exercise can be considered an important regulatory mechanism of glycogenolysis because it means that the increased NAD + availability faci l i tates flux via mass action effect at the level of the GAPDH reaction. 120 FREE ADP AND ATP/ADP RATIO AND PHOSPHORYLATION  POTENTIAL Free cytosolic ADP does not become limiting to glycogenolysis during short-term high intensity exercise in either fish or rat "skeletal muscle (Tables 3.5., 4.8., & 4.9. & 4.10.). The increase in the concentration of free cytosolic ADP in both species was consistent with the apparent Km values of PGK and PK being in the low mM range (Veech et al., 1979). The levels of free ADP fal l in the most sensitive range of each of the respective enzyme's ADP saturation curve. As emphasized throughout this thesis, one consequence of increasing free ADP is that both the ATP/ADP and ATP/ADP Pf ratios fal l . For each of the rat skeletal muscles both ratios decreased dramatically to about 30% of their pre-exercise values (Tables 4.8., 4.9. & 4.10.). This effect was further magnified in trout white skeletal muscle because of the 55% decrease in ATP (Table 3.3.). One conclusion to be drawn from the study on the rat during the 2 min high intensity run, was that anaerobic glycogenolysis and running performance was maintained in spite of the large percentage swings in the cytoplasmic ATP/ADP ratio or phosphorylation potential in both fast - and s low-twitch skeletal muscles. 121 CHARACTERIZATION OF THE GLYCOGENOLYTIC PATHWAY  AND REGULATION FOLLOWING ENDURANCE EXERCISE TO  FATIGUE AND EXHAUSTION The general etiology of fatigue for both the trout and the rat following exercise to complete exhaustion and fatigue respectivley, was the near-depletion of intramuscular stores of glycogen (Tables 3.3. and 4.1., 4.2. & 4.3.). Compared to the 10 min burst swimming trout, those trout selected to swim the endurance protocol to exhaustion displayed no apparent crossovers in white muscle with the exception at the combined GAPDH-PGK equilibrium which has already been discussed in detail in the previous section. As noted in Chapter 3, the sum of all the glycolytic intermediates from G 6-P to pyruvate decreased by a dramatic 80% compared to the 25% decrease in white muscle for the 10 min burst swimming fish. This near total depletion of glycolytic intermediates was accompanied by a 20 fold reduction in the ATP/ADP ratio, a 38 fold reduction in the phosphorylation potential, and a 2.5 fold incresase in the NADVNADH ratio (Table 3.5.). Singly, the most striking change of any single parameter in this study was the dramatic 80% fal l in ATP in trout white muscle which places this species in a unique position among the vertebrates (Table 3.3.). in a parallel set of studies, we have evidence that the concentration of ATP may fal l to levels as low as 0.50 u mol/g wet wt at exhaustion, which represented about 5% of 122 the total ATP content measured in the pre-exercise state (Dobson, Mommsen and Hochachka, work in progress). The reason why greater percentage reductions in ATP concentration occurred in trout fast-twitch muscle relative to the fast-twitch muscles of the rat, must obviously be linked to differences in the degree of PCr depletion accompanying exercise. As PCr stores become depleted, the creatine kinase reaction can no longer maintain ATP constant, and a mismatch between ATP supply and ATP usage develops. This proposal was supported from the data presented in Table 3.3. and Tables 4.1. & 4.2. showing that the PCr levels in rat fast - twitch muscles decreased by about 75%, compared to a 96% reduction in trout white muscle. That fish can nearly deplete their high energy stores, and drop their ATP levels to low levels, probably relates to their high degree of locomotory specialization designed to achieve fast-start performances in predator-prey interactions, and permitting supra-maximal feats while swimming against strong currents or up waterfalls during long migratory journeys. Another possibility may be the introduction of a psychological component to exercise performance in higher vertebrates, with the rat simply refusing to run "when the going gets tough". A recent study on thoroughbred racehorses galloping maximally to fatigue, provides additional support for a psychological component to exercise, because in these animals, muscle ATP was shown to fa l l by over 50% (Snow et al., 1985). The 'w i l l to win" in these elite performers is undoubtedly a key factor to racing success. The dramatic percentage reduction in ATP in white muscle 123 after both 10 and 30 minutes of swimming in trout was stoichiometricallu matched with a concomitant rise in IMP. As PCr supply in muscle becomes limiting, the subsequent rise in free cytosolic ADP (Fig. 3.5.) leads to the activation of the myokinase reaction via mass action effect (Noda, 1973). The AMP formed then provides substrate for the AMP deaminase reaction, and in the presence of a low pH (pH optimum for AMP deaminse is 6.1 to 6.5; Setlow and Lownestein, 1967) leads to the subsequent conversion to IMP and NH 4 + (Lowenstein, 1972). The free AMP in trout white muscle in the pre-exercise state was calculated to be 0.5 n moles/gm cell water and increased 58- and 122 fold following the 10 min and 30 min swimming protocols to fatigue and exhaustion respectively. These free estimates represent 0.8, 1 1 and 25% of the total AMP content measured in trout white skeletal muscle (Table 3.3.). Even though no such dramatic fal ls in the concentration of ATP were reported for each of the rat skeletal muscles, activation of AMP deaminase however was apparent, and discussed in detail in Chapter 4. One difference however between the trout and rat, was that the total