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The influence of adrenaline and adrenochrome on oxygen consumption of liver homogenates
DRENALINE
AND ADREN(
PTION OF LIVER
ON
HOMOG]~
by SMA A~D H. L. GOLTERMAN try o] the Municipal University o/ Amst
'lands)
~.HLGREN1 reported in 1925 t h a t the reduction of methylene me 1 ~cle-pulp O I ; I O 15 cuo was accelerated by adrenaline in concentration, mtrations from I : .~rous publications on this subject h~ve since then appeared : s results confirmed b y VON EULER z and LILJESTRAND amon~g others, stigators L, however, no influence of adrenaline i n vitro, neith~ either on red ethylene nor on oxygen-consumption of muscle-pulp or other othq tissues in the ,urg-apparatus (references in GRIFFITI~3). ~rRIFFITH3 has surveyed the literature of the last 2 5 y e a r s , ject. He ~aline in ions some 5o publications which reported no influence influen whate )ublications in lhibiting ng concentrations, an almost equal number of public mblications " which an inhibitir on methylene blue-reduction or oxygen-consumption )tion is described and about twice twi~ as mare :Ions my publications in which stimulation b y adrenaline : w was found from concentratioI between I : IOe and I : io 15. asonable No ~1o explanation has been given of these contradictor)y results. I t seems reasonab to the fact that, when who to supl ppose t h a t the differences can, at least partly, be ascribed as~ surviving tissue-preparations, oI, n e experimentin imenting with tissue pulp, tissue-slices or other survi bout is never 7er exactly informed about the kind of substrates which w are oxidised, nor abol .lene the q uestion which of the m a n y chemical reactions that are implied in the methyler is the rat~ rateblue-reduction or the oxygen-consumption by the tissue-preparations tissl the~ limiting reaction• I t seems quite possible that adrenaline stimulates some of these [ reactions and inhibits others•• Moreover, adlenaline is a labile sul~stance, which can be nce ther compounds possessing biological activity, for instan¢ transformed in tissues into other msformations too might vary in tissue preparations in affl a adrenochrome, and these trans uncontl ollable way. he influence of adrenaline and ot adrenochrome in tissuetissu, We have investigated the IOnS systems in which the nature of the substrates oxidised and the rate-limiting reactioi of oxygen-consumption could~t be varied in a controllable way. homogenates of rat-liver, which were diluted to concenconcel For this purpose we usedd homo ated ed trations in which they showed no spontaneous oxygen-consumption, and investigate nd adrenochrome on the oxygen-consumption obtained b,y the influence ot adrenaline and -factors. adding various substrates andd adding or omitting cytochrome c and various co-factor z.w.o.) Supported by a subsidy from1the Netherlands Organisation for Pure Scientific Research (Z.W.C References p. 86.
TECHNICAL DETAILS m o x - h e a r t . W e us e d t h e KEILII~ AND s o l u t i o n of c y t o c h r o m e w h i c h containec w i t h 2 ml phosphate buffer-solution, t t i n c t i o n c o n s t a n t s of c y t o c h r o m e c gi me m l of our s o l u t i o n is 1.o6 mg, t h a t
aethod as ry r e s i d u e Lt 550 m/~ IKIN 5 , t h e he p u r i t y
PN) w a s p r e p a r e d from b a k e r s y e a s t . " used t h e m e t h o d d e s c r i b e d b y LE PAGEe
ic technics
Io % NaOH solution to a diluted solut ed w a t e r t h e n w i t h a c e t o n e a n d d r y i n g me we used a m o d i f i c a t i o n of VEERS 7 m e t iOIVt~U Ill IO 1111 I ) I U I 3 L I I I U U w ~ £ t t ~ l ,r. T J. h IIe U ~ 1~ s ~ Ih i ~a Kk ~ Ie I n w W ]i t h 2 5 - 5 0 sUoI lUuL ItOi IoI n is ute. A f t e r t h e re d color of a d r e n o c h r o m e h a s d e v e l o p e d , t h e Ag~O is c t e m p e r a t u r e t h e r e d color fades o n l y v e r y slowly. I n t h e B ] eckmann a b s o r p t i o n m a x i m a a t 30o m/z a n d a t 485 m/z, m i n i m a a t 26o 26 m/z a n d w i t h t h a t of CHAIX, o n l y o u r m a x i m u m a t 485 m/~ w a s bbroa r o ad e r t h a n t h "~iver-homogenates were p r e p a r e d b y h o m o g e n i z i n g fresh rrat-1 a t - l i v e r in ice. )n in a W a r i n g - b l e n d o r . W e u s e d Ca-free K r e b s - R i n tggeerr--pphhooss]p h a t e solul )xygen-consumption w a s m e a s u r e d w i t h t h e W a r b u r g - a p p~a ar ar at ~t u s a t 38o C [ t h r e e m a n o m e t e r s w e r e u s e d for e a c h d e t e r m i n a t i o n . The fig1ares in t h e ee v a l u e s . H a l f of t h e m a x i m a l d e v i a t i o n f r o m t h e a v e r a g e m il t h e thre~
one g r a m
, washing Ldrenaline for 0. 5 t o • Even at m e t e r we spectrum by CHAIXs. )hosphate periments Le a v e r a g e [esignated
EXPERIMENTAL RESULTS
ence o/adrenaline and adrenochrome on the oxygen-cons'~ en-consumption o s to which no substrate has been added
d homog-
1For ~o r these experiments we used lO% and 20% ho~ homogenates, which showed an oxygen ~n consumption of about 60 and 15o/A, respectivel y, in 60 minutes by 3 ml of home ~genate. No influence of adrenaline and adrenochn ~nochrome on oxygen-consumptk )tion could be detected.
