BIOCHIMIE, 1978, 60, 823-826.
Glucose inhibition of oxygen utilization by isolated rat lung cells. Matilde S. AYUS0-PARRI,LLA *, Julio PER.EZ-D,IAZ *, Angeles MARTIN * and R o b e r t o P~ARRILLA **.
(25-7-1977).
Introduction. The i n h i b i t i o n of glycolysis by the a v a i l a b i l i t y of oxygen was o r i g i n a l l y d e s c r i b e d by P a s t e u r and is a m e t a b o l i c feature of p r a c t i c a l l y u n i v e r s a l o c c u r e n c e . C r a b t r e e [1] d e s c r i b e d the r e v e r s e phen o m e n o n , this is the i n h i b i t i o n of oxygen utilization by glycolysis. This effect, k n o w n as Crabtree effect, is r a t h e r u n u s u a l and so far has been circ u m s c r i b e d m a i n l y , if not exclusively, to r a p i d l y g l y c o l y z i n g ceils, mostly ~umoral cells. Isolated rat lung cells, w h i c h have in c o m m o n w i t h t u m o r a l cells to display high g l y c o l y t i c fluxes [2, 3] h a v e also been f o u n d to s h o w glucose i n h i b i t i o n of oxygen uptake [3]. T h e results s h o w e d h e r e i n are an a t t e m p t to c h a r a c t e r i z e this p h e n o m e n o n .
" Department o[ Metabolism, Institute G. Mara~6n, C.S.I.C., Madrid-6, Spain. "" Department of Biochemistry and Biophysics, Facultad de Medicina, Universidad Aut6noma, Madrid-34, Spain.
p e n s i o n w a s p l a c e d in an a i r t i g h t m i c r o c h a m b e r and kept u n d e r c o n t i n u o u s s t i r r i n g at 36.5°C. E a c h figure r e p r e s e n t s the results from a single e x p e r i m e n t e v e n t h o u g h the results w e r e c h e c k out at least four times. The oxygen uptake v a r i e d f r o m 100-200 n atoms × h r -1 × 106 cells -1 b e i n g the average value 126 --4---4 n atoms × hr-1 × 106 cells -1 (50 d e t e r m i n a t i o n s ) . Glucose a n d lactate w e r e m e a s u r e d e n z y m i c a l l y a c c o r d i n g to p r e v i o u s l y d e s c r i b e d p r o c e d u r e s [5].
TABLI~ I.
E[[ect of mannoheptulose on glucose utilization by lung cells and on glucose phosphorylation by a lung homogenate. Lung cells
Methods. Male W i s t a r rats, 2'5.0 g in b o d y w e i g h t w e r e used in all the e x p e r i m e n t s . T h e p r o c e d u r e for the isolation of the rat lung cells has been described before [3]. T h e t e n d e n c y of these cells to aggregate was p r e v e n t e d by t h e i r t r e a t m e n t w i t h d e o x y r i b o n u c l e a s e [4]. The ceils suspension w a s m a d e up in IKrebs-Rin~er b i c a r b o n a t e buffer satur a t e d w i t h 95 p e r cent 02 : 5 p e r cen,t CO 2 gas m i x t u r e . In a t y p i c a l e x p e r i m e n t four pairs of rat lungs w e r e p o o l e d for each digestion p r o c e d u r e a n d the final p r e p a r a t i o n y i e l d e d 8 ml of 2-3 × 106 cells × ml-L Oxygen uptake w a s d e t e r m i n e d p o l a r o g r a p h y cally using a Clark type oxygen e l e c t r o d e (Yellow Spring, Ohio, U.S.A.). One m i l l i l i t e r of cells sus-
To whom all correspondence should be addressed.