content of AMP in skeletal muscle significantly increased in trout white muscle, but not in either of the fast-twitch skeletal muscles of the rat. An explanation for this difference is not readily forthcoming, and requires further studies on the role of AMP compartmentation in vertebrate skeletal muscle at different activity states. Lastly, it is important to emphasize that the primary function of the purine nucleotide 124 cycle in vertebrate skeletal muscle must unequivocally be to provide a sink for the dramatic loss of adenine nucleotide at a time when the muscle fa i l s to balance ATP supply to ATP demand during high rates of contraction. This is achieved by an elaborate set of equilibrium and non-equilibrium reactions that conserve the loss of adenine nucleotide in the form of IMP, and thereby maintaining the total nucleotide pool constant. Restoration of ATP levels in muscle after strenuous exercise occurs during recovery (see Driedzic and Hochachka, 1978). In contrast to trout following 30 min of exhaustive exercise, a potential control site was apparent at the Hk locus for each of the rat skeletal muscles after endurance running for 30 min (Figs. 4.2., 4.3. & 4.4.). That Hk appeared to be facil itated at this time was s imilar to the case described for trout white muscle after 10 minutes of swimming (Fig. 3.2.). On the basis of the present data it is d i f f icult to decide the preferential route for glucose phosphorylation occurring in each of the muscle types. However, in view of the fact that no other apparent crossovers were identified along the glycolytic pathway, and that the concentration of many of the glycolytic intermediates fe l l below 50% their control values in the fast - twitch skeletal muscles (Tables 4.5 and 4.6.) and to a lesser extent in the soleus (Table 4.7.), suggests that activation of glycolysis at this time may have been precluded. Thus the role of glucose phosphorylation was probably glycogen replenishment. In support of this proposal, Hollozy and coworkers have demonstrated increases in glucose uptake (up to 10 fold) lasting many hours after 125 exercise for both skeletal muscles of the frog and rat hindlimb, and concluded on the basis of glycogen accumulation, that the major fate of glucose disposal was glycogen replenishment (Hollozy and Narahara, 1965; Ivy and Hollozy, 1981). To complete the analysis of glycogenolytic control in rat skeletal muscle, the NAD VNADH and ATP/ADP ratios, and the phosphorylation potential in each muscle type following 30 min of treadmill running to fatigue changed in s imilar directions to that described in detail for the 2 min running group except that the values for the ATP/ADP ratio and ATP/ADP P{ were considerably lower (Tables 4.8., 4.9. & 4.10). In summary, it may be speculated that in conjunction with the low ATP/ADP ratio and phosphorylation potential in rat skeletal muscle following the 2 min run, and certainly following the 30 min run to fatigue, the glycogenolytic pathway may become limited by NAD* 126 CHAPTER 7: SUMMARY AND CONCLUSIONS 127 CHAPTER 7: SUMMARY AND CONCLUSIONS It was clear from this study that the highest ATP turnover in skeletal muscle was supported solely by PCr hydrolysis. Despite anaerobic glycogenolysis possessing a lower maximal ATP generating potential than PCr hydrolysis, it has the advantage of not being so constrained by time, and can be recruited to extend muscle performance at sub-maximal workloads from seconds to minutes. Fatigue after 10 min of burst swimming in trout was related to the near-depletion of glycogen in white muscle. The glycolytic pathway appeared to be functional at this time with control sites being identified at Hk, PFK-1 and PK. PFK-1 did not appear to be inhibited by low muscle pH (6.6), and was considered not causal to the onset of fatigue. In addition to the presence of a number of positive modulators (AMP, P{ & NH 4 +), the pH-dependent ATP inhibition of PFK-1 catalysis was partially offset by the lower ATP concentration (a 55% decrease). Furthermore, inhibition of glycogenolysis at fatigue was not due to ADP or NAD + availability. One consequence of the decrease in ATP and increase in ADP concentrations, was the concomitant fa l l in both the ATP/ADP ratio and phosphorylation potential (70 to 80%). Total exhaustion after 30 min of burst swimming was similarly related to near-glycogen depletion, but differed from the 10 min group by showing a dramatic 80% compared to a 25% reduction in the sum of glycolytic 128 intermediates from G 6-P to pyruvate. No control sites were identified along the pathway except at PK. Moreover, ATP concentration in white muscle dramatically decreased by 80%. Despite the large decrease in this adenine nucleotide, the total nucleotide pool remained constant through the activation of myokinase and AMP deaminase with the subsequent formation of IMP and NH 4 +. Like the 10 min burst swimming group, the free cytoplasmic NADVNADH ratio and ADP increased in white muscle of trout after 30 min of burst swimming, and was considered not l imiting to glycogenolytic flux at this time. Associated with these changes was a marked displacement of the PGK and combined GAPDH-PGK reactions from thermodynamic equilibrium. It was suggested that displacement from equilibrium may be due to the effect of the low ATP/ADP ratio on the PGK reaction. The conclusion to be drawn from this study on rainbow trout white muscle was that inhibition of glycogenolytic flux accompanying fatigue was not due to the inhibition of PFK-1, or to the availability of either ADP or NAD+. The second phase of the study was designed to investigate whether s imilar patterns of change occurred in three different types of rat skeletal