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Creatine binding in white muscle of the rainbow trout Mossey, Mark Kenneth Philip. 1995

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CREATINE BINDING IN WHITE MUSCLE OF THE RAINBOW TROUT by Mark Kenneth Philip Mossey B.Sc. Hons. Zoology, The University of Western Ontario, 1992 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept this thesis as conforming to thgjequired standard THE UNIVERSITY OF BRITISH COLUMBIA March, 1995 © Mark Kenneth Philip Mossey, 1995 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 Vancouver, Canada Date DE-6 (2/88) Abstract The subcellular distribution of creatine (Cr) in white muscle of the rainbow trout (Oncorhynchus mykiss) was examined. Rainbow trout were cannulated via the dorsal aorta and injected with [amidino-14C] Cr. Following a 2 hour incubation period, fish were either sampled in the resting state (resting group) or exercised according to one of two different exercise protocols prior to sampling (buffering and depleting groups). The specific activity of phosphocreatine (SApcr) was over three times the SAcr within resting white muscle, and the SApc^SAc, ratio did not decrease towards 1 in muscle sampled from fish following either exercise protocol. This indicates; i) that a significant part of the pool of total Cr (the 'bound' pool) does not have access to creatine kinase (CK) in resting white muscle and ii) that this fraction of the Cr pool does not gain access to CK and become metabolically active during bouts of intense exercise. The S Apci/S Ac, ratio increased significantly with decreasing Cr charge and thus the pool of Cr which was interacting with CK was not mixing homogeneously in all parts of the cell. This suggests limited subcellular mobility of PCr and Cr. The finding that a significant part of the pool of total Cr is not active in energy metabolism affects calculations of free adenosine diphosphate (ADP) through the CK equilibrium. Calculations of free ADP which take the limited access of Cr to CK into consideration imply a significant role for ADP as a regulator of metabolism. iii Table of Contents Abstract ii Table of Contents iii List of Tables and Figures v Acknowledgments vi Introduction The Creatine Kinase Equilibrium in Muscle Metabolism 1 Links to Metabolism 2 Energy Buffering 5 Energy Transport 8 A Unifying Principle of C K Function 19 ADP - Roles in Metabolic Regulation 22 Creatine Distribution - Relevance to Metabolism 25 Materials and Methods Experimental Animals 30 Resting Experiments 30 Exercise Experiments 32 Tissue Analyses 33 E C F V Corrections 35 Statistics 36 i Results ECFV Plasma [14C] Clearance Metabolites Specific Activities Discussion Resting Muscle Exercise Groups and Extended Incubations Cr 'Binding' Quantification of Cr Binding Implications for Energy Metabolism SApo/SAcr and Cr Charge - Intracellular Mobility of Cr Summary References V List of Tables and Figures Table 1. Expression of miCK relative to CS for a variety of muscle fibre types in rats 51 Table 2. White muscle metabolite concentrations 52 Table 3. Specific activity of PCr and Cr in rainbow trout white muscle incubated with [14C] labeled Cr 53 Figure 1. Metabolite concentrations normalized to total Cr as a function of energy status in muscle 54 Figure 2. Plasma SAc r and Cr concentration 56 Figure 3. SApo/SAcr as a function of Cr charge in rainbow trout white muscle 58 Figure 4. SApo/SAcr as a function of Cr charge in isolated rat atria 60 Acknowledgments I have received generous assistance and support from a number of sources over the course of my MSc studies. Firstly, I would like to thank Dr. Peter Hochachka for an endless infusion of energy, enthusiasm and ideas. In retrospect, I also owe Peter thanks for allowing me total independence: learning through trial and (primarily) error may not be terribly efficient, but I have found it to be highly effective. I wish to acknowledge the time, energy and resources invested by Dr. Tony Farrell and Dr. Kurt Gamperl in work done at SFU. Technical assistance from Dr. Tim (Timmalimma) West, Dr. Steve (Bland) Land, Jim (F) Staples, Dr. Peter (little Peter) Arthur, and Joelle Harris was greatly appreciated. These people, in conjunction with my lab mates Gary (G) Burness, Grant (Granite) McClelland, Petra (PM) Mottishaw, Carole (with an e) Stanley, Sheila (I've been everywhere) Thornton and Mark (I've been more places than Sheila) Trump have provided many helpful ideas and discussions. Special thanks go to Tim, Jim, Gary, Grant and Carole for supporting an environment which was a sincere pleasure within which to work. I thank my parents, without whom I could never have come to Vancouver. The support which they have provided has come in many forms and was offered without question. I will not forget their example in the upbringing of my own children. This work was supported by an NSERC postgraduate fellowship and my ma and pa. 1 Introduction The Creatine Kinase Equilibrium in Muscle Metabolism Biochemical aspects of the regulation of energy metabolism have received considerable attention over the past twenty or thirty years. The roles played by the enzyme creatine kinase (CK) are central to many aspects of energy metabolism and thus much research has targeted C K and the equilibrium which it catalyzes. Creatine kinase is found in a number of different tissues including spermatozoa (studied in sea urchin, rooster, and man), the electric organ of several species of the family of electric fish Torpedinidae, vertebrate kidney, nervous tissue, retina and photoreceptor cells, intestinal epithelial cells, and all types of muscle (see Wallimann et al, 1992 for references). All of these tissues have metabolic rates which are very high during at least some phase of physiological activity. Their metabolism may be at a sustained high rate such as that of renal cells, or show dramatic fluctuations from a relatively low basal level to very high levels during activation (i.e. electrocytes). The fact that C K is specific to these tissues suggests an important role for this enzyme in relation to high rates of energy turnover. Creatine kinase (CK) catalyzes the following reaction: PCr + ADP + l f < - » Cr + ATP where PCr is phosphocreatine, ADP is adenosine diphosphate, Cr is creatine and ATP is adenosine triphosphate. This is considered a near equilibrium reaction, with a K ^ of 185 (pH 7, free [Mg2 +] = 1.0 mM, I = 0.25 M , temp.= 38°C) (Teague and Dobson, 1992). Intricate linkage of C K with energy metabolism is revealed through the reactants of this equilibrium. 2 Links to Metabolism A proton (tt) is consumed in the resynthesis of ATP from PCr and thus cellular pH is affected by changes in the C K equilibrium. Extensive acidosis is associated with muscle fatigue and can actually cause inhibition of the myosin ATPases through buildup of diprotonated phosphates (H2PO4) (Noesek et al, 1987) and pH also has important effects on phosphofructokinase (PFK) and a number of enzymes of glycolysis, and the pathways of glycogenolysis and oxidative phosphorylation (Hochachka, 1993). Therefore any effects which C K may have in modulation of pH will have widespread influence upon energy metabolism. The most profound effect of C K activity on cellular pH is seen early in a bout of high intensity work in skeletal muscle. The transient alkalosis often observed in this situation is attributed to the resynthesis of ATP through C K activity accompanied by the consumption of a proton. This proton consumption acts to reduce the acidosis seen with glycolytic activation in this type of work (Hochachka and Mommsen, 1981), thus minimizing the transient pH drop and abating the inhibitory effects of low pH on the contractile proteins. The subcellular localization of a specific isoenzyme of C K within the I-band of sarcomeres along with the enzymes of glycolysis may account directly for these observations (Wallimann etal, 1984; Krause and Jacobus, 1992). Studies of skeletal muscle depleted of Cr through feeding of the Cr analog P-guanidinopropionic acid (J3-GPA) offer further support to C K function in the control of pH balance (Meyer et al, 1989). Conversely, changes in pH affect the C K equilibrium. Work with anoxic teleost white muscle led van Waarde et al (1990) to argue that pH serves as a functional couple between C K 3 and glycolysis during anoxia, with protons released through glycolysis serving to push the C K reaction further in the direction of ATP synthesis. This helps to maintain the supply of energy for cellular function under these conditions. C K activity is also linked to inorganic phosphate (Pi), another important intermediate of energy metabolism. This relationship is revealed through a summation of the equations of C K in the direction of ATP synthesis and ATPases in the direction of ATP hydrolysis; PCr + ADP + FT <-> Cr + ATP ATP <-> ADP + FT + P; to yield; PCr <-> Cr + Pi as the net reaction. A direct stoichiometric relationship between PCr hydrolysis and Pi accumulation during non-steady state muscle work is in fact revealed by phosphorous nuclear magnetic resonance spectroscopy (MRS) work. The energy produced by cellular metabolism is captured directly in high energy phosphate bonds. Likewise, it is these bonds which are hydrolyzed to release the energy which drives cellular function. As a result of this, Pi affects energy metabolism at virtually every level through mass action. Pi is particularly important in the regulation of the rate of glycolysis. Meyer et al (1986) demonstrated that the stimulation of glycolytic rate by P; released from PCr by C K activity during high intensity work is an important role of the C K reaction. There is also an as yet unresolved role for Pi in the regulation of the rate of oxidative phosphorylation (Balaban, 1990). A detailed review of the roles FT and P; is beyond the scope of this report, however even the brief description above gives some idea of how central both molecules are as effectors of metabolism. Although this makes the relationships between FT, Pi, and C K of significant interest in themselves, they do not account for the primary link of the C K equilibrium to energy turnover within muscle. As mentioned above, the exchange of a high energy phosphoryl group between ATP and PCr is catalyzed by CK. This reaction is dead end for both PCr and Cr in that they do not participate in any other known reactions within the cell. In this context Cr can be thought of as an acceptor of the high energy y-phosphate group of ATP, and PCr as a donor of high energy phosphate to ADP. Adenosine triphosphate hydrolysis directly provides the overwhelming majority of energy consumed by a cell; ATP <-> ADP + Pi + FT and thus the C K equilibrium is coupled to virtually all of the synthesis and consumption of energy which occurs within muscle. Numerous studies in muscle support the idea that C K is a near equilibrium enzyme for which the rates of the forward and reverse reactions are much greater than any net changes observed in the concentrations of reactants (Yoshizaki et al, 1990; Bittl and Ingwall, 1985; Shoubridge et al, 1984). Thus C K has the ability to catalyze either the synthesis or hydrolysis of ATP depending on the local metabolic conditions within a muscle fibre at any given time. This coupling with ATP hydrolysis also results in an intimate relationship between C K function and ADP concentrations. ADP has long been considered an important regulator of many aspects of energy metabolism, providing a direct regulatory significance to C K function. This direct association between C K activity and the adenylates (ADP and ATP) results in a direct association between C K activity and virtually every aspect of energy metabolism. 