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Metabolic regulation of skeletal muscle energy metabolism during exercise Parkhouse, Wade Stephen 1986

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METABOLIC REGULATION OF SKELETAL MUSCLE ENERGY METABOLISM DURING EXERCISE by Wade Stephen Parkhouse B . P . E . , The Univer s i ty of A l b e r t a , 1980 M . P . E . , The Univer s i ty of B r i t i s h Columbia, 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Zoology, A p r i l 1986) We accept t h i s thes i s as conforming to the required standard' THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 19B6 (c) Wade Stephen Parkhouse, 1986 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by t h e head o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f ^r^Xncxy The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) II ABSTRACT The metabolic and biochemical factors involved in the regulat ion of fuel and pathway s e l e c t i o n , at appropriate rates and times, during elevated metabolic demands remains to be reso lved . Therefore, the purpose of t h i s inves t i ga t ion was to examine the metabolic i n t e r r e l a t i o n s h i p s involved in energy provi s ion and i t s regula t ion during exercise at d i f f e rent i n t e n s i t i e s . S p e c i f i c a l l y , fuel s e l e c t i o n and i t s contro l by adenine nucleot ides and c y t o s o l i c redox and were invest igated in red and white muscle of rainbow trout during various progress ive i n t e n s i t i e s of exerc i se . The control of g l y c o l y s i s was examined with respect to g l y c o l y t i c enzyme d i s e q u i l i b r i u m . The ef fects of substrate l i m i t a t i o n s and end product accumulations were examined with regard to f a t igue . F i n a l l y , the buffer ing capacity and various potent ia l buf fer ing const i tuents were inves t iga ted . It would appear that the energy turnovers required to perform the varied exercise i n t e n s i t i e s in t h i s study were achieved by se lec t ing the f iber type, fuel and pathway for opt imizing ATP production rate versus substrate and proton accumulation. The purine nucleot ide cyc le was found to be operat ional within both f iber types. Fuel s e l ec t ion appeared to be int imate ly re lated to m y o f i b r i l l a r ATPase a c t i v a t i o n with free ADP acting as the metabolic s ignal to coordinate the phasing in of appropriate fuels/pathways at appropriate ra tes . HK, phos, PFK and PK were i d e n t i f i e d as regulatory enzymes in both f iber types. As w e l l , the GPDH.PEK complex also appeared to exhib i t regula t ion when glycogen was l i m i t i n g and t h i s regula t ion appeared to have been induced by a decreased ATP/ADP* r a t i o . The redox state of the NAD couple became more oxidized in both t i s sues when muscle glycogen was low. This f ind ing was a t t r ibuted to an induced s h i f t in the equ i l ibr ium of LDH in the d i r e c t i o n of NAD and l a c t a t e . I l l The simultaneous ATP/ADP* induced d i s e q u i l i b r i u m of the PGK react ion would i n h i b i t flux through the GPDH.PGK complex. Skeleta l muscle buffering was found to be dominated by p r o t e i n , inorganic phosphate and h i s t i d i n e re la ted compounds. Thus, these metabolic and biochemical adjustments, allowed a coordinated in tegra t ion of f iber type, fuel and pathway s e l e c t i o n , to achieve the appropriate coupling of m y o f i b r i l l a r ATPase a c t i v i t y to ATP turnover , while minimizing substrate deplet ion and proton accumulations. IV TABLE OF CONTENTS P9 Abstract II Table of Contents IV L i s t of Tables v l L i s t of F i g u r e s . . . . . . IX L i s t of Abbrevia t ions . XI Acknowledgements XV Chapter 1 I n t r o d u c t i o n . . . . 1 Chapter 2 Mater ia l s and Methods 10 Animals • IB Swimming Assessment 10 Experimental Protocol 11 Biochemical Analyses 13 Buffering Analyses 26 Chapter 3 Results 31 Fuel Se lect ion • • 31 G l y c o l y t i c Intermediates 44 Metabolic R e g u l a t i o n . . . 47 Energy T u r n o v e r . . . . . 67 Fat igue. . . • 70 Proton Seqestering Mechanisms 80 In Vivo Intramuscular Buffering 90 Chapter 4 D i s c u s s i o n . . . • 9 7 Fuel S e l e c t i o n . • 97 Fat i gue. 122 V Skeleta l Muscle Buffering 129 Chapter 5 Summary and Conclusions • 137 R e f e r e n c e s . . . . . 140 Appendix I Determination of creat ine and phosphocreatine in ske le ta l muscle t i s sues at rest and during exercise by i s o c r a t i c anion exchange high performance l i q u i d chromatography. . . . 154 Appendix II Assessment of inorganic phosphate l eve l s in freeze clamped frozen and perch lo r i c acid extracts of trout white muscle • 164 Appendix III A c t i v i t i e s of g l y c o l y t i c enzymes in rainbow trout red and white muscle 169 Appendix IV S t a t i s t i c a l Analyses 170 VI L i s t of Tables P9 Table 1. D e f i n i t i o n s and values of equ i l ibr ium constants 18 Table 2. H* d i s o c i a t i o n constants of the h i s t i d i n e re la ted compounds, g l y c o l y t i c intermediates , inorganic and organic phosphates 19 Table 3. C e l l water content of rainbow trout white muscle 22 Table 4. Content of metabolites and pH in rainbow trout white m u s c l e . . . . 37 Table 5. Content of metabolites and pH in rainbow trout red muscle 38 Table 6. Content of metabolites in rainbow trout l i v e r 39 Table 7. Content of g l y c o l y t i c intermediates in rainbow trout white m u s c l e . . . . . 45 Table B. Content of g l y c o l y t i c intermediates in rainbow trout red muscie 46 Table 9. Measured and ca lcu la ted free cytoplasmic ADP content, ATP/ADP r a t i o and c y t o s o l i c phosphorylation potent i a l s in rainbow trout muscle 49 Table 10. Calculated free c y t o s o l i c redox state and PCr/Pi r a t i o ' s in rainbow trout muscle 50 Table 11. Estimated oxygen uptake and aerobic energy turnover (umol ATP/g/min) of rainbow trout 68 Table 12. Estimated energy turnover (umol ATP/g/min) from fa t s , g l y c o l y t i c and high energy phosphagen sources in rainbow trout red and white muscle during the PSS-30 and PSS-7 69 Table 13. Rainbow trout ion concentrat ions 72 Table 14. Measured and hypothet ica l rainbow trout white and red muscle i n t r a c e l l u l a r and e x t r a c e l l u l a r ion di f ference 74 VII Table IS. Calculated rainbow trout red and white muscle membrane po tent i a l s 75 Table 16. Buffer capacity of various f r ac t ions separated from white and red muscle of marlin and trout 83 Table 17. Comparison of p red ic ted , t i t r a t e d and ca lcula ted h i s t i d i n e re la ted compound and phosphate buffer capac i t i e s of marlin white muscie 84 Table 18. Buffer capacity of h i s t i d i n e re la ted compounds, phosphate, m y o f i b r i l l a r p r o t e i n , so luble prote in and taurine in white and red muscles of marlin and trout 85 Table 19. Percent r e l a t i v e contr ibut ions of various buffer ing const i tuents to to ta l t i s sue buffer ing in marlin and trout white muscle 86 Table 20. Percent r e l a t i v e contr ibut ions of the various buffer ing const i tuents to t o t a l t i s sue buffer ing in marlin and trout red muscle 86 Table 21. Concentrations of h i s t i d i n e re la ted compounds found in white and red muscle of trout and marlin 87 Table 22. Concentrations of p r o t e i n , inorganic phosphate and taur ine found in the white and red muscle of marlin and trout 87 Table 23. Estimates of proton absorbing potent ia l of rainbow trout white muscle due to a s soc ia t ion of H* ions with bases during the PSS-7 92 Table 24. Estimates of proton absorbing potent ia l of rainbow trout white muscle due to enzymatic a c t i v i t y during the PSS-7 93 VIII Table 25. Comparison of t i t r a t e d , ca lcu la ted ( lactate) and estimated (pK c h a r a c t e r i s t i c s ) buffer capac i t i e s of trout white muscle over the pH range 7.0 to 6.0 94 IX L i s t of Figures P9 Figure 1. F rac t iona t ion scheme used to separate the d i f fe rent t i s sue components. 30 Figure 2. The adenylate pool (sum of ATP+ADP+AMP) and adenylate plus IMP pool in rainbow trout muscle 41 Figure 3. A l t e r a t i o n s in metabolite contents of l i v e r , white and red muscle of trout 43 Figure 4. Deviat ions of trout white muscle g l y c o l y t i c enzymes from equi l ibr ium based on compensated metabolite data 53 Figure 5. Deviat ions of trout white muscle g l y c o l y t i c enzymes from equ i l ib r ium based on measured metabolite data 55 Figure 6. Deviat ions of trout red muscle g l y c o l y t i c enzymes from equi l ibr ium based on compensated metabolite data 57 Figure 7. Deviations of trout red muscle g l y c o l y t i c enzymes from equi l ibr ium based on measured metabolite data 59 Figure 8. Crossover p lots showing the i n t e r a c t i o n s caused by increas ing workloads on trout white muscle g l y c o l y s i s 62 Figure 9. Crossover p lo t s showing the in te rac t ions caused by increas ing workloads on trout red muscle g l y c o l y s i s . . . . 65 Figure 10. Metabolic a l t e r a t i o n s in P C r 2 - , L a - , P i 2 - and pH in rainbow trout muscle 77 Figure 11. Metabolic end product accumulations of c rea t ine , inorganic phosphate and l ac ta te in rainbow trout muscle 79 Figure 12. Percent r e l a t i v e contr ibut ions of the various buffer ing const i tuents to to ta l t i s sue buffer ing in marlin and trout muscle 89 X Figure 13. Comparison of the percent r e l a t i v e contr ibut ions of the various buffer ing const i tuents to t i t r a t e d , estimated and ca lcula ted to ta l buffer capac i t i e s 96 XI L i s t of Abbreviat ions Acid-Base B buffer capacity CH crude homogenate HMW high molecular weight HRC h i s t i d i n e re la ted compounds IDe ion di f ference e x t r a c e l l u l a r IDr ion di f ference red muscle i n t r a c e l l u l a r IDw ion di f ference white muscle i n t r a c e l l u l a r LMW low molecular weight NMR nuclear magnetic resonance SN supernatant Chemical Compounds AMP adenosine monophosphate ADP adenosine diphosphate ATP adenosine tr iphosphate CoASH acetyl-coenzyme A Cr creat ine C r c compensated creat ine DNFB dini trof luorobenzene E6TA e thyleneg lyco l -b i s - (B-amino-ethyl ether) N , N ' - t e t r a a c e t i c acid DHAP dihydroxyacetone phosphate DPS 1,3-d iphosphoglycerate XII FDP fructose-1,6-phosphate F2,6BP iructose-2,6-bisphosphate F6P fructose-6-phosphate GAP glyceraldehyde-3-phosphate GAP* free glyceraldehyde-3-phosphate GP g lycero l 3-phosphate GTP guanosine tr iphosphate G1P glucose-l-phosphate G6P glucose-6-phosphate IAA iodoacet ic acid IMP inosine monophosphate La~ l ac ta te NAD, NADH nicotinamide adenine d inuc leot ide (oxid ized , reduced) NADP, NADPH nicotinamide adenine d inuc leot ide phosphate (ox id ized , reduced) NH 3 ammonia NhU ammonium ion PCA p e r c h l o r i c acid PCr phosphocreatine PCr c compensated phosphocreatine PEP phosphoenolpyruvate Pi inorganic phosphate Pic compensated inorganic phosphate PMSF phenolmethylsulfonyl f l o u r i d e PYR pyruvate 3PG 3-phosphoglycerate 2PG 2-phosphoglycerate XIII Enzymes Aid aldolase CS c i t r a t e synthase enol enolase GPDH glyceraldehyde 3-phosphate dehydrogenase HK hexokinase ICDH i s o c i t r a t e dehydrogenase LDH lactate dehydrogenase OGDH oxyglutarate dehydrogenase ( ketoglutarate dehydrogenase) PFK phosphofructokinase PGI phosphoglucoisomerase PGK phosphoglycerate kinase PGM phosphoglyceromutase PGM* phosphoglucomutase phos phosphorylase PK pyruvate kinase TPI triose phosphate isomerase Locomotory BS burst swimming EMG electromyographic ES exhaustive swim PS prolonged swimming PSS prolonged steady swimming PSS-30 30 minute prolonged steady swim PSS-7 7 minute prolonged steady swim PUS prolonged unsteady swimming ss sustained swimming TBF t a i l beat -frequency U c r i t c r i t i c a l swimming v e l o c i t y Physi cal and Thermodynamic Em membrane potent ia l G Gibbs Free Energy H enthalpy Keq equ i l ibr ium constant Kd d i s s o c i a t i o n constant pK d i s s o c i a t i o n constant R gas constant XV Acknowledgements I would l i k e to thank Dr. Peter Hochachka for his guidance and f r i e n d s h i p , as well as the prov i s ion of a s t imulat ing academic environment which has to be a t t r ibuted in part to everyone associated with the l ab . I would also l i k e to thank Dr. Don McKenzie for both hi s encouragement and f r iendship as well as his a b i l i t y to put science in perspect ive . A spec ia l thanks i s extended to my f r iend and col league, Geoffrey Dobson, for a l l his help in these and other inves t iga t ions as well as many l i v e l y and sometimes inebr ia ted discuss ions on metabolic r egu la t ion . Thanks are also due to Dr. Hiroki Abe and Dr. U l i Hoeger for a l l t h e i r help on the HPLC and h i s t i d i n e re la ted compound study. I would l i k e to thank the members of my research committee for the i r c r i t i c i s m s and comments on my t h e s i s . F i n a l l y I would l i k e to thank my parents for imparting to me the desire to pursue an education. 1 INTRODUCTION Fuel Se lect ion and Fatigue A var ie ty of constra int s are involved in the s t ruc tura l and biochemical design for regula t ion of fuel s e l ec t ion at various exercise i n t e n s i t i e s . Di f ferent f iber types exis t with the i r va r i a t i ons in u l t r a s t r u c t u r e , metabolite l eve l s are a l t e r e d , enzyme a c t i v i t i e s are adjusted or occur as s p e c i f i c isozymes and mechanisms exi s t to negate metabolical1y generated de le ter ious end products . These cons t ra int s serve to integrate metabolic funct ioning during work to prevent muscular dysfunction due to f a t igue . Although fat igue has many e t i o l o g i e s , i t can be defined in funct ional terms as an i n a b i l i t y to generate s u f f i c i e n t power to continue to perform work at a given ra te . High i n t e n s i t y exercise requires high rates of ATP turnover and eventual ly r e s u l t s in f a t igue , with the time course of fat igue being re la ted to work i n t e n s i t y . These high ATP turnovers are achieved by phosphagen hydro lys i s and anaerobic g l y c o l y s i s which re su l t in substrate deplet ion and metabolic end product accumulations of c r e a t i n e , inorganic phosphate, l ac ta te and hydrogen (H*) ions . Of these, substrate deplet ion and proton accumulations have been the most thoroughly invest igated with regard to f a t i gue , but osmotic, i on ic and charge disturbances which accompany t h i s energy prov i s ion must be considered (Hochachka 1985). Substrate deplet ion i s recognized as a major contr ibutor to fa t igue during long term endurance work (Hermansen et a l . 1967; Hultman 1967; Hultman 1978) and during very high i n t e n s i t y exercise which i s p r i m a r i l y fueled by phosphagen hydro lys i s (McGilvery 1975). Proton accumulation has been impl icated in the fat igue process during short term high i n t e n s i t y exercise 2 (Toews et a l . 1970; Dawson et a l . 1978; Sutton et a l . 1981). Although the source of the protons during anaerobic g l y c o l y s i s has yet to be resolved (Krebs et a l . 1975; Gevers 1977, 1979; Hochachka and Mommsen 1983; Portner et a l . 1984), there appears to be no question as to the 1:1 s to ich iometr i c r e l a t i o n s h i p between l ac ta te and H* product ion. Increased H* concentrat ions have been associated with a l t e r a t i o n s to both the c o n t r a c t i l e and g l y c o l y t i c machinery r e s u l t i n g in decreased times to fat igue ( F i t t s and Holloszy 1976; Stevens 1980). However, Hochachka (1985) has recent ly suggested that protons per se, may play important ro le ( s ) during oxygen (02) l i m i t i n g per iods . It was suggested that protons may; (1) create more favourable condi t ions for the unloading of oxygen; (2) f a c i l i t a t e phosphagen hydro ly s i s and g l y c o l y s i s ; and (3) enhance l ac ta te e f f lux . Thus the importance of inve s t i ga t ing the ef fects of end product accumulations in exercise studies which require high rates of ATP turnover i s emphasized. The myotomal muscle mass of f i s h provides an ideal model to inves t iga te the metabolic i n t e r r e l a t i o n s h i p s involved in energy provi s ion and regula t ion during exercise at d i f f e rent i n t e n s i t i e s . This i s because i t comprises e s s e n t i a l l y two s p a t i a l l y and f u n c t i o n a l l y d i f f e rent f iber types, red and white (Johnston 1981). White muscle, which comprises the bulk of the myotomal muscle mass, i s predominantly a f a s t - twi tch g y l c o l y t i c t i s sue that i s thought to be recru i ted during a c t i v i t y which requires high rates of metabolic energy turnover (Johnston 1981). Conversely, red muscle appears as a l ong i tud ina l s t r i p running beneath the l a t e r a l l i n e and i s an aerobic s low-twitch oxidat ive t i s sue that i s thought to be recru i ted during longer term steady state swimming (Johnston 1981). Within many species , inc luding salmonids, the white muscle i s in r e a l i t y a mosaic muscle as red f iber s cons t i tu te a small percentage ( i e . 1 percent in trout) of t h i s muscle mass 3 (Proctor et a l . 1980) and must be considered when i n t e r p r e t i n g phys io log ica l and metabolic data. The evidence for t h i s d i v i s i o n in function comes from a var ie ty of sources. M y o f i b r i l l a r and sarcoplasmic ret iculum ATPase a c t i v i t i e s have shown that white and red muscle have the biochemical propert ies of f a s t - twi tch and slow-twitch muscle re spec t ive ly (Johnston 1982b). As w e l l , other d i f ferences between these two f iber groups with respect to f iber diameter, u l t r a s t r u c t u r e , enzyme a c t i v i t i e s , c a p i l l a r i z a t i o n , myoglobin and number of mitochondria support t h i s d i v i s i o n of function (Bone 1966; B i l i n s k i 1974; Johnston 1982a). Intramuscular Acid-Base Regulation A dominant theme in the evolut ion of vertebrate acid-base regula t ion i s the physicochemical basis for maintenance of the conf igurat ion and charge of prote ins necessary for the regulat ion of c e l l u l a r pH over a narrow range (about 1 pH unit from n e u t r a l i t y ) . Three major changes have evolved to minimze the impact of changes in i n t r a c e l l u l a r pH. These include (1) mechanisims to effux protons from the c e l l into the blood and/or regulate the inf lux of n e u t r a l i z i n g ions into the c e l l (Koch et a l . 1981), (2) u t i l i z a t i o n of the protons by metabolic processes so that the net proton production i s matched by the net proton u t i l i z a t i o n (Krebs et a l . 1975) and (3) absorbing protons by i n t r a c e l l u l a r buffers (Parkhouse and McKenzie 1984). These f u n c t i o n a l l y d i f f e rent muscle f iber types are associated with a d i f f e r e n t i a l capacity for generating and consuming protons. In genera l , white muscle metabolism can be considered a net proton generator, whereas red muscle i s not because the protons generated are s t o i c h i o m e t r i c a l l y matched to proton consumption by mitochondrial ox idat ive phosphorylation (Krebs et a l . 4 1975). This i s one reason why white muscle i s thought to have a higher buffer ing capacity than red muscle as was implied from e a r l i e r s tudies on a wide range of vertebrates ( C a s t e l l i n i and Somero 1981). Although the major buffer ing components have r a r e l y been adequately i d e n t i f i e d , a great deal of a t tent ion has focused on the imidazole moiety of h i s t i d i n e , p a r t i c u l a r l y in dipept ides such as anserine and carnosine (Davey 1960; Somero 1981; Morris and Baldwin 19B4; Parkhouse et a l . 1985). However, the precise ro le s of h i s t i d i n e re la ted compounds in vertebrate ske le t a l muscle buffer ing remains perp lex ing , in part because a l l species (Christman 1976; C a s t e l l i n i and Somero 1981; Morris and Baldwin 1984), inc lud ing f i shes (Abe 1981, 1983a, 1983b), show a wide v a r i a t i o n in both the i r concentrat ions and t h e i r choice of dominant h i s t i d i n e re la ted compounds. The major buffer ing const i tuents have been c l a s s i f i e d into three components: physico-chemical buf fe r ing , consumption or production of n o n - v o l a t i l e acids and transmembrane f luxes of protons or bicarbonate (Sies jo and Messeter 1971). The buffer ing capacity of in v i t r o preparations cons i s t s of the physico-chemical buf fer ing component, which includes the buffer ing within the c e l l as a consequence of proton as soc ia t ion with bases (Roos and Boron 1981). As such, most of the c r i t i c i s m surrounding the use of the crude homogenate t i t r a t i o n method (in v i t r o ) for assessing buffer capacity have in ferred that i t does not represent in vivo buf fer ing . It neglects the transmembrane f luxes of protons and bicarbonate as well as metabolic react ions associated with enzymatic a c t i v i t y . Within human vastus l a t e r a l i s muscle, Sahl in (1978) has suggested that bicarbonate could contr ibute as much as 15 to 18 percent (12 umol/g/pH) of to ta l t i s sue buffer capacity in vivo during exerc i se . Whereas these c r i t i c i s m s may be true for most animals, f i sh white muscle may provide the exception due to i t s a b i l i t y to r e t a in l ac ta te and protons (Holeton et a l . 1983; Turner et a l . 1983; l i i l l i g a n and Wood 1985) as well as possessing low bicarbonate l e v e l s (Heis ler 1978). Conversely, f i s h red muscle, because of i t s higher bicarbonate content (Heis ler 1978) and greater c a p i 1 1 a r i z a t i o n , would be subject to these same c r i t i c i s m s . Within ske le ta l muscle, the major buffer ing const i tuents have been i d e n t i f i e d as prote in (Bate-Smith 1938; Woodbury 1965), h i s t i d i n e re la ted compounds (Davey 1960; Somero 1981) and inorganic phosphate (Burton 1978). Assuming e i ther l i t t l e or no p r o t e o l y s i s during short-term exerc i se , as i s thought to be the case during high i n t e n s i t y , short duration swimming in f i sh (Driedzic and Hochachka 1978), the buffer ing const i tuents prote in and h i s t i d i n e re la ted compounds would contr ibute equivalent buffer ing power when assessed e i ther in vivo or in v i t r o . However, the inorganic phosphate cont r ibut ion to buffer ing would depend on i t s metabolic production during exerc i se . Using the fact that to ta l buffer capacity i s the sum of the i n d i v i d u a l buffer ac t ions , a t o t a l in vivo buffer capacity can be estimated based on the sum of the physico-chemical buffer ing components plus the production or consumption of acids or bases. S i m i l a r i l y , the 1:1 s to ich iometr i c r e l a t i o n s h i p between l ac ta te and protons allows an a l t e r n a t i v e in vivo buffer capacity value to be ca lcu la ted based on the intramuscular pH change and l ac ta te accumulation during exercise provided a l l the l ac ta te i s accounted for (Sahlin 1978). Regulation of Metabolism The regula t ion of metabolism in f i s h muscle appears to be s i m i l a r to mammalian muscle. Fish muscles possess a f u l l complement of enzymes for fermentative and oxidat ive metabolism, with the r e l a t i v e capac i t i e s of these 6 pathways varying between f iber types as expected (Crabtree and Newsholme 1972; Johnston 1977; Walton and Cowey 1982). F i sh possess l i t t l e adipose t i s sue and l i p i d s are stored within the l i v e r and muscle with red muscle stores being twice those of white muscle (Bone et a l . 1966; Lin et a l . 1974). ATP i s stored in s imi l a r quant i t i e s to mammalian muscle (Driedzic and Hochachka 1978) but PCr l eve l s were higher than most other animals. However, unl ike mammalian muscle, f i sh red muscle contained an equivalent or even greater amount of endogenous glycogen than white muscle (Johnston 1981). Glycogen deplet ion and l ac ta te accumulations during high i n t e n s i t y exercise in f i s h white muscle suggested an act ive g l y c o l y t i c pathway (Driedzic and Hochachka 1976; Wokoma and Johnston 1977; Guppy et a l . 1978; Dr iedz ic et a l . 1981). This pathway appeared to be under standard regulatory control s imi la r to mammalian muscle (Driedzic and Hochachka 1976). The conversion of phos b to phos a was mediated by C a * * (Yamamoto 1968) while PFK a c t i v i t y has been shown to be modulated by a var ie ty of factors inc luding adenylates (Freed 1971; Snudgen and Newsholme 1975; Newsholme et a l . 1977) and fructose 2,6 bisphosphate <F2,6BP) (Dobson et a l . 1986) but unl ike mammalian muscle, l ac ta te and proton efflux i s slow from t h i s t i s sue (Holeton et a l . 19B3; Turner et a l . 1983; Mi 11igan and Wood 1985). However, no information ex i s t s on the extent and control of g l y c o l y s i s in red muscle, while most of the information gathered on white muscle was from studies conducted at very high i n t e n s i t y workloads. The purine nucleot ide cyc le appeared to be ac t ive within f i s h white muscle (Driedzic and Hochachka 1976) but the extent of i t s act ion in red muscle i s unknown. A great deal of evidence has suggested that adenine nucleot ide metabolism plays a prominent ro le in metabolic r e g u l a t i o n . It has been shown to be involved i n ; (1) mitochondrial r e sp i ra tory control (Chance and Will iams 7 1955, 1956; S later et a l . 1973; Jacobus et a l . 1982); (2) the modulation of many g l y c o l y t i c enzymes (Bloxham and Lardy 1973; Sols 1979,1981; Racker 1981); (3) phosphorylation and dephosphorylation react ions (Randle 1981); and (4) the coordinat ion of m y o f i b r i l l a r ATPase a c t i v i t y with appropriate metabolic pathway funct ioning (Hochachka 1985). It has also been suggested that ox idat ive phosphorylation i s the master process c o n t r o l l i n g the c y t o s o l i c redox state of the NAD couple as a consequence of the c y t o s o l i c phosphorylation potent ia l (ATP/ADP.Pi) (Stubbs et a l . 1972). In t h i s regard, decreased oxygen tension at the mitochondrial l e v e l , as occurs during exercise in human muscle, i s r e f l ec ted in a more reduced c y t o s o l i c NAD couple (Sahlin 1985). Within f i s h muscle, large a l t e r a t i o n s in adenine nucleot ides were found to accompany exercise (Driedzic and Hochacka 1976) and as such could p o t e n t i a l l y be involved in the regula t ion of energy metabolism through e i ther d i rec t e f fec t s on the above processes or i n d i r e c t l y v ia a l t e r a t i o n s in the c y t o s o l i c redox state of the NAD couple. As such, f i sh muscle presents an exce l lent model to study the design and regula t ion of fuel s e l e c t i o n for exercise at various power outputs. Locomotion and Muscle Function Swimming in f i s h has been c l a s s i f i e d as sus ta ined, prolonged or burst and re fer s to both the durat ion pr io r to fat igue and the type of swimming performance with v e l o c i t y and t a i l beat frequency (TBF) being re la ted to the length of the f i s h (Hoar and Randall 1978; Stevens 1979). In genera l , sustained swimming (SS) i s character ized by long duration (greater than 200 minutes) , low i n t e n s i t y , steady swimming at a constant TBF. Prolonged swimming (PS) re fers to higher i n t e n s i t y swimming which i s of greater than 20 seconds and less than 200 minutes durat ion . This type of swimming i s 8 character ized by both steady (PSS) and unsteady (PUS) swimming with the degree of steady swimming being d i r e c t l y re la ted to pr ior a c t i v i t y and duration while being inverse ly re la ted to i n t e n s i t y . Burst swimming (BS) re fers to very high i n t e n s i t y swimming which i s of less than 20 seconds duration and i s character ized by b u r s t / g l i d e patterns of propuls ion (Hoar and Randall 1978; Stevens 1979). The swimming v e l o c i t y at which a f i s h can no longer maintain PS behavior has been i d e n t i f i e d as the c r i t i c a l swimming v e l o c i t y (Ucri t ) and i t appears that red muscle can support locomotion up to 80 percent of t h i s v e l o c i t y . Above 80 percent , the mosaic muscle has been shown to be act ivated (Johnston et a l . 1977b). As swimming v e l o c i t y approached and exceeded t h i s c r i t i c a l v e l o c i t y , there appeared to be an increased r e l i a n c e on white muscle (Johnston et a l . 1977b; Bone 1978) which may at f i r s t be aerobic but which in the l a t t e r phases required increased g l y c o l y t i c energy product ion. Therefore, t h i s Ucr i t appears to be analogous to maximal oxygen consumption whereby aerobic metabolism can support the energy needs up to approximately 80 percent of maximal oxygen uptake with the demands of higher work i n t e n s i t i e s being met by increased g l y c o l y t i c funct ion ing . Few inve s t i ga t ions have examined metabolic energy prov i s ion during PS as the v e l o c i t y surpasses the U c r i t in f i sh red and white muscle. However, electromyographic (EMG) evidence (Hudson 1973; Greer-Walker and Pu l l 1973; Kiceniuk 1975; Johnston et a l . 1977b; Bone 1978) and t r a i n i n g s tudies (Johnson and Moon 1980a,b) suggested that red muscle was r ec ru i t ed during SS with an increased r e l i ance on white muscle as swimming i n t e n s i t y was increased (Bone 1978; Rome et a l . 1984). These phys io log i ca l data have been supported with biochemical inves t i ga t ions at sustained (Johnson and Goldspink 1973; B i l i n s k i 1974) and at burst swimming v e l o c i t i e s (Stevens and Black 9 1966; Drei dz i c and Hochachka 1976; Wokoma and Johnston 1981). Thus, t h i s v e l o c i t y provides the opportunity to inves t iga te the designs and cons t ra int s of energy prov i s ion during high i n t e n s i t y exercise within red and white muscle as the system i s progress ive ly s t ressed . However, when f i s h in a respirometer were subjected to a large increase in work i n t e n s i t y , there was an immediate recruitment of high energy phosphagen sources (Dobson et a l . 1986) and propuls ion i s character ized by b u r s t / g l i d e pat terns , u n t i l e i ther fat igue or u n t i l the TBF i s matched to work demands (Hoar and Randall 1978). To achieve steady swimming performance, the work increments must be small and of long enough duration to prevent any lapses into unsteady swimming behavior. This steady swimming there fore , provides a model which can be progres s ive ly s t res sed , i n s t i g a t i n g a l t e r a t i o n s in fuel s e l ec t ion which must conform to the s t ruc tura l and biochemical make-up of the animal. Therefore the purpose of t h i s inve s t i ga t ion was to examine the metabolic i n t e r r e l a t i o n s h i p s involved in energy prov i s ion and i t s regula t ion during exercise at d i f f e rent i n t e n s i t i e s . S p e c i f i c a l l y , fuel s e l e c t i o n and i t s control by adenine nucleot ides and c y t o s o l i c redox were invest igated in red and white muscle of rainbow trout during progress ive i n t e n s i t i e s of exerc i se . The control of g l y c o l y s i s was examined with respect to g l y c o l y t i c enzyme d i s e q u i l i b r i u m . The e f fects of substrate l i m i t a t i o n s and end product accumulations were examined with regard to fat igue under the d i f f e rent exercise i n t e n s i t i e s . F i n a l l y , the proton sequestering capacity and ro les of various potent ia l buffer ing const i tuents were inves t iga ted . 