15.4. 1975
Specialia
c o n c e n t r a t i o n of 50 ~xg/ml C P Z e l i m i n a t e d t h e R - f a c t o r f r o m 1 5 % of t h e cells of a p o l y r e s i s t e n t s t r a i n of E. coli, w h i l s t 100 ~g e t h i d i u m - b r o m i d e , u n d e r t h e s a m e experim e n t a l c o n d i t i o n s , e l i m i n a t e d t h e p l a s m i d f r o m 10.5% of t h e cells. T h e a u x o t r o p h i c p r o p e r t i e s of t h e R - cells were t h e s a m e as t h e R+ ceils. I t is possible t h a t C P Z h a s a m u l t i f o c a l a c t i o n o n b a c t e r i a , since i t i n h i b i t s d i f f e r e n t e n z y m e s in m a m m a l i a n tissues e.g. g l u t a m a t e d e h y d r o g e n a s e 3, s u c c i n o x y d a s e , s u c c i n o d e h y d r o g e n a s e L a l t e r s t h e p e r m e a b i l i t y of biomembranes and inhibits oxydative-phosphorylation 5 and p h a g o c y t o s i s of h u m a n leucocytes in vitro".
Zusammen/assung. N a c h w e i s der a n t i m i k r o b i e l l e n Wirkung der bekannten Neuroleptika Chlorpromazin, Levom e p r o m a z i n u n d P r o m e t h a z i n , wobei C h l o r p r o m a z i n die b e s t e b a k t e r i z i d e W i r k u n g h a t t e . Die g r a m p o s i t i v e n
445
B a k t e r i e n s t ~ m m e w a r e n e m p f i n d l i c h e r gegen Chlorp r o m a z i n als die g r a m n e g a t i v e n , w ~ h r e n d C h l o r p r o m a z i n keinerlei YVirkung a u f die V e r m e h r u n g y o n Pseudomonas aeruginosa h a t t e . J. MOLNAR, J. I'{IRALY a n d Y v E ~ r n MANDI
Institute o/Microbiology, University Medical School, Dora tdr 70, H-6720 Szeged (Hungary), 18 November 7974. O. A. SttEMISA and L. A. FAHIEN, Molec. Pharmac. 7, 8 (1971). 4 •. W. HELPER, M, J. CARVER, H. P. JACOB and J. A. SMITH, Arch. Biochem. Biophys. 76, 354 (1958).
a F. LETERIER, J. CANVAand J. F. MARIAWD,C.r. hebd. S6anc. Acad. Sci. Paris. 273, 2668 (1971). 6 B. KVARSTEIN and H. STOR~mRKEN, Biochem. Pharmae. 20, 119 0971).
S i m u l t a n e o u s Recording of Heat and Fluorescence Following Contraction of Isolated Cardiac Muscle I t h a s b e e n e s t a b l i s h e d t h a t w h e n c a r d i a c m u s c l e is i r r a d i a t e d w i t h U V - l i g h t t h e i n t e n s i t y of f l u o r e s c e n t e m i s s i o n is l i n e a r l y r e l a t e d t o t h e a m o u n t of r e d u c e d n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e ( N A D H ) p r e s e n t 1. I n fluorescence s t u d i e s o n a m p h i b i a n s k e l e t a l m u s c l e JOBSlS a n d DUFFIELD 2 h a v e s u g g e s t e d t h a t t h e a r e a enclosed b y a fluorescence t r a n s i e n t w a v e f o r m fAFl.dt, s h o u l d b e l i n e a r l y p r o p o r t i o n a l to t h e flux of a d e n o s i n e diphosphate (ADP) through the respiratory chain during mitochondrial oxidative phosphorylation. This means t h a t t h e t i m e i n t e g r a l of a fluorescence c h a n g e following a m u s c l e c o n t r a c t i o n s h o u l d b e p r o p o r t i o n a l to t h e t o t a l e n e r g y cost of t h e c o n t r a c t i o n . I n o r d e r t o t e s t t h i s p o s t u l a t e we h a v e m a d e s i m u l t a n e o u s r e c o r d i n g s of h e a t p r o d u c t i o n a n d fluorescence c h a n g e s in i s o l a t e d c a r d i a c m u s c l e p e r f o r m i n g i s o m e t r i c a n d isotonic c o n t r a c t i o n s . TO TRANSDUCER
710,,Hg ARC ~ .
366nm
BEANSPLITTER
I >39Ohm
FLUORESCENCE ,REFLECTANCE
P a p i l l a r y muscles f r o m t h e r i g h t v e n t r i c l e s of s t u n n e d r a b b i t s were i s o l a t e d a f t e r p e r f u s i n g t h e c o r o n a r y c i r c u l a t i o n w i t h 20 t o 30 m l of p h y s i o l o g i c a l s o l u t i o n c o n t a i n i n g (raM) NaC1, 118.0; KC1, 4.75; M g S O 4 . 7 H 2 0 , 1.18; KH2PO4, 1.18; CaCI~.2H20 , 2.54; NaItCOa, 24.8 a n d a e r a t e d w i t h 9 5 % 0 2 - 5 % CO v T h i s s o l u t i o n was also used for b a t h i n g t h e i s o l a t e d m u s c l e w i t h t h e a d d i t i o n of 10 m M s o d i u m p y r u v a t e ( p H 7.4). The myothermic apparatus has been described previo u s l y 3. I t consists of a t h e r m o p i l e m a d e u p of o v e r 100 a c t i v e s i l v e r - c o n s t a n t a n j u n c t i o n s . T h e s e j u n c t i o n s are c o n t o u r e d i n t o a groove in w h i c h t h e p a p i l l a r y muscle sits. C e r t a i n m o d i f i c a t i o n s were m a d e to t h e t h e r m o p i l e t o f a c i l i t a t e fluorescence m e a s u r e m e n t s : in o r d e r t o m i n i m i z e b a c k g r o u n d fluorescence t h e t h e r m o p i l e a n d f r a m e were coated with black non-reflective, non-fluorescent paint e v e r y w h e r e e x c e p t for a s t r i p a b o u t 0.5 m m wide e x t e n d i n g d o w n t h e m i d d l e of t h e groove occupied b y t h e p a p i l l a r y muscle. T h i s g r o o v e also h o u s e d a f l a t t e n e d piece of p l a t i n u m a b o u t 4 m m in l e n g t h w h i c h was used as a s t i m u l a t i n g electrode a t t h e t e n d i n o u s e n d of t h e muscle. T h e o t h e r s t i m u l a t i n g electrode was m o u n t e d on a screw w h i c h could b e r o t a t e d so as t o p o s i t i o n t h e t i p of t h e electrode a g a i n s t t h e v e n t r i c u l a r e n d of t h e muscle as it l a y in t h e groove of t h e t h e r m o p i l e . T h i s a r r a n g e m e n t was used in order t o m i n i m i z e o p t i c a l a r t i f a c t s d u r i n g fluorom e t r i c r e c o r d i n g b u t it also allowed lower s t i m u l u s s t r e n g t h s t o be used t h a n w i t h t h e p r e v i o u s m e t h o d s w h e r e b o t h electrodes were c a n t i l e v e r e d f r o m a b o v e a. H e a t loss f r o m t h e m u s c l e - t h e r m o p i l e s y s t e m was p r a c t i c a l l y e x p o n e n t i a l so t h a t h e a t records could b e electrically c o r r e c t e d w i t h v e r y little error, b u t t o e n s u r e a c c u r a c y t h e u n c o r r e c t e d h e a t records were c o m p a r e d g r a v i m e t r i c a l l y w i t h a reference s t a n d a r d o b t a i n e d b y l i b e r a t i n g a k n o w n a m o u n t of e n e r g y i n t o t h e musclet h e r m o p i l e system.