muscle following a s imilar set of exercise protocols. Even though the 2 min high intensity run was not directly comparable to the 10 min burst swim of trout, a number of interesting s imi lar it ies and differences emerged, particularly at PFK-1. Coordination of glycogenolytic flux in skeletal muscle of the non-fatigued rat running for 2 min run was achieved through the 129 hierarchical control of glycogen phosphorylase and PFK-1. Glycogen was the principal fuel and no apparent crossover occurred at Hk. This was likely due to the inhibitory effect of increasing concentrations of G 6-P on Hk. Accompanying the increase in glycogenolytic flux was an increase in F 6-P and a decrease in F 1,6-BP. A point of emphasis was that PFK-1 was fully operational despite an intracellular pH of 6.6 in rat fast - twitch skeletal muscles. Maintenance of flux, and running performance, was accompanied by about a 3 fold increase in free cytosolic ADP, a 70 to 80% decrease in ATP/ADP ratio and phosphorylation potential, and a 50% decrease in the NADVNADH ratio. Similar percentage changes were apparent in the s low-twitch soleus muscle. A slight fa l l in ATP occurred in both fast-twitch skeletal muscles, and was matched with a stoichiometric rise in IMP and NH 4 +. No change in ATP occurred in the soleus despite a heavy reliance on anaerobic ATP generating processes. In contrast to the 10 min burst swimming fish, the glycogen concentration in each of the rat skeletal muscles remained high (50% decrease). In conjunction with a parallel series of experiments demonstrating that fatigue was indeed fast-approaching (another 15 to 30 sec), it seems highly unlikely that glycogen supplies would become limiting at this time. Other l imiting factors must be involved in the fatigue process after 2 min. Of the regulatory parameters examined, the cytoplasmic ATP/ADP, phosphorylation potential, or l imiting supplies of NAD+ may be likely candidates. In accord with the etiology of fatigue and exhaustion of trout after 30 min of endurance swimming, was the 130 finding of near-depletion of glycogen in each of the three skeletal muscles of the rat following the endurance run. Crossover analysis revealed faci l i tat ion of Hk at this time with the concomitant switch in fuel from glycogen to exogenous glucose. The concentration of ATP continued to fa l l , while free ADP continued to rise, resulting in large percentage reductions in the ATP/ADP ratio and phosphorylation potential. The NADVNADH ratio was low in the gastrocnemius and plantaris but not stat ist ical ly different from the condition after the 2 min high intensity run. The third phase of the study was to understand how PFK-1 can achieve high catalytic rates in working muscle despite fall ing pH. Evidence was presented that strongly suggested that inhibition of PFK-1 activity was not causal to fatigue in trout white muscle after 10 min of burst swimming, and that the enzyme was fully operational at an intramuscular pH of about 6.6 in the two rat fast -twitch skeletal muscles accompanying short-term high intensity running. From a detailed kinetic analysis on purified rabbit muscle PFK-1, it was concluded that any modulator that increases the ratio of unprotonated to protonated forms of the tetrameric enzyme, could supply the muscle cell with the means of controlling the pH-dependent ATP inhibition of PFK catalysis. An important conclusion to be drawn from this study was that H + ion should not be thought of as inhibiting PFK-1 per se. but more appropriately, should be viewed as a specific mechanism of control. It was shown that positive modulators exert their effect on PFK-1 catalysis in such a way as to broaden the pH profile of the enzyme 131 Into the physiological range that has often been considered inhibitory. One of the most challenging aspects of the present study was to provide an explanation for the contrasting cytosolic redox potentials in skeletal muscle of trout and the rat accompanying short-term high intensity exercise. The redox state of the cytosol in trout white muscle became more oxidized, while that in both rat fast -twitch skeletal muscles became more reduced. It was concluded that this opposite direction of change with exercise relates to species-specific differences in the abil ity of skeletal muscle to retain lactate. When lactate is retained, as in the case of trout white muscle, pyruvate increases due to mass action effect at the LDH equilibrium. Indeed, a literature survey showed at the onset of heavy exercise, mammalian skeletal muscle may behave as a trout white muscle, with the the muscle cytosol becoming more oxidized. This finding was considered an important, and often overlooked, mechanism of glycogenolytic control. In effect, it means that during short-term sub-maximal workloads, the increased availability of NAD+ may be considered as a catalytic potentiator of flux through mass action effect at the GAPDH reaction. Another consequence of lactate retention in skeletal muscle, was the apparent crossover identified at PK in fish white skeletal muscle at both fatigue states. This was misleading and caused by rising pyruvate brought about by the same mechanism decribed above for the redox changes. Another contrasting feature between the f ish and the rat 132 accompanying exercise was the precipitous fa l l in ATP concentration in trout fast -twitch skeletal muscle. The reason for this large difference must obviously be linked to differences in the degree of PCr depletion in skeletal muscle. As PCr stores become limiting, the creatine kinase reaction can no longer maintain ATP constant, and a mismatch between ATP supply and ATP usage develops. Evidence was provided that this was indeed the case, with PCr concentrations fall ing by 75% and over 95% in the rat and f ish skeletal muscles respectively. It was concluded that this difference in the degree of PCr depletion may reflect the locomotory specialization of trout to its l ifestyle, but species-specific differences in the psychological response to high intensity running performance can not be discounted. A general conclusion to be drawn from this comparative analysis of glycogenolytic control in trout white, and in three types of rat skeletal muscle, was that fatigue should be viewed as a multi-component process in response to l imiting glycogen, and not leveled at any one particular step of the glycogenolytic pathway. 