10 MATERIALS AND METHODS ANIMALS Metabol i te , Tissue and Plasma Ion Studies . Rainbow trout (150 f i sh) weighing 200-250 grams, 26-28 cm. in length , were obtained from a s ing le stock of a loca l s u p p l i e r . They were kept in c i r c u l a t i n g (10 cm/s) dechlorinated tap water at 1 0 - 1 2 ° C for one month pr ior to the experiments and fed every other day with high prote in trout p e l l e t s . H i s t i d i n e Related Compound Study. Rainbow trout and P a c i f i c blue marlin were used for t h i s i n v e s t i g a t i o n . The rainbow trout weighed 250-300 grams and were obtained from a l o c a l s u p p l i e r . They were kept in dechlor inated tap water at 8 - 1 2 ° C for at least two months p r io r to the s tar t of the experiment. During t h i s time, they were fed every other day with high prote in trout p e l l e t s . The P a c i f i c blue mar l in , weighing 50-100 kg were caught off Hawaii during the Annual B i l l - f i s h Tournament in August 1983. SWIMMING ASSESSMENT A l l f i s h used in the metabol i te , t i s sue and plasma ion studies were assessed for t h e i r swimming c a p a b i l i t i e s to obtain a homogeneous experimental popula t ion . During the month pr ior to the experiments, a l l f i s h were f ami la r i zed with the respirometer and those demonstrating incorrec t swimming behavior d i scarded. On a second occas ion, a l l remaining f i s h were subjected to an exercise test to fat igue to assess t h e i r swimming c a p a b i l i t i e s . This test consisted of the f o l l o w i n g : a 5 minute period at 0.8 body 1engths/second (BL/s) followed by 10 minute periods of 2.4, 2 .6, 2.8 and 3.0 BL/s . This was immediately followed by a f i n a l period at 3.2 BL/s u n t i l f a t igue . Fatigue was taken as an i n a b i l i t y to maintain the given v e l o c i t y and thus avoid the shock g r i d . The f i s h were grouped according to the i r swimming capac i t i e s and only those f i s h fa t iguing after 3 minutes at 3.0 BL/s and before 4 minutes at 3.2 BL/s were re ta ined . A t o t a l of 29 f i s h demonstrated swimming capac i t i e s within t h i s range. Three animals from t h i s group were assessed for the i r c r i t i c a l swimming v e l o c i t y (Ucri t ) (Brett 1964). E s s e n t i a l l y t h i s test cons i s t s of swimming the animals for 30 minutes at various incremental speeds ( i n i t i a l 2.0 BL/s ; increments of 0.2 BL/s every 30 minutes) u n t i l the animal was unable to complete the 30 minute work i n t e r v a l at a given speed. Then the U c r i t was determined according to the formula: U c r i t = Ui + ( t i / t i i x U i i ) where Ui i s the highest v e l o c i t y maintained for the prescribed time, U i i i s the v e l o c i t y increment, t i i s the duration of the fat igue v e l o c i t y and t i i i s the prescr ibed period of swimming. The Ucr i t was found to be 2.88+0.03 BL/s (mean+SE). A l l these exercise tes t s were completed at least one week pr io r to the i n i t i a t i o n of the experiments. EXPERIMENTAL PROTOCOL Metabolite Studies . Pr ior to t e s t i n g , f i s h were placed in a darkened p l ex ig l a s s box maintained with dechlorinated tap water at 1 0 + 1 ° C overnight . Animals were t ransferred from t h i s box to the respirometer in the dark. Struggl ing was minimal and frequently non-exis tent . A l l exercise tests were completed in 3 days and performed in low l i g h t between 1100 and 1600 hours. The experiment consisted of a 5 minute (27 cm/s; 1 BL/s) re-fami 1ar iza t ion period followed by a PSS of 30 minutes duration (PSS-30). This PSS-30 consis ted of three 10 minute work i n t e r v a l s at 65, 70 and 75 cm/5 (2.4, 2 .6, 2.8 BL/s) r e spec t ive ly and was designed to maximally s tress the red musculature without inducing unsteady swimming behavior. These v e l o c i t i e s correspond to approximately 83, 90 and 97 percent of the i r U c r i t . The PSS-30 was immediately followed by a second prolonged steady swim at 85 cm/s (3.2 B l / s ; 108 percent of Ucr i t ) u n t i l t h i s speed could not be maintained (mean+SE; 7.0+1.3 min) and w i l l be referred to as PSS-7. This protocol allows d i rec t comparison to the previous metabolic state (PSS-30), providing in s ight s into the metabolic regula t ion of fuel s e l ec t ion associated with an increased power output, substrate deplet ion and fa t igue . Immediately fo l lowing t h i s swim bout, f i s h were subjected to a prolonged unsteady swim bout which was designed to e l i c i t exhaustion. To achieve t h i s , they were made to swim at the maximal speed they could perform u n t i l they could no longer maintain a v e l o c i t y (55 cm/s; 2 BL/s) equivalent to low aerobic swimming (meaniSE; 42*3 min). The f i s h demonstrated a swimming behavior of repeated bursts interspersed steady swimming and t h i s bout w i l l be re ferred to as the exhaustive swim (ES). This protocol was designed to allow d i rec t comparison between d i f f e rent types of fat igue associated with the metabolic consequences of fuel and pathway se l ec t ion to achieve appropriate energy turnovers . F i sh were s a c r i f i c e d pr io r to the exercise (pre-exjPE; n=5) after the 30 minute PSS-30 (n=6) , when the f i s h were unable to maintain 85 cm/s (PSS-7; n=5) or 55 cm/s (ES; n=5). Fish were quickly decapitated upon completion of the experimental protocol (usual ly less than 8 seconds) with l i v e r , red and white muscle being r ap id ly exci sed . These t i s sues were immediately freeze clamped with s t a i n l e s s s teel tongs cooled to the temperature of l i q u i d n i t rogen. White and red muscle were always obtained pos ter ior to the caudal f i n and frozen within 20 and 45 seconds, r e s p e c t i v e l y . L iver s were frozen within 40 seconds in a l l cases. Samples 13 were stored at -80 0 C u n t i l ana ly s i s . Plasma Ion Study. Rainbow trout being used for t h i s inve s t i ga t ion were cannulated 24-48 hours p r io r to the experiment <n=5). A PE-50 polyethylene catheter was inserted into the dorsal aorta under MS-222 ( t r i c a i n e methane su l fonate , 1:15,000 w/v so lut ion) anaesthesia. Following cannula t ion , a l l f i s h were placed in a darkened p lex ig l a s s box maintained with dechlorinated tap water at 10+1 C u n t i l i n i t i a t i o n of the experiment. F i sh were t rans ferred to the respirometer in the dark. The experiment consisted of a 5 minute, 27 cm/s r e - f a m i l i a r i z a t i o n period followed by a burst swim at 85 cm/s u n t i l the f i s h could not maintain th i s v e l o c i t y and thus avoid the shock g r i d . Blood samples were obtained pre- and post exerc i se . H i s t i d i n e Related Compound Study. Five non-exercised rainbow trout were k i l l e d by decapi tat ion with the red and white muscle being quickly exci sed . These t i s sues were then frozen in isopentane (2-methylbutane) cooled to the temperature of l i q u i d n i t rogen. This was to maintain the i n t e g r i t y of the prote ins upon f r e e z i n g . The t i s sues were stored at - 8 0 ° C u n t i l a n a l y s i s . Upon capture, the red and white muscle was quick ly excised from f ive P a c i f i c blue mar l in , placed on ice and frozen at - 8 0 ° C as soon as pos s ib le . BIOCHEMICAL ANALYSES Metabol i te E x t r a c t i o n . Metabolite extrac t ion was by a modif icat ion of the method of Bergmeyer (1974). B r i e f l y , t i s sue sect ions were powdered under l i q u i d nitrogen and added to ice cold 0.6 N pe rch lo r i c acid (PCA) to a f i n a l d i l u t i o n of 6 volumes (v/w). The PCA extracts were homogenized for 2 x 15 seconds at maximum speed with a t i s sue homogenizer (Polytron PT-10). Two 100 ul a l iquot s were immediately frozen in l i q u i d nitrogen for l a ter determination of glycogen. The remaining PCA extract was centr i fuged for 3 minutes (white) and 5 minutes (red) at 11,000 g in a microcentri fuge (Eppendorf) at 4 e C . The supernatant was neutra l i zed with saturated trizma base (Tris ) and immediately frozen in l i q u i d n i t rogen. The neutra l i zed extracts were stored at - 8 8 ° C u n t i l ana lys i s ( less than 48 hours) . Chromatography. High performance l i q u i d chromatography (HPLC) was used to measure PCr, Cr , the nucleot ides and h i s t i d i n e re la ted compounds. For these determinations , three separate chromatographic procedures were employed on a HPLC system (Spectra Physics 8000B). U l t r a - v i o l e t l i g h t absorbing compounds were monitored with a Spectraflow 773 (Kratos A n a l y t i c a l Instruments) detector u n i t . PCr, Cr and nucleot ide separations were performed on a Brownlee (4.6 mm x 22 cm) Aquapore AX-300 column, 5 urn p a r t i c l e s with a Brownlee (4.6 mm x 3 cm) MPLC, AX-300, 10 urn p a r t i c l e guard column. PCr, Cr , and nucleot ide monophosphates were determined on a s ing le run. A 2 ml/min (800 psi) i s o c r a t i c flow rate was used with a 50 mil KHaPCU (pH 3.0) mobile phase at 55 C with detect ion at 210 nm. The column was cleaned of bound d i - and tr iphosphates once every hour with 600 mM KH 2 P0« (pH 2 .5 ) , at a flow rate of 2 ml/min (800 psi) for 10 minutes. Regeneration was complete in 20 minutes. Nucleotide d i - and tr iphosphates were determined with a gradient mobile phase and detect ion at 254 nm. E l u t i o n was i s o c r a t i c at 55*C for 4 minutes using 50 mM KH2PCU (pH 2.3) followed by a l i n e a r gradient (50-600 mM K H 2 P 0 4 , pH 2.5) for 10 minutes. This was followed by a f i n a l i s o c r a t i c mobile phase (600 mM K H 2 P O 4 , pH 2.5) for 10 minutes at a flow rate of 2 ml/min (1000 p s i ) . H i s t i d i n e re la ted compounds were determined as described previous ly (Abe 1961) using a Zipax SCX column (0.21 x 50 cm), 10 ul sample loop and UV detect ion at 210 nm. Elu t ion was i s o c r a t i c at 35"C for 10 minutes followed by a l inear gradient (12-30 mM phosphate) at a flow rate of 1 ml/min. The coe f i c i en t of va r i a t i on in analyses was always less than 5 percent. Spectrophotometry. A l l determinations were performed on a Pye Unicam SP8-400 spectrophotometer in dupl ica te with standards being analyzed for each metabolite measured. The c o e f f i c i e n t of v a r i a t i o n in analyses was always less than 5 percent. The metabolites were measured enzymatical ly by l i n k i n g them to react ions using NADH/NAD* or NADPH/NADP* and fo l lowing the react ion at 340 nm. Lab i l e phosphates were analyzed f i r s t . Unless otherwise s ta ted , the assays used are e s s e n t i a l l y those of Bergmeyer (1974) with s l i g h t modi f i ca t ions . Inorganic phosphate (Pi) was determined c o l o r i m e t r i c a l l y by the method of Black and Jones (19B3). Glycogen was digested according to the procedure of Keppler and Decker (1974) with standards being run to test the e f fect iveness of t h i s procedure. Protein l eve l s were determined using a modi f icat ion of the method of Lowry et a l . (1951). The modif icat ion incorporated s o l u b i l i z a t i o n of prote ins with 1 percent sodium dodecyl su l f a t e . Taurine l eve l s were determined on 1 ml. a l iquot s of the supernatants (HRC study) . These supernatant f r ac t ions were deproteinized by the addi t ion of 130 ul of 8 percent PCA. After c e n t r i f u g a t i o n , a l iquot s were applied to a clean up column to remove other amino acids (Stabler and Siegal 1981) and the e lutate and three 1 ml washings (water) c o l l e c t e d . The combined e lutates and washings were neutra l i zed with 2N KOH and a l iquot s of t h i s so lu t ion analyzed for taur ine by the phtalaldehyde-urea method. Calculated Metabolite Concentrat ions . The NMR studies have found PCr content t D be B5 (Meyer et a l . 1982; Meyer et a l . 1985; Shoubridge et a l . 1984) and 68 (Meyer et a l . 1982; Meyer et a l . 1985) percent of to ta l PCr plus Cr at r e s t i n w h i t e a n d r e d m u s c l e r e s p e c t i v e l y . W h i t e a n d r e d m u s c l e P C r , C r a n d P i c o n t e n t s w e r e c o m p e n s a t e d f o r on t h i s b a s i s , w i t h t h e c h a n g e i n P C r c o n t e n t b e i n g c o n s i d e r e d t o b e s i m i l a r u n d e r t h e d i f f e r e n t e x e r c i s e l o a d s i n a s p e c i f i c t i s s u e t y p e u n t i l ATP c o n t e n t was f o u n d t o d e m o n s t r a t e a l a r g e d e c l i n e . T h e s e c o m p e n s a t e d v a l u e s w e r e t h e n u s e d t o c a l c u l a t e a m i n i m u m f r e e ADP c o n t e n t i n e a c h f i b e r t y p e . M e t a b o l i t e c o n c e n t r a t i o n s w h i c h o c c u r a t t o o l o w l e v e l s a n d / o r a s f r e e / b o u n d c o m p o u n d s c a n b e c a l c u l a t e d f r o m t h e i r e q u i l i b r i u m c o n s t a n t s ( K e q ) o r b y . a c o m b i n a t i o n o f e q u i l i b r i u m c o n s t a n t s u s i n g e a s i l y m e a s u r e d m e t a b o l i t e s p r o v i d e d t h e r e a c t i o n ( s ) a r e i n e q u i l i b r i u m . E q u i l i b r i u m c o n s t a n t s a r e c a l c u l a t e d u n d e r s t a n d a r d c o n d i t i o n s a t pH 7.0 a n d a r e known t o b e a f f e c t e d by many f a c t o r s i n c l u d i n g i o n i c s t r e n g t h , t e m p e r a t u r e , f r e e Mg4""* a n d pH. I n t h e s e i n v e s t i g a t i o n s , w i t h t h e e x c e p t i o n o f t e m p e r a t u r e e f f e c t s on t h e LDH r e a c t i o n , o n l y t h e e f f e c t s o f pH on t h e e q u i l i b r i u m c o n s t a n t s w e r e c o n s i d e r e d a s a n y a t t e m p t t o c o r r e c t f o r a l l t h e s e v a r i a b l e s w o u l d l e a d t o s u b s t a n t i a l e r r o r . T h e u s e o f t h e G i b b s F r e e E n e r g y e q u a t i o n : A G 0 ' = - R T l n K e q *' a l l o w s e s t i m a t i o n o f a new K e q " a t a d i f f e r e n t pH. To c a l c u l a t e a new K e q " t h e G i b b s F r e e E n e r g y e q u a t i o n c a n be r e w r i t t e n : A G 0 " - A G ° ' = l n ( l + K s / l x l B - 7 ) - l n ( 1 + K p / l x 1 B ~ 7 ) RT ( 1 + K S / A H * ) ( 1 + K p M H * ) w h e r e : Ks=H* d i s s o c i a t i o n c o n s t a n t o f t h e s u b s t r a t e s Kp=H* d i s s o c i a t i o n c o n s t a n t o f t h e p r o d u c t s u p o n r e a r r a n g i n g : A G C " = RT l n ( 1 + K s / I x l 8 - 7 ) - l n ( 1 + K P / l x I B - 7 ) + A G ° ' ( 1 + K S / A H * ) ( 1 + K p / A H + ) t h e r e f o r e : A G ° " = - R T l n K e q ° " a t t h e new pH upon rearranging: ln Keq'>"'=-AG' ,"/RT K e q ° " * l / l n Keq*" The LDH react ion was also corrected for temperature s ince a Keq was ava i l ab le at 16 C (Keq = 1.11 x10" 1 2 ) (Hakala 1956) and the measured enthalpy of the react ion (Curtin and Woledge 1978) gave a s i m i l a r value at 10 C suggesting that the enthalpy was a l inear r e l a t i o n s h i p for t h i s r e a c t i o n . Temperature was corrected for by the use of the Gibbs-Helmholtz equation: log Keq= - H/2.3RT + C where: H=enthalpy, R=gas constant , T=temperature <°K) and C i s a constant. The apparent equ i l ibr ium constants of the PGK, CK and PK react ions are Mg* independent above approximately 1 mM concentrat ions . The equ i l ib r ium constants under standard condi t ions and at pH 6.5 are given in Table 1. These values were ca lcu la ted from the H* d i s s o c i a t i o n constants found in Table 2. Table 1. D e f i n i t i o n s and values of equ i l ibr ium constants . Apparent Constant pH 7.0 Keq pH 6.5 (ATP)(Cr) (ADP)(PCr)(H*) KHK= (G6P)(ADP*) (Glu)(ATP) K P G „ = (66P) (G1P) K P G I - (F6P) (G6P) K P F K = (FDP)(ADP.) (F6P)(ATP) K A I d 3 (DHAP)(GAP*) (FDP) K T P I = (DHAP) (GAP*) I S G P D H - (DPBMH*) NADH/NAD (GAP*)(Pi) (ATP)(3PG) (ADP*)(DPG) U D H (GAP*)(ADP*)(Pi)(Pyr) KPGM«= (2P6) (3PG) K.„oi» (PEP) (2PG) K P K = (ATP) (Pvr) (ADP*)(PEP) (Pvr)(NADH)(H*) (Lac)(NAD) 1.66x10' M"' 5.25x10" M"' 5 . 5 x l 0 3 0.055 0.430 800 1x10 12 0.51X10" 1 3.6x10* (ATP)(3P6)(H*) NADH/NAD 1.83x10' (ADPf)(GAPf)(Pi) (3PG)(ATP) (Lac) 1.68x10* M" 0.170 3.0 2.98x10" 1.09xl0- , 1 M 0.057 0.430 822 1.92x10-* 9.3 1.13xl0- 7 1.79xIB 3 2.02x10 - 4 0.59x10 s M" 0.219 2.59 2.04x10" 3 .44xl0- , 2 M Values are from Burton (1957), Noltman (1972), Bohme (1975), Cornel l et (1979), Veech et a l . (1979), Connett (1985). Table 2. H* d i s s o c i a t i o n constants o-f h i s t i d i n e re la ted compounds, g l y c o l y t i c intermediates , inorganic and organic phosphates. Compound pK kd P i 2 - 6.81 1.55xl0- 7 MgATP" 5.21 6. 17x10-* A T P 3 - 6.95 1.12xl0" 7 MgADP- 5.319 5.01x10-* ADP 2 " 6.78 1.66xl0- 7 AMP" 6.45 3 .55x l0 " 7 P C r 2 " 4.50 3.16x10- = Cr 4.10 8.00x10"= IMP- 6.25 5.62X10" 7 NHs 9.50 3 . 1 6 x l 0 - 1 B H i s t i d i n e 6.00 1.00x10-* Anserine 7.03 9 .33x l0 " B Carnosi ne 6.83 1 .48xl0" 7 SIP 6. 13 7.41X10" 7 G6P 6.11 7.76X10" 7 F6P 6.11 7 .76x l0 " 7 FDP 5.95,6.15 1 .12x l0- * , 7 .0Bx l0- 7 DHAP 6.52 3 .02x l0- 7 GAP 6.82 l . S l x l O " 7 DPG 7.40,7.99 3 . 9 B x l 0 - s , i . 0 2 x l 0 - Q 3PG 6.20 6.31X10" 7 2PG 6.65 2 .24x l0 " 7 PEP 6.40 3 .98x l0 " 7 PYR 2.55 2.82K I B " 3 20 La 3.66 2.19x10-* Values are from Bate-Smith 19385 P h i l l i p s et a l . 1965; Alberty 1969; Curt in and Woledge 197B; Sadian et a l . 1981. The concentrat ion of GAP in t i s sues i s very low and s imi l a r in magnitude to the concentrat ion of enzymes for which i t i s a substrate (Veech et a l . 1979; Ottaway and Mowbray 1977). Thus the amount of bound and free c y t o s o l i c GAP must be c a l c u l a t e d . Estimates of free GAP have been made using the measured DHAP, which has been shown to represent the free c y t o s o l i c concentrat ion (Veech et a l . 1979; Connett 1985) and the Keq of TPI which has been determined in vivo (Connett 1985) and corrected for pH changes (Table 1). KTPI=(DHAP)/(GAP) Though to ta l c y t o s o l i c ADP concentrat ion i s e a s i l y measured the amount considered to be free i s much lower (Veech et a l . 1979; Jacobus et a l . 1982; Shoubridge et a l . 1984; Meyer et a l . 1985). Estimates of free ADP have been made by using the Keq of the CK react ion (Table 1). K C K M A T P ) (Cr)/(ADP) (PCr) (H*") Free ADP was ca lcu la ted using the measured (ADP*„,> and compensated (ADPfC) contents of the reactants . A l l metabolite values measured are reported as umol/g w/w of t i s sue . In order to make the metabolite r a t i o ' s d i r e c t l y comparable to the Keq's measured in v i t r o , a l l metabolite values were converted to umol/g c e l l water. The percentage of t i s sue weight taken to be c e l l water was 78 percent under a l l cond i t ions . Percent water was assessed on pre and post freeze dryed, freeze clamped white muscle (Table 3). This value was very s i m i l a r to previous values determined for l i v e r and muscle (Krebs and Veech 1969). Table 3. C e l l water content of rainbow trout white muscle. 22 n Pre-ex (5) PSS-30 (6) PSS-7 (5) ES (5) C e l l Water 77.8 +0.5 77.6 +0.3 77.7 +0.6 78.3 +0.5 Values are means+SE expressed as percent. Energy Turnover. Estimated energy turnovers (umol ATP/g/min) from f a t s , g l y c o l y t i c and high energy phosphagen sources were ca lcu la ted for rainbow trout red and white muscle during the PSS-30 and PSS-7. Glucose fermentation cont r ibut ion was based on l ac ta te production not a t t r i b u t a b l e to muscle glycogen fermentation with 2 ATP being generated per glucose unit fermented. Glucose oxidat ion cont r ibut ion was based on l i v e r glycogen and blood glucose u t i l i z a t i o n (PSS-30=3.7 umol/ml; PSS-7=3.7 umol/ml) not accounted for by glucose fermentation with 36 ATP being generated per glucose unit ox id i zed . Blood glucose values are estimated from a previous inves t i ga t ion (Dobson, liommsen and Hochachka, unpublished observations) where blood glucose values decreased from 19.1 to 8.2 umol/ml with an exhaustive exercise regimen s i m i l a r to the present one. The glycogen fermentation contr ibut ion was based on the change in muscle glycogen accounted for by lac ta te production with 3 ATP being generated per g lucosyl unit fermented. The glycogen oxidat ion cont r ibu t ion was based on the change in muscle glycogen not accounted for by l ac ta te production with 37 ATP being generated per g lucosyl unit ox id i zed . Fat oxidat ion cont r ibut ion was based on the oxygen uptake data of Randall and Daxdoek (1979) assuming a l inear r e l a t i o n s h i p between workload and oxygen uptake with U c r i t being equivalent to maximal oxygen uptake. The muscle oxygen uptake was converted to moles of 02 by: PV=nRT where P = 140 mmHg at 1 0 ° C , V=0.00371 1, R=0.08205 1 atm deg" 1 mo l " 1 and T - 2 8 3 ° K . Since 1 mole of 02 produces 6 mol ATP, an energy turnover due to oxidat ion of substrate can be ca lcu la ted for muscle. The energy due to combustion of fats was assumed to be equal to the d i f ference between the to ta l muscle oxidat ion derived ATP turnover and the red plus white muscle glucose plus glycogen oxidat ion derived ATP turnover. Values are based on a 200 g f i s h which would contain 132 g white muscle, 2 g red muscle and 2 g l i v e r (Randall and Daxboeck 1982). It i s assumed that only half the red or white muscle i s ac t ive at any time. The stored e l a s t i c component was not taken into account. Blood volume was assumed to be 5 ml/100g (Stevens 1968) with 83 and 9.4 percent of t h i s volume during exercise being d i s t r i b u t e d within white and red muscle re spec t ive ly (Neumann et a l . 1983). The red muscle was assumed to have a 20 fo ld higher capacity to oxid ize f a t s / p r o t e i n s than white muscle based on maximal enzyme a c t i v i t i e s (Crabtree and Newsholme 1972) and free fa t ty acid oxidat ion rates ( B i l i n s k i 1963; Jonas and B i l i n s k i 1964). Intramuscular pH. Tissue sect ions were powdered under l i q u i d nitrogen and added to an ice cold sa l t so lu t ion (pH 7.2) containing in mmol/1: 145 KC1, 10 NaCl , 1 iodoacet ic acid (IAA) , 20 NaF~, 5 EGTA, 5 DNFB and 1 PMSF in a 1:10 d i l u t i o n (v/w). This so lu t ion had a very low buffer ing capac i ty . These extracts were homogenized for 2 x 15 seconds at maximum speed with a polytron PT-10 t i s sue homogenizer. The suspensions were then centri fuged in an eppendorf microcentri fuge at 11,000 g for 30 seconds. Al iquot s of t h i s supernatant were used for pH determination at 10"C on a d i g i t a l acid-base analyzer (Radiometer PHM 72) equipped with a microelectrode unit (Radiometer Type E 5021). V a l i d i t y of t h i s technique was ascertained by comparison with 24 pH values determined on the suspensions. A l l determinations were performed in dup l i ca te . Cytoso l i c Redox P o t e n t i a l . The free c y t o s o l i c redox potent ia l was ca lcu la ted from the temperature and pH adjusted Keq of the LDH react ion as out l ined by Williamson et a l . (1967). K i _ D H a ( P y r ) (H+) / (lac) x (NADH) / (NAD) Upon rearranging! NAD = (Pyr)(H>) NADH (Lac)K L D H Equi1ibrium/Nonequi1ibriurn. Mass act ion r a t i o s were ca lcu la ted and divided by t h e i r respect ive equ i l ib r ium constants for the g l y c o l y t i c react ions under a l l exercise states in white and red muscle. A plot of the log of these r e s u l t s (log (mass act ion rat io /Keq)) was used to demonstrate the amount of devia t ion from equi l ibr ium for a given r e a c t i o n . Regulatory Enzymes. The crossover theorem provides a method for l o c a l i z i n g i n t e r a c t i o n s or regulatory s i t e s in complex enzyme systems (Williamson 1969). For an enzyme to be regula tory , i t must be nonequil ibrium and demonstrate a change in substrate in the opposite d i r e c t i o n to f l u x . However, caution must be exercised when i n t e r p r e t i n g crossover p lo t s as errors in the i d e n t i f i c a t i o n of regulatory enzymes can occur due to modulators (Williamson 1969; Rol les ton 1972). Crossover p lots are presented for the g l y c o l y t i c intermediates of red and white muscle under a l l exercise cond i t ions . Ion Analyses , D i s t r i b u t i o n and Di f ferences . Blood samples were centri fuged in a microcentrifuge (Eppendorf) at 11,000 g to separate the plasma. An a l iquot of t h i s plasma was deproteinized with 200 ul of 0.6 N PCA and centr i fuged at 11,000 g for l ac ta te determinations. The remaining plasma was frozen in l i q u i d nitrogen and stored at - 8 0 ° C for determination of Na* , K* , C a * * , Mg + + and C l ~ . A l iquot s of muscle extracts were also analyzed for these ions with the Tris /PCA contamination being taken into account. Na* and K* were analyzed by with a flame photometer. C a * * and Mg** l eve l s were determined with an Atomic Absorption Spectrophotometer. Measurement of C l - was e s s e n t i a l l y by the method of Ramsay (1955) employing t i t r a t i o n with s i l v e r n i t r a t e . C e l l u l a r and e x t r a c e l l u l a r ion concentrat ions were ca lcu la ted according to the procedure of Macchia and Pol imini (1982). This procedure requires four va r i ab l e s : t i s sue ion content, plasma ion concentra t ion , wet and dry t i s sue weights. The e x t r a c e l l u l a r compartment was assumed to be 94.1 ml/kg (Mi l l i gan and Wood 1985). The ion di f ference was the d i f ference between the concentrat ions of cat ions and anions measured in mEq/1 (Stewart 1981). In t h i s inve s t i ga t ion the ion di f ference was defined as: IDe» <Na*)+<K*)+<Ca" )/2+(Mgw ) 12-(Cl" ) - (La") IDw=(Na*) + (K*) + (Ca~ ) /2+<Mg~ ) /3B-(Cl" ) - (La" )- (PCr r ") IDr=(Na*) + (K*) + (Ca ")/2+<Mg~ )/20-<Cr ) - (La") - (PCr l " ) The concentrat ions of plasma d iva lent cat ions C a * * and Mg** and i n t r a c e l l u l a r C a * * were d iv ided by 2 assuming that 58 percent were bound to molecules such as prote in (Jackson and Heis ler 1982). The content of c e l l u l a r Mg** was divided by 38 (white) or 20 (red) r e spec t ive ly to compensate for the amount bound by i n t r a c e l l u l a r proteins (assuming a free Mg** of approximately 1 mM; Velsco et a l . 1973; Connett 1985). It was assumed that during exerc i se , the content of prote in bound d iva lent cat ions remained constant. Any change in C a * * or Mg** was therefore in the form of free ions and/or Ca- or Mg- l ac ta te complexes (Cannon and Kibr i ck 1938). This assumption was based on the counteract ing ef fects of ac idos i s and hypercalcemia (Herbert and Jackson 1985). Ca- and Mg-lactate complexes 26 do not af fect the ion di f ference c a l c u l a t i o n . BUFFERING ANALYSES Tissue F r a c t i o n a t i o n . For the h i s t i d i n e re la ted compound study, red and white muscle were separated into a ser ie s of f r ac t ions with each f r a c t i o n containing one less buffer ing const i tuent than the preceeding f r a c t i o n . To achieve t h i s , a crude homogenate (CH) was prepared by homogenizing (Polytron PT-10) 2-3 grams of muscle t i s sue in 10 volumes of ice cold s a l t so lu t ion containing (in mM) 145 KC1, 10 NaCl and 5 iodoacet ic a c i d . The homogenate was centri fuged at 0 ° C for 20 minutes at 48,000 g. The p e l l e t was homogenized in the, same volume and processed as above. This procedure was repeated twice and the washings and supernatant (SN) were combined. The p e l l e t was reta ined and resuspended in 10 ml. of s a l t s o l u t i o n . Al iquot s of 5-10 ml. of the combined supernatants (SN plus washings) were applied to a Sephadex G-25 (coarse) column (1.9 x 25 cm.) and eluted at room temperature with sa l t s o l u t i o n . E l u t i o n of the high molecular weight f r a c t i o n (HMW) was followed at 280 nm; for the low molecular weight f r a c t i o n 260 nm was used as the i n d i c a t o r . The c o l l e c t e d f r a c t i o n s were stored at 4 °C and analyzed as soon as poss ib le ( less than 24 hr s ) . H i s t i d i n e Related Compounds. Method A. Tissue extracts were prepared with s l i g h t modi f icat ions according to Stein and Moore (1958). B r i e f l y , 1 g of t i s sue was homogenized (Polytron PT-7) in 5 ml of p i c r i c a c i d . After c e n t r i f u g a t i o n , the t i s sue was resuspended in 5 ml of p i c r i c acid and the procedure repeated. The supernatants were combined and applied to a Dowex 2X8 (200-400 mesh, C l - form) column (0.5 x 1.5 cm.) . The column was washed with 0.01 N HC1 and the e lutate stored at 4 ° C . The e lutates were 27 evaporated to dryness and brought to 2 ml (trout) and IE) ml (marlln) with high performance l i q u i d chromatography (HPLC) grade water. H i s t i n e Related Compounds. Method B. One gram of marlin white muscle was homogenized with 5 ml. of 8 percent PCA and centr i fuged . The p r e c i p i t a t e was homogenized in the same volume of PCA and centr i fuged . The combined supernatants were neutra l i zed with 2 N KOH and stored at 4 ° C as prote in- f ree ex t rac t s . The supernatant was applied to a 2.5 x 10 cm. copper-sephadex 6-25 (fine) column (prepared by e q u i l i b r a t i n g the Sephadex overnight in 0.08 M copper sulphate) to s p e c i f i c a l l y bind h i s t i d i n e re la ted compounds. The column was washed with water and the e luate , assumed to contain other amino acids and phosphates, was c o l l e c t e d . The h i s t i d i n e re la ted compounds were eluted with 0.01 N HC1. Both f r ac t ions were stored at 4 ° C . The concentrat ions of h i s t i d i n e re la ted compounds were determined as described previous ly (above). The content of h i s t i d i n e re la ted compounds of the marlin white muscle PCA extracts were s imi l a r to that of the p i c r i c acid extracts suggesting comparabi l i ty of the two methods. Buffer Capaci ty . Buffer capac i t i e s were determined on a l l i so l a ted f r a c t i o n s . To further assess the cont r ibut ion of h i s t i d i n e re la ted compounds to to ta l c e l l u l a r bu f fe r ing , s a l t so lu t ions containing the experimental ly determined marlin white muscle t i s sue l e v e l s of h i s t i d i n e re la ted compounds were prepared. In the same way, representat ive taurine concentrat ions (see below) were also prepared. These sa l t so lu t ions containing t h e i r respect ive h i s t i d i n e re la ted compounds or taur ine l eve l s were e m p i r i c a l l y t i t r a t e d for the c a l c u l a t i o n of the i r buffer c a p a c i t i e s . Buffer capacity was assessed by a modif icat ion of the method of Davey et a l . (1960). A l l preparations were adjusted to pH 6.0010.05 and t i t r a t e d to pH 8.0010.05 with NaOH. R e l i a b i l i t y was ascertained by r e t i t r a t i o n of the extracts fo l lowing pH readjustment to 6.00+0.05. Buffer capacity was determined as the number of DH ions required per gram of t i s sue to change the pH 1 u n i t . Buffer capac i t i e s were determined over the pH ranges 6 .0-7 .0 , 6.5-7.5 and 7 .0-B.0 . A schematic representat ion of the h i s t i d i n e re la ted compound study i s contained in Figure 1. A l t e r n a t i v e l y , an in vivo estimate of trout white muscle buffer capacity was ca lcu la ted during the PSS-7. This was achieved by assuming a 111 s to ich iometr i c r e l a t i o n s h i p between lac ta te and H+. By knowing the change in intramuscular pH, the buffer capacity was ca lcu la ted asi B= La-MpH As w e l l , a further estimate of trout white muscle buffer ing was made by c a l c u l a t i n g the buffer ing due to a s soc ia t ion of H* with bases and the buffer ing due to enzymatic a c t i v i t y . 29 Figure 1. F rac t iona t ion scheme used to separate the d i f f e rent t i s sue components, be, measurement of buffer capac i ty . deproteinize ( 8 % PCA) taurine MUSCLE 10 * vol. medium j crude homogenate Method A Method B S x vol. picric acid(1%) protein free fraction T 1 8 % PCA Oowex 2X8 (CU Cu-Sephadex G-25 phosphate other amino acids 0 phosphate protein protein histidine compounds histidine compounds 0 RESULTS FUEL SELECTION The metabolic a l t e r a t i o n s associated with the various work i n t e n s i t i e s in l i v e r , white and red muscle are i l l u s t r a t e d in Figure 3 and out l ined in d e t a i l in Tables 4, 5 and 6 r e s p e c t i v e l y . 30 Minute Prolonged Steady Swim (PSS-30) The r e s u l t s are presented r e l a t i v e to pre-exercise values. Phosphagen and Nucleotide Metabolism. During the 30 minute prolonged steady swim, red muscle demonstrated a high rate of phosphagen and nucleot ide metabolism r e l a t i v e to white muscle (Figure 3) . Large decrements in PCr (96 percent) and ATP (40 percent) were observed for red muscle while changes in these metabolites were minor in white muscle. Associated with these a l t e r a t i o n s were increases of 1.8 and 1.3 fo ld in red muscle ADP and AMP l e v e l s r e s p e c t i v e l y . These changes resul ted in a decrement of 40 percent in the to ta l adenylate pool (Figure 2) with the IMP increases (1.5 fold) accounting for the decrease in adenine nucleot ides (Figure 2) . White muscle to ta l adenylate pool remained r e l a t i v e l y constant (Figure 2) despite increases of 80 percent in IMP content. BTP l e v e l s f e l l approximately 40 percent in both t i s sues while Pi increased 4 and 8 fo ld in white and red muscle, r e s p e c t i v e l y . Glycogenolys i s . The 30 minute prolonged steady swim resul ted in an act ive recruitment of red f i b e r s motor units as evidenced by the large dec l ine in stored glycogen (97 percent) to very low l eve l s (0.6 umol/g). As w e l l , white muscle g l y c o l y t i c energy prov i s ion and to a far lesser degree, l i v e r derived glucose, contr ibuted to the energy requirements of t h i s workload (Figure 3). White muscle glycogen content was found to have decreased 31 percent while l i v e r glycogen had decl ined 7.3 umol/g. Associated with these dec l ines in l i v e r glycogen, were increases of SB and 23B percent in white and red muscle glucose contents . The anaerobic g l y c o l y t i c end product l ac ta te was found to have increased 34B and 7B percent in white and red muscle r e s p e c t i v e l y . These increases in l a c t a t e , were less than would be expected, had a l l the glycogen been fermented anaerobicai1y. Krebs Cycle Intermediates. Malate and fumarate l eve l s increased dramat ica l ly in both t i s sue types e x h i b i t i n g , increases of 1.3 to 4.5 f o l d . However, c i t r a t e l e v e l s were found to remain r e l a t i v e l y s table during t h i s workload. 