THERHOPILE ,TO FEEDBACK
Fig. 1. Schematic diagram of apparatus for simultaneous recording
1 B. CHANCE, J. R. WILLIAMSON, D. JAMIESOI~ and B. SCHOENEB,
of heat and fluorescence. Filters are labelled according to the wavelengths transmitted. The physiological solution was drained from the muscle-thermophile chamber for myothermic and optical recording.
Biochem. Z. 341, 357 (1965). F. F. JOBSlS and J. C. DUFFIELD, J. gen. Physiol. 50, 1009 (1967). 3 C. L, GIBBS, W. F. H. M, MOMMAERTSand N. V. RICCNIUTI, J. Physiol., Loud. 791, 25 (1967).
446
Specialia
T h e f l u o r o m e t r i c a p p a r a t u s was s i m i l a r t o t h a t described p r e v i o u s l y 4 w i t h a d d i t i o n a l f e e d b a c k c o m p e n s a t i o n for i n s t a b i l i t y of t h e arc l a m p 5. As c a n b e seen in F i g u r e 1 t h e p r e p a r a t i o n was i l l u m i n a t e d w i t h 366 n m r a d i a t i o n f r o m t h e e p i - i l l u m i n a t i o n a t t a c h m e n t of a microscope. T h e i n t e n s i t y of t h e e m i t t e d l i g h t w h i c h p e a k s a t 480 n m was r e c o r d e d w i t h a p h o t o m u l t i p l i e r t u b e . T h e s.c. o u t p u t f r o m t h e p h o t o m u l t i p l i e r t u b e was d e m o d u l a t e d a n d d i s p l a y e d as a d.c. oscillographic signal. T h e level of 366 n m reflected l i g h t was also m o n i t o r e d t o serve as a c h e c k o n t h e o p t i c a l a l i g n m e n t of t h e p r e p a r a -
b
t]
I
II11111111
Ililllllll
i !
t
Pig. 2. Original records fromisometric (a) and isotonic (b) contractions of a papillary muscle. From above down the traces show heat production, thermoelectric output, fluorescence, reflectance, tension and muscle length. The calibrations from above down are 60 mJ/g of muscle, 5 EzV, 20% of the resting optical baseline, 25 mN/mm -2, 0.5 mm" The horizontal line calibration is 30 sec.
500 ~~
~
o
400 o
"~=300
o
9
~
9
i
9
o9
if_
200
IO0
io
80
Iio I~o HEAT (ma/gl
26o
Fig. 3. Relationships between fluorescence-time integral (fAF1/dt) and heat productioi1 obtained from 4 papillary muscles and corrected for stimulus heat artifact. Least square regression lines have been fitted with correlation coefficients of 9 0.93; J,, 0.96; A, 0.85; 0, 0.77.
I~XPERIENTIA 31/4
t i o n in t h e l i g h t field. If, following a c o n t r a c t i o n , t h i s r e f l e c t a n c e d i d n o t r e t u r n to b a s e l i n e t h e n t h e r e c o r d w a s rejected. T h e critical p r o b l e m in c o m b i n i n g t h e t w o r e c o r d i n g t e c h n i q u e s is t o allow for t h e l i m i t e d w o r k i n g d i s t a n c e f r o m t h e f r o n t of t h e microscope o b j e c t i v e (16 m m ) a n d still h a v e a d e q u a t e t h e r m a l i s o l a t i o n of t h e t h e r m o p i l e f r o m t h e l a b o r a t o r y s u r r o u n d i n g s . T h i s p r o b l e m was o v e r c o m e b y u s i n g a c y l i n d r i c a l stainless steel c o n t a i n e r as t h e reference w a t e r b a t h c o n t a i n i n g well stirred w a t e r c i r c u l a t e d t o i t f r o m a n e x t e r n a l t h e r m o s t a t 6. T h e c o n t a i n e r was s u r r o u n d e d b y a wall of p l a s t i c f o a m w h i c h was held in place b y a n o u t e r h o u s i n g m o u l d e d f r o m fibreglass. A hole large e n o u g h to a c c o m o d a t e t h e microscope o b j e c t i v e was m a d e in t h e i n s u l a t i n g layers in line w i t h a q u a r t z w i n d o w fixed to t h e inside of t h e stainless steel cylinder. A n o t h e r q u a r t z w i n d o w was also f i t t e d i n t o t h e f r o n t of t h e glass o r g a n b a t h c o n t a i n i n g t h e preparation and bathing medium. The clearance between t h e 2 q u a r t z w i n d o w s was o n l y 2 m m b u t t h i s space, occupied b y t e m p e r a t u r e - s t a b i l i z e d flowing water, w a s sufficient t o allow a d e q u a t e t h e r m a l isolation (Figure 1). W h e n t h e physiological s o l u t i o n was d r a i n e d f r o m t h e o r g a n b a t h tile h e a t i n g effect of t h e arc t a m p was f o u n d to be equivalent to about half of that due to the resting m e t a b o l i s m of t h e muscle. T h e s t e a d y t e m p e r a t u r e r e s u l t i n g f r o m t h e s e t w o h e a t sources f o r m e d t h e r e s t i n g b a s e l i n e a g a i n s t w h i c h t h e a c t i v e h e a t p r o d u c t i o n of c o n t r a c t i n g muscles was m e a s u r e d . F i g u r e 2 shows m y o t h e r m i c , optical, a n d m e c h a n i c a l signals r e c o r d e d oscillographically d u r i n g i s o m e t r i c a n d l i g h t l y l o a d e d isotonic c o n t r a c t i o n s of a p a p i l l a r y muscle a t 22~ I n e a c h of t h e t w o sets of records t h e m u s c l e was s t i m u l a t e d electrically to c o n t r a c t t e n t i m e s a t a r a t e of 0.25 Hz. T h e r e c o r d i n g c o n d i t i o n s were a) i s o m e t r i c c o n t r a c t i o n , b) isotonic c o n t r a c t i o n a g a i n s t a load of 9.8 m N . T h e i n d i v i d u a l t r a c e s s h o w n in F i g u r e 2 are, f r o m a b o v e down, h e a t p r o d u c t i o n (corrected for h e a t loss), t h e r m o e l e c t r i c o u t p u t (uncorrected), fluorescence ( > 390 nm), r e f l e c t a n c e (366 nm), t e n s i o n a n d m u s c l e l e n g t h . T h e c o r r e c t e d h e a t records were o b t a i n e d b y r e c o r d i n g t h e thermoelectric output on magnetic tape and replaying the signals t h r o u g h a h e a t loss corrector. T h e n e w l y - g a i n e d a d v a n t a g e of s i m u l t a n e o u s r e c o r d i n g is t h a t i t affords a c a l i b r a t i o n of t h e f l u o r o m e t r i c t e c h n i q u e h i t h e r t o u n a v a