ence o~ adrenaline and adrenochrome on the oxygen-tons, Influence en-tonsumption o/diluted homogenat ares + succin ccinate with and without addition o/ cytochrome c Accordin kccording to SCHNEIDER AND POTTER 9, in a system cof this kind, without addition additk 4-~^'k . . . . . V'k . . . . ~ . - 1 ~ - - ~ . - I . . ^ ~ - : ~ _ _ ^t __..~_L. . . . . . of cytochrome e, -the oxido-reducti reduction of cytochrome c 2zs_ the rate-limiting reaction of oxygen-consumption• When cytochrome c is added in high concentration, the rate of dehydrogenation of succinate ~.e determines the rate of oxygen-consumption. We four found no influence of adrenaline and adrenochrome on the oxygen-consumption of thee s e systems. Influence o[ adrenaline and admnochr chrome on the oxygen-consumption o/diluted homogenat ~renochrome ares + cyto~hrome c + ascorbate When ascorbate is added ,,d to diluted homogenates in high concentration, the cyt 7tochrome c in the homogenate is kept continually in reduced state. Under these clrcumcircur stances the oxido-reduction • of cytochrome-oxidase is the rate-limiting reaction for f oxygen-consumption. In this system, as is shown ~hown in Table I, adrenochrome stimulated oxygen-co en-consumption, adrenaline had no effect. Re/erences p. 86. IO
TABLE
I
OF ADRENOCttROME ON THE OXYGENgRATES ..L CYTOCHROME C-.~- ASCORBAT
I OF
genate, o. 5 ml o.i 31 ascorbate, o. 5 ml t ome-solution), pH 7.4.
6 - 10 -4 3 i
O~ consumption in yl adrenaline 20 ,/]ml
II20
a,
4 ° ~:: ~ 35 ~: I
3o
(
26 ~ I 44 ± 2
5
28 =E I 48 ± 2
ks a d r e n o c h r o m e also s t i m u l a t e d o x y g e n - c o n s u m p t~tion ic when c o u l d n o t be ascribed to a c c e l e r a t i o n ot t h e o x i d o - r eMuction dt of ( ?re f o u n d t h a t a d r e n o c h r o m e also c a t a l y s e d t h e ox::ygen-con, ~hate solution to which ascorbate was a d d e d (Table II). I
d d e d its oxidase. F Ringer
TABLE II FLUENCE
OF A D R E N O C H R O M E ON OXYGEN-CONSUMPTION AND O F R I N G E R - P H O S P H A T E - S O L U T I O N
"oral v o l u m e 2.5 ml: o. 5 m l O.l M buffer solution or h o m o g e n a t e .
in minutes
adrenochrome 2o ~,/ml
Io
133
60
343
BATE
ml H~O,
drenochrome-s¢ ascorbate, o. 5 m l adren(
Homogenate e.5% x. 5 mt oxygen-consumption in #I
Time
OF H O M O G E N I -~- ASCORBATE At
n 5 ml Ringer-phosphate solution oxygen-consumption in i~l
HsO
adr enochrome zo F/ml
16 62
124 464
H20
IO
56
solutio 7he catalytic effect of a d r e n o c h r o m e on the o x i d a t i o n oof ascorbate in Ringer solution Th, Table I I I ) . be d e t e c t e d in c o n c e n t r a t i o n s as l o w as o.15 y m l (T could b, TABLE RELATION
III
~TION O F A D R E N O C H R O M E AND EFFECT B E T W E E N CONCENTRATION F ASCORBATE IN R I N G E R SOLUTION OF C o n c e n t r a t~i ot ino n o f 1 / 6 0 M a s c o r b a t e , Concen~rat%n of adrenochrome
(w)
1.5' I°-e 10-6 IO-7
0 3 consumption
(#) 183 86 2o
ON OXYGEN-CONSUMPTION
t i m e 2o m i n u t e s .
Concentration of adrenochrome
(w)
io -s IO -11 10 -14
0 z consumption (td)
2±5 5~=6 o=t=6
adrem Our results confirm thosee of FALKl° who described the catalytic influence of adrenochrome on the oxidation of atscorbic acid b y molecular oxygen in 1949. Re#rences p. 86.
r adrenaline was observed on the ch ascorbate was added in cone, ble IV).
tzxAx~ L~ul~.
,hich a r e
TABLE IV kDRENOCHROME DDED
IN
ON OXYGEN-CONSUMPTI
CONCENTRATIONS
D HOMOG-
NORMALLY
q TISSU]~S
s u c c i n a t e , 0. 5 m l i o -$ M a s c o r b a t e , o. cgenate.
tdrenaline
Ot consumption in #1 adrenaline I5 ~/ml
HtO
35 35 35 35
14o 152 59 112
2.5 2.5 2.5 5
• ~ 4±
3 2 2 4
a
62 4- 2 112 -¢- 3
It m a y be supposed, that although an increased oxyg en consul d not be ted with these small concentrations of ascorbate, yet tl ascorb Tet the lytically sed in the homogenate system b y adrenochrome. We W, argued ~rstem in a ascorbic acid catalyses oxygen-consumption, the addition ad( of ae might ,, inhibition of oxidation by destruction of ascorbic ac:id. ~s is shown in Table V, ascorbic acid does not catal catalyse the c umption ~mogenates and of diluted homogenates to which ssuccinate has been added a s ~ U U ~ L rate. I i:l,.t~. • TABLE V INFLUENCE
OF
ASCORBATK ON
AND
OXYGEN-CONSU ; UMMPPTTI O I O: N
OF HOMOGENATK
OF HOMOGENATE
~- S U U CCCCIINNAATT E Oxygen-consumption in Id
Time in minutes
35 35
P,.a,ation
h o m o g e n a t e IO % h o)m moo g e n a t e 2.5 % + succinate
no
ascorbate
z95 -¢- 2
5o+I
0. 5 ml i o- s M ascorbate
193 ± 2 47 • 2
We therefore investigated whether ascorbic acid had a catalytic influence oo n oxygen-consumption of diluted ed liver homogenates to which other substrates than sm cinate were added. We found ld that the oxidation of lactate by our homogenates w w~a s stimulated by ascorbic acid and the oxygen-consumption of the system: homogenat enate + lactate + ascorbic acid was as indeed inhibited b y adrenochrome. The oxygen-consumption ,n of the system homogenate + lactate + ascorbate w wea s small, it was increased by the addition of DPN. The inhibition of the oxygen-coi en-consumption of this system b y adrenocl lochrom~ is shown in Table VI. For these experimenl tdrenochrome nts Warburg-vessels with two side-arm, rms were used. The lactate was always added to tll de-arms the homogenate in the flask. Re]erences p. 86.
