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Respiratory properties of mitochondria from heart and mosaic muscle of rainbow trout (Salmo gairdneri)… Donaldson, Judith Margaret 1985

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RESPIRATORY PROPERTIES OF MITOCHONDRIA FROM HEART AND MOSAIC MUSCLE OF RAINBOW TROUT (Salmo q a i r d n e r i ) s SUBSTRATE UTILIZATION AND RESPONSE TO TEMPERATURE AND EXTRAMITOCHONDRIAL pH By JUDITH MARGARET DONALDSON B.Sc., Mount A l l i s o n U n i v e r s i t y , N.B., 19B1 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES ZOOLOGY DEPARTMENT We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA O c t o b e r 1985 ©Judith M a r g a r e t D o n a l d s o n , 1985 90 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Zoology  The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date October 12, 1985 DE-6(3/81) i i ABSTRACT Mitochondria were i so la ted -from heart and mosaic muscle o-f rainbow t rout (Salmo qai rdner i R. ) . State 3 resp i ra to ry rates were determined at 5 and 15°C using pyruvate, malate, l a c t a t e , glutamate or acety l—carn i t ine as substrate . The -f inal three substrates were used to generate pH p r o f i l e s . Pyruvate was ox id ized at high rates in a l l cases , i n d i c a t i n g good potent ia l for aerobic carbohydrate metabolism. At 15°C, malate was an equal ly good substrate for heart mitochondria, while a l l substrates were ox id ized at s i m i l a r rates to pyruvate in muscle mitochondria. Maximal ox idat ion ra tes of heart mitochondria were greater than or equal to those of muscle. State 3 D i o for ox idat ion of most substrates in heart was approximately 2 , except fo r malate which had a Q i o of 3 . Mitochondrial ox idat ion tended to be more s e n s i t i v e to decreased temperature in muscle than in hear t , p a r t i c u l a r l y with respect to acety l—carn i t ine and glutamate oxidat ion which in muscle had Q ± o values of 4 and 7, respec t i ve l y . Based on RCR values at 5 and 15*= ,^ there was no i n d i c a t i o n that membrane permeabi l i ty to H"*" ions was a l te red by a 10C 0 change in temperature in mitochondria from e i ther t i s s u e . At pH above 7.6 resp i ra to ry rates decreased with increasing pH. State 3 resp i ra to ry ra te increased in heart mitochondria as pH decreased, below 7.6 while in muscle m i t o c h o n d r i a , no s u c h pH d e p e n d e n c e was o b s e r v e d . RCR v a l u e s were above 4 i n a l l e x p e r i m e n t s e x c e p t a t h i g h pH. M u s c l e m i t o c h o n d r i a were t h e more s e n s i t i v e t o e x t r e m e pH w i t h r e s p e c t t o RCR. H e a r t m i t o c h o n d r i a had h i g h e r o x i d a t i v e r a t e s t h a n t h o s e o f m u s c l e and were l e s s s e n s i t i v e t o d e c r e a s e d t e m p e r a t u r e , i n k e e p i n g w i t h t h e t h e g r e a t e r o x i d a t i v e demands o f t h a t t i s s u e r e l a t i v e t o m o s a i c m u s c l e . M u s c l e m i t o c h o n d r i a w h i c h t y p i c a l l y f a c e l a r g e r f l u c t u a t i o n s i n e x t r a m i t o c h o n d r i a l pH i n v i v o t h a n do t h o s e o f h e a r t , were l e s s s e n s i t i v e t o pH i n v i t r o . I t was c o n c l u d e d t h a t s u b s t r a t e u t i l i z a t i o n p a t t e r n s and r e s p o n s e t o c h a n g e s i n t e m p e r a t u r e and e x t r a m i t o c h o n d r i a l pH i n t h e two m i t o c h o n d r i a l p o p u l a t i o n s was d i f f e r e n t and r e f l e c t e d b o t h t h e i n t r a c e l l u l a r e n v i r o n m e n t o f t h e m i t o c h o n d r i a and t h e d i f f e r e n t n e e d s o f e a c h t i s s u e f o r a e r o b i c e n e r g y s u p p l y . i v TABLE QF CONTENTS L i s t of F igures . . . . . . v L i s t of Tables . . . . . . . v i Acknowledgements . . . . . . v i i Introduction . . . . . . . 1 Mater ia ls and Methods . . . . . 7 Chemicals . . . . . . . 7 I so lat ion of Intact Mitochondria . . 8 Protein Determination . . . . 10 0 3 Consumption Rates . . . . 11 Spetrophotometric Check of LDH Reaction . 15 Results . . . . . . . . 16 Substrate U t i l i z a t i o n . . . . 20 Temperature . . . . . . 39 E f fec t of pH . . . . . 51 Discussion . . . . . . . 64 Substrate U t i l i z a t i o n . . . . 65 Temperature . . . . . . 75 E f f e c t of pH . . . . . Bl Conclusions . . . . . . 90 L i t e r a t u r e C i ted . . . . . . 93 V LIST OF FIGURES 1. 0 3 consumption — exogenous NADH . . . . 2 1 2. 0 3 consumption — succinate . . . . . 23 3. Mitochondrial i n h i b i t o r s and uncouplers . . . 25 4. Os consumption — malate "spark" . . . . 2 7 5. State 3 0 3 consumption and RCR at 15 0C . . 30 6. State 4 0 2 consumption at lS^C . . . . 3 2 7. State 3 consumption at 15°Cs Heart vs . mosaic muscle . . . . . 35 8. State3 0 2 consumption vs . pyruvate concentrat ion . . . . . . . 37 9. D 2 consumption — l i m i t i n g concentrat ions o-f pyruvate . . . . . . . 40 10. State 3 D 2 consumption at 5 and 15 0C . . . 42 11. State 4 D 3 consumption and RCR at 5°C . . . 4 5 12. State 3 0 3 consumption and RCR at 5 and 15°C: Heart vs . mosaic muscle . . 49 13. State 3 Q 3 consumption pH p r o f i l e s : Acety l—carn i t ine and glutamate . . . 52 14. State 3 0=. consumption pH p r o f i l e s : Lactate . . . . . . . . 54 15. Lactate ox idat ion at var ious pH values . . . 57 16. State 4 0 Z consumption and RCR pH p r o f i l e s - . 59 17. Malate ox idat ion as a funct ion of pH . . . 62 vi LIST OF TABLES 1. State 3 O z consumption, RCR and ADP/O at 15DC . . . . . . . . 17 2. Lactate s ta te 3 oxidat ion rates with 2 and lOmM NAD* . . . . . . IS 3. Conversion of l a c t a t e to pyruvate as a funct ion of CNAD~3 . . . 19 4. State 3 and s ta te 4 Q , 0 values . . . . 4 8 5. Some s tate 3 oxidat ion rates from var ious species . . . . . . 72 6. Comparison of s tate 3 oxidat ion rates from blowfly and locust fa t bodies . . . 73 ACKNOWLEDGEMENTS I wish f i r s t to thank my superv isor , Peter Hochachka, for h i s advice and support throughout the course of t h i s work. I would a lso l i k e to thank the members of my committee, Drs. John Gosl ine and John P h i l l i p s , for many he lpfu l suggestions on the manuscript. Thanks are due a lso to the members of The Lab, p a r t i c u l a r l y Je f f Dunn who i s no longer in The Lab, who provided both encouragement and technica l ass i s tance . Specia l thanks to Tom Mommsen for h i s invaluable ass is tance both with the techn ica l aspects of the study and for h i s unl imited patience in d iscuss ing the manuscript. F i n a l l y , I would e s p e c i a l l y l i k e to thank my husband, Michael C a s t e l l i n i , for h i s pat ience in helping me with the thousand l i t t l e chores involved in producing the f i n a l manuscript and for h i s tremendous support throughout the course of my s tud ies . 1 INTRODUCTION Modulation and adaptation of metabolic pathways i s a well estab l ished means for al lowing t i s s u e s to f u l f i l l s p e c i f i c funct ions . In order to accomplish t h i s the c e l l u l a r environment in which metabolism occurs must be taken in to cons iderat ion . Two major environmental fac to rs a f f e c t i n g poiki lotherms - temperature and i n t r a c e l l u l a r pH - have f a r - r e a c h i n g e f f e c t s on enzyme k i n e t i c s (Somero, 1981p White and Somero,1982), membrane s t ructure (Hazel and Prosser ,1974; Hazel ,1984) , t ransport (Halestrap,1978; Simpson,19BO) and, consequently, metabolic r a t e . The energy required for t i s s u e funct ion i s derived e i ther a e r o b i c a l l y or g l y c o l y t i c a l l y and the aim of t h i s study i s to compare ox idat ive metabolism as an energy source for two t i s s u e types with d i f f e r e n t ox idat ive requirements and determine how t h i s funct ion i s a f fected by environmental fac to rs such as temperature and pH. In order to accomplish t h i s , several aspects of mitochondrial r e s p i r a t i o n w i l l be invest igated . The f i r s t i s whether mitochondria from one t i s s u e exh ib i t higher maximal rates of r e s p i r a t i o n and how t h i s r e l a t e s to ox idat ive capac i ty . Secondly, which substrates are burned best ( ie . fas test ) in i s o l a t e d mitochondria? T h i r d l y , what are the e f f e c t s of temperature and pH on ox idat ive phosphorylation? F i n a l l y , are the aforementioned proper t ies t i s sue s p e c i f i c ? Apart from the d i r e c t e f f e c t of temperature on physical chemistry and enzyme Michaelis—Menten constants <K m >, temperature induced a l t e r a t i o n in membrane s t ructure may d isrupt the a c t i v i t i e s o-f membrane associated enzymes and transport processes (Hazel and Prosser ,1974; Hochachka and Somero,1973; Hirano et a l . , 1 9 8 1 ; T h i l o et a l . , 1 9 7 7 ) . Th is i s s i g n i f i c a n t s ince t ransporters and many mitochondrial enzymes are membrane-bound. Modulation of membrane-bound enzymes i s often ind icated by d i s c o n t i n u i t i e s in Arrhenius p l o t s which have been documented for some mitochondrial membrane-bound enzyme systems in poiki lotherms (Wodtke,1976; I rv ing and Watson,1976). The potent ia l e f f e c t s of a l te red i n t r a c e l l u l a r pH on mitochondrial r e s p i r a t i o n are many. Lowered intramitochondr ia l pH would a f f e c t mitochondrial enzymes which tend to have high pH optima (Mela et a l . , 1 9 7 2 ; D i P r i s c o , 1 9 7 5 ; ) . H* ions are involved in the exchange of ADP/ATP across the inner mitochondrial membrane (see Hinkle and Yu,1979) as well as other t ransport processes (Halestrap,1978; Nicholas et a l . , 1 9 7 4 ; Simpson,1980). The H* ion gradient p lays a fur ther r o l e by coupl ing e lect ron transport to ATP formation (Mitchel1,1961; Mi tche l l and Moyle,1968). Rate of ox idat ive phosphorylation i s d i r e c t l y re la ted to the magnitude of the pH gradient (see Hinkle and McCarty,1978). T issues are known to ox id ize substrates d i f f e r e n t i a l l y and mitochondria i s o l a t e d from d i f f e r e n t sources often have d i f f e r e n t resp i ra to ry proper t ies which r e f l e c t in v ivo t i s s u e func t ion . For example seal heart not only has fewer mitochondria per gram of t i s s u e than dog heart but the 3 mitochondria themselves have lower ac t i ve resp i ra to ry rates and lower cytochrome contents (Sordahl et a l . , 1 9 8 3 ) . Even when r e s p i r a t i o n i s expressed as a -function of cytochrome content , mitochondria i s o l a t e d from the same t i s s u e in d i f f e r e n t species may produce d i f f e r e n t maximal rates as has been observed in fa t body of blowfly and locust (Ballantyne and Storey,1983) . S imi la r observations were made with var ious t i s s u e s from a s i n g l e spec ies . Guinea pig white muscle mitochondria exhib i ted slower ra tes of r e s p i r a t i o n than red with a l l substrates except a -g lycerophsophate, i n d i c a t i n g a r e l a t i v e l y ac t i ve a-glycerophosphate shut t le in white muscle (Blanchaer,1964). Mitochondria from rabbi t red muscle exhib i ted higher resp i ra to ry rates than those from white muscle with e i ther acety l—carn i t ine or p a l m i t o y l - c a r n i t i n e as substrate (Pande and Blanchaer,1971), i n d i c a t i n g that fa t metabolism plays a larger r o l e in red muscle. It has been s i m i l a r l y demonstrated in fa t body of blowfly and locust (Ballantyne and Storey,1983) and locust rectum (Chamberlin and P h i l l i p s , 1 9 8 3 ) that t i s s u e funct ion and substrate preference are r e f l e c t e d in mitochondrial substrate u t i 1 i z a t i on. The t i ssues chosen for t h i s study were heart and mosaic muscle from rainbow trout (Salmo q a i r d n e r i ) . Oxidat ive metabolism i s the major energy source in t rout heart . Heart t i s s u e demands a constant energy supply and therefore the a b i l i t y to maintain high rates of ox idat ive phosphorylation i s mandatory. The bulk of the mosaic 4 muscle i s composed o-f white -f ibres which are interspersed with small -diameter red -f ibres (Boddeke et a l . , 1 9 5 9 ) . According to ATPase and succ in inc dehydrogenase a c t i v i t i e s , these mosaic muscle red -f ibres const i tu te a d i f f e r e n t f i b r e type than those of s u p e r f i c i a l muscle (Johnston et a l . , 1 9 7 5 ) . In genera l , the s u p e r f i c i a l red muscle of the te leos t i s ac t i ve at slow c r u i s i n g speeds (Hudson,1973). At high c r u i s i n g speeds mosaic muscle i s a l so rec ru i ted (Webb,1970; Hudson,1973) and der ives energy from ox idat ive sources. White f i b r e s are a lso capable of producing energy anaerobica l l y for short per iods . C l e a r l y , depending on the a c t i v i t y of the f i s h , mosaic muscle may or may not require energy from ox idat ive phosphorylat ion. N u t r i t i o n a l l y , carbohydrates seem to play a l e s s important r o l e in f i s h than in mammals. Many f i s h , inc lud ing t r o u t , th r i ve on h igh -prote in d i e t s and der ive energy from oxidat ion of l i p i d s or glucose derived through gluconeogenesis from amino ac ids (Love,1980). Teleost hearts contain the enzymes necessary to u t i l i z e carbohydrates, f a t t y ac ids and acetoacetate (Zammit