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The effect of cyclic AMP on glycogenolysis and glycolysis in human platelets
280
BBA
BIOCHIMICA ET BIOPHYSICA ACTA
26702
T H E E F F E C T OF CYCLIC AMP ON GLYCOGENOLYSIS AND GLYCOLYSIS IN HUMAN PLATELETS* PHILIP D. ZIEVE AND MORTON SCHMUKLER Department of Jtledieine, Baltimore City Hospitals and Johns Hopkins University, Baltimore 2I 224 ; and Baltimore Cancer Research Center, National Cancer Institute, Baltimore, Md. 2 1 2 1 1 ( U . S . A . )
(Received June I4th, 1971)
SUMMARY
Cyclic AMP increased glycogenolysis and stimulated phosphorylase in sonicates of human platelets. Paradoxically, lactate formation was reduced by cyclic AMP in platelet lysates as well as by dibutyryl cyclic AMP and prostaglandin E 1. This decrease in lactate formation appears to be mediated b y an inhibition of phosphofructokinase. These metabolic effects of cyclic AMP m a y be related to the inhibition of aggregation of platelets by this compound and by agents which stimulate its production.
INXRODUCTION
A d e n o s i n e - 3 ' , 5 ' - c y c l i c monophosphate (cyclic AMP) and adenyl cyclase, the enzyme which catalyzes its formation, have been demonstrated in human platelets 1-3. Cyclic AMP, dibutyryl cyclic AMP, and prostaglandin E1 (an agent which increases activity of adenyl cyclase', 2 and thereby increases levels of cyclic AMP within platelets 4) have been sbown to inhibit aggregation of these cellsa-L Conversely substances which decrease activity of adenyl cyclase 1, 2 and thereby decrease levels of cyclic AMP within platelets 3 have been shown to promote aggregation s. I t has been suggested therefore that cyclic AMP is involved in the regulation of platelet aggregation. Since aggregation is accompanied by a considerable increase in lactate formation in platelets 9-1~, the effect of cyclic AMP on glycogenolysis and glycolysis was studied in the experiments to be described. METHODS
Platelets were isolated from blood drawn from healthy volunteers, washed twice, and resuspended in 0.07 M phosphate (KH2PO4-Na2HPO~) buffer (pH 7.4) 13. The final number of cells in each sample was approx. I . lO9 (see ref. 14). In some experiments the cells were disrupted by sonication (IO % intensity for 30 sec, Biosonik sonicator). Glycogen was isolated from and measured 15in the sonicates prior to and after incubation with and without I mM cyclic AMP for 15 min at 37 °. * A preliminary abstract of this work appeared in Blood, 36 (197o) 853. Biochim. Biophys. Acta, 252 (197I) 28o-284
EFFECT OF CYCLIC A M P ON HUMAN PLATELETS
281
P r e l i m i n a r y e x p e r i m e n t s showed t h a t t h e r a t e of glycogenolysis was linear for 3o min. P h o s p h o r y l a s e a c t i v i t y was a s s a y e d in h o m o g e n a t e s of p l a t e l e t s p r e p a r e d b y freezing a n d t h a w i n g cells s e d i m e n t e d from p l a t e l e t rich plasma. M e a s u r e m e n t s were m a d e in t h e direction of glycogen b r e a k d o w n b y observing the r a t e of r e d u c t i o n of N A D P + s p e c t r o p h o t o m e t r i c a l l y in a coupled a s s a y s y s t e m 16. T o t a l p h o s p h o r y l a s e a c t i v i t y was m e a s u r e d in the presence of 4 m M A M P (final c o n c e n t r a t i o n ) ; phosp h o r y l a s e a (the active enzyme) was m e a s u r e d in t h e absence of a d d e d AMP. Cyclic A M P (I raM) was p r e i n c u b a t e d at 3o ° for IO min with the h o m o g e n a t e s prior to analysis; controls were p r e i n c u b a t e d w i t h buffer. T h e reaction r a t e s were linear a n d prop o r t i o n a l to the a m o u n t of tissue added. I n o t h e r e x p e r i m e n t s w a s