5 Research has however tended to centre on two general functional contexts for C K with respect to ATP turnover: i) energy buffering in which C K catalyzes the net hydrolysis of PCr stores in order to maintain the supply of ATP following the stimulation of ATPase activity and ii) energy transport in which C K activity mediates transfer of high energy phosphoryl groups generated at the mitochondria to sites of energy consumption. Energy Buffering This is the original function ascribed to C K within muscle (see Bessman and Geiger, 1981). C K mediated energy buffering may be defined as the hydrolysis of PCr to maintain ATP concentrations in the face of increased ATPase activity The effect may be quite dramatic as in fast glycolytic (FG) fibres under burst work conditions or more subtle such as that observed with steady state contraction or moderate transitions in work rate. In either case, the primary importance of energy buffering is generally thought to be maintenance of ATP levels near those of the resting muscle, and thus the maintenance of function for energy consuming processes such as ion pumping and myosin ATPase activity. This role is facilitated by a number of factors: i) Most resting muscle tissue has a larger concentration of PCr than ATP, and thus the energy stored as PCr is greater than that in ATP. The energy contained within the cellular PCr pool may serve as a short term energy reserve during brief bouts of intense energy consumption, ii) C K is always found with activity that surpasses that of the maximum ATPase rate of a muscle fibre, and is thus able to maintain near equilibrium conditions with respect to its reactants during periods of high or maximal ATPase activity, iii) C K has a low Km for ADP (about 20 to 30 uM; calculated resting ADP varies between 5 and 40 6 uM depending on fibre type and preparation) which makes the reaction highly sensitive to changes in ADP. ii) and iii) together allow the C K equilibrium to respond instantly to maintain ATP levels in the face of any change in ATP consumption rate, iv) Numerous studies have demonstrated the specific localization of isoenzymes of C K at sites of energy consumption in muscle. These include binding to the sarcoplasmic reticulum to support C a 2 + ATPase pumping (Rossi et al, 1990), binding at the sarcolemma to support Na + /K + ATPase work (Saks et al, 1977), and binding at the M-line and I-band (along with the glycolytic enzymes) of sarcomeres to provide energy directly to the contractile proteins (Wallimann et al, 1984; Krause and Jacobus, 1992). This allows immediate resynthesis of ATP from PCr upon stimulation of ATPases. Note also that all of these factors result in the maintenance of high local ATP/ADP ratios at sites of ATP consumption, thereby maximizing the free energy released by ATP hydrolysis and avoiding net loss of adenylates to inosine monophosphate (IMP) through the actions of adenylate kinase (a) and adenosine monophosphate (AMP) deaminase (b); 2 ADP <-> ATP + AMP (a) AMP <H> IMP + N H 4 + (b). This buffering role is thought to be of primary importance in F G fibres with high myosin ATPase catalytic potential and relatively low aerobic capacity. These fibres provide an extreme example in that they can demonstrate large and rapid changes in energy consumption upon stimulation. Under these conditions ATP levels remain remarkably stable through the actions of CK, as PCr hydrolysis supports metabolism in the early stages of contraction while glycolysis and oxidative phosphorylation have not yet been fully activated. Depletion of PCr stores is associated with fatigue and an accompanying drop in ATP levels if high intensity stimulation continues. 7 Thus the hydrolysis of PCr serves to 'buffer' the levels of ATP and allow continued muscle function under the energy depleting conditions imposed by dramatic increases in the rate of energy consumption. This effect is also seen to a lesser extent in more oxidative skeletal muscle fibres with lower ATPase rates for which changes in energy consumption are not as profound as those of FG fibres. What about steady state work? Funk et al (1989) calculated a significant bufFering effect even under these circumstances. They concluded that in the face of the small changes in ATP concentration seen with steady state work in the absence of CK, the resulting fluctuations in ADP and AMP levels would potentially have significant effects upon metabolism through regulatory effects on other pathways and loss of adenylates to IMP. Thus energy bufFering may even be important under these conditions. This may also be the primary buffering role played by CK in aerobic muscles such as heart or slow oxidative (SO) skeletal fibres. Although large scale bufFering of ATP levels through extensive PCr hydrolysis becomes important in unusual situations such as work to exhaustion or ischaemic contraction (where the energy stored in PCr may be directly related to survival of the organism in the case of cardiac muscle) (see Miller et al, 1993), varying the level of steady state work with which these fibres are normally associated in vivo reveals that these fibres are difficult to fatigue, and levels of high energy phosphates including PCr will show relatively little (SO; Kushmerick et al, 1992) or no (cardiac; Heineman and Balaban, 1990) variation. Work state transitions in these muscle types are typically not as dramatic as those seen in FG fibres. ThusCK mediated energy bufFering in these muscles is primarily involved in minimizing fluctuations of ADP and AMP levels with steady state work. 8 C K function in direct maintenance of a relatively constant supply of ATP is therefore of consequence under virtually all physiological work states in muscle. Not only does buffering maintain the energy supply for cellular ATPase function, but it serves to maintain local ATP/ADP ratios and thus maximize the free energy released by ATP hydrolysis, and to minimize unnecessary regulatory signaling and net loss of the adenylate pool to IMP. The energy buffering function ascribed to C K is thereby of considerable significance to muscle function. What then is the role played by C K in energy transport, and of what significance is C K function in this context? Energy Transport The linkage of C K activity with ATP and ADP along with a number of observations of the subcellular design of muscle lend obvious support to an association of C K with energy transport. It has unfortunately proven difficult to ascertain the nature of this association, and numerous studies have attempted to solve this problem without arriving at any definitive conclusions. A detailed review of available experimental data does however provide insight into the nature of C K mediated energy transport. The subcellular organization of C K within muscle is central to its role in energy transport, and thus a brief description is necessary prior to a the review of relevant experimental data. C K can be found in a number of separate isoenzymes. Cytosolic variants are all dimeric, composed of the M (muscle) and B (brain) monomers. Although both types of monomer are expressed within the same tissue, BB dimers are found to be the primary cytosolic isoenzyme in nervous tissue while the same applies for the M M variant in striated muscle. Most cytosolic C K 9 activity is found in homodimeric variants, however MB heterodimers may be expressed at low levels in certain tissues or during certain developmental stages. The third major type of CK isoenzyme is mitochondrial CK (miCK) which is reversibly bound on the outer surface of the inner mitochondrial membrane. The total amount of CK activity and the relative proportions of miCK and MMCK activity vary with the type of muscle fibre studied. Large FG fibres with a high glycolytic potential, high total Cr content, and relatively low oxidative potential demonstrate the highest levels of CK expression. Almost 90% of CK activity present is in the form of MMCK. In the smaller diameter oxidative fibres (SO, cardiac) there exists a lower glycolytic capacity, lower total Cr, and CK activity which is one half of that in FG fibres. Also, proportionate expression of CK isoenzymes is different. Oxidative fibres have over 2 times the absolute expression of miCK and less than one half of the MMCK activity seen in fast fibres, and thus the relative activity of miCK is 4 times greater in slow fibres (Yamashita and Yoshioka, 1991). This increase in miCK expression with increased oxidative capacity and the localization of miCK within the intermembrane space of mitochondria is indicative of an association with oxidative phosphorylation. This relationship has stimulated extensive research into the structural and functional aspects of this isoenzyme. Although the basic miCK unit is dimeric, the more active form of the enzyme exists as an octamer which is found concentrated at sites of contact between the inner and outer mitochondrial membranes. It seems that miCK has preferential access to mitochondrially generated ATP, and a number of experimental approaches have supported a functional coupling with oxidative phosphorylation (Jacobus, 1985; Gellerich et al, 1987; Aliev and Saks, 1993). Several lines of evidence have led Wallimann et al (1992) to 10 suggest the existence of a multienzyme complex linking the adenine nucleotide transporter (ANT) to octameric miCK which they hypothesize is in turn linked to the mitochondrial pores of the outer membrane. This could form an 'energy channel' at sites of contact between the two membranes, with the y-phosphate of mitochondrially generated ATP (moved across the inner membrane by the ANT) transferred to Cr (by miCK) which would then exit to the cytoplasm through the pore as PCr. Note that miCK has a higher affinity for Cr and ATP and a lower affinity for PCr and ADP than those of the cytoplasmic C K isoenzymes, making it more suited to this role by favouring the synthesis of PCr from ATP (Basson et al, 1985). Mitochondrial C K may thus function to rapidly regenerate ADP from oxidative ATP and maintain a relatively low ATP/ADP ratio at the outer face of the ANT. The maintenance of this constant and locally relatively large pool of ADP at the outer face of the A N T favours an increase in the rate of ANT turnover, resulting in the maintenance of a lower ATP/ADP ratio within the mitochondria where ATP synthesis occurs. The overall result is thus an increase in the thermodynamic efficiency of oxidative phosphorylation due to this decrease in the intramitochondrial ATP/ADP ratio. This information, taken in conjunction with the previously described subcellular localization of the cytosolic isoenzymes at sites of energy consumption, provides the framework for the first model to attempt a description of C K mediated energy transport: the 'PCr shuttle hypothesis' (Bessman and Geiger, 1981). This model holds that the flow of energy from the mitochondria is directed through miCK while its consumption is mediated primarily through M M -C K bound at sites of high energy consumption in support of ATPase activity. In turn, this essentially divides ATP and ADP into two separate functional pools; a mitochondrial pool and a 11 pool localized at sites of energy consumption. Energy generated by oxidative phosphorylation must then travel to sites of consumption as PCr rather than ATP, and it follows that the feedback to the mitochondria must be Cr rather than ADP. The theoretical framework for this model seems relatively straightforward, however it does not in itself explain why a specific alternative system of energy transport is needed. What possible benefits could this system confer upon a cell? The basis of the argument in favour of the PCr shuttle hypothesis is that energy transport in the form of PCr and return in the form of Cr must provide an increase in the efficiency of metabolism. Although the subcellular localization of the various isoenzymes of C K described above accounts for a large increase in metabolic efficiency through maintenance of advantageous local ATP/ADP ratios, this is not dependent upon energy shuttling through PCr for function and as such does not provide direct support of the hypothesis. Proponents of the shuttle hypothesis argue that direct evidence for the existence of the PCr shuttle may be found in the study of ADP flux. In a comparison of potential flux rates of ADP and Cr as feedback signals (and substrates) to heart mitochondria by Jacobus (1985), the calculated diffusion coefficient for ADP was