and Newsholme,1979; Dr iedz ic and Stewart,19B2; Hansen and S ide l1 ,1983) . In addi t ion te leos t plasma contains mi l l imolar concentrat ions of g lucose, l a c t a t e and f a t t y ac ids (Zammit and Newsholme,1979; Larsson and Fange,1977). S i g n i f i c a n t c o n t r a c t i l e f a i l u r e in fue l -depr i ved sea raven hearts could be prevented by inc lus ion of lOmM glucose or l.OmM palmitate in the perfusion medium (Driedzic and Hart ,1984). Lactate ox idat ion in f i s h hearts appears to be c o n t r o l l e d by l a c t a t e o x i d a s e a c t i v i t y ( D r i e d z i c e t a l . , 1984). The m o s a i c m u s c l e o f r a i n b o w t r o u t , t h o u g h l a r g e l y w h i t e m u s c l e , i s c a p a b l e o f some o x i d a t i v e m e t a b o l i s m . P e r f u s i o n o f t h e h i n d p a r t o f r a i n b o w t r o u t showed t h a t e x o g e n o u s g l u c o s e , a c e t o a c e t i c a c i d and 3 - h y d r o x y - b u t y r i c a c i d were c o n v e r t e d t o C 0 Z more q u i c k l y t h a n t h r e e amino a c i d s ( i s o l e u c i n e , a l a n i n e , g l u t a m i c a c i d ) , a l t h o u g h t h e main s o u r c e o f c a r b o n f o r C Q a p r o d u c t i o n was endogenous (Moen and K l u n g s o y r , 1 9 8 1 ) . M o s a i c m u s c l e may h a v e c o n t r i b u t e d o n l y m i n i m a l l y t o t h i s t o t a l C Q Z p r o d u c t i o n s i n c e t h e p r o p o r t i o n o f m i t o c h o n d r i a i n r e d t o m o s a i c m u s c l e i s 7:3 (Nag,1972) and s i n c e v a s c u l a r i z a t i o n o f m o s a i c m u s c l e i s l i m i t e d r e l a t i v e t o r e d . M i t o c h o n d r i a l s u b s t r a t e p r e f e r e n c e may s h e d more l i g h t on o x i d a t i v e s u b s t r a t e u t i l i z a t i o n i n m o s a i c m u s c l e . B o t h h e a r t and m u s c l e e x p e r i e n c e t h e same d e g r e e o f t e m p e r a t u r e f l u c t u a t i o n s , however, m e t a b o l i c - m o d u l a t i o n may v a r y s i n c e t h e t i s s u e s h a v e d i f f e r e n t o x i d a t i v e r e q u i r e m e n t s . B l o o d and i n t r a c e l l u l a r pH may v a r y w i t h t e m p e r a t u r e i n p o i k i l o t h e r m s (Reeves,1972; Rahn e t a l . , 1 9 7 5 ; R e e v e s , 1 9 7 7 ) . In f i s h t h e c h a n g e o f pH w i t h a c h a n g e o f t e m p e r a t u r e (ApH/AT) t e n d s t o be h i g h e r i n w h i t e m u s c l e t h a n i n h e a r t ( s e e H e i s l e r , 1 9 8 4 ) . W h i t e m u s c l e a l s o e x p e r i e n c e s l a r g e r i n t r a c e l l u l a r pH c h a n g e s d u r i n g e x e r c i s e s i n c e i t r e l i e s much more h e a v i l y on a n a e r o b i c m e t a b o l i s m t h a n d o e s h e a r t . In a d d i t i o n w h i t e m u s c l e i s n o t a s w e l l p e r f u s e d a s h e a r t , t h e r e b y r e s t r i c t i n g c l e a r a n c e o f p r o t o n s and o t h e r e n d — p r o d u c t s . 6 Thus condi t ions -for mitochondrial -function in t rout d i f f e r s u b s t a n t i a l l y in mosaic muscle and heart . From previous work one might a n t i c i p a t e that the two mitochondrial populat ions would d isp lay d i f f e r e n t substrate preferences. A d d i t i o n a l l y one might expect heart mitochondria to be l e s s temperature s e n s i t i v e in keeping with a constant demand for ox idat ive metabolism in heart . Since the two t i s s u e s are subject to d i f f e r e n t f l u c t u a t i o n s in i n t r a c e l l u l a r pH, one might expect d i f f e r e n t pH s e n s i t i v i t i e s between the two mitochondrial populat ions. Accordingly , mitochondrial r e s p i r a t i o n w i l l be character ized according to maximal ra tes of ox idat ion , substrate u t i l i z a t i o n pat terns , and response to temperature and extramitochondrial pH. An attempt w i l l be made to r e l a t e the q u a l i t i e s of mitochondrial r e s p i r a t i o n to the s p e c i f i c funct ion of the t i s s u e in v ivo . 7 MATERIALS AND METHODS Mitochondria were i s o l a t e d from heart and mosaic muscle of rainbow trout (Salmo qai rdner i R. ) . Trout of both sexes weighing 250-500g were obtained from a commercial f i s h farm. They were kept in aerated running water at 5 - 15 0C and fed d a i l y with 1/4 inch Clark f i s h p e l l e t s (Moore-Clark C o . , Sa l t Lake C i t y , Utah). In a l l experiments except those designed to generate pH p r o f i l e s , the bulk of the data were c o l l e c t e d during the summer months - from l a t e May to mid-September. The data from each t i s s u e do inc lude , however, a few measurements made in ear l y sp r ing . The data for heart pH p r o f i l e s were c o l l e c t e d in May and June of 1984 while the analogous measurements fo r mosaic muscle were made l a t e the fo l lowing winter . Because of pass ib le seasonal d i f fe rences in the pH study, absolute ra tes of mitochondrial r e s p i r a t i o n for each t i s s u e are never compared d i r e c t l y — merely the i r pattern of change with changing pH. CHEMICALS A c e t y l - D L - c a r n i t i n e HC1, d in i t rophenol (DNP), o l igomycin , rotenone, sodium s a l t of adenosine 5 ' -d iphosphate (ATP), B—nicotinamide adenine d inuc leot ide (NAD"*") , B—nicotinamide adenine d inuc leot ide (reduced) (NADH), pyruvate, L—malate, L—glutamate, succ inate , l a c t a t e (free a c i d ) , l a c t a t e dehydrogenase (porcine heart) (LDH), protease (P5255), bovine serum albumin (Fract ion V e s s e n t i a l l y f a t t y ac id free) (BSA) and sucrose (ribonuclease—free) were purchased from Sigma Chemical C o . , 8 S t . L o u i s MO. A l l o t h e r c h e m i c a l s u s e d were p u r c h a s e d -from v a r i o u s c o m m e r c i a l s o u r c e s and were o f a n a l y t i c a l g r a d e . ISOLATION OF INTACT MITOCHONDRIA T r o u t were k i l l e d by d e c a p i t a t i o n and t h e i r h e a d s were i m m e d i a t e l y p i t h e d . A l l i s o l a t i o n s t e p s beyond d i s s e c t i o n were c a r r i e d o u t a t 0 - 4«=*C. H e a r t s were removed e a s i l y w i t h f o r c e p s and washed i n i c e - c o l d i s o l a t i o n b u f f e r . The b u l b u s a r t e r i o s u s was trimmed away and d i s c a r d e d . H e a r t s f r o m e i g h t f i s h were p o o l e d t o o b t a i n a t i s s u e wet w e i g h t o f 2 — 3 g. The t i s s u e was f i n e l y m i n c e d and r i n s e d w i t h more i s o l a t i o n b u f f e r . M o s a i c m u s c l e was d i s s e c t e d f r o m t h e t r u n k o f t h e t r o u t , a b o v e t h e l a t e r a l l i n e , b e g i n n i n g d i r e c t l y b e h i n d t h e o p e r c u l u m and e x t e n d i n g a l o n g 2/3 t h e l e n g t h o f t h e t r o u t t r u n k . T h r o u g h o u t t h e d i s s e c t i o n , t h e m u s c l e was b a t h e d i n i c e — c o l d i s o l a t i o n b u f f e r i n an a t t e m p t t o keep i t a s c o l d a s p o s s i b l e . The m o s a i c m u s c l e was c a r e f u l l y s e p a r a t e d f r o m t h e s u p e r f i c i a l r e d m u s c l e w h i c h e x t e n d s a l o n g t h e l a t e r a l l i n e . A l a y e r o f p i g m e n t was a l s o removed f r o m t h e s u r f a c e o f t h e m u s c l e . The m u s c l e was p a r t i a l l y m i n c e d w h i l e s t i l l i n t h e f i s h , t h e n t r a n s f e r r e d t o i c e - c o l d i s o l a t i o n b u f f e r . Any t r a c e s o f p i g m e n t , r e d m u s c l e o r b l o o d were c a r e f u l l y trimmed away and t h e m u s c l e was t h o u r o u g h l y m i n c e d t h e n r i n s e d o n c e more w i t h b u f f e r . M u s c l e f r o m two f i s h was p o o l e d t o o b t a i n a t i s s u e wet w e i g h t o f 50 g. The i s o l a t i o n b u f f e r was e s s e n t i a l l y t h e same f o r b o t h h e a r t and m u s c l e m i t o c h o n d r i a . I t c o n s i s t e d o f lOOmM T r i s - H C l , 210mM m a n n i t o l , 70mM s u c r o s e , lOmM EDTA and, i n 9 the case of muscle, 5mM MgCl 2 and 0.IX BSA at pH 7 . 3 . The f i n a l mitochondrial suspension from heart a lso contained 0.1% BSA but t h i s was added as the very l a s t step of i s o l a t i o n in order to increase the s t a b i l i t y and durat ion of coupled r e s p i r a t i o n . The addi t ion of BSA at the s t a r t of i s o l a t i o n may fur ther increase the s t a b i l i t y of the f i n a l product (personal observation) but t h i s was not r e a l i z e d when the experiments were begun. The next step in mitochondrial i s o l a t i o n was t i s s u e homogenization. The t i s s u e was f i r s t incubated in a general protease (Sigma P5255) and s t i r r e d o c c a s i o n a l l y . In the case of hear t , 2 - 3 g of t i s s u e were incubated in 30 mis of i s o l a t i o n buffer and 22.4 mg protease for 10 minutes. The t i s s u e was then homogenized very gently in a Patter -E lvehjem homogenizer with a l o o s e - f i t t i n g t e f l o n p e s t l e . In the case of muscle, 50 - 55 g of t i s s u e were d iv ided in to two l o t s and each l o t was incubated in 60 mis of i s o l a t i o n buffer and 44.8 mg protease for 14 minutes. The buffer was then decanted and replaced with protease—free bu f fe r . Homogenization proceeded using t i s sue port ions of 8 - lOg t i s s u e in 30 mis of buf fe r . Mitochondria were then i s o l a t e d by d i f f e r e n t i a l c e n t r i f u g a t i o n . Crude t i s s u e homogenates were f i r s t spun in a Sorva l l RCB-2 at 600g for 10 minutes at 4°C. The c e l l u l a r debr is was discarded and the supernatant was centr i fuged at 9000g for 10 minutes at 4°C. The r e s u l t i n g mitochondrial p e l l e t was resuspended in buffer by gent le asp i ra t ion with a Pasteur p ipet te and recentr i fuged at 9000g. This process was repeated f i v e t imes. The f i n a l p e l l e t was resuspended 10 in 1.0 ml i s o l a t i o n bu f fe r . Two 60 ul a l iquo ts were removed and frozen at -80=0 for l a t e r prote in determination. A l iquots of the white muscle i s o l a t i o n buffer (containing BSA) were a lso frozen for prote in determination. 1.0 ml of i s o l a t i o n buffer was added to the remaining mitochondrial suspension. In the case of the heart p reparat ion , t h i s second addi t ion of i s o l a t i o n buffer contained 0.2% BSA to br ing the f i n a l concentrat ion of BSA to O. 1'/.. F ina l mitochondrial prote in concentrat ions of these preparat ions were genera l ly between 7 - 20 mg per ml. Preparat ions were s tab le for at leas t two hours when kept on i c e . PROTEIN DETERMINATION Mitochondrial prote in concentrat ion was determined according to a modi f icat ion of the Lowry technique in which the prote in i s p r e c i p i t a t e d by 6% TCA in the presence of Na—deoxycholate (Bensadoun and Weinstein, 1976). This technique el iminated in ter ference by sucrose, Tris—HC1 and EDTA. The s e n s i t i v i t y of t h i s technique i s in the range of 5 — 50 ug p ro te in . The technique was fur ther modified to allow for s o l u b i l i z a t i o n of mitochondrial membranes. Prote in a l i q u o t s (30 — 50 ul) were d i lu ted to a f i n a l volume of 100 ul with BSA-free i s o l a t i o n buf fe r . 100 ul of 10% Na-deoxycholate was added to s o l u b i l i z e mitochondrial membranes. A 10 ul a l iquot of t h i s so lu t ion was d i lu ted up to 3 mis with d i s t i l l e d water to give a f i n a l Na-deoxycholate concentrat ion of 167 ug/ml. Samples were mixed v igorously and allowed to stand for 15 minutes. Protein was then p r e c i p i t a t e d by addi t ion of 6% TCA ( f ina l 11 concentration) -Followed by centri-fLigation at 3300g for 30 minutes in a swinging bucket ro to r . The supernatant was completely removed using a Pasteur p ipet te a s p i r a t o r . The resu l tant prote in p e l l e t was d isso lved in 3 mis of Lowry reagent. Af ter mixing v igorously for 30 seconds, 1.5 mis of F o l i n - C i o c a l t e u reagent, d i l u t e d 1:1 with water, was added and the colour allowed to develop in the dark for 45 minutes (Bensadoun and Weinstein, 1976). Absorbance was measured at 660 nm in a Pye-Unicam spectrophotometer. Bovine serum albumin was used as the standard. Q 2 CONSUMPTION RATES Assays were conducted po larograph ica l l y i n a Gi lson oxygraph with a C la rk - t ype 0=. e lect rode and a water- jacketed chamber. Temperature was maintained at e i ther 5 or 15DC with a constant water bath and c i r c u l a t o r . C a l i b r a t i o n was achieved by al lowing water to e q u i l i b r a t e with a i r at the experimental temperature. Values for 0 2 content in 100% air—saturated water at var ious temperatures are a v a i l a b l e in Davis , 1975. Sodium s u l f i t e was added to determine zero O z content. F ina l assay volume was 2 .0 mis inc lud ing 200 — 400 ul of mitochondrial suspension containing 1.5 - 3 .0 mg mitochondrial p ro te in . The assay buffer consisted of lOOmM T r i s - H C l , 210mM mannitol , 70mM sucrose, lOmM EGTA and lOmM KH3P0^ at pH 7 . 