h e d p l a t e l e t s were r e s u s p e n d e d in distilled w a t e r a n d allowed to s t a n d for 30 min at o ° a f t e r which t h e s a m p l e s were frozen a n d t h a w e d once. T h e n an equal volume of o.14 M p h o s p h a t e buffer was a d d e d ; a n d t h e samples were i n c u b a t e d w i t h a n d w i t h o u t I mM cyclic A M P for 30 min at 37 °- E n d o g e n o u s l a c t a t e f o r m a t i o n as well as the f o r m a t i o n of l a c t a t e in t h e presence of various g l y c o l y t i c i n t e r m e d i a t e s was m e a s u r e d iv. O p t i m u m conditions in these e x p e r i m e n t s r e q u i r e d 0.5 mM A T P a n d IO mM N A D ÷. All of the chemicals used in these e x p e r i m e n t s were p u r c h a s e d from S i g m a Chemical C o m p a n y , St. Louis, Mo. RESULTS
Effect of cyclic A M P on glycogenolysis and phosphorylase activity (Table I) Cyclic A M P increased the r a t e of glycogenolysis in p l a t e l e t lysates b y 42 °o a n d the level of p h o s p h o r y l a s e a b y 33 %. I n control experiments, p h o s p h o r y l a s e a was 50 %, of t o t a l p h o s p h o r y l a s e a c t i v i t y . TABLE I EFFECT
OF CYCLIC A~{P
ON GLYCOGENOLYSIS
AND PHOSPHORYLASE
ACTIVITY IN HUMAN
PLATELETS
Values shown are means ± S.E. ; numbers in parentheses are numbers of experiments.
Rate of glycogenolysis Level of phosphorylase a
Control
± z mM cyclic A ~]IP (% increase)
272 ± 24 (I9) (ILg/h per lO9 cells)
q 42 ± s (s)
1.2"I0
-2 ±
0.2"10
-2 (II)
(#moles glucose-i-P/min per io 9 cells)
33 ±
II
(II)
Effect of cyclic A M P on lactate formation Cyclic A M P was a p o t e n t i n h i b i t o r of endogenous l a c t a t e f o r m a t i o n in p l a t e l e t lysates, showing an effect a t c o n c e n t r a t i o n s as low as I . 10 -9 M (Table II). T a b l e I I I shows t h e results of e x p e r i m e n t s wherein p l a t e l e t lysates were inc u b a t e d w i t h various g l y c o l y t i c s u b s t r a t e s in t h e presence or absence of cyclic AMP. Mean endogenous l a c t a t e f o r m a t i o n was 0.65 /zmole per lO 9 cells p e r h a n d was reduced, in t h e presence of I mM cyclic AMP, b y 0.27 /,mole. W h e n l y s a t e s were i n c u b a t e d w i t h g l y c o l y t i c i n t e r m e d i a t e s , l a c t a t e f o r m a t i o n was s t i m u l a t e d . This
Biochim. Biophys. Acta, 252 (1971) 280-284
282
P. D. ZIEVE, M. SCHMUKLER
"FABLE II EFFECT
OF VARIOUS
HUMAN
PLATELETS
CONCENTRATIONS
OF
CYCLIC
AMP
ON
ENDOGENOUS
LACTATE
FORMATION
IN
T h e r e s u l t s of a single e x p e r i m e n t are s h o w n . Cyclic A 2VIP conch. (M)
Lactate formation (ltmoles/h)
o
0.6 4
I " 10 -3
0.I0
I " I0
6
0.I6
I • IO
9
0.32
0.7o o.64
I • IO -12 I " IO -15
TABLE III EFFECT OF CYCLIC A M P ON LACTATE FORMATION IN HUMAN PLATELETS Values s h o w n are m e a n s ± S.E. ; n u m b e r s in p a r e n t h e s e s are n u m b e r s of e x p e r i m e n t s A ddition
ltmoles/h Control
E n d o g e n o u s (18) Glucose (6) G l u c o s e - 6 - P (6) F r u c t o s e 6 - P (6) F r u c t o s e - I , 6 - P 2 (6) G l y c e r a l d e h y d e - 3 - P (6) 3 - P h o s p h o g l y c e r a t e (6) P y r u v a t e (5)
0.65 l.i 3 1.76 2.44 o.84 o.93 1.o 7 1.22
= ~ ± ! ± ~ ± ~
~- Cyclic A 31P* 0.06 o.12 o.2o o.14 o. i i o.io o.2o o.ii
0.27 0.66 o.73 o.76 o.o 4 +o.o3 o.38 o.41
I ± ~ i ~ i ± ~
0.03 o.13 o.18 o-I5 o.i2 o.o7 o.o 4 o.05
* Values indicate d e v i a t i o n f r o m control. T A B L E IV EFFECT IN
OF DIBUTYRYL
HUMAN
CYCLIC
A~iP
AND
PROSTAGLANDIN
Addition
LACTATE
FORMATION
ttmoles / h Expt. : 1
Endogenous D i b u t y r y l cyclic A M P (I raM) P r o s t a g l a n d i n E 1 (4 ng/ml) Cyclic A M P (I mM)
stimulation phate, and