about one half of that for Cr. He then took the concentrations of these metabolites into consideration in order to calculate maximal flux rates. ADP measurements through conventional tissue extraction are not representative of the true free (and thus metabolically active) concentration of ADP within a muscle cell due to the binding of most ADP to intracellular proteins. Calculations of the free unbound ADP (based on the C K equilibrium) give low concentrations within muscle at rest (5-40 uM) and at work (up to 500 uM). In contrast, Cr is always available at concentrations which are two or more orders of magnitude higher than that of ADP. Based on this, Jacobus determined that flux of ADP in 12 myocardium would begin to limit the rate of oxidative phosphorylation at about 80% of maximal work rates, whereas Cr flux would not impose limitations upon metabolism at any work rate. In addition to this, more recent studies have suggested subcellular compartmentation of the adenylates and thus further limitation of their mobility (Gellerich et al, 1987; Zeleznikar and Goldberg, 1991), leading Wallimann et al (1992) to conclude that the internal geometry of muscle fibres would lead to limitation of cardiac oxidative metabolism by diffusive flux of the adenylates in the absence of energy shuttling through CK. Contradictory evidence is provided by Yoshizaki et al (1990), who performed an MRS study of bullfrog skeletal muscle to determine the relative diffusivities of the reactants of the C K equilibrium. They then determined flux through C K to determine in vivo life times and used this in conjunction with the diffusivity data to calculate diffusion lengths for the reactants. This analysis led to the conclusion that ADP diffusion would not limit the rate of cardiac oxidative metabolism. The determination of ADP flux and its potential to control or limit metabolism remains an issue of contention. Note that calculations of adenylate flux which do suggest a limiting role assume that 100% of energy transport and feedback to mitochondria is in the form of ATP and ADP respectively. This is not the case within cells containing CK, where PCr and Cr should provide most of the diffusive flux through simple facilitated diffusion (Meyer et al, 1984) even in the absence of any specific shuttling. Adenylate flux must therefore not be limiting to the rate of metabolism within C K containing muscle fibres, independent of PCr shuttling. Thus the PCr shuttle hypothesis has a reasonable theoretical framework for which the practical justification through limitations imposed upon mitochondrial metabolism by ADP flux is 13 uncertain. The hypothesis remains controversial in large part because the study of the relevance of this model to in vivo muscle metabolism has proven difficult. One technique that has offered insight is the use of the Cr analog B-guanidinopropionic acid (B-GPA) to deplete cellular Cr, thereby allowing the study of muscle function in the face of drastically lowered concentrations of PCr and Cr. Feeding of 0-GPA in the diet of animals results in a blockage of the uptake of Cr into muscle and a subsequent depletion of muscle Cr stores. Creatine levels drop to anywhere from 20% (Zweier etal, 1991) to 5% (Moerland and Kushmerick, 1994) of pre-treatment levels after 6 to 8 weeks of B-GPA feeding. Although B-GPA is a substrate for C K and is phosphorylated to P-GPAP within muscle, it has a high K„, and a low Vmax relative to those of Cr. Thus the consequence of B-GPA feeding is the elimination of most of the flux through the C K reaction. Studies of heart and skeletal muscle have repeatedly shown little or no effect of Cr depletion on performance per unit of cross sectional area (i.e. Shoubridge et al, 1985b; Meyer, 1989; Moerland and Kushmerick, 1994). However, F G fibres respond to Cr depletion with a decrease in size, resulting in a decrease in absolute force production. C K is generally thought to act primarily in the role of energy bufFering in F G fibres, and hence the obvious conclusion to be drawn from this is that the primary function of C K must simply be that of an energy buffer within large diameter fibres with high total Cr concentrations, high ATPase and glycolytic capacities and low oxidative capacity. This observation is unfortunately confounded by the fact that Cr depletion induces a series of biochemical changes in muscle fibres. In general, fibres undergo a shift in their structure and components which leaves them with a more oxidative metabolism; increases in mitochondria, miCK expression and the enzymes of oxidative phosphorylation, and 14 decreases in fibre diameter, total ATPase and C K activity, all accompanied by a shift in myosin isoforms from fast to slow (Shoubridge et al, 1985a; Moerland et al, 1989). This leads to the decrease in the size and absolute force production of treated fibres mentioned above, along with more rapid recovery and smaller changes in metabolite concentrations with work; attributes which are characteristic of oxidative fibres. This shift in metabolism is most pronounced in F G fibres, with only slight changes seen in F O G fibres and little if any change seen in SO or cardiac fibres (Moerland etal, 1989; Zweier etal, 1991). These adaptations are in themselves interpreted by Wallimann et al (1992) as proof of the significance of the shuttle based on their theory that miCK is more tightly linked to mitochondrial ATP synthesis in oxidative fibres with high levels of expression of miCK and that shuttling is therefore more important in these fibres. They suggest that Cr depleted skeletal muscles increase their shuttling capacity as a compensatory mechanism for the loss of energy buffering capacity caused by Cr (and PCr) depletion, and thus these muscle fibres become more dependent on oxidative metabolism and miCK activity. This argument is based upon calculations indicating that there is enough energy turnover supplied through hydrolysis of the remaining PCr and the phosphorylated analog (P-GPAP) to sustain observed ATP turnover through energy shuttling from the mitochondria. Recent work by Moerland and Kushmerick (1994) provides evidence which contradicts the idea that C K flux is still able to support ATP turnover in 0-GP A treated muscle. They measured recovery O2 consumption in both fast (EDL) and slow (soleus) skeletal muscle of mice treated with P-GPA to give a total Cr reduction of 95% and found that turnover of remaining PCr along with P-GPAP turnover could sustain an absolute maximum of 50% of observed ATP 15 consumption rates. A study by van Deursen et al (1994) reveals that the maximal possible flux through CK shuttling in Moerland and Kushmerick (1994) must have been even lower than that calculated. In mice which had been genetically altered to block expression of MMCK activity, no phosphorylation of P-GPA was found. P-GPA must then have no access to miCK, without which it cannot participate in shuttling. Therefore the total possible flux through CK depends purely upon PCr in P-GPA treated skeletal muscle. Energy transport in the study of Moreland and Kushmerick (1994) could not have been primarily through PCr shuttling in direct contrast to the increase in shuttling with Cr depletion suggested by Wallimann et al (1992). Furthermore, the claim of Wallimann et al (1992) that increased miCK expression is indicative of increased PCr shuttling through tighter coupling of miCK to oxidative phosphorylation is in itself questionable. Citrate synthase (CS) is an exclusively mitochondrial enzyme whose activity is close to the maximal flux seen through oxidative phosphorylation in skeletal muscle, although this may not hold true in heart (Cooney et al, 1981). Thus CS activity may be considered representative of the oxidative capacity (and thus mitochondrial density) within a given muscle. See Table 1 for a comparison of miCK and CS activity in a range of muscle fibre types. The ratio of miCK/CS activity is lowest in heart, but again, CS activity may not be representative of maximal flux through oxidative phosphorylation in this tissue. Consideration of miCK expression relative to that of CS does not reveal any specific upregulation of miCK beyond that expected through increased oxidative capacity. Therefore although miCK activity does increase with oxidative capacity and thus there is more miCK present to support PCr synthesis from mitochondrially generated ATP in more oxidative muscle, the increase in miCK activity 16 appears to be proportionate to that of the oxidative capacity. This does not support a tighter coupling of miCK with oxidative ATP synthesis in oxidative fibre types. Therefore it seems that the PCr shuttle model of Bessman and Gieger (1981) is inappropriate for skeletal muscle. Metabolism must not depend upon PCr and Cr alone to provide flux to and from mitochondria. An alternative model of the in vivo function of CK with respect to energy transport which is supported by data for skeletal muscle is that of Meyer etal (1984) who suggest that energy transport mediated through CK is best described by facilitated diffusion rather than a distinct shuttling mechanism. Calculations based on this model for skeletal muscle agree with the shuttle hypothesis in that they indicate that almost the entire flux of oxidative energy to and from sites of consumption must be in the form of PCr and Cr respectively rather than ATP and ADP, thereby overcoming any diffiisional limitations which may be imposed on the rate of oxidative phosphorylation by ADP flux alone. However, this is proposed to come as a result of facilitated diffusion along with ATP and ADP rather than through a separate shuttling system. The primary advantage offered to muscle cells through this model is a large reduction in required concentration gradients for ATP and ADP diffusion in order to support energy flux from the mitochondria to the sites of consumption. This allows the maintenance of a high ATP/ADP ratio throughout the cell, keeping the thermodynamic efficiency of ATP hydrolysis high (maximal energy yield) and minimizing net loss of the adenylate pool to IMP. Thus the advantages to metabolism derived through CK mediated facilitated diffusion are identical to those of the shuttle hypothesis but without the requirement for the high degree of subcellular compartmentation implicit in the latter. 