V o l u m e 85, n u m b e r i

FEBS LETTERS

INI-I[I~ITION OF PLATELET

ADENYLATE

CYCLASE

J a n u a r y 1978

BY EPINEPHRINE

REQUIRES

GTP

Karl H. J A K O B S , Wilhelm S A U R and Giinter S C H U L T Z Pharmakologisches lnstitut der Universitiit Heidelberg, bn Areuenheimer Fe!d 366, D-6900 Heidelberg, FRG

Received 31 October 1977

~. Introduc~or~ Epinephrine and norephaephrine cause aggregation o f h u m a n piatelets. This e f f e c t is b l o c k e d b y o~-adrenergic blocking agents such as phentolarnine and d i h y d r o e r g o t a m J n e and, therefore, is assumed to involve a - a d r e n e r ~ c receptors [ 1 - - 3 ] . Epinephrine is capable o f lowering p r o s t a ~ a n d i n E ~-stimulated cyclic AMP levels, an effect that involves ~-adrenergic

particles were o b t a i n e d b y 20 rain centrifugation o f the lysates at 30 0 0 0 × g a n d 0°C and were resuspended in the above buffer. Adenylate cyclase activity was d e t e r m i n e d as described [9] with 0 . i m M [CL-32P]A T P (purified, essentially free o f G T P [ 10 ] , 0.3--0.8 taCi/tube), 5 m M MgC!2, 0.1 m M et~tylene~ycol-bis(/3-aminoethy!ether) N , N ' t e t r a a c e t i c acid, 1 mM 3 - i s o b u t y ! - ! - m e t h y l x a n t h i n e , 5 mM creatine p h o s p h a t e and 0.4 m g / m l creatine kinase in 50 m M

receptors

triethartolamine--HCi

[4--8].

We have recently d e m o n s t r a t e d an inhibitory effect o f epinephrine and n o r e p i n e p h r i a e o n adenylate cyclase (EC 4.6.1.1) in cell-free preparations o f h u m a n platelets [ 9 ] . This effect was e n h a n c e d b y ~-adrenergic bloc ;king agents such as p r o p r a n o l o l and pindolol and was reversed b y ~ - a d r e n e r ~ c blocking agents such as p h e n t o l a m i n e and d i h y d r o e r g o t a m i n e . Inhibition b y e~-adrenergic agonists was seen w i t h the unstimulated, 'basal' f o r m o f the e n z y m e and with the e n z y m e activated b y sodium fluoride, prostag!andins [9] and adenosine (K. H. J_, W. S. and R. A. J o h n s o n , unpublished observations) b u t n o t after stimulation b y g u a n y l y l i m i d o d i p h o s p h a t e . The above studies were p e r f o r m e d in whole lysates o f platelets. We r e p o r t here t h a t G T P as a c o n s t i t u e n t o f the c y t o s o l is required for epinephrine-induced inhibition o f m e m b r a n e - b o u n d adenylate cyclase.

buffer, pH 7.4, at 37°C in total

vol. 0.1 ml. Reactions were initiated b y the addition o f platelet particles ( 2 0 - - 1 0 0 / ~ g p r o t e i n ) and conducted for 10 rain or as indicated. Cyclic AMP f o r m e d was purified as described [ 9 ] . U n d e r these c o n d i t i , ns cyclic AMP f o r m a t i o n was linear as a f u n c t i o n o f t i m e for at least 20 rain and as f u n c t i o n o f platetet protein. The ~-adrenergic blocking agent, pindolol (10/zM), was present under each condition. When L-epinephrine was added, its c o n c e n t r a t i o n was 30 pM. C o m p a r a b l e results to those s h o w n in the figures were o b t a i n e d in at least t w o separate e x p e r i m e n t s in each case. Standard deviations o f triplicate d e t e r m i n a t i o n s were less t h a n 5% o f the means. Protein was d e t e r m i n e d according to [11] using bovine serum albumin as standard.

3. R e s u l ~ 2. rdatefials a n d m e ~ o d s Materials and m e t h o d s used were essentially as described [ 9 ] . Isolated h u m a n platelets were !ysed b y rapid freezing ha liquid nitrogen and subsequent thawhag in a b o u t 50 vol. 10 m M t r i e t h a n o l a m i n e - - ~ l buffer, p H 7.4, containing t 50 m M NaCI. Platelet Elsevier/North-Holland Biomedical Press

Epineprhine b y its ~-adrenergic c o m p o n e n t reduced adenylate cyclase activity b y m a x i m a i l y 50--60% w h e n added to iysates o f hurnan platelets [ 9 ] . The epinephrine

e f f e c t w a s l a r g e l y r e d u c e d ( 0 - - 1 9 % ;_nhibi-

tion) in particulate preparations. Re-addition o f supern a t a n t fluid restored the i n h i b i t o r y effect o f epinephrine. T r e a t m e n t o f the s u p e r n a t a n t E a ~ w i t h heat, [ 67

Volume 85, number I

FEBS L E t t E R S

c h a r c o a l o r zdkaline p h o s p h a t a s e r e d u c e d o r a b o l i s h e d its c a p a b i l i t y o f r e s t o r i n g e p i n e p h r k , :-reduced i n h i b i tion (data not shown).

January 1978

300

Therefore, we considered the possibility that a nucleotide other khan ATP was a constituent o f the supernatant fluid that was required for the epinephrine effect. Of various nuc!eoside tdphosphates tested, G T P was m o s t e f f e c t i v e in r e s t o r i n g t h e e f f e c t o f

epinephrine on adeny!ate cycla~e in particulate preparations (fig.l). Maximal inhibition (about 45%) was seen with 1 0 - s - 1 0 -4 M GTP; 10 -~ M GTP was halfmaximally effective. Deoxy-GTP was slightly less e f f e c t i v e , a n d I T P w a s a b o u t o n e - t e n t h as p o t e n t as G T P . U T P , C T P a n d X T P w e r e f a r less e f f e c t i v e . W i t h G D P a d d e d at 10 -s a n d 10 -4 M in t h e a b s e n c e o f a n u c l e o s i d e t r i p h o s p h a t e - r e ~ e n e r a t i n 8 system~ e p i n e p h r i n e - i n d u c e d i n h i b i t i o n w a s o n l y 10% a n d

20%, respectively. Adenylate cyclase activity measured in the absence o f epinephrine was not signific-

%-

250

E

E

~-

200

. . . . .

0

Inhibition of platelet adenylate cyclase by epinephrine requires GTP.

V o l u m e 85, n u m b e r i FEBS LETTERS INI-I[I~ITION OF PLATELET ADENYLATE CYCLASE J a n u a r y 1978 BY EPINEPHRINE REQUIRES GTP Karl H...
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