Glucose utilization
Lactate production
Lung homogenate hexokinase
activity 1~moles/g/mio
nmoles/t0e eells/h Control
212 -+- 3
250 ~___ 4
0.54
Mannohept u l o s e (5 mM)
220 __+ 7
278 -~- 10
0.06
Lung cells were prepared as described in Methods, 1 ml of cells suspension were suppl.emented with 4 mM glucose, placed in 25 ml Erlenmeyer flaskss sealed with a 95 per cent O~ : 5 per cent CO._, mixture. The flasks were incubated at 36.5°C, in a rotary shaker for 1 hour at the end of which the ine'~bation was terminated by adding perchloric acid (6 per cent final concentration). The precipitate was removed by centrifugation and the clear supernatant brought to pH 6.5, with potassium carbonate. Glucose utilization was assessed by determining the dissapearence of glucose from the incubation medium. For the det~ermination of the hexokinase activity the assay was carried out under conditions in which maximal activity was observed. Lung homogenates (1:10) were prepared in 50 mM phosphate buffer pH' 7.2. The assay of hexokinase was carried out at 30°C in a filter fluorometer as described before [5].
M. S. Aguso-ParrilIa and coil.
824
All the reagents c h e m i c a l s w e r e o b t a i n e d t h r o u g h Sigma (St. Louis, Missouri, U.S.A.) and enzymes from B o e h r i n g e r (Mannheim, Germany).
I
2.5 I
GLUCOSEmM
o
it w a s able of i n h i b i t i n g glucose p h o s p h o r y l a t i o n b y 87 p e r cent in a lung h o m o g e n a t e (table I), that is w h e n p e r m e a b i l i t y r e s t r i c t i o n of the plasma membrane had dissapeared.
I ,ot
IAA(O
{raM)
200 IO0 ~
150
80
/
GLUCiSE(25raM) ~
GLU~,OSE(SmM)
\
I00
6O I
0 175 0
I
l
o
c
I
I0
20
i
5O
a. --
GLUCOS i 5 mM DNP0.25raM
Z
I
J¢ ~.
X o 0 c
150
5O
120
I
GLUCOSE(5raM) 1
125
IAA (O.ImM)
I00 I00
80
75
I 0
I
5 TIME (minutes)
I
I0
FIG.-1. - - Glucose i n h i b i t i o n of the isolated rat lung cells respiration. R e l e a s e b y d i n i t r o p h e n o l (DNP). 1 m l
6O
of cells, s u s p e n d e d i n K r e b s - R i n g e r b i c a r b o n a t e b u f f e r c o n t a i n i n g 1 m g / m l of d e f a t t e d b o v i n e s e r u m a l b u m i n , w e r e i n c u b a t e d a t 36.5'°C i n a t h e r m o s t a t e d a i r t i g h t microchamber under continuous stirring. Oxygen uptake was continously recorded using a Clark type platinum electrode.
0
q
t
5
I
iO
TIME (minutes) F1o. 2. - - C o m b i n e d effects of glucose and iodoacetale (IA,A) on the rates of respiration of isolated rat lung cells s u s p e n s i o n s . E x p e r i m e n t a l d e t a i l s a s i n f i g u r e 1.
Results. The i s o l a t e d r a t lung cells used in this s t u d y w e r e f u n c t i o n a l l y i n t a c t as s h o w n b y the fact t h a t m o r e t h a n 95 p e r cent e x c l u d e d vital colorants. The intactness of the p l a s m a m e m b r a n e w a s also sugested b y the o b s e r v a t i o n s t h a t A DP d i d not stimulate oxygen uptake by a suspension of these cells. F u r t h e r m o r e , m a n n o h e p t u l o s e (5 raM) h a d no effect on glucose u t i l i z a t i o n b y the cells w h i l e
BIOCHIMIE, 1978, 60, 11° 8.