7 Minute Prolonged Steady Swim (PSS-7) Under the condi t ions of t h i s experiment, a l l animals had performed the PSS-3B immediately pr ior to t h i s exercise i n t e n s i t y . The re su l t s are therefore presented r e l a t i v e to the PSS-30 metabolite concentrat ions unless otherwise s p e c i f i e d . Phosphagen and Nucleotide Metabolism. During the higher i n t e n s i t y (10B percent Ucr i t ) 7 minute prolonged steady swim which followed the PSS-30, white muscle high energy sources were a c t i v e l y recru i ted while red muscle PCr and ATP appeared to i n i t i a t e replenishment. Within white muscle, PCr content was found to have decreased 62 percent (13 umol/g) to 20.8 umol/g, 55 percent lower than the pre-exercise value. This corresponded to an average rate of PCr deplet ion (1.86 umol/g/min) which was 14 fo ld greater than the PSS-30 ra te . ATP l eve l s had decreased a further 11 percent r e s u l t i n g in the ATP content being reduced 20 percent from pre-exercise l e v e l s . ADP l eve l s were increased 1.6 fo ld to 1.15 umol/g. AMP content increased 1.9 fo ld such that i t s concentrat ion was now 3.5 times i t s pre-exercise l e v e l s . A 3.3 fo ld e levat ion in IMP content to 1.8 umol/g was observed with t h i s l eve l of IMP being 6 times the pre-exercise value. GTP content remained at a constant value 35 percent lower than i t s pre-exercise concentrat ions . Inorganic phosphate and NH4 contents were elevated 3.3 (Table 4) and 2.2 fo ld r e spec t ive ly such that these values were 12.9 and 3.1 times the i r pre-exerc i se l e v e l s . In contras t , red muscle PCr values , although remaining 94 percent below pre-exercise values were found to have increased 1.6 fo ld while ATP l e v e l s were elevated 12 percent. Associated with these a l t e r a t i o n s were decreases in ADP of 20 percent, increases in AMP of 50 percent and increases of 18 percent (3 umol/g) for inorganic phosphate. IMP values increased 20 percent br inging the to ta l increase to 7.2 times i t s pre-exercise value. GTP content increased 50 percent (0.016 umol/g) to a value only 17 percent less than pre-exercise l e v e l s . Glycogenolys i s . The higher i n t e n s i t y 7 minute prolonged steady swim resul ted in a white muscle glycogen content decrement of 65 percent to 5.6 umol/g, a value 75 percent lower than i t s pre-exercise concentrat ion . The average rate of glycogen deplet ion (1.49 umol/g/min) exceeded the PSS-30 rate by 6.2 f o l d . Lactate concentrat ion increased 3.3 fo ld to 33 umol/g, an 11 fo ld e levat ion from pre-exercise values with i t s rate of accumulation being 25 times the PSS-30 ra te . In red muscle, glycogen content remained low (0.5 umol glucosyl uni t s /g) while l ac ta te and glucose decreased 17 (1.5 umol/g) and 38 (1.27 umol/g) percent r e s p e c t i v e l y . Glucose l eve l s s t i l l remained 45 percent higher than pre-exercise concentrat ions . During the PSS-7, l i v e r glycogen values decreased 11 percent (18.6 umol/g), a deplet ion amount 3 times greater than was achieved during the PSS-30. The f i n a l glycogen values were 15 percent (25.9 umol/g) less than pre-exercise content. This deplet ion of l i v e r glycogen occurred at a mean rate (2.66 umol/g/min) 11 times greater than during the PSS-30. Associated with t h i s dec l ine in l i v e r glycogen, were increases of 2.4 fo ld to 11 umol/g in l i v e r glucose, a value 3.2 times i t s pre-exercise concentrat ion. Despite t h i s increase in a v a i l a b i l i t y of blood glucose, white muscle glucose l eve l s remained r e l a t i v e l y constant. Krebs Cycle Intermediates. During the PSS-7, both white and red muscle demonstrated increased contents of malate, fumarate and c i t r a t e such that these intermediates were now elevated 1.3 to 6 fo ld above pre-exercise 1evels . Exhaustive Swim (ES) The ES protocol immediately followed the PSS-30 and PSS-7 exercise regimens. The r e s u l t s are therefore presented r e l a t i v e to the PSS-7 metabolite contents unless otherwise s p e c i f i e d . Phosphagen and Nucleotide Metabolism. The exhaustive swim resul ted in a further recruitment of white and red muscle high energy sources. In white muscle, PCr was found to have decreased 87 percent with t h i s deplet ion r e s u l t i n g in PCr l eve l s at exhaustion being only 5 percent (1.8 umol/g) of the pre-exerc i se concentrat ion . ATP l eve l s decreased 54 percent such that ATP content was reduced 65 percent (4.61 umol/g) from pre-exercise l e v e l s . No s i g n i f i c a n t change was found in ADP content but AMP values increased 2 f o l d ; ADP and AMP being 1.5 and 7.2 times the i r pre-exercise l e v e l s . IMP increased 2.4 fo ld br inging the to ta l e leva t ion to 14.5 fo ld (4.3 umol/g). The t o t a l adenylate pool decreased 513 percent but the to ta l adenylate plus IMP pool was unchanged (Figure 2) . NH* concentrat ion was increased 2 fo ld such that ES l e v e l s (6.37 umol/g) were elevated 6.4 times pre-exercise values . Phosphate l e v e l s increased 88 percent (26 umol/g) while GTP l eve l s were decreased 19 percent (0.007 umol/g). GTP content was depressed 40 percent (0.024 umol/g) from pre-exercise l e v e l s . In red muscle, further decrements in PCr (67 percent) and ATP (31 percent) occurred during t h i s work i n t e n s i t y (ES) with f i n a l concentrat ions being 2 (0.4 umol/g) and 46 (1.6 umol/g) percent of pre-exerc i se values r e s p e c t i v e l y . Associated with these changes were increases in ADP (32 percent) , AMP (55 percent) and inorganic phosphate (6 percent) (Table 5) r e s u l t i n g in l e v e l s 1.9, 3.0 and 9.9 times t h e i r pre-exercise values . IMP content increased 77 percent to a f i n a l concentrat ion (2.33 umol/g) 3.2 fo ld greater than i t s pre-exercise l e v e l . The to ta l adenylate pool was decreased 45 percent but the to ta l adenylate plus IMP pool M a s unchanged from pre-exercise values (Figure 2) . GTP l eve l s were decreased 31 percent (0.015 umol/g) with the f i n a l content (0.034 umol/g) being 42 percent lower than i t s pre-exercise value. Glycogenolys i s . White and red muscle glycogen contents decl ined to values less than 1 percent of t h e i r pre-exercise concentrat ions (Tables 4 and 5) . Associated with these dec l ines in glycogen were increases in white and red muscle l ac ta te l e v e l s . White muscle l ac ta te concentrat ion increased 1.3 fold to 43 umol/g, a 14.3 fo ld increase from pre-exercise content. In red muscle, l a c ta te content increased 50 percent to 10.8 umol/g, a value 2.1 times the pre-exerc i se l e v e l . L iver glycogen decreased 23 percent (36.6 umol/g) while glucose l e v e l s remained constant. F ina l glycogen concentrat ions were decreased 34 percent (62.5 umol/g) and glucose l eve l s increased 3213 percent o-f the i r respect ive pre-exercise values . White and red muscle glucose contents remained r e l a t i v e l y constant despite the large increase in glucose a v a i l a b i l i t y . However, the white muscle lac ta te l eve l s observed, were greater than would have been expected from the amount of glycogen fermented suggesting that some lac ta te must have been derived from glucose. Krebs Cycle Intermediates. In both white and red muscle, malate and fumarate contents were found to be elevated s u b s t a n t i a l l y from pre-exercise l eve l s (3 to 1(9 f o l d ) . However, c i t r a t e contents were found to be e q u i v i l e n t to pre-exerc i se values for both t i s sue types. Table 4. Content of metabolites and pH in rainbow trout white muscle. Metabol i te Pre-ex PSS-30 PSS-7 ES n (5) (6) (5) (5) PCr 19.9+1.6 15.9+0.9 2.9+0.7 1.810.6 PCr = 37.8+1.6 33.8+0.9 20.810.7 1.8+0.6 Cr 24.6+1.0 29.7+1.4 42.710.2 43.4+1.8 C r c 6.7+0.3 11.8+1.4 24.810.2 43.411.8 ATP 7.26+0.11 6.57+0.22 5.8210.5 2.6510.25 ADP 0.70+0.01 0.68+0.04 1.1510.07 1.0510.05 AMP 0.021+0.001 0.039+0.007 0.07310.012 0.15210.035 Pi 21. 1 + 2.6 26.8 + 1.-9 47.6+3.2 55.711.8 Pic 2.3+0.4 8.9+1.4 29.712.8 55.7+1.8 IMP 0.30+0.05 0.53+0.09 1.78+0.27 4.3410.26 N H ; 1.04+0.05 1.43+0.15 3.2010.42 6.3710.19 BTP 0.054+0.012 0.039+0.003 0.03710.004 0.03010.007 Glucose 1.02+0.14 1.86+0.55 2.1910.51 2.16+0.44 Glycogen 23.3+1.0 16.0+1.6 5.6+1.1 0.210.04 Lactate 3.0+0.4 10.1+1.1 33.010.6 42.9+3.0 Malate 0 . 1 3 ± 0 . 0 7 0.2510.07 0.25+0.03 0.39+0.06 Fumarate 0.01+0.002 0.0410.01 0.0610.01 0.0B10.01 C i t r a t e 0.30+0.01 0.28+0.02 0.3510.05 0.2810.03 P H 6.97+0.04 6.93+0.03 6.65+0.03 6.5610.04 Values are means + SE expressed in umol/g (w/w). Blycogen was ca lcu la ted glucose u n i t s , c, compensated metaboli te . Table S. Content o-f metabolites and pH in rainbow trout red muscle. Metaboli te Pre-ex PSS-30 PSS-7 ES n (5) (6) (5) (5) PCr 5.2+0.7 0.8+0.4 1.210.2 0.410.2 PCr c 18.6+0.8 0.8+0.4 1.210.2 0.4+0.2 Cr 22.1+1.0 26.5+0.8 26.011.9 26.5+0.3 C r c B.B+0.4 26.5+0.8 26.0+1.9 26.5+0.3 ATP 3.43+0.18 d 2.02+0.27 2.2610.32 1.57+0.32 ADP 0.65+0.07 1. 18+0.08 0.84+0.04 1.1110.17 AMP 0.106+0.012 0.137+0.014 0.207+0.023 0.32110.055 Pi 14.1+1.2 21.1+2.9 21.5+2.4 22.712.1 P i c 2.3+1.3 21.1 + 2.9 21.5+2.4 22.7+2.1 IMP 0.73+0.11 1.09+0.18 1.3110.15 2.3310.28 N H ; 1.60+0.14 1.57+0.16 1.7110.08 2.8810.30 GTP 0.059+0.003 0.033+0.007 0.04910.002 0.03410.003 Glucose 1.46+0.19 3.38+0.55 2.1110.14 2.9510.05 Glycogen 1 8 . 1 ± 2 . 5 0.610.3 0.510.2 <0. 1 Lactate 5.2+0.8 8.8+1.1 7.310.9 10.811.4 Malate 0.2010. 10 0.32+0.03 0.4010.05 0.6510. 1 Fumarate 0.03+0.01 0.0410.01 0.0810.02 0.0810.02 C i t r a t e 0.40+0.05 0.34+0.05 0.44+0.05 0.4410.09 PH 6.89+0.02 6.92+0.04 6.88+0.02 6.81 (1) Values are means + SE expressed as umol/g (w/w). Glycogen was ca l cu la ted glucose u n i t s , c, compensated metabol i tes . Table 6. Content of metabolites in rainbow trout l i v e r . lietabol i t e Pre-ex PSS-30 PSS-7 ES n (5) (6) (5) (5) Glycogen 183.6+35.6 176.3+34.1 157.7*36.8 121.118.5 Glucose 3.53+JJ.B5 4.69+0.51 11.14+1.22 11.44+1.12 G6P 0.12 + 0.03 0.20+0.04 0.7710.06 0.8210.07 F6P 0.02+0.01 0.04t0.01 0.1210.01 0.1410.02 2PG 0.09+0.01 0.08+0.01 0.0810.01 0 . 0 8 ± 0 . 0 1 PEP 0.11+0.01 0.1110.02 0.0810.004 0.06+0.004 Pyruvate 0.17+0.01 0.15+0.01 0.1610.01 0.14+0.01 Lactate 1.5+0.2 2.210.1 2.1+0.3 4 . 0 ± 0 . 6 Alani ne 3.54+0.41 3.0310.94 1.5510.28 2.48+0.39 Values are mean + SE expressed as umol/g (w/w). Glycogen was ca lcu la ted glucosyl u n i t s . 40 Figure 2. The adenylate pool (sum of ATP+ADP+AMP) and adenylate pi IMP pool in rainbow trout muscle. A=ADPmj B=ADP*m| C=ADP«c. • T - rx.. P E PSS-7 PSS-JO ES White Muscle Adenylates PE PSS-7 PSS-80 ES Red Muscle • 1> Adenylates • IMP 42 Figure 3. A l t e r a t i o n s in metabolite contents of l i v e r , white and red muscle of t rou t . A. A l t e r a t i o n s in rainbow trout white muscle glycogen, PCr, ATP, l ac ta te and IMP contents . B. A l t e r a t i o n s in rainbow trout red muscle glycogen, PCr, ATP, l ac ta te and IMP contents . C. A l t e r a t i o n s in rainbow trout l i v e r glycogen, glucose, l ac ta te and alanine contents, ( o g l y c o g e n ; 4 glucose; • PCr; A ATP; 0 l a c t a t e ; A IMP; ^ a l a n i n e ) time (min) 44 GLYCOLYTIC INTERMEDIATES White and red muscle g l y c o l t i c intermediate contents are contained in Tables 7 and 8 r e s p e c t i v e l y . During the PSS-30, white muscle G1P, 66P, F6P, FDP and pyr l eve l s were found to increase from 48 to 233 percent. The remaining white muscle g l y c o l y t i c intermediates remained at pre-exercise l e v e l s . When white muscle g l y c o l y t i c energy prov i s ion was furher act ivated (PSS-7), G1P, G6P, F6P and PYR concentrat ions demonstrated higher accumulations. As w e l l , GP and 3PG demonstrated increases in content while FDP content decl ined although remaining 32 percent above pre-exercise values . DHAP, GAP, GAP*, DPG, 2PG and PEP concentrat ions were found to remain r e l a t i v e l y constant. During the ES which resul ted in very low l eve l s of glycogen (Table 4) , a l l white muscle g l y c o l y t i c intermediates with the exception of GP, DPG and PYR exhibi ted lower contents than those of the PSS-30 although 61P, G6P and F6P l eve l s were s t i l l elevated when compared to pre-exerc i se values . FDP content was found to have f a l l e n to values 75 percent lower than pre-exercise l e v e l s . During the PSS-30, red muscle g l y c o l y t i c intermediate contents demonstrated r e l a t i v e l y minor va r i a t i ons (Table 8) despite a large dec l ine in glycogen concentrat ion (Table 5) . G1P was found to decl ine while G6P content increased and FDP l eve l s decreased to 47 percent of pre-exercise l e v e l s . BP and pyr contents increased while 2PG content decreased. During the PSS-7, red muscle demonstrated dec l ines in the contents of G6P and F6P while FDP remained depressed as GP and pyr l eve l s were increas ing . The ES resul ted in decreases in a l l red muscle g l y c o l y t i c intermediates with the exceptions of GAP, BAP* and DPS which a l l remained at pre-exercise l eve l s and BP and PYR contents which were e levated. 45 Table 7. Content of g l y c o l y t i c intermediates in rainbow trout white muscle. Intermediate Pre-ex n (5) PSS-30 (6) PSS-7 (5) ES (5) SIP 0.25+0.06 0.40+0.08 0.6410.04 0.3110.07 G6P 0.59*0.97 0.9710.16 1.7110.21 0.7310.24 F6P 0.07+0.01 0.1510.03 0.2710.04 0.1510.05 FDP 1.28+0.13 1.9010.12 1.6910.31 0.3210.08 DHAP 0.21+0.03 0.1910.02 0.2310.03 0.1710.02 GP 0.50+0.03 0.5210.08 0.8010.02 0.9510.11 GAP 0.05+0.01 0.0610.02 0.00310.005 0.0310.01 GAP* 0.018+0.002 0.01710.002 0.022+0.003 0.017+0.002 DPG 0.06+0.01 0.0810.02 0.0610.01 0.16+0.03 3PG 0.55+0.05 0.5910.03 0.96+0.11 0.32+0.01 2PG 0.08+0.02 0.0810.02 0.0610.01 0.0110.004 PEP 0.06+0.004 0.0410.01 0.04*0.003 0.01*0.003 PYR 0.03+0.005 0.1010.02 0.1410.02 0.3310.05 Values are mean+SE expressed as umol/g w/w. 46 Table 8. Content of g l y c o l y t i c intermediates in rainbow trout red muscle. Intermedi ate Pre-ex n (5) PSS-30 (6) PSS-7 (5) ES (5) B1P 0.41+0.02 0.20+0.06 0.1810.06 0.1410.03 66P 0.45+0.08 0.54+0.10 0.31+0.04 0.33+0.06 F6P 0.10+0.01 0.08+0.02 0.0410.004 0.02+0.004 FDP 0.55+0.09 0.26+0.02 0.25+0.03 0.1210.03 DHAP 0.19+0.05 0. 19+0.05 0.15+0.06 0.1410.02 GP 0.62+0.10 0.71+0.16 0.98+0.55 1.2110.32 GAP 0.03+0.02 0.04+0.01 0.0410.02 0.06+0.01 GAP* 0.017+0.002 0.016+0.004 0.01310.005 0.01510.002 DPG 0.06+0.01 0.0510.01 0.0510.01 0.06+0.01 3PG 0.16+0.03 0.2010.03 0.2010.05 0.11+0.05 2P6 0.11+0.02 0.0510.01 0.0510.01 0.0310.01 PEP 0.04+0.01 0.0310.01 0.03+0.01 0.0210.01 PYR 0.05+0.01 0.0710.01 0.10+0.02 b 0.1110.002 Values are meantSE expressed as umol/g w/w. 47 METABOLIC REGULATION Free ADP contents were ca lcu la ted for both the measured and compensated metabolite data of the CK r e a c t i o n . Free ADP pre-exercise contents were very s i m i l a r between f iber types when ca lcu la ted using e i ther the measured or compensated metabolite data, although the ADP + m contents were 8-10 fo ld higher than the ADP* C values (Table 9) . Free ADP contents were found to increase in both t i s sues with increas ing exercise i n t e n s i t y . Following the ES, both red and white muscle free ADP contents were s imi la r and represent approximately 20 percent of the to ta l ADP. An inconsis tency occurred as free ADP in white muscle was found to demonstrate i t s largest increase during the PSS-7 <ADP*m) and ES (ADP*C) r e s p e c t i v e l y . However, red muscle free ADP increased extens ively during the PSS-30 when using e i ther the measured or compensated metabolite data (Table 9) . ATP/ADP r a t i o s were ca lcu la ted for the to ta l (measured), free measured and free compensated ADP contents (Table 9) . Pre-exercise white muscle ATP/ADP r a t i o s were approximately 2 fo ld greater than red muscle. S imi lar patterns of dec l ine in the ATP/ADP r a t i o were exhibi ted for both t i s sues between the various procedures of c a l c u l a t i n g t h i s r a t i o (ADPm; ftDP*mj ADP*c) . The ATP/ADP r a t i o demonstrated by white muscle after the ES was s i m i l a r to the ATP/ADP r a t i o in red muscle fo l lowing the PSS-30, PSS-7 and ES. Phosphorylation po tent i a l s (ATP/ADPxPi) were ca lcu la ted for the t o t a l , free measured and free compensated ADP contents (Table 9) . The pre-exercise phosphorylation po tent i a l s were 1.4 to 2.9 fo ld larger in white than red muscle. S imi lar decreases in phosphorylation po tent i a l s between the methods 4B of assessment were noted for white and red muscle. The ES phosphorylation po tent i a l s were very s i m i l a r between t i s sue s . The free c y t o s o l i c redox state of the NAD couple was ca lcu la ted in rainbow trout muscle from the measured contents of pyr and lac ta te employing the equ i l ib r ium constant of the LDH react ion (Table 1) and the values are presented in Table 10. White and red muscle NAD/NADH r a t i o s remained at pre-exercise l eve l s u n t i l after the ES and PSS-30, r e s p e c t i v e l y . During these exercise i n t e n s i t i e s , the NAD/NADH r a t i o increased 2.8 to 3.6 fo ld in white and red muscle r e s p e c t i v e l y . PCr/Pi r a t i o s were ca lcu la ted for the measured and compensated metabolite data (Table 10). Measured PCr/Pi r a t i o s were very low for a l l experimental s tates in both t i s sue s . The compensated PCr/Pi r a t i o ' s were 16.8 and 8.1 for white and red muscle pre-exercise values r e s p e c t i v e l y . These r a t i o ' s ( P C r c / P i c ) decl ined to very low values fo l lowing the ES in white and the PSS-30 in red muscle. 49 Table 9. Measured and ca lcu la ted free cytoplasmic ADP content, ATP/ADP r a t i o and c y t o s o l i c phosphorylati on po tent i a l s in rainbow trout muscle. Muscle Pre-ex PSS-30 PSS-7 ES n (5) (6) (5) (5) ADPm W 0.70+0.01 0.6810.04 1.15+0.07 1.0510.09 (umol/g) R 0.6510.01 1.18+0.08 0.83910.04 1.1110.17 ADP* m W 0.057+0.004 0.06510.004 0.290+0.063 0.203+0.056 (umol/g) R 0.07310.012 0. 18910.010 0.22910.022 0.229+0.021 ADP* = W 0.007+0.001 0.01210.002 0.019+0.002 0.20310.056 (umol/g) R 0.00810.001 0.189+0.010 0.229+0.022 0.229+0.021 ATP/ADPm W 10.410.2 9.810.2 5.110.4 2.610.2 R 5.410.5 1.7+0.2 2.710.4 1.510.4 ATP/ADP*m W 129.619.5 103.014.5 24.5+5.9 18.115.1 R 52.418.9 10.611.3 10.411.6 7.512.1 • A T P/ADP, c W 1025198 582166 312121 18.115. 1 R 457124 10.611.3 10.411.6 7.512. 1 ATP W 553173 371124 110112 4613 A D P m . P i (M _ 1 ) R 383133 149121 140120 74123 ATP W 6950+1100 39381367 5001164 316181 A D P , m . P i (M-M R 36821513 813196 5421103 3701127 ATP W 451200144800 6090018900 1100011400 316181 ADP* = . P U (M _ 1 ) R 155000130700 813196 5421103 3701127 Values are mean+SE. W, white muscle; R, red muscle. 50 Table 10. Calculated free c y t o s o l i c redox state and PCr/Pi r a t i o ' s in rainbow trout muscle. Muscle Pre-ex n (5) PSS-30 (6) PSS-7 (5) ES (5) NAD/NADH 749+153 961+145 1068+82 888+132 828+106 2027+173 2076+458 1525+142 PCr/Pi R 1.04+0.12 0 . 3 7 ± 0 . 0 4 0.6+0.06 0.04+0.02 0.0610.02 0.0610.01 0.03+0.01 0.02+0.01 PCr. Pic 16.811.5 8.1+0.4 3.410.3 0.0410.02 0 . 7 ± 0 . 1 0.06+0.01 0 . 0 3 ± 0 . 0 1 0.0210.01 Values are mean+SE. W, white muscle; R, red muscle. Equi l ib r ium versus Nonequi1ibrium The degree of devia t ion from equ i l ib r ium was assessed for the g l y c o l y t i c enzymes using both the measured and compensated metabolite data in white (Figures 5 and 6) and red (Figures 7 and 8) muscle. S imi lar r e su l t s were noted within each f iber type for the measured and compensated metabolite data. Within both t i s sue types, the enzymes HK, PGM, PFK, Aid and PK appear to be out of equ i l ib r ium under a l l experimental cond i t ions . Of these, HK, PFK and PK demonstrate the largest dev ia t ion from equ i l ib r ium in both t i s sue s . With increas ing workload, these nonequi1ibrium enzymes demonstrate r e l a t i v e l y s imi l a r patterns of d e v i a t i o n . However, the equ i l ib r ium enzymes GPDH and PGK which act together in vivo (Lehninger 1975), demonstrate a devia t ion from equ i l ib r ium as exercise i n t e n s i t y increases . This change to nonequil ibrium occurred during the ES for white muscle and for a l l exercise i n t e n s i t i e s in red muscle. The combined 6PDH.PGK/LDH react ion demonstrated s i m i l a r devia t ions from equi l ibr ium as the GPDH.PGK r e a c t i o n . 52 Figure 4. Deviat ions of trout white muscle g l y c o l y t i c enzymes from equi l ibr ium based on compensated metabolite data. A=Pre-exs B=PSS-30j C=PSS-7; D=ES. £3 G P D H P G K P G M * P F K T P I G P D H P G K L D H Enol H K P G I A i d G P D H P G K P G M P K • T I A -8 A • T B I - i ' 1 1 ' i c J ' »--8 J-8 i • 1 54 Figure 5. Deviat ions of trout white muscle g l y c o l y t i c enzymes •from equ i l ib r ium based on measured metabolite data. A=Pre-ex- B=PSS-30| C=PSS-7{ D=ES. 56 Figure 6. Deviat ions of trout red muscle g l y c o l y t i c enzymes from equ i l ib r ium based on compensated metabolite data. A=Pre-exj B=PSS C=PSS-7j D=ES. S 7 GPDH PGK P G M * PFK TPI GPDHPGK LDH Enol HK PGI Aid GPDH PGK PGM PK 'IA 58 Figure 7; Deviat ions of trout red muscle g l y c o l y t i c enzymes from equi l ibr ium based on measured metabolite data. A=Pre-ex; B=PSS-S0; C=PSS-7; D=ES. HK GPDH PGK PGM* PFK TPI GPDH PGK LDH Enol PGI Aid GPDH PGK PGM PK A < • —1 1 '—' 1—' 1 i i « • -8 A 8T IB 5 - 8 * 1 — . — —1 U - 4 _ • i c -8-L 'ID -8 60 Regulatory Enzymes Potent ia l regulatory enzymes were assessed using the crossover theorem as out l ined by Williamson (1969) and discussed in d e t a i l by Rol les ton (1972). Pathway intermediates are p lo t ted as a percent of control values on the ordinate with the sequence of intermediates on the abscissa and potent i a l regulatory enzymes are i d e n t i f i e d whenever the axis i s crossed. However, not a l l regulatory enzymes demonstrate a crossover and not a l l crossovers denote a regulatory enzyme (Rolleston 1972). A l l g l y c o l y t i c intermediate contents were p lo t ted versus t h e i r preceeding exercise values and versus the pre-exercise contents (Figures 9 and 10). Within white muscle, phos and PK were i d e n t i f i e d as po tent i a l regulatory enzymes under a l l condi t ions with the exception of phos in the ES versus PSS-7 condi t ion (Figure 8) . No apparent regula t ion was found to occur for HK and PFK within white muscle while the GPDH.P6K complex was found to exhib i t an apparent regulatory i n t e r a c t i o n fo l lowing the ES (Figure 8) . Red muscle in contra s t , demonstrated no regula t ion at phos and apparent regula t ion at the 6PDH.P6K complex under a l l exercise states (Figure 9). HK was found to be p o t e n t i a l l y regulatory fo l lowing glycogen deplet ion to less than 1 umol/g (Figure 9) while PK demonstrated s imi l a r regulatory potent i a l as in white muscle (Figures 9 and 10). 61 Figure 8. Crossover p lots showing the in te rac t ions caused by increas ing workloads on trout white muscle g l y c o l y s i s . A=PSS-30 versus Pre-ex; B=PSS-7 versus Pre-ex; C=ES versus Pre-ex; D=PSS-7 versus PSS-30; E=ES versus PSS-7. 6 2 63 0 I Glyc G6P FDP GAP, 3PG PEP L a G1P F6P DHAP 1 DPG 2PG PYR 64 Figure 9. Crossover p lo t s showing the in te rac t ions caused by increas ing workloads on trout red muscle g l y c o l y s i s . A=PSS-30 versus Pre-ex; B=PSS-7 versus Pre-ex; C=ES versus Pre-ex; D=PSS-7 versus PSS-30; E=ES versus PSS-7. 300 T 200 + 100 0 ' " Glyc Glu G1P G6P F6P FDP DHAP GAP, DPG SPG 2PG PEP La PYR 67 Energy Turnover The maximal cont r ibu t ion of aerobic metabolism to energy turnover in t h i s animal i s estimated in Table 11. This aerobic cont r ibut ion i s 2.2 fo ld greater in red as opposed white muscle with mean values of 138.1 and 153.4 umol ATP/g muscle/min during the PSS-30 and PSS-7 r e s p e c t i v e l y . Estimated metabolic energy turnover from a l l sources and the r e l a t i v e contr ibut ions of these sources to energy turnover in red and white muscle are presented in Table 12. During the PSS-30, aerobic energy turnover predominated both red and white muscle energy metabolism. Glycogen and glucose oxidat ion accounted for 14 percent of the energy turnover achieved by red muscle with t h i s oxidat ion being e s s n t i a l l y a t t r i b u t a b l e to glycogen ox ida t ion . Fat oxidat ion accounted for 86 percent of the energy turnover . In white muscle, 7 percent of the energy turnover was derived from glycogen and glucose oxidat ion with the glucose oxidat ion cont r ibut ion being i n s i g n i f i c a n t when expressed r e l a t i v e to per gram of muscle. Fat oxidat ion accounted for 92 percent of the energy turnover. The t o t a l ATP production for the PSS-30 equalled 18,624 umol ATP/min for the exerc i s ing muscle. During the PSS-7, red and white muscle energy turnover increased 10 and 20 percent r e spec t ive ly (Table 12). However t h i s increased white muscle energy turnover was less than was achieved by red muscle at e i ther exercise i n t e n s i t y (Table 12). Fat oxidat ion accounted for 99+ and 92 percent of the energy turnover generated by red and white muscle r e s p e c t i v e l y . Within white and red muscle glucose oxidat ion increased 6.5 and 5.2 fo ld r e s p e c t i v e l y . White muscle glycogen fermentation increased 8.0 umol ATP/g/min, a 12.3 fo ld increase . The to ta l ATP production during the PSS-7 was equal to 22,330 umol ATP/min for the exerc i s ing musculature. Table 11. Estimated oxygen uptake and aerobic energy turnover (umol ATP/g/min) of rainbow t r o u t . Pre-ex Total 02 uptake 0.41 (ml/min/Kg) Muscle 02 uptake 0.22 (ml/min/Kg) Muscle 02 uptake a 0.33 (ml/min/Kg muscle) Muscle 02 uptake increase (ml/min/Kg) Muscle 02 uptake increase a (ml/min/Kg muscle) Energy Turnover by muscle b (umol ATP/g muscle/min) Energy Turnover by RM c Energy Turnover by WM c 807. 90"/. 100% U c r i t Ucr i t U c r i t 2.97 3.34 3.71 2.59 2.88 3.24 3.87 4.36 4.84 2.37 2.56 2.84 3.54 3.82 4.24 122.7 138.1 153.4 267.5 301.1 334.5 120.5 135.6 150.7 Data are from Randall and Daxdoeck 1979. RM, red muscle; WM, white muscle, a Assumes white muscle cons t i tu te s 66 percent and red muscle 1 percent of the f i shes t o t a l body mass (200g). b Assumes that PV=nRT where P=140 mmHg, V=volume (1), R=0.08205 1 atm/deg/mol and T = 2 8 3 ° K . c Assumes red muscle oxid izes substrates at a rate 20 fo ld greater than white muscle but blood flow to white muscle i s 9 fo ld greater than red muscle. 69 Table 12. Estimated energy turnover (umol ATP/g/min) from f a t s , g l y c o l y t i c and high energy phosphagen sources in rainbow trout red and white muscle during the PSS-30 and PSS-7. Fuel/Pathway PSS-30 PSS-7 R W R W glucose fermentation a - - - 0.62 glucose oxidat ion b 0. 17 0.02 0.89 0.13 glycogen fermentation c 0.78 0.72 - 8.86 glycogen oxidat ion d 41.32 9.32 - -PCr hydro ly s i s 0.41 0.13 - 3.72 ATP hydro ly s i s 0.20 0.04 - 0.22 Fat oxidat ion e 259.61 126.26 333.61 150.57 Total Energy Turnover 302.8 136.5 334.5 164.1 (umol ATP/g/min) Total ATP Production 18,624 22,330 (umol ATP/g / f i sh muscle) a Based on l ac ta te production not a t t r i b u t a b l e to muscle glycogen fermentation with 2 ATP being generated per glucose fermented, b Based on l i v e r glycogen and blood glucose u t i l i z a t i o n (PSS-30=3.7 umol/ml| PSS-7=3.7 umol/ml) not accounted for by glucose fermentation with 36 ATP being generated per glucose ox id ized . c Based on the change in muscle glycogen accounted for by l ac ta te production with 3 ATP being generated per g lucosyl unit fermented. d Based on the change in muscle glycogen not accounted for by l ac ta te production with 37 ATP being generated per g lucosyl unit ox id i zed . e Based on the increase in oxygen uptake of the working muscle and assuming that the red muscle ox id izes substrates at a rate 20 fo ld greater than red muscle. Notes Values are based on a 200g f i sh which would contain 132g white muscle, 2g red muscle and 2g l i v e r (Randall and Daxboeck 1982). It i s assumed that only half of the red and white muscle i s ac t ive at any time. The stored e l a s t i c component was not taken into account. Blood volume was assumed to be 5 mls/100g (Stevens 1968) with 83.2 and 9.4 percent of t h i s volume during exercise being d i s t r i b u t e d within white and red muscle re spec t ive ly (Neumann et a l . 1983). R, red muscle; W, white muscle. FATIGUE Within white muscle, PCr deplet ion occurred as l ac ta te and inorganic phosphate accumulated (Figure 10). S i m i l a r i l y , red muscle PCr contents decreased as inorganic phosphate and l ac ta te concentrat ions increased, although the e levat ion in l ac ta te was low compared to white muscle increments (Figure 10). White muscle ATP l eve l s decreased s l i g h t l y during the PSS-30 and PSS-7 while dec l in ing to low values (2.65 umol/g) after the ES (Table 1). Red muscle ATP content decreased to low l eve l s during the PSS-30 and remained low for both the PSS-30 and PSS-7 (Table 2) . Simultaneously, ADP content increased in both f iber types as ATP concentrat ions decreased (Tables 1 and 2) . Glycogen l eve l s were found to be low and to remain depressed in red muscle after the PSS-30 (Table 2). In contras t , white muscle glycogen l eve l s remained r e l a t i v e l y high u n t i l after the ES (Table 1). The corresponding muscle pH values refected these perturbat ions in metabolism (Figure 10). White muscle pH was maintained during the PSS-30 and dropped to values of 6.65+0.04 for the PSS-7 and 6.56*0.04 for the ES regimes (Figure 10). A l t e r n a t i v e l y , red muscle pH was maintained during both the PSS-30 and PSS-7 protocols demonstrating a s l i g h t decrease during ES (Figure 10). The metabolic end products accumulations of Cr , Pi and l ac ta te in white and red muscle are presented in Figure 11. Creatine accumulation was roughly e q u i v i l e n t to the amount of PCr hydrolyzed in both the red and white muscle (Tables 4 and 5) . Inorganic phosphate l eve l s increased r e l a t i v e l y p r o p o r t i o n a l l y to Cr content in red and white muscle during a l l exercise s tates in the former and after the PSS-30 in the l a t t e r (Figure 11). However, inorganic phosphate l eve l s increased to a greater extent than could be accounted for by ATP and PCr hydro ly s i s in white muscle after the PSS-7 71 and ES bouts (Tables 1 and 3). Lactate accumulations were far greater in white than red muscle under a l l exercise conditions (Figure 11). No differences existed between the pre-exercise and any exercise state for tissue water content which averaged 78 percent (Table 3). Plasma, t i s s u e , e x t r a c e l l u l a r and i n t r a c e l l u l a r ion contents are reported in Table 13. Only K* and lactate were observed to change within the plasma and e x t r a c e l l u l a r compartments. Na"*, K"* and lactate were found to increase while C l " and P C r 2 - decreased in the white muscle i n t r a c e l l u l a r compartment. Na*, K*, Ca**, C l ~ and lactate content increased while P C r 2 - content decreased in the red muscle i n t r a c e l l u l a r compartment. Measured and hypothetical white and red muscle i n t r a c e l l u l a r and e x t r a c e l l u l a r ion differences are,reported in Table 14. Hypothetical ion difference, were calculated assuming only changes in lactate or lactate and P C r 2 - with a l l other ions remaining at t h e i r pre-exercsie l e v e l s . Pre-exercise values were found to be 51.1, 85.5 and 11.2 for white muscle, red muscle and the e x t r a c e l l u l a r compartments, resp e c t i v e l y . The la c t a t e accumulations and P C r 2 - hydrolysis resulted in large perturbations in charge within a l l three compartments. Accumulations and/or s h i f t s of Na*, K*, Ca**, Mg** and C l - f a i l e d to compensate for these metabolicai1y induced perturbations in charge. In f a c t , a l t e r a t i o n s in these ions resulted in the i n t r a c e l l u l a r compartment becoming more p o s i t i v e in both tissues and less p o s i t i v e or even negative in the e x t r a c e l l u l a r compartment. Despite these large changes in charge and ionic composition, the calculated membrane potenti a l s , though more negative, remained r e l a t i v e l y constant (Table 15). Table 13. Rainbow trout ion concentrat ions . Ion Compartment Muscle Pre-ex Exercise State PSS-30 PSS-7 ES Na* Ca*" Mg** P E T I T I P E T I T I P E T I T I P E T I T I 135.1+3.4 141.1+3.7 29.2+2.2 12.3+0.9 34.1+2.1 17.2+1.1 1.78+0.17 1.85+0.17 149.413.9 149.1+3.9 138.9+9.0 138.619.0 4.57+0.03 4.77+0.03 5.6210.74 5.0410.66 4.2110.45 3.6810.39 1.3410.03 1.4110.03 38.711.7 38.611.7 20.410.4 20.3+0.4 134.1+2.7 140.112.7 36.511.8 19.711.0 40.112.8 23.311.6 2.4910.27 2.5910.27 171.8+7.4 171.517.4 167.117.4 166.817.1 4.7410.10 4.9410.10 5.15+0.67 4.5610.59 3.3310.15 2.7310.12 1.3810.03 1.4410.03 35.311.5 35.211.5 17.910.6 17.810.6 132.712.7 138.412.7 30.012.4 13.311.1 49.111.0 32.410.7 3.1910.27 3.3310.27 159.016.8 158.716.8 147.215.8 146.915.8 4.9110.13 5.1110.13 5. 1810.41 4.5610.36 4.3810.47 3.7710.40 1.3810.03 1.44+0.03 38.310.9 3B.210.9 20.510.5 20.410.5 134.112.7 140.112.7 35.610.5 18.910.3 51.412.2 34.611.5 3.1910.27 3.3310.27 187.216.4 186.916.4 169.2111.5 159.9111.5 4.9110.13 5.1110.13 4.6210.33 4.0010.29 6.5610.94 5.9510.85 1.3810.03 1.4410.03 37.110.4 37.010.4 20.910.4 20.8+0.4 73 C l " P E W T I R T I La- P E W T I R T I P C r 2 " P E W T I R T I 127.7+4.0 133.3+4.0 25.9+4.1 13.1+2.1 37.213.6 1 8 . B H . B 1.4+0.4 1.5+0.4 3.9+0.5 3.7+0.5 6.7+1.0 6.5+1.0 97.0+4.2 97.0+4.2 47.8+2.0 47.8+2.0 131.0+3.4 137.1+3.4 23.4+4.1 10.2+1.8 32.1+0.8 14.0+0.4 2.8+0.5 2.9+0.5 1 2 . 9 ± 1 . 4 12.6+1.4 11.3+1.4 10.9+1.4 B6.6+2.4 86.612.4 2.011.0 2.011.0 135.1+2.7 141.012.7 19.411.5 7.5+0.6 33.8+5.6 13.1+2.2 4.110.6 4.310.6 42.310.8 41.810.8 9.4+1.2 8.8+1.1 53.4+1.8 53.411.8 3.010.6 3.010.6 135.112.7 141.0+2.7 21.311.5 8.210.6 44.6+6.7 17.2+2.6 7.810.7 8.110.7 55.013.8 54.013.7 13.8+1.8 12.811.7 4.6+1.6 4.611.6 1.010.6 1.010.6 Values are mean+SE expressed in mEq/1. PSS-30 plasma values were assumed to be equal to the mean Df the pre-ex and PSS-7 values . The ES plasma values were assumed to be equ iv i l en t to PSS-7 values with the exception of l ac ta te which i s taken from a separate inves t i ga t ion (Dobson, Mommsen and Hochachka unpublished observat ions) . Plasma water, t i s sue water and e x t r a c e l l u l a r volume were assumed to be 95.8 percent (Mi l l i gan and Wood 1985), 78 percent (Table 3) and 9 percent ( M i l l i g a n and Wood 1985) under a l l cond i t ions . P, plasma; E , e x t r a c e l l u l a r ; T, t i s sue ; I, i n t r a c e l l u l a r ; W, white muscle; R, red muscle. Table 14. Measured and hypothet ica l rainbow trout white and red muscle i n t r a c e l l u l a r and e x t r a c e l l u l a r ion d i f ferences . Condit ion Control Hypothetical Measured Pre-ex (La") (La" + P C r 2 - ) White Muscle I n t r a c e l l u l a r PSS-30 51.1 42.2 52.6 85.1 PSS-7 51.1 13.B 56.6 72.6 ES 51.1 B.8 93.2 142.B Red Muscle I n t r a c e l l u l a r PSS-3B 85.5 81.1 126.9 165.6 PSS-7 85.5 83.2 128.B 157.3 ES 85.5 79.2 126.B 167.5 E x t r a c e l l u l a r PSS-3B 11.2 9.8 - 5.9 PSS-7 11.2 8.4 - -B.3 ES 11.2 4.6 - -2.4 IDe=(Na*)+(K*)+(Ca**/2)+(Mg**/2)-(Cl-)-(La-) IDw=<Na*)+(K*)+(Ca**/2)+(Mg**/38)-<Cl-)-(La-)-<PCr 2") IDr=(Na*) + (K*) + (Ca**/2) + (Mg* * /2B) - (C l - ) - (La- ) - (PCr 2*> Hypothet ical ion d i f ferences are ca lcu la ted assuming only changes in L a _ or L a - plus P C r 2 - from pre-exercise values . IDe, ion di f ference e x t r a c e l l u l a r ; IDw, ion d i f ference white muscle; IDr, ion d i f ference red muscie. Table IS. Calculated rainbow trout red and white muscle membrane p o t e n t i a l s . Muscle Pre-ex PSS-30 PSS-7 ES White -65 -71 -76 -76 Red -56 -64 -64 -59 Values are means expressed as m i l l i v o l t s . Values are ca lcu la ted from the Goldman Hodgekin Katz equationi Em=RT/Fxln PK (K*)o+PNa <Na*)o+PCl ( C l ' ) i / PK (K*)i+PNa (NaMi+PCl ( C l ' ) o where PK*, PNa* and P C l - represent the r e l a t i v e permeabi l i ty c o e f i c i e n t s . These values were taken from frog s a r tor ius muscle and are 0.5, 0.005 and 1 r e spec t ive ly (Hodgkin and Horowicz 1959). Mean K* , C l ~ and Na* contents were used for a l l c a l c u l a t i o n s . O o , concentrat ion outs ide ; ( ) i , concentrat ion i n s i d e . 