TABLE VI HROME ON THE SYSTEM: D I L U T E D HO~ LACTATE-]- DPN-ASCORBATE
mogenate, o, 5 rnl 1/4 M lactate 3 M ascorbate, 0. 5 ml adrenochromc s(
ai
(I
mgl.
Oxygen-con
mD~ules
Experimen
iment 1I
le-arm I1
79 ± 55 _. ~ 4° ffz
±i -= I
HO Ascorbate + adrenochrome 2o ?/ml H20
45~ 35 ± 3I ±
±I.5 ±i ±i
Ascorbate + DPN Ascorbate + DPN H20 + DPN
25 ± 55 _± 30 ±
±i ±o -LI
) ~nochrome 20 ?/ml )
ochrome
ffzL
.'ctive of is f( ?he i n h i b i t i o n of o x y g e n c o n s u m p t i o n b y a d r e n~nochrome ochr t h e side ombined. The a y in w h i c h t h e c o m p o n e n t s of t h e s y s t e m are ccombi: thermow e r e i n t r o d u c e d in t h e flasks a i t e r t h e e q u i l i b r a t i o n of ¢ temper~ by the All t h e e x p e r i m e n t s s h o w also t h e s t i m u l a t i o n of o x y g e n - c ion of a s c o r b a t e . ochrome m-consumpti, :o t e s t o u r h y p o t h e s i s t h a t t h e i n h i b i t i o n of oxygen-co a c t i o n of a d r e n o c h: hr roor m r e a b o v e - m e n t i o n e d e x p e r i m e n t s w a s c a u s e d b y t h e catalytic cat~ ht e T a b l e V I I s h o w s t h a t in t o n t h ~e e a s c o r b a t e in t h e s y s t e m , we o m i t t e d a s c o r b a t e . : enm h o m o g e n a t e + , a c t a t e + D P N a d r e n o c h r o m e h a d a stimulating effect on o x y g e l system consumption. T A B L E VII N-CONSUMPTION O F THE SYSTEM: I N F L:,UENCE U E N C E OF A D R E N A L I N E AND OF ADRENOCHROME ON OXYGEN D I L U T E D HOMOGENATE ~- LACTATE -~ DPN
In flask: I. 4 ml 2.5% homogenate, o. 5 ml I/4 M lactate. In side-arm I: o.I ml DPN-sol. (i rag) + 4 mg nicotinamide. In side-arm II: o. 5 ml solution of adrenaline or of ( adrenochrome, or o. 5 ml H2~O. Ox>ygen-consumption in/zl in 2o minutes H,O
24-bi
31±1 30 ± 2 36:~4 31±1 46 ~22 28±4
adrenaline 20 ?[ml
adrenochrome ~o y / m l
adrenochrome 2 y/ml
46iI
I7~:2 24±1 3I~_2
48±I 50±2 5o±I 7o:~I
33-4-1 36:~I 53~ I
27 :J= 2
DISCUSSION had O u r figures s h o w t h a t a d r ee n o c h r o m e , in c o n c e n t r a t i o n s f r o m IOO y t o I o y / m l , h', in s p o n t a n e o u s o x y g e n c o n s u m p t i o n of l i v e r h o m o g e n a t e s no d e t e c t a b l e effect on t h e
Re/erences p. 86.
9n, nor on the oxygen-consumpti. uccinate + ascorbate are added. tion of diluted homogenates to w] 1 oxygen-consumption is stimulat -consumption in diluted homogen~ oxygen-consumption of adrenoc~ drenochrome in 3 ml Ringer solut: en in 75 minutes. When adrenocl ntained 0.5 ml of lO -3 M ascorbic cl 6.6/A of oxygen, exactly the qu~ ~tion ot ascorbic acid to dehydroascorbic acid. I t is evident ( t 'bic acid by adrenochrome plays no part in the oxygel en-consum --
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0 - - - - ,
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IIUIIIU
)me
con-
has been ~bic acid h lactate can be ~d in the tdded to Warburg lated for tation of systems
Fhis does not, in itself, prove that the oxygen-consum ption in I to which bate is added, is not partly caused by oxidation of a~ ascorbic ac cts other dehydroascorbic acid. ~,ddition of 2"IO -4 M ascorbate to homogenate o r to the h( uccinate m does not enhance oxygen-consumption, evidently ascorbic oxidized is system in appreciable quantity. On the other hamd additiol acid to homogenate lactate or homogenate lactate D P N sy~stem im gen-con)tion. This m a y mean that ascorbic acid catalyses oxidation oxi of it might mean that the oxidation of ascorbic acid to product, )roducts other tt )ascorbic is catalysed by the system homogenate lactate. Ho~ However, since homogenate arnd )genate + succinate certainly do not show such a cat~flytic action on the breakdo~ )wn of ascorba corbate, it seems more probable that the stimulatic)n of oxygen-consumption of ( the s 3ystem homogenate lactate D P N by the addition of ascorbic a,, acid is really a stlmustiml lation1 of the oxidation of lactate by ascorbic acid. The i~ influence of adrenochrome on c the ox~ xygen consumption of this system can then be expla cid ~lained as follows: ascorbic acJ catal 37ses the oxidation of lactic acid by homogenate + ]DPN, adrenochrome oxidizes oxidiz( and inactivates ascorbic acid and thereby inhibits the oxygen-consumption of the tt homc )genate D P N lactate ascorbic acid system. Our 3ur figures also show that adrenochrome increases tthe h oxygen-consumption of the tt system homogenate + lactate :e DPN. A similar effect of adrenochrorr ['ome on the oxidation of lactic acid was described b,y lochrome GREEN AND RICHTER u . These ~se authors found that adrenaline catalysed the oxygeI;nconsumption of a system consistin asisting of lactic acid dehydrogenase (which they prepare ~ared from pigs heart) + lactic acid id + DPN + KCN, and showed that this catalytic effe( ffect was in reality an effect of adrenoch )chr, Jrenochrome, adrenaline being transformed in this systel: m into adrenochrome. This transforma Lsformation of adrenaline into adrenochrome evidently do¢ does not occur in our homogenate systems, adrenaline had no effect or a slightly inhibitin ~g effect in our experiments (Table Lble VI). We have, until now, not )t further analysed the mechanism of the inhibiting anad accelerating effects of ascorbic ion. ic acid and of adrenochrome described in this publicatioi As adrenaline can be transformed )rmed into adrenochrome in living tissues, our experimem ,iments point out a possible cause o[ the different effects o! adrenaline on the oxygen-consumptio ~tion o] tissue preparations, which have ~sti~ave been described by different investigators. In our invest Re[erences p. 86.