17 The fundamental purpose of C K mediated energy transport is the movement of energy from the mitochondria to sites where it is consumed. The prediction of Meyer et al (1984) that the role played by C K in energy transport should be most important in large diameter F G fibres where diffusion distances are relatively long and mitochondrial abundance is low is consistent with this idea. It is these F G fibres that undergo significant metabolic changes (discussed above) upon depletion of intracellular Cr in P-GPA feeding experiments. Fast glycolytic fibres may therefore be adjusting their metabolism to compensate for limitations imposed by Cr depletion and the accompanying loss of capacity for energy transport through facilitated diffusion. Thus the metabolic changes which F G fibres undergo as a result of Cr depletion are explained as a compensation for the loss of energy transport through C K rather than an increased reliance on C K mediated transport as predicted by supporters of the shuttle hypothesis. Smaller oxidative fibres with short diffusion distances and dense mitochondria are relatively unaffected in these experiments. Diffusional limitations for energy transduction must not be as significant in smaller fibres depleted of Cr. Note that this is in direct contrast to the shuttle hypothesis which predicts that energy transport through C K should be of maximal importance in the smaller oxidative fibres based on the high level of miCK expression. What about heart muscle? Results in heart muscle are variable and difficult to interpret. Rat hearts severely depleted of both ATP (undetectable by NMR) and PCr (15% of control values) through 2-deoxyglucose treatment maintain contractility to 65% of the level of control hearts (Hoerter etal, 1988), leading the authors to conclude that PCr must play at least some role in energy transport due to the dramatic decrease of ATP concentrations seen in the preparation. Rat hearts depleted of Cr through analog feeding alternately show no change in performance 18 (Shoubridge etal, 1985b) or a decrease in developed pressure with maintenance of contractility (Zweier et al, 1991). Neither study claims to offer support for the PCr shuttle hypothesis. Note that P-GPAP may be more metabolically active in heart than in skeletal muscle (Conley and Kushmerick, 1990). Calculations of flux through C K in relation to ATP turnover are thus uncertain, leaving no definite answer as to the role of C K in energy transduction within heart muscle. The model of Meyer et al (1984) is, however, in agreement with the data. Observations of reduced heart muscle performance in the face of decreased flux through C K may be explained via the restricted capacity for facilitated diffusion, an explanation which holds true whether or not CK flux is below the level of ATPase activity. In contrast, the shuttle hypothesis requires that C K flux be at or above the level of ATPase activity if energy is to be transported as PCr. This is not in agreement with all acquired data. Creatine kinase function in terms of energy transport is thus apparently best explained in all muscle fibre types by the model of C K mediated facilitated diffusion (Meyer et al, 1984). Although PCr shuttling may occur to some extent, it is clear that not all energy must pass through a muscle fibre as PCr in order to support metabolism. Phosphocreatine shuttling is not obligatory for cellular function. The shuttling model relies upon a higher degree of complexity in the intracellular environment and restriction of adenylate diffusion, the support for which is uncertain. In contrast, the model of C K mediated facilitated diffusion is consistent with results for all fibre types and offers a more parsimonious explanation of the phenomenon of energy transport within muscle. 19 A Unifying Concept of C K Function A review of C K function in terms of both energy bufFering and energy transport which are generally considered separately reveals what may be considered a singular principle to describe C K function within muscle: maximization of thermodynamic efficiency through control of local metabolite concentrations. A brief explanation of the concept of thermodynamic efficiency in relation to energy metabolism in muscle is first required in order to develop this argument. Briefly, maximizing thermodynamic efficiency in the context of muscle energy metabolism means consuming minimal energy to synthesize high energy compounds and releasing maximal energy from their hydrolysis. Since muscle function depends primarily upon energy derived through ATP hydrolysis, it is important to maximize the amount of energy released from each molecule of ATP and also to minimize the amount of energy consumed for the production of each molecule of ATP. This will maintain the highest possible thermodynamic efficiency in both energy consumption and energy production. In terms of energy consumption, this is described through changes in the free energy of ATP hydrolysis (AG), given by the equation; A G = A G 0 + RT In [ADP] [P;] [FT] / [ATP] where A G 0 is the standard free energy change of ATP hydrolysis, R is the gas constant, T is the absolute temperature, and the terms in brackets are reported in terms of tissue concentrations. It is obvious from this equation that the value of A G is directly dependent upon the concentrations of the reactants. A negative A G value indicates energy released by the reaction, and thus in order to maximize the energy released through ATP hydrolysis the concentrations of the products should be minimized relative to that of ATP. That is, maintenance of low concentrations of ADP, 20 Pi, and IT at sites where ATP hydrolysis occurs will result in the maximal release of energy to drive cellular functions (i.e. ion pumping, contractile protein function) per molecule of ATP consumed. Thus the rapid removal of products upon activation of ATPases is crucial in order avoid a decrease in the free energy of ATP hydrolysis. The inverse is of course true for ATP synthesis which can be considered the reverse reaction of ATP hydrolysis. Here a relatively low ATP/ADP ratio allows ATP production with less free energy consumed per molecule of ATP manufactured, and rapid removal of ATP and resupply of ADP are advantageous. Creatine kinase function within muscle is crucial in this context. Upon the hydrolysis of a molecule of ATP by cellular ATPases, C K will catalyze the rapid rephosphorylation of ADP to ATP at the expense of a molecule of PCr, consuming a proton (FT) in the process. This maintains a high ATP/ADP ratio and thereby keeps the energy released through ATP hydrolysis at a maximum. The opposite is true of C K at the outer face of the inner mitochondrial membrane where C K assists in the maintenance of a low ATP/ADP ratio and minimizes the energy required for ATP synthesis. Thus the thermodynamic efficiency of energy metabolism is increased in both production and consumption of ATP by C K activity. This holds true for both C K mediated energy buffering and energy transport. As discussed above, energy buffering contributes to maintenance of relatively high ATP/ADP ratios at sites of energy consumption, and C K mediated facilitated diffusion reduces the concentration gradients required for ATP and ADP diffusion and thus also serves to maintain a high cellular ATP/ADP ratio. Also consider the role of miCK in generating a low ATP/ADP ratio at the outer face of the ANT in this context. Meyer et al (1984) point out that in the context of the facilitated diffusion model, energy transport and energy buffering through C K are directly linked by their role in 21 maintenance of high local ATP/ADP ratios. Consequently, these two functions which are generally considered as distinct roles played by the CK equilibrium can be considered as one. The roles played by the various isoenzymes of CK within muscle energy metabolism may then be summarized through the following model: Mitochondrial CK maintains a relatively low ATP/ADP ratio at the outer surface of the ANT in order to maintain a constant and relatively large supply of ADP to the ANT for translocation. This maximizes the efficiency of ANT function and thus ultimately increases the efficiency of ATP synthesis (lowering the free energy required for ATP synthesis) through maintenance of a low ATP/ADP ratio within the mitochondria. Phosphocreatine and ATP diffuse from the mitochondria and Cr and ADP return from sites of ATP hydrolysis in a mode best described by facilitated diffusion mediated by cytosolic CK. This minimizes the concentration gradients required to maintain diffusive flux of ATP and ADP to and from the mitochondria and indirectly allows local ATP/ADP ratios to remain high at sites of energy consumption. Direct maintenance of ATP/ADP ratios in areas of high ATPase activity is mediated by isoenzymes of CK functionally coupled to ATPases (ion pumps, contractile proteins). Again, the high ATP/ADP ratios at sites of ATP hydrolysis allow a greater free energy release for each molecule of ATP consumed. Two recent studies provide excellent demonstrations of this concept. In the context of steady state work where PCr concentrations remain constant and the energy transport role of CK is prominent in normal hearts, Zweier et al (1991) saw an increase over controls in the O2 consumption of myocardium of Cr depleted rats at the same work rate. They attributed this to the elevation of free ADP at sites of energy consumption, causing a decreased ATP/ADP ratio and thereby a decrease in the free energy released through ATP hydrolysis. Therefore the treated 22 hearts consumed more ATP per unit of work than controls, and demonstrated a corresponding increase in the rate of mitochondrial ATP synthesis through an increase in oxygen consumption. Complementary to this is work by van Deursen et al (1993) which provides an excellent demonstration of the importance of C K in maintenance of local ATP/ADP ratios in the context of energy buffering. They introduced a null mutation in the M C K gene of mice, resulting in the loss of M M C K function. Skeletal muscles of these mice actually had higher resting PCr levels (Cr phosphorylated by miCK), and showed no difference from controls in initial absolute force development. However treated muscles could not perform burst work; developed force dropped off rapidly from initial levels with repeated stimulation. This can be directly attributed to a large increase in local free ADP at sites of high ATPase activity in the absence of M M C K , and thus significant drops in local ATP/ADP ratios upon the initiation of muscle contraction. The efficiency of energy metabolism is thereby increased both at sites of production and consumption through C K mediated control of local metabolite concentrations. This model encompasses all aspects of C K function within muscle. Although C K is linked to energy metabolism at multiple sites and in a number of capacities, the purpose of C K function is best described through increased efficiency in all contexts. ADP - Roles in Metabolic Regulation The regulation of metabolism in muscle has been a field of intensive research for decades however the overall picture remains unclear in many respects (see Balaban, 1990). There are many variables which interact within energy metabolism, and it has proven extremely difficult to determine the in vivo role of any particular metabolite in the control of metabolism. This complex 23 interdependence may actually make studies which attempt to isolate the effects of individual variables misleading in terms of their actual physiological roles in normal muscle. Studies of metabolic regulation have centred around ADP control since the early kinetic model for ADP regulation of oxidative phosphorylation was proposed by Chance and Williams (1956). Adenosine diphosphate is easily envisioned operating as a direct feedback to stimulate oxidative phosphorylation in that it is a direct product of ATP hydrolysis and a substrate for ATP synthesis. Research on a variety of skeletal muscle fibre types indicates that resting ADP concentrations are at or below the Km for oxidative phosphorylation, lending further support to a possible regulatory role for ADP. While this model has not proven able to describe most observations of in vivo muscle metabolism, it is still accepted that ADP does play at least some role in the regulation of energy metabolism within all muscle. In vitro work with heart mitochondria has shown that under certain conditions, oxidative metabolism may be regulated by changes in ADP. Work with isolated perfused hearts also reveals some dependence of metabolic rate on ADP concentrations, however this relationship in turn depends on which substrate is provided in the perfusate (From et al, 1990). In contrast, in vivo MRS work has demonstrated little or no change in ADP levels with significant changes in work rate (Ffeineman and Balaban, 1990). Since such small changes are observed in the levels of ADP during physiological work in heart, it appears that the role played by ADP in regulation of metabolism within heart is minimal. In an extensive review of myocardial energy metabolism, Balaban (1990) concludes that changes in ADP and Pj play a role in fine tuning of metabolism, while large scale changes in metabolic rate must be mediated through other regulatory parameters. 