76 F i g u r e 10. M e t a b o l i c a l t e r a t i o n s i n P C r 2 - , L a - , P i 2 -a n d pH i n r a i n b o w t r o u t m u s c l e . Whitt Muiclt R t l MMSCII 78 Figure 11. Metabolic end product accumulations of c rea t ine , inorganic phosphate and l ac ta te in rainbow trout muscle. 50 -40 •• 30 •• u m t l / i 20 •• 10 0 1 1 — 1 — 1 — 1 1 — 1 — 1 — 1 1 — 1 — 1 — 1 1 — 1 — 1 — 1 1 — 1 — 1 — 1 1 — 1 — • — 1 C r Ri L a C r Pi L a PSS-30 PSS-7 ES .A Whiti Muscle Ref Muscle PROTON SEQUESTERING MECHANISMS Crude Homogenate Buffer Capaci ty . Marl in red and white muscle buffer capaci ty was about 2 fo ld higher than the respect ive muscles of trout and within each species the buffer ing capacity of white muscle was twice that of red muscle (Table 16). In both species the buffer ing capacity of white muscle was found to be r e l a t i v e l y constant over the pH range 6.(3-7.5. Trout red muscle appeared to maintain i t s a b i l i t y to absorb protons up to pH 8.2, whereas marlin red muscle did not (Table 16). H i s t i d i n e Related Compounds. Comparable r e s u l t s were obtained for the pred ic ted , t i t r a t e d and ca lcu la ted h i s t i d i n e re la ted compound buffer capacity of marlin white muscle (Table 17). The ca lcu la ted h i s t i d i n e re la ted compound buffer capaci ty was equal to the d i f ference between the low molecular weight and phosphate plus other amino acid f r a c t i o n buffer c a p a c i t i e s . The predicted h i s t i d i n e re la ted compound buffer capacity was based on the t i s sue content and pK c h a r a c t e r i s t i c s . Since no d i f ferences were found between these methods, a predicted h i s t i d i n e re la ted compound buffer capacity was assessed for marlin red and trout red and white muscle (Table 18). Based on these f i n d i n g s , the h i s t i d i n e re la ted compounds contr ibute from as high as 65 percent for marlin white muscle over the pH range 7.0-8.0 (Table 19) to as low as 5 percent for trout red muscle over the pH range 7.0-8.0 (Table 20). The h i s t i d i n e re la ted compound buffer capaci ty of white and red muscle was about s i x f o l d higher in marlin than t r o u t , regardless of the pH range. This d i f ference i s r e f l ec ted in the r e l a t i v e t i s sue l eve l s of the h i s t i d i n e re la ted compounds (Table 17). A general trend of increased h i s t i d i n e re lated compound buffer ing in the pH range 6.5-7.5 was observed for a l l t i s sues (Table 18). 81 Prote in Buf fer ing . The buffer ing capci ty of the p e l l e t f r a c t i o n , assumed to cons i s t of m y o f i b r i l l a r p r o t e i n , was comparable in a l l t i s sues (Table 16) and remarkably s i m i l a r to the ca lcu la ted m y o f i b r i l l a r prote in buffer ing (Table 18). This ca lcu la ted m y o f i b r i l l a r prote in buffer ing was based on the d i f ference between the crude homogenate and supernatant f r a c t i o n buffer capac i t i e s (Table 16). The s i m i l a r i t y in values i s taken as evidence that the f r a c t i o n a t i o n procedure did not a l t e r the charge or pK c h a r a c t e r i s t i c s of the m y o f i b r i l l a r p r o t e i n . The comparable values found in a l l t i s sues can be a t t r ibuted to the s imi l a r prote in concentrat ions of the i r p e l l e t f r a c t i o n s (Table 22). When the buffer ing due to m y o f i b r i l l a r prote in was expressed r e l a t i v e to the crude homogenate buffer capac i ty , values ranged from a low of 8 percent over the pH range 7.0-8.0 for marlin white muscle (Table 19) to a high of 39 percent over the pH range 6.0-7.0 for trout red muscle (Table 20). This cont r ibu t ion was found to vary inverse ly with maximal buffer capacity of the muscle t i s sue . In a l l t i s s u e s , the buffer ing contr ibut ion a t t r i u t a b l e to m y o f i b r i l l a r prote in decreased as pH increased from 6.0-8.0 (Table IB). The buffer capacity of the high molecular weight f r a c t i o n , assumed to consis t of mainly soluble pro te ins , was 1.5 fo ld greater in both white and red muscle of marlin than in trout (Table 16). These values were very s i m i l a r to the ca lcu la ted soluble prote in buffer ing (Table IB) assessed as the d i f ference between the supernatant and low molecular weight f r a c t i o n buffer c a p a c i t i e s . Due to the s i m i l a r values , i t would appear that the f r a c t i o n i z a t i o n procedure did not a l t e r the charge and pK c h a r a c t e r i s t i c s of the so luble p r o t e i n . When expressed r e l a t i v e to to ta l t i s sue (crude homogenate) buffer capac i ty , the soluble prote in contr ibutes only 6 to 16 percent for a l l t i s sues (Tables 19 and 20). The buffer capacity of the 82 so luble prote in appears to r e f l e c t the d i f ferences found in the supernatant prote in content of the various f r ac t ions (Table 22). Phosphate Buf fer ing . Marl in white muscle phosphate buffer ing capci ty was assessed on the f r a c t i o n i so l a ted from the q u a n t i f i c a t i o n method B for h i s t i d i n e re la ted compounds. The contr ibut ion of other amino acids to t h i s buffer ing was considered to be n e g l i g i b l e . An estimate of the other t i s s u e ' s phosphate buffer ing capacity was ca lcu la ted as the d i f ference between the buffer capacity of the low molecular weight f r a c t i o n and the h i s t i d i n e re la ted compound plus taur ine buffer ing (Table 18). The buffer ing that could be assigned to phosphate showed higher values in the white than in red muscle for both species (Table 18). These d i f ferences seem to r e f l e c t those found in t i s sue phosphate content (Table 22). The a b i l i t y of the phosphate to buffer the a l k a l i load decreased with increas ing pH in a l l t i s sues (Table 18). The r e l a t i v e cont r ibut ion of phosphate to to ta l t i s sue buffer capacity ranged from 14 percent in marlin white muscle over the pH range 7.0-8.0 to 50 percent for trout white muscle in the pH range 6.0-7.0 (Tables 19 and 20). Taurine Buf fer ing . The red muscles of trout and marlin demonstrated high l eve l s of taur ine (Table 22). With increas ing pH, the buffer ing a b i l i t y of taur ine increased (Table 18) to l eve l s where i t could contr ibute 27 percent of trout red muscle to ta l t i s sue buffer capacity in the pH range 7.0-8.0 (Table 20). The r e l a t i v e cont r ibut ion of the various buffer ing const i tuents to to ta l t i s sue buffer ing i s i l l u s t r a t e d in Figure 12. Table 16. Buffer capacity of various f r ac t ions muscle of marlin and t rou t . separated from white and Frac t ion Muscle pH 6-7 Marl in 6.5-7.5 7-8 6-7 Trout 6.5-7.5 7-8 Crude Homogenate W 96.4 +8.5 97.3 +5.8 76.1 +4.4 63.3 ± 3 . 7 56.7 +2.2 43.4 + 1.7 R 56.8 +5.3 54.6 +2.9 43.4 + 1.7 30.1 +2.1 29.0 +2.2 27.3 13.0 P e l l e t W 12.0 + 1.9 8.9 + 1.3 6.5 + 1.0 14.9 +2.7 13.3 + 2.2 10.7 + 1.5 R 16.8 +2.2 14.3 + 1.7 12.1 + 1.9 11.8 +0.5 10.4 +0.5 8.5 +0.4 Supernatant W 84.4 +8.9 88.5 +6.3 69.6 +4.5 48.4 + 1.5 43.7 + 3.2 32.7 +0.9 R 40.0 +7.2 39.5 +3.6 30.8 +2.7 18.3 + 1.9 18.6 + 2.2 18.4 ± 2 . 7 High Molecular W Weight 11.1 + 1.8 9.9 + 1.6 7.4 + 1.2 7.0 + 1.0 5.5 + 1.0 4.2 +0.9 R 8.0 +0.7 6.2 +0.7 4.4 +0.7 4.2 +0.2 3.3 +0.2 2.3 ± 0 . 2 Low Molecular W Weight 74.1 +6.8 77.9 +5.3 59.9 +4.2 40.2 + 1.2 36.2 + 1.4 25.9 + 1.6 R 32.0 +2.7 31.2 +2.9 27.9 +4. 1 16.5 +2.0 15.9 t l . 9 15.3 ± 1 . 6 Values are means+SD expressed in umol NaOH/g/pH. W, white muscle; R, red muscle. B4 Table 17. Comparison of p red ic ted , t i t r a t e d and ca lcu la ted h i s t i d i n e re lated compound and phosphate buffer capac i t i e s of marlin white muscle. HRC pH 6-7 pH 6.5-7.5 pH 7-8 Predicted a 50.2*5.7 5 9 . 3 ± 6 . B 46.4*5.4 T i t r a t e d b 52.3+8.2 66.018.5 57.617.5 Calculated c 39.6+8.9 60.6+5.7 49.2+6.7 Phosphate plus 3 3 . 2 ± 9 . 5 21.4+8.9 11.0+4.5 Other Amino Acids Values are meanslSD expressed in umol NaOH/g/pH. HRC, h i s t i d i n e re l a ted compounds. a Based on pK c h a r a c t e r i s t i c s of anserine (pK=7.03), carnosine (pK=6.83) and h i s t i d i n e (pK=6.0) and the t i s sue HRC contents . b Representative marlin white muscle HRC concentrat ions were t i t r a t e d . c Low molecular weight f r a c t i o n - phosphate plus other amino acid f r a c t i o n 85 Table 18. Buffer capacity of h i s t i d i n e re la ted compounds, phosphate, m y o f i b r i l l a r p r o t e i n , soluble prote in and taurine in white and red muscle of marlin and t rou t . Compound Muscle PH 6-7 Marl in 6.5-7.5 7-8 6-7 Trout 6.5-7.5 7-8 HRC a 1*1 39.6 +8.9 68.6 +5.7 49.2 ± 6 . 7 7.9 ± 0 . 3 10.5 ± 0 . 4 7.5 ± 0 . 3 R 11.3 +3.3 12.3 + 3.8 9.4 +3.0 2.3 +0.6 2.0 +0.7 1.4 ± 0 . 6 Phosphate b W 34.5 ± 8 . 8 17.3 ± 6 . 2 10.7 ± 4 . 5 32.3 ± 1 . 4 25.7 ± 1 . 9 18.4 + 1.8 R 19.6 +4.4 16.9 ± 7 . 8 12.3 ± 8 . 8 12.9 ± 1 . 9 11.6 ± 1 . 9 6.5 ± 1 . 8 M y o f i b r i l l a r Protein W 12.13 ± 1 . 2 c 8.8 ± 0 . 9 6.5 ± 0 . 6 17.9 ± 2 . 1 13.0 ± 1 . 5 10.7 ± 1 . 2 R 16.8 ± 1 . 9 15.1 ± 1 . 7 12.6 ± 1 . 4 11.8 ± 1 . 8 10.4 ± 1 . 6 8.9 ± 1 . 4 Soluble Prote in W d 119.4 ± 1 . 2 10.6 ± 1 . 2 9.7 + 1.1 8.2 ± 0 . 5 7.5 ± 0 . 4 6.B ± 0 . 4 R 7.9 + 1.6 8.3 ± 1 . 7 2.9 +0.6 1.8 ± 0 . 4 2.7 ± 0 . 6 3.1 +0.7 Taurine e W - - - - - -R 1.1 + 0.2 2.0 ± 0 . 4 6.2 ± 1 . 3 1.3 ± 0 . 2 2.3 ± 0 . 3 7.4 ± 0 . 9 Values are means±SD expressed in umol NaOH/g/pH. HRC, h i s t i d i n e re la ted compounds, a) Marl in white muscle values are based on the d i f ference between low molecular weight (LMW) and phosphate plus taur ine buf fe r ing . Mar l in red and trout red and white values are based on the pK's and t i s sue l e v e l s of these compounds (Table 17). b) Phosphate buffering=LMW-HRC plus taur ine buf fer ing , c) M y o f i b r i l l a r prote in buffering=crude homogenate-supernatant buffer ing (Table 20). d) Soluble prote in b u f f e r i n g=5upernatant - L M W buf fer ing , e) Values are based on the r e l a t i v e t i s sue contents to t i t r a t e d taur ine buffer capac i ty . 86 Table 19. Percent r e l a t i v e contr ibut ions of various buffer ing const i tuents to to ta l t i s sue buffer ing in marlin and trout white muscle. Buffer ing Marl in Trout Constituent 6-7 6.5-7.5 7-8 6-7 6.5-7.5 7-8 Tissue 100 1019 100 100 100 100 M y o f i b r i l l a r Prote in 12 9 8 27 23 25 Soluble Protein 11 11 13 12 13 16 Phosphate 36 18 14 50 45 42 HRC 41 62 65 11 19 17 Taurine HRC, h i s t i d i n e re la ted compounds. The cont r ibu t ion of other amino acids was considered to be n e g l i g i b l e . Table 20. Percent r e l a t i v e contr ibut ions of the various buffer ing const i tuents to to ta l t i s sue buffer ing in marlin and trout red muscle. Buffering Consti tuent 6-7 Mar 1 in 6.5-7.5 7-8 6-7 Trout 6.5-7.5 7-8 Ti ssue 100 100 100 100 100 100 M y o f i b r i l l a r Prote in 30 28 29 39 36 33 Soluble Prote in 14 15 7 6 9 11 Phosphate 34 31 28 43 40 24 HRC 20 22 22 8 7 5 Taurine 2 4 14 4 8 27 HRC, h i s t i d i n e re la ted compounds. The cont r ibut ion of other amino acids was considered to be n e g l i g i b l e . Table 21. Concentrations of h i s t i d i n e re la ted compounds found in white and red muscle of trout and mar l in . Species Muscle His L-MeHis Car Ans Total Trout W 2.57 + + 17.16 ± 0 . 8 3 19.73 +0.68 R B.59 + 0. IB + + 3.B2 + 0.57 3.61 +0.72 Marl in W 15.86 +16.70 + 2.65 ± 2 . 4 1 104.80 ± 1 1 . 8 7 124.SB ± 1 3 . 9 4 R 4.79 .+0.48 + +' 21.13 +7.35 26.12 + 7.25 Values are means+SD expressed in umol/g (w/w). + re fers to trace amount Table 22. Concentrations of p r o t e i n , inorganic phosphate and taur ine found in the white and red muscle of marlin and t rou t . Compound F r a c t i on Marl in Trout W R W R Prote i n (mg/g) Crude Homogenate 125.4 + 10.6 108.3 ± 8 . 6 115.5 ± 1 2 . 2 99. 1 ± 8 . 3 P e l l e t 61.1 +5.9 54.0 +6. 1 54.0 +6.2 49.9 ± 7 . 6 Supernatant 69.6 +7.9 61.5 ± 1 2 . 5 58.4 ± 4 . 3 43.7 ± 9 . 4 Inorganic Phosphate (umol/g) Supernatant 62.4 ± 6 . 8 45. 1 + 2.4 64.4 + 4.3 34.5 ± 7 . 9 Taurine (umollq) Supernatant <3.0 44.7 ± 8 . 5 <6.0 53.4 ± 6 . 0 Values are mean±SD. 88 F i g u r e 12. P e r c e n t r e l a t i v e c o n t r i b u t i o n s o f t h e v a r i o u s b u f f e r i n g c o n s t i t u e n t s t o t o t a l t i s s u e b u f f e r i n g i n m a r l i n a n d t r o u t m u s c l e . 100 T 80 g 80 40 20 •• 7A / 1 3 7 \ 7A / / \ 7 i z \ i . Taurine HRC • Pi Soluble Protein Myofibrillar Protein Marlin White .Trout White Marlin Red Trout Red pH Range (6.0-7.0,8.5-7.5,7.0-8.0) Buffer Capacity 0Q 90 IN VIVO INTRAMUSCULAR BUFFERING Tissue l eve l s of the white muscle buffer ing const i tuents ATP, ADP, AMP, IMP and Pi are contained in Table 4, while the content of the g l y c o l y t i c intermediates are found in Table 7. Tissue contents of the h i s t i d i n e re lated compounds are reported in Table 21. An estimate of white muscle buffer ing was made by c a l c u l a t i n g the buffer ing due to a s soc ia t ion of protons with bases (Table 23) and the buffer ing due to enzymatic a c t i v i t y (Table 24) fo l lowing the burst exercise regime. Estimates of bicarbonate buffer ing are based on the low white muscle t i s sue contents (Heis ler 1978) while the prote in contr ibut ion to buffer ing (26.1 umol/g/pH) was assumed to be equ iv i l en t to that of the h i s t i d i n e re lated compound study (Table 18). The to ta l buffer ing of a l l the const i tuents analyzed due to proton as soc ia t ion with bases was 40.9 umol H*/g/pH (Table 23). The major contr ibutor to th i s buffer ing appears to be prote in which accounted for 64 percent of the to ta l physico-chemical buf fer ing . H i s t i d i n e re la ted compounds contr ibuted 6.7 umol HVg/pH (16 percent) of t h i s buffer ing while the inorganic phosphate was capable of absorbing 4 umol H*7g/pH (10 percent) . These three buffer ing const i tuents account for 90 percent of the buffer ing due to a s soc ia t ion of protons with bases. The other buffer ing cons t i tuent s : bicarbonate , ATP, IMP and the g l y c o l y t i c intermediates account for the remaining 10 percent of the physico-chemical buf fer ing . The to ta l buffer ing of a l l the const i tuents analyzed due to enzymatic a c t i v i t y was 25.3 umol HVg/pH (Table 24). The major contr ibutor to t h i s buffer ing was the inorganic phosphate released during phosphate hydro lys i s reac t ions . Release of Pi from the breakdown of PCr and ATP could account for 12.1 umol H"7g/pH, while the phosphate released from the other react ions could account for 10.4 umol H* /g /pH. Phosphate therefore accounted for 89 percent of the buffer ing due to production or consumption of acids or bases (enzymatic a c t i v i t y ) . Ammonia production would absorb 1.8 umol H*/g/pH such that phosphate and ammonia would account for 96 percent of the buffer ing due to enzymatic a c t i v i t y . The remaining buffer ing can be p r i n c i p a l l y a t t r ibuted to the accumulation of g l y c o l y t i c intermediates , in p a r t i c u l a r 66P. The t o t a l in vivo buffer capacity was estimated to be 66.2 umol H*7g/pH (Table 25). Physico-chemical buffer ing accounted for 62 percent of t h i s buffer ing p o t e n t i a l . The p r i n c i p a l buffers were assessed to be prote in (39 percent ) , h i s t i d i n e re la ted compounds (10 percent) and inorganic phosphate (40 percent) . The remaining buffer ing was a t t r ibuted to other physico-chemical and enzymatic buffer ing (6 and 4 percent, r e spec t ive ly ) (Figure 13). The t i t r a t e d in v i t r o buffer capacity was found to be 63.3+3.7 umol H*/g/pH (Table 16). The r e l a t i v e contr ibut ions of prote in (39 percent ) , h i s t i d i n e re la ted compounds (11 percent) and inorganic phosphate (50 percent) to t o t a l t i t r a t e d buffer capacity are presented in Figure 13. The in vivo ca lcu la ted ( lactate) buffer capacity was found to be 88.1 umol H+/g/pH (Table 25). A comparison of percent r e l a t i v e contr ibut ions of the various buffer ing const i tuents to t i t r a t e d , estimated and ca lcu la ted to ta l buffer capac i t i e s i s contained in Figure 13. 92 Table 23. Estimates of proton absorbing potent ia l of rainbow trout white muscle due to a s soc ia t ion of H+ ions with bases during the PSS-7. Buffering H+ Buffer Capacity Constituent (umol/g) (umol H+/g/pH) HCC-3 a -0.26 1.0 ATP -0.09 0.86 IMP -0.06 0.12 Pi b -1.32 3.96 NH 3 Ans c -2.35 6.19 His c -0.19 0.53 Prote in d -6.78 26.10 G1P -0.04 0.19 G6P -0.09 0.47 F6P -0.01 0.07 FDP -0.16 0.82 DHAP -0.03 0.10 3P6 -0.06 0.30 GP -0.08 0.25 Total -11.52 40.9 Only those buffer ing const i tuents contr ibut ing greater than 0.01 buffer ing uni t s (umol H+/g/pH) have been inc luded . a Calculated from estimated change in i n t r a c e l l u l a r bicarbonate during exercise (Heis ler 1978). A l l other values are ca lcu la ted base on the lowest content measured and the change in concentrat ions of H+ buffer ing const i tuent associated with the i r respect ive pK's (Table 2) . b Calculated from the Pi content after the PSS-30 and a pK of 6.81. c Calculated from the respect ive white muscle t i s sue contents (Table 21) and t h e i r respect ive pK's (Table 2) . d Assumed to be equ iv i l en t to the ca lcu la ted soluble plus m y o f i b r i l l a r prote in buffer ing (Table 18). Table 24. Estimates of proton absorbing potent ia l of rainbow trout white muscle due to enzymatic a c t i v i t y during the PSS-7. Buffering Metabolite H+ Buffer Constituent Content Capacity (umol/g) (umol/g) (umol H+/g/pH) PCr a 13.0 -7.68 11.55 ATP 0.75 -0.03 0.50 IMP 1.25 -0.36 0.22 Pi b 7.05 -4. 16 6.26 HHA 1.8 -1.8 1.80 GiP 0.25 . -0.06 0.15 G6P 0.74 -0.17 0.45 F6P 0. 12 -0.03 0.07 FDP -0.21 0.05 -0.13 DHAP 0.04 -0.02 0.03 3PG 0.37 -0.10 -0.24 GP 0.29 -0.14 0.24 Total 25.51 -12.69 21.1 Only those buffer ing const i tuents contr ibut ing greater than 0.01 buffer ing uni t s (umol H"7g/pH) have been inc luded . a Calculated from the change in PCr content and a pK of 6.81 for P i . b Calculated from the change in Pi not a t t r i b u t a b l e to PCr and ATP h y d r o l y s i s . A l l other values are based on t h e i r change in concentrat ion and t h e i r respect ive pK c h a r a c t e r i s t i c (Table 2) . 94 Table 25. Comparison of t i t r a t e d , ca lcu la ted ( lactate) and estimated (pK c h a r a c t e r i s t i c s ) buffer capac i t i e s of trout white muscle over the pH range 7.0 to 6.0. Buffer Capacity T i t r a t e d Calculated Estimated (umol H*/g/pH) 63.3*3.7 a 88.1 b 62.0 c a Value i s taken from the trout white muscle crude homogenate buffer capacity (Table 16). b Value i s ca l cu la ted assuming a 1:1 coupling of H* ion production to l ac ta te accumulation. B= H"7dpH c Value i s estimated as the buffer ing due to proton as soc ia t ion with bases plus the buffer ing due to enzymatic a c t i v i t y . 95 F i g u r e 13. C o m p a r i s o n of t h e r e l a t i v e c o n t r i b u t i o n s o f t h e v a r i o u s b u f f e r i n g c o n s t i t u e n t s t o t h e t i t r a t e d , c a l c u l a t e d and e s t i m a t e d b u f f e r c a p a c i t i e s of t r o u t w h i t e m u s c l e o v e r t h e pH r a n g e 7.0 t o 6.0. 100 t 80 •• J * 60 C 40 20 •• 2 nm • •. • • • • • • • • * 2 Enzyme •J Physico-chemical H R C E3 P i • Soluble Protein S Myofibrillar Protein T i t r a t e d Est imated C a l c u l a t e d Buffer Capacity 97 DISCUSSION T h e s e - f i n d i n g s d e m o n s t r a t e d t h e i n t e g r a t i v e n a t u r e of f u e l s e l e c t i o n i n s k e l e t a l m u s c l e s of r a i n b o w t r o u t d u r i n g e x e r c i s e . The r e g u l a t i o n of t h i s i n t e g r a t i v e f u e l s e l e c t i o n , a p p e a r e d t o r e v o l v e a r o u n d a d e n i n e n u c l e o t i d e m e t a b o l i s m w i t h c l a s s i c a l g l y c o l y t i c c o n t r o l . F a t i g u e was a s s o c i a t e d w i t h s u b s t r a t e and end p r o d u c t l i m i t a t i o n s i n e n e r g y t u r n o v e r s , t o s u s t a i n t h e g i v e n work i n t e n s i t y . However, a s u b s t a n t i a l p r o t o n s e q u e s t e r i n g c a p a c i t y w i t h i n f i s h m u s c l e , due p r e d o m i n a n t l y t o i n o r g a n i c p h o s p h a t e , p r o t e i n and h i s t i d i n e r e l a t e d compounds, a l l o w e d h i g h a c c u m u l a t i o n s o f l a c t a t e w i t h c o m p a r a t i v e l y m i n o r p e r t u r b a t i o n s t o i n t r a m u s c u l a r pH. FUEL S E L E C T I O N P h o s p h a g e n and A d e n y l a t e M e t a b o l i s m PCr h a s an i m p o r t a n t r o l e i n e n e r g y m e t a b o l i s m as t h i s m e t a b o l i t e i s i n v o l v e d i n t h e g e n e r a t i o n o f ATP v i a t h e r e a c t i o n c a t a l y z e d by c r e a t i n e k i n a s e (CK) and ATP t h u s f o r m e d m a i n t a i n s ATP l e v e l s d u r i n g p e r i o d s of h i g h e n e r g y t u r n o v e r ( H o c h a c h k a 1 9 8 5 ) . To a c h i e v e t h i s f u n c t i o n , i t s P g r o u p must be t r a n s f e r a b l e t o ADP a t h i g h r a t e s and a t a p p r o p r i a t e t i m e s as d i c t a t e d by m y o s i n A T P a s e a c t i v a t i o n due t o t h e i r f u n c t i o n a l c o u p l i n g . T h i s i s a c c o m p l i s h e d by h a v i n g v e r y h i g h a c t i v i t i e s of t h e c y t o s o l i c (MM) i s o z y m e of CK w i t h i t s a p p r o p r i a t e k i n e t i c p r o p e r t i e s (Dawson e t a l . 1978; B a d i a n e t a l . 1981; H o c h a c h k a e t a l . 1 9 8 3 ) . In t h i s r e g a r d , t h e Kd v a l u e s f o r PCr a r e 72 and 32 mM f o r t h e b i n a r y and t e r n a r y c o m p l e x e s r e s p e c t i v e l y , w h i l e f o r ADP t h e y a r e 0.2 and 0.06 mM r e s p e c t i v e l y ( J a c o b s and Kuby 1 9 8 0 ) . CK has been i d e n t i f i e d as t h e most a b u n d a n t s a r c o p l a s m i c p r o t e i n f o u n d i n f i s h m u s c l e (Gosselin-Rey et a l . 1968) with the MM isozyme being found in both red and white muscle of carp (Watts 1973). This enzyme i s l a rge ly c o n t r o l l e d by the concentrat ion of adenylates and pH (Watts 1973). However, increas ing concentrat ions of the g l y c o l y t i c intermediates G6P, F6P, PEP, pyruvate and l ac ta te have a l l been shown to i n h i b i t f i sh muscle CK a c t i v i t y (Taame et a l . 1979). Based on the k i n e t i c s , PCr content under most condit ions would not be sa turat ing and the enzyme i s therefore maximally responsive to changes in PCr content (Jacobs and Kuby 1980). A l t e r n a t i v e l y , recent evidence suggested that the ADP (free ADP; ADP*) ava i l ab le to p a r t i c i p a t e in t h i s and other react ions occurs in much lower concentrations than i s measured enzymat ica l ly . Estimates of re s t ing free ADP general ly f a l l in the range from 1 ( less than 0.01 umol/g; Jacobus et a l . 1982; Shoubridge et a l . 19B4) to 10 (approximately 0.07 umol/g) percent of measured ADP concentrat ions with the majority of inves t iga tor s c a l c u l a t i n g free ADP to be at the l a t t e r end of t h i s range (Dawson et a l . 1977; Wilk ie 1981; Meyer et a l . 1985). In the present i n v e s t i g a t i o n , free ADP was ca lcu la ted for both the measured and compensated metabolite data r e s u l t i n g in values ranging from 7 to 70 uM for pre-exercise red and white muscles (Table 9) . Remarkably s imi la r values of free ADP were demonstrated between f iber types when ca lcula ted with e i ther the measured or compensated metabolite data. Free ADP contents were found to increase in both f iber types with exerc i se , these increases being minimal u n t i l PCr contents were low as would be expected due to the high CK and AK a c t i v i t i e s found in f i s h muscle (Noda et a l . 1975; Johnston 1982a). Therefore the evidence from t h i s study suggests that free ADP would not be sa turat ing to the CK react ion allowing responsiveness to the free ADP increases which accompany exerc i se . As w e l l , the high a f f i n i t y of CK for ADP would make i t very competit ive for ADP such that during the ear ly stages of work the CK react ion would be driven in the d i r e c t i o n of ATP formation (Gadian et a l . 1981). Thus both enzyme content and enzyme k i n e t i c propert ies favour high rates of ~ P transfer to ADP. However, since PCr l eve l s are not sa turat ing and since they diminish during high energy turnover , these rates must r a p i d l y decl ine (Hochachka 1985). The di f ferences between red and white muscle in the i r a b i l i t y to maintain ATP l eve l s (Tables 4 and 5) may be due to the d i f ferences in PCr l eve l s (Shoubridge et a l . 1984; Meyer et a l . 19B5) , the d i f ferences in enzyme a c t i v i t i e s (Hochachka et a l . 1983) and to the large equ i l ib r ium constant for ATP formation of the CK react ion (Dawson et a l . 1978; Gadian et a l . 1981). This allows PCr to be almost completely converted to ATP pr ior to any dec l ine in ATP concentrat ions . In red muscle, very low l e v e l s of PCr remained after the PSS-30 K I umol/g) and the ATP content was decreased. In contras t , white muscle at the end of the PSS-7 p r o t o c o l , s t i l l contained modest PCr contents and therefore a r e l a t i v e l y high ATP content. The ES resul ted in both red and white muscle PCr deplet ion to very low values with the resul tant ATP dec l ine s . Enzymatic determination of PCr content in freeze clamped t i s sues has resul ted in much lower concentrat ions than are determined by nuclear magnetic resonance (NMR) (Busby et a l . 1978; Kushmerick and Meyer 1985; Meyer et a l . 1985). It has been found that f reezing per se r e s u l t s in PCr hydro ly s i s but not ATP hydro lys i s (Meyer et a l . 1985). The NMR inves t iga t ions have found PCr content to be 85 (Meyer et a l . 1982; Shoubridge et a l . 1984; Meyer et a l . 1985) and 68 (Meyer et a l . 1982 s Meyer et a l . 1985) percent of to ta l PCr and Cr at rest in white and red muscle r e s p e c t i v e l y . These values are in agreement with the human values of 75-7B percent for t h i s heterogeneous muscle (Dawson et a l . 1978; Chance et a l . 1981). These NMR inves t i ga t ions use these facts to assess the true re s t ing PCr content by measuring the ATP and PCr plus Cr contents in freeze clamped t i s sues and r e l a t i n g t h i s information to the i r NMR spectras . In the present i n v e s t i g a t i o n , white and red muscle PCr contents were assessed based on these percentages (Tables 4 and 5) and found to be greater than previous ly recorded mammalian (Chance et a l . 1981; Shoubridge et a l . 1984; Kushmerick and Meyer 1985; Meyer et a l . 1985) and f i s h (Dreidzic et a l . 1981) values due to the i r greater t o t a l Cr plus PCr pool . PCr contr ibuted only a small percentage of the metabolic energy turnovers required for these exercise i n t e n s i t i e s in both red and white muscle (Table 12). It i s therefore apparent that the ro le of PCr during t h i s type of exercise i s one of ATP buf fer ing . The compensated PCr/Pi r a t i o s observed in t h i s study (Table 10) are s i m i l a r to previous ly reported res t ing values (Chance et a l . 1981; Dawson et a l . 1977; Meyer et a l . 1985) and demonstrate s imi l a r r e l a t i o n s h i p s with increas ing workload to the e l e c t r i c a l l y st imulated gastrocnemius muscle of rats (Shoubridge et a l . 1984). Chance et a l . (1981) observed a l inear r e l a t i o n s h i p between the PCr/Pi r a t i o and workload down to a r a t i o of approximately 1. They suggested that mitochondrial r e s p i r a t i o n was responsive to e i ther these changes or the increas ing Pi content. However, deplet ion of PCr resul ted in lower r a t i o s than have been reported previous ly due to the high i n i t i a l PCr l e v e l s and the exhaustive exercise regime performed (Table 10). The l inear r e l a t i o n s h i p observed by Chance et a l . (1981) no longer ex i s t s at these lower r a t i o s demonstrating an uncoupling of m y o f i b r i l l a r ATPase a c t i v i t y to mitochondrial r e s p i r a t i o n . The decreases in white muscle ATP content noted in t h i s study (Table 4) are of s i m i l a r magnitude to values found in previous studies on exerc i s ing •fish (Dreidzic and Hochachka 1976). No previous s tudies of f i s h exerc i s ing have reported ATP decrements in red muscle (Table 5) but, hypoxia has been found to e l i c i t t h i s response fol lowing one hour of decreased oxygen tension (20 Torr) (Dunn 1985). Accompaning these decreases in ATP are increases in ADP and AMP, although t h e i r e levat ions are i n s u f f i c i e n t to maintain the adenylate pool (Figure 2). Increasing ADP concentrations ac t iva te the adenylate kinase (AK) react ion by mass act ion such that the ADP increases are low (Dreidzic and Hochachka 1978). Therefore, AK functions to minimize a l t e r a t i o n s in the r a t i o ADP x P i / A T P , thus maintaining a high free energy of ATP hydro lys i s (Lowenstein 1972; Dre idz ic and Hochachka 1978). However, although free ADP changes are minimized, concurrent a l t e r a t ions in the contents of ATP, PCr and Pi resul ted in large changes in phosphorylation potent i a l (ATP/ADP,.Pi) (Table 9) and a subsequent decl ine in the free energy ava i l ab le from ATP h y d r o l y s i s . Purine Nucleotide Cycle At these workloads, which exceeded the t i s sues aerobic c a p a b i l i t i e s , GTP l e v e l s were found to d e c l i n e . The GTP i n h i b i t i o n of 5'-AMP deaminase i s therefore removed while increas ing ADP a c t i v a t i o n i s occurr ing . The concerted act ion of AK and 5'-AMP deaminase in response to the removal of BTP i n h i b i t i o n and ADP a c t i v a t i o n account for the decrease in the adenylate pool and the e levat ions of IMP and ammonia (Tables 4 and 5). The 5'-AMP deaminase react ion i s one step in the react ion span termed the purine nucleot ide cycle (Lowenstein 1972). According to th i s c y c l e , IMP further reacts with GTP and aspartate to form adenylsuccinate which in turn i s converted to AMP and fumarate by the act ions of adenylsuccinate synthetase and adenylsuccinase (Lowenstein 1972). It has been shown that t h i s cycle functions in concert with g l y c o l y s i s in mammalian white but not red muscle (Meyer and Terjung 1979; Meyer et a l . 1980). Furthermore, ac idos i s has been found to ac t ivate AMP deaminase in white muscle (Dudley and Terjung 1985). However, Dr iedz ic and Hochachka (1978) suggested that in te leos t white muscle the cyc le acts as two separate arms with; (1) IMP accumulation during a c t i v i t y and (2) replenishment of the adenylate pool during recovery. On the other hand, the present inves t i ga t ion demonstrated decrements in the adenylate pool (Figure 2) as IMP accumulated while simultaneously AMP and fumarate were found to increase (Tables 4 and 5) . As w e l l , IMP continued to accumulate in red muscle during the PSS-7 while PCr, ATP, AMP and fumarate l eve l s a l l increased (fumarate demonstrating i t s largest increase) (Table 5) . It thus appears that 5'-AMP deaminase a c t i v i t y predominates over adenylsuccinate synthetase a c t i v i t y during exercise r e s u l t i n g in an accumulation of IMP. During the PSS-7, red muscle GTP content returned to pre-exercise values and would i n h i b i t 5'-AMP deaminase serving to ac t iva te adenylsuccinate synthetase. Thus i t would appear that the purine nucleot ide cycle i s operative in f i s h red and white muscle but the production of IMP and i t s reconversion to AMP occur at d i f f e rent rates during exercise and recovery. This d i f ference between mammalian and trout red muscle in the funct ioning of the purine nucleot ide c y c l e , may be re la ted to the higher c o n t r a c t i l e (Johnston 1982b) and g l y c o l y t i c (Johnston 1977) capac i t i e s of f i s h muscle. Although many ro le s for the purine nucleot ide cyc le have been postulated (Aragon and Lowenstein 1980), i t would appear that i t s primary function i s conservation of the adenine nucleot ide pool (Figure 2) . These f indings in regard to the purine nucleot ide c y c l e , AK and CK stress two important functions of adenylate metabolism during exerc i se . F i r s t , a mechanism ex i s t s which attempts to prevent a reduction in the free energy of 103 ATP hydro ly s i s by contro l ing ADP increases when ATP i s reduced and secondly, the importance of ADP in the control of energy metabolism becomes apparent. Phospagen Replenishment Although t h i s f ind ing i s tenuous, i t was i n t e r e s t i n g to f ind an i n i t i a t i o n of phosphagen replenishment within red muscle during the PSS-7 (Table 5) as electromyograph (EMS) recordings suggest that t h i s muscle i s s t i l l a c t i v e l y involved in force production (Hudson 1973} Bone 1978). To achieve t h i s , the energy turnover requirements would have to be met by an increased white muscle energy turnover cont r ibut ion which would allow a small percentage of the red muscle energy provi s ion to be used for substrate replenishment. This appears to have occurred in the present i n v e s t i g a t i o n , although the metabolic costs are c lo se ly matched to the metabolic requirements such that the phosphagen replenishment i s minor (Table 12). It i s i n t e r e s t i n g to note that during the PSS-7, red muscle content of ADP decreased and GTP increased (Table 5) which could e f f e c t i v e l y i n h i b i t AK and S'-AMP deaminase. As w e l l , c i t r a t e l eve l s were increased, p o t e n t i a l l y act ing to i n h i b i t PFK along with decreasing ADP. Carbon flux through anaerobic g l y c o l y s i s appeared to be reduced as evidenced by the lower lac ta te l e v e l s . It i s therefore postulated that a small percentage of the red muscle energy turnover i s used to rep leni sh PCr and ATP while the increased white muscle energy prov i s ion suppl ies the necessary energy for the elevated workload. These red muscle ATP costs appear to be met c h i e f l y by l i v e r derived glucose and/or f a t ty acids s ince a l l other precursors were s u b s t a n t i a l l y depleted (Table 12). P r i o r i t y of replenishment appears to have been given to ATP and PCr as glycogen l eve l s remained low (Figure 3). 