terns in which adrenochrome
had r
, v ~ v / j t, vvj
i n g effect, d e p e n d i n g u p o n t h e u s e
" normal
r as catalysing agents for oxygen-
Ft.
SUMMARY tion of a s c o r b a t e in R i n g e r - K r e b s solutic p e r ml. in c o n c e n t r a t i o n s f r o m ioo ~ to io ~ p m consisting of diluted h o m o g e n a t e + ns f r o m diluted • A d r e n o c h r o m e in c o n c e n t r a t i o n s f r o m aaption of a s y s t e m consisting of diluted **t" . . . . . . . . .
d .................
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IOO y to io ~ per ml constantl~ b o m o go e n a t e of r a t liver + D P N mll , c o n s t a n t l y ioo }, to io y per m P N + lactat~ homogenate + D DPI~ .
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~.r o x y g e n influence aseorbate oxygen+ lactate. e oxygen-
Rt~SUMt~ • L ' a d r 6 n o c h r o m e A des c o n c e n t r a t i o n s comprises entre I / /zg zg e t o . i/~g uide de Ringer-Kre p a r l'oxyg~ne mol6culaire de l ' a s c o r b a t e dans le liquide l • L'adr6naline et l ' a d r 6 n o c h r o m e ~ des c o n c e n t r a t i o n s allant allant de ioo/z! rst~me form6 influence sur la c o n s o m m a t i o n d'oxyg~ne d ' u n syst~me form~ d'homog~ ~corbate ~ la c o n c e n t r a t i o n de 15o/zg p a r ml. • L ' a d r 6 n o c h r o m e , ~, des c o n c e n t r a t i o n s de ioo lug g i o / ~ g p9aT ml, inh ~ommation d'oxyg~ne d ' u n syst~me form6 d ' h o m o g 6 n a t dilu6 dde foie de rs actate. p a r ml, augm, • L ' a d r 6 n o c h r o m e , h des c o n c e n t r a t i o n s de i o o / z g g i o / zug g pm de D: s o m m a t i o n d'oxyg~ne d ' u n syst~me form6 d ' u n h o m o g 66nat n a t dilu6, d
gse l ' o x y ml n ' o n t succinate ~onstante ascorbate ~onstante ire.
ZUSAMMENFASSUNG bsl6sui I •• Die O x y d a t i o n y o n a s c o r b i n s a u r e n Salzen m i t m o l e k u l a r e m Sauerstoff in R i n g e r - K r eebsl6sung wird y~on o n A d r e n o c h r o m in einer K o n z e n t r a t i o n zwischen I y - o.I p r o ml katalysiert. ~ - iv ~ p r o ml h a t t e n keim ~emen l 2 .• Adrenalin u n d A d r e n o c h r o m in K o n z e n t r a t i o n e n zwischen ioo orbat ss auf den V e r b r a u c h y o n Sauerstoff eines aus v e r d f i n n t e m H eo m o g e n a t + Succinat + Ascorb Einfluss ~er K o n z e n t r a t i o n y o n 15o ~ p r o ml b e s t e h e n d e n Systems. bei einer uerstoff3-• A d r e n o c h r o m in K o n z e n t r a t i o n e n y o n ioo ~ - io ? p r o ml hemmte k o n s t a n t den S a u e r s t o stehenden aus v e r d i i n n t e m R a t t e n l e b e r - h o m o g e n a t + D P N -+ A s c o r b a t + L a k t a t bestehend, v e r b r ainch u c h eines at Systems. Sauerm] stimulierte k o n s t a n t den Saue 4-.. A d r e n o c h r o m in K o n z e n t r a t i o n e n v o n ~oo 7 - io ? pro ml a k t a t b e s t e h e n d e n Systems• ~rbrauch eines aus v e r d i i n n t e m H o m o g e n a t + D P N + L Lakt~ stoffverbrauc REFERENCES 1 G. AHLGREN, Shand. Arch. Physiol., vsiol., 47 (1925) 1. S . V O N E U L E R , Arch. exptl. Pathol. Pharmakol., 17I (1933) 186. s F• R. GRIFFITH, Physiological Reviews Reviews, 3I (1951) 151. 2II. ethnics and tissue metabolism, b y W. W. UMBREIT et a l . , 1951, p. 21 4 V. R. POTTER, I n Manometric te~ 5 D. L. DRABKIN, J. Biol. Chem. , 171 (1947) 409 • 2I e G. A. LE PAGE, I n Manometric technics and tissue metabolism b y W. W. UMBREIT et al., 1951, p. 215. 7 W. L. C. VEER, Rec. tray. chim.!., 61 (1942) 643. s p. CHAIX, J. CHAtWET AND J. JEZEQUEL, Biochim. Biophys. Acta, 4 (195 °) 471" J. Biol. Chem., 149 (1943) 217; 177 (1949) 893. 9 W. SCHNEIDER A N D V . R . P O T TTER, ER 10 j . E . F A L H , Biochem. J., 44 (1949) 949) 129, 369. 11 D . E . GREEN AND D . RICHTERt, Biochem. J., 31 (1937) 5962 U.