i l able e x c e p t for a m p h i b i a n s k e l e t a l m u s c l e 7. T h e tissue fluorescence r e c o r d e d u n d e r t h e p r e s e n t c o n d i t i o n s arizes largely f r o m t h e fluorescence of r e d u c e d n i c o t i n a m i d e a d e n i n e dinucleotide, N A D H 2,4, ~. If, as h a s b e e n a r g u e d for c a r d i a c m u s c l e 4, t h i s fluorescence is m i t o c h o n d r i a l in origin, t h e n i t is possible t o use t h e fluorom e t r i c m e a s u r e m e n t s to e s t i m a t e t h e e n e r g y c o n s u m p t i o n of c a r d i a c muscle c o n t r a c t i o n . F o u r p a p i l l a r y muscles were s t i m u l a t e d to c o n t r a c t i s o m e t r i c a l l y or i s o t o n i c a l l y a g a i n s t v a r i o u s loads a f t e r t h e p a t t e r n i l l u s t r a t e d in F i g u r e 2. Tile t i m e - i n t e g r a l s of t h e r e s u l t i n g fluorescence c h a n g e s were m e a s u r e d b y t r a c i n g t h e fluorescence w a v e f o r m s a n d u s i n g l i n e a r interpolation during the optical artifacts occurring during e a c h m e c h a n i c a l e v e n t . T h e t r a c i n g s were c u t o u t a n d weighed, a n d t h e w e i g h t s were p l o t t e d a g a i n s t t h e w e i g h t s of s i m i l a r t r a c i n g s of t h e u n c o r r e c t e d t h e r m o electric signals. T h e s e l a t t e r w e i g h t s were verified to b e p r o p o r t i o n a l w i t h i n 2 % error t o t h e a m p l i t u d e s of t h e c o r r e c t e d records of h e a t p r o d u c t i o n . J. B. C H A P M A N , J. gen. Physiol. 59, 135 (1972). a F. F. JOBSlS and W. N. STAIWSBY, Respir. Physiol. 4, 292 (1968). GC. L. GIBBS and W. R. GIBSON, J. gen. Physiol. 56, 732 (1970). 7 A. GODFRAIXD-DE BECKER, J. Physiol., Lond. 223, 719 (1972).
15.4. 1975
447
Specialia
I n F i g u r e 3 we s h o w d a t a f r o m t h e 4 e x p e r i m e n t s w i t h p y r u v a t e as s u b s t r a t e w h e r e f l u o r e s c e n c e - t i m e i n t e g r a l has been plotted against the total energy production. F o r e a c h m u s c l e t h e e n e r g y p r o d u c t i o n was v a r i e d b y allowing the muscle to contract isotonically against a r a n g e of afterloads. Use of a t-test r e v e a l e d t h a t n o n e of t h e i n t e r c e p t s was s i g n i f i c a n t l y d i f f e r e n t f r o m zero. T h e r e s u l t s are clearly c o m p a t i b l e w i t h t h e a s s u m p t i o n of J o B s l s a n d DUFFIELD 2. P r e l i m i n a r y e x p e r i m e n t s also show t h a t t h i s l i n e a r p r o p o r t i o n a l i t y b e t w e e n fluorescencet i m e i n t e g r a l a n d e n e r g y p r o d u c t i o n is f o u n d in o t h e r subs t r a t e s , b u t t h a t t h e p r o p o r t i o n a l i t y f a c t o r for a p a r t i c u l a r muscle depends on the substrate present. I n t w i t c h c o n t r a c t i o n s of s k e l e t a l muscle a s i m i l a r relationship between energy production and time-integral of fluorescence p r o b a b l y exists since in t o a d s a r t o r i u s m u s c l e t h e v a r i a t i o n in f l u o r e s c e n c e - t i m e i n t e g r a l w i t h load s is v e r y s i m i l a r t o t h e e n e r g y v a r i a t i o n w i t h load 9 T h e r e l a t i o n s h i p a p p a r e n t l y b r e a k s d o w n for r e t a i n as s h o w n b y t h e s i m u l t a n e o u s h e a t a n d fluorescence m e a s u r e m e n t s of GODFRAIND-DE ]~ECKER 7 on a m p h i b i a n s k e l e t a l m u s c l e a n d f r o m m e a s u r e m e n t s we h a v e m a d e on m a m m a l i a n s k e l e t a l muscle 10. I n m e t a b o l i c a l l y i n h i b i t e d p r e p a r a t i o n s several a u t h o r s h a v e d e m o n s t r a t e d t h a t a l i n e a r r e l a t i o n s h i p exists b e t w e e n p h o s p h o c r e a t i n e (PC) b r e a k - d o w n a n d e n e r g y p r o d u c t i o n ( h e a t + work) in m a n y t y p e s of c o n t r a c t i o n s n-13. H o w e v e r r e c e n t t e t a n i c e n e r g y b a l a n c e s t u d i e s 14,15 h a v e s h o w n a g r e a t e r i n i t i a l e n e r g y p r o d u c t i o n in m e t a b o l i c a l l y i n h i b i t e d a m p h i b i a n s k e l e t a l muscle t h a n w o u l d h a v e b e e n p r e d i c t e d o n t h e b a s i s of m e a s u r e d PC a n d A T P b r e a k d o w n . T h e comp l i c a t e d fluorescence d a t a o b t a i n e d a t h i g h s t i m u l u s r a t e s or u n d e r t e t a n i c c o n d i t i o n s m a y reflect c o m p l e x i t i e s p r o d u c e d b y h i g h glycolytic a c t i v i t y as s u g g e s t e d b y JOBSlS a n d DUFFIELD 2 a n d GODFRAIND-DE 13ECKER 7, o r m a y reflect t h e p o s s i b i l i t y t h a t u n k n o w n r e a c t i o n s cont r i b u t e t o t o t a l e n e r g y p r o d u c t i o n . H o w e v e r , in a e r o b i c c a r d i a c muscle, w h e r e t o t a l e n e r g y p r o d u c t i o n (initial a n d o x i d a t i v e r e c o v e r y h e a t ) is b e i n g m e a s u r e d 16-1s t h e
p o s t u l a t e of JOBSlS a n d DUFFIELD 2 does seem t o be confirmed. T h e t e c h n i q u e s d e s c r i b e d in t h i s r e p o r t are n o w available to study the relationship between pyridine n u c l e o t i d e fluorescence a n d t o t a l e n e r g y p r o d u c t i o n in i s o l a t e d p a p i l l a r y muscle. T h i s s h o u l d allow t h e fluorescence m e a s u r e m e n t s to be c a l i b r a t e d a c c o r d i n g to t h e prevailing metabolic conditions and hence provide much n e e d e d i n f o r m a t i o n as t o t h e n a t u r e a n d e x t e n t of t h e c o n d i t i o n s u n d e r w h i c h t h e f l u o r e s c e n c e - t i m e i n t e g r a l is proportional to total energy turnover.