104 G l y c o l y t i c Energy Provi s ion G l y c o l y t i c energy provi s ion can be achieved e i ther anaerobicai1y or a e r o b i c a l l y with the concominant enhancement in ATP y i e l d . Most of the g l y c o l y t i c enzymes have been found in red and white muscles of trout (Johnson 1977, Walton and Cowey 1982) and both t i s sues have demonstrated the capacity to u t i l i z e glycogen, although at varying rates (Johnston and Goldspink 1973; Johnston and Moon 1980a,b). Red muscle glycogen decreased to very low values after a l l three exercise protocols (<1 umol/g) and could not be accounted for by l ac ta te production (Table 5) . Previous inves t iga tor s haVe shown increased blood concentrat ions of l a c t a t e , presumably from white muscle and to a lesser extent red muscle (Johnston and Moon 1980a,b), but these l eve l s (even assuming a l l blood lac ta te came from red muscle) plus t i s sue contents , s t i l l could not account for the amount of glycogen broken down in t h i s t i s s u e . Glycogen was found to contr ibute 14 percent of the energy turnover generated by red muscle (Table 12). Glycogen breakdown in white muscle occurred at a l l exercise i n t e n s i t i e s to values below 1 umol/g after the ES. The white muscle l ac ta te accumulation could account for a l l the glycogen breakdown during the PSS-7 and ES, but not during the PSS-30 (Table 5). Since the release of l ac ta te from white muscle i s slow (Holeton et a l . 1983; Turner et a l . 1983; M i l l i g a n and Wood 1985), th i s descrepancy in l ac ta te accumulation to glycogen breakdown i s taken as evidence that white muscle can oxid ize glycogen. Two p o s s i b i l i t i e s exis t to explain t h i s descrepancy; (1) l ac ta te oxidat ion within white muscle (glycogen oxidat ion i n d i r e c t l y ) and/or (2) glycogen oxidat ion d i r e c t l y . The question as to whether t h i s glycogen oxidat ion occurs d i r e c t l y and/or i n d i r e c t l y v ia l ac ta te oxidat ion i s uncer ta in . Evidence for the white muscle capacity to oxid ize fuels comes from i so la ted mitochondrial s tud ie s , perfused trout hind parts and i so l a ted muscle s l i c e s . In these 105 i n v e s t i g a t i o n s , w h i t e m u s c l e m i t o c h o n d r i a o x i d i z e d p y r u v a t e a t a r a t e r o u g h l y e q u i v a l e n t t o h e a r t m i t o c h o n d r i a ( D o n a l d s o n 1985) and t h e p e r f u s e d h i n d p a r t s o x i d i z e d g l u c o s e a t a r a t e of 80 n m o l / 1 0 0 g / m i n (Moen and K l u n g s o y r 1 9 8 1 ) . L a c t a t e h a s a l s o been shown t o be o x i d i z e d i n i s o l a t e d w h i t e m u s c l e s l i c e s a t a r a t e o f 1 / 1 0 t h t o 1/5th t h a t o f r e d m u s c l e ( B i l i n s k i and J o n a s 1 9 7 2 ) . W h a t e v e r t h e m e c h a n i s m , t h i s a e r o b i c c o m b u s t i o n of g l y c o g e n a p p e a r s t o c o n t r i b u t e a p p r o x i m a t e l y 7 p e r c e n t of t h e w h i t e m u s c l e and t o t a l e n e r g y t u r n o v e r s d u r i n g t h e PSS-30 ( T a b l e 1 2 ) . R e g u l a t i o n of G l y c o l y s i s The f u n c t i o n a l c o u p l i n g of m y o f i b r i l l a r A T P a s e t o t h e e n e r g y p r o d u c i n g p a t h w a y s s h o u l d be e m p h a s i z e d b e f o r e i n i t i a t i n g any d i s c u s s i o n of t h e r e g u l a t i o n of g l y c o l y s i s . In t h i s r e g a r d , ATP t h e s u b s t r a t e of t h e f o r m e r i s t h e p r o d u c t of t h e l a t t e r and d u r i n g m u s c u l a r a c t i v i t y t h e r a t e of ATP r e q u i r e d by t h e m y o f i b r i l l a r A T P a s e s , d i c t a t e s w h i c h e n e r g y p r o d u c i n g p a t h w a y s and t o what e x t e n t e a c h i s a c t i v a t e d . E v i d e n c e f o r t h i s comes f r o m r e c o n s t i t u t e d g l y c o l y t i c s y s t e m s where g l y c o l y t i c f l u x c a n be i n c r e a s e d by s i m p l y a d d i n g A T P a s e s (Wu and D a v i s 1 9 8 1 ) . The m y o f i b r i l l a r A T P a s e a c t i v i t y of t r o u t w h i t e m u s c l e i s a p p r o x i m a t e l y 3 f o l d h i g h e r t h a n r e d , w i t h t h e m y o f i b r i l l a r p r o t e i n c o n s t i t u t i n g 2 t i m e s t h e c e l l v o lume i n w h i t e as o p p o s e d t o r e d m u s c l e ( J o h n s t o n and Moon 1 9 B 0 b ) . T h e r e f o r e t h e p o t e n t i a l r a t e of ATP t u r n o v e r due t o m y o f i b r i l l a r A T P a s e a c t i v i t y w o uld be 6 f o l d h i g h e r i n t r o u t w h i t e m u s c l e and i s r e f l e c t e d i n t h e h i g h e r g l y c o l y t i c enzyme a c t i v i t i e s ( J o h n s t o n 1 9 7 7 ) . The r e g u l a t i o n of g l y c o l y s i s was e x a m i n e d i n b o t h t i s s u e s by t r a d i t i o n a l t e c h n i q u e s as o u t l i n e d by R o l l e s t o n (1972) u s i n g a n a l y s i s of enzyme d i s e q u i l i b r i u m and app l i ca t ions of the crossover theorem. The crossover theorem i d e n t i f i e s regulatory s i t e s in complex enzyme systems and was f i r s t used by Chance and Will iams (1955) to i d e n t i f y crossover points for the i n t e r a c t i o n of ADP with the re sp i ra tory chain of coupled mitochondria . An enzyme w i l l be i d e n t i f i e d as regulatory i f ; (1) i t i s nonequi1ibrium, (2) i t s slope changes in the same d i r e c t i o n as f l u x , (3) i t demonstrates a crossover and (4) i t s substrate concentrat ion changes in the opposite d i r e c t i o n to flux (Williamson 1969).. However, s t r i c t adherence to these rules in multimodulated enzyme systems can lead to errors in the i d e n t i f i c a t i o n of regulatory s i t e s due to the high complexity of pathway r e g u l a t i o n . The i n t e r a c t i o n of various modulators at the d i f f e rent regulatory s i t e s can mask the apparent crossover l o g i c . This has led to the development of the Fault Theorem which assesses the a l l o s t e r i c modulators inf luence on the react ion as well (Rolleston 1972). Phos, HK, PFK and PK have been i d e n t i f i e d by in v i t r o techniques as regulatory enzymes when e i ther glycogen or glucose acts as the substrate for g l y c o l y s i s (for reviews see: Bloxham and Lardy 1973; Sols 1979, 19B1; Claus et a l . 1984). S i m i l a r i l y , numerous inves t i ga t ions have i d e n t i f i e d these enzymes as regulatory in g l y c o l y s i s in red blood c e l l s , tumor a s c i t i e s c e l l s , heart , red and white muscle. In the present i n v e s t i g a t i o n , HK, PFK and PK have been i d e n t i f i e d as potent ia l regulatory enzymes due to the i r degree of d i s e q u i l i b r i u m (Tables 4-7). S i m i l a r i l y , phos may act as a regulatory enzyme due to i t s assumed nonequi1ibrium status (Krebs 1981). However, i t was s u r p r i s i n g to f ind the 6PDH.P6K complex exh ib i t ing nonequi1ibrium k i n e t i c s in both t i s sues with increas ing exercise i n t e n s i t y (Tables 4-7). Since th i s f ind ing contrasts that found in the r a t , where the GPDH.PGK complex within the gastrocnemius, soleus and p l a n t a r i s maintains equ i l ibr ium with increas ing exercise i n t e n s i t y (Dobson et a l . 1986), i t i s apparent that they may cons t i tu te a further regulatory complex within f i s h muscle. The i d e n t i f i c a t i o n of control by the two enzymes (phos and HK) i n i t i a t i n g entry of substrate into the g l y c o l y t i c pathway i s complicated by the merging of t h e i r carbons at G6P. Increasing G6P concentrat ions i n h i b i t both HK and phos a c t i v i t i e s while serving to ac t ivate PFK (Sols 1981). Since G6P contro l s i t s own rate of formation from HK, G6P contents are thought to always change in the opposite d i r e c t i o n to the rate of i t s formation from HK and therefore demonstrate no crossover point for t h i s react ion (Rolleston 1972). When f i s h muscle m y o f i b r i l l a r ATPases are act ivated at rates which require g l y c o l y t i c energy p r o v i s i o n , muscle glycogen i s the preferred fuel due to the much higher enzyme a c t i v i t i e s of phos than HK (Johnston 1977). Providing muscle glycogen i s not l i m i t i n g , the ac t iva t ion of phos by C a * * , cAMP and epinephrine, which have been shown to accompany exercise in f i sh (Nakano and Tomlinson 1967), ensures that phos i s act ivated at an appropriate rate and G6P accumulates and i n h i b i t s HK. Therefore, phos demonstrates regulatory propert ies provided muscle glycogen l eve l s are not l i m i t i n g as was demonstrated for trout white muscle (Figure 8) . When muscle glycogen l eve l s are l i m i t i n g , HK may demonstrate regulatory propert ies as was observed for the trout red muscle (Figure 9) . The observed slope which opposes f l u x , would tend to ind ica te i n h i b i t i o n at the HK r e a c t i o n . However, the large d i f ference in maximal enzyme a c t i v i t y between HK and PFK (Johnston 1977) would allow a new lower steady state of G6P to exist by matching the carbon flux through the two reac t ions . PFK has repeatedly been shown to be regulatory for g l y c o l y s i s (for reviews see: Bloxham and Lardy 1973; Ramaiah 1976; Goldhammer 1979; Sols IBB 1979, 1981; Claus et a l . 1984) demonstrating in v i t r o multimodulation by substrates , products and co-factors. However, no apparent regula t ion was demonstrated at PFK -for e i ther t i s sue in t h i s study (Figures 8 and 9). This i s misleading since FDP contents were -found to increase when glycogen l eve l s were not l i m i t i n g suggesting a c t i v a t i o n of PFK. Furthermore, the a l l o s t e r i c modulators ATP, ammonia, P i , ADP and AMP (Bloxham and Lardy 1973) were found to be a l tered in the appropriate d i r e c t i o n to cause a c t i v a t i o n . However, F6P content has frequently been observed to change in the same d i r e c t i o n as flux through the PFK react ion owing to the in f lux of hexose phosphates from glycogen (Williamson 1965; Dr iedz ic and Hochachka 1976; Buppy et a l . 1979). Phos demonstrates a 2.5 fo ld greater a c t i v i t y than PFK in both t i s sues and therefore could p o t e n t i a l l y account for these elevated F6P contents . As w e l l , within red muscle, F6P contents were below pre-exercise values as would be expected when glycogen l eve l s were l i m i t i n g the inf lux of hexose phosphates (Figure 9) . Furthermore, the F6P contents observed in t h i s i n v e s t i g a t i o n (Tables 4 and 5) serve to s t a b i l i z e the PFK aggregates and thereby amplify a c t i v a t i o n (Bloxham and Lardy 1973). The F6P saturat ion curve i s sigmoidal demonstrating a Km in the B . l mM range and an increase in i t s concentrat ion would lead to an increase in i t s rate of u t i l i z a t i o n by PFK while revers ing ATP substrate i n h i b i t i o n (Danforth 1965). The f a l l i n g ATP l e v e l s would lead to reduced substrate i n h i b i t i o n and to an increased a f f i n i t y for F6P while the observed increased FDP and ADP contents , would serve to s t imulate the i r own rate of formation r e s u l t i n g in a further a c t i v a t i o n of PFK (Sols 1981). Insu l in or epinephrine induced a c t i v a t i o n of g l y c o l y s i s in the perfused rat hind limb found F2,6BP to increase 2 to 4 fo ld (Blackmore et a l . 1982). F2,6BP has been shown to be the most potent ac t iva tor of PFK thus far known (Furuya and Uyeda 1982) demonstrating several c r i t i c a l e f fect s on PFKt (1) It increases the a f f i n i t y for FAP while decreasing the strength of F6P s i t e - s i t e cooperative i n t e r a c t i o n s thereby making the F6P saturat ion curves less sigmoidal by moving them to the l e f t ; (2) It increases the a f f i n i t y for the co-substrate ATP while revers ing the i n h i b i t i o n caused by high l eve l s of ATP; (3) It reverses i n h i b i t i o n by c i t r a t e and i s s y n e r g i s t i c in i t s e f fect s with other p o s i t i v e modulators ( i e , AMP and Pi) (Uyeda et a l . 1981). Nevertheless , the p o s s i b i l i t y that F2,6BP i s involved in PFK a c t i v a t i o n within f i s h muscle remains to be inves t iga ted . PFK a c t i v i t y i s integrated with PK and PGK a c t i v i t i e s s ince the ADP thus formed can act as a substrate for these reac t ions . Furthermore, FDP i s a feed forward ac t iva tor of PK in lower vertebrates and serves to couple PFK and PK func t ion ing , a s i t u a t i o n whish i s potentiated as pH decreases (Storey and Hochachka 1974). PK demonstrates marked p o s i t i v e coopera t iv i ty in the binding of i s substrates , with the Kd for ADP binding being lowered from the non-phys io log ica l range to 8.2 to 8.3 mM by the addi t ion of PEP. S i m i l a r i l y , the Kd for PEP i s also lowered from the non-phys io log ica l range to values between 0.81 and 1 mM on the addi t ion of ADP (Dann and B r i t t o n 1978). Therefore the binding of one substrate leads to an order of magnitude increase in the a f f i n i t y for i t s co-substrate with PK and PFK being integrated due to FDP and ADP a c t i v a t i o n of PK. The evidence for PK a c t i v a t i o n comes from the crossover ana lys i s whereby PEP l eve l s are lowered and PYR accumulates in both red and white muscle (Figures 8 and 9). PGK demonstrates high a c t i v i t i e s in muscle and d i sp lays a high a f f i n i t y for DPG (uM range) which suggests that t h i s enzyme i s saturated with substrate under most condi t ions (Krietsch and Bucher 1978). However, i t s a f f i n i t y for i t s co-substrate (ADP) has an apparent Km in the phys io log i ca l range and would therefore be maximally responsive to changes in ADP content (Krietsch and Bucher 1970). In v i v o , t h i s enzyme functions as a complex with GPDH and i s thought to be in equ i l ib r ium under most condit ions (Lehninger 1975). However, within the erythrocyte , ouabain has been shown to induce i n h i b i t i o n of flux through t h i s complex (Minakami and Yoshikawa 1966). In that i n v e s t i g a t i o n , an increased ATP/ADP r a t i o suggested the p o s s i b i l i t y of an ADP l i m i t a t i o n at the PGK s i t e . Increased Pi concentrat ions have also been shown to decrease flux through the GPDH.PGK complex r e l a t i v e to increased flux through PFK in the erythrocyte (Rose et a l . 1964). Here i t was suggested that the high Pi concentrat ion sh i f ted the mass act ion r a t i o of GPDH in favour of DPG and NADH, e f f e c t i v e l y i n h i b i t i n g GPDH. In the present i n v e s t i g a t i o n , the GPDH.PGK complex was found to be out of equ i l ib r ium and to demonstrate crossover points (Figures B and 9) when PCr and glycogen contents were depleted to very low l eve l s in both t i s sues (Figure 3) . At these times, free ADP (Table 9) , Pi (Tables 4 and 5) and NAD/NADH r a t i o s (Table 10) were high while ATP leve l s were low. However, the NAD/NADH r a t i o does not appear to be involved in the regula t ion at t h i s s i t e as the combined GPDH.PGK/LDH react ion which factors out redox from the c a l c u l a t i o n (Table 1), was also found to be displaced from equi l ibr ium (Figures 4-7). Therefore, i t appears that the low ATP/ADP* r a t i o (Table 9) r e s u l t s in the d i s e q u i l i b r i u m of the PGK react ion by s h i f t i n g i t s mass act ion r a t i o in the d i r e c t i o n of DPG and ADP, e f f e c t i v e l y i n h i b i t i n g flux through the PGK.GPDH complex. An uncoupling of m y o f i b r i l l a r ATPase to g l y c o l y s i s would have occurred due to an i n a b i l i t y to maintain the correct ATP/ADP r a t i o as a consequence of an i n s u f f i c i e n t rate of carbon substrates . Within the red muscle, although control of g l y c o l y s i s would appear to have been shared by HK, PFK, GPDH.PGK and PK as in white muscle, the low glycogen content •following the PSS-30 resul ted in HK acting as the flux generator. Because of i t s low a c t i v i t y r e l a t i v e to the other g l y c o l y t i c enzymes and due to , the increased flux as indicated by the elevated pyruvate l e v e l s , a l l intermediates appear to have es tabl i shed a lower steady state (Figure 9) . This s i t u a t i o n seems to have been re f l ec ted in the f i s h white muscle fo l lowing the exhaustive swim (Figure 8) . Redox Balance It was i n t e r e s t i n g to note that both the red and white muscle c y t o s o l i c compartments became more oxidized (increased NAD/NADH r a t i o ) , a t t a in ing maximal values around 2000 when muscle glycogen was depleted (Table 10). These f ind ings are s i m i l a r to those of Jobsis and Stainsby (1968) on the gastrocnemius-plantar is group and g r a c i l i s muscles of the dog fo l lowing e l e c t r i c a l l y st imulated 5 twitch per second contrac t ions . Jobsis and Stainsby (1968) a t t r ibuted the oxidat ion of NADH + H* to a temporary imbalance between the rates of pyruvate production by aerobic g l y c o l y s i s and pyruvate u t i l i z a t i o n by the Krebs c y c l e . In contras t , Sahl in (1985) has found human quadriceps femoris muscle to become more reduced fo l lowing exhaustive exercise performed at maximal oxygen uptake which would suggest e i ther l o c a l hypoxia, an i n a b i l i t y of the re sp i ra tory chain to ox id ize NADH + H* at a s u f f i c i e n t rate and/or a l i m i t a t i o n to mitochondrial membrane s h u t t l i n g . During anaerobic g l y c o l y s i s , there i s a need to continuously reox id ize NADH formed at the GPDH r e a c t i o n . This i s usual ly thought to be achieved by a 111 funct ional coupling between GPDH and LDH, although condi t ions during 112 the i n i t i a l stages of g l y c o l y s i s have been found to be unsuitable for NAD regeneration by LDH (Guppy and Hochachka 1978). I n i t i a l l y the pH i s h igh , making the Km for pyruvate h igh , while pyruvate content i s low. As pH decreases and pyruvate increases , LDH serves to maintain redox due to the a c i d i c pH optimum of LDH in the forward d i r e c t i o n and a lowering of the Km for pyruvate (Guppy and Hochachka 1978). However, when glycogen l eve l s are low, these k i n e t i c propert ies of LDH could continue to convert the elevated pyruvate to l ac ta te due to : (1) the much higher LDH than PDH a c t i v i t y and (2) the induced s h i f t of the LDH equ i l ib r ium in the d i r e c t i o n of l ac ta te and NAD formation. Since pyruvate l eve l s are s t i l l elevated and GAP content depressed under t h i s condi t ion (Tables 7 and 8) , an imbalance could have occurred between the funct ional coupling of GPDH and LDH react ions r e s u l t i n g in the free c y t o s o l i c NAD couple becoming more ox id ized . L i p i d Metabolism L i p i d s as a fuel source were not accounted for in th i s study but i t has been demonstrated that free fa t ty acids (FFA) derived from t r i g l y c e r i d e s serve as a major aerobic fuel for energy metabolism in mammalian (Shaw et a l . 1975j Hickson et a l . 1977) and f i s h muscle (Krueger et a l . 1968). The d i s p o s i t i o n of l i p i d in f i s h i s d i f f e rent than mammals as f i s h possess n e g l i g i b l e adipose t i s sue and the l i p i d s are stored within the l i v e r and muscle (Farkus 1967} B i l i n s k i 1969). As w e l l , the fat content of red muscle i s usua l ly twice that of white muscle (Bone 1966; Lin et a l . 1974) with the fat being stored both i n t r a - and e x t r a c e l 1 u l a r i y in red muscle. Furthermore, these red muscle i n t r a c e l l u l a r l i p i d s may be t o t a l l y surrounded by mitochondria (Lin et a l . 1974). While fat i s a poor fuel for white muscle, red muscle demonstrates a high a b i l i t y to u t i l i z e f a t , demonstrating at least 113 ID fo ld higher rates of FFA oxidat ions in salmonids ( B i l i n s k i 1963; Jonas and B i l i n s k i 1964). As w e l l , high a c t i v i t i e s of c a r n i t i n e palmitoyl transferase have been found in f i s h red muscle with the a c t i v i t y of t h i s enzyme being 20 fo ld greater in red as opposed white muscle (Crabtree and Newsholme 1972). To assess the r e l a t i v e cont r ibut ion of the various substrates to energy provi s ion of locomotion at d i f f e rent v e l o c i t i e s , an estimate of the cont r ibut ion of fats to energy turnover was ca lcu la ted based on the maximal oxygen consumption data of f i s h muscle (Randall and Oaxboeck 1979). Extrapola t ing the i r oxygen uptake data (for f i sh of comparable s i ze to t h i s study) to 100 percent U c r i t gave a maximal oxygen uptake of 3.71 ml 02/min/kg with 3.24 ml 02/min/kg being a t t r i b u t a b l e to exerc i s ing muscle, an increase of 2.84 ml 02/min/kg (17.2 mmol 02/min/kg) above re s t ing values . These values are very s imi la r to the reported oxygen uptake value of Neumann et a l . (1983) fo l lowing strenuous exercise (3.1 ml 02/min/kg). Assuming a formation of 6 umol ATP/mmol 02/g of muscle and subtract ing the glucose plus glycogen derived aerobic energy turnover , gave the energy turnover due to the oxidat ion of f a t s . The oxidat ion of fats predominated the energy provi s ion in both red and white muscle at these workloads (Table 12). The c i t r a t e synthase a c t i v i t i e s found in f i s h muscle are more than s u f f i c i e n t to account for these observed oxidat ion rates in both red and white muscle (Walton and Cowey 1982). 114 Regulation of Krebs Cycle Flux Within t h i s study, further evidence for an a c t i v a t i o n of aerobic combustion of fuels within white and red muscle were the increases in the Krebs cycle intermediates malate and fumarate. Aspartate and alanine aminotransferase enzymes have been i d e n t i f i e d in f i s h muscle (Bel l 1968) and could serve to augment Krebs cyc le intermediates by forming oxaloacetate (Hochachka and Storey 1975) from which the carbon can be r e d i s t r i b u t e d throughout the intermediates . Augmentation of Krebs cycle sp inning , as would occur during exerc i se , could be accomplished in three ways; (1) Having mitochondrial isozymes with appropriate c a t a l y t i c and regulatory proper t ie s ; (2) Having enzymes which occur in low and high a c t i v i t y s tates ; and (3) Having enzymes which are not saturated with substrate (Hochachka et a l . 1983). The Krebs cyc le enzymes do not appear to occur as isozymes but c i t r a t e synthase (CS) , i s o c i t r a t e dehydrogenase (ICDH) and 2-oxoglutarate dehydrogenase (OGDH) do appear to undergo a t r a n s i t i o n from a low to high a c t i v i t y s ta te . These enzymes are c o l l e c t i v e l y under a l l o s t e r i c regula t ion by the adenylates and CoASH (Hochachka et a l . 1983). CS i s i n h i b i t e d by ATP and the decreasing ATP l eve l s (Tables 4 and 5) along with increas ing CoASH would serve to ac t iva te the enzyme. The observed increases in ADP (Table 9) would ac t iva te ICDH since ADP i s a s p e c i f i c ac t iva tor of t h i s enzyme. As w e l l , CS i s l imi ted by low concentrat ions of oxaloacetate while ICDH and OGDH are probably l imi ted by i s o c i t r a t e and 2-oxoglutarate a v a i l a b i l i t y , r e spec t ive ly (Hochacka and Somero 1984). Since CS, ICDH and OGDH appear to be l imi ted by low l eve l s of substrates , the increased concentrat ions of Krebs cycle intermediates serve to increase Krebs cycle sp inning . Therefore, the regula t ion of Krebs cyc le flux during exercise would appear to be under the control of the adenylates, 115 CoASH and l eve l s o-f Krebs cyc le intermediates . Respiratory Control Respiratory control re fers to the coordinat ion of mitochondrial oxidat ive phosphorylation with the ATP demands of the cytoplasm. I n i t i a l l y , the graded rates of r e s p i r a t i o n between State 3 and State 4 were preposed to be a funct ion of ADP a v a i l a b i l i t y (Chance and Will iams 1955; 1956). Subsequently, Klingenberg (1961) suggested that re sp i ra tory control was regulated by the cytoplasmic phosphorylation potent ia l (ATP/ADP.Pi) and th i s theory has subs tant ia l support (Wilson et a l . 1974; N i s h i k i et a l . 1978; Stubbs et a l . 1978s van der Meer et a l . 1978). F i n a l l y , r e sp i ra tory control was postulated to be simply a function of the ATP/ADP r a t i o (Slater et a l . 1973; Davis and Lumeng 1975; Lemasters and Sowers 1979; Bohnensack 1981). The lower estimates of ADP (free ADP) ava i l ab le to p a r t i c i p a t e in react ions has cast considerable doubt on the l a t t e r two theor ie s . The estimates of free ADP (Veech et a l . 1979; Jacobus et a l . 1982; Shoubridge et a l . 1984; Meyer et a l . 1985) are s imi l a r to t h i s inve s t i ga t ion and give r i s e to phosphorylation po tent i a l s and ATP/ADP r a t i o s (Table 9) which are from 1 to several orders of magnitude higher than previous estimates (Veech et a l . 1979). S imi lar ATP/ADP* r a t i o s were found in t h i s study but estimates of phosphorylation po tent i a l s were even higher due to the lower estimates of Pi (compensated). These new values f a l l in the ranges where only low rates of mitochondrial r e s p i r a t i o n are measured and the question ar i se s as to how mitochondria can a c t i v e l y re sp i re under presumably i n h i b i t o r y condit ions of high phosphorylation po tent i a l s or high ATP/ADP r a t i o s ? To resolve th i s quest ion, Jacobus et a l . (1982) experimentally separated the a v a i l a b i l i t y of ADP to the Fl-ATP synthetase and the exogenous ATP/ADP r a t i o . The rate of State 3 r e s p i r a t i o n was d i r e c t l y cont ro l l ed by the ADP concentra t ion , with l i t t l e or no c o r r e l a t i o n to e i ther c y t o s o l i c phosphorylation po tent i a l s or c y t o s o l i c ATP/ADP r a t i o s . This suggested that re sp i ra tory control i s regulated by a v a i l a b i l i t y of ADP to the Fl-ATP synthetase mediated by the k i n e t i c propert ies of the adenine nucleot ide translocase (Lemasters and Sowers 1979). It was i n t e r e s t i n g to note that the Km values for heart mitochondria were in the 15 uM range (Jacobus et a l . 1982). The values observed for free ADP (ADP*C) in t h i s inve s t i ga t ion f e l l in t h i s range and would suggest that mitochondrial r e s p i r a t i o n would be maximally responsive to increases in free ADP concentrat ion . These r e s u l t s lend further support to the l i m i t a t i o n of ox idat ive phosphorylation by the adenine nucleot ide t rans loca tor ra te . However, other factors inc luding oxygen, P i , substrate a v a i l a b i l i t y and matrix ATP/ADP r a t i o s must be considered when examining the c e l l u l a r control mechanisms involved in the regula t ion of ox idat ive phosphorylat ion. Protein Metabolism Prote ins are known to contr ibute up to 10 percent of the to ta l energy requirements during long-term exercise in mammals. The protein contr ibut ion to the energy requirements of exercise were not considered in t h i s i n v e s t i g a t i o n , but the use of amino acids as fuels has been shown during s tarvat ion (Johnston and Goldspink 1973) and migration (Mommsen et a l . 1980) in f i s h . The decreased alanine content and increased g l y c o l y t i c intermediates within the l i v e r suggested that the alanine may be act ive within the trout providing further hepatic glucose as f u e l . However, s ince no s tudies have shown amino acid catabolism within f i s h during exercise and because of the high rates of work (greater than 85 percent Ucr i t ) performed in t h i s study, i t would appear safe to assume that the prote in cont r ibut ion to energy provi s ion was minimal. However, t h i s does not preclude the p o s s i b i l i t y that prote in catabolism could contr ibute s u b s t a n t i a l l y to sustained swimming energy requirements. Hepatic Glucose It was i n t e r e s t i n g to note that l i v e r glycogen values decreased very l i t t l e during the PSS-30 when red muscle glycogen values were f a l l i n g rather markedly (Figure 3). This i s in accord with the evidence that f i s h muscle does not u t i l i z e blood glucose very well as fuel (Walton and Cowey 1782). However, blood glucose l eve l s have been found to decrease during exercise (Dobson, liommsen and Hochachka, unpublished observations) and experiments using rad ioac t ive label s have demonstrated glucose uptake c a p a b i l i t i e s in both red and white muscle (Moen and Klungsoyr 1981). The increased red muscle glucose content fo l lowing the PSS-30 (Table 5) suggested that hepatic glucose was involved in the energy provi s ion s ince only low l e v e l s of glucose 6-phosphatase have been found in f i sh muscle (Walton and Cowey 1982). As w e l l , the elevated glucose l e v e l s in both red and white muscles under a l l exercise states suggested a further involvement of blood glucose in energy production within the exerc i s ing muscles (Tables 4 and 5) . Further evidence for t h i s came from the high rate of l i v e r glycogen breakdown during the PSS-7 (Figure 3) . A previous inves t i ga t ion found blood glucose to decrease from 19.5 to 8.2 umol/ml during an exhaustive swim s imi l a r to the present study (Dobson, Mommsen and Hochachka, unpublished observat ions) . The l i v e r glycogen decrement observed in that study would have increased blood glucose l e v e l s 8.7 umol/ml assuming a l i v e r weight of 2 grams and a blood volume of 5 mls/100g. Based on these values , approximately 210 umol o-f glucose would have been consumed over the whole swim bout (PSS-30+PSS-7+ES) r e s u l t i n g in a glucose removal rate o-f 2.7 umol/min. Though the hexokinase l eve l s are low in f i s h muscle (white muscle 0.03-1.6; red muscle 0.14-2.6 umol glucose/min/g) (Johnston and Moon 1980a, Walton and Cowey 1982), they are s u f f i c i e n t to account for t h i s hypothet ica l rate of removal when blood d i s t r i b u t i o n i s taken into account (Neumman et a l . 1983). Energy Turnover It was of in te re s t to f ind that ; (1) The energy turnover required to perform the PSS-7 was approximately 1.2 times that of the PSS-30; and (2) The rate of energy turnover generated in white muscle was less than red muscle. In the former, the increase in the rate of work was 1.2 fo ld (mean v e l o c i t y PSS-30=90 percent U c r i t ; PSS-7=108 percent Ucr i t ) demonstrating a remarkable matching of energy turnover to power output. In the l a t t e r case, high speed burst exercise has been found to e l i c i t a energy turnover of 375-1200 umol ATP/g/min in white muscle, far greater than red muscle (Dobson et a l . 1986). It would appear that the metabolic energy turnovers required to perform the varied exercise i n t e n s i t i e s in t h i s study were achieved by se l ec t ing the f iber type, fuel and pathway for opt imizing ATP production rate versus substrate deplet ion and proton accumulation. Role of ADP in Integrat ing Fuel Se lect ion The phasing in of the appropriate metabolic pathways at appropriate rates as exercise i n t e n s i t y increases , appears to be int imate ly re la ted to the rate of m y o f i b r i l l a r ATPase a c t i v i t i e s . Although many regulatory s igna l s may be involved (Hochachka and Somero 1984), a l t e r a t i o n s in free ADP with increas ing m y o f i b r i l l a r ATPase a c t i v i t i e s appears to play a prominant ro le through i t s act ions on: (1) mitochondrial r e s p i r a t i o n ; (2) the g l y c o l y t i c enzymes PGK and PK; and (3) CK. It should be emphasized that a competition ex i s t s for ADP between a l l enzymes for which i t i s a substrate as these enzymes general ly occur in the I B - 4 M range while free ADP occurs in the I B " 6 M range (Ottaway and Mowbray 1977). I n i t i a l l y , when mitochondria of non-working muscle are in State 4, f lux through the e lec t ron transfer system i s low because of l i m i t i n g ADP or Pi content. As muscle work begins, mitochondria enter State 3 demonstrating a l i n e a r r e l a t i o n s h i p in mitochondrial r e s p i r a t i o n and phosphorylation as ADP and/or Pi contents increase (Jacobus et a l . 