As m a y be seen in figure 1, glucose i n d u c e d a dose d e p e n d e n t i n h i b i t i o n of oxygen utilization. U n c o u p l i n g of r e s p i r a t i o n from o x i d a t i v e phosp h o r y l a t i o n b y d i n i t r o p h e n o l (.fig. 1), in a g r e e m e n t w i t h p r e v i o u s studies on n e o p l a s t i c tissues [6-9], r e l e a s e d the i n h i b i t o r y effect of glucose on lung cells r e s p i r a t i o n . Glucose, on the other h a n d , was unable of i n h i b i t i n g oxygen u p t a k e in the presence of d i n i t r o p h e n o l (results not shown). P r e t r e a t ment of the cells w i t h an i n h i b i t o r of glycolysis,
Glucose
inhibition
of
oxygen
like iodoacetate, d i d not p r e v e n t the glucose ind u c e d i n h i b i t i o n of oxygen utilization (fig. 2). I o d o a c e t a t e w a s effective as j u d g e d b y its i n h i b i t o r y effects on endogenous r e s p i r a t i o n as w e l l as b y its effect i n h i b i t i n g b y 75 p e r cent the rate of lactate p r o d u c t i o n from glucose (results not shown). This o b s e r v a t i o n (fig. 2), in a g r e e m e n t w i t h p r e v i o u s r e p o r t s utilizing other systems [6, 10] seems to s u p p o r t the p o i n t of view that a normal rate of glucose b r e a k d o w n is not s t r i c t l y nec e s s a r y for the i n h i b i t i o n of oxygen utilization b y isolated rat lung cells.
uptake
by lung
cells.
825
The i n h i b i t i o n of lung ceils r e s p i r a t i o n by rotenone (10 ~M) d i d not i n t e r f e r e w i t h the manifestation of the glucose effect on oxygen uptake (fig. 3). H o w e v e r , i n c u b a t i o n of the cells w i t h a t r a c t y l o s i d e , a d r u g w h i c h blocks a d e n i n e nucleotides t r a n s l o c a t i o n across the m i t o c h o n d r i a l m e m b r a n e [11], c o m p l e t e l y p r e v e n t e d the glucose effect, eventhough, glucose w a s a d d e d at high doses (fig. 4). A t r a c t y l o s i d e , on the other h a n d , i n h i b i t e d endogenous r e s p i r a t i o n (fig. 4).
Discussion. ROTENONE(IOgM) I00
GLUCOSE(SmM)
a__o
Z) ',.. LU~
O r i g i n a l l y C r a b t r e e thought that the effect of glucose i n h i b i t i n g r e s p i r a t i o n was specific of tum o r a l tissues [1]. The same effect has been seld o m d e s c r i b e d u n d e r c e r t a i n c o n d i t i o n s in norreal cells [12-15].
6C
\
\
2O I
(~
I
5
I0
TIME (minutes)
FiG. 3. - - Effect of glucose on the rates of exygen utilization by isolated rat lung cells pretreated w i t h rotenone. Experimental conditions as described in
figure 1.
160Ii
ATRACT LOSIOEI (2raM) Y
120 in F-O n~
GLUCOSE (5raM)
8C
Z ~ o
~0 s= 4C
The finding that u n c o u p l i n g of m i t o c h o n d r i a l p h o s p h o r y l a t i o n from o x i d a t i o n released the Crabtree effect (fig. 1) suggests, t a k i n g into a c c o u n t the high rate of glycolisis in these cells, that a comp e t i t i o n m a y exist b e t w e e n glucose b r e a k d o w n a n d the r e s p i r a t o r y chain for a c o m m o n p h o s p h o r y l a t e d i n t e r m e d i a t e . H o w e v e r , the fact that the i n h i b i t i o n of the glucose b r e a k d o w n b y i o d o a c e tare does not i n t e r f e r e w i t h the effect of glucose on oxygen c o n s u m p t i o n (fig. 2) seems to i n d i c a t e that o t h e r factors are involved. The results r e p o r t e d h e r e i n s h o w i n g that a t r a c t y l o s i d e p r e v e n t s the glucose i n d u c e d i n h i b i t i o n of r e s p i r a t i o n , c l e a r l y i n d i c a t e that the t r a n s l o c a t i o n of a d e n i n e n u e l e o t i d e s a c r o s s the m i t o c h o n d r i a l m e m b r a n e is i n v o l v e d in the process. A t r a c t y l o s i d e alone w a s f o u n d to i n h i b i t r e s p i r a t i o n to an extent s i m i l a r to glucose alone (fig. 4) suggesting that both, glucose a n d a t r a e t y l o s i d e , m a y act in s i m i l a r w a y b y p r e v e n t i n g e y t o s o l i c ADP to pass into the mitoe h o n d r i a . Thi~ p o s s i b i l i t y is in a g r e e m e n t w i t h p r e v i o u s r e p o r t s [6, 16] suggesting that ATP becomes t r a p p e d w i t h i n the m i t o c h o n d r i a due to an i n i t i a l i n c r e a s e d rate of glucose o x i d a t i o n a n d acc o m p a n y i n g changes in m i t o e h o n d r i a l p e r m e a b i lity.