24 Skeletal muscle fibre types vary with respect to the importance of the role played by ADP in regulation of the rate of metabolism. Slow twitch oxidative muscle does not show a simple relationship between ADP concentration and O2 consumption with changes in submaximal work (Kushmerick etal, 1992; Connett and Honig, 1989). This argues against direct kinetic control of oxidative phosphorylation through an ADP feedback mechanism within this fibre type, however ADP levels do still seem to play some significant regulatory role as a part of a complex regulatory system. The same experiment yielded a first order relationship between ADP concentration and O2 consumption for fast twitch muscle, suggesting that control of oxidative phosphorylation in fast twitch glycolytic muscle may indeed be explained by the kinetic model for ADP at low work rates. Other studies have supported these observations for skeletal muscle, but again only at low work rates (see Nioka et al, 1992). Hochachka and Matheson (1992) point out that observed changes in ADP concentration are nowhere near large enough to explain the increases in oxidative metabolism encountered at high work rates for skeletal muscle, and thus the regulatory role played by ADP at these work rates must be small relative to that of other parameters. In summary then, present knowledge of muscle metabolism supports ADP as a regulator of oxidative phosphorylation but only to a limited extent. At low work rates in skeletal muscle, and particularly in F G fibres, ADP seems to play an important role in the control of metabolic rate. The situation changes in muscle fibres of a more oxidative nature, where the role of ADP regulation is downscaled relative to that of other parameters as the coupling of energy consumption to aerobic energy production becomes tighter. ADP cannot be considered a primary regulator of muscle energy metabolism at maximal or near maximal work rates. 25 Creatine Distribution - Relevance to Metabolism Tissue ADP concentrations measured through conventional tissue extraction techniques give an overestimate of the actual 'free' (metabolically active) ADP due to extensive subcellular binding of ADP to a variety of proteins. The knowledge of free ADP is important to the study of metabolic regulation, and the problem of binding has been circumvented through the use of the C K equilibrium to calculate metabolically active free ADP concentrations: A D P ^ = [ATP] [Cr] / [PCr] (K^ ) (10"pH). Thus free ADP concentrations are calculated through measurement of ATP, PCr, Cr, and pH in tissue extracts and the knowledge of the equilibrium constant for CK. An underlying assumption of this calculation is that all other reactants are not binding to subcellular structures to any significant degree and thus that their measured concentrations are representative of their in vivo activities. Unrecognized binding of any of these measured metabolites would introduce error into the calculation. The specific activity (S A) of a metabolite is a ratio of the activity of radiolabel contained in the compound of interest to the total tissue concentration of that compound following incubation of the tissue with the radiolabeled metabolite. This parameter may then be considered an index of the extent of mixing of the radiolabel with the total pool of this compound. Hence a ratio of the specific activities of two compounds which exist in equilibrium provides an indication of mixing of radiolabel with the pool of one compound to the mixing of label with the next. For example, for the equilibrium: A<->B 26 the ratio of S A A / S A B offers an indication of the relative access of radiolabel to the pools of A and B within the tissue sampled. If access of label to both pools is equal, then the S A A / S A B ratio should be equal to 1. In terms of the present study it is the ratio of SApo/SAcr which can be considered in this context. Early work from Lee and Visscher (1961) found that after incubating rabbit heart with [1-1 4 C] Cr, the specific activity ratio of PCr to Cr was greater than 1 ( S A P O / S A O > 1). This indicates that the labeled Cr apparently had access to a larger proportion of the PCr pool than the Cr pool, leading the authors to postulate a bound pool of Cr which does not participate in the C K equilibrium. The controversy surrounding the PCr shuttle hypothesis prompted Savabi (1988) to investigate this question further. Using isolated rat atria incubated with [1-14C] Cr and a series of treatments involving anoxia and reoxygenation, Savabi calculated that approximately 20% of Cr measured through tissue extracts (9% of total Cr) is actually free to react with C K within the muscle. This leaves 80% of unphosphorylated Cr (36% of total Cr) bound. The bound pool did not unbind with anoxic treatment or show a tendency to mix with the free pool over the time course employed (up to 45 minutes). Thus the majority of unphosphorylated Cr within normoxic rat heart exists within a slow turnover 'bound' pool which does not participate in the C K reaction. If only 20% of measured Cr participates in the reaction, then calculations of free ADP concentrations based on Cr determined in tissue extracts are overestimated by a factor of five. Correction of current numbers then puts free ADP levels well below the Km for oxidative phosphorylation, suggesting that ADP has the potential to play a more important regulatory role than is currently thought. Also note that the magnitude of calculated changes in free ADP concentrations is underestimated without consideration of Cr binding, due to the fact that the free 27 Cr is only a portion of that measured. This may not be of consequence with respect to the role of ADP in regulation of metabolism in heart where high energy phosphates show little variation within the range of physiological work rates, however in skeletal fibres where ADP plays a more important role this may have a significant effect on current thinking. Unitt et al (1992) followed up on Savabi's study with MRS work on isolated rat heart. Study with MRS can only detect free metabolites and thus whatever is detectable by MRS is thought to be free within the cell. Quantification of the free pools of PCr and Cr through the use of an internal standard allowed direct comparison of MRS data with that gathered through conventional analyses. The MRS measurements of unphosphorylated Cr were just over 10% (8% of total Cr) lower than those determined through tissue extractions, and this difference was not statistically significant. This led Unitt et al to conclude that Cr binding is not significant in rat heart, and that the binding observed by Savabi (1988) was the result of an artefact induced by some unknown factor. It is however possible that the so-called bound fraction of Cr is MRS visible, which would suggest that it is free in solution but somehow not accessible to CK. No clear solution has thus been obtained with regard to the binding of Cr within muscle tissue. Also, research to date has only been applied to mammalian heart, where studies of Cr depletion have indicated that the function of the C K equilibrium may not be absolutely essential to function. Fast glycolytic fibres are more directly dependent on the C K equilibrium to support energy metabolism than the smaller more oxidative fibre types, as is demonstrated by the significant metabolic adjustments which these fibres undergo in Cr depleted animals. Fast fibres are known to maintain large pools of total Cr and thus have large concentrations of PCr present in the resting 28 state. High ATPase activity and relatively low oxidative capacity characterize this type of muscle fibre, and thus energy buffering through PCr hydrolysis is more pronounced than in other fibre types. Creatine kinase mediated energy transport through facilitated diffusion should also be of greater importance in F G fibres due to the large fibre diameters and high ATPase activity which would result in large concentration gradients for diffusion of the adenylates in the absence of the C K equilibrium (Meyer et al, 1984). In addition to this high level of dependence on C K activity, ADP based regulation of oxidative phosphorylation appears to be more significant in F G fibres than other muscle fibre types. These factors combine to make F G fibres an excellent model for the study of Cr binding. If a large part of the pool of total Cr measured through tissue extracts is actually not participating in energy metabolism in vivo, then this has implications for current thinking about C K function in terms of both energy buffering and energy transport. Creatine binding will also affect calculations of free ADP through the use of the C K equilibrium, and thus may be important to models of metabolic regulation via ADP control of oxidative phosphorylation. The present study investigates further the phenomenon of Cr binding using rainbow trout white muscle. Although the white muscle of rainbow trout is described as a mosaic of F G and SO fibres, it is composed largely of F G type muscle fibres and is far more homogeneous in this respect than mammalian muscles which are often used as models of F G fibre function (i.e. gastrocnemius or extensor digitorum longus). This model system also has the advantage of being ectothermic, and having a slower metabolic rate than that of mammalian preparations. Metabolic processes are therefore more easily analyzed. 29 We injected [amidino-14C] Cr into the circulation of rainbow trout (Oncorhynchus mykiss). Following an initial incubation period of 2 hours, white muscle was sampled either in the resting state or following partial recovery from one of two exercise protocols which were designed to present differing levels of energy depletion. Our findings are similar to those of Savabi (1988) in that we found significant binding of Cr in the resting state and this binding did not seem to change with exercise. The data also suggests some subcellular compartmentation of the metabolically active pool of total Cr, a finding which is inconsistent with the PCr shuttle hypothesis. 30 Materials and Methods Experimental Animals Juvenile rainbow trout (Oncorhynchus mykiss) of both sexes were obtained from West Creek Trout Farms, Aldergrove, B C and kept in aerated dechlorinated tap water in an outdoor circular holding tank. Feeding to satiation with commercial trout pellets was performed 3 times per week. Fish were cannulated via the dorsal aorta (Soivio et al, 1972) and transferred to black holding boxes with a continuous flow of aerated dechlorinated water (temp. 8-12 °C). A continuous flow of dechlorinated water containing MS222 (0.1 g/1) buffered with sodium bicarbonate (0.2 g/1) and aerated with 100% 02 was maintained over the gills throughout surgeries. Fish were allowed to recover for three days following surgery before experimentation. A l l experiments were carried out during the fall of 1994. Resting Experiments Twelve hours before extraction of muscle tissue, fish (mean mass = 469.1 ± 36 g; SEM; n = 7) were injected with a 0.5 ml bolus of [3H] polyethyleneglycol (PEG; M W = 4000; New England Nuclear) suspended in Cortland's saline at a dosage of 20 uCi/kg. This was followed at 2 hours prior to tissue sampling with a bolus injection of 0.5 ml/kg of [amidino-14C] Cr hydrate (Amersham) suspended in Cortland's saline at an activity of 60 uCi/ml, resulting in a dosage of 30 uCi/kg of [amidino-14C] Cr hydrate per fish. Cannulae were flushed with 0.5 ml of saline followed by 0.3 ml of heparinized saline to fill the dead volume of the cannulae after each injection. Five hundred ul of whole blood was collected into 1.5 ml Eppendorf tubes at 2, 5, 15, 30, 60, and 120 minutes after [amidino-14C] Cr hydrate injection. Cannulae were again flushed with 0.3 ml of heparinized saline after each sample. Blood samples were held on ice until being spun in a microfuge for 10 minutes. One hundred ul of plasma was then drawn off of each sample for scintillation counting while the rest of the plasma was saved for assay of plasma Cr concentration. The red cell pellets were discarded. Two hours after injection of isotope, 0.5 ml of whole blood was drawn, followed by the injection of sodium pentobarbital (1 ml/kg). As soon as opercular movement stopped the fish were pulled from the boxes without struggle and a block (approx. 