R e c e i v e d J u n e I 7 t h , I9~153
AND ADREN(
PTION OF LIVER
ON
HOMOG]~
by SMA A~D H. L. GOLTERMAN try o] the Municipal University o/ Amst
'lands)
~.HLGREN1 reported in 1925 t h a t the reduction of methylene me 1 ~cle-pulp O I ; I O 15 cuo was accelerated by adrenaline in concentration, mtrations from I : .~rous publications on this subject h~ve since then appeared : s results confirmed b y VON EULER z and LILJESTRAND amon~g others, stigators L, however, no influence of adrenaline i n vitro, neith~ either on red ethylene nor on oxygen-consumption of muscle-pulp or other othq tissues in the ,urg-apparatus (references in GRIFFITI~3). ~rRIFFITH3 has surveyed the literature of the last 2 5 y e a r s , ject. He ~aline in ions some 5o publications which reported no influence influen whate )ublications in lhibiting ng concentrations, an almost equal number of public mblications " which an inhibitir on methylene blue-reduction or oxygen-consumption )tion is described and about twice twi~ as mare :Ions my publications in which stimulation b y adrenaline : w was found from concentratioI between I : IOe and I : io 15. asonable No ~1o explanation has been given of these contradictor)y results. I t seems reasonab to the fact that, when who to supl ppose t h a t the differences can, at least partly, be ascribed as~ surviving tissue-preparations, oI, n e experimentin imenting with tissue pulp, tissue-slices or other survi bout is never 7er exactly informed about the kind of substrates which w are oxidised, nor abol .lene the q uestion which of the m a n y chemical reactions that are implied in the methyler is the rat~ rateblue-reduction or the oxygen-consumption by the tissue-preparations tissl the~ limiting reaction• I t seems quite possible that adrenaline stimulates some of these [ reactions and inhibits others•• Moreover, adlenaline is a labile sul~stance, which can be nce ther compounds possessing biological activity, for instan¢ transformed in tissues into other msformations too might vary in tissue preparations in affl a adrenochrome, and these trans uncontl ollable way. he influence of adrenaline and ot adrenochrome in tissuetissu, We have investigated the IOnS systems in which the nature of the substrates oxidised and the rate-limiting reactioi of oxygen-consumption could~t be varied in a controllable way. homogenates of rat-liver, which were diluted to concenconcel For this purpose we usedd homo ated ed trations in which they showed no spontaneous oxygen-consumption, and investigate nd adrenochrome on the oxygen-consumption obtained b,y the influence ot adrenaline and -factors. adding various substrates andd adding or omitting cytochrome c and various co-factor z.w.o.) Supported by a subsidy from1the Netherlands Organisation for Pure Scientific Research (Z.W.C References p. 86.
TECHNICAL DETAILS m o x - h e a r t . W e us e d t h e KEILII~ AND s o l u t i o n of c y t o c h r o m e w h i c h containec w i t h 2 ml phosphate buffer-solution, t t i n c t i o n c o n s t a n t s of c y t o c h r o m e c gi me m l of our s o l u t i o n is 1.o6 mg, t h a t
aethod as ry r e s i d u e Lt 550 m/~ IKIN 5 , t h e he p u r i t y
PN) w a s p r e p a r e d from b a k e r s y e a s t . " used t h e m e t h o d d e s c r i b e d b y LE PAGEe
ic technics
Io % NaOH solution to a diluted solut ed w a t e r t h e n w i t h a c e t o n e a n d d r y i n g me we used a m o d i f i c a t i o n of VEERS 7 m e t iOIVt~U Ill IO 1111 I ) I U I 3 L I I I U U w ~ £ t t ~ l ,r. T J. h IIe U ~ 1~ s ~ Ih i ~a Kk ~ Ie I n w W ]i t h 2 5 - 5 0 sUoI lUuL ItOi IoI n is ute. A f t e r t h e re d color of a d r e n o c h r o m e h a s d e v e l o p e d , t h e Ag~O is c t e m p e r a t u r e t h e r e d color fades o n l y v e r y slowly. I n t h e B ] eckmann a b s o r p t i o n m a x i m a a t 30o m/z a n d a t 485 m/z, m i n i m a a t 26o 26 m/z a n d w i t h t h a t of CHAIX, o n l y o u r m a x i m u m a t 485 m/~ w a s bbroa r o ad e r t h a n t h "~iver-homogenates were p r e p a r e d b y h o m o g e n i z i n g fresh rrat-1 a t - l i v e r in ice. )n in a W a r i n g - b l e n d o r . W e u s e d Ca-free K r e b s - R i n tggeerr--pphhooss]p h a t e solul )xygen-consumption w a s m e a s u r e d w i t h t h e W a r b u r g - a p p~a ar ar at ~t u s a t 38o C [ t h r e e m a n o m e t e r s w e r e u s e d for e a c h d e t e r m i n a t i o n . The fig1ares in t h e ee v a l u e s . H a l f of t h e m a x i m a l d e v i a t i o n f r o m t h e a v e r a g e m il t h e thre~
one g r a m
, washing Ldrenaline for 0. 5 t o • Even at m e t e r we spectrum by CHAIXs. )hosphate periments Le a v e r a g e [esignated
EXPERIMENTAL RESULTS
ence o/adrenaline and adrenochrome on the oxygen-cons'~ en-consumption o s to which no substrate has been added
d homog-
1For ~o r these experiments we used lO% and 20% ho~ homogenates, which showed an oxygen ~n consumption of about 60 and 15o/A, respectivel y, in 60 minutes by 3 ml of home ~genate. No influence of adrenaline and adrenochn ~nochrome on oxygen-consumptk )tion could be detected.