E 1 ON ENDOGENOUS
PLATELETS
o. 7 ° o.38 0.22 o.27
2
3
1. o6 o.2i 0.42 o.21
i. 32 o.3i 0.53 o.37
was considerably reduced, in the presence of glucose, glucose-6-phosfructose-6-phosphate, by cyclic AMP. In the presence of fructose-x,6-
diphosphate and glyceraldehyde-3- phosphate, cyclic AMP had no effect. Stimulation of lactate formation produced by 3-phosphoglycerate and pyruvate was also inhibited b y c y c l i c A M P b u t t h e d e g r e e o f i n h i b i t i o n w a s s l i g h t a n d m o s t o f it c o u l d b e a c c o u n t ed for by a reduction in the endogenous rate. Biochim. Biophys. Acta, 252 {1971) 280-284
EFFECT OF CYCLIC
AMP
ON HUMAN PLATELETS
283
In additional experiments endogenous lactate formation in lysates was also found to be inhibited by dibutyryl cyclic AMP and by prostaglandin E 1 (Table IV). DISCUSSION
One of the earliest recognized effects of cyclic AMP is the stimulation of glycogenolysis in liver is and muscle 19. This effect has been shown to be mediated by the activation of an enzyme, phosphorylase kinase kinase 2° which results ultimately in the activation of phosphorylase. The present experiments show t h a t cyclic AMP also increases glycogenolysis and activates phosphorylase in human platelets. Phosphorylase a was found by us to be 5o % of total phosphorylase, a figure similar to that reported by others 21, ~2. Under the conditions of our experiments, cyclic AMP increased phosphorylase a by 33 %. In contrast, DEISSEROTH et al. 22 reported no eflect on phosphorylase a in whole platelets or in subcellular fractions after 2 min of pre-incubation with dibutyryl cyclic AMP. I t is not clear that these 2 sets of observations are conflicting since the conditions of the experiments were different. Paradoxically, despite stimulation of glycogenolysis, the present experiments show that cyclic AMP, at low concentrations, inhibits endogenous lactate formation in platelet lysates. In an attempt to localize the site or sites of inhibition, lactate formation was also measured after lysates were incubated with various glycolytic intermediates. The data suggest that the primary site of inhibition is the conversion of fructose-6-phosphate to fructose-l, 6-diphosphate, a reaction catalyzed by the enzyme, phosphofructokinase. This reaction has been identified as a probable rate controlling step in glycolysis in platelets 2a and is also a reaction wherein cyclic AMP has been shown to have an effect in other tissues 24-27. The apparent inhibitory effect of cyclic AMP on phosphofructokinase in platelets is somewhat surprising, however, since the cited studies all report stimulation of this enzyme by cyclic AMP, suggesting that phosphofructokinase in platelets is activated b y a different mechanisnl than is present in other tissues. Dibutyryl cyclic AMP, an analogue with generally similar biologic effects to cyclic AMP, was shown in the current experiments to inhibit lactate formation in platelet lysates to the same extent as cyclic AMP. Prostaglandin E t also inhibited lactate formation in these studies, presumably b y stimulating the formation of cyclic AMP1, 2. Aggregation of platelets is usually accompanied by a marked increase in lactate production. Since cyclic AMP, dibutyryl cyclic AMP, and prostaglandin E 1 decrease lactate formation in platelets, it is possible that the inhibition of aggregation by these substances is related to their effect on glycolysis. WOLFE AND SHULMAN28 have shown, in this regard, that cyclic AMP blocks thrombin-stimulated increase of lactate production in platelets. ACKNOWLEDGEMENT
This work was supported by a grant, H E - o 9 I o 2 , from the National Heart Institute, Bethesda, Md.