5 g) of dorsal epaxial muscle was excised with a razor blade and freeze-clamped with aluminum tongs pre-cooled in liquid N2. Samples were then stored at -80°C until processing. Although use of a biopsy drill would have allowed more rapid freezing of tissue and thus presumably less hydrolysis of PCr (see Botker, 1993), this technique does not provide enough tissue for the analyses required by this study (Arthur et al, 1992). The incubation period chosen for this experiment was based on the work of Danulat (1988) with starry flounder (Platichthys stellatus) which indicated rapid initial uptake of label into white muscle. Preliminary experiments found no difference in S Ap&/S Acr ratios for incubation periods of 1 hour, 2 hours, 6 hours, and 6 days, with the specific activities rising rapidly within the first two hours and then showing no more large increase. Thus a 2 hour incubation was long enough both to maximize uptake of label and to allow equilibration of label between the PCr and Cr fractions. 32 Exercise Experiments These experiments were designed to test the effects of exercise on creatine binding. The exercise protocols employed were intended to expose the different groups to distinct degrees of metabolic challenge. Connett (1988) describes two phases of energy depletion; i) a 'buffering' phase in which PCr levels decrease while ATP remains relatively constant and ii) a 'depleting' phase during which ATP levels can no longer be maintained through PCr hydrolysis and ATP levels subsequently fall (Figure 1). The buffering protocol was designed to cause hydrolysis of most of the PCr stores without a large drop in ATP, while the depleting protocol was intended to achieve maximal depletion of both stores. Exercise was followed by recovery periods which were designed to give both groups a similar Cr charge ([PCr] / ([PCr] + [Cr])) at the time of sampling in order to facilitate comparisons of SApo/SAc* between groups. Appropriate recovery periods were determined through preliminary experiments in which fish were exercised according to either of the two protocols and then allowed to recover for various periods of time prior to tissue sampling. Fish were exercised in a Brett type swim tunnel equipped with an 8 volt electrical grid. Thirty minutes prior to exercise fish were transferred to the tunnel and allowed to equilibrate at a low flow speed (1 body length per second (bl/s)). Isotope was injected 2 hours prior to exercise in the manner previously described. Ucrit was determined in a preliminary set of experiments at 2.5 + 0.3 bl/s (n = 6). White muscle recruitment is poor at swimming speeds below Ucrit (Johnston, 1981), and thus fish for the buffering group (mean mass = 437.7 ± 24.8 g; SEM; n = 9) were swum at 120% of Ucrit in order to insure full white muscle recruitment. All fish required burst swimming to maintain their position in the swim tunnel at this speed. Swimming at 120% of Ucrit was continued for 7 minutes, followed by recovery at a flow rate of 0.5 bl/s for 10 minutes. This protocol was designed based on the work of Dobson et al (1987) who found depletion of ATP stores after 10 minutes of swimming at this intensity. Preliminary work demonstrated that fish swum for 7 minutes at 120% of Ucrit did not suffer significant depletion of white muscle ATP while there was a large depletion of PCr stores (Table 2). Note that anesthetic was injected at the end of the exercise and took approximately 1 to 2 minutes to take full effect, thus the levels given are for tissue sampled following partial recovery. A 10 minute recovery period with flow down to 0.5 bl/s then followed prior to blood sampling, injection of anesthetic and tissue sampling. The depleting group fish (mean mass = 4 1 7 . 5 ± 3 7 . 1 g ; SEM; n = 6) were swum to exhaustion through the exercise protocol of Schulte et al (1992), with the total exercise time varying between 25 and 35 minutes per fish. Exhaustion was defined by an inability to maintain 2 bl/s and a lack of struggle upon grasping. Fish sampled at exhaustion showed depletion of both ATP and PCr stores (Table 2). The recovery period for this group was 30 minutes at 0.5 bl/s before blood sampling, injection of anesthetic and tissue sampling. Tissue Analyses Approximately 500 mg of white muscle were chipped from the epaxial sample block under liquid nitrogen. This tissue was then transferred into preweighed tubes containing 2.0 ml of ice cold perchloric acid (PCA), which were then reweighed to determine the precise mass of tissue analyzed. Samples were homogenized with an Ultra Turrax homogenizer for 3 x 15 seconds with about a 30 second wait between grinds to allow cooling. During homogenization the tubes were 34 held in a beaker containing an ice and salt water slurry which maintained the temperature between -5 and 0° C. The samples were then centrifuged at lOOOOxg for 10 minutes and neutralized with 3 M K2CO3, 100 mM triethanolamine-HCl to a pH of 7.6. Neutralization was followed by centrifugation at lOOOOxg for 10 minutes and the supernatant was separated into 0.5 ml aliquots, frozen in liquid nitrogen, and stored at -80°C until analysis. A set of 4 preliminary samples were run in which a known amount of PCr was added to the test tube prior to homogenization. Recovery of PCr was above 95% for all extracts. Extracts were assayed for ATP, PCr, andCr on a Perkin Elmer Lambda 2 UV/visual spectrophotometer and a Titertek Multiskan MCC/340 plate spectrophotometer using enzyme assays adapted from Lowry and Passoneau (1993). All assays were run in duplicate and validated by the use of appropriate standards. Plasma samples were assayed for Cr alone. Creatine and PCr fractions were separated by high pressure liquid chromatography (HPLC) using a modified version of the method of Harmsen et al (1982). Two hundred and fifty ul of each extract was injected onto a Whatman Partisil 10-SAX column using a Waters 625LC system. Initial solvent was 0.01 M phosphoric acid at pH 2.85, changing to 0.75 M KH2PO4 after 5 minutes, all at a constant flow of 1.5 ml per minute and a temperature of 25°C Total run time was 21 minutes. Six ml were collected directly into scintillation vials for both theCr and PCr fractions. Fractions collected from preliminary runs with unlabeled PCr and Cr were assayed to determine the appropriate collection protocol. The fractions were then dried down for 24 hours at 80°C and suspended in ACS TI scintillation cocktail (Amersham). Once the scintillation cocktail was added the fractions were stirred for 15 minutes on a magnetic stirrer and then stored in the dark for five days prior to 35 counting in an L K B 1214 Rackbeta scintillation counter. The amount of quench was found to undergo quite a large change over the first few days following suspension of samples in scintillant. A series of external standards were run with known amounts of [amidino-14C] Cr hydrate suspended in 6 ml of HPLC buffer, dried down and then resuspended in scintillant and stirred. The activity of label used was varied across the range observed for samples. This work indicated that the amount of quenching was consistent in samples containing different amounts of [1 4C] activity and that quench was different for each buffer. The amount of quenching stabilized in samples from both HPLC buffers by the fifth day following suspension in scintillant. Appropriate corrections were applied to sample dpm values. E C F V Corrections Extracellular fluid volume (ECFV) was calculated according to the method of Milligan and Wood (1986); E C F V (ml/g wet weight) = Tissue [3H1 (dpm/g^ x plasma % H?Q Plasma [3H] (dpm/g) The marker used was [3H] P E G of M W = 4000 (New England Nuclear). Munger et al (1991) found this to be the most conservative and reliable in a study in which they compared a variety of extracellular space markers in rainbow trout white muscle. Plasma % H 2 0 was determined by drying 200 ul of plasma to constant weight in previously weighed 1.5 ml Eppendorf centrifuge tubes. 36 Statistics Values are reported as mean + SEM. Group by group comparisons are performed through the application of a Student's t-test where F-tests are not significant. In cases where significant heterogeneity of variance between groups is indicated by an F-test, comparisons are made by means of a one-way ANOVA. Data are log transformed where necessary. The level of significance was set at p < 0.05. Calculation of the ratio of the specific activity of PCr over the specific activity of Cr (SApc/SAcr) following ECFV corrections gave some extreme outlying values. These were removed prior to calculation of means and graphing of data. 37 Results Extracellular Fluid Volume The E C F V value determined for samples from resting fish (ECFV = 0.0398 ± 0.0045 ml/g wet weight; SE; n = 7) is slightly higher than those for the buffering group (ECFV = 0.0362 ± 0.0031 ml/g wet weight; SE; n = 9) and the depleting group (ECFV = 0.0251 ± 0.0012 ml/g wet weight; SE; n = 6). This decrease in E C F V following exercise is consistent with that seen by Milligan and Wood (1986). Plasma [1 4C] Clearance Plasma [1 4C] clearance and Cr concentration are reported in Figure 2. Radioactivity is rapidly cleared from the plasma, falling over 90% over the first 2 hours of incubation. Plasma Cr concentration remains stable over this time period. Metabolites Refer to Table 2 for white muscle metabolite concentrations. Adenosine triphosphate and PCr concentrations from the resting group compare well with literature values for rainbow trout white muscle (Dobson et al, 1987; Schulte et al, 1992). Note however that the PCr concentrations determined through this method of tissue sampling are not those seen within resting white muscle in vivo due to activation of the muscle during excision (Botker et al, 1994). Nuclear magnetic resonance spectroscopy work consistently shows PCr content to be approximately 75% of total Cr within resting skeletal muscle (i.e. Cr charge = 0.75), 38 whereas the resting Cr charge determined for this study is 0.47. For the purpose of comparisons between groups these numbers remain valid, as tissue for all groups was extracted in identical fashion. Fish sampled after 7 minutes of burst swimming showed a large reduction in PCr with only a small drop in ATP concentrations. It appears that this treatment did work the white muscle within the bufFering phase of energy depleting work described by Connett (1988) (Figure 1). Note that it took approximately 1 to 2 minutes for anaesthetic to take effect prior to freeze clamping and therefore by the time of tissue sampling, the muscle had already undergone partial recovery. Due to this, the PCr concentration reported is probably slightly higher than the actual value within the muscle upon completion of the exercise protocol. The ATP concentration immediately following exercise should not have been different from that reported considering that recovery of ATP in this tissue follows a much slower time course than that observed for PCr (see Schulte etal, 1992). Fish from the bufFering group had ATP and PCr concentrations at 10 minutes of recovery which were not significantly different from controls, indicating rapid recovery from work of this intensity. The depleting protocol (fish swum to exhaustion) significantly depleted both ATP and PCr in white muscle. Phosphocreatine concentrations increased over the 30 minute recovery period while ATP remained low over this period, consistent with previous studies of recovery of teleost white muscle from exhaustion (Schulte et al, 1992) and anoxia (van Waarde et al, 1990). Both metabolites remained below resting levels at the time of sampling. Total Cr did not vary between groups. 39 Specific Activities Table 3 contains specific activity data gathered in this study. No significant differences were found between groups with respect to the SApc r or the SAc r. Note that the SApo/SAcr ratios reported are greater than 1 for all groups. Consideration of Cr charge (Table 2) in conjunction with S Apa/S A c r ratios suggests a relationship between these two parameters. A linear regression of SApo/SAcr against Cr charge for trout white muscle gives a significant negative slope of -6.10 at p = 0.014, r 2 = 0.27 (Figure 3). Although there is considerable variability in the data, it is apparent that SAp C l /SAcr increases with decreasing Cr charge consistent with the results of Savabi (1988). Due to this variation of SApct/SAcr with Cr charge, comparisons of SApo/SAcr between groups first required that groups be tested for significant differences in Cr charge. These data were log transformed to accommodate heterogeneity of variance between groups, and then compared through a one-way ANOVA. The test revealed that the three groups are significantly different with respect to Cr charge, making direct comparison of mean S A p o / S A o among groups difficult to interpret. 