3 . The pH of the assay mixture was measured with a radiometer pH—meter a f te r addi t ion of mitochondria — then the chamber was sea led . Assay pH was allowed to d r i f t with temperature so at 15°C the pH was 7.4 and at 5°C the pH 12 was 7.6—7.7. C e l l c o n t e n t s were mixed w i t h a m a g n e t i c s t i r r e r and t e f l o n - c o a t e d b a r . A l l s u b s t r a t e s were d i s s o l v e d i n a s s a y b u f f e r and a d j u s t e d t o pH 7.3. • l i g o m y c i n , r o t e n o n e and DNP were d i s s o l v e d i n e t h a n o l . S u b s t r a t e s , u n c o u p l e r s and i n h i b i t o r s were i n j e c t e d i n t o t h e chamber t h r o u g h a s m a l l , r e m o v a b l e p o r t u s i n g l O u l H a m i l t o n s y r i n g e s . M i t o c h o n d r i a l r e s p i r a t i o n was i n i t i a t e d by 400 nmole ADP p u l s e s a d m i n i s t e r e d by H a m i l t o n s y r i n g e . 0 2 c o n s u m p t i o n , RCR and ADP/O were c a l c u l a t e d a c c o r d i n g t o E s t a b r o o k ( 1 9 6 7 ) . R e s p i r a t o r y s t a t e s a r e d e f i n e d by Chance and W i l l i a m s ( 1 9 5 6 ) . M i t o c h o n d r i a l v i a b i l i t y was c o n f i r m e d by a number o f methods. Membrane i n t e g r i t y was t e s t e d by m e a s u r i n g 0 Z c o n s u m p t i o n i n t h e p r e s e n c e o f NADH and ADP. ADP/O was measured u s i n g e i t h e r p y r u v a t e o r s u c c i n a t e a s s u b s t r a t e , RCR was m o n i t o r e d and s e v e r a l i n h i b i t o r s and u n c o u p l e r s were a d m i n i s t e r e d , i n c l u d i n g r o t e n o n e , o l i g o m y c i n o r DNP, i n o r d e r t o c h a r a c t e r i z e c o u p l e d r e s p i r a t i o n . Once t h e i n t e g r i t y o f t h e m i t o c h o n d r i a l p r e p a r a t i o n s f r o m e a c h t i s s u e had been e s t a b l i s h e d , t h r e e d i f f e r e n t e x p e r i m e n t a l r e g i m e s were im p o s e d . The A D P — p u l s e method was u s e d t h r o u g h o u t . The f i r s t r e g i m e i n v o l v e d measurement o f maximal r a t e s o f D 2 c o n s u m p t i o n f o r s p e c i f i c s u b s t r a t e s a t 15°C. The s u b s t r a t e s i n c l u d e d 5mM p y r u v a t e , 5mM m a l a t e , lOmM l a c t a t e , 4mM a c e t y l - c a r n i t i n e and 5mM g l u t a m a t e . A l l e x c e p t m a l a t e were a c c o m p a n i e d by 0.05 mM m a l a t e t o " s p a r k " t h e TCA c y c l e . L a c t a t e measurements a l s o r e q u i r e d t h e a d d i t i o n o f 2 u n i t s o f LDH, s i n c e no LDH a c t i v i t y was o b s e r v e d i n t h e m i t o c h o n d r i a l s u s p e n s i o n . P o r c i n e h e a r t LDH 13 suspended in g lycero l was used. It appeared that i n c l u s i o n o-f 2mM NAD* in the assay buffer produced maximal rates of ox idat ion in heart preparat ions but subsequent experiments have ind icated that these assays were NAD*-1imited. Muscle assays were ca r r ied out in the presence of lOmM NAD*. The second regime was a repeat of the f i r s t but ca r r ied out at 5°C rather than 15°C. In the case of l a c t a t e ox idat ion , addi t ion of LDH was increased to 4 units/2 mis assay volume. Sample s i z e was extremely va r iab le in these experiments because some preparat ions were more s tab le than others . Results are expressed as mean + SEM. Oxidation ra tes were measured for 3 ADP pulses per assay. The rates were va r iab le from one pulse to another in each assay, but they did not vary according to any p red ic tab le pat tern . Each mean represents the average of a l l observat ions and inc ludes 2 or 3 assays per preparat ion and 3 - 6 preparat ions for each experimental cond i t ion . As an aside to these f i r s t two sets of experiments, the rate of 0=> consumption at var ious concentrat ions of pyruvate was measured at lS^C. The malate "spark" and ADP pulse were administered before the addi t ion of pyruvate in order to be sure of the i n i t i a l pyruvate concentrat ion at the onset of s tate 3 r e s p i r a t i o n . Pyruvate concentrat ion ranged from 0.01 — lOmM. The t h i r d experimental regime monitored mitochondrial ox idat ion as a funct ion of pH. Mitochondrial r e s p i r a t i o n was assayed at lS^C and a var ie ty of pH values . The pH of the assay suspension was measured before addi t ion of substrates or ADP. The pH was measured again at the end of 14 each assay to ensure that i t had not changed markedly during the course of the experiment. In the few cases where pH var ied by more than 0.1 pH uni t during the course of an assay the data were d iscarded. Substrates included a c e t y l - c a r n i t i n e , glutamate and l a c t a t e . Assay buffer pH values var ied from 5.2 - 8 . 3 . Addit ion of 200 - 400uls of buffered mitochondrial suspension a l te red f i n a l assay pH s ince i t was a s i g n i f i c a n t f r a c t i o n of t o t a l assay volume. The f i n a l assay pH values could not be dupl icated accurately from one mitochondrial preparat ion to another so each point on the pH p r o f i l e p l o t s usual ly represents data from a s i n g l e assay. Values are expressed as mean + SEM. Means represent average values from 3 - 4 ADP pulses per assay. Where pH values from separate assays did not vary by more than 0.02 pH u n i t s , values from the assays were combined to represent a s i n g l e data po in t . These occurrences are noted on the p l o t s . Experimental pH values were staggered among preparat ions so as not to create a r t i f i c i a l trends as a r e s u l t of any preparat ion having an inherent ly higher or lower resp i ra to ry rate than others . A t o t a l of 18 and 16 preparat ions were used to generate the heart and muscle data r e s p e c t i v e l y . Malate ox idat ion as a funct ion of pH was measured using a s i n g l e preparat ion of heart mitochondria. The r e s u l t s have been included for i l l u s t r a t i v e reasons which w i l l be d iscussed. "Each point on the p H - p r o f i l e p lot represents the mean from 4 ADP pulses in a s i n g l e assay except where noted. The order in which the measurements were made was pH 6 . 8 , 7 . 3 , 7 . 8 , 8 .2 and 6 . 4 . 15 Mitochondrial aging r e s u l t s in slowed resp i ra to ry rates and diminished resp i ra to ry c o n t r o l . If s igns of mitochondrial aging were apparent ( i f RCR and ADP/O were suddenly low and the preparat ion was more than one hour old) the data were d iscarded. Data were analysed using one—way ANOVAs fol lowed by the Student-Newman-Keuls (SNK) t e s t . Transformations were performed as required in order to meet the appropriate s t a t i s t i c a l assumptions. SPECTROPHOTOMETRIC CHECK OF LDH REACTION The LDH react ion was measured spectrophotometr ical ly at room temperature using mitochondrial assay condi t ions . The assay buffer was i d e n t i c a l to that used for 0 3 consumption measurements and included lOmM l a c t a t e and 2-10mM NAD"*". Addit ion of 2 un i ts of LDH i n i t i a t e d the r e a c t i o n . The conversion of NAD"*" to NADH was fol lowed to completion at 340 nm in a Pye—Unicam spectrophotometer. Results were not a l te red by increas ing LDH or l a c t a t e concentrat ion. RESULTS Mitochondria i s o l a t e d from heart and mosaic muscle of rainbow t rout exhib i ted resp i ra to ry c o n t r o l , as evidenced by RCR values and ADP/O r a t i o s (Table 1) and were s tab le for at least two hours. Table 1 shows the s ta te 3 rate of oxygen consumption of mitochondria at 15°C, ox id i z ing saturat ing concentrat ions of var ious subst rates . Mean rates of s tate 3 oxidat ion ranged from 70-124 natoms 0 m i n - 1 mg p r o t e i n - 1 in heart mitochondria, with highest rates being observed when e i ther pyruvate or malate were suppl ied as subst rate . The rate of heart mitochondrial l a c t a t e ox idat ion in the presence of 2 un i ts of LDH and 1-2 mM NAD* was lower than that of pyruvate. It was found upon subsequent inves t iga t ion that increas ing NAD* concentrat ion to lOmM increased l a c t a t e ox idat ion rates r e l a t i v e to those of pyruvate (Table 2 ) . The LDH react ion was checked spectrophotometr ical ly using buf fers and assay condi t ions ( s p e c i f i c a l l y pH) which were i d e n t i c a l to those of the mitochondrial ox idat ion assays (Table 3 ) . The react ion was found to be slow in t h i s assay buffer and was NAD*—1imited. Mean rates of s tate 3 oxidat ion ranged from 67-120 natoms O m i n - 1 mg p r o t e i n - 1 in mosaic muscle mitochondria, with highest rates being observed with e i ther pyruvate or l a c t a t e as substrate (Table 1). It was demonstrated that by increas ing CNAD*] from 2-10mM, l a c t a t e conversion in mitochondrial assay buffer was increased from 0.02 umoles/ml to 0.05 umoles/ml (Table 3 ) . Reported values for l ac ta te 17 TABLE 1: State 3 0 2 consumption, RCR, ADP/O for mitochondria i s o l a t e d from heart and mosaic muscle of rainbow trout at 15°C. Sample s i z e as described in Mater ia ls and Metods. Mean ± SEM STATE 3 RCR (natoms O/min/mg protein) HEART ADP/O Pyruvate(5mM) Malate(5mM) Lactate(lOmM) 124.49±7.17 117.79±6.25 8 2 . 2 4 ± 7 . 0 6 Acetyl -carnit ine(4mM) 7 3 . 6 9 ± 5 . 7 2 Glutamate(5mM) 7 0 . 3 3 ± 4 . 5 4 7 . 6 8 ± 0 . 5 4 5 . 8 5 ± 0 . 5 3 7 . 1 3 ± 0 . 6 8 7 . 6 0 ± 0 . 4 2 6 . 0 5 ± 0 . 3 7 2 . 5 4 ± 0 . 0 6 2 . 2 6 ± 0 . 0 4 2 . 5 6 ± 0 . 0 5 2 . 3 6 ± 0 . 1 3 2 . 2 9 ± 0 . 1 0 MUSCLE Pyruvate(5mM) Malate(5mM) Lactate(5mM) 95 .33±6.71 6 7 . 0 8 ± 6 . 13 120.34±12.71 Acetyl -carnit ine(4mM) 7 8 . 5 3 ± 7 . 1 8 Glutamate(5mM) 8 7 . 4 6 ± 6 . 8 8 6 . 0 0 ± 0 . 4 4 4 . 8 4 ± 0 . 2 8 4 . 3 9 ± 0 . 1 3 5 . 6 4 ± 0 . 2 7 4.6S+0.19 2 . 8 2 ± 0 . 0 9 2 . 7 9 ± 0 . 1 4 2 . 5 8 ± 0 . 0 4 2 . 4 5 ± 0 . 0 7 2 . 8 0 ± 0 . 0 7 18 TABLE 2: State 3 ox idat ion rates with lac ta te as substrate , in the presence o-f 2mM or lOmM NAD*, compared to s tate 3 ox idat ion rates with saturat ing concentrat ions of pyruvate. Mean ± SEM. Sample s i z e in parentheses. Assays included lOmM l a c t a t e , 2 un i ts LDH and 0.05mM malate. State 3 0 2 Consumption Rate (natoms D m i n - 1 mg p r o t e i n - 1 ) NAD* <2mM) 88.06 ± 2.98 (4) NAD* (lOmM) 115.98 ± 0.92 (6) Pyruvate (lOmM) H O . 88 ± 3.09 (6) 19 TABLE 3: Conversion of l a c t a t e to pyruvate as a funct ion of NAD*. Assay condi t ions described in Mater ia ls and Methods. I n i t i a l l ac ta te concentrat ion was lOumoles/ml in a react ion volume of 1ml. CNAD-3 Lactate -> Pyruvate (mM) (umoles) 2 0.021 4 0.033 9 0.042 10 0.054 20 ox idat ion in muscle were assayed in the presence o-f 2 un i ts LDH and lOmM NAD*. Mitochondrial i n t e g r i t y was assessed by a number of standard t e s t s . In the absence of shut t le systems neither heart nor mosaic muscle mitochondria could ox id i ze NADH as an exogenous subst rate , confirming that mitochondrial membranes were i n t a c t ( F i g . l ) . High RCR values (Table 1) a lso suggest that the majority of r e s p i r i n g mitochondria were i n t a c t . ADP/0 r a t i o s were 2 .3 - 2 .9 (Table 1> with NAD"*"-1 inked substrates . Rotenone appeared to i n h i b i t NAD*-1inked r e s p i r a t i o n (F ig . 2) and ADP/0 r a t i o s for the FAD*-1inked subst rate , succ inate , were 1.4 - 1.9 (F ig . 2 ) . Oligomycin completely i n h i b i t e d ADP-stimulated r e s p i r a t i o n and an uncoupler of ox idat ive phosphorylat ion, DNP, re l ieved t h i s i n h i b i t i o n (F ig . 3 ) . Oxidation of a l l substrates required the presence of a malate "spark" (0.05mM) to augment depleted TCA intermediates. Although both mitochondrial populat ions were capable of ox id i z ing malate as a so le subst ra te , the concentrat ion of malate used for "sparking" was too low to contr ibute s i g n i f i c a n t l y as a d i rec t substrate for 0 3 consumption (F ig . 4 ) . SUBSTRATE UTILIZATION Figure 5 compares substrate preference between heart and muscle mitochondria at lS^C. Figure 5a shows s tate 3 ox idat ion rates in heart mitochondria using a var ie ty of exogenous substrates . The rates were highest with e i ther pyruvate or malate as subst rate . The rate of l ac ta te 21 FIGURE Is Representative mitochondrial 0 2 consumption t races . Concentration of each addi t ion in a f i n a l volume of 2 .0 miss NADH - 2mM, ADP - 200uM, malate - 0.05mM, glutamate - 5mM, Pyruvate - 5mli A. Heart; 1.75 mg p ro te in ; lS^C B. Muscle; 2.33 mg p ro te in ; 15°C NADH I ADP NADH N3 23 FIGURE 2: Representative mitochondrial 0 2 consumption t races . Concentrations for each addi t ion in a f i n a l volume of 2.0 miss ADP - 200uM; rotenone -lmg/ml; succinate — 5mM A. Heart; 1.05 mg p ro te in ; 15°C ADP/D=1.9 B. Muscle; 4.00 mg p ro te in ; 5°C ADP/0=1.4 5mM PYRUVATE SmM MALATE I ADP ROTENONE 25 FIGURE 3: Representative mitochondrial 0 2 consumption t races . Concentrations for each addi t ion in a f i n a l volume of 2.0 mis: ADP — 200uM; oligomycin - 5 ug/ml; DNP - 0.04uM A. Heart; 1 .29 mg p ro te in ; 5°C B. Muscle; 3.63 mg p ro te in ; 5°C 27 FIGURE 4s Representative mitochondrial D 2 consumption t races . A. Hearts 1.38 mg proteins 15*=*C; concentrat ion of each addi t ion in a f i n a l volume of 2.0 miss malate - O.OSmM, ADP - 200uM, pyruvate - 0.05mM B. Muscle; 2.40 mg p ro te in ; 15°C; concentrat ion for each addi t ion in a f i n a l volume of 2.0 miss malate - O.OSmM, ADP - 200uM, pyruvate - 0.02mM 28 ADP | PYRUVATE 29 ox idat ion was s i g n i f i c a n t l y lower than that of e i ther pyruvate or malate, although l a c t a t e ox idat ion rates were not maximized under these condi t ions of low CNAD*3. Both a c e t y l - c a r n i t i n e and glutamate were burned at the same r a t e , which was s i g n i f i c a n t l y lower than that of pyruvate or malate. There were no s i g n i f i c a n t d i f fe rences among any of the RCR values with t h i s treatment - where s ta te 3 rates were h igh , s ta te 4 rates were s i m i l a r l y elevated <Fig. 6) . The pattern of substrate preference in mosaic muscle mitochondria was qui te d i f f e r e n t from that of heart at the same temperature (F ig . 