Biochim. Biophys. Mcta, 252 (1971) 28o-284
284
P.D.
Z l E V E , M. SCHMUKLER
REFERENCES i 2 3 4 5 6 7 8 9 io ti 12 13 14 15 [6 t7 18 19 20 21 22 23 24 25 26 27 28
S. M. WOLFE AND N. R . SHULMAN, Biochem. Biophys. Res. Commun., 35 (1969) 265. P. D. Z1EVE AND W . B. GREENOUGH, Biochem. Biophys. Res. Commun., 35 (1969) 462. E . W . SALZIVlAN ANn L. L. NERI, Nature, 224 (1969) 609. N. R. MARQUIS, 1{. L. VIGDAHL AND D. A. TAVORMINA, Biochem. Biophys. Res. Commttn., 36 (1969) 965 • J- KLOEZE, Proc. 2nd Nobel Nymp., Stockholm, I966, A h n q u i s t a n d \ V i k s e l l , S t o c k h o l m , I9~7, p. 241. P. R. EMMONS, J. R. ~{AMPTON, ~[. J. G. HARRISON, A. J. HONOUR AND J. I~. A, MITCHELL, Br. 1~led. J . , 2 (1967) 468. Y. H . ABDULLAH, J. Atherosclerosis Res., 9 (1969) 171. A. J. MARCUS AND M. G. ZUCKER, Tt~e Physiology of Blood Platelets, G r u n e a n d S t r a t t o n , N e w Y o r k , 1965 , pp. 52 , 53. M. CORN, J./~ppl. Physiol., 21 (1966) 62. A. E. WARSHAV¢, L. EASTER AND ~NT. R. SHULMAN, J . C/in. Invest., 45 (1966) I923. S. KARPATKIN AND I~. ,'~. LANGER, jr. Clin. Invest., 47 (1968) 2158. P. B. LODER, J. HIRSH AND G. C. DEGRUCHY, Br. Jr. Haematol., 14 (1968) 563. P. D. ZIEVE, H . M. SOLOMON AND J. R. I%REVANS, J. Cell. Physiol., 67 (1966) 271. 13. S. BULL, 1~. A. SCHNEIDERMAN AND G. 13RECHER, Am. J. Clin. Pathol., 44 (1965) 67~. J. H. ROE AND R. E. DAILEY, Anal. Biochem., 15 (1966) 245. C. C. CHILDRESS AND B. SAKTOR, J. Biol. Chem., 245 (197 ° ) 2927 . E. P. MARBACH AND M. U . \ ¥ E I L , Clin. Chem., 13 (1967) 314. T. \V. I~ALL AND E. \ ¥ . SUTHERLAND, J . Biol. Chem., 232 (1958) lO65. E. G. I(REBS, D. J. GRAVES AND E . H . FISCHER, J. Biol. Chem., 234 (1959) 2867. D. A. WALSH, J. P. PERKINS AND t'~. G. I{REBS, J. Biol. Chem., 243 (1968) 3763 . S. KARPATK1N AND R. M. LANGER, Biochim. Biophys. Acta, 185 (1969) 35 o. A. DEISSEROTH, S. M. WOLFE AND N. 1~. SHULMAN, Biochem. Biophys. Res. Commt*n., 39 (197 ° ) 551 • T. C. DETWlLER AND R, V. ZIVKOVlC, Bioehim. Biophys. Acta, 197 (197 o) 117J. V. PASSONNEAU AND O. U. LOWRY, Biochem. Biophys. Res. Commztn., 7 (1962) io. T. E. MANSOUR AND J. M. MANSOUR, J. Biol. Chem., 237 (1962) 629. R . G. KEMP AND E . G. KREBS, Biochemistry, 6 (1967) 423 . T. E. MANSOUR AND C. E. AHLFORS, J. Biol. Chem., 243 (1968) 2523. S. M. WOLFE AND N. R. SHULMAN, Biochem. Biophys. R e s Commun, 41 (197 o) 128.