Zusammen/assung. Nachweis, dass bei i s o m e t r i s c h e r u n d bei i s o t o n e r A r b e i t des P a p i l l a r m u s k e l s des K a n i n c h e n s die V e r / i n d e r u n g d e r F l u o r e s z e n z d i r e k t e IRiickschltisse a u f die d u r c h die A r b e i t v e r b r a u c h t e E n e r g i e erlaubt. J. t3. CI~AP~AN, C. L. GIBBS a n d H.
VOGELSANGER 19
Department o/Physiology, Monash University, Clayton 3168 (Victorla, Australia), 30 December 1974.
8 F. F. JoBsis and J. C. DUFEIELD, Science 756, 1388 (1967). 9 j . B. CHAPMANand C. L. GIBRS, Biophys. J. 72, 215 (1972). 10 I. WEXDT, Ph.D. Thesis, Monash University, Clayton (1974). 11 E. LUNDSGAARD, Bioehem. Z. 233, 322 (1931). 13 F. D. CARLSON and A. SIGER, J. gem Physiol. 43, 301 (1959). x3 D. R. WILKIE, J. Physiol., Lond. 795 (1968). 14 C. GILBERT, K. M. KRETZSCV:MAR, D. R. WILXlE and R. C. WOLEDGE, J. Physiol., Lond. 218, 163 (1971). 15 p. CANFIELD,J. LEBACQand G. MARECHAL,J. Physiol., Lond. 232, 467 (1973). is C. L. GIBBS, in Comparative Physiology o/ the Heart (Ed. F. V. McCA~r~; Birkh/iuser Verlag, Basel 1969), p. 78. 1T j. B. CI~AP~IANand C. L. GIBBS, Cardiovasc. Res. 8, 656 (1974). 18 j. B. ChAPMAn',in Advances in Cardiology (1974), vol. 12, p. 128. 19 This work was supported by a Grant-In-Aid from the National Heart Foundation of Australia.
Temperature Sensitivity of Bromosulphophthalein Clearance by the Liver C l e a r a n c e of b r o m o s u l p h o p h t h a l e i n ( B S P ) is used as a n i n d e x of h e p a t i c f u n c t i o n 1 a n d for t h e i n d i r e c t m e a s u r e m e n t of h e p a t i c b l o o d flow in e x p e r i m e n t a l animals2, ~ a n d m a n 4. I n a p e r f u s e d r a t liver, t h e t r a n s p o r t m a x i m u m for B S P in t h e f o r m a t i o n of bile is r e d u c e d b y decrease in t e m p e r a t u r e 5, 6. T h e r e f o r e t h e e v a l u a t i o n of B S P c l e a r a n c e in h y p o t h e r m i c a n i m a l s m a y b e a f f e c t e d b y t h e t e m p e r a t u r e d e p e n d e n c e of t h i s p r o c e d u r e . T h i s p a p e r r e p o r t s a n i n v e s t i g a t i o n of t h e c l e a r a n c e of B S P b y a n i s o l a t e d p e r f u s e d r a t l i v e r a t 37~ a n d 27~ in r e l a t i o n to t h e e s t i m a t i o n of h e p a t i c b l o o d flow. Materials and methods. L i v e r d o n o r s were fed m a l e W i s t a r a l b i n o r a t s w e i g h i n g b e t w e e n 250 a n d 300 g. T h e l i v e r p e r f u s i o n s y s t e m was b a s e d on t h e m e t h o d used b y HEMS et al. ~ a n d i n c o r p o r a t e d t h e m o d i f i c a t i o n s suggested b y HEMS a n d WtIITTON s. B o v i n e s e r u m a l b u m i n used in t h e p e r f u s a t e was d i a l y z e d a g a i n s t 3 c h a n g e s of K r e b ' s b i c a r b o n a t e b u f f e r ( p H 7.4) o v e r a 48 h p e r i o d to r e m o v e small vasoactive peptides. A water-jacket on the bulbo x y g e n a t o r e n s u r e d t h a t t h e t e m p e r a t u r e of t h e p e r f u s a t e was m a i n t a i n e d c o n s t a n t . T h e p e r i u s i o n p r e s s u r e (14 c m w a t e r ) was d e t e r m i n e d b y t h e h e i g h t of t h e p e r f u s a t e r e s e r v o i r in t h e o x y g e n a t o r a b o v e t h e liver. P e r f u s a t e flow r a t e was m e a s u r e d d i r e c t l y b y collecting t h e o u t f l o w in a g r a d u a t e d cylinder. C a n n u l a t i o n of t h e bile d u c t allowed
d i r e c t collection a n d m e a s u r e m e n t of bile flow. P e r f u s a t e s a m p l e s were c e n t r i f u g e d a t 1000 g a n d t h e s u p e r n a t a n t used. L a c t a t e was a s s a y e d u s i n g l a c t a t e d e h y d r o g e n a s e ( W o r t h i n g t o n ) " a f t e r p r e c i p i t a t i o n of p r o t e i n w i t h p e r c h l o r i c acid. S a m p l e s for ]3SP e s t i m a t i o n (0.2 E l ) were a d d e d t o 0.5 m l of 0.05 N s o d i u m h y d r o x i d e a n d t h e o p t i c a l d e n s i t y m e a s u r e d a t 565 n m . R e s u l t s are e x p r e s s e d as m e a n zh s t a n d a r d e r r o r of t h e m e a n a n d are c o m p a r e d b y S t u d e n t ' s t-test.
1 N. B. JAVITT, Progr. Liver Dis. 3, 110 (1970). 2 j . p. GILMORE, Am. J. Physiol. 795, 465 (1958). 8 W. C. SHOEMAKER, J. appl. Physiol. 15, 473 (1960). 4 S. ]~. BRADLEY, F. J. INGLEFINGER, G. P. BRADLEY and J. J.
CURRY, J. clin. Invest. 2d, 890 (1945). 5 R. J. ROBERTS, C. D. I{LAASSENand G. L. PLAA, Proc. Soe. exp.
Biol. Red. 125, 313 (1967). 6 j. L. BOYER, R. L. SC•EIG and G. K. KLATSKIN,J. elin. Invest. 49, 206 (1970). 7 R. HEMS, B. D. Ross, M. N. BERRY and H. A. t(REBS, Biochem. J. 101,284 (1966). s D. A. HEMS and P. D. WPIITTON,J. Physiol., Lond. 223, 6P (1972). 9 0 . WEILAND, in Methods in Enzymatic Analysis (Ed. H. U. BERGMEYER; Academic Press, New York 1963), p. 271.