Acknowledgements. I
I
I L0
TIME (minutes) FIO. 4. - - Differential effects of glucose on the rates
of respiration of isolated rat lung cells in the presence of atractgloside. Experimental conditions as described
in figure 1.
BIOCHIMIE, 19'78, 60, n ° 8.
This w o r k was supported in part by grants f r o m Comisi6n Asesora para el Desarrollo de la Investigaci6n and Essex Espafia S.A.J.P.D. and A.M. are recipients of research f e l l o w s h i p s f r o m the S p a n i s h Secretary of Education and Science. Authors w i s h to t h a n k Mr. Tomds Fonlela, Mr. Fernando Martin and Miss M. N. Gareia f o r devoted and s k i l f u l l technical assistance.
M. S. Ayaso-Parrilla
826 REFERENCES.
1. Crabtree, H. G. (19299 Biochem. J., 23, 5~36-545. 2. Ayuso-Parrilla, M. S., Fisher, A. B., Parrilla, R. ,¢ W i l l i a m s o n , J. R. (19.73) Am. J. Physiol., 225, 1153-11"60.
3. Pdrez~Diaz, J., Garhallo, B., Ayuso-Parrilla, M. S. a Parrilla R. (1977') Biochimie, 59, 411-416. 4. Wolfe, B. M. J., Rubinstein, D. a Beck, J. C. (1968) Can. J. Biochem., ~6, 151-154. 5. Pdrez-Diaz, J., Martin, A., Ayuso-Parrilla, M. S. Parrilla, R. (1977) Am. J. Physiol., 232, E394E'400. 6. Racker, E. (1956) Ann. N.Y. Acad. Sci., 63, 10171021. 7. Kun, E., Talalay, P. ,¢ Williams-Ash.man, H. G. (1951,) Cancer Res., 11, 855-893.
BIOCHIMIE, 1978, 60, n ° 8.
and
coll.
8. Acs, G. & Straub, F. B. (1.954") Doklady Akad. Naak S.S.S.R., 95, 1,0~1-1024. 9. Wenner, D. F. (197~4) Fed. Proc., 33, 2232-2~2~37. 10. Slechta, L., Jakubovic, A. a Sorm, F. (1965) Collect Cesk. Chem. Commun., 20, 86,3~869. 11. Heldt, H. W. (1969) in . Bucher, Th. a Sies, H., eds. pp. 301317, Springer Verlag, Berlin. 12. Belitzer, W. A. (19'36) Biochem. Ztschr., 283, 339-342. 13. Rosenthal, O., Bowie, M. A. & Wagoner, G. (1940) Science, 92, 3'8,2-393. I4. Cohen, L. H. (1957) Fed. Proc., 16, 165. 15. Estes, F. L., Smith, S. & Cast, G. H. (19.57) Fed. Proc., 16, 178. 16. Chance, B. & Hess, B. (195.6) Ann. N.Y. Aead. Sci., 63, 1008-1016.
Glucose inhibition of oxygen utilization by isolated rat lung cells. Matilde S. AYUS0-PARRI,LLA *, Julio PER.EZ-D,IAZ *, Angeles MARTIN * and R o b e r t o P~ARRILLA **.
(25-7-1977).
Introduction. The i n h i b i t i o n of glycolysis by the a v a i l a b i l i t y of oxygen was o r i g i n a l l y d e s c r i b e d by P a s t e u r and is a m e t a b o l i c feature of p r a c t i c a l l y u n i v e r s a l o c c u r e n c e . C r a b t r e e [1] d e s c r i b e d the r e v e r s e phen o m e n o n , this is the i n h i b i t i o n of oxygen utilization by glycolysis. This effect, k n o w n as Crabtree effect, is r a t h e r u n u s u a l and so far has been circ u m s c r i b e d m a i n l y , if not exclusively, to r a p i d l y g l y c o l y z i n g ceils, mostly ~umoral cells. Isolated rat lung cells, w h i c h have in c o m m o n w i t h t u m o r a l cells to display high g l y c o l y t i c fluxes [2, 3] h a v e also been f o u n d to s h o w glucose i n h i b i t i o n of oxygen uptake [3]. T h e results s h o w e d h e r e i n are an a t t e m p t to c h a r a c t e r i z e this p h e n o m e n o n .