5b). L ike hear t , muscle exhib i ted high rates of ox idat ion with pyruvate as subst ra te , however malate produced the lowest mean ra tes in muscle. In muscle assays, where lac ta te ox idat ion was maximized, s ta te 3 rates of l a c t a t e ox idat ion were at l eas t as high as those of pyruvate. In f a c t , the mean rate was higher fo r l a c t a t e , although the d i f fe rence was not s i g n i f i c a n t . While for heart mitochondria acety l—carn i t ine and glutamate were poor substrates r e l a t i v e to pyruvate (F ig . 5a ) , for muscle a l l three substrates were equal ly good, based on maximal ra tes of s ta te 3 ox idat ion . Other than the d i f fe rence between malate and l a c t a t e ox idat ion ra tes (F ig . 5 ) , there was no s i g n i f i c a n t d i f fe rence in muscle s ta te 3 ra tes among any of the substrates examined. There appeared to be s i g n i f i c a n t d i f fe rences among the RCR's for d i f f e r e n t substrates in mosaic muscle at lS^C (F ig . 5b). The usual transformations did not render the data homogeneous, however, and the conclusions of the SNK tes t must be viewed caut ious l y . State 4 ra tes were clearer. 30 FIGURE 5: State 3 0 2 consumption at 15°C. Sample s i z e as described in Mater ia ls and Methods (mean ± SEM). S i g n i f i c a n t d i f fe rence among means (P<0.05) ind icated by * (0 3 consumption) or + (RCR) A. Heart B. Muscle State 3 0 2 consumption RCR 31 - A ir f f -h 12 BO - B f f 12 3D A C E T Y L -CARNITINE GLUTAMATE MALATE PYRUVATE LACTATE 32 FIGURE 6s State 4 0 2 consumption at 15°C. Sample s i z e as described in Mater ia ls and Methods (mean ± SEM). S i g n i f i c a n t d i f fe rence between means (P < 0.05) ind icated by * . A. Heart B. Muscle S T A T E 4 O , CONSUMPTION (natonta O/mln/mg protein) S T A T E 4 O , CONSUMPT ION (natoma O/mln/ms prolalnj T T T > o OD State 4 rates were the same for a l l substrates except l a c t a t e which had a s i g n i f i c a n t l y higher rate (F ig . 6 ) . Lactate ox idat ion produced the lowest mean RCR. At 15°C, maximal s ta te 3 oxidat ion ra tes were always at l eas t as high in heart mitochondria as in muscle and in the case of both malate and pyruvate, the rates were s i g n i f i c a n t l y higher in heart (F ig . 7 ) . In the cases where heart mitochondria had a higher rate of ox idat ive phosphorylation than muscle there was no s i g n i f i c a n t d i f fe rence in RCR, so an elevated s ta te 3 rate was accompanied by an elevated s ta te 4 ra te (F ig . 6 ) . This higher s ta te 4 rate was only s i g n i f i c a n t with malate as substrate . RCR values in heart mitochondria were s i g n i f i c a n t l y higher than in muscle for e i ther a c e t y l - c a r n i t i n e or glutamate (F ig . 7 ) . Although muscle and heart had the same maximal rate of s tate 3 ox ida t ion , the s ta te 4 ra te for both substrates was s i g n i f i c a n t l y higher in muscle (F ig . 6 ) . Although the d i f fe rence was not always s i g n i f i c a n t , the mean RCR was higher fo r heart than for muscle mitochondria for a l l substrates at lo^C. Pyruvate was suppl ied at a concentrat ion of 5mM to ensure maximal rates of ox idat ion . Since malate or l a c t a t e must f i r s t be converted to pyruvate in order to be oxidized by mitochondria, the concentrat ion of d i r e c t l y ox id i zab le substrate (pyruvate) was unknown when e i ther of these substrates was suppl ied . Pyruvate concentrat ion was var ied for each t i s s u e at 15°C in order to discover what concentrat ion was required to maintain maximal rates of s ta te 3 oxidat ion (F ig . 8 ) . Each point represents only one 35 FIGURE 7: State 3 0 2 consumption at 15°C. Sample s i z e as described in Mater ia ls and Methods (mean ± SEM). S i g n i f i c a n t d i f fe rence between p a i r s (P < 0.05) ind icated by * (0 a consumption) or + (RCR) Heart s ta te 3 0 3 consumption Muscle s tate 3 0 3 consumption RCR 37 FIGURE Ss State 3 0 2 consumption as a funct ion of pyruvate concentrat ion. Values were obtained from a t o t a l of 4 mitochondrial preparat ions for each t i s s u e . Number of assays are ind icated in parentheses and values are expressed as mean ± SEM. A. Heart B. Muscle 38 [PYRUVATE] (mM) 39 s ta te 3 measurement made during the f i r s t pulse of ADP, when the i n i t i a l pyruvate concentrat ion was known. It was apparent that at pyruvate concentrat ions as low as O.OSmM maximal rates of s ta te 3 0 3 consumption could be atta ined by both populat ions (F ig . 8) . State 3 0 3 consumption rates were only obviously l im i ted when the i n i t i a l pyruvate concentrat ion was O.OlmM. A pyruvate concentrat ion of O.OlmM in 2 mis of so lu t ion i s equivalent to 20 nmoles of pyruvate. This was not s u f f i c i e n t substrate to allow complete phosphorylation of 400 nmoles of ADP - the amount contained in one pulse (F ig . 9 ) . A second addi t ion of 20 nmoles of pyruvate resu l ted in an equivalent rate of •=> consumption as was obtained with the f i r s t pulse of pyruvate. Typical 0 2 consumption, given 400 nmoles of ADP and saturat ing pyruvate, was 0.08 umoles D 2. If 5 oxygen atoms were consumed for each molecule of pyruvate ox id i zed , then t h i s 0 2 consumption i s equivalent to the ox idat ion of 0.032 umoles of pyruvate. In a 2.0 ml volume, 0 .05 , 0.025 and 0.01 mM pyruvate are equivalent to 0 . 1 , 0.05 and 0.02 umoles of pyruvate, respec t i ve l y . C l e a r l y , O.OlmM pyruvate in t h i s volume would not susta in 0 3 consumption to phosphorylate the e n t i r e ADP pulse TEMPERATURE For mitochondria i s o l a t e d from e i ther t i s s u e the pattern of substrate preference was d i f f e r e n t at 5°C than at 15 aC (F ig . 10a,b) . For hear t , pyruvate remained the substrate of preference at 5°C. Malate, which had been equal ly good at 15 Q C, produced s i g n i f i c a n t l y lower s tate 3 40 FIGURE 9s Representative 0 3 consumption t race from heart ; 1 . 3 8 mg p ro te in ; 15°C. Concentration of each addi t ion in a f i n a l volume of 2.0 miss malate - 0.05mM ADP - 200uM; pyruvate - O.Olmli M A L A T E ADP P Y R U V A T E ADP P Y R U V A T E ADP P Y R U V A T E 42 FIGURE 10: State 3 0 2 consumption at 5 and 15°C. Sample s i z e as described in Mater ia ls and Methods (mean ± SEM). S i g n i f i c a n t d i f fe rences among means (P < 0.05) ind icated by * (15=C), + ( 5 ° C ) . A. Heart B. Muscle 15°C STATE 3 O, CONSUMPTION (natoms O/min/mg protein) 44 rates than pyruvate at 5°C (F ig . lOa). The rates with malate were equivalent to those produced with e i ther a c e t y l - c a r n i t i n e or glutamate as substrate . RCR values for heart at 5°C were qui te v a r i a b l e . RCR was highest for the ox idat ion of pyruvate. Lactate was character ized by a s i g n i f i c a n t l y higher s ta te 4 rate than any other substrate (F ig . 11) and, consequently, a r e l a t i v e l y low RCR. There was no s i g n i f i c a n t d i f fe rence in RCR between 5 and 15°C for any given substrate . The pattern of mosaic muscle mitochondrial substrate preference a lso changed as a funct ion of temperature (F ig . 10b). At 5°C pyruvate and l a c t a t e continued to be the substrates which produced the highest s ta te 3 Q 2 consumption ra tes . Malate was ox id ized s i g n i f i c a n t l y more slowly than pyruvate or l a c t a t e , while a c e t y l - c a r n i t i n e and glutamate produced rates which were s i g n i f i c a n t l y lower than those of the other subst rates . This i s qui te d i f f e r e n t from the pattern observed at 15°C (F ig . 5b) in which there was no rea l d i f fe rence in s tate 3 rates among subst rates . RCR did not vary s i g n i f i c a n t l y among substrates at 5°C , except for a c e t y l - c a r n i t i n e which exhib i ted a remarkably high degree of resp i ra to ry c o n t r o l . State 4 oxidat ion ra tes mimicked s ta te 3 rates at 5°C (F ig . 10b, F i g . 11). Oxidation of pyruvate and lac ta te proceeded most r a p i d l y , fol lowed by malate, while glutamate and acety l—carn i t ine produced the lowest s ta te 4 ra tes . With a c e t y l - c a r n i t i n e as substrate the RCR was s i g n i f i c a n t l y higher at 5°C than at lS^C. Otherwise there was no s i g n i f i c a n t d i f fe rence in RCR between 5 and 15 0C for any substrate . 45 FIGURE 11: State 4 0 2 consumption and RCR at 5°C. Sample s i z e as described in Mater ia ls and Methods (mean ± SEM). S i g n i f i c a n t d i f fe rence among means (P < 0.05) ind icated by + (0 2 consumption) or + (RCR) A. Heart B. Muscle State 4 0 2 consumption RCR S T A T E 4 O , CONSUMPTION (natoms O/mln/mg protein) S T A T E 4 O , CONSUMPTION (natoms O/mln/mg protein) o <•> a ro o •> « n I I 1 1 1 1 r ' i 1 i 1 1 -r •P-boa uou 47 Q i o values -for s tate 3 and s tate 4 Q 2 consumption rates are shown in Table 4. Q i 0 values are simply based on the d i f fe rence between mean O z consumption rates at 5 and lS^C. They should therefore be considered as approximations only . State 3 Qjo values for heart mitochondria were approximately 2 for a l l substrates except malate which was higher (3.08). State 3 Q t o values fo r muscle mitochondria tended to be higher than for hear t , although Q x o values fo r malate and pyruvate were approximately 2 in muscle. Muscle s tate 3 Q i o ' s were p a r t i c u l a r l y high for acety l—carn i t ine or glutamate ox idat ion . The dramatic e f f e c t of temperature on a c e t y l - c a r n i t i n e and glutamate oxidat ion was a lso r e f l e c t e d in s t r i k i n g l y high s ta te 4 Q t o va lues. Except with malate or l a c t a t e , heart mitochondria exhib i ted the higher rates of ox idat ive phosphorylation at 5°C (F ig . 12). In f a c t , at both 5 and 15°C, heart s ta te 3 ra tes were always at leas t as h igh , and often higher than the corresponding muscle r a t e , except for ox idat ion of l a c t a t e at 15°C (Table 1). At 5°C, heart RCR values were higher than those of muscle with e i ther malate or pyruvate as subst rate . Otherwise no s i g n i f i c a n t d i f fe rence occurred between RCRs of d i f f e r e n t t i s sues (F ig . 12). In conjunction with t h i s , there were no s i g n i f i c a n t d i f fe rences between heart or muscle s ta te 4 ra tes with e i ther malate or pyruvate as subst rate , even though heart had s i g n i f i c a n t l y higher s tate 3 rates under these cond i t ions . 48 TABLE 4: State 3 and s tate 4 D 3 consumption Q 1 0 values based on mean D z consumption rates at 5 and 15 0C STATE 3 Q 1 0 STATE 4 Q 1 0 Heart Muscle Heart Muscle A c e t y l - c a r n i t i n e 2.25 4.09 2.01 7.19 Glutamate 1.99 6.89 1.69 7.51 Malate 3.08 2.03 3.38 1.98 Pyruvate 1.75 2.30 2.34 1.76 Lactate 1.87 2.75 1.34 2.88 49 FIGURE 12: State 3 0 2 consumption and RCR of heart and muscle mitochondria at 5°C. Sample s i z e as described in Mater ia ls and Methods (mean ± SEM). S i g n i f i c a n t d i f fe rence between means (P < 0.05) ind icated by * (0 3 consumption) or + (RCR). Heart s ta te 3 0 3 consumption Muscle s tate 3 0 3 consumption RCR 51 EFFECT OF pH Three substrates - a c e t y l - c a r n i t i n e , glutamate and l a c t a t e - were used to generate pH p r o f i l e s for each t i s s u e . F igures 13a and b show the pH p r o f i l e fo r heart burning a c e t y l - c a r n i t i n e and glutamate, respec t i ve l y . As pH decreased, s ta te 3 0 2 consumption rate increased. As pH decreased to low phys io log ica l pH (6.8) and beyond, t h i s increase of s tate 3 rate appeared to leve l o f f , although more measurements are required below pH 6.8 to be conc lus ive . State 3 r e s p i r a t i o n in mosaic muscle showed no c lea r pH-dependence below pH 7.4 (F ig . 13c,d) . At pH above 7.4 or 7.6 s ta te 3 rate decreased. Mitochondrial r e s p i r a t i o n was measured at lower pH in muscle than in heart . There was no s i g n i f i c a n t change in s ta te 3 rate as pH dropped as low as 6 . 3 . Even at pH 5 . 8 , well beyond the phys io log ica l range, s ta te 3 rate was within the range of rates measured at h igher , more phys io log ica l pH values (F ig . 13c). Although the pH p r o f i l e s using l a c t a t e as substrate d i f f e r e d from those of a c e t y l - c a r n i t i n e or glutamate, c e r t a i n s i m i l a r i t i e s were evident (F ig . 14a,b) . State 3 D = consumption was pH—dependent between pH 7.0 — 8 .0 in heart mitochondria (F ig . 14a), while muscle showed no c lear pH-dependence between pH 7.0 - 7.6 (F ig . 