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Inhibition by sulfhydryl agents of arachidonic acid-induced platelet aggregation and release of potential inflammatory substances
I N H I B I T I O N BY S U L F H Y D R Y L A G E N T S OF A R A C H I D O N I C P L A T E L E T A G G R E G A T I O N AND R E L E A S E OF P O T E N T I A L SUBSTANCES B.B. with
V a r g a f t i g , Y. the t e c h n i c a l MERRELL
T r a n i e r and M. C h i g n a r d c o l l a b o r a t i o n of J. K i n t z
ACID-INDUCED INFLAMMATORY
and M.L.
Part
INTERNATIONAL RESEARCH CENTER 16, rue d ' A n k a r a 67000 S T R A S B O U R G , F r a n c e
ABSTRACT Sulfhydryl agents (mercaptoethanol, thioglycerol, dithiot r e i t o l , and s o d i u m d i e t h y l d l t h i o c a r b a m a t e ) prevented a g g r e g a t i o n of r a b b i t p l a t e l e t s and the a c c o m p a n y i n g g e n e r a t i o n of p h a r m a c o l o g i c a l l y a c t i v e s u b s t a n c e s due to arac h i d o n i c acid. I n h i b i t i o n was also found a f t e r in vivo a d m i n i s t r a t i o n of the a n t a g o n i s t s . This a n t a g o n i s m was s u p p r e s s e d if the i n h i b i t o r s w e r e r e m o v e d f r o m the p l a t e l e t s u s p e n s i o n by w a s h i n g p r o c e d u r e s , w h e r e a s i n h i b i t i o n by i n d o m e t h a c i n was i r r e v e r s i b l e . A m i n o - t h i o l r e a g e n t s e i t h e r f a i l e d to a n t a g o n i z e the e f f e c t s of AA or p o t e n t i a t e d them. CuCI 2 i n c r e a s e d the a m o u n t s of p h a r m a c o l o g i c a l l y a c t i v e s u b s t a n c e s g e n e r a t e d in i n c u b a t e s of intact p l a t e lets w i t h a r a c h i d o n i c acid, and r e v e r s e d the i n h i b i t i o n due to thiol agents, but did not i n t e r f e r e w i t h i n h i b i t i o n by i n d o m e t h a c i n . P l a t e l e t s s u s p e n d e d in T y r o d e s o l u t i o n generated unstable pharmacologically active substances u p o n i n c u b a t i o n w i t h a r a c h i d o n i c acid ; s t a b i l i t y of these s u b s t a n c e s c o u l d be m a i n t a i n e d at 4°C. G e n e r a t i o n of this t e m p e r a t u r e - s e n s i t i v e m a t e r i a l was i n h i b i t e d by i n d o m e t h a c i n and by thiol agents. I n t e r f e r e n c e w i t h a c o p p e r c o n t a i n i n g c o m p o n e n t of PG s y n t h e t a s e or r e d u c t i o n of an i n t e r m e d i a t e l i p o p e r o x i d e a p p e a r as two p o s s i b l e m e c h a n i s m s of a c t i o n of thiol agents.
~ccepted
September
7,
1974.