Biochim. Biophys. Acta, 252 (1971) 2 8 0 - 2 8 4
BBA
BIOCHIMICA ET BIOPHYSICA ACTA
26702
T H E E F F E C T OF CYCLIC AMP ON GLYCOGENOLYSIS AND GLYCOLYSIS IN HUMAN PLATELETS* PHILIP D. ZIEVE AND MORTON SCHMUKLER Department of Jtledieine, Baltimore City Hospitals and Johns Hopkins University, Baltimore 2I 224 ; and Baltimore Cancer Research Center, National Cancer Institute, Baltimore, Md. 2 1 2 1 1 ( U . S . A . )
(Received June I4th, 1971)
SUMMARY
Cyclic AMP increased glycogenolysis and stimulated phosphorylase in sonicates of human platelets. Paradoxically, lactate formation was reduced by cyclic AMP in platelet lysates as well as by dibutyryl cyclic AMP and prostaglandin E 1. This decrease in lactate formation appears to be mediated b y an inhibition of phosphofructokinase. These metabolic effects of cyclic AMP m a y be related to the inhibition of aggregation of platelets by this compound and by agents which stimulate its production.
INXRODUCTION
A d e n o s i n e - 3 ' , 5 ' - c y c l i c monophosphate (cyclic AMP) and adenyl cyclase, the enzyme which catalyzes its formation, have been demonstrated in human platelets 1-3. Cyclic AMP, dibutyryl cyclic AMP, and prostaglandin E1 (an agent which increases activity of adenyl cyclase', 2 and thereby increases levels of cyclic AMP within platelets 4) have been sbown to inhibit aggregation of these cellsa-L Conversely substances which decrease activity of adenyl cyclase 1, 2 and thereby decrease levels of cyclic AMP within platelets 3 have been shown to promote aggregation s. I t has been suggested therefore that cyclic AMP is involved in the regulation of platelet aggregation. Since aggregation is accompanied by a considerable increase in lactate formation in platelets 9-1~, the effect of cyclic AMP on glycogenolysis and glycolysis was studied in the experiments to be described. METHODS
Platelets were isolated from blood drawn from healthy volunteers, washed twice, and resuspended in 0.07 M phosphate (KH2PO4-Na2HPO~) buffer (pH 7.4) 13. The final number of cells in each sample was approx. I . lO9 (see ref. 14). In some experiments the cells were disrupted by sonication (IO % intensity for 30 sec, Biosonik sonicator). Glycogen was isolated from and measured 15in the sonicates prior to and after incubation with and without I mM cyclic AMP for 15 min at 37 °. * A preliminary abstract of this work appeared in Blood, 36 (197o) 853. Biochim. Biophys. Acta, 252 (197I) 28o-284
EFFECT OF CYCLIC A M P ON HUMAN PLATELETS
281
P r e l i m i n a r y e x p e r i m e n t s showed t h a t t h e r a t e of glycogenolysis was linear for 3o min. P h o s p h o r y l a s e a c t i v i t y was a s s a y e d in h o m o g e n a t e s of p l a t e l e t s p r e p a r e d b y freezing a n d t h a w i n g cells s e d i m e n t e d from p l a t e l e t rich plasma. M e a s u r e m e n t s were m a d e in t h e direction of glycogen b r e a k d o w n b y observing the r a t e of r e d u c t i o n of N A D P + s p e c t r o p h o t o m e t r i c a l l y in a coupled a s s a y s y s t e m 16. T o t a l p h o s p h o r y l a s e a c t i v i t y was m e a s u r e d in the presence of 4 m M A M P (final c o n c e n t r a t i o n ) ; phosp h o r y l a s e a (the active enzyme) was m e a s u r e d in t h e absence of a d d e d AMP. Cyclic A M P (I raM) was p r e i n c u b a t e d at 3o ° for IO min with the h o m o g e n a t e s prior to analysis; controls were p r e i n c u b a t e d w i t h buffer. T h e reaction r a t e s were linear a n d prop o r t i o n a l to the a m o u n t of tissue added. I n o t h e r e x p e r i m e n t s w a s h e d p l a t e l e t s were r e s u s p e n d e d in distilled w a t e r a n d allowed to s t a n d for 30 min at o ° a f t e r which t h e s a m p l e s were frozen a n d t h a w e d once. T h e n an equal volume of o.14 M p h o s p h a t e buffer was a d d e d ; a n d t h e samples were i n c u b a t e d w i t h a n d w i t h o u t I mM cyclic A M P for 30 min at 37 °- E n d o g e n o u s l a c t a t e f o r m a t i o n as well as the f o r m a t i o n of l a c t a t e in t h e presence of various g l y c o l y t i c i n t e r m e d i a t e s was m e a s u r e d iv. O p t i m u m conditions in these e x p e r i m e n t s r e q u i r e d 0.5 mM A T P a n d IO mM N A D ÷. All of the chemicals used in these e x p e r i m e n t s were p u r c h a s e d from S i g m a Chemical C o m p a n y , St. Louis, Mo. RESULTS
Effect of cyclic A M P on glycogenolysis and phosphorylase activity (Table I) Cyclic A M P increased the r a t e of glycogenolysis in p l a t e l e t lysates b y 42 °o a n d the level of p h o s p h o r y l a s e a b y 33 %. I n control experiments, p h o s p h o r y l a s e a was 50 %, of t o t a l p h o s p h o r y l a s e a c t i v i t y . TABLE I EFFECT
OF CYCLIC A~{P
ON GLYCOGENOLYSIS
AND PHOSPHORYLASE
ACTIVITY IN HUMAN
PLATELETS
Values shown are means ± S.E. ; numbers in parentheses are numbers of experiments.