133 REFERENCES Ainsworth, S and N. MacFarlane (1973). A kinetic study of rabbit muscle pyruvate kinase. Biochem. J. 131 223-236. Aragon, J.J. and J.M. Lowenstein (1980). The purine nucleotide cycle. Eur. J. biochem. 110:371-377. Ariano, M.A., R.B. Armstrong and V.R. Edgerton (1973). Hindiimb muscle fiber populations of five mammals. J. histochem. Cytochem. 21(0 51-55. Armstrong, R.B., C.W. Saubert, W.L. Sembrowich., R.E. Shepherd and P.D. Gollnick (1974). Glycogen depletion in rat skeletal muscle fibers at different intensities and durations of exercise. Pflugers arch. 352: 243-256. Armstrong, R.B. and M.H. Laughlin (1985). Metabolic indicators of fiber recruitment in mammalian muscles during locomotion. J. exp. Biol. 1 15:201-213. Bailey, I.A. and A.M. Seymour (1983). Glycolysis is not inactivated by pH in the Langendorff heart at pH values above 5.7. Biochem. soc. Trans. Ii: 278-288. Baldwin, K.M., J.S. Reitman., R.L. Terjung., W.W. Winder and J.O. Hollozy (1973). Substrate depletion in differernt fiber types of muscle and liver during prolonged running. Am. J. Physiol. 225: 1045-1050. Baldwin, K.M., R.H. Fitts., F.W. Booth., W.W. Winder and J.O. Hollozy (1975). Depletion of muscle and liver glycogen during exercise: protective effect of training. Pflugers arch. 354:203-212. , Baldwin, K.M., V. Valdez., R.E. Herrick., A.M. Macintosh and R.R. Roy (1982). Biochemical properties of overloaded fast-twitch skeletal muscle. J. appl. Physiol. 52(2) 467-472. 134 Bendall, J.R. (1961). A study of the kinetics of the f ibr i l lar adenosine triphosphatase of rabbit skeletal muscle. Biochem. J. 8J: 520-534. Bergmeyer, H.A. (1983) Methods of Enzymatic Analusis: Metabolites: I., Carbohydrates: Vol. 6. N.Y. Academic Press., 3rd Edition. Bessman, 5.P. and CL. Carpenter (1985). The creatine-creatine phosphate shuttle. Ann. Rev. Biochem. 54:831 -862. Bie-tner, R. (1979). The role of glucose 1,6-bisphosphate in the regulation of carbohydrate metabolism in muscle. Trends, biochem. Soc. 4 228-230. Bigland-Ritchie, B., C.G. Kukulka., O.C.J. Lippold and J.J. Woods (1982). The absence of neuromuscular transmission failure in sustained maximal voluntary contractions. J. Physiol. 330: 265-278. Black, E.C., A.R. Conner., K.C. Lam and W.G. Chiu (1962). Changes in glycogen, pyruvate and lactate in rainbow trout (Salmo  gairdneri) during and following muscular activity. J. Fish. Res. Board. Can. ±9:409-436. Black, M.J. and M.E. Jones (1983). Inorganic phosphate determination in the presence of a labile organic phosphate: assay for carbamyl phosphate phosphatase activity. Anal. Biochem. 135: 233-238. Bloxham, D.P. and H.A. Lardy (1973). Phosphofructokinase. In: The  Enzymes. Edited by P.D. Boyer. N.Y. Academic Press, 1973, Vol 9. p. 239-278. Bock, P.E. and C. Frieden (1974). pH-induced cold labil ity of rabbit muscle phosphofructokinase. Biochemistry 13:4191-4196. Bock, P.E. and C. Frieden (1976a). Phosphofructokinase. I. Mechanism of pH-dependent inactivation and reactivation of the rabbit 135 muscle enzyme. J. biol. Chem. 251: 5630-5636. Bock, P.E. and C. Frieden (1976b). Phosphofructokinase. II. Role of ligands in pH-dependent structural changes of the rabbit muscle enzyme. J. biol. Chem. 251: 5637-5643. Bohme, H.J., W. Schellenberger and E. Hofmann (1975). Mikrolkalorimetrische bestimmung der thermodynamischen parameter der phosphofructokinase-reaction. Acta biol. med. Germ. 34: 15-20. Bolitho-Donaldson, 5.K. and L. Hermansen (1978). Differential, direct effects of H + on Ca + + -activated force of skinned fibers from the soleus, cardiac and adductor magnus muscles of rabbits. Pflugers arch. 376: 55-65. Bone, Q., J. Kicenuik and D.R. Jones (1978). On the role of the different fiber types of f ish myotomes at intermediate swimming speeds. Fish. bull. 76: 691-699. Bosca, L, J.J. Aragon and A. Sols (1985). Modulation of muscle phosphofructokinase at physiological concentrations of enzyme. J. biol. Chem. 260(4)2100-2107. Brett, J.R. (1964). The respiratory metabolism and swimming performance of young sockeye salmon. J. Fish. Res. Board Can. 21: 1183-1226. Bucher, T. and W. Russman (1964). Equilibrium and non-equilibrium in the glycolytic system. Angew. chem. 19: 426-439. Burt, C.T., T. Glonek and M. Barany (1976). Analysis of phosphorus metabolites, the intracellular pH, and the state of adenosine triphosphate in intact muscle by phosphorus nuclear magnetic resonance. J . biol. Chem. 251(9) 2584-2591. Burton, K (1957). Free energy data of biological interest. In: Energy  Transformations in Living Matter. Edited by H.A. Krebs and H.L. Kornberg. Springer Verlag, Berlin. 1957 p 282. 136 Cain, D.F. and R.E. Davies (1962). Breakdown of adenosine triphosphate during a single contraction of working muscle. Biochem. biophys. res. Commun. 8:361-366. Chance, B. and CR. Williams (1956). The respiratory chain and oxidative phosphorylation. Adv. enzymol. rel. areas mol. Biol. 12. 