" Department o[ Metabolism, Institute G. Mara~6n, C.S.I.C., Madrid-6, Spain. "" Department of Biochemistry and Biophysics, Facultad de Medicina, Universidad Aut6noma, Madrid-34, Spain.
p e n s i o n w a s p l a c e d in an a i r t i g h t m i c r o c h a m b e r and kept u n d e r c o n t i n u o u s s t i r r i n g at 36.5°C. E a c h figure r e p r e s e n t s the results from a single e x p e r i m e n t e v e n t h o u g h the results w e r e c h e c k out at least four times. The oxygen uptake v a r i e d f r o m 100-200 n atoms × h r -1 × 106 cells -1 b e i n g the average value 126 --4---4 n atoms × hr-1 × 106 cells -1 (50 d e t e r m i n a t i o n s ) . Glucose a n d lactate w e r e m e a s u r e d e n z y m i c a l l y a c c o r d i n g to p r e v i o u s l y d e s c r i b e d p r o c e d u r e s [5].
TABLI~ I.
E[[ect of mannoheptulose on glucose utilization by lung cells and on glucose phosphorylation by a lung homogenate. Lung cells
Methods. Male W i s t a r rats, 2'5.0 g in b o d y w e i g h t w e r e used in all the e x p e r i m e n t s . T h e p r o c e d u r e for the isolation of the rat lung cells has been described before [3]. T h e t e n d e n c y of these cells to aggregate was p r e v e n t e d by t h e i r t r e a t m e n t w i t h d e o x y r i b o n u c l e a s e [4]. The ceils suspension w a s m a d e up in IKrebs-Rin~er b i c a r b o n a t e buffer satur a t e d w i t h 95 p e r cent 02 : 5 p e r cen,t CO 2 gas m i x t u r e . In a t y p i c a l e x p e r i m e n t four pairs of rat lungs w e r e p o o l e d for each digestion p r o c e d u r e a n d the final p r e p a r a t i o n y i e l d e d 8 ml of 2-3 × 106 cells × ml-L Oxygen uptake w a s d e t e r m i n e d p o l a r o g r a p h y cally using a Clark type oxygen e l e c t r o d e (Yellow Spring, Ohio, U.S.A.). One m i l l i l i t e r of cells sus-
To whom all correspondence should be addressed.
Glucose utilization
Lactate production
Lung homogenate hexokinase
activity 1~moles/g/mio
nmoles/t0e eells/h Control
212 -+- 3
250 ~___ 4
0.54
Mannohept u l o s e (5 mM)
220 __+ 7
278 -~- 10
0.06
Lung cells were prepared as described in Methods, 1 ml of cells suspension were suppl.emented with 4 mM glucose, placed in 25 ml Erlenmeyer flaskss sealed with a 95 per cent O~ : 5 per cent CO._, mixture. The flasks were incubated at 36.5°C, in a rotary shaker for 1 hour at the end of which the ine'~bation was terminated by adding perchloric acid (6 per cent final concentration). The precipitate was removed by centrifugation and the clear supernatant brought to pH 6.5, with potassium carbonate. Glucose utilization was assessed by determining the dissapearence of glucose from the incubation medium. For the det~ermination of the hexokinase activity the assay was carried out under conditions in which maximal activity was observed. Lung homogenates (1:10) were prepared in 50 mM phosphate buffer pH' 7.2. The assay of hexokinase was carried out at 30°C in a filter fluorometer as described before [5].
M. S. Aguso-ParrilIa and coil.
824
All the reagents c h e m i c a l s w e r e o b t a i n e d t h r o u g h Sigma (St. Louis, Missouri, U.S.A.) and enzymes from B o e h r i n g e r (Mannheim, Germany).