1982). This l inear r e l a t i o n s h i p probably accounts for the l inear r e l a t i o n s h i p observed by Chance et a l . (1981) between work rates and PCr/Pi r a t i o s . When State 3 capacity i s surpassed, the further increases in ADP and Pi create condi t ions favouring CK and g l y c o l y t i c competition for substrate . I n i t i a l l y the high a c t i v i t y of CK and i t s a f f i n i t y for ADP ensures that t h i s pathway predominates (Hochachka 1985). However, as work continues PGK and PK demonstrate a higher a f f i n i t y for ADP than CK and a funct ional coupl ing occurs between g l y c o l y s i s and myosin ATPase. This i s achieved in the case of PK by lowering i t s Kd for PEP from a non-phys io log ica l range to values between B.B1 and 1 umol/g on the addi t ion of ADP. As w e l l , PEP has the same effect on the binding of ADP (Dann and B r i t t o n 1978). These cons t ra int s are recognizable in t h i s study where free ADP l e v e l s were o r i g i n a l l y around those necessary for low rates of mitochodrial r e s p i r a t i o n . Subsequently, free ADP demonstrated a d i f f e rent rate of increase between f iber types which would lead to a coordinated a c t i v a t i o n of pathways and f iber recruitment . Therefore , the rate of m y o f i b r i l l a r ATPase induced a l t e r a t i o n s in ADP a v a i l a b i l i t y which accompany exercise ensures the phasing in of appropriate 120 pathways at appropriate ra tes . Regulation of Exercise Metabolism by Compartmentation The evidence for compartmentation of metabolism comes from a var ie ty of sources and supports the concept that c e l l metabolism i s a highly organized matrix of pathways where phys ica l pos i t ion of enzymes and changes in enzyme a c t i v i t y af fect metabolite a c t i v i t i e s . With regard to g l y c o l y s i s , evidence ex i s t s demonstrating s p e c i f i c l o c a l i z a t i o n and order of enzymes in the I-bands of the muscle f iber (Arnold et a l . 1969; Segal and Pette 1969) with the equ i l ib r ium between bound and soluble forms being highly regulated . This in i t s e l f , suggests l o c a l i z a t i o n of g l y c o l y t i c enzymes into 2 compartments (soluble and bound), which have been demonstrated within a s ing le smooth muscle c e l l (Lynch and Paul 1983; Paul 1983). In th i s system, two pathways of g l y c o l y s i s are f u n c t i o n a l l y and d i sc re te enough so that the glycogen derived glucosyl units do not enter the same pool of g l y c o l y t i c intermediates that are used in the glucose derived l ac ta te and vice versa . Thus, two lac ta te forming pathways e x i s t , which are semi independent and use d i f f e rent precursors , while forming non-mixing pools of i d e n t i c a l intermediates . This c a l l s for a far more complicated metabolic control system than the t r a d i t i o n a l regulatory models of g l y c o l y s i s . The c e l l u l a r concentrat ions of g l y c o l y t i c enzymes far exceeds (approximately 100 fold) the c e l l u l a r concentrat ions of g l y c o l y t i c intermediates , which i s opposite to condit ions for most in v i t r o studies of enzyme k i n e t i c s . Based on t h i s premise, Sr ivastava and Berhnard in a ser ies of inve s t i ga t ions (Weber and Berhnard 19B2; 1985a,b) have demonstrated the d i rec t t ransfer of the g l y c o l y t i c intermediate DPG to GPDH and the GPDH t ransfer of NADH to LDH with these react ions proceeding at a s u b s t a n t i a l l y faster rates than when NADH and DPG were supplied from s o l u t i o n . These rates were further enhanced 20 fo ld by saturat ing 3PG concentrat ion which acted as a s p e c i f i c ef fector of the t ransfer process i t s e l f . Although t h i s d i scuss ion i s far from encompassing, i t serves to i l l u s t r a t e the potent i a l advantage of d i r e c t t ransfer of metabol i tes . It i s postulated that during act ivated g l y c o l y s i s , GPDH may be involved in a d i r ec t coupling for the transfer of DPG and NADH to PGK and LDH, e f f e c t i v e l y acce le ra t ing carbon flux through g l y c o l y s i s by s h i f t i n g the equ i l ib r ium from soluble to bound enzyme system. In t h i s regard, i t was i n t e r e s t i n g to note that hypoxia and ischemia in mammalian heart , resul ted in an increased binding of g l y c o l y t i c enzymes leading to increased a c t i v i t y and flux (Clarke et a l . 1984). This system would not preclude the operation of a so luble enzyme g l y c o l y t i c pathway under the t r a d i t i o n a l regulatory control but provide a second, bound g l y c o l y t i c enzyme pathway (or parts of the g l y c o l y t i c pathway), where the in tegra t ion of the two pathways would achieve a coordinated t r a n s i t i o n from low to high flux ra tes . The f indings in t h i s study are interpreted as being based a so luble enzyme system under c l a s s i c a l metabolic regula t ion which requires that the flux changes and concentrat ion changes be q u a l i t a t i v e l y and q u a n t i t a t i v e l y cons i s tent with a so luble model. The current ly ava i l ab le NMR data suggest that those phosphate compounds in high concentrat ion behave as i f they are free in s o l u t i o n . The a s soc ia t ion of enzymes may not preclude a free so lu t ion equ i l ib r ium approach to the ana lys i s s ince the ef fects are p r imar i ly to increase rates at low substrate concentrat ions and promote combined equ i l ib r ium condi t ions . Thus t h i s approach to the data analys i s i s , in l i g h t of the current state of knowledge, the most reasonable. However, simple c a l c u l a t i o n of free/bound g l y c o l y t i c intermediate concentrat ions from equ i l ib r ium constants reveals how dramatic changes in the concentrat ion of free intermediates has very l i t t l e effect on enzyme d i s e q u i l i b r i u m . As w e l l , the evidence that mitochondrial re sp i ra tory control i s mediated by free ADP concentrat ions lends support to the concept that the equ i l ibr ium between soluble and bound enzyme/metabolite complexes serves not only to affect metabolite concentrat ions but as a potent ia l regulatory mechanism i t s e l f . S i m i l a r i l y , the amount of bound ADP may be important in d r i v i n g the mi tochondr ia l , m y o f i b r i l l a r and sarcoplasmic ret iculum CK isozymes which have been found to l i e in close proximity to the mitochondrial t r a n s l o c a t o r , M-l ine and C a * * ATPase, r e spec t ive ly (Jacobus and Lehninger 1973; Bessman and Geiger 1961; Many and Kay 1978; L e v i t s k i i et a l . 1977). Thus, the importance of compartmentation i s apparent and subsequent inves t i ga t ions into metabolic regula t ion of exercise must address these f ind ings . However, these f indings in no way i n v a l i d a t e e i ther previous or the present inves t i ga t ion into the regulat ion of metabolism during exerc i se , as these studies r e f l e c t whatever i s occurr ing in the t i s s u e , s ing le or mul t ip le pools . FATIGUE As the energy turnover requirements increased, there was a greater r e l i ance on white f iber s and an increased use of phosphagen and anaerobic g l y c o l y t i c energy production to meet these needs (Tables 4 and 5). This was evidenced by the large drops in red muscle phosphagens and glycogen during the PSS-30 exercise while white muscle remained r e l a t i v e l y inac t ive (Figure 3) . At the higher workloads, white muscle phosphagen and glycogen l eve l s were depleted while l ac ta te accumulated. The intramuscular pH values r e f l e c t e d these perturbat ions in metabolism (Figure 10). The same energy sources (ATP and PCr h y d r o l y s i s , anaerobic fermentations and ox idat ive metabolism) were used by both red and white muscle during exercise to achieve the metabolic energy turnovers necessary tD maintain muscular work at given rates (Table 12). Subsequent deplet ion of intramuscular glycogen has been found to co inc ide with exhaustion during prolonged exercise (Hermansen et a l . 1967; Hultman 1967; Hultman 1978). However, during e i ther intermit tent or short term high i n t e n s i t y exerc i se , fat igue was always found to occur pr ior to glycogen deplet ion (Sa l t in and Karlsson 1971; Hermansen and Vaage 1977). S imi lar f indings were demonstrated in the present inves t i ga t ion in both red and white t i s sues (Tables 4 and 5). PCr content was found to decrease in both red and white t i s sues during high i n t e n s i t y exercise (Figure 10). No r e l a t i o n s h i p was found between PCr l eve l s and tension development during intense exercise (Dawson et a l . 1978). As w e l l , during recovery experiments where PCr content was not re s tored , further contract ions could be e l i c i t e d although subsequent fat igue occurred at a great ly accelerated rate (Harris et a l . 1976; Sahl in et a l . 1979). It would appear that PCr p r i n c i p a l l y functions to defend changes in ATP content during high i n t e n s i t y exercise and i s d i r e c t l y involved in the fat igue process when very high rates of energy turnovers are required . Many inves t i ga tor s have found ATP content to dec l ine during intense exercise (Hultman 1967; Hermansen 1971; Vaage et a l . 1978) but few studies have assessed the time course of t h i s d e c l i n e . Wilk ie (1981) reported ATP l eve l s to remain r e l a t i v e l y constant u n t i l fat igue was very advanced at which time ATP content f e l l to 25 percent of control values in frog gastrocnemius muscle. This suggests that ATP decrements per se are not the major factor in muscle f a t igue , since ATP a l t e r a t i o n s did not p a r a l l e l the changes in force generat ion. As w e l l , ATP l eve l s at fat igue are not diminished to a leve l 124 which should impair muscular contract ions (Tables 4 and 5). Further evidence that ATP l eve l s are not a causal factor in fat igue came about in recovery experiments where recovery in the c o n t r a c t i l e parameters occurred without any change in ATP l eve l s (Harris et a l . 1976} Sahl in et a l . 1979). Associated with these decrements in ATP content are increases in ADP and inorganic phosphate concentrat ions (Tables 4 and 5) . Dawson et a l . (1978) found changes in force generation and cross-br idge c y c l i n g to p a r a l l e l a l t e r a t i o n s in ADP and inorganic phosphate contents . Hultman et a l . (1981) has suggested that the increased concentrat ion of ADP could both slow down the cross-bridge detachment and reduce the a c t i v i t y of the sarcoplasmic ret iculum C a * * ATPase. Inorganic phosphate has also been found to reduce the amplitude of s t re tch induced a c t i v a t i o n of insect f i b r i l l a r f l i g h t muscle (White and Thorson 1972) and increase the re laxa t ion rate of skinned smooth muscle (Guth and Junge 1982). It would appear that ADP and inorganic phosphate accumulations may play a ro le within the fat igue process. Host d i scuss ions of end-product accumulations during high i n t e n s i t y exercise have focussed on protons and l a c t a t e . An often overlooked fact i s that PCr hydro lys i s i s coupled to myosin ATPase and therefore r e s u l t s in the accumulation of the guanidino compound Cr (or in the case of molluscan muscle, arginine) and inorganic phosphate (Figure 11). As with protons and l a c t a t e , i t i s imperative that these metabolic end-products not be d e l e t e r i o u s . One reason why Cr i s not de le ter ious i s that i t s only metabolic fate i s reconversion to i t s o r i g i n a l phosphagen form (Walker 1979). However, inorganic phosphate i s involved in many enzyme react ions in intermediary metabolism and a need to control i t s accumulation may place l i m i t s on the amount of PCr which can be stored (Hochachka et a l . 1983). Fish white muscle 125 contains the highest PCr l eve l s reported and because of t h i s , may r e f l e c t the high l ac ta te and P i , contents of exhausted muscle. It would appear that deplet ion of PCr could lead to ion ic and charge perturbat ions s ince the breakdown of PCr which i s present in the form P C r 2 - would require production of a d iva lent counter ion. Cr i t s e l f i s uncharged and would therefore leave the c e l l in an anion gap. Hochachka (1985) has suggested that the coupling of the creat ine kinase react ion to myosin ATPase negates th i s problem as inorganic phosphate ( P i 2 - ) i s e q u i v i l e n t to P C r 2 - in terms of charge. As w e l l , he emphasizes that a second function of P i 2 - accumulation i s buffer ing during proton generating metabolism. This i s where the problem occurs , as P C r 2 - and P i 2 - though demonstrating s i m i l a r charge, have widely d i f fe rent pK values of 4.5 and 6.81 r e spec t ive ly (Table 2) . In the phys io log i ca l pH range, P C r 2 - would be f u l l y d i s soc ia ted while P i 2 - would bind protons thereby a l t e r i n g i t s charge. If P C r 2 - hydro lys i s occurs without any proton accumulations, then P i 2 - would indeed negate the anion gap. However, should proton l e v e l s increase while P C r 2 - hydro lys i s i s occurr ing (as would occur i f anaerobic g l y c o l y s i s were a c t i v a t e d ) , P i 2 -would buffer the protons p o t e n t i a l l y leaving the c e l l in an anion gap. For tunate ly , associated with the proton production i s the accumulation of the anerobic end product l ac ta te ( L a - ) . La~ demonstrates a low pK (Table 2) and would as with P C r 2 - be f u l l y d i s soc ia ted in the p h y s i o l o g i c a l pH range. In the present i n v e s t i g a t i o n , l ac ta te accumulations were i n s u f f i c i e n t to compensate for the changes in P C r 2 - content within red and white muscle (Figure 11). However, the P C r 2 - hydro ly s i s would re su l t in an equ iv i l en t release of P i 2 - . This P i 2 - in excess of the amount necessary to negate proton accumulations would be ava i l ab le to maintain charge as was suggested by Hochachka (1985). Therefore i t would appear that a combination of P C r 2 - hydro lys i s r e s u l t i n g in P i 2 -accumulations and l ac ta te production serve to maintain constancy of charge. Pre-exercise i n t r a c e l l u l a r and e x t r a c e l l u l a r concentrations of Na* , K* , Ca n , Mg ** and C l " were s imi l a r to previous ly reported values for f i s h plasma (Holeton et a l . 1983), frog (Hodgkin and Horowitz 1959) or mammalian muscle (Sembrowich et a l . 1983). Herbert and Jackson (1985) recent ly found a l t e r a t i o n s in these ions associated with anoxia to compensate the l ac ta te accumulations in t u r t l e plasma. However, in t h i s i n v e s t i g a t i o n , a l t e r a t i o n s in Na* , K*, Ca ** , Mg *• and Cl - associated with exercise (Table 13), resul ted in large changes in charge within the red and white muscle i n t r a c e l l u l a r and e x t r a c e l l u l a r compartments (Table 14). F l u i d s h i f t s could not account for these ion ic per turbat ions . However, the techniques used in th i s inve s t i ga t ion to assess i n t r a c e l l u l a r ion content may have masked any loca l i n t r a c e l l u l a r d i s t r i b u t i o n . As w e l l , the ion binding c h a r a c t e r i s t i c s may have been a l tered such that the content of free ions may be quite d i f f e r e n t . A l t e r n a t i v e l y , a change in prote in conf igurat ion and charge may occur negating the accumulation of po s i t i ve charge within the i n t r a c e l l u l a r compartment. Accepting these uncer ta in ie s , i t would appear from the present i n v e s t i g a t i o n that fat igue was not associated with any a l t e r a t i o n in charge. The evidence for t h i s conclusion comes from the f ind ing that the ion di f ference was greater in white muscle after the PSS-30 exercise than PSS-7 exercise pro toco l s . Despite these perturbat ions in charge and ion content, membrane potent ia l was remarkably s imi la r between f iber types and and after the various exercise i n t e n s i t i e s (Table 15). This was assuming a constant permeabi l i ty coef f icent •for each ion which may have been in error s ince various factors are known to a l t e r membrane permeabi l i ty . Lactate permeabi l i t i e s are affected by external l ac ta te concentrat ions (Koch et a l . 19B1) while Na* and K* permeabi l i ty are affected by external calcium concentrat ions (McWilliams and Potts 197B). However, e x t r a c e l l u l a r calcium concentrat ion remained constant and would therefore not affect the ion ic p e r m e a b i l i t i e s . Fatigue was not associated with the white muscle after the PSS-30 exercise despite a drop in membrane p o t e n t i a l . Thus i t would appear that fat igue was not due to any change in membrane p o t e n t i a l . However, the uncer t in ie s in t h i s c a l c u l a t i o n render t h i s conclusion somewhat quest ionable . Red muscle pH was r e l a t i v e l y constant during a l l exercise i n t e n s i t i e s while white muscle pH was only maintained during the PSS-30 exercise regime. Subsequent decreases in white muscle pH occurred during the PSS-7 and exhaustive exercise bouts (Figure 10). The values reported in t h i s i n v e s t i g a t i o n are s i m i l a r to both the re s t ing and working muscle pH values reported for human (Sahlin 1978), mammalian (Roos 1971; Hoult et a l . 1974; Hei s ler 1975) and frog (Malan et a l . 1976; Dawson et a l . 1977). However, higher values have been found by M i l l i g a n and Wood (1985) using the DM0 method on i so l a ted trout trunk preparations (pH approximately 7 .3) . Three mechanisms exi s t for minimizing the decrements in intramuscular pH; (1) Eff lux of protons from the c e l l into the blood and/or inf lux of n e u t r a l i z i n g ions into the c e l l (Koch et a l . 1981); (2) S to ichiometr ica i1y matching the protons generated to proton consumption by the aerobic combustion of fuels (Krebs et a l . 1975); and (3) Absorption of protons by i n t r a c e l l u l a r buffers (Parkhouse et a l . 1985; Abe et a l . 1985). At higher workloads, metabolic energy turnover cannot be matched by the aerobic combustion of fuels and proton production exceeds proton consumption with the resul tant drop in pH. 128 These drops in pH, i f of s u f f i c i e n t magnitude have been associated with reduced rates of g l y c o l y s i s (Toews et a l . 1970; Sutton et a l . 1981), decreased times to fat igue ( F i t t s and Hol loszy 1976; Stevens 1980) and decreased force generation by i so l a ted muscle preparations (Dawson et a l . 1978). However, T r i v e d i and Danforth (1966) have shown that increas ing AMP and F6P lowers the pH optimum for g l y c o l y s i s . As w e l l , Dobson et a l . (1986) has found F2,6BP increases to negate the i n h i b i t o r y ef fects of decreasing pH on PFK a c t i v i t y . Re-examination of the time courses of pH changes and aberations in muscle function reveal that : (1) proton accumulations can occur before s i g n i f i c a n t g l y c o l y t i c a c t i v a t i o n ( F i t t s and Holloszy 1976); (2) the highest rates of g l y c o l y s i s occur while protons are accumulating at the i r highest rates (Sahlin et a l . 1981); and (3) g l y c o l y t i c rate remains high and unchanged despite continued proton production and lac ta te formation (Dawson et a l . 1978). Recently , a carr ier-mediated l ac ta te transfer system has been found to operate in ske le ta l muscle (Koch et a l . 1981). This carr ier-mediated transfer of l ac ta te i s pH dependent and appears to be an ant iport system, l ac ta te anions exchanging for OH - ions (Dubinsky and Racker 1980; Koch et a l . 1981). Hochachka and Mommsen (1983) therefore argue that proton production may; (a) create more favourable condi t ions for the unloading of oxygen; (b) f a c i l i t a t e phosphagen h y d r o l y s i s ; (c) e s t ab l i sh a pH optimum for g l y c o l y s i s s ince i n h i b i t i o n of g l y c o l y s i s only occurs at low pH values; and/or (d) f a c i l i t a t e l ac ta te eff lux due to the pH dependency of the carr ier-mediated l ac ta te t r ans fe r . There can be no question as to the benef i t s of proton accumulation on the Bohr s h i f t or the rate of phosphagen h y d r o l y s i s , but the idea of e s t ab l i sh ing a pH optimum for g l y c o l y s i s i s mis leading. This impl ies that decreasing pH increases the g l y c o l y t i c ra te . It i s not the pH that optimizes the g l y c o l y t i c rate but the act ion of p o s i t i v e modulators which counteract the i n h i b i t o r y e f fects of proton accumulations. S i m i l a r i l y , within f i s h white muscle l ac ta te i s retained negating the potent ia l ro le of f a c i l i t a t e d l ac ta te eff lux in t h i s species . In the present i n v e s t i g a t i o n , the f i s h were unable to continue to function at the PSS-7 swimming speed while substrate (glycogen) l eve l s remained high. This i n a b i l i t y to maintain a s u f f i c i e n t l y high metabolic energy turnover could have resided in e i ther the c o n t r a c t i l e or g l y c o l y t i c machinery and appears to be re la ted to the proton, ADP, inorganic phosphate and/or l ac ta te accumulations. The matching of La~ production to P C r 2 - hydro lys i s and subsequent P i 2 - accumulations appears to negate potent ia l metabol ica l ly induced charge perturbat ions . However, i on ic a l t e r a t i o n s accompaning exercise were found to affect the i n t r a c e l l u l a r and e x t r a c e l l u l a r compartments charge c h a r a c t e r i s t i c s , although these changes did not appear to a l ter membrane p o t e n t i a l . Since proton i n h i b i t i o n of g l y c o l y t i c rate appears to be counteracted by increased p o s i t i v e modulators, the proton, ADP and Pi induced a l t e r a t i o n s in the c o n t r a c t i l e machinery appear to be the most probable cause of fat igue during the PSS-7 swim while substrate deplet ion appears to be the primary cause of fat igue during the ES. SKELETAL MUSCLE BUFFERING The PSS-7 workload resul ted in an i n a b i l i t y of the f i s h to maintain the required v e l o c i t y and appears to be int imate ly re la ted to the e f fects of proton accumulations on the c o n t r a c t i l e machinery. Thus the a b i l i t y of f i sh muscle to absorb protons generated during high i n t e n s i t y exercise was examined. High concentrat ions of imidazole based compounds ex i s t in f i sh 130 muscle and the r e s u l t s support the ro le of these h i s t i d i n e re la ted compounds as i n t r a c e l l u l a r buf fers . There are several l i n e s of evidence from t h i s i n v e s t i g a t i o n and others that support t h i s conc lus ion . (1) The s t r i k i n g d i f ferences between red and white muscle buffer ing capac i t i e s (Table 16) and the concentrat ions of h i s t i d i n e re la ted compounds (Table 21) with both species demonstrating s i m i l a r f i ve fo ld d i f ferences between the i r respect ive t i s sues in these parameters. (2) Marl in also showed six fo ld higher l eve l s of h i s t i d i n e re la ted compounds (Table 21) and buffer capac i t i e s in both i t s red and white muscle compared with trout (Table 16). (3) H i s t i d i n e re la ted compounds demonstrate pK c h a r a c t e r i s t i c s which are consistent with t h e i r phys io log i ca l ro le as buf fers . The pK values are 6 .0 , 6.83 and 7.04 ( h i s t i d i n e , carnos ine , anser ine, re spec t ive ly ) and in the case of anser ine, i t s pK c h a r a c t e r i s t i c s can account for the elevated buffer capac i t i e s found in the 6.5 to 7.5 pH range. (4) These two species operate in widely d i f f e rent water temperatures (marlin 2 5 ° C j trout 4 - 1 5 ° C ) and therefore i t i s paramount that i f s i m i l a r , t h e i r choice of buffers not be temperature s e n s i t i v e , s ince temperature a l t e r s the protonation state of many compounds. The imidazole based h i s t i d i n e re la ted compounds are per fec t ly sui ted for t h i s function as they conserve t h e i r protonation state under a l l condi t ions of temperature v a r i a t i o n , thereby re ta in ing the i r buffer ing potent ia l over the p h y s i o l o g i c a l pH range (Somero 1981). The higher buffer capacity observed for marlin ske le ta l muscle t i s sue was expected based on the swimming behavior of t h i s species . These values are as high as any reported for f i s h species ( C a s t e l l i n i and Somero 1981) and r e f l e c t the i r h i s t i d i n e re la ted compound l e v e l s . The other p r i n c i p a l components of to ta l t i s sue buffer capacity appear to be prote in and phosphate, while taur ine may contr ibute within red muscle in the a l k a l i n e pH range (Table 18). Proteins have long been recognized as a major buffer within ske le ta l muscle (Woodbury 1965). The comparable m y o f i b r i l l a r prote in content of the t i s sues accounts for the i r s imi l a r non-soluble prote in buffer capac i t i e s (Table 22). Previous inves t iga tor s have shown t h i s m y o f i b i l l a r prote in buffer ing to be h i s t i d i n e based (Woodbury 1965; Morris and Baldwin 1984); t h i s f ind ing appears to be confirmed in t h i s study, because the buffer ing capacity of t h i s f r a c t i o n decreased with increas ing pH (Table 16). However, compared to soluble h i s t i d i n e re la ted compounds, the m y o f i b r i l l a r prote in h i s t i d i n e cont r ibut ion to buffer ing i s small (Table 18). The soluble prote in buffer ing cont r ibut ion r e l a t i v e to to ta l t i s sue buffer ing capacity was remarkably s imi la r across the species . An inverse r e l a t i o n s h i p was demonstrated for the r e l a t i v e contr ibut ion of prote in buffer ing to to ta l t i s sue buf fe r ing . This suggests that buffer ing const i tuents other than prote in are responsible for the elevated buffer ing capac i t i e s of the marlin red and white t i s sues . Of these, the aforementioned h i s t i d i n e re la ted compounds are the most important but phosphate may have a potent ia l r o l e . Free phosphate can contr ibute to buffer ing s ince i t demonstrates a pK (6.61) in the phys io log i ca l pH range. During the t i s sue homogenization, hydro ly s i s of ATP and PCr would lead to an increase in free phosphate, thereby e levat ing the t i s sue buffer capacity and the phosphate buffer ing c o n t r i b u t i o n . Thus, d i f ferences in buffer ing may be p a r t i a l l y a t t r i b u t a b l e to the higher phosphagen l eve l s found in white as opposed to red t i s sues . Note that the elevated buffer capacity of marlin white and red muscle (Table 16) could be t o t a l l y a t t r ibuted to the i r higher t i s sue l eve l s of h i s t i d i n e re la ted compounds (Tables 21). The high concentrat ion of anserine in marlin white muscle p a r a l l e l s the s i t u a t i o n found in tuna in which s i m i l a r i l y high concentrat ions of h i s t i d i n e were found (80-90 umol/g) (Abe 1983b). It i s i n t e r e s t i n g that both species belong to the same r e l a t i v e sub-order group (Scombriodidei) , having s imi l a r swimming behavior and yet appear to have d i f fe rent h i s t i d i n e re la ted compounds acting as the i r p r i n c i p a l buffers . This may be misleading since these compounds are metabo l i ca l ly interconvertable (Aonouma et a l . 1969). In tuna, a s h i f t in the h i s t i d i n e re la ted compounds has been observed during s t a rva t ion . Free h i s t i d i n e decreased to 4 and 34 ( 5 and 12 days s t a r v a t i o n , re spec t ive ly ) percent of contro l s but, the concomitant increase in the l eve l s of anserine and carnosine led to a net decrease of only 30 to 40 percent of the control to ta l h i s t i d i n e re la ted compound pool (Abe and Hochachka 1986). S i m i l a r l y in sockeye salmon, in which white muscle i s the primary source of amino acids u t i l i z e d during spawning migration (Mommsen et a l . 1980), high anserine l eve l s (around 40 umol/g white muscle) are maintained throughout the spawning run. Free h i s t i d i n e , on the other hand, shows a more than 10 fo ld drop (from 3.6 to 0.3 umol/g) during migration (Mommsen, French and Hochachka 1980). These s h i f t s could be explained by a metabolic d i s p o s i t i o n of h i s t i d i n e to anserine and carnosine to prevent the degradation of free h i s t i d i n e during s t a r v a t i o n . Anserine synthesis requires the methylation of h i s t i d i n e , which i s in ef fect a way of protect ing t h i s reserve, s ince at least in r a t s , i t cannot be converted back to methylh i s t id ine (Aonouma et a l . 1969). Thus the marlin may simply convert i t s free h i s t i d i n e into the more metabol ica l ly s table anserine dipept ide to conserve i t s buffer ing p o t e n t i a l . There i s l i t t l e information on the phys io log i ca l functions of h i s t i d i n e re la ted compounds other than buf fer ing . Like t aur ine , h i s t i d i n e i s only slowly metabolized and therefore does not seem to play a ro le in energy metabolism. In rat muscle, the h a l f - l i f e of carnosine was 29 days and 200 fo ld that of h i s t i d i n e while the h a l f - l i f e of anserine could not be determined (Tamaki et a l . 1977). Recent studies ind ica te a slow turnover o-f h i s t i d i n e in trout muscle although i t s uptake -from blood into the t i s sue proceeds r e l a t i v e l y r a p i d l y (Abe and Hochachka 1986). The h i s t i d i n e re la ted compounds were -found to contr ibute s u b s t a n t i a l l y to t o t a l t i s sue buffer capacity (Tables 19 and 20). It i s known that h i s t i d i n e re la ted compounds can complex copper (Brown 1981) and that t h i s chela t ion would negate t h e i r potent ia l proton absorbing capac i ty . As w e l l , other ro les for h i s t i d i n e re la ted compounds have been proposed: they have been i d e n t i f i e d as myosin ATPase ac t iva to r s (Avena and Bowen 1969), as a c t iva to r s of sarcolemma Na+,K+ ATPase (Boldyrev and Petukhov 1978), as s t imulators of fructose 1,6 bisphosphatase (Ikeda et a l . 1980) and as a mechanism for t ransport ing copper for the a c t i v a t i o n of cytochrome oxidase (Brown 1981). Thus the ent i re pool of h i s t i d i n e re la ted compounds may not be free to p a r t i c i p a t e as buffers . However, in f i sh species which possess high concentrat ions of these compounds, i t i s apparent that t h e i r dominant ro le i s that of phys io log i ca l buffers with the d i f ferences in l eve l s of these compounds p r i n c i p a l l y accounting for the buffer ing capacity d i f ferences found between the species and f iber types. In Vivo Buffering Capacity S imi lar values were found for the trout white muscle t i t r a t e d (in v i t ro ) and estimated (in vivo) buffer capac i t i e s (Table 25). At f i r s t glance, t h i s f ind ing would appear to suggest that f i s h white muscle crude homogenate ( t i t r a ted ) buffer capac i t i e s represent in vivo buf fer ing . However, the in v i t r o crude homogenate t i t r a t i o n technique assesses the maximal potent ia l buffer capaci ty of the t i s s u e s , whereas the in vivo estimation of buffer capacity only assess the buffer ing which occurred during the exerc i se . This implies that some potent ia l buffers are unavai lable to negate proton accumulations during the exerc i se . I n t u i t i v e l y t h i s makes sense, as intermediary metabolism generates weak acids ( g l y c o l y t i c intermediates and inorganic phosphate) as glycogen, PCr and ATP are depleted. PCr and ATP generated inorganic phosphate can p o t e n t i a l l y be the most important buffering const i tuent during exercise (Burton 197B). As w e l l , during periods of high i n t e n s i t y exerc i se , PCr hydro lys i s serves to defend ATP dec l ines (Hochachka 1985). Therefore t h i s potent ia l buffer (Pi) i s made ava i l ab le during periods of high proton generat ion. The evidence for t h i s hypothesis comes from the examination of the cont r ibut ion of inorganic phosphate to buf fer ing . As was suggested by Burton (1978), the cont r ibut ion of inorganic phosphate to buffer ing increases great ly once exercise has been i n i t i a t e d . In the present i n v e s t i g a t i o n , inorganic phosphate demonstrated minor v a r i a t i o n in i t s cont r ibut ion to bu f f e r ing , 22.4 in vivo (Tables 23 and 24) versus 32.3 in v i t r o ( T a b l e IB) (umol H+/g/pH). However, PCr and ATP contents at the end of the PSS-7 exercise were s t i l l 28.8 and 5.8 (umol/g) r e spec t ive ly (Table 4). During the ES, these values were found to decl ine to 1.8 and 2.7 umol/g for PCr and ATP r e s p e c t i v e l y (Table 4). These a l t e r a t i o n s would increase the in vivo inorganic phosphate contr ibut ion to buffer ing approximately 21 umol H+/g/pH and the to ta l t i s sue buffer ing to about 83 umol H+/g/pH. Therefore i t would appear that the in vivo buffer capacity i s great ly infuenced by the metabolic s tatus of the muscle. The cont r ibut ion of prote in buffer ing was considered to be equivalent in both the t i t r a t e d and estimated buffer c a p a c i t i e s . This was based on the 135 assumption of no p ro teo ly s i s during the exercise p ro toco l . This assumption may be in error as f i s h muscle has the potent ia l to use proteins as fuels for work (Johnston and Goldspink 1973). However, these f indings have usual ly been found in combination with s tarvat ion (Johnston and Goldspink 1973; Mommsen et a l . 