65-134. Chance, B., J.S. Leigh., B.J. Clark., J . Maris., J. Kent, 5. Nioka and D. Smith (1985). Control of oxidative metabolism and oxygen delivery in human skeletal muscle: A steady state analysis of work/energy cost transfer reaction. Proc. Natl. Acad. Sci. USA 82: 8384-8388. Cheetham, M.E., L.H. Boobis and C. Williams (1985). Human muscle metabolism during treadmill sprinting. Clin. Physiol. 5: Suppl. 4. No. 154. Cleland, W.W. (1967). Enzyme kinetics. Ann. Rev. Biochem. 36 77-112 Cohen, P. (1983). Protein phosphorylation and the control of glycogen metabolism in skeletal muscle. Phil. Trans. R. Soc. Lond. B 302: 13-25. Collowick, S.P. (1973). The hexokinases. In: The Enzymes. Edited by P.D. Boyer. N.Y. Academic Press, Vol IX, 3rd Ed. 1973. p 1-46. Conlee, R.K., M. J. Rennie and W.W. Winder (1976). Skeletal muscle glycogen content: diurnal variations and effects of fasting. Am. J. Physiol. 231:614-618. Connett, R. J., T.E.J. Gayeski. and CR. Honig (1984). Lactate accumulation in fully aerobic, working, dog gracil is muscle. Am. J. Physiol 246 (Heart Circ. Physiol. 15): H120-H128. Connett, R.J. (1985). In vivo glycolytic equilibria in dog gracilis 137 muscle. J. biol. Chem. 260.(6)3314-3320. Cori, C.F. (1956). Regulation of enzyme activity in muscle during work. In: Enzymes: Units of Biological Structure and Function. Edited by O.H. Gaebler. N.Y. Academic Press. 1956. p 573-583. Cori, G.T., and C. F. Cori (1933). Changes in hexose phosphate, glycogen and lactic acid during contraction and recovery of mammalian muscle. J . biol. Chem. 99 493-505 Costi l l , D.L., R.L. Sharp., W.J. Fink and A. Katz (1982). Determination of human muscle pH in needle biopsy specimens. J. appl. Physiol. 53 (5) 1310-1313. Danforth, W.H. (1965). Activation of glycolytic pathway in muscle. In: Control of Energy Metabolism. Edited by B. Chance., R.W. Estabrook and J.R. Williamson. N.Y. Academic Press. 1965. p 287-297. Davison, W., G. Goldspink and I.A. Johnston (1976). Division of labour between fish myotomal muscles during swimming. J. Physiol. (Lond.) 263: 185-186. Dawson, J . M., D.R. Wilkie and D.G. Gadian (1980). The biochemical causes of fatigue studies by 3 1 P NMR. In: Exercise Bioenergetics  and Gas Exchange. Edited by P. Cerretelli and B.J. Whipp. Nth Holland. Elsevier 1980. p 25-34 Driedzic, W.R. and P. W. Hochachka (1976). Control of energy metabolism in fish white muscle. Am. J. Physiol. 230 (3) 579-582. Driedzic, W.R. and P. W. Hochachka (1978). Metabolism in fish during exercise. In: Fish Physiology. Edited by W.S. Hoar and D.J. Randall. N.Y. Academic Press. Vol. VII, p 503-543. Dudley, G.A. and R.L. Terjung (1985). Influence of acidosis on AMP deaminase activity in contracting fast-twitch muscle. Am. J. 138 Physiol. 248 (Cell Physiol. 17) C43-C50. Ebashi, S. (1980). Regulation of muscle contraction. Proc. Roy. Soc. Lond. 299: 465-484. Edington, D.W. (1970). Pyridine nucleotide oxidized to reduced ratio as a regulator of muscular performance. Experimentia 26: 601-602. "Edington, D.W., G.R. Ward and W.A. Saville (1973). Energy metabolism of working muscle: concentration profiles of selected metabolites. Am. J. Physiol. 224 (6) 1375-1380. Edwards, R.H.T. (1975). Muscle Fatigue. Post Grad. med. J. 51: 137-143. Eisenberg, E. and T.L. Hil l (1985). Muscle contraction and free energy transduction in biological systems. Science 227: 999-1006. Fabiato, A. and F. Fabiato (1978). Effects of pH on the myofilaments and the sarcoplasmic reticulum of skinned cells from cardiac and skeletal muscles. J . Physiol. (Lond.) 276: 233-255. Faulkner, J.A. (1983). Fatigue of skeletal muscle fibers. In: Proceedings of the third Banff International Hypoxia  Symposium. Edited by J.R. Sutton., C. S. Houston and N.L Jones. N.Y. Alan R. Liss Inc. 1983. Vol 136., p 243-255. Fischer, E.H., L.M.G. Heilmeyer and R.H. Haschke (1971). Phosphorylase and the control of glycogen degradation. In: Current Topics of  Cellular Regulation. Edited by B.L. Horecker and E.R. Stadtman. N.Y. Academic Press. Vol. 4. p211-251. Fitch, C.D. R. Chevli and M. Jellinek (1979). Phosphocreatine does not inhibit rabbit muscle phosphofructokinase or pyruvate kinase. J. biol. Chem. 254. 11357-11359. Fitts, R.H, J.B. Coutright., D.H. Kim and F.A. Witzmann (1982). Muscle fatigue with prolonged exercise: contractile and biochemical 139 alteration. Am. J. Physiol. (Cell Physiol.) C65-C73. Frieden, C, H.R. Gilbert., and P.E. Bock (1976). Phosphofructokinase. III. Correlation of the regulatory kinetic and molecular properties of the rabbit muscle enzyme. J. biol. Chem. 251: 5644-5647. Gadian, D. G., G.K. Radda., T.K.Brown., E.fi. Chance., M.J. Dawson and D.R. Wilkie (1981). The activity of creatine kinase in frog skeletal muscle studied by saturation-transfer nuclear magnetic resonance. Biochem. J. 195:1-14. Gardiner, K.R., P.F. Gardiner and V.R. Edgerton (1982). Guinea pig soleus and gastrocnemius electromyograms at varying speeds, grades and loads. J. appl. Physiol. 52:451 -457. Garetto, L.P. and R.B. Armstrong (1983). Influence of circadian rhythms on rat muscle glycogen metabolism during and after exercise. J . exp. Biol. 10221 1-222. Goldhammer, A.R. and H.H. Paradies (1979). Phosphofructokinase: structure and function. In: Current Topics of Cellular  Regulation. Edited by B. L. Horecker and E.R. Stadtman. N.Y. Academic Press. 1979 Vol 15, p 109-141. Gollnick, P. D., R.B. Armstrong., W.L. Sembrowich., R.E. Shepherd and B. Saltin (1973). Glycogen depletion pattern in human skeletal muscle fibers after heavy exercise. J . appl. Physiol. 3 4 615-618 Gollnick, P. D. (1982). Perpheral factors as limitations to exercise capacity. Can. J . appl. sport Sci. 7(1) 14-21. Goodman, M.N. and J.M. Lowenstein (1977). The purine nucleotide cycle: studies of ammonia production by skeletal muscle in situ and in perfused preparations. J . biol. Chem. 252 (14) 5054-5060. Greer-Walker, M. and G.A. Pull (1975). A survey of red and white skeletal muscle in marine fish. J. fish Biol. 7:295-300. 