ence o~ adrenaline and adrenochrome on the oxygen-tons, Influence en-tonsumption o/diluted homogenat ares + succin ccinate with and without addition o/ cytochrome c Accordin kccording to SCHNEIDER AND POTTER 9, in a system cof this kind, without addition additk 4-~^'k . . . . . V'k . . . . ~ . - 1 ~ - - ~ . - I . . ^ ~ - : ~ _ _ ^t __..~_L. . . . . . of cytochrome e, -the oxido-reducti reduction of cytochrome c 2zs_ the rate-limiting reaction of oxygen-consumption• When cytochrome c is added in high concentration, the rate of dehydrogenation of succinate ~.e determines the rate of oxygen-consumption. We four found no influence of adrenaline and adrenochrome on the oxygen-consumption of thee s e systems. Influence o[ adrenaline and admnochr chrome on the oxygen-consumption o/diluted homogenat ~renochrome ares + cyto~hrome c + ascorbate When ascorbate is added ,,d to diluted homogenates in high concentration, the cyt 7tochrome c in the homogenate is kept continually in reduced state. Under these clrcumcircur stances the oxido-reduction • of cytochrome-oxidase is the rate-limiting reaction for f oxygen-consumption. In this system, as is shown ~hown in Table I, adrenochrome stimulated oxygen-co en-consumption, adrenaline had no effect. Re/erences p. 86. IO
TABLE
I
OF ADRENOCttROME ON THE OXYGENgRATES ..L CYTOCHROME C-.~- ASCORBAT
I OF
genate, o. 5 ml o.i 31 ascorbate, o. 5 ml t ome-solution), pH 7.4.
6 - 10 -4 3 i
O~ consumption in yl adrenaline 20 ,/]ml
II20
a,
4 ° ~:: ~ 35 ~: I
3o
(
26 ~ I 44 ± 2
5
28 =E I 48 ± 2
ks a d r e n o c h r o m e also s t i m u l a t e d o x y g e n - c o n s u m p t~tion ic when c o u l d n o t be ascribed to a c c e l e r a t i o n ot t h e o x i d o - r eMuction dt of ( ?re f o u n d t h a t a d r e n o c h r o m e also c a t a l y s e d t h e ox::ygen-con, ~hate solution to which ascorbate was a d d e d (Table II). I
d d e d its oxidase. F Ringer
TABLE II FLUENCE
OF A D R E N O C H R O M E ON OXYGEN-CONSUMPTION AND O F R I N G E R - P H O S P H A T E - S O L U T I O N
"oral v o l u m e 2.5 ml: o. 5 m l O.l M buffer solution or h o m o g e n a t e .
in minutes
adrenochrome 2o ~,/ml
Io
133
60
343
BATE
ml H~O,
drenochrome-s¢ ascorbate, o. 5 m l adren(
Homogenate e.5% x. 5 mt oxygen-consumption in #I
Time
OF H O M O G E N I -~- ASCORBATE At
n 5 ml Ringer-phosphate solution oxygen-consumption in i~l
HsO
adr enochrome zo F/ml
16 62
124 464
H20
IO
56
solutio 7he catalytic effect of a d r e n o c h r o m e on the o x i d a t i o n oof ascorbate in Ringer solution Th, Table I I I ) . be d e t e c t e d in c o n c e n t r a t i o n s as l o w as o.15 y m l (T could b, TABLE RELATION
III
~TION O F A D R E N O C H R O M E AND EFFECT B E T W E E N CONCENTRATION F ASCORBATE IN R I N G E R SOLUTION OF C o n c e n t r a t~i ot ino n o f 1 / 6 0 M a s c o r b a t e , Concen~rat%n of adrenochrome
(w)
1.5' I°-e 10-6 IO-7
0 3 consumption
(#) 183 86 2o
ON OXYGEN-CONSUMPTION
t i m e 2o m i n u t e s .
Concentration of adrenochrome
(w)
io -s IO -11 10 -14
0 z consumption (td)
2±5 5~=6 o=t=6
adrem Our results confirm thosee of FALKl° who described the catalytic influence of adrenochrome on the oxidation of atscorbic acid b y molecular oxygen in 1949. Re#rences p. 86.
r adrenaline was observed on the ch ascorbate was added in cone, ble IV).
tzxAx~ L~ul~.
,hich a r e
TABLE IV kDRENOCHROME DDED
IN
ON OXYGEN-CONSUMPTI
CONCENTRATIONS
D HOMOG-
NORMALLY
q TISSU]~S
s u c c i n a t e , 0. 5 m l i o -$ M a s c o r b a t e , o. cgenate.
tdrenaline
Ot consumption in #1 adrenaline I5 ~/ml
HtO
35 35 35 35
14o 152 59 112
2.5 2.5 2.5 5
• ~ 4±
3 2 2 4
a
62 4- 2 112 -¢- 3
It m a y be supposed, that although an increased oxyg en consul d not be ted with these small concentrations of ascorbate, yet tl ascorb Tet the lytically sed in the homogenate system b y adrenochrome. We W, argued ~rstem in a ascorbic acid catalyses oxygen-consumption, the addition ad( of ae might ,, inhibition of oxidation by destruction of ascorbic ac:id. ~s is shown in Table V, ascorbic acid does not catal catalyse the c umption ~mogenates and of diluted homogenates to which ssuccinate has been added a s ~ U U ~ L rate. I i:l,.t~. • TABLE V INFLUENCE
OF
ASCORBATK ON
AND
OXYGEN-CONSU ; UMMPPTTI O I O: N
OF HOMOGENATK
OF HOMOGENATE
~- S U U CCCCIINNAATT E Oxygen-consumption in Id
Time in minutes
35 35
P,.a,ation
h o m o g e n a t e IO % h o)m moo g e n a t e 2.5 % + succinate
no
ascorbate
z95 -¢- 2
5o+I
0. 5 ml i o- s M ascorbate
193 ± 2 47 • 2
We therefore investigated whether ascorbic acid had a catalytic influence oo n oxygen-consumption of diluted ed liver homogenates to which other substrates than sm cinate were added. We found ld that the oxidation of lactate by our homogenates w w~a s stimulated by ascorbic acid and the oxygen-consumption of the system: homogenat enate + lactate + ascorbic acid was as indeed inhibited b y adrenochrome. The oxygen-consumption ,n of the system homogenate + lactate + ascorbate w wea s small, it was increased by the addition of DPN. The inhibition of the oxygen-coi en-consumption of this system b y adrenocl lochrom~ is shown in Table VI. For these experimenl tdrenochrome nts Warburg-vessels with two side-arm, rms were used. The lactate was always added to tll de-arms the homogenate in the flask. Re]erences p. 86.