Specialia
c o n c e n t r a t i o n of 50 ~xg/ml C P Z e l i m i n a t e d t h e R - f a c t o r f r o m 1 5 % of t h e cells of a p o l y r e s i s t e n t s t r a i n of E. coli, w h i l s t 100 ~g e t h i d i u m - b r o m i d e , u n d e r t h e s a m e experim e n t a l c o n d i t i o n s , e l i m i n a t e d t h e p l a s m i d f r o m 10.5% of t h e cells. T h e a u x o t r o p h i c p r o p e r t i e s of t h e R - cells were t h e s a m e as t h e R+ ceils. I t is possible t h a t C P Z h a s a m u l t i f o c a l a c t i o n o n b a c t e r i a , since i t i n h i b i t s d i f f e r e n t e n z y m e s in m a m m a l i a n tissues e.g. g l u t a m a t e d e h y d r o g e n a s e 3, s u c c i n o x y d a s e , s u c c i n o d e h y d r o g e n a s e L a l t e r s t h e p e r m e a b i l i t y of biomembranes and inhibits oxydative-phosphorylation 5 and p h a g o c y t o s i s of h u m a n leucocytes in vitro".
Zusammen/assung. N a c h w e i s der a n t i m i k r o b i e l l e n Wirkung der bekannten Neuroleptika Chlorpromazin, Levom e p r o m a z i n u n d P r o m e t h a z i n , wobei C h l o r p r o m a z i n die b e s t e b a k t e r i z i d e W i r k u n g h a t t e . Die g r a m p o s i t i v e n
445
B a k t e r i e n s t ~ m m e w a r e n e m p f i n d l i c h e r gegen Chlorp r o m a z i n als die g r a m n e g a t i v e n , w ~ h r e n d C h l o r p r o m a z i n keinerlei YVirkung a u f die V e r m e h r u n g y o n Pseudomonas aeruginosa h a t t e . J. MOLNAR, J. I'{IRALY a n d Y v E ~ r n MANDI
Institute o/Microbiology, University Medical School, Dora tdr 70, H-6720 Szeged (Hungary), 18 November 7974. O. A. SttEMISA and L. A. FAHIEN, Molec. Pharmac. 7, 8 (1971). 4 •. W. HELPER, M, J. CARVER, H. P. JACOB and J. A. SMITH, Arch. Biochem. Biophys. 76, 354 (1958).
a F. LETERIER, J. CANVAand J. F. MARIAWD,C.r. hebd. S6anc. Acad. Sci. Paris. 273, 2668 (1971). 6 B. KVARSTEIN and H. STOR~mRKEN, Biochem. Pharmae. 20, 119 0971).
S i m u l t a n e o u s Recording of Heat and Fluorescence Following Contraction of Isolated Cardiac Muscle I t h a s b e e n e s t a b l i s h e d t h a t w h e n c a r d i a c m u s c l e is i r r a d i a t e d w i t h U V - l i g h t t h e i n t e n s i t y of f l u o r e s c e n t e m i s s i o n is l i n e a r l y r e l a t e d t o t h e a m o u n t of r e d u c e d n i c o t i n a m i d e a d e n i n e d i n u c l e o t i d e ( N A D H ) p r e s e n t 1. I n fluorescence s t u d i e s o n a m p h i b i a n s k e l e t a l m u s c l e JOBSlS a n d DUFFIELD 2 h a v e s u g g e s t e d t h a t t h e a r e a enclosed b y a fluorescence t r a n s i e n t w a v e f o r m fAFl.dt, s h o u l d b e l i n e a r l y p r o p o r t i o n a l to t h e flux of a d e n o s i n e diphosphate (ADP) through the respiratory chain during mitochondrial oxidative phosphorylation. This means t h a t t h e t i m e i n t e g r a l of a fluorescence c h a n g e following a m u s c l e c o n t r a c t i o n s h o u l d b e p r o p o r t i o n a l to t h e t o t a l e n e r g y cost of t h e c o n t r a c t i o n . I n o r d e r t o t e s t t h i s p o s t u l a t e we h a v e m a d e s i m u l t a n e o u s r e c o r d i n g s of h e a t p r o d u c t i o n a n d fluorescence c h a n g e s in i s o l a t e d c a r d i a c m u s c l e p e r f o r m i n g i s o m e t r i c a n d isotonic c o n t r a c t i o n s . TO TRANSDUCER
710,,Hg ARC ~ .
366nm
BEANSPLITTER
I >39Ohm
FLUORESCENCE ,REFLECTANCE
P a p i l l a r y muscles f r o m t h e r i g h t v e n t r i c l e s of s t u n n e d r a b b i t s were i s o l a t e d a f t e r p e r f u s i n g t h e c o r o n a r y c i r c u l a t i o n w i t h 20 t o 30 m l of p h y s i o l o g i c a l s o l u t i o n c o n t a i n i n g (raM) NaC1, 118.0; KC1, 4.75; M g S O 4 . 7 H 2 0 , 1.18; KH2PO4, 1.18; CaCI~.2H20 , 2.54; NaItCOa, 24.8 a n d a e r a t e d w i t h 9 5 % 0 2 - 5 % CO v T h i s s o l u t i o n was also used for b a t h i n g t h e i s o l a t e d m u s c l e w i t h t h e a d d i t i o n of 10 m M s o d i u m p y r u v a t e ( p H 7.4). The myothermic apparatus has been described previo u s l y 3. I t consists of a t h e r m o p i l e m a d e u p of o v e r 100 a c t i v e s i l v e r - c o n s t a n t a n j u n c t i o n s . T h e s e j u n c t i o n s are c o n t o u r e d i n t o a groove in w h i c h t h e p a p i l l a r y muscle sits. C e r t a i n m o d i f i c a t i o n s were m a d e to t h e t h e r m o p i l e t o f a c i l i t a t e fluorescence m e a s u r e m e n t s : in o r d e r t o m i n i m i z e b a c k g r o u n d fluorescence t h e t h e r m o p i l e a n d f r a m e were coated with black non-reflective, non-fluorescent paint e v e r y w h e r e e x c e p t for a s t r i p a b o u t 0.5 m m wide e x t e n d i n g d o w n t h e m i d d l e of t h e groove occupied b y t h e p a p i l l a r y muscle. T h i s g r o o v e also h o u s e d a f l a t t e n e d piece of p l a t i n u m a b o u t 4 m m in l e n g t h w h i c h was used as a s t i m u l a t i n g electrode a t t h e t e n d i n o u s e n d of t h e muscle. T h e o t h e r s t i m u l a t i n g electrode was m o u n t e d on a screw w h i c h could b e r o t a t e d so as t o p o s i t i o n t h e t i p of t h e electrode a g a i n s t t h e v e n t r i c u l a r e n d of t h e muscle as it l a y in t h e groove of t h e t h e r m o p i l e . T h i s a r r a n g e m e n t was used in order t o m i n i m i z e o p t i c a l a r t i f a c t s d u r i n g fluorom e t r i c r e c o r d i n g b u t it also allowed lower s t i m u l u s s t r e n g t h s t o be used t h a n w i t h t h e p r e v i o u s m e t h o d s w h e r e b o t h electrodes were c a n t i l e v e r e d f r o m a b o v e a. H e a t loss f r o m t h e m u s c l e - t h e r m o p i l e s y s t e m was p r a c t i c a l l y e x p o n e n t i a l so t h a t h e a t records could b e electrically c o r r e c t e d w i t h v e r y little error, b u t t o e n s u r e a c c u r a c y t h e u n c o r r e c t e d h e a t records were c o m p a r e d g r a v i m e t r i c a l l y w i t h a reference s t a n d a r d o b t a i n e d b y l i b e r a t i n g a k n o w n a m o u n t of e n e r g y i n t o t h e musclet h e r m o p i l e system.