40 Discussion Resting Muscle Savabi (1988) reports a SApa/SAcr ratio of 1.87 for normoxic isolated rat atria, at a Cr charge of 0.56. This is similar to that reported by Lee and Visscher (1961) of 1.61 at a Cr charge of 0.56 with perfused rabbit heart. Thus it seems that in both of these preparations relatively more label is found in the PCr fraction, and hence access of label to the PCr pool is greater than that to the Cr pool. All of cellular PCr is thought to be free to interact with C K within muscle (Zeleznikar and Goldberg, 1991), leading to the conclusion that there must be at least two pools of unphosphorylated Cr; one which has access to C K and one which does not. The nature of this second inaccessible pool is unknown and will be referred to as 'bound'. Our results indicate that there is significant binding of Cr in trout white muscle. The ratio of the SApa/SAcr is greater than 1 for all groups sampled (Table 3) and thus the [1 4C] Cr taken up by this muscle only had access to part of the pool of unphosphorylated Cr. Analysis of resting rainbow trout white muscle following 2 hours of incubation with [1 4C] Cr reveals a SApC l/SAc r ratio of 3.10 at a Cr charge of 0.47; a value notably higher than those quoted above for similar experiments with normoxic mammalian hearts. There are a couple of possible explanations for this discrepancy. Firstly, as previously discussed, the tissue excision technique used in this study causes considerable hydrolysis of PCr prior to freezing and therefore this is not representative of the true resting state for this tissue. The relatively high value for the SApo/SAcr ratio obtained with resting fish white muscle may then be explained by the apparent increase in this ratio with decreased Cr charge (see Figures 3 and 4). Also, while the numbers reported in these other 41 studies were for normoxie hearts at resting work rates (spontaneously beating, Savabi 1988; low flow rate, Lee and Visscher 1961), the hearts were contracting and not quiescent as is the case for resting white muscle. This fundamental contrast in the physiology of the two muscle fibre types may contribute to the observed difference. Exercise Groups and Extended Incubations The second major conclusion which may be drawn from our data is that exercise does not cause significant mixing of the metabolically active pool of Cr (that which has access to CK) with the bound pool. Neither of the two exercise protocols drove the S A p o / S A c r ratio towards 1, which would be expected if the bound pool became accessible to C K and thus mixed with the pool containing the [1 4C] Cr label. Thus it seems that the size of the metabolically active pool of total Cr is relatively constant through a variety of physiological states. Furthermore, turnover of the bound pool of Cr appears to be very slow. Fish in which the incubation with [1 4C] Cr was extended to 6 days did not show a decrease in S A p C l / S A c r relative to the resting fish incubated for only 2 hours (Table 3). Again, turnover of the bound pool would be expected to drive the ratio towards 1 as a larger proportion of the total pool of Cr gains access to label. The observed ratio suggests very slow turnover of the bound pool of Cr, or possibly a distinct pathway for turnover which does not involve mixing with the metabolically active pool. C r ' Binding' What might be the nature of the binding of Cr observed in muscle? Exhaustive exercise did not cause mixing of the bound and free pools. When considered along with the lack of 42 decrease in the SApa/SAcr ratio offish incubated with radiolabel for 6 days, it seems that the bound pool of Cr is not easily released or rapidly turned over. Kinetic studies such as the present study and those of Savabi (1988) and Lee and Visscher (1961) suggest a high degree of binding, leaving a large portion of total Cr inactive in metabolic terms. Somewhat surprisingly, an MRS study by Unitt et al (1992) found no significant difference between the concentration of Cr in isolated perfused rat heart determined through MRS and that determined through standard biochemical analyses. To be visible to MRS study, molecules must be 'free-tumbling' and relatively mobile. This suggests that the motion of the so called bound pool of Cr may not be restricted to the extent that it is no longer visible to MRS. This conflict between the kinetic and MRS data could be caused by the existence of the so-called bound fraction of Cr in solution within a distinct subcellular compartment which is not in contact with the major metabolic pathways. The slow turnover and lack of change observed in the bound fraction with exercise would however tend to contradict this idea. A more plausible explanation is that the metabolically inactive fraction of Cr is bound to another small molecule (i.e. a small protein) which allows mobility but not reaction with CK. This is a area which requires further investigation. Quantification of Cr Binding Although precise quantification of the extent of binding in rainbow trout white muscle is not possible in this study, for the purposes of discussion an attempt can be made through a modified version of the calculation used by Savabi (1988). Values obtained for the resting group may be used as a reference against those of the exercise groups in order to calculate the size of 43 the free and bound pools of Cr. The ratio of SApc/S Acr can be thought of as an index of the relative access of [14C]-Cr to the total pools of both metabolites: SApo/SAcr = access of [14C] Cr to PCr pool/ access of [14C] Cr to Cr pool. The slow uptake of [14C] Cr into trout white muscle relative to that of mammalian heart preparations required long incubation times prior to sampling, and thus for the purposes of the calculation it must be assumed that isotope had access to the entire pool of PCr. Access of labeled Cr to the unphosphorylated pool of Cr can be represented in the following terms: SApa/SAcr=l/[(S + C)/D] where S is the percentage of total Cr which interacts with CK (i.e. not bound) and which is unphosphorylated at rest, C is the percentage of total Cr which is phosphorylated at rest but remains unphosphorylated at the time of sampling a group which has undergone partial recovery from exercise, and D is the percentage of total Cr measured as unphosphorylated Cr in tissue extracts at the time of sampling a group which has undergone partial recovery from exercise. The value of S determined through this method should represent the proportion of total Cr which is unphosphorylated and interacts with CK in the true resting state of the muscle (Cr charge = 0.75). The reference numbers used for the calculation of Cr pool sizes are those from Table 2 for samples excised as described in materials and methods. As previously discussed, samples for all groups were excised in identical fashion and thus although the absolute values determined for PCr and Cr (and thus SApcr /SAc r ) are not those within the muscle prior to excision, relative differences between groups should not be affected. It is these relative differences that are used in the calculation of Cr pool sizes, and thus the values calculated for S should be reflective of the actual resting muscle (Cr charge = 0.75). 44 Applying this calculation to data for the buffering group (see Tables 2 and 3) gives a value for S (unbound Cr which is free to interact with C K in the resting state) of 7.6% of total Cr. This agrees well with calculations for rat atria, with S determined at 9% of total Cr (Savabi, 1988). The same calculation applied to the depleting group results in a value of -7.3% for S. Accepting the shortcomings of data gathered from an in vivo system, it does appear that the S pool may be of approximately the same size in resting trout white muscle (below 10% of total Cr) as in normoxic rat atria (8 or 9% of total Cr). This then suggests that approximately 15 to 20% of the total Cr measured in trout white muscle is bound and not accessible to C K in the resting state. The values calculated through this method for this study should only be interpreted as rough estimates due to two factors which make precise quantification of pool sizes difficult: i) there is considerable variability in the S A p o / S A c r data which may cause the observed variability in calculations of the free pool of Cr, and ii) although tissue excision was identical in all three groups, different degrees of PCr hydrolysis may have occurred in sampling different groups due to the variation in metabolic status between groups at the time of sampling (see Table 2). Thus the use of the resting data as a reference against data for fish which were recovering from exercise may not be appropriate. Implications for Energy Metabolism The sequestration of a significant part of the pool of total Cr into a bound fraction which does not seem to participate in energy metabolism means that the level of free Cr is lower than that determined through standard analyses. This may mean that the level of free Cr in resting trout white muscle is actually in the range of only 2 to 5 mM (based on 5 to 10% of total Cr free 45 at rest). While this would still be enough to easily support the energy transport function ascribed to C K (see Yoshizaki et al, 1990), reference to the equilibrium equation reveals that calculations of free ADP will be affected. Calculations of free ADP for trout white muscle give a concentration of just over 20 uM at rest (Schulte etal, 1992) based on PCr and free Cr concentration determined through standard analysis of tissue extracts (Cr charge = 0.5). If, as our results suggest, 10 to 20% of total Cr (approximately 20 to 40% of unphosphorylated Cr for Cr charge = 0.5) is bound within fish white muscle, then free ADP concentrations must be 20 to 40% lower than those previously calculated. This would put resting free ADP in the range of 12 to 16 uM. If these values are then corrected to a Cr charge of 0.75 which is probably representative of the true metabolic status of resting white muscle, then free ADP must be only 4 to 6 uM. Resting ADP concentrations may then be well below the Km of oxidative phosphorylation for ADP (25 to 30 uM), where small changes in ADP concentrations may have relatively large effects on the rate of oxidative energy production. This suggests greater significance for the role played by ADP as a regulator of oxidative metabolism in the resting state and at low work rates. Also note that the magnitude of calculated changes in free ADP concentrations with work are affected byCr binding. Since the total pool of metabolically active Cr is smaller than that assumed in previous work, Cr released through PCr hydrolysis represents a larger proportion of total active Cr. Thus per-fold changes in free Cr are larger and this is reflected in calculations of free ADP. Assuming a total Cr pool of 45 mM of which 7 mM is bound (15% of total assuming 10% free Cr at resting Cr charge of 0.75), and if resting free Cr is actually only 5 mM, then the change in free Cr seen with severe PCr depletion (2 mM PCr remaining, 36 mM free Cr) is somewhere around 7 fold, rather than the just under 4 fold change determined without consideration of Cr binding. This translates into a 3 fold increase in free ADP over values previously calculated for this extreme metabolic condition of almost total PCr depletion. However, Schulte al (1992) found free ADP at exhaustion to be equal to that at rest and thus within rainbow trout white muscle the effect of Cr binding on per fold changes in the calculation of free ADP is negligible. The low resting free ADP concentration of 4 to 6 uM also suggests that feedback to the mitochondria in the