140 Griff iths, J.R., R.A. Dwek and G.K. Radda (1976). Conformational changes in glycogen phosphorylase studied with a spin-label probe. Eur. J. Biochem. 61; 237-242. Guppy, M, W.C. Hulbert and P.W. Hochachka (1979). Metabolic sources of heat and power in tuna muscles. II. Enzyme and metabolite profiles. J. exp. Biol. 82: 303-320. Hakala, M.T., AJ.Glaid and G.W. Schwert (1956). Lactate dehydrogenase. II. Variations of kinetic and equilibrium constants with temperature. J . biol. Chem. 221: 191-209. Harris, R.C., B. Essen and E. Hultman (1976). Glycogen phosphorylase in biopsy samples and single muscle fibers of musculus quadraceps femoris of man at rest. Scan. J. d in . lab. Invest, 36: 521-526. Heinrich, R., S.M. Rapoport and T.A. Rapoport (1977). Metabolic regulation and mathematical models. Prog, biophys. mol. Biol. 32: 1-82. Helmreich, E., W.H. Danforth., S. Karpatkin and C.F. Cori (1965). The response of the glycolytic system of anaerobic frog sartorius muscle to electrical stimulation. In: Control of Energy  Metabolism. Edited by B. Chance., R.W. Estabrook and J.R. Williamson. N.Y. Academic Press. 1965. p 299-31 1. Hermansen, L. (1981). Effect of metabolic changes on force generation in skeletal muscle during maximal exercise. In: Human Muscle Fatigue: Physiological mechanisms. Edited bu R. Porter and J. Whelan, Cibia Foundation Symposium 82., 1980, p 75-88. Hers, H.G. and E. VanShaftigen (1982). Fructose 2,6-bisphosphate two years after its discovery. Biochem. J. 206: 1-12. Hess, B. (1962). Control of metabolic rates. In: Control Mechanisms  in Respiration and Fermentation. Edited by B. Wright. N.Y. p 141 333-350. Hintz, C.S., M.M.Y. Chi., R.D. Fell., J.L. Ivy., K.K. Kaiser., C.V. Lowry and O.H. Lowry (1982). Metabolite changes in individual rat muscle fibers during stimulation. Am. J. Physiol. 242 (Cell Physiol. 11) C218-C228. Hoar, W.5. and D.J. Randall (1978). Terminology to describe swimming activity of fish. In: Fish Physiology. Edited by W.5. Hoar and D.J. Randall. N.Y. Academic Press. 1978, vol vii., p xi i i -x iv. Hochachka, P.W., G.P. Dobson and T.P.Mommsen (1983). Role of isozymes in metabolic regulation during exercise: insights from comparative studies. In: Isozymes: Current Topics in  Biological and Medical Research. Edited by M.C. Rattazzi., J.G. Scanalios and G.S. Whitt. N.Y. Alan R. Liss Inc. 1983. Vol 8. p 91-113. Hofer, H.W. and D. Pette (1968) Wirkungen und Wechselwirkungen von Substraten and Effektoren an der Phosphofrctokinase des Kaninchen-skeletmuskles. Hoppe-Seylers Z. Physiol. Chem. 349: 1378-1392. Hofmann, E. (1976). The significance of phosphofructokinase to the regulation of carbohydrate metabolism. Rev. physiol. biochem. Pharmacol. 75: 1-68. Hohorst, H.J., M. Reim and H. Bartels (1962). Studies on the creatine kinase equilibrium in muscle and the significance of ATP and ADP levels. Biochem. Biophys. Res. Commun. 7: 142-146. Hollozy, J.O. and H.T. Narahara (1965). Studies of tissue permeability. X. Changes in permeability to 3-methylglucose associated with contraction of isolated frog muscle. J . biol. Chem. 240: 3493-3500. Hollozy, J.O. and F.W. Booth (1976). Biochemical adaptations to endurance exercise in muscle. Ann. Rev. Physiol. 38: 273-291. 142 Hollozy, J.O., W.W. Winder., R.H. Fitts., M.J. Rennie., R.C. Hickson and R.K. Conlee (1978). Energy production during exercise. In: Third  International Symposium on Biochemistry of Exercise. Edited by F. Landry and W.AR. Orban. Miami. 1978. p 61-74. Hollozy, J.O. and E.F. Coyle (1984). Adaptations of skeletal muscle to endurance exercise and their metabolic consequences. J . Appl. Physiol. (Respirat. Environ. Exercise Physiol.) 56 (4) 831-838. Hue, L., P.F. Blackmore., H. Shikama, A. Robinson-Steiner and J.H. Exton (1982). Regulation of fructose 2,6-bisphosphate content in rat hepatocytes, perfused hearts, and perfused hindlimbs. J . biol. Chem. 257: 4308-4313. Huxley, H.E. (1985). The cross bridge mechanism of muscular contraction and its implications. J . exp. Biol. 1 15 17-30. Ivy, J . L. and J.O. Hollozy (1981). Persistent increase in glucose uptake by rat skeletal muscle following exercise. Am. J. •Physiol. 241 (Cell Physiol. 10) C200-C203. Jobsis, F.F. and J.C. Duffield (1967). Oxidative and glycolytic recovery metabolism in muscle. Flourometric observations on their relative contributions. J . gen. Physiol. 50: 1009-1047. Jobsis F.F. and W.N. Stainsby (1968). Oxidation of NADH during contractions of circulated mammalian skeletal muscle. Respir. Physiol. 4: 292-300. Jacobus, W.E., R.W. Moreadith and K.M. Vandegaer (1982). Mitochondrial respiratory control. Evidence against the regulation of respiration by extra-mitochondrial phosphorylation potentials or by ATP/ADP ratios. J. biol. Chem. 257: 2397-2402. Johnston, I.A. (1977). A comparative study of glycolysis in red and white muscles of the trout (Salmo Gairdneri) , and mirror carp 143 (Cyprinus carpio). J. f ish Biol. IT 575-588. Johnston, I. A. (1981). Structure and function of f ish muscles. Symp. zool. soc. Lond. 48:71 -113. Johnston, I. A. (1985). Sustained force development specializations and variations among the vertebrates. J . exp. Biol. 115: 239-251. Jones, D.R. and D.J. Randall (1978). The respiratory and circulatory systems during exercise. In: Fish Physiology. Edited by W.5. Hoar and D.J. Randall. N.Y. Academic Press. 