1980) and are therefore thought to be u n l i k e l y in t h i s i n v e s t i g a t i o n . Protein buffer ing has been demonstrated to be h i s t i d i n e based (Woodbury 1965; Morris and Baldwin 1984) and the content of h i s t i d i n e necessary to produce the 26.1 umol H+/g/pH of buffer ing i s approximately 125 umol/g or 13 percent. As w e l l , h i s t i d i n e re la ted compounds were found to contr ibute remarkably s imi l a r amounts to the t i t r a t e d ( 7 . 9 ± 0 . 3 umol H+/g/pH; Table 18) and estimated (6.7 umol H+/g/pHj Table 23) buffer c a p a c i t i e s . Thus i t i s apparent that prote ins and h i s t i d i n e re la ted compounds cons t i tu te consis tent and important buffer ing components. The ca lcu la ted ( lactate) buffer capacity exceeded both the t i t r a t e d and estimated buffer capacity values demonstrating a value s imi l a r to the estimated maximal white muscle buffer ing capacity (Table 25). However, the ES demonstrated afurther decrease in pH to approximately 6.55 and an increase in l ac ta te content to 43 umol/g. These values would re su l t in a ca lcu la ted buffer capacity of approximately 110 umol H"*, far in excess of the t h e o r e t i c a l maximal buffer capac i ty . This f ind ing was s imi l a r to that of Sahl in et a l . (1978) for human vastus l a t e r a l i s muscle and was not unexpected based on the pH range over which the buffer capacity was determined. This pH range (6.91-6.65) revolves around the two p r i n c i p a l buf fers , anserine (pK 7.04) and inorganic phosphate (pK 6.81). Thus the change in pH during the PSS-7 exercise centered around the point of greatest buffering potent i a l within the c e l l and would produce a f a l s e l y high buffer value. It would therefore appear that r e s t r a i n t must be exercised when attempting to imply that the crude homgenate t i t r a t e d buffer capacity r e f l e c t s in vivo buf fer ing . However, the dominance of buffer ing by p r o t e i n , h i s t i d i n e re la ted compounds and inorganic phosphate does allow a reasonable estimate of maximal po tent i a l in vivo buf fe r ing . These buffer ing const i tuents represent approximately 90 and 80 percent of the maximal po tent i a l buffer ing capacity in white and red muscle r e s p e c t i v e l y . The lower percentage for red i s due to the greater cont r ibut ion of ammonia to i t s bu f fe r ing . Based on these f i n d i n g s , buffer capaci ty values of 83 and 55 (umol H*/g/pH) are estimated for rainbow trout white and red muscle r e s p e c t i v e l y . These values are r e l a t i v e l y high and r e f l e c t the capacity of t h i s animal to accumulate l ac ta te and protons. 137 SUMMARY AND CONCLUSIONS ATP h y d r o l y s i s , phosphagen h y d r o l y s i s , ox idat ive and anaerobic fermentative based energy prov i s ion occurred at a l l exercise i n t e n s i t i e s with swimming v e l o c i t y d i c t a t i n g the extent to which each f iber type, fuel and pathway was used. Thus, the metabolic energy turnovers required to perform these workloads were achieved by using the same fuels (ATP, PCr, glycogen, glucose and fats) and varying the metabolic pathway cont r ibut ion in both red and white muscle. The i n a b i l i t y to generate s u f f i c i e n t energy turnover to continue to work resul ted in fat igue under the PSS-7 and ES regimes. However, the e t i o l o g i e s of the fat igue were d i f f e r e n t . During the ES and PSS-7 regimes, substrate and end product l i m i t a t i o n s r e s p e c t i v e l y , would appear to have been re la ted to the i n a b i l i t y to continue func t ion ing . During both the PSS-30 and PSS-7, white muscle was found to be act ivated to a lesser extent than red muscle. However, the PSS-7 resul ted in a further a c t i v a t i o n of white muscle g l y c o l y t i c energy provi s ion while red muscle energy turnover requirements were achieved by predominantly aerobic combustion of fue l s . The purine nucleot ide cyc le was found to be operat ional in both f iber types although the formation of IMP and replenishment of adenine nucleot ides appear to operate at d i f f e r e n t i a l ra tes . L i p i d metabolism dominated energy prov i s ion at these power outputs although complete deplet ion of phosphagen and glycogen accompanied the ES in both red and white muscle. It would appear that the metabolic energy turnovers required to perform the varied exercise i n t e n s i t i e s in t h i s study were achieved by se l ec t ing the f iber type, fuel and pathway for opt imizing ATP production rate versus substrate and proton accumulation. The regula t ion of fuel s e l e c t i o n appears to be int imate ly re la ted to m y o f i b r i l l a r ATPase a c t i v a t i o n with free ADP acting as a metabolic s ignal to coordinate the phasing in of appropriate fuels/pathways at appropriate ra tes . This metabolic control exerted by free ADP i s accomplished in the case of high energy turnovers by the competition of CK and the g l y c o l y t i c enzymes PGK and PK for t h i s substrate . During lower i n t e n s i t y workloads, mitochondrial r e s p i r a t i o n exerts a high a f f i n i t y for ADP and because of the low concentrat ions of free ADP, i s maximally responsive to changes in the concentrat ions of t h i s metabol i te . As m y o f i b r i l l a r ATPase a c t i v i t y increases , free ADP content a v a i l a b i l i t y increases creat ing condi t ions favouring CK and g l y c o l y t i c competition for the substrate . However, concurrent a l t e r a t i o n s in ATP, PCr and Pi re su l t in large changes in phosphorylation potent ia l (ATP/ADP.Pi) and a subsequent decl ine in the free energy ava i l ab le from ATP h y d r o l y s i s . HK, phos, PFK and PK are a l l subject to a l l o s t e r i c regula t ion by adenine nucleot ides and were i d e n t i f i e d as regulatory enzymes in both f iber types. However, the GPDH.PGK complex also appears to exhib i t regula t ion when glycogen i s l i m i t i n g and t h i s regula t ion appears to be induced by a decreased ATP/ADP, r a t i o . An uncoupling of m y o f i b r i l l a r ATPase to g l y c o l y s i s would have occurred due to an i n a b i l i t y of g l y c o l y s i s to maintain an appropriate ATP/ADP, r a t i o as a consequence of i n s u f f i c i e n t carbon substrate . The free c y t o s o l i c redox state of the NAD couple became more oxidized in both t i s sues when muscle glycogen was l i m i t i n g and t h i s f ind ing was a t t r ibuted to an induced s h i f t in the Keq of LDH in the d i r e c t i o n of NAD and lac ta te by the elevated pyruvate and low pH. The simultaneous ATP/ADP, induced d i s e q u i l i b r i u m of the PGK react ion would i n h i b i t f lux through the GPDH.PGK complex and allow an imbalance to occur in the free c y t o s o l i c redox state of the NAD couple. It would therefore appear that adenine nucleot ides play a centra l ro le in the coordinat ion of metabolism during exerc i se . 139 P r o t o n i n d u c e d a l t e r a t i o n s i n t h e c o n t r a c t i l e m a c h i n e r y a p p e a r e d t o be t h e most p r o b a b l e c a u s e of f a t i g u e d u r i n g t h e 7 m i n u t e h i g h e r i n t e n s i t y p r o l o n g e d s t e a d y swim. H i s t i d i n e r e l a t e d compounds were f o u n d t o p l a y a d o m i n a n t r o l e i n b u f f e r i n g w i t h i n t r o u t and m a r l i n m u s c l e w i t h d i f f e r e n c e s i n t h e l e v e l s of t h e s e compounds p r i n c i p a l l y a c c o u n t i n g f o r t h e b u f f e r i n g c a p a c i t y d i f f e r e n c e s f o u n d b e t w e e n t h e s p e c i e s and f i b e r t y p e s . However, i n v i v o b u f f e r c a p a c i t y was d o m i n a t e d by p r o t e i n and t h e i n o r g a n i c p h o s p h a t e r e l e a s e d d u r i n g p h o s p h a t e h y d r o l y s i s and t r a n s f e r r e a c t i o n s . H i s t i d i n e r e l a t e d compounds c o n t r i b u t e d a f i x e d s m a l l p e r c e n t a g e of t h e t r o u t w h i t e m u s c l e b u f f e r i n g d u r i n g t h e e x e r c i s e . T h u s , t h e s e m e t a b o l i c and b i o c h e m i c a l a d j u s t m e n t s , a l l o w e d a c o o r d i n a t e d i n t e g r a t i o n of f i b e r t y p e , f u e l and pathway s e l e c t i o n , t o a c h i e v e t h e a p p r o p r i a t e e n e r g y t u r n o v e r s f o r t h e c o u p l i n g of m y o f i b r i l l a r A T P a s e a c t i v i t y t o ATP t u r n o v e r , w h i l e o p t i m i z i n g ATP p r o d u c t i o n r a t e v e r s u s s u b s t r a t e d e p l e t i o n and p r o t o n a c c u m u l a t i o n s . 140 References Abe, H. 1981. Determination of L - h i s t i d i n e re la ted compounds in f i s h muscles using high-performance l i q u i d chromatography. Bui 1 . J p n . S o c . S c i . F i s h . 47s139. Abe, H. 1983a. D i s t r i b u t i o n of free L - h i s t i d i n e and i t s re la ted compounds in marine f i she s . Bui 1 . J p n . S o c . S c i . F i s h . 49:1683-1687. Abe, H. 1983b. D i s t r i b u t i o n of free L - h i s t i d i n e and re la ted dipeptides in the muscle of fresh-water f i she s . Comp.Biochem.Physiol. 76B:35-39. Abe, H . , 6 .P. Dobson, U. Hoeger and W.S. Parkhouse. 1985. Role of h i s t i d i n e re la ted compounds to i n t r a c e l l u l a r buffer ing in f i s h ske le ta l muscle. A m . J . P h y s i o l . 249:R449-R454. Abe, H. and P.W. Hochachka. 1986. Turnover of 1 4 C - l a b e l l e d L - h i s t i d i n e and i t s incorporat ion into carnosine and anserine in rainbow t rou t . C a n . J . Z o o l . (in press ) . A l b e r t y , R.A. 1969. Standard Gibbs Free Energy, enthalpy, and entropy changes as a function of pH and pMg for several rac t ions involv ing adenosine phosphates. J .B io l .Chem. 244: 32913-3302. Aonuma, S. , T. Hama, N. Tamaki and H. Okumura. 1969. Orotate as a B-alanine donor for anserine and carnosine biosynthes i s and ef fects of actinomycin D and azaurac i l on the i r pathway. J .Biochem. 66:123-132. Aragon, J . J . and J . H . Lowenstein. 1980. The purine nucleot ide c y c l e . E u r . J . B i o c h . 110:371-377. Arnold , H. and D. Pette . 1968. Binding of g l y c o l y t i c enzymes to s t ructure prote ins of the muscle. Eur . J .Biochem. 6:163-171. Avena, R.M. and W.J. Bowen. 1969. Ef fects of carnosine and anserine on muscle tr iphosphatases . J .B io l .Chem. 244:1600-1604. Bate-Smith, E . C . 1938. The buffer ing of muscle in r i g o r , p r o t e i n , phosphate and carnosine . J . P h y s i o l . (Lond.) 92:336-343. B e l l , G.R. 1968. D i s t r i b u t i o n of transaminases in the t i s sues of P a c i f i c salmon with emphasis on the propert ies and diagnost ic use of GOT. J . F i s h . R e s . B d . C a n . 25:1247-1268. Bergmeyer, H.V. 1974. Methods of Enzymatic A n a l y s i s . New York: Academic Press . Bessman, S.P. and P . J . Geiger. 1981. Transport of energy in muscle: the phosphoryl-creat ine shu t t l e . Science 215:295-296. B i l i n s k i , E. 1963. U t i l i z a t i o n of l i p i d s by f i s h I. Fatty acid oxidat ion by t i s sue s l i c e s from dark and white muscle of rainbow trout (Salmo G a i r d n e r i ) . Can . J .B iochem.Phys io l . 41:107-112. 141 B i l i n s k i , E. 1969, L i p i d catabolism in f i s h . In: Fish in Research. O.W. Neuhaus and J . E . Halver (Eds . ) . New York: Academic Press , pp. 135-151. B i l i n s k i , E. and R . E . E . Jonas. 1972. Oxidation of l ac ta te to carbon dioxide by rainbow trout (Salmo ga i rdner i ) t i s sues . J . F i s h . R e s . B d . C a n . 29:1467-1471. B i l i n s k i , J . 1974. Biochemical aspects of f i sh swimming. In: Biochemical and Biophys ica l Perspect ives in Marine Bio logy . D.C. Malins and J .R. Sargent (Eds). New York: Academic Press , 1:239-288. Black, M. J . and M.E. Jones. 1983. Inorganic phosphate determination in the presence of l a b i l e organic phosphate: Assay for carbamyl phosphate phosphatase a c i t i v i t y . A n a l . B i o c h . 135:233-238. Blackmore, P . F . , L. Hue, H. Shilama et a l . 1982. Regulation of fructose 2,6-bisphosphate content in rat hepatocytes, perfused hearts and perfused hindl imbs. Fed.Proc . (abstract) 41:1678. Bloxham, D.P. and H.A. Lardy 1973. Phosphofrutokinase. In: The Enzymes. P.D. Boyer ( E d . ) , New York, Academic Press , pp. 239-278. Bohroe, H . - J . , W. Schel1enberger and E. Hofmann. 1975. Mikrokalor imetr i sche bestimmung der thermochemischen parameter der phosphofruktokinase-reaktion. Acta BiolMed.Germ. 34:15-20. Bohnensack, R. and W. Kunz. 197B. Mathematical model of regula t ion of ox idat ive phosphorylation in in tac t mitochondria . Acta B io l .Med .Ger . 37:97-112. Boldyrev, A . A . and V . B . Petukhov. 1978. L o c a l i z a t i o n of carnosine ef fect on fat igued muscle preparat ion. J .Gen.Pharmacol. 9:17-2(9. Bone, Q. 1966. On the function of two types of myotomal muscle f iber in elasmobranch f i s h . J . M a r . B i o l . A s s . U . K . 46:321-349. Bone, Q. 1978. Locomotor muscle. In: Fish Physiology. W.S. Hoar and D. J . Randall (Eds . ) . 7:361-424. Brown, C . E . 1981. Interact ions between carnosine, anserine, ophidine and copper in biochemical adaptat ion. J . T h e o r . B i o l . 88:245-256. Burton, K. 1957. Free energy data of b i o l o g i c a l i n t e r e s t . Appendix to : Energy transformations in l i v i n g matter. H.A. Krebs and H.L . Kornberg. Ergebnisse der P h y s i o l . 49:275-286. Burton, R .F . 1978. I n t r a c e l l u l a r bu f fe r ing . Resp .Phys io l . 33:51-58. Busby, S . J .W. , D.G. Gadian, 6.K. Radda et a l . 1978. Phosphorus nuclear-magnetic-resonance studies of compartmentation in muscle. Biochem.J. 170:103-114. Cannon, R.K. and A. K i b r i c k . 1938. Complex formation between carboxyl ic acids and d iva lent ca t ions . J.Am.Chem.Soc. 60:2314-2320. 142 C a s t e l l i n i , M.A. and 6.N. Somero. 1981. Buffering capacity of vertebrate muscles c o r r e l a t i o n s with po tent i a l s for anaerobic funct ion . J .Comp.Phys io l . 143s191-198. Chance, B. and G.R. Wil l iams . 1955. Respiratory enzymes in ox idat ive phosphorylation I. K i n e t i c s of oxygen u t i l i z a t i o n . J .B io l .Chem. 217i385-393. Chance, B. and G.R. Wil l i ams . 1956. The re sp i ra tory chain and oxidat ive phosphorylat ion. Adv.Enzymol. 17s 65-134. Chance, B . , S. E l e f f , J .S . Le igh , J r . , D. Sokolow and A. Sapega. 1981. Mitochondria l regula t ion of phosphocreat ine/ inorganic phosphate r a t i o s in exerc i s ing human muscle: A gated 31P NMR study. Proc.Nat 1 .Acad .Sc i . 78:6714-6718. Christman, A . A . 1976. Factors a f fec t ing anserine and carnosine l e v e l s in ske le ta l muscles of various animals. Int . J .Biochem. 7:519-527. C larke , F . M . , P. Stephen, G. Huxham et a l . Metabolic dependence of g l y c o l y t i c enzyme binding in rat and sheep heart . Eur . J .Biochem. 138:643-649. Claus , T . H . , M.R. El-Maghrabi , D.M. Regan et a l . 1984. The ro le of fructose2,6-bisphosphate in the regula t ion of carbohydrate metabolism. C u r r . T o p . C e l 1 . R e g u l . 23:57-86. Connett, R . J . In vivo g l y c o l y t i c e q u i l i b r a in dog g r a c i l i s muscle. J .B io l .Chem. 260:3314-3320. C o r n e l l , N.W., M. Leadbetter and R.L. Veech. 1979. Ef fects of free magnesium concentrat ion and ion ic strength on equ i l ib r ium constants for glyceraldehyde phosphate dehydrogenase and phosphoglycerate kinase reac t ions . J .B io l .Chem. 254:6522-6527. Cowey, C . B . , M. De La Higurea and J.W. Adron. 1977. The ef fect of d ie tary composition and of i n s u l i n on gluconeogenisis in rainbow t rout . B r . J . N u t r . 38:385-395. Crabtree , B. and E .A . Newsholme. 1972. A c t i v i t i e s of phosphorylase, hexokinase, phosphofructokinase, l ac ta te dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase in muscles from vertebrates and inver tebra tes . Biochem.J. 126:49-58. C u r t i n , N.A. an R.C. Woledge. 197B. Energy changes and muscular cont rac t ion . Phys io l .Rev . 58:690-761. Danforth, W.H. 1965. A c t i v a t i o n of g l y c o l y t i c pathway in muscle. In: Control of Energy Metabolism. B. Chance, R.W. Estabrook and J.R. Williamson (Eds . ) . New York:Academic Press , pp.287-297. Dann, L . G . and H.G. B r i t t o n . 1978. K i n e t i c s and mechanism of act ion of muscle pyruvate kinase. Biochem.J. 169:39-54. Davey, C L . 1960. The s i g n i f i c a n c e of carnosine and anserine in s t r i a t e d ske le ta l muscle. Arch.Biochem.Biophys. 89:303-308. 143 Davis , E . J . and L. Lumeng. 1975. Relat ionships between the phosphorylation po tent i a l s generated by l i v e r mitochondria and re sp i ra tory state under condi t ions of adenosine diphosphate c o n t r o l . J .B io l .Chem. 2513:2275-2282. Dawson, M . J . , D.G. Gadian and D.R. W i l k i e . 1977. Contract ion and recovery of l i v i n g muscles studied by 3 1 P nuclear magnetic resonance. J . P h y s i o l . 267: 7(33-735. Dawson, M . J . , D.G. Gadian and D.R. W i l k i e . 1978. Muscular fat igue invest igated by nuclear magnetic resonance. Nature (London) 274i861-866. Dobson, G . P . , E. Yamamoto and P.W. Hochachka. 19B6. Phosphofructokinase control in muscle: nature and reversa l of pH-dependent ATP i n h i b i t i o n . A m . J . P h y s i o l . 250:R71-R76. Dobson, G . P . , P.W. Hochachka, A . N . Belcastro and W.S. Parkhouse. 1986. A re-eva luat ion of metabolic control and fat igue in rat fast and slow twitch muscle fo l lowing spr int and endurance running, (in preparation) Donaldson, M. 1985. Respiratory propert ies of mitochondria from heart and mosiac muscle of rainbow trout (Salmo g a i r d n e r i ) : substrate u t i l i z a t i o n and response to temperature and extramitochondrial pH. MSc. Thes i s , Univ. of B r i t i s h Columbia, 1985. D r e i d z i c , W.R. and P.W. Hochachka. 1976. Control of energy metabolism in f i sh white muscle. A m . J . P h y s i o l . 230:579-582. D r i e d z i c , W.R. and P.W. Hochachka. 1978. Metabolism in f i s h during exerc i se . In: Fish Physiology. W.S. Hoar and D. J . Randall (Eds . ) . 7:503-543. D r i e d z i c , W.R., G. McGuire and M. Hatheway. 1981. Metabolic a l t e r a t i o n s associated with increased energy demand in f i sh white muscle. J .Comp.Phys io l . 141:425-432. Dubinsky, W.P. and E. Racker. 1978. The mechanism of l ac ta te transport in human erythrocytes . J .Memb.Biol . 44:25. Dudley, G.A. and R.L . Terjung. 1985. Influence of ac idos i s on AMP deaminase a c t i v i t y in contract ing f a s t - twi tch muscle. A m . J . P h y s i o l . 248:C43-C50. Dunn, J . F . Metabolic adjustments to acute hypoxia in the Afr ican lungf i sh and rainbow t rout . Ph.D. Thes i s , Univ. of B r i t i s h Columbia, 1985. F i t t s , R.H. and J .O . Hol lo szy . 1976. Lactate and c o n t r a c t i l e force in frog muscle during develoement of fat igue and recovery. A m . J . P h y s i o l . 231:430-433. Freed, J .M. 1971. Propert ies of muscle phosphofructokinase of c o l d - and warm-acclimated Carassius auratus. Comp.Biochem.Physiol.B 39:747-764. Furuya, E. and K. Uyeda. 1982. Regulation of phosphofructokinase by a new mechanism. An ' a c t i v a t i o n f a c t o r ' binding to the phosphorylated enzyme. J .B io l .Chem. 255:11656-11659. 144 Gadian, D . G . , G.K. Radda, T .K . Brown, E.M. Chance, M.J . Dawson and D.R. W i l k i e . 1981. The a c t i v i t y o-f c ra t ine kinase in -frog ske le ta l muscle studied by sa tura t ion- t rans fer nuclear magnetic resonance. Biochem.J. 194:215-228. Gadian, D . G . , G.K. Radda, R .E . Richards and P . J . Seeley. 1979. 3 1 P NMR in l i v i n g t i s sue : the road from a promising to an important tool in b io logy . In: B i o l o g i c a l App l i ca t ions of Magnetic Resonance. R.G. Shulman (Ed . ) . New York: Academic Press , pp. 463-535. Gevers, W. 1977. Generation of protons by metabolic processes in heart c e l l s . J . M o l . C e l l . C a r d i o l . 9:867-874. Gevers, W. 1979. Reply to W i l k i e , D .R. : Generation of protons by processes other than g l y c o l y s i s in muscle c e l l s : a c r i t i c a l view. J . M o l . C e l 1 . C a r d i o l . 11:328-330. Gossel in-Rey, C , 6. Hamoir and R,K, Scopes. 1968. L o c a l i z a t i o n of creat ine kinase in the s tarch-gel and moving-boundary e l ec t rophore t i c patterns of f i sh muscle. J . F i s h . R e s . B d . C a n . 25:2711-2714. Greer Walker, M. and G. P u l l . 1973. Skeleta l muscle function and sustained swimming speeds in the c o a l f i s h Gadus v irens L. Comp.Biochem.Physiol. 44A:495-501. Guppy, M. and P.W. Hochachka. 1978. C o n t r o l l i n g the highest l ac ta te dehydrogenase a c t i v i t y known in nature. A m . J . P h y s i o l . 234:R136-R140. Guppy, M. , W.C. Hulbert and P.W. Hochachka. 1979. Metabolic sources of heat and power in tuna muscles. II . Enzyme and metabolite p r o f i l e s . J . E x p t . B i o l . 82:303-320. Guth, K. and J.Junge. 1962. How C a 2 - impedes cross-bridge detachment in chemical ly skinned Taenia c o l i . Nature (London) 300:775-776. Hakala, M . T . , A . J . Gla id and G.W. Schwert. 1956. Lac t i c dehydrogenase. II . Var i a t i on of k i n e t i c and equ i l ib r ium constants with temperature. J .B io l .Chem. 221:191-209. H a r r i s , R . C . , R .H.T . Edwards, E. Hultman et a l . 1976. The time course of phosphorylcreat ine resynthes i s during recovery of the quadriceps muscle in man. Pfugers Arch. 367:137-142. H e i s l e r , N. 1975. I n t r a c e l l u l a r pH of i so l a t ed rat diaphragm muscle t i s sue determined by PCO2 e q u i l i b r a t i o n of homogenates. R e s p i r . P h y s i o l . 23:243-255. H e i s l e r , N. 1976. Bicabonate exchange between body compartments after changes of temperature in the larger spotted dogfish (SCYLIORHINUS STELLARIS). R e s p i r . P h y s i o l . 33:145-160. Herbert , C .V. and D.C. Jackson. 1985. Temperature e f fects on the responses to prolonged submergence in t u r t l e chrysemys p i c t a b e l l i i I Blood acid-base and i o n i c changes during and fo l lowing anoxic submergence. P h y s i o l . Z o o l . 58:655-669. 145 Hermansen, L . , E. Hultman and B. S a l t i n . 1967. Muscle glycogen during prolonged severe exerc i se . Acta Phys io l .Scand . 71:129-139. Hermansen, L. 1971. Lactate production during exerc i se . In: Muscle Metabolism During Exerc i se . B. Pernow and B. S a l t i n (Eds . ) . New York: Plenum Press , pp. 401-408. Hermansen, L. and 0. Vaage. 1977. Lactate disappearance and glycogen synthesis in human muscle after maximal exerc i se . A m . J . P h y s i o l . 233:E422-E429. Hickson, R . C . , M.J . Rennie, R.K. Conlee et a l . 1977. Ef fect s of increased plasma fa t ty acids on glycogen u t i l i z a t i o n and endurance. J . A p p l . P h y s i o l . 43:829-833. Hoar, and D. Randal l . 1978. Terminology to decribe swimming a c t i v i t y in f i s h . In: F i sh Physiology. W.S. Hoar and D. J . Randall (Eds . ) . 7 : x i i i - x i v . Hochachka, P.W. and K.B. Storey. 1975. Metabolic consequences of d iv ing in animals and man. Science 187:613-621. Hochachka, P .W. , 6 .P. Dobson and T . P . Mommsen. 1983. Role of isozymes in metabolic regula t ion during exerc i se : in s ight s from comparative s tudies . In: Isozymes-Current Topics in B i o l o g i c a l and Medical Research. M.C. R a t t a z z i , J . G . Scandalios and G . S . ' W h i t t (Eds . ) . New York: Academic Press , 8:91-113. Hochachka, P.W. and T . P . Mommsen. 19B3. Prtons and anaerobios is . Science 219:1391-1398. Hochachka, P.W. and G.N. Somero. 1984. Biochemical Adaptation. Pr inceton : Princeton Univ. Press. Hochachka, P.W. Fuels and pathways as designed systems for support of muscle work. J . E x p t . B i o l . 115:149-164, 1985. Hodgkin, A . L . and P. Horowitz. 1959. The inf luence of potassium and ch lor ide ions on the membrane po tent i a l s of s ing le muscle f i b e r s . J . P h y s i o l . 148:127. Holeton, G . F . , P. Neumann and N. H e i s l e r . 1983. Branchial ion exchange and acid-base regula t ion after strenuous exercise in rainbow trout (Salmo g a i r d n e r i ) . Resp .Phys io l . 51:303-318. Houl t , D . I . , S.J .W. Busby, D.G. Gadian et a l . Observations of t i s sue metabolites using 3 1 P nuclear magnetic resonance. Nature 252:285-287. Hudson, R . C . L . 1973. On the function of the white muscles in t e leos t s at intermediate swimming speeds. J . E x p t . B i o l . 58:509-522. Hultman, E. 1967. Studies on muscle metabolism of glycogen and act ive phosphate in man with spec ia l reference to exercise and d i e t . S c a n d . J . C l i n . L a b . I n v e s t . 19 (suppl.) 94:1-64. 146 Hultman, E. 1978. Regulation o-f carbohydrate metabolism in the l i v e r during rest and exercise with specia l reference to d i e t . In: The 3rd Internat ional Symposium on the Biochemistry of Exerc i se . F. Landry and W.A.R. Orban (Eds . ) . Symposia S p e c i a l i s t s , Miami, pp. 99-126. Hultman, E. , H. Sjoholm, K. Sahlin and L. Edstrom. 1981. G l y c o l y t i c and oxidat ive energy metabolism and contract ion c h a r a c t e r i s t i c s of in tac t human muscle. In: Human Muscle Fat igue: Phys io log ica l Mechanisms. Ciba Found.Symp. 82:19-40. Ikeda, T . , K. Kimura, T. Hama and N. Tamaki. 1980. A c t i v a t i o n of muscle f ructose- l ,6-bisphosphatase by h i s t i d i n e and carnosine. J .Biochem. 87:179-185. Jacobs, H.K. and S.A. Kuby. 1980. Studies on muscular dystrophy. A comparison of the steady-state k i n e t i c s of the normal human ATP-creatine transphosphorylase isozymes (creat ine kinases) with those from Duchenne muscular dystrophy. J ,B io l .Chem. 255:8477-8482. Jacobus, W.E. and A . L . Lehninger. 1973. Creatine kinase in rat heart mitochondria . J .B io l .Chem. 248:4803-4810. Jacobus, W . E . , R.W. Moreadith and K.M. Vandegaer. 1982. Mitochondrial r e sp i ra tory c o n t r o l . Evidence against the regula t ion of r e s p i r a t i o n by extramitochondrial phosphorylation potent i a l s or by ATP/ADP r a t i o s . J .B io l .Chem. 257:2397-2402. Jobs i s , F . F . and W.N. Stainsby. 1968. Oxidation of NADH during contract ions of c i r c u l a t e d mammalian ske le ta l muscle. R e s p i r . P h y s i o l . 4:292-300. Johnston, I .A . and G. Goldspink. 1973. Quantitative studies of muscle glycogen u t i l i z a t i o n during sustained swimming in crucian carp (Carassius carass ius L . ) . J . E x p t . B i o l . 59:607-615. Johnston, I .A. 1977. A comparative study of g l y c o l y s i s in red and white muscles of the trout (Salmo ga i rdner i ) and mirror carp (Cyprinus c a r p i o ) . J . F i s h B i o l . 11:575-588. Johnston, I .A . and T.W. Moon. 19B0a. Exercise t r a i n i n g in ske le ta l muscle of brook trout (Sa lvel inus f o n t i n a l i s ) . J . E x p t . B i o l . 87:177-195. Johnston, I .A . and T.W. Moon. 1980b. Endurance exercise t r a i n i n g in the fast and slow muscles of te leos t f i s h (Pol lachius v i r e n s ) . J . Comp. P h y s i o l . 135:147-156. Johnston, I .A . 1981a Structure and function of f i s h muscles. Symp.Zool .Soc.Lond. 48:71-113. Johnston, I .A. 1982a. Physiology of muscle in hatchery ra i sed f i s h . Comp.Biochem.Physiol. 73B:105-124. Johnston, I .A . 1982b. Biochemistry of myosins and c o n t r a c t i l e propert ies of f i s h ske le ta l muscle. M o l . P h y s i o l . 2:15-29. 147 Jonas, R . E . E . and E. B i l i n s k i . 1964. U t i l i z a t i o n of l i p i d s by f i sh III . Fatty acid oxidat ion by various t i s sues from sockeye salmon (Oncorhynchus nerka) . J . F i s h . R e s . B d . C a n . 21:653-656. Kiceniuk , J.W. 1975. Some aspects of exercise physiology in f i s h . Ph.D. Thes i s . Univ. B r i t i s h Columbia, Vancouver, B.C. Canada. Koch, A . , B. Webster and S. Lowel l . 19B1. C e l l u l a r uptake of L- lac ta te in mouse diaphragm. B iophys . J . 36:775-796. Krebs, E .G . 1981. Phosphorylation and dephosphorylation of glycogen phosphorylase: a prototype for r e v e r s i b l e covalent enzyme modi f i ca t ion . C u r r . T o p . C e l l . R e g u l . 18:401-419. Krebs, H.A. and R.L . Veech. 1969. The energy leve l and metabolic control in mitochondria . S. Papa, J .M. Tager, E. Q u a g l i a r i e l l o and E . C . S la ter (Eds . ) . A d r i a t i c a E d i t r i c e , Bari pp. 329-3B2. Krebs, H . A . , H.F . Woods and K.G. A l b e r t i . 1975. Hyperlactaemia and l a c t i c a c i d o s i s . Essays Med.Biochem. 1:81-103. K r i e t s c h , W.K.G. and T. Bucher. 3-phosphoglycerate kinase from rabbit s e l e t a l muscle and yeast. Eur . J .Biochem. 17:568-580. Kruger, H . M . , J .B . Saddler, G.A. Chapman et a l . 1968. B ioenerget ic s , exercise and fa t ty acids of f i s h . Am.Zool. 8:119-129. Kushmerick, M.J . and R.A. Meyer. 1985. Chemical changes in rat leg muscle by phosporus nuclear magnetic resonance. A m . J . P h y s i o l . 248:C542-C549. Lehninger, A . L . 1975. Biochemistry. New York: Worth P u b l , , 2nd Ed. Lemasters, J . J . and A . E . Sowers. 1979. J .B io l .Chem. 254:1248-1251. L e v i t s k i i , D . O . , T .S . Levchenko, V . A . Saks et a l . 1977. Funct ional coupling between Ca 2 *-ATPase and creat ine phosphokinase in sarcoplasmic ret iculum of myocardium. B iok imi ia 42:1389-1395. L i n , Y . , G.H. Dobbs and A . L . De V r i e s . 1974. Oxygen consumption and l i p i d content in red and white muscle of Anta rc t i c f i shes . J . E x p t . Z o o l . 189:379-385. Lopina, O.D. and A . A . Boldyrev. 1974. Influence of dipeptides carnosine and sarcoplasmic re t i cu lum. Dolk.Akad.Nauk.SSSR. 220:1218-1221. Lowenstein, J .M. 1972. Ammonia production in muscle and other t i s sues : the purine nucleot ide c y c l e . Phys io l .Rev . 52:382-414. Lowry, O . H . , N. Rosebrough, A. Farr and R, Randal l . 1951. Protein measurement with Fol in-phenol reagent. J .B io l .Chem. 193:265-275. Lynch, R.M. and R. J . Paul . 1983. Compartmentation of g l y c o l y t i c and g lycogenolyt ic metabolism in vascular smooth muscle. 222:1344-1346. 146 Macchia, D.D. and P . I . Pol imeni . 1982. A program in basic for c a l c u l a t i o n of t i s sue e x t r a c e l l u l a r space and ion d i s t r i b u t i o n s in vivo using morphometric and/or e x t r a c e l l u l a r tracer methods. Computors Biomed.Res. 15:592-597. Malan, A . , T . L . Wilson and R.B. Reeves. 1976. I n t r a c e l l u l a r pH in cold-blooded vertebrates as a function of body temperature. R e s p i r . P h y s i o l . 28:29-47. Mani, R.S. and C M . Kay. 1978. I so la t ion and charac te r i za t ion of the 165,000 dalton component of the M-l ine of rabbit ske le ta l muscle and i t s in te rac t ion with creat ine kinase. Biochem.Biophys.Acta 533:248-255. McGilvery , R.W. 1975. The use of fuels for muscular work. In: Metabolic Adaptation to Prolonged Physical Exerc i se . H. Howald and J .R. Poortmans (Eds . ) . Basel : Birkhauser Ver lag , pp. 12-30. McWilliams, P .G. and W.T.W. Potts . 1978. The e f fects of pH and calcium concentrat ions on g i l l po tent i a l s in brown t r o u t , Salmo t r u t t a . J.Comp. P h y s i o l . 126:277-286. Meyer, R.A. and R.L . Terjung. 1979. Differences in ammonia and adenylate metabolism in contract ing fast and slow muscle. Am. J .Phys io l . 237iC111-C118. Meyer, R.A. and R.L . Terjung. 1960. AMP deamination and IMP reamination in working ske le ta l muscle. A m . J . P h y s i o l . 239:C32-C3B. Meyer, R . A . , M.J . Kushmerick and T.R. Brown. 1982. App l i ca t ion of 31P NMR spectroscopy to the study of s t r i a t e d muscle metabolism. A m . J . P h y s i o l . 242:C1-C11. Meyer, R . A . , T.R. Brown and M.J . Kushmerick. 19B5. Phosphorus nuclear magnetic resonance of fas t - and slow-twitch muscle. A m . J . P h y s i o l . 24B:C279-C287. M i l l i g a n , C . L . and C M . Wood. 1985. I n t r a c e l l u l a r pH t rans ients in rainbow trout t i s sues measured by dimethadione d i s t r i b u t i o n . A m . J . P h y s i o l . 248:R668-R673. Minakami, S. and H. Yoshikawa. 1966. Studies of erythrocyte g l y c o l y s i s III The e f fects of ac t ive cat ion t ransport , pH and inorganic phosphate concentrat ion on erthrocyte g l y c o l y s i s . J .Biochem. (Tokyo) 59:145 Moen, K.A. and L. Klungsoyr. 1981. Metabolism of exogenous substrates in perfused hind parts of rainbow trout Salmo g a i r d n e r i . Comp.Biochem.Physiol. 68B:461-466. Mommsen, T . P . , C J . French and P.W. Hochachka. I960. S i tes and patterns of prote in and amino acid u t i l i z a t i o n during spawning migration of slamon. C a n . J . Z o o l . 58:1785-1799. Morr i s , G.M. and J . Baldwin. 1964. pH buffer ing capacity of invertebrate muscle: c o r r e l a t i o n s with anaerobic muscle work. M o l . P h y s i o l . 5:61-70. 149 Mosse, R . R . L . 1980. An i n v e s t i g a t i o n of gluconeogenesis in marine te leos t s and the ef fect of long term exercise on hepatic gluconeogenesis. Comp.Biochem.Physiol. 67B:583-592. Nakano, T. and N. Tomlinson. 1967. Catecholamine and carbohydrate concentrat ions in rainbow trout (Salmo ga irdner i ) in r e l a t i o n to phys ica l dis turbance. J . Fi sh. Res. Bd. Can. 24:17131-1715. Newsholme, E . A . , P .H. Snugden and T. Wil l i ams . 1977. Ef fect of c i t r a t e on the a c t i v i t i e s of 6-phosphofructokinase from nervous and muscle t i s sue from d i f f e rent animals and i t s r e l a t i o n s h i p to the regulat ion of g l y c o l y s i s . Biochem.J. 166:123-129. N i s h i k i , K . , li . Erecinska and D.F . Wilson. 1974. Homeostatic regula t ion of c e l l u l a r energy metabolism: experimental charac te4r iza t ion in vivo and f i t to a model. A m . J . P h y s i o l . 234:C82-CB9. Noda, L . , G .E . Schultz and I. Von Zabern. 