14b). At high pH (7 .8 ) , muscle s tate 3 rate decreased. Unl ike the patterns observed with the f i r s t two subst rates , the l a c t a t e pH p r o f i l e for each t i s s u e showed a d r a s t i c decrease in s ta te 3 resp i ra to ry rate below pH 7.0 In some muscle mitochondrial assays, pyruvate was added and s ta te 3 rates measured a f te r 52 FIGURE 13s State 3 D 3 consumption pH p r o f i l e for a c e t y l - c a r n i t i n e and glutamate oxidat ion at 15 0 C. Sample s i z e as described in Mater ia ls and Methods (mean ± SEM). A. Heart; acety l—carn i t ine B. Heart; glutamate C. Muscle; acety l—carn i t ine D. Muscle; glutamate STATE 3 O, CONSUMPTION (n.Iom. O/mln/mg prot.ln) « o • I-O ES 54 FIGURE 14: State 3 0 3 consumption pH p r o f i l e for l a c t a t e ox idat ion at 15 a C. Sample s i z e as described in Mater ia ls and Methods (mean ± SEM). A. Heart B. Muscle STATE 3 02 CONSUMPTION (natoms O/min/mg protein) _ * o> ro at o o o o -r • r r r • r r - -•'T > 1 o 1 b _ 1 Q 1 1—o—| r ' b M b I o CD b M '•*> > •oH b j i- L I 1 i l a c t a t e ox idat ion rates had been measured. At pH 7.S the ra tes of l ac ta te ox idat ion were the same as ox idat ion of pyruvate (F ig . 15). At pH 7.3 s ta te 3 l a c t a t e ox idat ion was somewhat reduced r e l a t i v e to pyruvate and at pH 6.5 t h i s reduct ion was fur ther accentuated. RCR and state 4 values at d i f f e r e n t pH's are shown in Figure 16. In most cases the RCR's and s ta te 4 rates were almost mirror- images of each other. When RCR was low, s tate 4 was high. The RCR patterns were not normally dupl icated by the s ta te 3 patterns (F ig . 13,14). The exception to t h i s was heart mitochondria burning l a c t a t e . In t h i s case RCR values r e f l e c t e d s ta te 3 patterns more c l o s e l y than state 4 (F ig . 13). State 4 rates f e l l in to a more narrow range in heart than in muscle. The lowest s tate 4 ra tes measured for both populat ions of mitochondria were 10 natoms 0 m i n - 1 mg p r o t e i n - 1 . Heart s tate 4 rates r a r e l y reached 20 natoms 0 m i n - 1 mg p r o t e i n - 1 , a l though, admittedly , the pH range was more narrow in heart than muscle. In several instances muscle s ta te 4 rates approached 30 natoms m i n - 1 mg p r o t e i n - 1 and were often well above 20, although with a c e t y l - c a r n i t i n e as substrate s tate 4 ra te remained r e l a t i v e l y low between pH 6.B — 7.4 With l a c t a t e as subst ra te , muscle mitochondria exhib i ted the highest s ta te 4 rates of a l l , with a l l measurements except one (pH 6.7) f a l l i n g between 24 - 52 natoms 0 m i n - 1 mg p r o t e i n - 1 . RCR values r a r e l y dropped below 4 in e i ther t i s s u e except at very high or very low pH. Based on data from muscle mitochondria, s ta te 4 rates were elevated at high pH (>7.5) and RCRs were cons i s ten t l y below 4. Oxidation with heart mitochondria was not cons is tent l y 57 FIBURE 15: Representative heart mitochondrial 0 2 consumption t races of l a c t a t e ox idat ion at var ious values. Assay buffer included lOmM NADH and 2 uni t LDH. Concentrations of each addi t ion in a f i n a l volume of 2 .0 mis: ADP — 200uM; pyruvate — 5mM. A. pH 6.54 B. pH 7.32 C. pH 7.84 10mM LACTATE O.OSmM MALATE o o 59 FIGURE 16: State 4 O a consumption and RCR pH pro- f i les at 15°C. Sample s i z e as described in Mater ia ls and Methods (mean ± SEM). A. Heart; a c e t y l - c a r n i t i n e B. Heart; glutamate C. Heart; l ac ta te D. Muscle; a c e t y l - c a r n i t i n e E. Muscle; glutamate F. Muscle; l a c t a t e State 4 0 2 consumption o o R C R STATE 4 Oa CONSUMPTION (natoma O/min/mg protaln) aoa 09 monitared at high enough pH to make a v a l i d comparison to muscle. The point that s tate 3 resp i ra to ry ra te increased with decreasing pH in heart was further emphasized by a s ing le set of assays in which 5mli malate was used as substrate (F ig . 17). It i s important to note that the assays were done in the fo l lowing order : pH 6 . 8 , 7 . 3 , 7 . 8 , 8 .2 and 6 . 5 . The f i n a l assay, at the lowest pH, y ie lded the highest s tate 3 rates and RCR's. 62 FIGURE 17s Malate ox idat ion <5mM> by heart mitochondria at lS^C as a -function o-f pH. Each point represents 4 observations -from a s i n g l e assay except where noted (*). Values expressed as mean ± SEM. A l l points were derived from a s i n g l e mitochondrial preparat ion . STATE 4 ) 8 CONSUMPTION (natoma O/mln/mg protein) ro o STATE 3 O, CONSUMPTION (natoma O/mln/mg protein) o T " T ro o T" 9 O T " M J L 64 DISCUSSION Mitochondria from both heart and mosaic muscle exhib i ted a high degree of resp i ra to ry c o n t r o l , i n d i c a t i n g that preparat ions from each t i s s u e were equal ly v i a b l e . The protocol fo r preparation of heart mitochondria did not vary s i g n i f i c a n t l y from that of Chappell and Hansford (1972). While the technique for i s o l a t i n g mitochondria from mosaic muscle of rainbow trout was an adaptation of that used for hear t , i t was the f i r s t such i s o l a t i o n from t h i s t i s s u e . The low density of mitochondria in te leos t white or mosaic muscle makes i s o l a t i o n of reasonable y i e l d s of undamaged mitochondria somewhat more d i f f i c u l t than from a mitochondr ia - r ich t i s s u e such as heart . The problem had r a r e l y been approached, presumably because the r o l e of mitochondria in energy production of white or mosaic muscle i s minor r e l a t i v e to that of other t i s s u e s . However, for s tud ies of t i s sue adaptation at the sub—cel lu lar l e v e l , i t i s i n t e r e s t i n g to see how the resp i ra tory proper t ies of mitochondria i s o l a t e d from mosaic muscle, which has a low ox idat ive requirement, d i f f e r from those of heart whose need for ox idat ive metabolism i s high. Mitochondrial v i a b i l i t y was judged on the bas is of exogenous NADH ox idat ion , RCR and ADP/O. Impermeability of the inner mitochondrial membrane i s a requirement of e f f i c i e n t ox idat ive phosphorylation (Mitchel1,1961) and ox idat ion of exogenous NADH should requi re a funct ional shut t le system such as the malate-aspartate or a~glycerophosphate shut t le (Williamson et a l . , 1 9 7 3 ; 65 Buecher,1964s Hochachka et a l . , 1 9 7 9 ) . In the experiments presented here, exogenous NADH was not ox id ized at measurable r a t e s , i n d i c a t i n g membrane i n t e g r i t y . Both populat ions of mitochondria had acceptable ADP/O r a t i o s , ranging from 2.3 - 2 .8 for NAD*-1inked substrates at both 5 and 15°C. The ADP/O r a t i o for the FAD*-1inked subst ra te , succ inate , was 1.4 - 1.9. Both sets of values f e l l somewhat below the theore t i ca l values of 3 for NAD--1inked and 2 fo r FAD--1inked substrates (Ochoa,1943; Hunter,1951; Coopenhaver and Lardy,1952; Estabrook,1967; Davis et a l . , 1 9 7 4 ) , although according to Hinkle and Yu (1979) these t r a d i t i o n a l values are probably not r e a l i z e d because H* ions are required fo r var ious inner membrane transport funct ions . P e r f e c t l y coupled mitochondria may have rea l ADP/O r a t i o s of 2.0 and 1.33 for NAD~-1inked and FAD"*"-1 inked subst rates , respec t i ve l y (Hinkle and Yu,1979). ADP/O r e s u l t s from t h i s study ind ica te that in both mitochondrial populat ions 0= consumption was t i g h t l y coupled to ATP product ion. This was confirmed by high RCR values (>4) . SUBSTRATE UTILIZATION At 15=*C a l l substrates (except NADH) produced high rates of ox idat ive phosphorylation in both mitochondrial populat ions. Pyruvate in p a r t i c u l a r had cons i s ten t l y high ra tes of ox ida t ion , not only at lS^C but a lso at 5°C , i n d i c a t i n g a good potent ia l for aerobic carbohydrate metabolism in both t i s s u e s . 6 6 Lactate i s a potent ia l ox idat ive substrate in heart and, in f a c t , te leos t hearts are known to burn l a c t a t e ox idat i ve l y (Lanctin et a l . , 1 9 8 0 ; Dr iedz ic et a l . , 1 9 8 4 ) . Lactate produced as an endproduct of anaerobic metabolism in mosaic muscle i s retained in the muscle fo r long periods (Jones and Randal1,1978; Turner et a l . ,1983) and i t s u l t imate fa te i s a matter of debate. A part of i t i s eventual ly transported v i a the blood to the l i v e r , while some f r a c t i o n may be converted to glycogen in s i t u or ox idized in s i t u (Batty and Wardle,1979; Donovan and Brooks,1983; Turner et a l , 1983). Thus l a c t a t e i s a potent ia l ox idat ive fue l fo r mosaic muscle as well as for heart . The ra te of l a c t a t e ox idat ion by heart mitochondria was s u r p r i s i n g l y low, assuming that l ac ta te was converted to pyruvate ext ramitochondr ia l l y . Doubling the concentrat ion of substrate or LDH and increas ing NAD"*" from 1-2 mM made no d i f fe rence to s ta te 3 ox idat ion ra tes . State 3 rates obtained under these condi t ions were assumed at the time to be maximal. However, in l a t e r assays, lOmM NAD"*" st imulated s ta te 3 ox idat ion rates beyond those produced in the presence of 1—2 mM NAD"*". The ra tes of l ac ta te ox idat ion achieved with lOmM NAD"*" were equal to those obtained with saturat ing concentrat ions of pyruvate. In a d d i t i o n , given mitochondrial assay cond i t ions , the LDH react ion converted s i g n i f i c a n t l y more l a c t a t e to pyruvate in the presence of lOmM NAD"*" than in the presence of 2mM NAD*. It i s qui te probable that the reported rates for l a c t a t e oxidat ion in heart mitochondria were NAD"*"—1 i mi ted . 67 In mosaic muscle, no s i g n i f i c a n t d i f fe rence occurred between s ta te 3 ra tes of ox idat ion of l a c t a t e or pyruvate. This i s what one would pred ic t i f l a c t a t e were being converted to pyruvate qu ick ly enough to supply saturat ing concentrat ions of pyruvate to r e s p i r i n g mitochondria. Since both populat ions were capable of burning pyruvate at high r a t e s , the a b i l i t y to use l a c t a t e as a substrate seemed to depend on the a b i l i t y to convert l a c t a t e to pyruvate extramitochondr ia l ly which would be consistent with lac ta te ox idat ion rate being dependent on l a c t a t e oxidase a c t i v i t y (Dr iedzic et a l . , 1 9 8 4 ) . The d iscuss ion of l a c t a t e ox idat ion to t h i s point has assumed that l a c t a t e was converted to pyruvate ext ramitochondr ia l ly and was transported and ox id ized as pyruvate. The p o s s i b i l i t y of an int ramitochondr ia l L D H should not be overlooked however. In such a case , the ox idat ive ra te may be a f fec ted not only by L D H k i n e t i c s but by t ransport of l a c t a t e in to the mitochondrial matrix. The only known intramitochondr ia l L D H i s L D H x , whose synthesis i s cont ro l led by a unique genetic locus and which occurs in s p e c i a l i z e d sperm mitochondria (Clausen,1969; Machado de Domenech et a l . , 1 9 7 2 ; Blanco et a l . , 1 9 7 5 ) . Van Dop et a l . (197B) have provided evidence that the pyruvate t rans locase of bovine sperm mitochondria has dual s p e c i f i c i t y for l a c t a t e and pyruvate. In cont ras t , the pyruvate t rans locase of rat l i v e r mitochondria does not t ransport l ac ta te (Halestrap and Denton,1974). While intramitochondr ia l LDH has only been observed in sperm c e l l s , S k i l l e t e r and Kun (1972) reported the 68 presence of LDH associated with the intermembrane space in rat l i v e r mitochondria. Brdiczka and Krebs <1973) demonstrated that LDH associated with the rat l i v e r mitochondrial f r a c t i o n was located on the outer membrane outer sur face . LDH bound to subce l lu la r p a r t i c l e s has been demonstrated in t rout s k e l e t a l muscle (Melnick and Hult in,1970) and s p e c i f i c a l l y mitochondrial -bound LDH has been observed in brain (Vdovichenko,1978) and ske le ta l muscle <Lluis,1984) of mammals and ske le ta l muscle of eel (Mattison et a l . , 1 9 7 2 ) . While any poss ib le phys io log ica l r o l e i s uncer ta in , such an o r ienta t ion may a id in the e f f i c i e n c y of mitochondrial substrate supply. It should be noted that LDH associated with rabbi t s k e l e t a l muscle mitochondria had somewhat lower a c t i v i t y in the bound form than s o l u b i l i z e d (L lu is ,1984) . There was no evidence of mitochondrial—associated LDH in e i ther t rout heart or mosaic muscle mitochondria in the present study. Lactate ox idat ion c l e a r l y required the addi t ion of exogenous LDH. While pyruvate was normally suppl ied at a concentrat ion of 5mM, a concentrat ion as low as 0.05mM produced maximal rates of s ta te 3 ox idat ion . Low rates of l a c t a t e oxidat ion would ind ica te minimal l a c t a t e conversion to pyruvate unless l a c t a t e i t s e l f was somehow i n t e r f e r i n g with pyruvate ox idat ion . In a study in which ox idat ive phosphorylation of malate/glutamate in rat brain mitochondria was i n h i b i t e d in the presence of l a c t a t e , i t was concluded that low pH was the cause of i n h i b i t i o n rather than the presence of lac ta te per se (H i l le red et a l , 1 9 8 4 ) . In the present study a l l substrates were neut ra l i zed p r io r to inc lus ion in the assay, 69 so there was no d i f fe rence in pH between l a c t a t e and pyruvate. State 3 i n h i b i t i o n in the study of H i l l e r e d et a l (19S4) was accompanied by loss of resp i ra to ry c o n t r o l . Respiratory contro l was high in the present study, even when the state 3 rate of l a c t a t e oxidat ion was low. F i n a l l y , addi t ion of 5mM pyruvate to assays in which muscle mitochondria were ox id i z ing l a c t a t e at sub-maximal r a t e s , resu l ted in maximal rates of pyruvate ox idat ion . It was concluded that l a c t a t e did not , in any way, i n h i b i t ox idat ive phosphorylation of pyruvate in t h i s study. Malate, when suppl ied as the so le exogenous subst rate , must a lso be converted to pyruvate in order to be oxidized by i s o l a t e d mitochondria. One would not expect maximal ra tes of malate s ta te 3 oxidat ion to be higher than those of pyruvate unless the pyruvate t ransporter were l i m i t i n g maximal rates of ox idat ion . In f a c t , ra tes of malate ox idat ion were never higher than pyruvate in t h i s study. Lower malate rates must have been a resu l t of r e s t r i c t i o n of pyruvate supply in t ramitochondr ia l l y e i ther because of slow transport of malate into the mitochondria or poor malate conversion v i a malic enzyme. Most mitochondria i s o l a t e d from animal t i s s u e s are not able to rap id l y ox id ize malate as the only substrate (see Skorkowski et a l . , 1 9 8 4 ) . This l i m i t a t i o n i s overcome in some organisms by a high malic enzyme a c t i v i t y . NADP-*"—! inked malic enzyme i s present in the mitochondria of many t i s s u e s (Henderson,1966; Simpson and Estabrook,1969; Brdiczka and Pette ,1971; Frenkel ,1971,1972; Bartholome et a l . , 1 9 7 2 ; L in and Davis,1974; Skorkowski et a l . , 1 9 7 7 ) . 