1978. Vol v i i , P425-499. Karlsson, J (1980). Localized muscular fatigue: role of muscular contraction and substrate depletion. Exercise Sport Sci. Rev. 7: 1-42. Kasvinsky, P.J. and W.C. Meyer (1977). The effect of pH and temperture on the kinetics of native and altered glycogen phosphorylase. Arch, biochem. Biophys. J8i: 616-631. Kemp, R.G. and L.G. Foe (1983). Al losteric properties of muscle phosphofructokinase. Mol. cell. Biochem. 5/7: 147-154. Krebs, H.A. and H.L. Kornberg (1957). Energy transformation in living  matter. Springer Verlag. Berlin. Kuby, 5.A., L. Noda and H.A. Hardy (1954). Adenosine triphosphate-creatine transphosphorylase. III. Kinetic studies. J. biol. Chem. 21Q 65-82. Kushmerick, M.J., T. Brown and M. Crow (1980). Rates of ATP:creatine phosphorytransferase in skeletal muscle by 3 1 P nuclear magnetic resonance. (Abstract) Fed. Proc. 39: 1934. Kushmerick, M.J. and R.A. Meyer (1985). Chemical changes in rat leg muscle by phosphorus nuclear magnetic resonance. Am. J. Physiol. 248 (Cell Physiol. 17). C542-C549. 144 Lad, P. M., D.E. Hill and G.G. Hammes (1973). Influence of allosteric ligands on the activity and aggregation of rabbit muscle phosphofructokinase. Biochemistry 12: 4303-4309. Lardy, H.A. and R.E. Parks (1956). Phosphofructokinase. In: Enzymes:  Units of Biological Structure and Function. Edited by O.H. Gaebler. N.Y. Academic Press., 1956. p 584-587. Lohmann, K (1934). Uber die enzymatische Aufspaltung der Kreatinphosphorsaure zugleich ein Beitrage zum Chemismus der Muskelkontraktion. Biochem. Z. 271 264-277. Lowenstein J.M. (1972). Ammonia production in muscle and other tissues: the purine nucleotide cycle. Physiol. Rev. 52:382-414. Lowry, 0. H. and J. V. Passoneau (1966). Kinetic evidence for multiple binding sites on phosphofructokinase. J . biol. Chem. 241: 2268-2279. Lueck, J.D. and H.J. Fromm (1974). Kinetics and regulation of rat skeletal muscle hexokinase. J . biol. Chem. 249 (5) 1341-1347. flansour, T.E. (1972). Phosphofructokinase. In: Current Topics of  Cellular Regulation. Edited by B.L. Horecker and E.R. Stadtman. N.Y. Academic Press. Vol 5, p 1-46. Margaria, R. (1972). The sources of muscular energy. Sci. Amer. 226 (3) 84-92. McGilvery, R.W. (1975). The use of fuels for muscular work. In: Metabolic Adaptation to Prolonged Physical Exercise. Edited by H. Howald and J.R. Poortmans. p 12-30. McGilvery, R.W. (1983). Biochemistry: a functional approach. W.B. Saunders Co. 3rd Ed. pp 909. Meyer, R.A. and R.L. Terjung (1979). Differences in ammonia and adenylate metabolism in contracting fast- and slow-muscles. 145 Am. J. Physiol. 237(Cell Physiol.6).Cl 1 1-C118. Meyer, R.A. and R.L. Terjung (1980). AMP deamination and IMP reamination in working skeletal muscle. Am. J. Physiol. 259 (Cell Physiol. 8) C32-C38. Meyer, R.E., M.J. Kushmerick and P.F. Dillon (1982a). Intracellular pH changes in contracting fast-and s low-twitch muscles (Abstract). Fed. Proc. 4J: 979. Meyer, R.A., M.J. Kushmerick and T.R. Brown (1982). Application of ' 3 1 P NMR spectroscopy to the study of striated muscle metabolism. Am. J. Physiol. 242 (Cell Physiol.11) C l -C l l . Midelfort, C.E. R.K. Gupta and I.A. Rose (1976). Fructose 1,6-bisphosphate: isomeric compositions, kinetics and substrate specificity for aldolase. Biochemistry 15: 2178-2185. Nakamaru, Y. and A. Schwarts (1972). The influence of hydrogen ion concentration on calcium binding and release by skeletal muscle sarcoplasmic reticulum. J. gen. Physiol. 59: 22-32. Nassar-Gentina, V., J.V. Passoneau., J.L. Vergara and 5.1. Rapoport (1978). Metabolic correlates of fatigue and of recovery from fatigue in single frog muscle fibers. J. gen. Physiol. 72: 593-606. Neumann, P., G.F. Holeton and N. Heisler (1983). Cardiac output and regional blood flow in gi l ls and muscles after exhaustive exercise in rainbow trout (Salrno gairdneri). J . exp. Biol. 105: 1-14. Noda, L. (1973). Adenylate kinase. In: The Enzymes. Edited by P.D. Boyer. Vol VIII. Part A. 3rd Ed. N.Y. Academic Press, p 279-304. Noltmann, E.A. (1972). Aldose-Ketose Isomerases. In: The Enzymes. Edited by P.D. Boyer. Vol VI. 3rd Ed. N.Y. Academic Press, p 271-354. 146 Ozand, P. and H.T. Narahara (1964). Regulation of glycolysis in muscle. J. biol. Chem. 239(10)3146-3152. Passoneau, J.V. and O.H. Lowry (1962). Phosphofructokinase and the pasteur effect. Biochem. Biophys. Res. Commun. 7 (1) 10-15. Perry, 5.V. (1979). The regulation of contractile activity in muscle. Trans, biochem. Soc. 7: 593-617. Peter, J.B., R.J. Barnard., V.R. Edjerton., C.A. Gillepsie and K.E. Stempel (1972). Metabolic profiles of three fiber types of skeletal muscle in guinea pigs and rabbits. Biochemistry H: 2627-2634. Pettigrew D.W. and C. Frieden (1979). Binding of regulatory ligands to rabbit muscle phosphofructokinase. A model for nucleotide binding as a funciton of temperature and pH. J. biol. Chem. 254: 1887-1895. Phillips, F.C. and S. Ainsworth (1977). Al losteric properties of rabbit muscle pyruvate kinase. Int. J. Biochem. 8: 729-735. Portzehl, H., P. Zaoralek and J. Gaudin (1969). The activation by C a + + of the ATPase of extracted muscle f ibr i l s with variation of ionic strength, pH and concentration of MgATP. Biochim. biophys. Acta. J89: 440-448. Rahim, Z.H.A., D. Perrett and J.R. Griff iths (1978). Regulation in vivo of phsophorylase b in skeletal muscle of phosphorylase kinase-def icient mice. Biochem. soc. Trans. 6: 164-166. Randle P.J. and P.K. Tubbs (1979). Carbohydrate and fatty acid metabolism. In: Handbook of Physiology. The Cardiovascular  System I. Chapter 23. p 805-844. Reynolds, 5.J., D.W. Yates and C.I. Pogson (1971). Dihyroxyacetone phosphate. Biochem. J. 122: 285-297. 