Rate of glycogenolysis Level of phosphorylase a
Control
± z mM cyclic A ~]IP (% increase)
272 ± 24 (I9) (ILg/h per lO9 cells)
q 42 ± s (s)
1.2"I0
-2 ±
0.2"10
-2 (II)
(#moles glucose-i-P/min per io 9 cells)
33 ±
II
(II)
Effect of cyclic A M P on lactate formation Cyclic A M P was a p o t e n t i n h i b i t o r of endogenous l a c t a t e f o r m a t i o n in p l a t e l e t lysates, showing an effect a t c o n c e n t r a t i o n s as low as I . 10 -9 M (Table II). T a b l e I I I shows t h e results of e x p e r i m e n t s wherein p l a t e l e t lysates were inc u b a t e d w i t h various g l y c o l y t i c s u b s t r a t e s in t h e presence or absence of cyclic AMP. Mean endogenous l a c t a t e f o r m a t i o n was 0.65 /zmole per lO 9 cells p e r h a n d was reduced, in t h e presence of I mM cyclic AMP, b y 0.27 /,mole. W h e n l y s a t e s were i n c u b a t e d w i t h g l y c o l y t i c i n t e r m e d i a t e s , l a c t a t e f o r m a t i o n was s t i m u l a t e d . This
Biochim. Biophys. Acta, 252 (1971) 280-284
282
P. D. ZIEVE, M. SCHMUKLER
"FABLE II EFFECT
OF VARIOUS
HUMAN
PLATELETS
CONCENTRATIONS
OF
CYCLIC
AMP
ON
ENDOGENOUS
LACTATE
FORMATION
IN
T h e r e s u l t s of a single e x p e r i m e n t are s h o w n . Cyclic A 2VIP conch. (M)
Lactate formation (ltmoles/h)
o
0.6 4
I " 10 -3
0.I0
I " I0
6
0.I6
I • IO
9
0.32
0.7o o.64
I • IO -12 I " IO -15
TABLE III EFFECT OF CYCLIC A M P ON LACTATE FORMATION IN HUMAN PLATELETS Values s h o w n are m e a n s ± S.E. ; n u m b e r s in p a r e n t h e s e s are n u m b e r s of e x p e r i m e n t s A ddition
ltmoles/h Control
E n d o g e n o u s (18) Glucose (6) G l u c o s e - 6 - P (6) F r u c t o s e 6 - P (6) F r u c t o s e - I , 6 - P 2 (6) G l y c e r a l d e h y d e - 3 - P (6) 3 - P h o s p h o g l y c e r a t e (6) P y r u v a t e (5)
0.65 l.i 3 1.76 2.44 o.84 o.93 1.o 7 1.22
= ~ ± ! ± ~ ± ~
~- Cyclic A 31P* 0.06 o.12 o.2o o.14 o. i i o.io o.2o o.ii
0.27 0.66 o.73 o.76 o.o 4 +o.o3 o.38 o.41
I ± ~ i ~ i ± ~
0.03 o.13 o.18 o-I5 o.i2 o.o7 o.o 4 o.05
* Values indicate d e v i a t i o n f r o m control. T A B L E IV EFFECT IN
OF DIBUTYRYL
HUMAN
CYCLIC
A~iP
AND
PROSTAGLANDIN
Addition
LACTATE
FORMATION
ttmoles / h Expt. : 1
Endogenous D i b u t y r y l cyclic A M P (I raM) P r o s t a g l a n d i n E 1 (4 ng/ml) Cyclic A M P (I mM)
stimulation phate, and
E 1 ON ENDOGENOUS
PLATELETS
o. 7 ° o.38 0.22 o.27
2
3
1. o6 o.2i 0.42 o.21
i. 32 o.3i 0.53 o.37
was considerably reduced, in the presence of glucose, glucose-6-phosfructose-6-phosphate, by cyclic AMP. In the presence of fructose-x,6-
diphosphate and glyceraldehyde-3- phosphate, cyclic AMP had no effect. Stimulation of lactate formation produced by 3-phosphoglycerate and pyruvate was also inhibited b y c y c l i c A M P b u t t h e d e g r e e o f i n h i b i t i o n w a s s l i g h t a n d m o s t o f it c o u l d b e a c c o u n t ed for by a reduction in the endogenous rate. Biochim. Biophys. Acta, 252 {1971) 280-284
EFFECT OF CYCLIC
AMP
ON HUMAN PLATELETS
283