70 Skorkowski et al (1984) demonstrated that the a b i l i t y o-f cod heart mitochondria to ox id ize malate at s i g n i f i c a n t l y higher ra tes than those of rabbi t or rat heart was l inked to higher combined a c t i v i t i e s of NADP"*"- and NAD*-1 inked malic enzyme in the cod mitochondria. In the present study, malate appeared to be e a s i l y transported across the inner mitochondrial membrane s ince the requirement of many substrates fo r a malate "spark" was met by extramitochondrial malate concentrat ions of 0.05mM. The "spark" was not of s u f f i c i e n t l y high concentrat ion to be ox id ized d i r e c t l y . Mitochondria from e i ther heart or mosaic muscle mitochondria of rainbow trout contained high enough malic enzyme a c t i v i t y to allow oxidat ion of 5mM malate at ra tes equal to those of pyruvate under some cond i t i tons . In cont ras t , t rout l i v e r mitochondria were incapable of burning malate at high ra tes (Suarez and Hochachka,1981a). It has been postulated by Davis and h i s co-workers (Davis et a l . , 1 9 7 2 ; Davis and Bremer,1973; L in and Davis,1974; Lee and Davis,1979; Hi ltunen and Davis,1981) that malic enzyme may be important in the regulat ion of the concentrat ion of TCA cyc le intermediates and in the regulat ion of energy metabolism in muscle. Furthermore, the a b i l i t y to convert TCA cyc le intermediates to pyruvate may play a spec ia l r o l e in the conversion of the carbon skeleton of branched chain amino ac ids to a lanine in s k e l e t a l muscle (Barber et a l . , 1 9 7 6 ; Goldstein and Newsholme,1976; Lee and Davis ,1979) . L i p i d s and amino ac ids (protein) are potent ia l f u e l s fo r both heart and mosaic muscle mitochondria of rainbow t rou t . A c e t y l - c a r n i t i n e , p a l m i t o y l - c a r n i t i n e and several 71 amino ac ids have proven good substrates for mitochondria i s o l a t e d from a number of sources (Table 5 , 6 ) . In order to assess r e l a t i v e va lue , ox idat ive phosphorylation of a c e t y l - c a r n i t i n e or glutamate was monitored for each t i s s u e . State 3 rates of ox idat ion of both substrates at lS^C were high in heart (see Table 5) although somewhat lower than those of pyruvate or malate. RCR values remained high (>6), ADP/O was unchanged and a l l substrates appeared to be equal ly e f f i c i e n t in terms of resp i ra to ry c o n t r o l . The potent ia l fo r using carbohydrate, l i p i d or prote in as an ox idat ive fue l source e x i s t s in hear t , based on mitochondrial substrate u t i l i z a t i o n . Carbohydrate would appear to be the best source, at leas t in terms of rate of ox idat ive phosphorylat ion. The high ra te of pyruvate u t i l i z a t i o n may have r e f l e c t e d a need to burn l a c t a t e ox ida t i ve l y . According to mitochondrial substrate u t i l i z a t i o n at lS^C, carbohydrate, l i p i d and prote in are a l l potent ia l ox idat ive substrates in t rout mosaic muscle. Both acety l—carn i t ine and glutamate produced s ta te 3 ra tes which were s i m i l a r to those of pyruvate. Although RCR values were v a r i a b l e , they were a l l greater than 4. State 4 resp i ra to ry ra tes were s i m i l a r fo r a l l substrates except l a c t a t e which produced s i g n i f i c a n t l y higher ra tes . This was r e f l e c t e d in the fac t that l a c t a t e had the lowest RCR. The reason for high s ta te 4 rates with l a c t a t e as substrate i s unknown. ADP/O r a t i o s were s i m i l a r to one another for a l l subst rates . In terms of rate and contro l of ox idat ive phosphorylat ion, 72 TABLE 5; State 5 O s Consumption of var ious species SUBSTRATE Q 2 Consumption SUBSTRATE Q z Consumption Pyruvate 5mM 5mH 5oH 5ffiH Pyruvate and Halate 5DIH 3sH 5mH 0.5aH iii>M Hal ate ifsM 3mH 5fflH Succinate 5IBH 5aiH 5ffiH + rotenone lOcifl + rotenone lOuH + rotenone 8.8" 74.2 + 17.0 b 330 i 8 C 335 j 13 d 50.8* 115.2 b 15.6 + 0.8" 20.2 + 0.4-8.7* 92.2 + 14,8" 64.3* 54.2 i 15.6" 81.0 + 11.2* 2188 38 h 9B l Glutamate 1(BH 5mH 5mH + 3mH malate 5sH + O.loH pyruvate + malate lOaH + 5BH i a l a t e 10aH + 5mH malate P r o l i n e 30mH IfflH 5mH + 0.05 fflH pyruvate P a l m i t o y l - c a r n i t i n e 50uM + 3mM malate 7.5 uH + 0.05aH malate 30uli 30uM A c e t y l - c a r n i t i n e 0.63iH 0.63mH 22.2 + 1.2-116.8 + 10.0 b 35.4* 33.4 + 8.5 f 2349 22" 55 1 209.0 i 22.4 b 27.6 + 1.2-71.2 + 15.6* 27.1* 94.8" 278 + 10 c 159 ± 7 d 244 t l l c 132 t 5* Oxygen consumption i n natoms Q/min/oq p r o t e i n a. Trout l i v e r , 15°, (Suarez and Hochachka, 1981a) b. l o c u s t rectum, 25°C, (Chamberlin and P h i l l i p s , 19835 c. r a b b i t red muscle, unknown T°, (Pande and Blanchaer,1971) d. r a b b i t white muscle, unknown T°,(Pande and Blanchaer, 1971) e. ribbed mussel, 25°, (Burcham et al.,1984! f. squid heart, 15°C, (Hommsen and Hochachka, 1981) g. dog heart, unknown T°,(Hukherjee et al.,1979) h. dog l i v e r , unknown T D, (Fry et a l . , 1980) i . dog kidney, unknown T°, (Fry et a l . , 1980) 73 TABLE 6: State 3 0=. consumption of locust and blowfly fa t body mitochondria at 30°C (Ballantyne and Storey,1983). Mean ± S E M SUBSTRATE Pyruvate(5.71mM) Pyruvate(5.71mM) malate(0.1ImM) Malate(5.71mM) Malate(5„71mM) pyruvate(0.1ImM) Succinate(5.71mM) rotenone Glutamate(5.71mM) malate(0.llmM) Proline(22.9mM) pyruvate(0.1ImM) Palmitoy l -carn i t ine( lOuM) malate<0.llmM) D 2 CONSUMPTION (natoms O/min/molecule cyt a) LOCUST N.D. N.D. 93.0 ± 30.4 101.0 ± 10.0 66.9 ± 37. 1 130.6 ± 35.2 N.D. 187.8 ± 21.8 BLOWFLY 195.8 ± 54.3 283.3 ± 82.6 130.3 ± 35.9 211.2 ± 70.2 424.7 ± 78.0 133.8 ± 71.5 165.O ± 41.5 594.3 ± 20.8 74 a c e t y l - c a r n i t i n e , glutamate and pyruvate were u t i l i z e d equal ly well by mosaic muscle mitochondria. State 3 rates for a l l substrates in both t i ssues compared favourably to ox idat ion rates measured by other workers (Table 5 ) , p a r t i c u l a r l y those for rainbow trout l i v e r (Suarez and Hochachka,1981a). At 15°C heart mitochondria exhibi ted higher rates of ox idat ive phosphorylation than did muscle mitochondria with e i ther pyruvate or malate as substrate . In terms of ox idat ive carbohydrate metabolism, heart not only has the advantage of more mitochondria per given mass of t i s s u e , but a lso a higher rate of substrate ox idat ion as a funct ion of mitochondrial p ro te in . The e f f i c i e n c y with which substrate u t i l i z a t i o n i s coupled to ATP production appears to be the same for both t i s s u e s . A c e t y l - c a r n i t i n e and glutamate were u t i l i z e d at the same rate by mitochondria from both t i s s u e s at 15°C. RCR values were higher in heart with these subst rates , i n d i c a t i n g a more e f f i c i e n t coupl ing of substrate u t i l i z a t i o n to ATP product ion. Rates of l a c t a t e u t i l i z a t i o n in the two mitochondrial populat ions could not be d i r e c t l y compared because of sub—maximal rates of l a c t a t e oxidat ion in heart . Substrate u t i l i z a t i o n in v ivo depends on substrate a v a i l a b i l i t y and competi t ion. Both the presence and ox idat ion of f a t t y ac ids can i n h i b i t g l y c o l y s i s at several l e v e l s , inc lud ing t ransport of glucose in to the c e l l , phosphofructokinase (PFK) and pyruvate dehydrogenase (see Neely and Morgan,1974). Oxidation of a c y l - c a r n i t i n e s , in 75 p a r t i c u l a r p a l m i t o y l - c a r n i t i n e , have been shown to i n h i b i t decarboxylat ion o-f pyruvate in rat heart (Bremer, 1965) , ra t l i v e r (Batenburg and 01sen,1976), t rout l i v e r (Suarez and Hochachka,19Blb) and chicken l i v e r (Jagow et a l . ,1968) mitochondria. This inh ib i ton was par t l y a l l e v i a t e d in rat heart by f ree c a r n i t i n e and may have been caused by increased l e v e l s of acyl -CoA and decreased l e v e l s of f ree CoA (Bremer,1965). In addi t ion to substrate a v a i l a b i l i t y and compet i t ion, power output p lays a r o l e in aerobic substrate preference. Glycogen can susta in high—power aerobic metabolism for a l imi ted period in man, while f a t s provide a more long—term energy supply at the cost of reduced power output. This i s presumably mediated by competit ion for ADP (Hochachka and Somero,1984). TEMPERATURE Temperature may a f f e c t mitochondrial r e s p i r a t i o n at several l e v e l s . Once a substrate has entered the TCA cyc le however, a l l steps to the f i n a l reduct ion of oxygen are the same no matter what the o r i g i n a l substrate . Any e f f e c t that temperature has on any of these steps would a f f e c t ox idat ion of a l l substrates equal l y . Each substrate used in t h i s study has i t s own d isc reet t ransporter and no two substrates use exact ly the same enzyme complement for entry in to the TCA c y c l e . The fac t that a l l of the t ransporters and some of the enzymes required for entry in to the TCA cyc le are membrane—bound increases the l i k e l i h o o d of temperature s e n s i t i v i t y s ince temperature change i s known to a l t e r 76 membrane - f l u i d i t y . The general phenomenon of temperature induced membrane res t ruc tu r ing in rainbow trout has been reviewed by Hazel (1984). Accl imation and environmental temperatures known to a-ffect the l i p i d compositon o-f membranes inc lud ing mitochondrial membranes, can inf luence a c t i v i t i e s of enzymes which are f u n c t i o n a l l y bound to these membranes (Hazel and Prosser ,1974; Hochachka and Bomero,1973p Smith, 1973,1974,1976). The e f f e c t of temperature on the transmitochondrial pH gradient i s not c l e a r . Change of i n t r a c e l l u l a r pH with temperature i s t i s sue dependent, but whether t h i s a f f e c t s the pH gradient i s uncer ta in . Temperature may d i r e c t l y a f f e c t int ramitochondr ia l pH. It i s important to determine i f the transmitochondrial pH gradient va r ies with temperature because the magnitude of t h i s gradient d i r e c t l y a f f e c t s the rate of ox idat ive phosphorylation (see Hinkle and McCarty,1978) . Temperature had an e f f e c t on the pattern of substrate u t i l i z a t i o n in mitochondria i s o l a t e d from both t i s s u e s . In heart mitochondria pyruvate remained the substrate of preference at 5 a C , while a c e t y l - c a r n i t i n e or glutamate were ox id ized at r e l a t i v e l y reduced ra tes . State 3 rates for malate were a lso s i g n i f i c a n t l y lower than for pyruvate at 5°C. This switch in substrate u t i l i z a t i o n was r e f l e c t e d by Q i o of s ta te 3 ox idat ion . State 3 Q 1 D values were c lose to 2 for a l l substrates except for malate, which produced a mean Q i 0 of 3.08 for s ta te 3 and 3.38 for s tate 4 r e s p i r a t i o n . It i s poss ib le that e i ther malate transport or malic enzyme a c t i v i t y were s i g n i f i c a n t l y reduced at low 77 temperature in t rout heart mitochondria. RCR did not vary s i g n i f i c a n t l y as a resu l t of temperature except with lac ta te as subst rate . Apparently , a change of temperature - at l eas t in the range of 5 - 15°C did not s i g n i f i c a n t l y a l t e r H"* ion permeabi l i ty . These r e s u l t s were consistent with those of Pye et a l . (1976) which ind icated no consistent change in RCR over a broad temperature range in e i ther muscle or l i v e r of tench (Tinea t i n e a ) . The reason for high s ta te 4 rates of l a c t a t e oxidat ion at 5 Q C in heart mitochondria i s unknown although may be re la ted to sub-maximal ox idat ion condi t ions in the assay. The net e f f e c t of temperature on heart mitochondria was that even at 5 D C , mitochondrial ox idat ion could be maintained at reasonable rates (for comparison see Table 5 ) . The maximal rates of ox idat ion of pyruvate, a c e t y l - c a r n i t i n e and glutamate a l l changed by approximately the same amount. As at 15 0 C, carbohydrate would appear to be the best ox idat ive fue l a v a