form of ADP alone could not support metabolism. Yoshizaki et al (1990) calculate a diffusion length of 1.8 um for ADP in bullfrog skeletal muscle based on a free concentration of 30 uM. They argue that this would not pose a problem in myofibrils of approximately 1 um diameter in the absence of C K mediated energy transport. Consideration of the lower resting free ADP in trout white muscle and the larger fibre diameter of 15 to 90 um (Johnston, 1981) reveals that ADP diffusion alone could not support energy metabolism. Creatine kinase mediated facilitated diffusion of high energy phosphates must therefore be an essential function of C K in rainbow trout white muscle. In this context, a study of the effects of Cr depletion on this tissue would be interesting. SAgfVSAr, and Cr Charge - Intracellular Mobility of Cr The recovery periods allowed prior to tissue sampling in the exercise protocols were intended to provide samples with the same Cr charge from the buffering and depleting groups. Analysis of the samples revealed that Cr charge was different in the white muscle of these two 47 groups at the time of sampling, thus making direct comparison of the SApo/SAo ratios difficult to interpret. This variation in Cr charge among groups may however offer an unexpected insight. A linear regression of S Apo/S A c r ratio against Cr charge reveals a significant relationship between these two parameters (Figure 3), with the negative slope indicating a trend towards an increase in the SApo/SAcr ratio with decreased Cr charge. Although this relationship explains just under one third of the variability in the data, note that the use of an in vivo model in this study makes a relatively high level of variability in this type of data inevitable. Also, since this study was not designed to investigate the relationship between these two variables, the data set only covers a narrow range of Cr charge values relative to that observed in vivo. Consideration of Savabi's (1988) data in this context supports the relationship observed in the present study. Savabi (1988) observed an increase in the S A P C / S A c r ratio with decreased Cr charge during treatment of rat atria with anoxia. As Cr charge decreases in rat atria, the SAp C l /SAcr ratio appears to increase in exponential fashion (Figure 4). Note that this also includes groups which were treated with anoxia and then reoxygenated, and thus PCr stores in these hearts underwent extensive hydrolysis and resynthesis yet ended up with a final SApo/SAcr ratio very close to that of the normoxic controls (1.98 and 1.95 versus 1.87 for the controls). The relationship between SApc/SAcr and Cr charge for our data is best described by a linear regression. This includes the buffering and depleting groups which were sampled during recovery from exercise, and in which the PCr pool underwent extensive hydrolysis and resynthesis in the presence of [1 4C] Cr label prior to tissue excision and freezing. Again, our data set does not contain values for the entire in vivo range of Cr charge and thus the possibility that the relationship between SApo/SAcr and Cr charge is also exponential in trout white muscle cannot be excluded. 48 This relationship is difficult to explain given current knowledge of muscle energy metabolism and subcellular design. The fact that the SApo/SAcr increases as PCr is hydrolyzed indicates that the relative amount of isotope in the PCr fraction versus the Cr fraction increases, and thus that the unlabeled cellular PCr is at least to some extent preferentially consumed over that which contains the [1 4C] Cr label. Of further interest is the fact that as the muscle recovers and PCr concentrations return towards resting values, the SApo/SAcr ratio also returns towards the value found in resting muscle which has not undergone any experimental perturbations. Thus PCr breakdown and resynthesis seem to follow an identical inverse course with respect to the SApc/SAcr ratio. Savabi (1988) cites this observation as evidence of a highly compartmentalized intracellular environment. This may be true, however the nature of the proposed compartmentation is entirely unknown at present. What is apparent from the relationship observed in both trout white muscle and rat atria (Savabi, 1988) between the SApo/SAcr ratio and Cr charge is that even the pool of total Cr (both PCr and Cr) which is metabolically active does not mix homogeneously within the cell. If this pool were distributed throughout a muscle fibre via simple diffusion, then the [1 4C] Cr would equilibrate with the entire active pool of Cr. In this case the SApo/SAo ratio would be constant at the value seen in resting muscle, independent of Cr charge. Deviation of the ratio from a value of 1 would then solely be due to the lack of equilibration of label with the bound Cr pool. The fact that the ratio of SApc/SAc r increases with decreasing Cr charge indicates that the [ 1 4C] Cr is not equilibrated with the entire metabolically active portion of total Cr. This may actually be interpreted to suggest the presence of more than one pool of active PCr and Cr which have different levels of Cr charge within muscle during periods when 49 metabolism is stimulated above the resting level. Could one of the pools be that found between the inner and outer mitochondrial membranes? There is experimental support for the dynamic compartmentation in the intermembrane space of a pool of ATP and ADP with an ATP/ADP ratio different from that of the extramitochondrial space (Gellerich et al, 1987). It is also possible that the Cr charge in the intermembrane space is different from that outside the mitochondria. Two factors argue against this as an explanation of our results. Firstly, the difference in ATP/ADP ratio between the intermembrane space and the extramitochondrial space was small, and the two ratios varied together (Gellerich et al, 1987). If the increase in S A p c / S A c r seen with decreasing Cr charge is to be explained through compartmentation in the intermembrane space, then the difference between the Cr charge in the intermembrane space and that outside the mitochondria would have to be large and increase with decreasing Cr charge. A second reason why compartmentation of PCr and Cr in the intermembrane space is not a likely explanation is that the pool of PCr and Cr within this compartment which represents a vanishingly low proportion of the total intracellular volume would of necessity be very small. Thus effects of a separate pool of PCr and Cr within this compartment on results determined through extraction of whole muscle would be negligible. The intermembrane space of mitochondria would not serve as a functional compartment of enough significance to explain the observed increase in S A p o / S A c r with decreased Cr charge. In the absence of any other evidence for compartmentation of PCr and Cr, this must be left as an open question. Supporters of the PCr shuttle hypothesis lean heavily on arguments favouring the increased mobility of PCr and Cr over ATP and ADP within muscle as support for energy transport through PCr shuttling. Thus they propose that energy transport from, and feedback to 50 the mitochondria must be in the form of PCr and Cr respectively, molecules whose diffusion would not be limiting to energy metabolism. The nature and extent of the restriction which keeps the [14C] Cr from equilibrating with the entire metabolically active pool of Cr are unknown. It is, however, obvious that any significant barrier to the mobility of PCr and Cr within muscle would argue directly against energy transport through PCr shuttling, although not necessarily that through facilitated diffusion. MRS saturation transfer studies of the mobility of Cr and PCr within muscle have not indicated any restrictions beyond those imposed through the basic properties of the intracellular environment (Yoshizaki et al, 1990). This type of study cannot however distinguish between separate functional pools within a fibre (A . Mackay, pers. com.). The determination of the intracellular mobility of these metabolites remains an area which requires further exploration. Summary In conclusion, it does seem that a significant portion of the pool of total Cr within rainbow trout white muscle is metabolically inactive through some unknown mechanism. This fraction is referred to as 'bound' for lack of further information. The bound fraction does not become accessible to CK during exercise and seems to be turned over at a very slow rate. The fact that the ratio of SApcr/SAcr increases with decreasing Cr charge suggests that even the metabolically active portion of total Cr is not equilibrating throughput the cell. This may be indicative of restricted intracellular movement for PCr and Cr, which would argue against energy transport through PCr shuttling. 51 Enzyme Activities, (umol/min-g wet weight, 25° C) Total CK miCK CS miCK/CS Heart 340 102* 111 0.92 SO 260 36 ** 20 1.8 FOG 606 34 ** 28 1.2 FG 904 28** 13 2.2 Table 1. Expression of miCK relative to CS for a variety of muscle fibre types in rats. Abbreviations: SO, slow oxidative (soleus); FOG, fast oxidative glycolytic (red gastrocnemius); FG, fast glycolytic (superficial gastrocnemius); CK, creatine kinase; miCK, mitochondrial CK; CS, citrate synthase. Data are from Shoubridge et al (1985). Calculations of miCK activity are based on ratios calculated from the work of Ingwall et al (1990) * and Yamashita and Yoshioka (1991) **. 52 ATP PCr Cr Total Cr Cr Charge Group Resting n = 7 7 min. 120% Ucrit n = 4 Buffering n = 9 Exhaustion n = 5 Depleting n = 6 7.61 ±0.19 6.45 ±0.23*° 7.22 ±0.13 2.86 ± 0.82*° 3.52 ±0.46*° 22.2 ± 0.84 8.27 ± 1.9*° 19.8 ±1.3 2.21 ±0.71*°' 11.6 ± 1.1*° 25.2 ±1.1 36.9 ±1.7*° 29.0 ± 1.6 45.3 ± 1.3*°A 38.0 ± 1.9*° 47.4 ± 1.0 45.3 ± 1.3 48.7 ± 1.2 47.5 ± 1.7 49.6 ± 1.5 0.468 ± 0.020 0.183 ±0.041*° 0.406 ± 0.026 0.045 ± 0.014*°' 0.236 ± 0.024*° * Significantly different from resting value (p < 0.05). ° Significantly different from buffering value (p < 0.05). " Significantly different from depleting value (p < 0.05). Table 2. White muscle metabolite concentrations. Values are reported as mean ± S.E. (umol/g wet weight). Abbreviations: ATP, adenosine triphosphate; PCr, phosphocreatine; Cr, creatine; Total Cr = (PCr + Cr); Cr charge = PCr/Total Cr. 7 minute 120% Ucrit and exhaustion groups were sampled immediately after exercise. Buffering group was sampled after 10 minutes of recovery from 7 minutes of swimming at 120% of Ucrit and depleting group was sampled after 30 minutes of recovery from exhaustive exercise. 53 SApCr SAQ. SApo/SAcr Resting 985 ±155 366 ±96 3.10 ±0.29 n = 7 Buffering 1216 ±164 271 ±48 4.74 ±0.31 n = 9 Depleting 1033 ±53 212 ±30 5.45 ±0.50 n = 6 6 Days Resting 1337 ±85 268 ±25 5.0 ±0.15 n = 2 Table 3. Specific activity of PCr and Cr for rainbow trout white muscle incubated with [14C] labeled creatine. Units for specific activity are mean ± S.E. (dpm/umol). Abbreviations: SApcr, specific activity of phosphocreatine; SAcr, specific activity of creatine. No significant differences between groups with respect to SApcr or SAcr were found. Animals for the buffering group were exercised at 120% Ucrit for 7 minutes and then allowed to recover for 10 minutes prior to sampling. The depleting group was exercised to exhaustion, followed by a 30 minute recovery period. 54 Figure 1. Metabolite concentrations normalized to total Cr as a function of energy status in muscle. Abbreviations: RATP = [ATP]/[total adenylates], F c = Cr charge = [PCr]/[PCr + Cr]. Figure taken from Connett (1988). 56 Figure 2. Specific activity of creatine (SAcr) and Cr concentration in plasma of rainbow trout following bolus injection of 30 uCi/kg of [amidino-14C] Cr into the dorsal aorta. 57 Time (min.) 58 Figure 3. The ratio of SApo/SAcr as a function of Cr charge ([PCr]/[PCr + Cr]) in rainbow trout white muscle. Each data point represents an individual animal. See materials and methods for a description of groups. A linear regression yields a slope of = -6.10 at p = 0.014, r 2 = 0.27. 60 Figure 4. 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