TABLE VI HROME ON THE SYSTEM: D I L U T E D HO~ LACTATE-]- DPN-ASCORBATE
mogenate, o, 5 rnl 1/4 M lactate 3 M ascorbate, 0. 5 ml adrenochromc s(
ai
(I
mgl.
Oxygen-con
mD~ules
Experimen
iment 1I
le-arm I1
79 ± 55 _. ~ 4° ffz
±i -= I
HO Ascorbate + adrenochrome 2o ?/ml H20
45~ 35 ± 3I ±
±I.5 ±i ±i
Ascorbate + DPN Ascorbate + DPN H20 + DPN
25 ± 55 _± 30 ±
±i ±o -LI
) ~nochrome 20 ?/ml )
ochrome
ffzL
.'ctive of is f( ?he i n h i b i t i o n of o x y g e n c o n s u m p t i o n b y a d r e n~nochrome ochr t h e side ombined. The a y in w h i c h t h e c o m p o n e n t s of t h e s y s t e m are ccombi: thermow e r e i n t r o d u c e d in t h e flasks a i t e r t h e e q u i l i b r a t i o n of ¢ temper~ by the All t h e e x p e r i m e n t s s h o w also t h e s t i m u l a t i o n of o x y g e n - c ion of a s c o r b a t e . ochrome m-consumpti, :o t e s t o u r h y p o t h e s i s t h a t t h e i n h i b i t i o n of oxygen-co a c t i o n of a d r e n o c h: hr roor m r e a b o v e - m e n t i o n e d e x p e r i m e n t s w a s c a u s e d b y t h e catalytic cat~ ht e T a b l e V I I s h o w s t h a t in t o n t h ~e e a s c o r b a t e in t h e s y s t e m , we o m i t t e d a s c o r b a t e . : enm h o m o g e n a t e + , a c t a t e + D P N a d r e n o c h r o m e h a d a stimulating effect on o x y g e l system consumption. T A B L E VII N-CONSUMPTION O F THE SYSTEM: I N F L:,UENCE U E N C E OF A D R E N A L I N E AND OF ADRENOCHROME ON OXYGEN D I L U T E D HOMOGENATE ~- LACTATE -~ DPN
In flask: I. 4 ml 2.5% homogenate, o. 5 ml I/4 M lactate. In side-arm I: o.I ml DPN-sol. (i rag) + 4 mg nicotinamide. In side-arm II: o. 5 ml solution of adrenaline or of ( adrenochrome, or o. 5 ml H2~O. Ox>ygen-consumption in/zl in 2o minutes H,O
24-bi
31±1 30 ± 2 36:~4 31±1 46 ~22 28±4
adrenaline 20 ?[ml
adrenochrome ~o y / m l
adrenochrome 2 y/ml
46iI
I7~:2 24±1 3I~_2
48±I 50±2 5o±I 7o:~I
33-4-1 36:~I 53~ I
27 :J= 2
DISCUSSION had O u r figures s h o w t h a t a d r ee n o c h r o m e , in c o n c e n t r a t i o n s f r o m IOO y t o I o y / m l , h', in s p o n t a n e o u s o x y g e n c o n s u m p t i o n of l i v e r h o m o g e n a t e s no d e t e c t a b l e effect on t h e
Re/erences p. 86.
9n, nor on the oxygen-consumpti. uccinate + ascorbate are added. tion of diluted homogenates to w] 1 oxygen-consumption is stimulat -consumption in diluted homogen~ oxygen-consumption of adrenoc~ drenochrome in 3 ml Ringer solut: en in 75 minutes. When adrenocl ntained 0.5 ml of lO -3 M ascorbic cl 6.6/A of oxygen, exactly the qu~ ~tion ot ascorbic acid to dehydroascorbic acid. I t is evident ( t 'bic acid by adrenochrome plays no part in the oxygel en-consum --
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d
0 - - - - ,
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U
IIUIIIU
)me
con-
has been ~bic acid h lactate can be ~d in the tdded to Warburg lated for tation of systems
Fhis does not, in itself, prove that the oxygen-consum ption in I to which bate is added, is not partly caused by oxidation of a~ ascorbic ac cts other dehydroascorbic acid. ~,ddition of 2"IO -4 M ascorbate to homogenate o r to the h( uccinate m does not enhance oxygen-consumption, evidently ascorbic oxidized is system in appreciable quantity. On the other hamd additiol acid to homogenate lactate or homogenate lactate D P N sy~stem im gen-con)tion. This m a y mean that ascorbic acid catalyses oxidation oxi of it might mean that the oxidation of ascorbic acid to product, )roducts other tt )ascorbic is catalysed by the system homogenate lactate. Ho~ However, since homogenate arnd )genate + succinate certainly do not show such a cat~flytic action on the breakdo~ )wn of ascorba corbate, it seems more probable that the stimulatic)n of oxygen-consumption of ( the s 3ystem homogenate lactate D P N by the addition of ascorbic a,, acid is really a stlmustiml lation1 of the oxidation of lactate by ascorbic acid. The i~ influence of adrenochrome on c the ox~ xygen consumption of this system can then be expla cid ~lained as follows: ascorbic acJ catal 37ses the oxidation of lactic acid by homogenate + ]DPN, adrenochrome oxidizes oxidiz( and inactivates ascorbic acid and thereby inhibits the oxygen-consumption of the tt homc )genate D P N lactate ascorbic acid system. Our 3ur figures also show that adrenochrome increases tthe h oxygen-consumption of the tt system homogenate + lactate :e DPN. A similar effect of adrenochrorr ['ome on the oxidation of lactic acid was described b,y lochrome GREEN AND RICHTER u . These ~se authors found that adrenaline catalysed the oxygeI;nconsumption of a system consistin asisting of lactic acid dehydrogenase (which they prepare ~ared from pigs heart) + lactic acid id + DPN + KCN, and showed that this catalytic effe( ffect was in reality an effect of adrenoch )chr, Jrenochrome, adrenaline being transformed in this systel: m into adrenochrome. This transforma Lsformation of adrenaline into adrenochrome evidently do¢ does not occur in our homogenate systems, adrenaline had no effect or a slightly inhibitin ~g effect in our experiments (Table Lble VI). We have, until now, not )t further analysed the mechanism of the inhibiting anad accelerating effects of ascorbic ion. ic acid and of adrenochrome described in this publicatioi As adrenaline can be transformed )rmed into adrenochrome in living tissues, our experimem ,iments point out a possible cause o[ the different effects o! adrenaline on the oxygen-consumptio ~tion o] tissue preparations, which have ~sti~ave been described by different investigators. In our invest Re[erences p. 86.