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INTRODUCTION Platelet a g g r e g a t i o n and the a c c o m p a n y i n g release of p h a r m a c o l o g i c a l l y active substances due to a r a c h i d o n i c acid +(AA) are blocked by n o n s t e r o i d a n t i i n f l a m m a t o r y drugs (AID) (],2,3). A r a c h l d o n i c acid is the p r e c u r s o r of prostaglandins E 2 and F2~ (PGE 2 and PGF2~) ; as p r o s t a g l a n d i n s do not a g g r e g a t e p l a t e l e t s (4), the h y p o t h e s i s was raised that an i n t e r m e d i a t e is r e s p o n s i b l e for the platelet effect; the cyclic e n d o p e r o x i d e i n t e r m e d i a t e s formed during bioc o n v e r s i o n of AA into PG have in fact been suggested to be a g g r e g a n t a g e n t s ( 1 , 3 , 5 ~ 6 , 7 ) . These i n t e r m e d i a t e s are supposed to account at least in part for the c o n t r a c t i o n s of the isolated rabbit aorta strip, induced by incubates of blood or of platelet s u s p e n s i o n s with AA or with slow reacting substance C (SRS-C), an incubate of egg yolk with p h o s p h o lipase A 2 (8,9) that shares in vivo p h a r m a c o l o g i c a l activities with AA (9,|0,11). The m a t e r i a l g e n e r a t e d in incubates of platelets with AA or with SRS-C was named rabbit aorta c o n t r a c t i n g substance (RCS) and can be also obtained from g u i n e a - p i g lungs during a n a p h y l a c t i c shock (12), or injected with AA or SRS-C (13). Blockade by sulfhydryl agents of the in vivo effects a t t r i b u t e d to a c t i v a t i o n of PG s y n t h e t a s e that f o l l o w the a d m i n i s t r a t i o n of AA and of SRS-C (11) lead us now to study the i n t e r a c t i o n of those sulfhydryl agents with the g e n e r a t i o n of p h a r m a c o l o g i c a l l y active substances in incubates of p l a t e l e t s and AA. It was d e m o n s t r a t e d that those thlol agents that prevent the in vivo effects of AA and of SRS-C interfere with the g e n e r a t i o n of p h a r m a c o l o g i c a l l y active substances in incubates of PRP with AA and prevent AA induced platelet aggregation. The m e c h a n i s m of such an i n h i b i t i o n may involve r e d u c t i o n of the l l p o p e r o x i d e i n t e r m e d i a t e to inactive h y d r o x y - f a t t y acids, or i n t e r a c t i o n with a metal c o m p o n e n t of the PG s y n t h e t a s e complex.
+ A b b r e v i a t i o n s used : AA, a r a c h i d o n i c acid; PGE 2 and PGF2~ , r e s p e c t i v e l y prostaglandins E 2 and F2~; RCS, rabbit aorta c o n t r a c t i n g substance; SRS-C, slow reacting substance C; 5HT, 5 - h y d r o x y tryptamine c r e a t i n i n e sulfate (serotonin); AID, non steroid acidic a n t i i n f l a m m a t o r y drugs; ME, 2 - m e r e a p t o e t h a n o l ; TG, 2 - t h i o g l y c e r o l ; DTT, d i t h i o t h r e i t o l ; DEDTC, sodium d i e t h y l d i t h i o c a r b a m a t e ; GSH, reduced g l u t a t h i o n e ; EGTA, ethyleneglycol bis (amino-2 ethylether) N, N - t e t r a c e t a t e , d i s o d i u m salt; PRP and PPP, r e s p e c t i v e l y platelet rich and platelet poor plasma; O.D., optical d e n s i t y
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MATERIALS
AND M E T H O D S