THERHOPILE ,TO FEEDBACK
Fig. 1. Schematic diagram of apparatus for simultaneous recording
1 B. CHANCE, J. R. WILLIAMSON, D. JAMIESOI~ and B. SCHOENEB,
of heat and fluorescence. Filters are labelled according to the wavelengths transmitted. The physiological solution was drained from the muscle-thermophile chamber for myothermic and optical recording.
Biochem. Z. 341, 357 (1965). F. F. JOBSlS and J. C. DUFFIELD, J. gen. Physiol. 50, 1009 (1967). 3 C. L, GIBBS, W. F. H. M, MOMMAERTSand N. V. RICCNIUTI, J. Physiol., Loud. 791, 25 (1967).
446
Specialia
T h e f l u o r o m e t r i c a p p a r a t u s was s i m i l a r t o t h a t described p r e v i o u s l y 4 w i t h a d d i t i o n a l f e e d b a c k c o m p e n s a t i o n for i n s t a b i l i t y of t h e arc l a m p 5. As c a n b e seen in F i g u r e 1 t h e p r e p a r a t i o n was i l l u m i n a t e d w i t h 366 n m r a d i a t i o n f r o m t h e e p i - i l l u m i n a t i o n a t t a c h m e n t of a microscope. T h e i n t e n s i t y of t h e e m i t t e d l i g h t w h i c h p e a k s a t 480 n m was r e c o r d e d w i t h a p h o t o m u l t i p l i e r t u b e . T h e s.c. o u t p u t f r o m t h e p h o t o m u l t i p l i e r t u b e was d e m o d u l a t e d a n d d i s p l a y e d as a d.c. oscillographic signal. T h e level of 366 n m reflected l i g h t was also m o n i t o r e d t o serve as a c h e c k o n t h e o p t i c a l a l i g n m e n t of t h e p r e p a r a -
b
t]
I
II11111111
Ililllllll
i !
t
Pig. 2. Original records fromisometric (a) and isotonic (b) contractions of a papillary muscle. From above down the traces show heat production, thermoelectric output, fluorescence, reflectance, tension and muscle length. The calibrations from above down are 60 mJ/g of muscle, 5 EzV, 20% of the resting optical baseline, 25 mN/mm -2, 0.5 mm" The horizontal line calibration is 30 sec.
500 ~~
~
o
400 o
"~=300
o
9
~
9
i
9
o9
if_
200
IO0
io
80
Iio I~o HEAT (ma/gl
26o
Fig. 3. Relationships between fluorescence-time integral (fAF1/dt) and heat productioi1 obtained from 4 papillary muscles and corrected for stimulus heat artifact. Least square regression lines have been fitted with correlation coefficients of 9 0.93; J,, 0.96; A, 0.85; 0, 0.77.
I~XPERIENTIA 31/4
t i o n in t h e l i g h t field. If, following a c o n t r a c t i o n , t h i s r e f l e c t a n c e d i d n o t r e t u r n to b a s e l i n e t h e n t h e r e c o r d w a s rejected. T h e critical p r o b l e m in c o m b i n i n g t h e t w o r e c o r d i n g t e c h n i q u e s is t o allow for t h e l i m i t e d w o r k i n g d i s t a n c e f r o m t h e f r o n t of t h e microscope o b j e c t i v e (16 m m ) a n d still h a v e a d e q u a t e t h e r m a l i s o l a t i o n of t h e t h e r m o p i l e f r o m t h e l a b o r a t o r y s u r r o u n d i n g s . T h i s p r o b l e m was o v e r c o m e b y u s i n g a c y l i n d r i c a l stainless steel c o n t a i n e r as t h e reference w a t e r b a t h c o n t a i n i n g well stirred w a t e r c i r c u l a t e d t o i t f r o m a n e x t e r n a l t h e r m o s t a t 6. T h e c o n t a i n e r was s u r r o u n d e d b y a wall of p l a s t i c f o a m w h i c h was held in place b y a n o u t e r h o u s i n g m o u l d e d f r o m fibreglass. A hole large e n o u g h to a c c o m o d a t e t h e microscope o b j e c t i v e was m a d e in t h e i n s u l a t i n g layers in line w i t h a q u a r t z w i n d o w fixed to t h e inside of t h e stainless steel cylinder. A n o t h e r q u a r t z w i n d o w was also f i t t e d i n t o t h e f r o n t of t h e glass o r g a n b a t h c o n t a i n i n g t h e preparation and bathing medium. The clearance between t h e 2 q u a r t z w i n d o w s was o n l y 2 m m b u t t h i s space, occupied b y t e m p e r a t u r e - s t a b i l i z e d flowing water, w a s sufficient t o allow a d e q u a t e t h e r m a l isolation (Figure 1). W h e n t h e physiological s o l u t i o n was d r a i n e d f r o m t h e o r g a n b a t h tile h e a t i n g effect of t h e arc t a m p was f o u n d to be equivalent to about half of that due to the resting m e t a b o l i s m of t h e muscle. T h e s t e a d y t e m p e r a t u r e r e s u l t i n g f r o m t h e s e t w o h e a t sources f o r m e d t h e r e s t i n g b a s e l i n e a g a i n s t w h i c h t h e a c t i v e h e a t p r o d u c t i o n of c o n t r a c t i n g muscles was m e a s u r e d . F i g u r e 2 shows m y o t h e r m i c , optical, a n d m e c h a n i c a l signals r e c o r d e d oscillographically d u r i n g i s o m e t r i c a n d l i g h t l y l o a d e d isotonic c o n t r a c t i o n s of a p a p i l l a r y muscle a t 22~ I n e a c h of t h e t w o sets of records t h e m u s c l e was s t i m u l a t e d electrically to c o n t r a c t t e n t i m e s a t a r a t e of 0.25 Hz. T h e r e c o r d i n g c o n d i t i o n s were a) i s o m e t r i c c o n t r a c t i o n , b) isotonic c o n t r a c t i o n a g a i n s t a load of 9.8 m N . T h e i n d i v i d u a l t r a c e s s h o w n in F i g u r e 2 are, f r o m a b o v e down, h e a t p r o d u c t i o n (corrected for h e a t loss), t h e r m o e l e c t r i c o u t p u t (uncorrected), fluorescence ( > 390 nm), r e f l e c t a n c e (366 nm), t e n s i o n a n d m u s c l e l e n g t h . T h e c o r r e c t e d h e a t records were o b t a i n e d b y r e c o r d i n g t h e thermoelectric output on magnetic tape and replaying the signals t h r o u g h a h e a t loss corrector. T h e n e w l y - g a i n e d a d v a n t a g e of s i m u l t a n e o u s r e c o r d i n g is t h a t i t affords a c a l i b r a t i o n of t h e f l u o r o m e t r i c t e c h n i q u e h i t h e r t o u n a v a