terns in which adrenochrome
had r
, v ~ v / j t, vvj
i n g effect, d e p e n d i n g u p o n t h e u s e
" normal
r as catalysing agents for oxygen-
Ft.
SUMMARY tion of a s c o r b a t e in R i n g e r - K r e b s solutic p e r ml. in c o n c e n t r a t i o n s f r o m ioo ~ to io ~ p m consisting of diluted h o m o g e n a t e + ns f r o m diluted • A d r e n o c h r o m e in c o n c e n t r a t i o n s f r o m aaption of a s y s t e m consisting of diluted **t" . . . . . . . . .
d .................
o
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IOO y to io ~ per ml constantl~ b o m o go e n a t e of r a t liver + D P N mll , c o n s t a n t l y ioo }, to io y per m P N + lactat~ homogenate + D DPI~ .
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~.r o x y g e n influence aseorbate oxygen+ lactate. e oxygen-
Rt~SUMt~ • L ' a d r 6 n o c h r o m e A des c o n c e n t r a t i o n s comprises entre I / /zg zg e t o . i/~g uide de Ringer-Kre p a r l'oxyg~ne mol6culaire de l ' a s c o r b a t e dans le liquide l • L'adr6naline et l ' a d r 6 n o c h r o m e ~ des c o n c e n t r a t i o n s allant allant de ioo/z! rst~me form6 influence sur la c o n s o m m a t i o n d'oxyg~ne d ' u n syst~me form~ d'homog~ ~corbate ~ la c o n c e n t r a t i o n de 15o/zg p a r ml. • L ' a d r 6 n o c h r o m e , ~, des c o n c e n t r a t i o n s de ioo lug g i o / ~ g p9aT ml, inh ~ommation d'oxyg~ne d ' u n syst~me form6 d ' h o m o g 6 n a t dilu6 dde foie de rs actate. p a r ml, augm, • L ' a d r 6 n o c h r o m e , h des c o n c e n t r a t i o n s de i o o / z g g i o / zug g pm de D: s o m m a t i o n d'oxyg~ne d ' u n syst~me form6 d ' u n h o m o g 66nat n a t dilu6, d
gse l ' o x y ml n ' o n t succinate ~onstante ascorbate ~onstante ire.
ZUSAMMENFASSUNG bsl6sui I •• Die O x y d a t i o n y o n a s c o r b i n s a u r e n Salzen m i t m o l e k u l a r e m Sauerstoff in R i n g e r - K r eebsl6sung wird y~on o n A d r e n o c h r o m in einer K o n z e n t r a t i o n zwischen I y - o.I p r o ml katalysiert. ~ - iv ~ p r o ml h a t t e n keim ~emen l 2 .• Adrenalin u n d A d r e n o c h r o m in K o n z e n t r a t i o n e n zwischen ioo orbat ss auf den V e r b r a u c h y o n Sauerstoff eines aus v e r d f i n n t e m H eo m o g e n a t + Succinat + Ascorb Einfluss ~er K o n z e n t r a t i o n y o n 15o ~ p r o ml b e s t e h e n d e n Systems. bei einer uerstoff3-• A d r e n o c h r o m in K o n z e n t r a t i o n e n y o n ioo ~ - io ? p r o ml hemmte k o n s t a n t den S a u e r s t o stehenden aus v e r d i i n n t e m R a t t e n l e b e r - h o m o g e n a t + D P N -+ A s c o r b a t + L a k t a t bestehend, v e r b r ainch u c h eines at Systems. Sauerm] stimulierte k o n s t a n t den Saue 4-.. A d r e n o c h r o m in K o n z e n t r a t i o n e n v o n ~oo 7 - io ? pro ml a k t a t b e s t e h e n d e n Systems• ~rbrauch eines aus v e r d i i n n t e m H o m o g e n a t + D P N + L Lakt~ stoffverbrauc REFERENCES 1 G. AHLGREN, Shand. Arch. Physiol., vsiol., 47 (1925) 1. S . V O N E U L E R , Arch. exptl. Pathol. Pharmakol., 17I (1933) 186. s F• R. GRIFFITH, Physiological Reviews Reviews, 3I (1951) 151. 2II. ethnics and tissue metabolism, b y W. W. UMBREIT et a l . , 1951, p. 21 4 V. R. POTTER, I n Manometric te~ 5 D. L. DRABKIN, J. Biol. Chem. , 171 (1947) 409 • 2I e G. A. LE PAGE, I n Manometric technics and tissue metabolism b y W. W. UMBREIT et al., 1951, p. 215. 7 W. L. C. VEER, Rec. tray. chim.!., 61 (1942) 643. s p. CHAIX, J. CHAtWET AND J. JEZEQUEL, Biochim. Biophys. Acta, 4 (195 °) 471" J. Biol. Chem., 149 (1943) 217; 177 (1949) 893. 9 W. SCHNEIDER A N D V . R . P O T TTER, ER 10 j . E . F A L H , Biochem. J., 44 (1949) 949) 129, 369. 11 D . E . GREEN AND D . RICHTERt, Biochem. J., 31 (1937) 5962 U.
R e c e i v e d J u n e I 7 t h , I9~153