i l a b l e at 5°C. The advantage of carbohydrate ox idat ion may be emphasized as a r e s u l t of the large decrease in ox idat ion rate of a l l substrates at 5 0 C . Movement of f i s h to lower temperature genera l ly r e s u l t s in a major decrease in metabolic rate (Prosser,1973). Recent ly , M o f f i t t and Crawshaw (1983) demonstrated that heart ra te dropped in carp (Cyprinus carpi o L.) in conjunction with metabolic rate with small drops in environmental temperature. Both the response and recovery were r a p i d . No compensation occurred over a period of one hour. However while the heart rate was diminished, and presumably i t s demand for energy was slowed, the heart must 7 8 have s t i l l continued funct ion ing a e r o b i c a l l y . Teleost heart cannot maintain a workload using anaerobic energy (see Dr iedz ic and H a r t , 1 9 8 4 ) . If heart must maintain energy supply at low temperature i t may depend more heavi ly on a substrate which can produce high rates of ox idat ive phosphory1ati on. The substrate s p e c i f i c i t y of the temperature response ind ica tes s p e c i f i c act ion of temperature on substrate t rans locato rs or on the biochemical steps required for entry in to the TCA c y c l e . It has been prev ious ly stated that these funct ions may be af fected by membrane s t ructure which i s a l te red by temperature. In contrast to mammals, poiki1otherms have been reported to show l i n e a r Arrhenius p l o t s for mitochondrial enzymes with a constant energy of a c t i v a t i o n over the e n t i r e range studied (Lyons and Ra ison , 1 9 7 0 s McMurchie et a l . , 1 9 7 3 ; S m i t h , 1 9 7 3 ) . Smith ( 1 9 7 6 ) has suggested that the greater f l e x i b i l i t y of f i s h mitochondrial enzymes at low temperature might be a property which, together with the absence of a phase change in membrane l i p i d s , would account for the maintenance of a low and constant a c t i v a t i o n energy over a wide range of temperature. However, more recent data has challanged t h i s general view of phase t r a n s i t i o n , with discontinuous Arrhenius p l o t s being reported fo r succinate oxidase in three species of t r o p i c a l f i s h ( Irving and Watson,1 9 7 6 ) and succinate oxidase and cytochrome oxidase in l i v e r and red muscle of carp (Wodtke,1 9 7 6 ) . The pattern of substrate u t i l i z a t i o n was more dramat ica l l y a f fected by temperature in muscle mitochondria 79 than in heart . At 15°C a l l substrates appeared t c be equal ly good -for aerobic muscle metabolism based on maximal s ta te 3 r a t e s . As in heart mitochondria malate was burned s i g n i f i c a n t l y more slowly than pyruvate at 5 a C , i n d i c a t i n g that supply of pyruvate from malate was somehow r e s t r i c t e d at low temperatures. The degree to which malate oxidat ion was af fected by temperature was l e s s in muscle than in heart . In genera l , TCA cyc le enzymes do not occur in t i s sue s p e c i f i c isozymic forms (see Hochachka and Somero,1984). Mal ic enzyme i s not , s t r i c t l y speaking, a TCA cyc le enzyme, but i t i s c l o s e l y associated with the c y c l e . A comparison of the temperature k i n e t i c s of t h i s enzyme and the malate t ransporter from both t i s s u e s might expla in the d i f fe rence in Q i o values observed between the two t i s s u e s . While acety l—carn i t ine and glutamate were u t i l i z e d by muscle mitochondria at 15°C, both substrates produced very low rates of ox idat ive phosphorylation at 5°C. This extreme drop in ox idat ion was r e f l e c t e d in very high s ta te 3 and s ta te 4 Q i o va lues. A s i m i l a r s i t u a t i o n has been observed in freeze—tolerant g a l l f l y (Eurosta s o l i d a q i n i s ) larvae where l i p i d metabolism was switched off in cold—acclimated larvae (Ballantyne and Storey,1984a). Since oxidat ion of other substrates was not so d r a s t i c a l l y a f f e c t e d , i t was concluded by the authors that i n h i b i t i o n did not l i e at the leve l of the TCA c y c l e , the e lect ron transport system or ox idat ive phosphorylation and that the s i t e of i n h i b i t i o n would occur between the t ransfer of palmitoyl—L—carnit ine in to mitochondria and c i t r a t e synthase. A s i m i l a r explanation seems l i k e l y in t h i s study given that 80 a c e t y l - c a r n i t i n e and glutamate oxidat ion rates were much more d r a s t i c a l l y lowered than pyruvate, l a c t a t e or malate. According to mitochondrial substrate u t i l i z a t i o n , carbohydrate would be the best aerobic fue l source for muscle at 5°C. The slow rate of a c e t y l - c a r n i t i n e or glutamate ox idat ion taken in conjunction with low mitochondrial density ind ica tes that these would be poor substrates fo r t h i s t i s s u e . No substrate had an advantage over the other in terms of RCR or ADP/O except a c e t y l - c a r n i t i n e which had a higher mean RCR than any other substrate at e i ther temperature. This may have been a r e s u l t of temperature i n h i b i t i o n of overa l l a c e t y l - c a r n i t i n e ox idat ion . L ike heart mitochondria, there did not appear to be any change in membrane i n t e g r i t y , s p e c i f i c a l l y H"*" ion permeabi l i t y , over t h i s temperature range. In a d d i t i o n , ADP/O values were e s s e n t i a l l y unchanged for a l l substrates at 5°C. Pye et a l . (1976) measured s ta te 3 ra te as a funct ion of temperature in tench. As with the present r e s u l t s and those of Bal lantyne and Storey (1984a) they discovered that the r e l a t i o n s h i p var ied according to substrate and t i s s u e , d isp lay ing a wide range of Q » o values . In t r o u t , temperature had a more d r a s t i c e f f e c t on mitochondrial r e s p i r a t i o n in muscle than in heart . State 3 Q i 0 values were higher for muscle except with malate as substrate . The imp l i ca t ion of t h i s i s that at low temperature ox idat ive metabolism in mosaic muscle of rainbow trout was s i g n i f i c a n t l y c u r t a i l e d . Siven the low mitochondrial density and somewhat lower resp i ra tory rates with some 81 substrates in t h i s t i s sue r e l a t i v e to hear t , i t i s poss ib le that mosaic muscle -f ibres were not rec ru i ted fo r aerobic work during acute temperature drop. Unl ike hear t , aerobic metabolism in mosaic muscle i s not the major component of t o t a l muscle energy supply. Other energy sources which may be a v a i l a b l e at low temperature inc lude anaerobic metabolism in mosaic muscle and aerobic metabolism in s u p e r f i c i a l red muscle, whose mitochondrial population may have somewhat d i f f e r e n t resp i ra to ry p roper t ies . It was noted that at lo^C maximal ra tes of ox idat ive metabolism in heart as a funct ion of mitochondrial protein were always equal to or greater than in muscle. At 5°C maximal oxidat ion rates of a l l substrates except malate were higher in heart than in muscle. Note that l a c t a t e ox idat ion i s not included in t h i s statement s ince i t was not maximally st imulated in heart mitochondria. C l e a r l y , muscle has not compensated for low mitochondrial density by increas ing mitochondrial a c t i v i t y . It has been shown by e lect ron microscopy that muscle mitochondria tend to have l e s s e laborate c r i s t a e (Nag,1972). A small inner membrane surface area r e l a t i v e to heart mitochondria may ind icate a lower cytochrome content , r e s u l t i n g in lower resp i ra to ry ra tes per given mass of mitochondrial prote in (Sordahl et a l . , 1 9 8 3 ) . EFFECT OF pH A c r i t i c a l fac to r which may a f f e c t rate of ox idat ive phosphorylation i s extramitochondrial pH. Synthesis of ATP i s thought to be dr iven by movement of H"*" ions in to the 82 mitochondrion in response to an electrochemical gradient (Mi tche l l ,1961; Mi tche l l and Moyle,1968). Part of t h i s gradient i s a r e s u l t of a transmitochondrial pH gradient . If t h i s gradient i s increased, ATP synthes is , coupled to 0 3 consumption i s dr iven more qu ick ly (see Hinkle and McCarty,1978). I n t r a c e l l u l a r pH may be a l te red in several ways. Increased temperature often r e s u l t s in decreased i n t r a c e l l u l a r pH in poiki1otherms (Reeves and Wilson,1970; Malan and Reeves,1973). Data from a number of f i s h species suggest that the change would be l e s s fo r heart than for white muscle (see He is le r ,1984) . In a d d i t i o n , a c i d i c endproducts from anaerobic metabolism a l t e r i n t r a c e l l u l a r pH, at leas t t r a n s i e n t l y . This would be a factor p a r t i c u l a r l y in mosaic muscle where anaerobic metabolism i s the major energy source. C l e a r l y , mosaic muscle would be expected to experience the larger s h i f t s in i n t r a c e l l u l a r pH. The next question i s - how do changes in i n t r a c e l l u l a r pH a f f e c t the transmitochondrial pH gradients? It has recent ly been demonstrated that the presence of phosphate or bicarbonate i s required in order for a decrease of extramitochondrial pH to r e s u l t in an increase of the pH gradient of non—respiring rabbi t kidney cortex , heart or l i v e r mitochondria (Simpson and Hager,1984). The assay buffer used in t h i s study contained lOmM phosphate. According to M i t c h e l l ' s chemiosmotic theory (1961), a pH gradient i s produced in a c t i v e l y r e s p i r i n g mitochondria v i a coupl ing of ADP phophorylation to e lect ron t ransport . It i s not c lear i f decreased i n t r a c e l l u l a r pH r e s u l t s in an increased pH gradient in such mitochondria. With e i ther acety l—carn i t ine or glutamate as substrate heart mitochondria displayed a progressive decrease of s ta te 3 rate as pH increased above 7 .0 . This i s what one might expect i f a decreased pH gradient was a f f e c t i n g substrate t ransport and rate of ox idat ive phosphorylat ion. A small sample s i z e and high v a r i a b i l i t y between preparat ions made determination of t r a n s i t i o n points d i f f i c u l t , but at a pH between 6.8 and 7.1 s ta te 3 rates began to leve l o f f . The highest rates of ox idat ive phosphorylation occurred at low to medium phys io log ica l pH. No sharp pH optimum was observed as in t i s s u e s of other organisms such as marine clam (fiercenaria mercenaria) (Ballantyne and Storey , 1984b) . RCR values were qu i te va r iab le and tended to mirror s tate 4 r a t e s . Again there was no c lear pH optimum. As fa r as one can judge on the bas is of RCR va lues , membrane permeabi l i ty to H"*" ions was not s i g n i f i c a n t l y a f fected by extramitochondrial pH at leas t within the normal phys io log ica l range. RCR and s tate 4 v a r i a b i l i t y could be a t t r ibu ted as e a s i l y to v a r i a b i l i t y among ind iv idua l preparat ions . It i s i n t e r e s t i n g to note that , regardless of the mechanism by which pH a f f e c t s s tate 3 r a t e s , between pH 7.4 — 7.0 absolute s ta te 3 rates did not change d r a s t i c a l l y . They ranged between 60 - 80 natoms m i n - 1 mg p r o t e i n - 1 for e i ther substrate . The v a r i a b i l i t y in measured parameters of ox idat ive phosphorylation was qui te high from one preparat ion to the next. This d i f f i c u l t y i s normally overcome by repeating 84 s p e c i f i c experimental condi t ions on several mitochondrial preparat ions . In the case of t h i s study, only four separate assays could be performed from each preparat ion . The pH could only be approximated p r io r to assay and confirmed once the assay was in progress or f i n i s h e d . This resul ted in an i n a b i l i t y to repeat s p e c i f i c pH values accurate ly which i s why each point on the pH p r o f i l e usual ly only represents a s i n g l e preparat ion . The pH values were scattered randomly among the preparat ions in order to avoid generation of a r t i f i c i a l t rends. The consequence of t h i s was poss ib le masking of rea l trends becauseof v a r i a b i l i t y between populat ions. This i s i l l u s t r a t e d by the malate pH p r o f i l e which was generated from a s i n g l e mitochondrial preparat ion . Measurements were made as described in Mater ia ls and Methods, taking care that a decrease in s ta te 3 ra te was not caused p r imar i l y by the e f f e c t of mitochondrial aging. It seems c lear from t h i s p r o f i l e that the rate of s ta te 3 ox idat ion of malate in heart increased with decreasing pH. In a d d i t i o n , RCR did not vary very much except at high pH where low RCR and high s ta te 4 rates occurred. These r e s u l t s were s i m i l a r to those observed with other substrates in the same t i s s u e , where the v a r i a b i l i t y of the response was increased by the need to use several preparat ions in order to encompass a wide range of pH. Mosaic muscle mitochondria responded d i f f e r e n t l y to changes in pH than did those of heart . With e i ther acety l—carn i t ine or glutamate as substrate there was a c lear decrease in s tate 3 rate above pH 7 .6 . Otherwise, there was no c lear pH dependence of s ta te 3 except at very low pH 85 where s tate 3 rates appeared to decrease somewhat. There was no i n d i c a t i o n that an increased