147 Rolleston, F.S. (1972). A theoretical background to the use of measured concentrations of intermediates in study of the control of intermediary metabolism. In: Current Topics of  Cellular Regulation. Edited by B.L. Horecker and E.R. Stadtman. N.Y. Academic Press. Vol 5. p 47-76. Rovetto, M.J., W.F. Lamberton and J.R. Neely (1975). Mechanisms of glycolytic inhibition in ischemic rat hearts. Circ. Res. 37: 742-751. Sahlin, K., R.C. Harris., B. Nylind and E. Hultman (1976). Lactate content and pH in muscle samples obtained after dynamic exercise. Pflugers Arch. 367: 143-149. Sahlin, K (1978). Intracellular pH and energy metabolism in skeletal muscle of man. Acta, physiol. Scan. Suppl. 455: 1-56. Sahlin, K., L. Edstrom., H. Sjoholm and E. Hultman (1981). Effects of lactic acid accumulation and ATP decrease on muscle tension and relaxation. Am. J. Physiol. 240(Cell Physiol. 9)C121 -C126. Scopes, R.K. (1973). 3-phosphoglycerate kinase. In: The Enzymes. Edited by P.D. Boyer. N.Y. Academic Press. Vol. VIII Part A, 3rd Ed. p 335-351. Setlow, B. and J. M. Lowenstein (1967). Adenylate deaminase. II. Purification and some regulatory properties of the enzyme from calf brain. J. biol. Chem. 242: 607-615. Srutton M.C. and M.F. Utter (1968). The regulation of glycolysis and gluconeogenesis in animal tissues. Ann. Rev. Biochem. 37: 249-302. Shoubridge, E.A., R.W. Briggs and G.K. Radda (1982). 3 1 P NMR saturation transfer measurements of the steady state rates of creatine kinase and ATP synthetase in the rat brain. FEBS Lett. 140: 288-292. 146 Slater, E.C. (1976). Intra- and extra-mitochondrial phosphorylation potentials: In: Use of Isolated Liver Cells and Kidney in  metabolite studies. Edited by J.fi. Tager, H.D. Soling and J.R. Williamson. 1976. Nth Holland Co. Amsterdam, p 65-77. Snow, D.H., R.C. Harris and S.P. Gash (1985). Metabolic response of equine muscle to intermittent maximal exercise. J. appl. Physiol. 58 (5) 1689-1697. Soderling, T.R. and CR. Park (1974). Recent advances in glycogen metabolism. In: Advances in Cyclic Nucleotide Research. Edited by P. Greengard and G.A. Robinson. N.Y. Raven. Vol.4, p 283-333. Sols, A (1981). Multimodulation of enzyme activity. In: Current  Topics of Cellular Regulation. Edited by B.L. Horecker and E.R. Horecker. N.Y. Academic Press. Vol 19. p 77-101. Spriet, L.L., CG. Masters., S.J. Peters., G.J.F. Heigenhauser and N.L. Jones (1985). Muscle metabolism and performance in the perfused rat hindquarter during heavy exercise. Am. J. Physiol. 248 (Cell Physiol. 17) C109-C118. Storey, K.B. (1983). Regulation of cockroach flight muscle phosphofructokinase by fructose 2,6-bisphosphate. Role in the activation of muscle metabolism during flight. FEBS Lett. 161:265-268. Taegtmeyer, H (1985). On the role of the purine nucleotide cycle in the isolated working rat heart. J . mol. cell. Cardiol. 17: 1013-1018. Terjung, R.L., K.M. Baldwin., W.W. Winder and J.O. Hollozy (1974). Glycogen repletion in different types of muscles and in the liver after exhaustive exercise. Am. J. Physiol. 226 (6) 1387-1391. Trivedi, B. and W.H. Danforth (1966). Effects of pH on the kinetics of frog muscle phosphofructokinase. J . biol. Chem. 241: 2268-2279. 149 Ui, M. (1966). Multiple inhibitor sites for ATP on muscle phosphofructokinase as influenced by a change of pH: a computer analysis of "non-linear" kinetic data. Biochim. biophys. Acta ±24 310-322. Uyeda, K (1979). Phosphofructokinase. Adv. enzymol. rel. areas, mol. Biol. 48: 193-244. Uyeda, K., E. Furuya, and L. J. Luby (1981). The effect of natural and synthetic D-fructose 2,6-bisphosphate on the regulatory and kinetic properties of liver and muscle phosphofructokinase. J . biol. Chem. 256: 8394-8399. Veech, R.L. (1980). Freeze-blowlng of brain and the interpretation of the meaning of certain metabolite levels. In: Cerebral  Metabolism and Neural Function. Edited by J.V. Passoneau, R.A. Hawkins., W.D. Lust and F.A. Welsh. Baltimore. Lond. Williams and Wilkins. 1980. p 34-41. Veech, R. L., J.W.R. Lawson., N.W. Cornell and H.A. Krebs (1979). Cytosolic phosphorylation potential. J. Biol. Chem. 254 (14) 6538-6547. Vergera, J.L., S.I. Rapoport and V. Nassar-Gentina (1977). Fatigue and post-tetanic potentiation of single muscle fibers of the frog. Am. J. Physiol. 232 (Cell Physiol. 1) C185-C190. Wahren, J . (1979). Metabolic adaptation to physical exercise. In: Endocrinology. Edited by L.J. DeGroot. Grune Stratton. N.Y. Vol. 3. 1979 p 1911-1920. Webb, P. W. (1978). Fast-start performance and body form in seven species of teleost fish. J . exp. Biol. 74:211-226. Wenger, H.A. and AT. Reed (1976). Metabolic factors associated with muscular fatigue during aerobic and anaerobic work. Can. J. appl. sports Sci. V. 43-48. 150 Wilkie, D.R. (1980). Equations describing power input by humans as a function of duration of exercise. In: Exercise Bioenergetics  and Gas Exchange. Edited by P. Cerretelli and R.J. Whipp. Elselvier Press. 1980. p 75-80. Williamson, D.H., P. Lund and H.A. Krebs (1967). The redox state of free nicotinamide-adenine dinucleotide in the cytoplasm and mitochondria of rat liver. Biochem. J. 103:514-527. Williamson, J.R. (1965). Metabolic control in the perfused rat heart. In: Control of Energy Metabolism. Edited by B. Chance., R.W. Estabrook and J.R. Williamson. N.Y. Academic Press. 1965. p 333-346. Williamson, J.R. (1970). General features of metabolic control as applied to the erythrocyte. In: Red Cell Metabolism and  Function. Edited by G. J . Brewer. N.Y. Plenum Press, p 117-136. Wilson, J.E., B. Sactor and CG. Tiekert (1967). In situ regulation of glycolysis in tetanized cat skeletal muscle. Arch, biochem. Biophys. 120: 542-546. Wu, T.F.L. and E.J. Davis (1981). Regulation of glycolytic flux in an energetically controlled cel l-free system: the effects of adenine nucleotide ratios, inorganic phosphate, pH and citrate. Arch, biochem. Biophys. 209: 85-99. 

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