i l able e x c e p t for a m p h i b i a n s k e l e t a l m u s c l e 7. T h e tissue fluorescence r e c o r d e d u n d e r t h e p r e s e n t c o n d i t i o n s arizes largely f r o m t h e fluorescence of r e d u c e d n i c o t i n a m i d e a d e n i n e dinucleotide, N A D H 2,4, ~. If, as h a s b e e n a r g u e d for c a r d i a c m u s c l e 4, t h i s fluorescence is m i t o c h o n d r i a l in origin, t h e n i t is possible t o use t h e fluorom e t r i c m e a s u r e m e n t s to e s t i m a t e t h e e n e r g y c o n s u m p t i o n of c a r d i a c muscle c o n t r a c t i o n . F o u r p a p i l l a r y muscles were s t i m u l a t e d to c o n t r a c t i s o m e t r i c a l l y or i s o t o n i c a l l y a g a i n s t v a r i o u s loads a f t e r t h e p a t t e r n i l l u s t r a t e d in F i g u r e 2. Tile t i m e - i n t e g r a l s of t h e r e s u l t i n g fluorescence c h a n g e s were m e a s u r e d b y t r a c i n g t h e fluorescence w a v e f o r m s a n d u s i n g l i n e a r interpolation during the optical artifacts occurring during e a c h m e c h a n i c a l e v e n t . T h e t r a c i n g s were c u t o u t a n d weighed, a n d t h e w e i g h t s were p l o t t e d a g a i n s t t h e w e i g h t s of s i m i l a r t r a c i n g s of t h e u n c o r r e c t e d t h e r m o electric signals. T h e s e l a t t e r w e i g h t s were verified to b e p r o p o r t i o n a l w i t h i n 2 % error t o t h e a m p l i t u d e s of t h e c o r r e c t e d records of h e a t p r o d u c t i o n . J. B. C H A P M A N , J. gen. Physiol. 59, 135 (1972). a F. F. JOBSlS and W. N. STAIWSBY, Respir. Physiol. 4, 292 (1968). GC. L. GIBBS and W. R. GIBSON, J. gen. Physiol. 56, 732 (1970). 7 A. GODFRAIXD-DE BECKER, J. Physiol., Lond. 223, 719 (1972).
15.4. 1975
447
Specialia
I n F i g u r e 3 we s h o w d a t a f r o m t h e 4 e x p e r i m e n t s w i t h p y r u v a t e as s u b s t r a t e w h e r e f l u o r e s c e n c e - t i m e i n t e g r a l has been plotted against the total energy production. F o r e a c h m u s c l e t h e e n e r g y p r o d u c t i o n was v a r i e d b y allowing the muscle to contract isotonically against a r a n g e of afterloads. Use of a t-test r e v e a l e d t h a t n o n e of t h e i n t e r c e p t s was s i g n i f i c a n t l y d i f f e r e n t f r o m zero. T h e r e s u l t s are clearly c o m p a t i b l e w i t h t h e a s s u m p t i o n of J o B s l s a n d DUFFIELD 2. P r e l i m i n a r y e x p e r i m e n t s also show t h a t t h i s l i n e a r p r o p o r t i o n a l i t y b e t w e e n fluorescencet i m e i n t e g r a l a n d e n e r g y p r o d u c t i o n is f o u n d in o t h e r subs t r a t e s , b u t t h a t t h e p r o p o r t i o n a l i t y f a c t o r for a p a r t i c u l a r muscle depends on the substrate present. I n t w i t c h c o n t r a c t i o n s of s k e l e t a l muscle a s i m i l a r relationship between energy production and time-integral of fluorescence p r o b a b l y exists since in t o a d s a r t o r i u s m u s c l e t h e v a r i a t i o n in f l u o r e s c e n c e - t i m e i n t e g r a l w i t h load s is v e r y s i m i l a r t o t h e e n e r g y v a r i a t i o n w i t h load 9 T h e r e l a t i o n s h i p a p p a r e n t l y b r e a k s d o w n for r e t a i n as s h o w n b y t h e s i m u l t a n e o u s h e a t a n d fluorescence m e a s u r e m e n t s of GODFRAIND-DE ]~ECKER 7 on a m p h i b i a n s k e l e t a l m u s c l e a n d f r o m m e a s u r e m e n t s we h a v e m a d e on m a m m a l i a n s k e l e t a l muscle 10. I n m e t a b o l i c a l l y i n h i b i t e d p r e p a r a t i o n s several a u t h o r s h a v e d e m o n s t r a t e d t h a t a l i n e a r r e l a t i o n s h i p exists b e t w e e n p h o s p h o c r e a t i n e (PC) b r e a k - d o w n a n d e n e r g y p r o d u c t i o n ( h e a t + work) in m a n y t y p e s of c o n t r a c t i o n s n-13. H o w e v e r r e c e n t t e t a n i c e n e r g y b a l a n c e s t u d i e s 14,15 h a v e s h o w n a g r e a t e r i n i t i a l e n e r g y p r o d u c t i o n in m e t a b o l i c a l l y i n h i b i t e d a m p h i b i a n s k e l e t a l muscle t h a n w o u l d h a v e b e e n p r e d i c t e d o n t h e b a s i s of m e a s u r e d PC a n d A T P b r e a k d o w n . T h e comp l i c a t e d fluorescence d a t a o b t a i n e d a t h i g h s t i m u l u s r a t e s or u n d e r t e t a n i c c o n d i t i o n s m a y reflect c o m p l e x i t i e s p r o d u c e d b y h i g h glycolytic a c t i v i t y as s u g g e s t e d b y JOBSlS a n d DUFFIELD 2 a n d GODFRAIND-DE 13ECKER 7, o r m a y reflect t h e p o s s i b i l i t y t h a t u n k n o w n r e a c t i o n s cont r i b u t e t o t o t a l e n e r g y p r o d u c t i o n . H o w e v e r , in a e r o b i c c a r d i a c muscle, w h e r e t o t a l e n e r g y p r o d u c t i o n (initial a n d o x i d a t i v e r e c o v e r y h e a t ) is b e i n g m e a s u r e d 16-1s t h e
p o s t u l a t e of JOBSlS a n d DUFFIELD 2 does seem t o be confirmed. T h e t e c h n i q u e s d e s c r i b e d in t h i s r e p o r t are n o w available to study the relationship between pyridine n u c l e o t i d e fluorescence a n d t o t a l e n e r g y p r o d u c t i o n in i s o l a t e d p a p i l l a r y muscle. T h i s s h o u l d allow t h e fluorescence m e a s u r e m e n t s to be c a l i b r a t e d a c c o r d i n g to t h e prevailing metabolic conditions and hence provide much n e e d e d i n f o r m a t i o n as t o t h e n a t u r e a n d e x t e n t of t h e c o n d i t i o n s u n d e r w h i c h t h e f l u o r e s c e n c e - t i m e i n t e g r a l is proportional to total energy turnover.