pH gradient was d r i v ing ox idat ive phosphorylation more rap id l y as extramitochondrial pH was decreased. RCR values were highest between pH 6.9 -7.4 with a c e t y l - c a r n i t i n e and pH 7.0 — 7.4 with glutamate i n d i c a t i n g that membranes tended to be more leaky at e i ther low or high pH. State 4 rates were higher in mosaic muscle than in heart with glutamate as substrate and, to some extent , with a c e t y l - c a r n i t i n e as we l l . RCR values tended to be lower at both low and high pH in muscle than in heart . In a d d i t i o n , substrate u t i l i z a t i o n data presented prev iously has ind icated that RCR tended to be lower in muscle mitochondria than in heart . According to these data , muscle mitochondrial membranes may have been more leaky than the i r heart counterparts and may have been more s e n s i t i v e to extreme pH. Low RCR and high s tate 4 rates may t rans la te d i r e c t l y to increased permeabi l i ty o-f H"1" ions which, l o g i c a l l y , would be t rans la ted to a reduced H~*~ ion gradient across the membrane. It i s p o s s i b l e , the re fo re , that reduced extramitochondrial pH did not r e s u l t in an increased pH gradient because of changes in membrane permeabi l i ty . This may have been the reason that s tate 3 rates did not appear pH—dependent except at high pH. At very high pH, ox idat ive phosphorylation invar iab l y proceeded slowly. Unfortunately , few data are a v a i l a b l e on RCR or ADP/0 r a t i o s because the slow rates of ox idat ion made s ta te 4 very d i f f i c u l t to measure. From the sparse data a v a i l a b l e i t would appear that there was a severe breakdown in resp i ra to ry contro l and poss ib ly membrane damage at high 86 pH. In u l t r a s t r u c t u r e s t u d i e s , mitochondrial—swel1ing was demonstrated at high pH (Chang and M e r g n e r , 1 9 7 3 ) » The DMO technique i s commonly used to measure transmitochondrial pH. This technique would be a useful tool for examining fur ther the e f f e c t of extrami tochondri a l pH on resp i ra tory a c t i v i t y . It could be used to determine i f decreased extramitochondrial pH does in fac t r e s u l t in an increased pH grad ient , as has been observed with other t i s s u e s (Simpson and Hager,1984), and i f t h i s e f f e c t i s the same in both heart and muscle. Another technique a v a i l a b l e fo r measuring pH gradients i s 3 1 P NMR (Ogawa et a l . ,1981>. RCR values and s ta te 4 ra tes only allow one to speculate on the nature of H"" ion permeabi l i ty and pH gradients and should , consequently be used caut ious ly . Regardless of the mechanism by which pH a f f e c t s mitochondrial metabolism, the r e s u l t i s that s tate 3 ox idat ion appears to be l e s s dependent on extramitochondrial pH in mosaic muscle than in heart . Muscle, which may experience s i g n i f i c a n t changes in i n t r a c e l l u l a r pH as a r e s u l t of temperature or exe rc i se , would not be expected to experience any s i g n i f i c a n t change in rate of ox idat ive phosphorylation as a r e s u l t . Heart would a lso not experience large v a r i a t i o n in ox idat ive phosphorylation ra tes because i n t r a c e l l u l a r pH i s l e s s var iab le than in muscle. Otherwise, pH-dependece of s tate 3 rates in heart would cause increased capaci ty fo r 0 Z f lux in heart as pH dropped. In phys io log ica l terms, one would only expect a large decrease in i n t r a c e l l u l a r pH during cardiac hypoxia or ischemia when ox idat ive metabolism i s already severely 87 Q 2—1imited. Increased ra tes of ox idat ive phosphorylation may be useful during recovery from such a s t a t e . The e f f e c t of a c i d o s i s on mitochondrial r e s p i r a t i o n has long been of in te res t to researchers , p a r t i c u l a r l y with respect to hypoxia and ischemic c e l l damage (Mela et a l . , 1 9 7 2 ; Kahles et a l ,1979 ; Fry et al,1980s H i l l e r e d et a l ,1984) . Ischemia i s often character ized by c e l l u l a r swel l ing and i r r e v e r s i b l e mitochondrial damage (see Chaudry,1985). It has been postulated that the accompanying a c i d o s i s may be p a r t i a l l y responsib le for t h i s damage. Mitochondria i s o l a t e d from ischemic heart have been shown to d isp lay reduced RCR and s tate 3 ra te (Jennings and Ganote,1976; Schwartz et a l . , 1 9 7 3 ; Peng et a l . , 1 9 7 7 ; Kahles et a l . , 1 9 7 9 ) . Numerous mammalian s tud ies have ind icated that s tate 3 r e s p i r a t i o n of many t i s s u e s i s s e n s i t i v e to extramitochondrial pH (Chance and Conrad,1959; Mela et a l . , 1 9 7 2 ; Mitchelson and Hird,1973; Sharyshev et a l . , 1 9 8 3 ; Fry et a l . , 1 9 8 0 ; H i l l e r e d et a l . , 1 9 B 4 ) . The general response i s a decrease in s tate 3 rate with decreasing pH, although Chance and Conrad (1959) observed a s i g n i f i c a n t decrease in s tate 3 rate of heart mitochondria only as pH increased above pH 7 . 1 . The work of Tobin et a l . (1972) suggested that while s ta te 3 decreased at extreme pH, mitochondrial pH was not very s e n s i t i v e to pH in the phys io log ica l range. Mukherjee et a l . (1979) demonstrated that dog heart mitochondria, incubated with l a c t a t e at pH 6.3 for 3 hours showed no change in s tate 3 although RCR and SB ADP/O were depressed with glutamate or succinate as substrate . The mechanism by which H"*" ion concentrat ion af fected s ta te 3 rates in these studies i s unknown. T y p i c a l l y , ADP/O was not depressed except at f a i r l y extreme pH (Tobin et a l . , 1972.; i i i tchelson and Hird ,1973; Fry et a l . , 1 9 8 0 ) . Fry et a l . (19B0) have found that succinate ox idat ion was the best preserved during a pH change and have suggested that i n h i b i t i o n occurred at the NAD*—NADH redox l e v e l . However, while the response appears to be substrate s p e c i f i c , succinate i s not always the least a f fec ted (Tobin et a l . , 1 9 7 2 ; Mukherjee et a l . ,1979) i n d i c a t i n g that the response i s not e n t i r e l y caused by a pH—induced change in the resp i ra to ry chain (Tobin et a l . , 1 9 7 2 ) . The fac t that many mitochondrial enzymes have high optimal pH has been suggested as a means by which pH may a f f e c t r e s p i r a t i o n (Mela et a l . , 1 9 7 2 ) . This would imply that a c i d i f i c a t i o n of the external medium would cause some degree of int ramitochondr ia l a c i d i f i c a t i o n . Other f a c t o r s which are a l te red by ischemia and which may a f f e c t mitochondrial r e s p i r a t i o n include ECa35*"] (Mela at a l . , 1 9 7 2 ) , [phosphate], C lactate3 and d i l u t i o n caused by c e l l u l a r swel l ing (Mukherjee et a l . , 1 9 7 9 ) . According to r e s u l t s from t h i s study there was no decrease of s tate 3 resp i ra to ry rate with decreasing pH in t rout heart or mosaic muscle. It seems u n l i k e l y that phys io log ica l a c i d o s i s alone would be capable of c reat ing i r r e v e r s i b l e mitochondrial damage and concommitant c e l l u l a r abnormal i t ies in these t i s s u e s in the same manner postulated 89 by some mammalian researchers . Mitochondria i s o l a t e d from f r e e z i n g - t o l e r a n t larvae of ga l l f l y (Eurosta s o l i d a q i n i s ) a lso had a broad pH optimum in the phys io log ica l range (Ballantyne and Storey,1984a). However, the same authors have demonstrated a pH pattern s i m i l a r to that observed in many mammalian s tud ies in mitochondria i s o l a t e d from the hepatopancreas of a marine clam (Mercenaria mercenaria) (Ballantyne and Storey,1984b). C l e a r l y , the e f f e c t of extramitochondrial pH on mitochondrial r e s p i r a t i o n i s not s t ra ight forward , varying from species to species and t i ssue to t i s s u e . The e f f e c t of pH on l a c t a t e ox idat ion was s t r i k i n g l y d i f f e r e n t at low pH than had been observed with other substrates in t h i s study. For heart mitochondria a d i s t i n c t increase in s tate 3 rate was observed as pH was decreased. Below pH 7 . 0 , however, r e s p i r a t i o n dropped suddenly. Muscle mitochondria had a broader pH optimum, in keeping with the general trend observed prev ious ly , but below pH 7.0 s ta te 3 rates rap id l y decreased. No such rapid drop at low pH had been prev iously observed. Since H* ions are a product of pyruvate formation from l a c t a t e , increased a c i d i f i c a t i o n would tend to s h i f t the react ion toward formation of l a c t a t e rather than pyruvate. In a d d i t i o n , heart LDH, which was used in t h i s study, has a pH optimum greater than 10.0 (Lindahl and Mayeda,1975). Rapid decreases in lactate -based s ta te 3 ra tes at low pH probably ind ica te l imi ted conversion of pyruvate from l a c t a t e . This in te rp re ta t ion was confirmed by the addi t ion of pyruvate to the muscle assay mixture a f te r l ac ta te metabolism had been measured. The higher the 90 pH, the c loser l ac ta te ox idat ion rates were to maximal pyruvate ox idat ion r a t e s , i n d i c a t i n g that pH i n h i b i t i o n of s ta te 3 oxidat ion was a d i r e c t r e s u l t of a reduced a b i l i t y to convert l a c t a t e to pyruvate. State 4 rates were low and unchanging over a wide pH range in heart ox id i z ing l a c t a t e . RCR values r e f l e c t e d changes in s tate 3 r a t e . In muscle, s ta te 4 rates were much higher than in hear t , in accordance with high s ta te 4 and low RCR values observed prev ious ly in muscle mitochondria. The s tate 4 rates seemed inord inate ly h igh , however, p a r t i c u l a r l y at high pH. The reason for low RCR and high s ta te 4 values during ox idat ion of l a c t a t e by muscle mitochondria i s not known but i s consistent with previous f ind ings at 5 and 15°C. CONCLUSIONS Mitochondrial substrate u t i l i z a t i o n at 15°C ind ica tes that carbohydrates have the potent ia l for highest rates of aerobic energy supply in heart . Lactate i s a potent ia l substrate depending on conversion of l a c t a t e to pyruvate by LDH. L i p i d and prote in are a lso potent ia l substrates based on high oxidat ion rates of acety l—carn i t ine or glutamate. A l l subst ra tes , p a r t i c u l a r l y a c e t y l - c a r n i t i n e and glutamate, produce a high degree of resp i ra to ry c o n t r o l . In mosaic muscle, a l l substrates have equal potent ia l at 15°C» At 5°C carbohydrate appears to be the preferred aerobic energy source based on s ta te 3 ox idat ion ra tes . In heart mitochondria the pattern i s s i m i l a r to that at 15°C, but 91 lower overa l l ra tes at the lower temperature suggest that higher pyruvate ox idat ion ra tes r e l a t i v e to other substrates may be se lec ted . In mosaic muscle mitochondria both l i p i d and glutamate metabolism are dramat ica l l y switched down, leaving carbohydrate as the preferred aerobic fue l source. Q i o values for s ta te 3 oxidat ion rates by heart mitochondria are approximately 2 for most subst rates . Muscle mitochondria tend to d isp lay a greater temperature dependence, p a r t i c u l a r l y for ox idat ion of a c e t y l - c a r n i t i n e or glutamate. This may r e f l e c t each t i s s u e ' s s p e c i f i c need to maintain aerobic energy generation at low temperature. There i s no change in membrane i n t e g r i t y over the 10C o temperature range inves t iga ted , which i s presumably why a decrease in temperature i s not accompanied by a pred ic tab le change in RCR. Given i t s higher mitochondrial density heart d isp lays a higher capaci ty for provid ing energy a e r o b i c a l l y than does mosaic muscle. Mosaic muscle metabolism, on the other hand, does not appear to be compensated to any degree by increas ing resp i ra to ry rates of the mitochondria themselves. If anything, heart has the advantage of higher mitochondrial s ta te 3 rates per mg of mitochondrial p ro te in . State 3 r e s p i r a t i o n i s l e s s pH—dependent in muscle than in heart where s ta te 3 ra tes increase as pH decreases to approximately pH 6 .8 . Mitochondria from both t i ssues d isp lay a l imi ted a b i l i t y to produce energy a e r o b i c a l l y at high pH. RCR and s ta te 4 data suggest a higher degree of H"*" ion leakage in muscle mitochondria at pH extremes. Such leakage may a f f e c t the maintenance of a pH gradient 92 r e s u l t i n g in a decreased pH dependence in muscle mitochondria. As in a l l spec ies , t rout heart has a constant need for high rates of ox idat ive metabolism. This i s r e f l e c t e d by higher ra tes of mitochondrial r e s p i r a t i o n as compared to mosaic muscle, which i s l e s s ox idat i ve . The point i s fur ther emphasized by the fac t that muscle mitochondria are more severely a f fected by a drop in temperature. At low temperature i t i s not as c r i t i c a l for t rout to maintain ox idat ive metabolism in mosaic muscle as in heart . Of the two t i s s u e s , mosaic muscle i s l i k e l y to experience the larger f l u c t u a t i o n s in i n t r a c e l l u l a r pH. Mitochondrial resp i ra to ry rates of t h i s t i s s u e are l e s s dependent on pH in the phys io log ica l range. State 3 rates of heart mitochondria increase as pH decreases. 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