1975. C r y s t a l l i n e adenylate kinase from carp muscle. Eur . J .Biochem. 51:229-235. Noltman, E .A . 1972. Aldolase-ketose isomerases. In: The Enzymes. P.D. Boyer ( E d . ) , New York: Academic Press , 3rd e d . , v o l . 7, pp. 271-354. Ottaway, J . H . and J. Mowbray. 1977. The ro le of compartmentation in the contro l of g l y c o l y s i s . 12:107-208. Parkhouse, W.S. and D.C. McKenzie. 1984. Poss ib le cont r ibut ion of ske le ta l muscle buffers to enhanced anaerobic performance: a br ie f review. Med.Sci .Sp . 16:32B-338. Parkhouse, W.S . , D.C. McKenzie, P.W. Hochachka and W.K. Ova l le . 1985. Buffer capacity of deproteinized human vastus l a t e r a l i s muscle. J . A p p l . P h y s i o l . 58:14-17. Paul , R . J . 1983. Functional compartmentation of oxidat ive and g l y c o l y t i c metabolism in vascular smooth muscle. A m . J . P h y s i o l . 244:C399-C409. P h i l l i p s , R . S . J . , P. Eisenberg, P. George and R. J . Rutman. 1965. Thermodynamic data for the secondary phosphate i o n i z a t i o n s of adenosine, guanosine, c y t i d i n e , and ur id ine nucleot ides and tr iphosphate . J .B io l .Chem. 240:4393-4397. P o r t n e r , H . O . , N. Hei s ler and M.K. Grieshaber. 1984. Anaerobiosis and acid-base status in marine inver tebra tes : a t h e o r e t i c a l ana lys i s of proton generation by anaer ibic metabolism. J .Comp.Phys io l . 155:1-12. Proc tor , C , P . R . L . Mosse and R . C . L . Hudson. 1980. A histochemical and u l t r a s t r u c t u r a l study of the development of the propuls ive musculature of brown t r o u t , Salmo t r u t t a L in r e l a t i o n to i t s swimming behavior. J . F i s h B i o l . 16:303-321. Racker, E. 19B1. Energy cyc les in health and disease, Curr .Top ic s C e l l . R e g u l . 18:361-376. 150 Ramsey, J .A . 1955. The excretory system of the s t i ck i n s e c t , Dixippus morosus (Orthoptera, Phasmidae). J . E x p t . B i o l . 32:183-199. Randa l l , D . J . and C. Daxboeck. 1982. Cardiovascular changes in the rainbow trout (Salmo ga i rdner i Richardson) during exerc i se . C a n . J . Z o o l . 60:1135-1140. Randle, P . J . 1981. Phosphorylation-dephosphorylation cycles and the regula t ion of fuel s e l ec t ion in mammals. C u r r . T o p . C e l 1 . R e g u l . 18:107-130. Ramaiah, A. 1976. Regulation of g l y c o l y s i s in ske le ta l muscle. L i f e S c i . 19:455-466. R o l l e s t o n , F .S . A t h e o r e t i c a l background to the use of measured concentrat ions of intermediates in study of the control of intermediary metabolism. Cur .Topics C e l l . R e g u l . 5:47-75. Rome, L . C . , P .T . Loughna and G. Goldspink. 1984. Muscle f iber a c t i v i t y in carp as a function of swimming speed and muscle temperature. A m . J . P h y s i o l . 247:R272-R279. Roos, A. I n t r a c e l l u l a r pH and buffer ing power of rat muscle. A m . J . P h y s i o l . 221:182-188. Roos, A. and W.R. Boron. 1981. I n t r a c e l l u l a r pH. P h y s i o l . Rev. 61:296-434. Rose, I . A . , J . V . B . Warms and E . L . O 'Conne l l . 1964. Role of inorganic phosphate in s t imulat ing the glucose u t i l i z a t i o n of human blood c e l l s . Biochem.Biophys.Res.Commun. 15:33 S a h l i n , K. 1978. Intrace l1a lar pH and energy metabolism in ske le ta l muscle of man. Acta Phys io l .Scand . (Suppl.) 455. S a h l i n , K. , R.C. Harr i s and E. Hultman. 1979. Resynthesis of creat ine phosphate in human muscle after exercise in r e l a t i o n to intramuscular pH and a v a i l a b i l i t y of oxygen. S c a n d . J . C l i n . L a b . Invest. 39:551-557. S a h l i n , K. 1985. NADH in human ske le ta l muscle during short-term intense exerc i se . Pflugers Arch. 403:193-196. S a l t i n , B. and J . Kar l s son. 1971. Muscle ATP, CP, and lac ta te during exercise after phys ica l c o n d i t i o n i n g . In: Muscle Metabolism During Exerc i se . B.Pernow and B. S a l t i n (Eds . ) , New York:Plenum Press , pp.395-399. Sembrowich, W . L . , E. Wang, T . E . Hutchinson and D. Johnson. 1983. E lectron microprobe analys i s of fatigued fas t - and slow-twitch muscle. In: Biochemistry of Exerc i se . H . J . Knuttgen, J . A . Vogel and J. Poortmans (Eds . ) . Champaign: Human K i n e t i c s P u b l . , pp. 571-576. Shoubridge, E . A . , J . L . Bland and G.K. Radda. 1984. Regulation of creat ine kinase during steady-state i sometric twitch contract ion in rat ske le ta l muscle. B ioch .B iophys .Acta . 805:72-78. S i e g a l , P. and D. Pette . 1969. I n t r a c e l l u l a r l o c a l i z a t i o n of g lycogenolyt ic and g l y c o l y t i c enzymes in white and red rabbit ske le ta l muscle. J .Histochem.Cytochem. 17:225-237. 151 Sies jQ, B.K. and K. Messeter. 1971. Factors determining i n t r a c e l l u l a r pH. Int Ion Homeostasis of the B r a i n , B.K. S ies jo and 5 .C . Sorenson (Eds . ) . New York: Academic Press , pp. 244-262. S l a t e r , E . C , J . Rossing and A. Mol. 1973. The phosphorylation potent ia l generated by r e s p i r i n g mitochondria . Biochem.Biophys.Acta 292:543-553. Snugden, P .H. and E .A . Newsholme. 1975. The ef fects of ammonium, inorganic phosphate and potassium ions on the a c t i v i t y of phosphofructokinase from muscle and nervous t i s sues of vertebrates and inver tebra tes . Biochem.J. 150:113-122. So l s , A. 1979. Multimodulation of enzyme a c t i v i t y . Phys io log ica l s i gn i f i c ance and evolut ionary o r i g i n . In: Modulation of Prote in Funct ion . New York:Academic Press , pp. 27-45. So l s , A. 1981. Multimodulation of enzyme a c t i v i t y . Curr .Topic s C e l l . R e g u l . 19:77-101. Somero, B.N. 1981. pH-temperature in te rac t ions on prote ins : p r i n c i p l e s of optimal pH and buffer system design. M a r . B i o l . L e t t . 2:163-178. Sr ivas tava , D.K. and S.A. Bernhard. 1985. Mechanism of transfer of reduced nicotinamide adenine d inuc leot ide among dehydrogenases. Biochem. 24:623-628. S r ivas tava , D . K . , S .A. Bernhard, R. Langridge and J . A . McClar in . 1985. Molecular basis for the transfer of nicotinamide adenine d inuc leot ide among dehydrogenases. Biochem. 24:629-635. S tab le r , T . V . and A . L . S i e g e l . 1981. Rapid l i q u i d chromatographic f luorometr ic method for taur ine in b i o l o g i c a l f l u i d s , invo lv ing p r e d e r i v i t i z a t i o n with f luorescamine. Cl in.Chem. 27:1771. S t e i n , W.H. and S. Moore. 1958. The free amino acids of human blood plasma. J .B io l .Chem. 211:915-926. Stevens, E .D. and E . C . Black. 1966. The ef fects of in termit tent exercise on carbohydrate metabolism in rainbow t r o u t , Salmo g a i r d n e r i . J . F i s h . R e s . B d . C a n . 23:471-495. Stevens, E .D. 1979. The ef fect of temperature on t a i l beat frequency of f i sh swimming at constant v e l o c i t y . C a n . J . Z o o l . 57:1628-1635. Stevens, E .D. 1980. Ef fect of pH on muscle fat igue in i so l a ted frog sa r tor ius muscle. Can . J .Phys io l .Pharmaco l . 58:568-570. Stewart, P .A. How to understand acid-base. A quant i t a t ive acid-base primer for biology and medicine. New YorksElsevier North Hol land. Storey, K.B. and P.W. Hochachka. 1974. Enzymes of energy metabolism in a vertebrate f a c u l t a t i v e anaerobe, Pseudemys s c r i p t a . Tur t l e heart pyruvate kinase. J .B io l .Chem. 249:1423-1427. 132 Stubbs, M. , R .L . Veech and H.A. Krebs. 1972. Control of redox state of the nicotinamide-adenine d inuce lo t ide couple in rat l i v e r cytoplasm. Biochem.J. 126:59-65. Stubbs, M . , P .V. Vignais and H.A. Krebs. 1978. Is the adenine nucleot ide t rans locator r a t e - l i m i t i n g for ox idat ive phosphorylat ion. Biochem.J. 172:333-342. Sutton, J . R . , N .L . Jones and C . J . Toews. 1981. Effect of pH on muscle g l y c o l y s i s during exerc i se . C l i n . S c i . 61:331-338. Taante, N. , V . E . Stefanov and S.N. Lyz lova . 1979. Regulatory propert ies of c reat ine kinase from white muscles of f i s h . Biokhimiya. 44:361-366. Tamaki, N . , M. Nakamura and M. Harada. 1977. Anserine and carnosine contents in muscular t i s sue of rat and r a b b i t . J . N u t r . S c i . V i t a m i n o l . 2 3 : 2 1 3 - 2 1 9 . Taussky, H.H. and R. Shorr. 1953. A micro-colormetr ic method for determination of inorganic phosphorous. J .B io l .Chem. 202:675-685. Toews, C . J . , C. Lowry and N.B. Rudermen. 1970. The regulat ion of gluconeogenesis. J .B io l .Chem. 245:818-824. T r i v e d i , . B. and W.H. Danforth. 1966. Ef fect of pH on the k i n e t i c s of frog muscle phosphofructokinase. J .B io l .Chem. 241:4110-4114. Turner, J . D . , C M . Wood and D. C l a r k . . 1983. Factors a f fect ing l ac ta te and proton efflux from pre-exerc i sed , i so lated-prfused rainbow trout trunks. J . E x p t . B i o l . 105:395-401. Uyeda, K. , E. Furuya and L . J . Luby. 1981. The ef fect of natural and synthet ic D-fructose 2,6-bisphosphate on the regulatory k i n e t i c propert ies of l i v e r and muscle phosphofructokinase. J .B io l .Chem. 256:8394-8399. Vaage, 0. , E .A . Newsholme, 0. Gronnerod and L. Hermansen. 1976. Muscle metabolites during recovery after maximal exercise in man. Acta Phys io l .Scand . 102:11A-12A. van der Meer, R. , R. Akerboom, A.K. Groen et a l . 1978. Relat ionships between oxygen uptake of perfused r a t - l i v e r c e l l s and cytosol phosphorylation state ca lcu la ted from ind ica tor metabolites and a redetermined equ i l ib r ium constant. Eur . J .Biochem. 84:421-428. Veech, R .L . , J.W. Randolph Lawson, N.W. Cornel l and H.A. Krebs. 1979. Cy to so l i c phosphorylation P o t e n t i a l . 254:6538-6547. Vel sco , D . , R.W. Guynn, M. Oskarsson and R.L . Veech. 1973. The concentrations of free and bound magnesium in rat t i s sues . Re la t ive constancy of free M g 2 - concentrat ions . J .B io l .Chem. 248:4811-4819. Walker, J .B . 1979. Creat ine : b iosynthes i s , regula t ion and func t ion . Adv.Enzymol. 50:177-242. Walton, M.J . and C B . Cowey. 1982. Aspects of intermediary metabolism in salmonid f i s h . Comp.Biochem.Physiol. 73B:59-79. 153 Watts, D.C. 1973. Creatine kinase (adenosine 5 ' - t r iphosphate-crea t ine phophotransferase). Ins The Enzymes. P.D. Boyer (Ed . ) . New York: Academic Press , 3rd e d . , 8:369-446. Webb, P.W. 1971a. The swimming energet ics of trout I. Thrust and power output at c r u i s i n g speeds. J . E x p t . B i o l . 55 s 489-520. Weber, J .P . and S.A. Bernhard. 19B2. Transfer of 1,3-diphosphoglycerate between glyceradehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase v ia an enzyme-substrate-enzyme complex. Biochem. 21:4189-4194. White, D.C.S . and J. Thorson. 1972. Phosphate s tarvat ion and the nonlinear dynamics of insect f i b r i l l a r f l i g h t muscle. J . G e n . P h y s i o l . 60:307-336. W i l k i e , D. 1981. Shortage of chemical fuel asa cause of f a t igue : s tudies by nuclear magnetic resonance and b i c y c l e ergometry. Ins Human Muscle Fat igue: phys io log i ca l mechanisms. London: Pitman Medica l , Ciba Symp. 82:102-119. Wil l iamson, J .R. 1965. Metabolic control in the perfused rat heart . In: Control of Energy Metabolism. B. Chance, R.W. Estabrook and J .R. Williamson (Eds . ) . New York: Academic Press , pp. 333-355. Wil l iamson, D . H . , P. Lund and H.A. Krebs. 1967. The redox state of free nicotinamide-adenine d inuc leot ide in the cytoplasm and mitochondria of rat l i v e r . Biochem.J. 103:514-527. Wil l iamson, J .R. 1969. General features of metabolic control as applied to the erythrocyte . Adv .Expt .Med .B io l . 6:117-136. Wilson, D . F . , M. Stubbs, R .L . Veech et a l . 1974. Equi l ibr ium measurements between the ox idat ion-reduct ion react ions and the adenosine tr iphosphate synthesis in suspensions of i so l a ted l i v e r c e l l s . Biochem.J. 140:57-64. Wokoma, A. and I .A. Johnson. 1981. Lactate production at high susta inable c r u i s i n g speeds in rainbow trout (Salmo gairdner i Richardson). J . E x p t . B i o l . 190:361-364. Woodbury, J.W. 1965. Regulation of pH. In: Physiology and B iophys ic s , Ed. C .T . Ruch and H.D. Patton. P h i l a d e l p h i a , P .A. Saunders, pp. 899-934. Wu, T - F . L . and E . J . Davis . 1981. Regulation of g l y c o l y t i c flux in an e n e r g e t i c a l l y cont ro l l ed c e l l - f r e e systems the ef fects of adenine nucleot ide r a t i o s , inorganic phosphate and c i t r a t e . Arch .B ioch . Biophys. 209 s 85-99, 1981. Yamamoto, M. 1968. Fish muscle glycgen phosphorylase. Can.J .Biochem. 46s 423-432. Appendix I Determination of creat ine and phosphocreatine l eve l s in ske le ta l muscle t i s sues at rest and during exercise by i s o c r a t i c anion exchange high performance l i q u i d chromatography. Introduction Phosphocreatine (PCr) and creat ine (Cr) are important components of ske le ta l muscle during bouts of high i n t e n s i t y exercise where PCr serves as an energy substrate . PCr i s very l a b i l e . a n d spec ia l care must be taken to prevent hydro lys i s during storage and preparation of b i o l o g i c a l samples. Lowry et a l . (1964) has described enzymatic procedures for the determination of PCr and Cr but, these methods involve mult ip le enzymes and co fac tor s , besides being tedious . Prev ious ly , Harmen et a l . (1982) has described a method for the separation of creat ine phosphate and the adenine nucleot ides by anion exchange high performance l i q u i d chromatography (HPLC), but each run took considerable time (28 minutes) and was followed by a long column regeneration time. As w e l l , no creat ine values were reported for t h i s method. This was because the bases adenosine, inosine and hypoxanthine overlap the Cr peak g iv ing f a l s e l y elevated values . More r e c e n t l y , McMahon and Lutz (1984) have been able to moniter PCr l eve l s in brain t i s sues by i s o c r a t i c reverse phase, ion pair HPLC. This procedure can be performed in a short time (7 minutes) and appears well sui ted for t i sues e x h i b i t i n g low phosphate l e v e l s . However, the high values of phosphate and phosphorylated compounds present in ske le ta l muscle render t h i s system inappropr ia te . As w e l l , no values are again reported for c r e a t i n e . 155 For the purpose of t h i s Inves t i ga t ion , we have chosen to examine exerc i s ing f i s h muscle s ince the body musculature comprises two f u n c t i o n a l l y and s p a t i a l l y d i f f e rent f iber types, red and white. As w e l l , these t i s sues possess a highly act ive purine nucleot ide cyc le which would re su l t in elevated l e v e l s of IMP and AMP during high i n t e n s i t y exerc i se . The fol lowing procedure describes a rapid and accurate method for the analys i s of PCr, Cr , NAD and the nucleot ide monophosphates in ske le ta l muscle t i s sue by i s o c r a t i c anion exchange HPLC. Methods and Mater ia l s (see Di s se r t a t ion methodology; Animals-Metabolite Studies ; Metabolite Studies ; Metabolite E x t r a c t i o n ; Chromatography) Results and Discuss ion Separation of Cr , PCr, inos ine , adenosine, NAD and nucleot ide monophosphate standards are presented in Figure 1. Figures 2 and 3 demonstrate the hydro lys i s of PCr, a s h i f t to creat ine and an e levat ion of IMP and AMP in white and red muscle r e s p e c t i v e l y , as a re su l t of vigorous phys ica l a c t i v i t y . As previous ly mentioned, f i s h muscle possesses a very act ive purine nucleot ide cyc le which i s responsible for the elevated IMP values which accompany exerc i se . Inosine l eve l s can be seen to have increased within red muscle during the exercise as a re su l t of adenosine deaminase a c t i v i t y , the enzyme responsible for converting adenosine into inos ine . Adenosine has become ava i l ab le during the exercise v ia the act ion of AMP deaminase and the purine nucleot ide c y c l e . However, the use of th i s procedure to assess inos ine l eve l s can only be q u a l i t a t i v e due to the overlapping of the hypoxanthine peak which would be expected to contr ibute approximately a 2-5 percent error at phys io log i ca l l e v e l s . 156 NAD l e v e l s were -Found to be comparable to reported mammalian values determined enzymatical ly U o b s i s and Stainsby 196B[ Sahlin 1985) and did not change in e i ther t i s sue during the exerc i se . Nevertheless , the PCA extrac t ion procedure employed in the t i s sue preparation would have converted a l l the reduced NAD into i t s oxidized form and therefore appropriate precautions must be employed i f t h i s metabolite i s to be assessed. NADH cannot be detected by t h i s procedure due to i t s low e x t i n c t i o n c o e f f i c i e n t at t h i s wavelength. The measured PCr and Cr l eve l s were compared to enzymatical ly determined values in Table 1. The l eve l s obtained by these two methods were comparable with no di f ferences being observed on samples stored at -80 C and those analyzed immediately after e x t r a c t i o n . Table 1. Resting Cr and PCr values in white muscle of rainbow t rou t . Method White Muscle n Cr <5) PCr (5) HPLC 24.6 +2.2 19.9 + 3.5 Enzymatic Assay 27.5 +3.9 17.5 +0.8 Values are mean±SD expressed as umol/g wet weight. By using a lower strength anion exchange column than was employed by Harmsen et a l . (1982), Cr i s separated from the i n t e r f e r i n g bases. As w e l l , the use of an i s o c r a t i c potassium phosphate elutant provides for a more accurate de ter ina t ion of nucleot ide monophosphates, due to lower background noise in comparison to the elevated potassium phosphate l eve l s required for the gradient mode chosen by Harmsen et a l . (19B2). The nucleot ide d i - and tr iphosphates are s trongly bound to the column and require higher concentrat ions of potassium phosphate to be e luted . However with time, these compounds begin to e lute even under the 50 mM potassium phosphate e lu tant . No inter ference from these compounds was observed i f the column was washed with 600 mM potassium phosphate (pH 2.5) for 10 minutes after every hour of use. This metod therefore allows a rapid and sens i t i ve method for the determination of Cr , PCr and nucleot ide monophosphates while s imultaneously monitering the funct ioning of the purine nucleot ide c y c l e . References Bergmeyer, H.U. 1974. Methods in enzymatic a n a l y s i s . New York: Academic Press , pp. 164-167. Harmsen, E . , P.Ph. De Tombe and J.W. De Jong. 1982. Simultaneous determination of myocardial adenine nucleot ides and creatine-phosphate by high-performance l i q u i d chromatography. J.Chromatogr. 230:131-136. Lowry, O . H . , J . V . Passonneau, F .X . Hasselberger and D.W. Schul tz . 1964. J .B io l .Chem. 239:18-30. McMahon, P.M. and P . L . Lutz . 1984. Determination of creat ine phosphate l eve l s in brain t i s sue by i s o c r a t i c reverse-phase, ion-paired high-performance l i q u i d chromatograpy. A n a l . B i o c h . 138:252-254. 158 Figure 1. Separation of Cr , PCr, adenosine, inos ine , NAD and nucleot ide monophosphates (113 u l ) . 1=13.1 mM adenosine; 2=1 mM Cr; 3=0.1 mM inos ine ; 4=1.5 mM CMP; 5=0.1 mM NAD; 6=1.5 mM AMP; 7=1.5 mM UMP; 8=1.5 mM IMP; 9=1 mM PCr; 10=1.5 mM GMP. 2 E c o UJ O Z < CD tr O CO < 8 9 t (min, 10 160 F i g u r e 2. S e p a r a t i o n of C r , P C r , a d e n o s i n e , i n o s i n e , NAD and n u c l e o t i d e m o n o p h o s p h a t e s i n t r o u t w h i t e m u s c l e . UHQIZ 30NV8H0S9V 162 Figure 3. Separation of Cr , PCr, adenosine, inos ine , NAD and nucleot ide monophosphates in trout red muscle. wu OIZ 3 0 N V 9 M O S 8 V Appendix II Assessment of inorganic phosphate l eve l s in freeze clamped frozen and p e r c h l o r i c acid extracts of trout white muscle. The high l e v e l s of inorganic phosphate observed in t h i s study, were considerably higher than expected based on 3 1 P NMR in vivo i n v e s t i g a t i o n s , while being r e l a t i v e l y s imi l a r to enzymatical ly determined inorganic phosphate (Pi) contents . Recently , Meyer et a l . (1985) demonstrated the elevated Pi contents determined enzymatical ly to be a t t r i b u t a b l e to PCr hydro ly s i s upon rapid f r eez ing . To assess t h i s p o s s i b i l i t y in these r e s u l t s , 3 1P NMR spectra of freeze clamped frozen and freeze clamped PCA extracts of trout white muscle were determined. Although these re su l t s are merely q u a l i t a t i v e , i t appears ce r t a in that the colormetr ic assay of Black and Jones (1983) re f l ec ted accurate ly the Pi content in the t i s sues after freeze clamping. Therefore the elevated Pi contents can be a t t r ibuted to the PCr hydro lys i s upon rapid freezing and/or pr io r handling (see attached spectra ) . References Black, M.J . and M.E. Jones. 1983. Inorganic phosphate determination in the presence of l a b i l e phosphate: Assay for carbamyl phosphate phosphatase a c t i v i t y . A n a l . B i o c h . 135:233-238. Meyer, R . A . , T.R. Brown and M.J . Kushmerick. 1985. Phosphorus nuclear magnetic resonance of fa s t - and slow-twitch muscle. A m . J . P h y s i o l . 248:C279-C287. 165 Figure 1. 3 1 P NMR spectra of rainbow trout freeze clamped white muscle at - 5 ° C . j 0 B N - 0 ^ ~ - - 8 5 - 3 2 0 W. P A R K H O U S E S A M P L E F R O Z E N M U S C L E P - 3 1 S T A N D A R D P A R A M E T E R S E X P 1 P U L S E S E Q U E N C E : D A T E 2 2 - U - 8 5 S O L V E N T D 2 0 F I L E P 3 1 S 2 P U L A C Q U I S I T I O N D E C . II V T T N 3 1 . 0 0 0 DN 1 5 0 0 SW 2 0 0 0 0 . 0 D O 0 A T 0 . 7 5 0 DM Y Y Y N P 3 0 0 1 6 DMM S PW 15 0 DMF 6 0 0 0 P I 0 D H P NMN D I 0 D L P 0 D 2 0 T E M P - 5 . 0 T O 0 V T C 2 2 0 NT 1 0 0 0 0 C T 3 0 0 0 P R O C E S S I N G A L F A 2 0 0 S E 0 0 6 4 P A D 0 . 5 0 0 L B 5 . 0 0 0 F B 1 1 0 0 0 F N 3 2 7 6 8 B S 1 0 0 M A T H F S S 0 I L N D I S P L A Y I N N S P - 5 5 8 5 1 D P Y WP 9 7 1 3 7 H S N N V S 1 5 0 S C 1 0 0 WC 4 0 0 I S 3 5 4 R F L 1 0 0 4 4 6 R F P 0 T H 2 0 I N S 1 0 0 0 D C <—\—i—r—|—i—i—i—i—|—i—i—i—i—| i—i i i——i—i—i—|—i—i—n—r 30 20 10 i | i i i i I i i i i | i i i i I i i i i | i i i i I i i i i | i i i ' I 1 1 1 ' | 1 1 1 ' 1 ' 0 -10 -20 -30 -40 PPM 167 Figure 2. 3 1 P NMR spectra of rainbow trout freeze clamped white muscle PCA extract at -5"C. J O B N O 8 5 - 3 1 9 3 1 p F T S P E C T R U M A T P A R K H O U S E p - 3 1 S T A N D A R D P A R A M E T E R S E X P 3 P U L S E S E Q U E N C E S 2 P U L D A T E 2 0 - 1 l - 8 b S O L V E N T H 2 0 F I L E P J 1 A C Q U I S I T I O N D E C &. VT T N 31 0 0 0 DN 1 . bOO SW 2 0 0 0 0 0 DO 0 AT 0 7 b 0 DM Y Y Y NP 3 0 0 1 6 DMM S PW 18 0 DMF 6 0 0 0 P1 0 DHP NNN D1 0 D L P 0 D2 0 T E M P - 5 0 T O 0 V T C 22. 0 NT 4 0 0 0 CT 1 6 0 0 P R O C E S S I N G A L F A 2 0 0 S E 0. 0 3 2 P A D 0 . bOO L B 1 0 . 0 0 0 F B 1 1 0 0 0 F N 3 2 7 6 8 B S 1 0 0 M A T H F S S 0 I L N D I S P L A Y I N N S P - 5 7 0 7 1 DP Y WP 9 7 13 7 HS NN V S 1 5 0 S C 100 WC 4 0 0 IS 3 5 4 R F L 9 b 9 b . b RF P 0 T H 2 0 I N S 1. 0 0 0 DC 1 . - , 0 - 2 0 - 3 0 - 4 0 PPM C0 169 Appendix III A c t i v i t i e s (umol/g wet weight/min) o-f g l y c o l y t i c enzymes in rainbow trout red and white muscle. Enzyme Red Muscle White Muscle Phosphorylase 14-22 48-70 Hexokinase 0.14-2.6 0.03-1.6 Phospho-f ructokinase 9 24 Pyruvate Kinase 110-1225 90-310 Lactate Dehydrogenase 340-800 23-200 Values are from Crabtree and Newsholme (1972)} Snugden and Newsholme (1973)} Johnston (1977). Appendix V S t a t i s t i c a l Analyses The r e s u l t s of t h i s i n v e s t i g a t i o n can only discussed in d e s c r i p t i v e terms because of the large number of var iab le s and r e l a t i v e l y small sample s i ze per group would render the v a l i d i t y of any s t a t i s t i c a l anayl s i s quest ionable . However, as a check of the trends discussed, an ana lys i s of variance was used to evaluate poss ib le intergroup d i f f e rences . The Tukey (HSD) test was used on var iab le s exh ib i t ing s i g n i f i c a n t omnibus F r a t i o s to i d e n t i f y where group di f ferences ex i s ted . Content of metabolites and pH in rainbow trout white muscle. Metabol i te Pre-ex PSS-30 PSS-7 ES n (5) (6) (5) (5) PCr 19.9*1.6 15.9*0.9 c 2 .9*0.7 b l . B * 0 . 6 b PCr = 37.8+1.6 c 33.8+0.9 c 20.8*0.7 c 1.8+0.6 c Cr 24.6+1.0 29.7*1.4 a 42.7*0.2 b 43.4*1.8 b C r c 6.7+0.3 c 11.8+1.4 c 24.8+0.2 c 43.4+1.8 c ATP 7.26+0.11 6.57+0.22 5.82+0.5 a 2.65+0.25 c ADP 0.70+0.01 0.68*0.04 1.15*0.07 b 1.05*0.05 b AMP 0.021+0.001 0.039*0.007 0.073*0.012 0.152*0.035 c Pi 21.1+2.6 c 26.8+1.9 c 47.6+3.2 c 55.7+1.8 c P i c 2.3+0.4 c 8.9+1.4 c 29.7+2.8 c 55.7+1.8 c IMP 0.30+0.05 0.53+0.09 1.78*0.27 c 4.34+0.26 c NH; 1.04+0.05 1.43*0.15 3.20*0.42 c 6.37*0.19 c GTP 0.054*0.012 0.039*0.003 b 0.037*0.004 b 0.030+0.007 b Glucose 1.02+0.14 1.86+0.55 2.19+0.51 2.16+0.44 Glycogen 23.3+1.0 c 16.0+1.6 c 5.6+1.1 c 0.2+0.04 c Lactate 3.0+0.4 c 10.1+1.1 c 33.0+0.6 c 42.9+3.0 c Malate 0. 13+0.07 0.25+0.07 0.25+0.03 0.39+0.06 b Fumarate 0.01+0.002 0.04+0.01 a 0.06+0.01 b 0.08+0.01 b C i t r a t e 0.30+0.01 0.28+0.02 0.35+0.05 0.2Bt0.03 pH 6.97+0.04 6.93+0.03 6.65+0.03 b 6.56+0.04 b Values are means + SE expressed in umol/g (w/w). Glycogen was ca lcu la ted glucose u n i t s , c, compensated metabol i te . a S i g n i f i c a n t l y (p<0.05) d i f f e rent from Pre-ex b S i g n i f i c a n t l y (p<0.05) d i f f e rent from Pre-ex and PSS-30 c S i g n i f i c a n t l y (p<0.05) d i f f e rent from other 3 groups Content of metabolites and pH in rainbow trout red muscle. Metabol i te n Pre-ex (5) PSS-30 (6) PSS-7 (5) ES (5) PCr 5.2+0.7 d 0.810.4 a 1.210.2 a 0.410.2 a PCr c 18.6+0.8 d 0.810.4 a 1.210.2 a 0.410.2 a Cr 22.111.0 d 26.510.8 a 26.011.9 a 26.510.3 a C r c 8.810.4 d 26.510.8 a 26.011.9 a 26.5+0.3 a ATP 3.43+0.18 d 2.02+0.27 2.2610.32 1.57+0.32 ADP 0.6510.07 1.1810.08 c 0.8410.04 1.1110.17 c AMP 0.10610.012 0.13710.014 0.20710.023 a 0.32110.055 d Pi 14.111.2 d 21.112.9 a 21.512.4 a 22.712.1 a Pic 2.311.3 d 21.112.9 a 21.512.4 a 22.712.1 a IMP 0.729+0. 1 10 1.090+0.180 1.31310.145 a 2.33010.278 d N H : 1.6010.14 1.5710.16 1.7110.08 2.8810.30 d GTP 0.05910.003 0.03310.007 c 0.04910.002 0.03410.003 c Glucose 1.4610.19 3.3810.55 a 2.1110.14 2.9510.05 a Glycogen 18.112.5 d 0.610.3 a 0.510.2 a (0 .1 a Lactate 5.210.8 8.811.1 c 7.310.9 10.B+1.4 c Malate 0.2010. 10 0.3210.03 0.4010.05 a 0.6510.1 d Fumarate 0.0310.01 0.0410.01 0.08+0.02 b 0.08+0.02 b C i t r a t e 0.40+0.05 0.3410.05 0.44+0.05 0.4410.09 P H 6.89+0.02 6.9210.04 6.8810.02 6.81 (1) Values are means 1 SE expressed as umol/g (w/w). Glycogen was ca lcu la ted glucose u n i t s , c, compensated metaboli tes . a S i g n i f i c a n t l y (p<0.05) d i f f e rent from Pre-ex b S i g n i f i c a n t l y (p<0.05) d i f f e rent from Pre-ex and PSS-30 c S i g n i f i c a n t l y (p<0.05) d i f f e rent from Pre-ex and PSS-7 d S i g n i f i c a n t l y d i f fe rent from other 3 groups Content of metabolites in rainbow trout l i v e r . Metabol i te Pre-ex PSS-30 PSS-7 ES n (5) (6) (5) (5) Glycogen 183.6+35.6 176.3+34.1 157.7136.8 121.118.5 b Glucose 3.53+0.85 4.69+0.51 11.1411.22 b 11.4411.12 b G6P 0.12+0.03 0 . 2 0 ± 0 . 0 4 0.7710.06 b 0.8210.07 b F6P 0.02+0.01 0.04+0.01 0.12+0.01 b 0.1410.02 b 2PG 0.09+0.01 0.08+0.01 0.0810.01 0.0810.01 PEP 0.11+0.01 0. 11+0.02 0.0810.004 0.0610.004 c Pyruvate 0.17+0.01 0.15+0.01 0.1610.01 0.1410.01 Lactate 1.5+0.2 2.210.1 2. 1+0.3 4.0+0.6 c Alanine 3.54+0.41 3.0310.94 1.5510.28 b 2.4810.39 Values are mean + SE expressed as umol/g (w/w). Glycogen was ca lcu la ted glucosyl u n i t s . a S i g n i f i c a n t l y (p<0.05) d i f f e rent from Pre-ex b S i g n i f i c a n t l y (p<0.05) d i f f e rent from Pre-ex and PSS-30 c S i g n i f i c a n t l y (p<0.05) d i f f e rent from other 3 groups Content of g l y c o l y t i c intermediates in rainbow trout white muscle. Intermedi ate Pre-ex PSS-30 PSS-7 ES n (5) (6) (5) (5) G1P 0.25+0.06 0.40+0.08 0.64+0.04 c 0.31+0.07 G6P 0.59*0.97 0.97+0.16 1.71+0.21 c 0.73+0.24 F6P 0.07+0.01 0.15 + 0.03 0.27+0.04 c 0.15+0.05 FDP 1.28+0. 13 1.90+0.12 a 1 . 6 9 ± 0 . 3 1 0.3210.08 DHAP 0.21+0.03 0.19 + 0.02 0.23+0.03 0.1710.02 GP 0.50+0.03 0.52+0.08 0.80+0.02 b 0.95+0.11 b GAP 0 . 0 5 ± 0 . 0 1 0 . 0 6 ± 0 . 0 2 0.003+0.005 0.0310.01 GAP, 0 . 0 1 8 ± 0 . 0 0 2 0.017+0.002 0.022+0.003 0.017+0.002 DPG 0.06+0.01 0.08+0.02 0.06+0.01 0.1610.03 c 3PG 0.5510.05 0.59+0.03 0.9610.11 0.32+0.01 c 2PG 0.08+0.02 0.08+0.02 0.06+0.01 0.01+0.004 PEP 0.06+0.004 0.04+0.01 0.0410.003 0.0110.003 PYR 0.03+0.005 0.10+0.02 a 0.14+0.02 a 0.3310.05 c Values are meanlSE expressed as umol/g w/w. a S i g n i f i c a n t l y (p<0.05) d i f f e rent from Pre-ex b S i g n i f i c a n t l y (p<0.05) d i f f e rent from Pre-ex and PSS-30 c S i g n i f i c a n t l y (p<0.05) d i f f e rent from other 3 groups Content of g l y c o l y t i c intermediates in rainbow trout red muscle. Intermedi ate Pre-ex PSS-30 PSS-7 ES n (5) (6) (5) (5) 61P 0.41+0.02 c 0.20+0.06 a 0.18+0.06 a 0.1410.03 a G6P 0.45*0.08 0.5410.10 0.31+0.04 0.33+0.06 F6P 0.1010.01 0.0810.02 0.0410.004 b 0.0210.004 b FDP 0.55+0.09 c 0.2610.02 a 0.2510.03 a 0.1210.03 a DHAP 0.19+0.05 0.1910.05 0.1510.06 0.1410.02 GP 0.62+0.10 0.7110.16 0.9810.55 1.2110.32 GAP 0.03+0.02 0.04+0.01 0.04+0.02 0.06+0.01 GAP, 0.01710.002 0.016+0.004 0.013+0.005 0.015+0.002 DPG 0.06+0.01 0.05+0.01 0.0510.01 0.0610.01 3PG 0.16+0.03 0.2010.03 0.20+0.05 0.1110.05 2PG 0.11+0.02 c 0.05+0.01 a 0.0510.01 a 0.0310.01 a PEP 0 . 0 4 ± 0 . 0 1 0.03+0.01 0.03+0.01 0.02+0.01 PYR 0.05+0.01 0.07+0.01 0.10+0.02 b 0.11+0.002 b Values are mean+SE expressed as umol/g w/w. a S i g n i f i c a n t l y <p<0.05) d i f f e rent from Pre-ex b S i g n i f i c a n t l y (p<0.05) d i f f e rent from Pre-ex and PSS-30 c S i g n i f i c a n t l y (p<0.05) d i f f e rent from other 3 groups Measured and ca lcu la ted free cytoplasmic ADP content, ATP/ADP r a t i o and c y t o s o l i c phosphorylation po tent i a l s in rainbow trout muscle. Muscle Pre-ex n (5) PSS-30 (6) PSS-7 (5) ES (5) ADPm W 0.7010.01 0.6810.04 1.1510.07 b 1.0510.09 (umol/g) R 0.6510.01 1. 18+0.08 c 0.839+0.04 1.1110.17 i ADP-f m W 0.05710.004 0.06510.004 0.290+0.063 b 0.203+0.056 (umollq) R 0.07310.012 c 0.189+0.010 a 0.22910.022 a 0.22910.021 ADP,= W 0.00710.001 0.012+0.002 0.019+0.002 a 0.203+0.056 (umol/g) R 0.00810.001 c 0.18910.010 a 0.229+0.022 a 0.229+0.021 ATP/ADPm W 10.410.2 9.810.2 5.1+0.4 c 2.6+0.2 c R 5.4+05 c 1.7+0.2 a 2.710.4 a 1.510.4 a ATP/ADP, m "W 129.6+9.5 103.014.5 24.515.9 b 18.1+5. 1 b R 52.4+8.9 c 10.6+1.3 a 10.411.6 a 7.5*2.1 a ATP/ADP, c W 1025198 c 582166 c 312121 c 1 B . H 5 . 1 c R 457+24 c 10.611.3 a 10.411.6 a 7.512.1 a ATP W 553173 c 371+24 c 110112 c 4613 c ADP m .P i (M - 1 ) R 383133 c 149121 a 140120 a 74123 a ATP W 6950+1100 c 39381367 c 500+164 b 316+81 b A D P , m . P i (M~1) R 36821513 c 813196 a 5421103 a 3701127 c ATP W 451200+44800 c 60900+8900 c 1100011400 c 316181 c A D P , c . P i c (M - 1 ) R 155000+30700 c 813196 a 5421103 a 3701127 b Values are meantSE. W, white muscle; R, red muscle. a S i g n i f i c a n t l y <p<0.05) d i f f e rent from Pre-ex b S i g n i f i c a n t l y (p<0.05) d i f f e rent from Pre-ex and PSS-30 c S i g n i f i c a n t l y <p<0.05) d i f f e rent from other 3 groups Calculated free c y t o s o l i c redox state and PCr/Pi r a t i o ' s in rainbow trout muscie. Muscle Pre-ex n (5) PSS-30 (6) PSS-7 (5) ES (5) NAD/NADH 749+153 961+145 1068+82 888+132 828+106 2027+173 c 2076+458 c 1525+142 c PCr/Pi W 1.04+0.12 c 0.6+0.06 c 0.06+0.02 b 0.0310.01 b R 0.3710.04 c 0.0410.02 a 0.0610.01 a 0.0210.01 a PCr. Pi = 16.811.5 c 8.110.4 c 3.410.3 c 0.0410.02 a 0.7+0.1 c 0.06+0.01 a 0.0310.01 0.0210.01 a Values are mean+SE. W, white muscle; R, red muscle. a S i g n i f i c a n t l y <p<0.05) d i f f e rent from Pre-ex b S i g n i f i c a n t l y (p<0.05) d i f f e rent from Pre-ex and PSS-30 c S i g n i f i c a n t l y (p<0.05) d i f f e rent from other 3 groups PUBLICATIONS 1. Abe, H., G.P. Dobson, U. Hoeger and W.S. Parkhouse. 1985. The role of h i s t i d i n e and h i s t i d i n e - r e l a t e d compounds to i n t r a c e l l u l a r buffering i n f i s h muscle. Am. J . Phy s i o l . 249:R449-R454. 2. Parkhouse, W.S., D.C. McKenzie, P.W. Hochachka and W.K. Ovalle. 1985. Buffer capacity of deproteinized human s k e l e t a l muscle. J . Appl. P h y s i o l . 58:14-17. 3. McKenzie, D.C, W.S. Parkhouse, E.C. Rhodes, W.K. Ovalle and S.L. Shinn. 1985. Anaerobic capacity and muscle f i b e r type. In: Current selected research i n exercise physiology. C O. Dotson and J.H. Humphrey (eds.), AMS Press Inc., 1:23-29. 4. Parkhouse, W.S. and D.C. McKenzie. 1984. Possible contribution of s k e l e t a l muscle buffers to enhanced anaerobic performance: a b r i e f review. Med. S c i . Sp. 16:328-338. 5. Parkhouse, W.S., D.C. McKenzie, P.W. Hochachka, W.K. Ovalle, T.P. Mommsen and S.L. Shinn. 1983. The re l a t i o n s h i p between carnosine l e v e l s , buffering capacity, f i b e r type and anaerobic capacity i n e l i t e a t h l e t e s . In: Biochemistry of Exercise. H. Knuttgen, J . Vogel and J. Poortmans (eds.), Human Kinetics Pub., Champaign, I I . , 590-594. 6. McKenzie, D.C, W.S. Parkhouse, E.C. Rhodes, P.W. Hochachka, W.K. Ovalle, T.P. Mommsen and S.L. Shinn. 1983. Skeletal muscle buffering capacity i n e l i t e a t h l e t e s . In: Biochemistry of Exercise. H. Knuttgen, J . Vogel and J . Poortmans (eds.), Human Kine t i c s Pub., Champaign, I I . , pp. 584-589. 7. Parkhouse, W.S., D.C. McKenzie, E.C. Rhodes, D. Dunwoody and P. Wiley. 1982. Cardiac frequency and anaerobic threshold: Implications for p r e s c r i p t i v e exercise programs. Eur. J . Appl. P h y s i o l . 50:117-123. 8. McKenzie, D.C, W.S. Parkhouse and W.E. Hearst. 1982. Anaerobic performance c h a r a c t e r i s t i c s of e l i t e Canadian 800 meter runners. Can. J . Appl. Sport S c i . 7:158-160. 

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