Zusammen/assung. Nachweis, dass bei i s o m e t r i s c h e r u n d bei i s o t o n e r A r b e i t des P a p i l l a r m u s k e l s des K a n i n c h e n s die V e r / i n d e r u n g d e r F l u o r e s z e n z d i r e k t e IRiickschltisse a u f die d u r c h die A r b e i t v e r b r a u c h t e E n e r g i e erlaubt. J. t3. CI~AP~AN, C. L. GIBBS a n d H.
VOGELSANGER 19
Department o/Physiology, Monash University, Clayton 3168 (Victorla, Australia), 30 December 1974.
8 F. F. JoBsis and J. C. DUFEIELD, Science 756, 1388 (1967). 9 j . B. CHAPMANand C. L. GIBRS, Biophys. J. 72, 215 (1972). 10 I. WEXDT, Ph.D. Thesis, Monash University, Clayton (1974). 11 E. LUNDSGAARD, Bioehem. Z. 233, 322 (1931). 13 F. D. CARLSON and A. SIGER, J. gem Physiol. 43, 301 (1959). x3 D. R. WILKIE, J. Physiol., Lond. 795 (1968). 14 C. GILBERT, K. M. KRETZSCV:MAR, D. R. WILXlE and R. C. WOLEDGE, J. Physiol., Lond. 218, 163 (1971). 15 p. CANFIELD,J. LEBACQand G. MARECHAL,J. Physiol., Lond. 232, 467 (1973). is C. L. GIBBS, in Comparative Physiology o/ the Heart (Ed. F. V. McCA~r~; Birkh/iuser Verlag, Basel 1969), p. 78. 1T j. B. CI~AP~IANand C. L. GIBBS, Cardiovasc. Res. 8, 656 (1974). 18 j. B. ChAPMAn',in Advances in Cardiology (1974), vol. 12, p. 128. 19 This work was supported by a Grant-In-Aid from the National Heart Foundation of Australia.
Temperature Sensitivity of Bromosulphophthalein Clearance by the Liver C l e a r a n c e of b r o m o s u l p h o p h t h a l e i n ( B S P ) is used as a n i n d e x of h e p a t i c f u n c t i o n 1 a n d for t h e i n d i r e c t m e a s u r e m e n t of h e p a t i c b l o o d flow in e x p e r i m e n t a l animals2, ~ a n d m a n 4. I n a p e r f u s e d r a t liver, t h e t r a n s p o r t m a x i m u m for B S P in t h e f o r m a t i o n of bile is r e d u c e d b y decrease in t e m p e r a t u r e 5, 6. T h e r e f o r e t h e e v a l u a t i o n of B S P c l e a r a n c e in h y p o t h e r m i c a n i m a l s m a y b e a f f e c t e d b y t h e t e m p e r a t u r e d e p e n d e n c e of t h i s p r o c e d u r e . T h i s p a p e r r e p o r t s a n i n v e s t i g a t i o n of t h e c l e a r a n c e of B S P b y a n i s o l a t e d p e r f u s e d r a t l i v e r a t 37~ a n d 27~ in r e l a t i o n to t h e e s t i m a t i o n of h e p a t i c b l o o d flow. Materials and methods. L i v e r d o n o r s were fed m a l e W i s t a r a l b i n o r a t s w e i g h i n g b e t w e e n 250 a n d 300 g. T h e l i v e r p e r f u s i o n s y s t e m was b a s e d on t h e m e t h o d used b y HEMS et al. ~ a n d i n c o r p o r a t e d t h e m o d i f i c a t i o n s suggested b y HEMS a n d WtIITTON s. B o v i n e s e r u m a l b u m i n used in t h e p e r f u s a t e was d i a l y z e d a g a i n s t 3 c h a n g e s of K r e b ' s b i c a r b o n a t e b u f f e r ( p H 7.4) o v e r a 48 h p e r i o d to r e m o v e small vasoactive peptides. A water-jacket on the bulbo x y g e n a t o r e n s u r e d t h a t t h e t e m p e r a t u r e of t h e p e r f u s a t e was m a i n t a i n e d c o n s t a n t . T h e p e r i u s i o n p r e s s u r e (14 c m w a t e r ) was d e t e r m i n e d b y t h e h e i g h t of t h e p e r f u s a t e r e s e r v o i r in t h e o x y g e n a t o r a b o v e t h e liver. P e r f u s a t e flow r a t e was m e a s u r e d d i r e c t l y b y collecting t h e o u t f l o w in a g r a d u a t e d cylinder. C a n n u l a t i o n of t h e bile d u c t allowed
d i r e c t collection a n d m e a s u r e m e n t of bile flow. P e r f u s a t e s a m p l e s were c e n t r i f u g e d a t 1000 g a n d t h e s u p e r n a t a n t used. L a c t a t e was a s s a y e d u s i n g l a c t a t e d e h y d r o g e n a s e ( W o r t h i n g t o n ) " a f t e r p r e c i p i t a t i o n of p r o t e i n w i t h p e r c h l o r i c acid. S a m p l e s for ]3SP e s t i m a t i o n (0.2 E l ) were a d d e d t o 0.5 m l of 0.05 N s o d i u m h y d r o x i d e a n d t h e o p t i c a l d e n s i t y m e a s u r e d a t 565 n m . R e s u l t s are e x p r e s s e d as m e a n zh s t a n d a r d e r r o r of t h e m e a n a n d are c o m p a r e d b y S t u d e n t ' s t-test.
1 N. B. JAVITT, Progr. Liver Dis. 3, 110 (1970). 2 j . p. GILMORE, Am. J. Physiol. 795, 465 (1958). 8 W. C. SHOEMAKER, J. appl. Physiol. 15, 473 (1960). 4 S. ]~. BRADLEY, F. J. INGLEFINGER, G. P. BRADLEY and J. J.
CURRY, J. clin. Invest. 2d, 890 (1945). 5 R. J. ROBERTS, C. D. I{LAASSENand G. L. PLAA, Proc. Soe. exp.
Biol. Red. 125, 313 (1967). 6 j. L. BOYER, R. L. SC•EIG and G. K. KLATSKIN,J. elin. Invest. 49, 206 (1970). 7 R. HEMS, B. D. Ross, M. N. BERRY and H. A. t(REBS, Biochem. J. 101,284 (1966). s D. A. HEMS and P. D. WPIITTON,J. Physiol., Lond. 223, 6P (1972). 9 0 . WEILAND, in Methods in Enzymatic Analysis (Ed. H. U. BERGMEYER; Academic Press, New York 1963), p. 271.