In additional experiments endogenous lactate formation in lysates was also found to be inhibited by dibutyryl cyclic AMP and by prostaglandin E 1 (Table IV). DISCUSSION
One of the earliest recognized effects of cyclic AMP is the stimulation of glycogenolysis in liver is and muscle 19. This effect has been shown to be mediated by the activation of an enzyme, phosphorylase kinase kinase 2° which results ultimately in the activation of phosphorylase. The present experiments show t h a t cyclic AMP also increases glycogenolysis and activates phosphorylase in human platelets. Phosphorylase a was found by us to be 5o % of total phosphorylase, a figure similar to that reported by others 21, ~2. Under the conditions of our experiments, cyclic AMP increased phosphorylase a by 33 %. In contrast, DEISSEROTH et al. 22 reported no eflect on phosphorylase a in whole platelets or in subcellular fractions after 2 min of pre-incubation with dibutyryl cyclic AMP. I t is not clear that these 2 sets of observations are conflicting since the conditions of the experiments were different. Paradoxically, despite stimulation of glycogenolysis, the present experiments show that cyclic AMP, at low concentrations, inhibits endogenous lactate formation in platelet lysates. In an attempt to localize the site or sites of inhibition, lactate formation was also measured after lysates were incubated with various glycolytic intermediates. The data suggest that the primary site of inhibition is the conversion of fructose-6-phosphate to fructose-l, 6-diphosphate, a reaction catalyzed by the enzyme, phosphofructokinase. This reaction has been identified as a probable rate controlling step in glycolysis in platelets 2a and is also a reaction wherein cyclic AMP has been shown to have an effect in other tissues 24-27. The apparent inhibitory effect of cyclic AMP on phosphofructokinase in platelets is somewhat surprising, however, since the cited studies all report stimulation of this enzyme by cyclic AMP, suggesting that phosphofructokinase in platelets is activated b y a different mechanisnl than is present in other tissues. Dibutyryl cyclic AMP, an analogue with generally similar biologic effects to cyclic AMP, was shown in the current experiments to inhibit lactate formation in platelet lysates to the same extent as cyclic AMP. Prostaglandin E t also inhibited lactate formation in these studies, presumably b y stimulating the formation of cyclic AMP1, 2. Aggregation of platelets is usually accompanied by a marked increase in lactate production. Since cyclic AMP, dibutyryl cyclic AMP, and prostaglandin E 1 decrease lactate formation in platelets, it is possible that the inhibition of aggregation by these substances is related to their effect on glycolysis. WOLFE AND SHULMAN28 have shown, in this regard, that cyclic AMP blocks thrombin-stimulated increase of lactate production in platelets. ACKNOWLEDGEMENT
This work was supported by a grant, H E - o 9 I o 2 , from the National Heart Institute, Bethesda, Md.
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Z l E V E , M. SCHMUKLER
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