I
2.5 I
GLUCOSEmM
o
it w a s able of i n h i b i t i n g glucose p h o s p h o r y l a t i o n b y 87 p e r cent in a lung h o m o g e n a t e (table I), that is w h e n p e r m e a b i l i t y r e s t r i c t i o n of the plasma membrane had dissapeared.
I ,ot
IAA(O
{raM)
200 IO0 ~
150
80
/
GLUCiSE(25raM) ~
GLU~,OSE(SmM)
\
I00
6O I
0 175 0
I
l
o
c
I
I0
20
i
5O
a. --
GLUCOS i 5 mM DNP0.25raM
Z
I
J¢ ~.
X o 0 c
150
5O
120
I
GLUCOSE(5raM) 1
125
IAA (O.ImM)
I00 I00
80
75
I 0
I
5 TIME (minutes)
I
I0
FIG.-1. - - Glucose i n h i b i t i o n of the isolated rat lung cells respiration. R e l e a s e b y d i n i t r o p h e n o l (DNP). 1 m l
6O
of cells, s u s p e n d e d i n K r e b s - R i n g e r b i c a r b o n a t e b u f f e r c o n t a i n i n g 1 m g / m l of d e f a t t e d b o v i n e s e r u m a l b u m i n , w e r e i n c u b a t e d a t 36.5'°C i n a t h e r m o s t a t e d a i r t i g h t microchamber under continuous stirring. Oxygen uptake was continously recorded using a Clark type platinum electrode.
0
q
t
5
I
iO
TIME (minutes) F1o. 2. - - C o m b i n e d effects of glucose and iodoacetale (IA,A) on the rates of respiration of isolated rat lung cells s u s p e n s i o n s . E x p e r i m e n t a l d e t a i l s a s i n f i g u r e 1.
Results. The i s o l a t e d r a t lung cells used in this s t u d y w e r e f u n c t i o n a l l y i n t a c t as s h o w n b y the fact t h a t m o r e t h a n 95 p e r cent e x c l u d e d vital colorants. The intactness of the p l a s m a m e m b r a n e w a s also sugested b y the o b s e r v a t i o n s t h a t A DP d i d not stimulate oxygen uptake by a suspension of these cells. F u r t h e r m o r e , m a n n o h e p t u l o s e (5 raM) h a d no effect on glucose u t i l i z a t i o n b y the cells w h i l e
BIOCHIMIE, 1978, 60, 11° 8.
As m a y be seen in figure 1, glucose i n d u c e d a dose d e p e n d e n t i n h i b i t i o n of oxygen utilization. U n c o u p l i n g of r e s p i r a t i o n from o x i d a t i v e phosp h o r y l a t i o n b y d i n i t r o p h e n o l (.fig. 1), in a g r e e m e n t w i t h p r e v i o u s studies on n e o p l a s t i c tissues [6-9], r e l e a s e d the i n h i b i t o r y effect of glucose on lung cells r e s p i r a t i o n . Glucose, on the other h a n d , was unable of i n h i b i t i n g oxygen u p t a k e in the presence of d i n i t r o p h e n o l (results not shown). P r e t r e a t ment of the cells w i t h an i n h i b i t o r of glycolysis,
Glucose
inhibition
of
oxygen
like iodoacetate, d i d not p r e v e n t the glucose ind u c e d i n h i b i t i o n of oxygen utilization (fig. 2). I o d o a c e t a t e w a s effective as j u d g e d b y its i n h i b i t o r y effects on endogenous r e s p i r a t i o n as w e l l as b y its effect i n h i b i t i n g b y 75 p e r cent the rate of lactate p r o d u c t i o n from glucose (results not shown). This o b s e r v a t i o n (fig. 2), in a g r e e m e n t w i t h p r e v i o u s r e p o r t s utilizing other systems [6, 10] seems to s u p p o r t the p o i n t of view that a normal rate of glucose b r e a k d o w n is not s t r i c t l y nec e s s a r y for the i n h i b i t i o n of oxygen utilization b y isolated rat lung cells.
uptake
by lung
cells.