Platelet a~re~ation B l o o d was c o l l e c t e d f r o m the c e n t r a l ear a r t e r y of u n a n a e s t h e t i z e d r a b b i t s d i r e c t l y into p l a s t i c tubes c o n t a i n i n g a 4 % w/v s o d i u m c i t r a t e s o l u t i o n (9 ml of b l o o d and | ml of s o d i u m c i t r a t e ) . A f t e r c e n t r i f u g a t i o n at r o o m t e m p e r a t u r e (220 g for |2 mln.) the tubes w e r e left for one h o u r on the bench. P l a t e l e t r i c h p l a s m a (PRP) was c o l l e c t e d and k e p t at r o o m t e m p e r a t u r e for the r e m a i n d e r of the day. A g g r e g a t i o n was s t u d i e d on two B r y s t o n a g g r e g o m e t e r s , w i t h t e m p e r a t u r e k e p t at 37°C. P l a t e l e t c o u n t s w e r e p e r f o r m e ~ m i c r o s c o p i c a l ly and c o n c e n t r a t i o n a d j u s t e d to 3 O O , O O O / m m with equal v o l u m e s of p l a s m a and n o n - p y r o g e n i c 0.9 % NaCI. S t i r r i n g was of I|00 rpm. A f t e r ]-3 m i n u t e s a l l o w e d to a d j u s t the t e m p e r a t u r e of the sample, AA (0.02 to 0.5 mM) was added. P o t e n t i a l a n t a g o n i s t s w e r e i n c u b a t e d w i t h PRP for one m i n u t e b e f o r e a d d i t i o n of AA. All a d d i t i o n s w e r e of |0-50 ~I. Washed platelets PRP was c e n t r i f u g e d for 15 m i n u t e s at 720 g. The p e l l e t was w a s h e d twice w i t h T y r o d e s o l u t i o n w i t h the f o l l o w i n g c o m p o s i t i o n ( g / l ) : N a C i , 8 ; KCI, 0.2; KH 9 PO&, 0.05; Mg Clp 6 H20, 0.]; g l u c o s e , I; N a H C O ~ , 0.035. F i r s t - w a s h i n g s o l u t i o n ( T y r o d e A) c o n t a i n e d also EGTA, 0 . 0 7 6 g/l, and b o v i n e s e r u m a l b u m i n , 3.5 g/l; s e c o n d w a s h i n g s o l u t i o n (Tyrode B) c o n tained no EGTA, w h e r e a s third w a s h i n g s o l u t i o n (Tyrode C) c o n t a i n e d no E G T A or a l b u m i n , CaCI 2 (0.2 g/l) b e i n g added. The p e l l e t o b t a i n e d f r o m PRP was r e s u s p e n d e d g e n t l y in T y r o d e A; a new p e l l e t was r e c o v e r e d a f t e r c e n t r l f u g a t i o n (15 m i n u t e s at 580 g), and r e s u s p e n d e d in T y r o d e B. A new c e n t r i f u g a t i o n (15 m i n u t e s at 470 g) a l l o w e d to r e c o v e r a final p e l l e t w h i c h was r e s u s p e n d e d in T y r o d e C in h a l f the o r i g i n a l v o l u m e of PRP ( 1 4 , ] 5 , ] 6 ) . W a s h e d p l a t e l e t s w e r e u s u a l l y k e p t at 37°C and gave r e p r o d u c i b l e r e s u l t s w h e n t e s t e d f r o m one to five a f t e r final s u s p e n s i o n . D o n o r and r e c i p i e n t c u v e t t e s One h y p o t h e s i s we a i m e d to c h e c k was that a p r o - a g g r e g a n t m a t e r i a l was f o r m e d from AA, w h e n a d d e d to p l a t e l e t s . A s y s t e m was thus d e v i s e d to d i s c r i m i n a t e b e t w e e n the d i r e c t e f f e c t of AA, and that of r e l e a s e d a g g r e g a n t m a t e r i a l s . In this system, p l a t e l e t s a m p l e s w e r e c o l l e c t e d f r o m the c u v e t t e s d u r i n g a g g r e g a t i o n due to ADP or to AA, w i t h and w i t h o u t a n t a g o n i s t s , and w e r e added (0.05 to 0.2 ml) to a n o t h e r s a m p l e of PRP, p r e p a r e d in a s e c o n d a g g r e g o m e t e r and c o n t a i n i n g e n o u g h i n d o m e t h a c i n (0.5 mM) to b l o c k c o m p l e t e l y any e f f e c t of AA. It was e x p e c t e d that a g g r e g a t i o n in the r e c i p i e n t c u v e t t e w o u l d thus not be due to a " p a r a s i t i c " e f f e c t of AA, t r a n s f e r r e d f r o m the d o n o r cuvette.