825
The i n h i b i t i o n of lung ceils r e s p i r a t i o n by rotenone (10 ~M) d i d not i n t e r f e r e w i t h the manifestation of the glucose effect on oxygen uptake (fig. 3). H o w e v e r , i n c u b a t i o n of the cells w i t h a t r a c t y l o s i d e , a d r u g w h i c h blocks a d e n i n e nucleotides t r a n s l o c a t i o n across the m i t o c h o n d r i a l m e m b r a n e [11], c o m p l e t e l y p r e v e n t e d the glucose effect, eventhough, glucose w a s a d d e d at high doses (fig. 4). A t r a c t y l o s i d e , on the other h a n d , i n h i b i t e d endogenous r e s p i r a t i o n (fig. 4).
Discussion. ROTENONE(IOgM) I00
GLUCOSE(SmM)
a__o
Z) ',.. LU~
O r i g i n a l l y C r a b t r e e thought that the effect of glucose i n h i b i t i n g r e s p i r a t i o n was specific of tum o r a l tissues [1]. The same effect has been seld o m d e s c r i b e d u n d e r c e r t a i n c o n d i t i o n s in norreal cells [12-15].
6C
\
\
2O I
(~
I
5
I0
TIME (minutes)
FiG. 3. - - Effect of glucose on the rates of exygen utilization by isolated rat lung cells pretreated w i t h rotenone. Experimental conditions as described in
figure 1.
160Ii
ATRACT LOSIOEI (2raM) Y
120 in F-O n~
GLUCOSE (5raM)
8C
Z ~ o
~0 s= 4C
The finding that u n c o u p l i n g of m i t o c h o n d r i a l p h o s p h o r y l a t i o n from o x i d a t i o n released the Crabtree effect (fig. 1) suggests, t a k i n g into a c c o u n t the high rate of glycolisis in these cells, that a comp e t i t i o n m a y exist b e t w e e n glucose b r e a k d o w n a n d the r e s p i r a t o r y chain for a c o m m o n p h o s p h o r y l a t e d i n t e r m e d i a t e . H o w e v e r , the fact that the i n h i b i t i o n of the glucose b r e a k d o w n b y i o d o a c e tare does not i n t e r f e r e w i t h the effect of glucose on oxygen c o n s u m p t i o n (fig. 2) seems to i n d i c a t e that o t h e r factors are involved. The results r e p o r t e d h e r e i n s h o w i n g that a t r a c t y l o s i d e p r e v e n t s the glucose i n d u c e d i n h i b i t i o n of r e s p i r a t i o n , c l e a r l y i n d i c a t e that the t r a n s l o c a t i o n of a d e n i n e n u e l e o t i d e s a c r o s s the m i t o c h o n d r i a l m e m b r a n e is i n v o l v e d in the process. A t r a c t y l o s i d e alone w a s f o u n d to i n h i b i t r e s p i r a t i o n to an extent s i m i l a r to glucose alone (fig. 4) suggesting that both, glucose a n d a t r a e t y l o s i d e , m a y act in s i m i l a r w a y b y p r e v e n t i n g e y t o s o l i c ADP to pass into the mitoe h o n d r i a . Thi~ p o s s i b i l i t y is in a g r e e m e n t w i t h p r e v i o u s r e p o r t s [6, 16] suggesting that ATP becomes t r a p p e d w i t h i n the m i t o c h o n d r i a due to an i n i t i a l i n c r e a s e d rate of glucose o x i d a t i o n a n d acc o m p a n y i n g changes in m i t o e h o n d r i a l p e r m e a b i lity.
Acknowledgements. I
I
I L0
TIME (minutes) FIO. 4. - - Differential effects of glucose on the rates
of respiration of isolated rat lung cells in the presence of atractgloside. Experimental conditions as described
in figure 1.
BIOCHIMIE, 19'78, 60, n ° 8.
This w o r k was supported in part by grants f r o m Comisi6n Asesora para el Desarrollo de la Investigaci6n and Essex Espafia S.A.J.P.D. and A.M. are recipients of research f e l l o w s h i p s f r o m the S p a n i s h Secretary of Education and Science. Authors w i s h to t h a n k Mr. Tomds Fonlela, Mr. Fernando Martin and Miss M. N. Gareia f o r devoted and s k i l f u l l technical assistance.
M. S. Ayaso-Parrilla
826 REFERENCES.
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