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I n t e r f e r e n c e of 5 - h y d r o x y t r y p t a m i n e (5 HT), k n o w n to be r e l e a s e d by AA f r o m p l a t e l e t s (17), was d i s c a r d e d by the use of m e t h y s e r g i d e , a r e c o g n i z e d i n h i b i t o r of 5 H T - i n d u c e d a g g r e g a t i o n (18), a d d e d e i t h e r to d o n o r or to r e c i p i e n t c u v e t t e s b e f o r e AA. Superfusion experiments I n c u b a t e s of AA and of p l a t e l e t p r e p a r a t i o n s were t e s t e d w i t h the s u p e r f u s i o n t e c h n i q u e (12,13). A r a b b i t a o r t a strip, to d e t e c t RCS, and a rat s t o m a c h strip to d e t e c t PGlike a c t i v i t y , w e r e s u p e r f u s e d w i t h K r e b s s o l u t i o n c o n t a i n ing a m i x t u r e of a n t a g o n i s t s to p r e v e n t any e f f e c t of c a t e c h o l a m i n e s , a c e t y l c h o l i n e , s e r o t o n i n and h i s t a m i n e . In a few e x p e r i m e n t s the rat c o l o n was a d d e d as a third tissue. The a s s a y o r g a n s w e r e s t i m u l a t e d w i t h PGE 2 and P G F 2 e (|OIOOng) to c o n t r a c t the g a s t r o - i n t e s t i n a l t i s s u e s , and w i t h n o r e p i n e p h r i n e (20-250 ng) to c o n t r a c t the r a b b i t a o r t a strip. The c o m p o s i t i o n of the i n c u b a t e s was i d e n t i c a l to those used for a g g r e g a t i o n . E x p e r i m e n t s w e r e run at r o o m t e m p e r a t u r e w i t h s t i r r i n g at I]OO rpm. In vivo e x p e r i m e n t s The i n h i b i t o r s of A A - i n d u c e d p l a t e l e t a g g r e g a t i o n w e r e inj e c t e d by the i n t r a v e n o u s r o u t e to r a b b i t s . S a m p l e s of b l o o d w e r e c o l l e c t e d b e f o r e and five m i n u t e s after t r e a t ment; p l a t e l e t rich p l a s m a s a m p l e s w e r e used as such or as w a s h e d p l a t e l e t s s u s p e n d e d in T y r o d e s o l u t i o n or in p l a s m a o b t a i n e d f r o m b l o o d c o l l e c t e d r e s p e c t i v e l y b e f o r e or a f t e r i n t r a v e n o u s t r e a t m e n t . T h e s e m a t e r i a l s were s t u d i e d in the a g g r e g o m e t e r , a f t e r w h i c h the c u v e t t e s w e r e t e s t e d on the isolated tissues. Druss The f o l l o w i n g r e a g e n t s w e r e p u r c h a s e d : p h e n o x y b e n z a m i n e h y d r o c h l o r i d e (Smith, K l i n e and F r e n c h , U . S . A . ) ; m e p y r a m i ne m a l e a t e ( R h S n e - P o u l e n c , F r a n c e ) ; a t r o p i n e s u l f a t e (Laboratolres Bruneau, France); propranolol hydrochloride ( A v l o c a r d y l R, L a b o r a t o i r e s A v l o n , F r a n c e ) ; 2 m e r c a p t o e t h a nol, t h i o g l y c e r o l , and r e d u c e d g l u t a t h i o n e (Fluka, S w i t z e r land); s o d i u m diethyldithiocarbamate ( S c h u c h a r d t , G e r m a n y ) ; d i t h i o t h r e i t o l ( C a l b i o c h e m . U . S . A . ) ; D L - p e n i c i l l a m i n e , tetraethylthiuram disulfide (disulflram), N-acetyl-L-cysteine, n o r e p i n e p h r i n e and 5 - h y d r o x y t r y p t a m i n e (serotonin) (Sigma, U . S . A . ) ; i n d o m e t h a c i n (Merck, Sharp and Dohme, U . S . A . ) ; and m e t h y s e r g i d e m a l e a t e (Sandoz, S w i t z e r l a n d ) ; salts for s o l u t i o n s w e r e p u r c h a s e d from u s u a l c o m m e r c i a l s o u r c e s . D r u g s i n s o l u b l e in 0.9 % NaCI w e r e s o l u b i l i z e d by a d d i n g g r a d e d a m o u n t s of 0.2 N N a O H or HCI. T e t r a e t h y l t h i u r a m d i s u l f i d e was s o l u b i l i z e d in p o l y e t h y l e n e g l y c o l 300 (Carl R o t h OHG), w h i c h had no p h a r m a c o l o g i c a l e f f e c t of its own on r a b b i t PRP. A r a c h i d o n i c acid (Sigma) was p r e p a red as d e s c r i b e d (|3).
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RESULTS
E f f e c t of a r a c h i d o n i c acid on p l a t e l e t s A d d i t i o n of AA ( 0 . 0 5 - 0 . 5 mM) to r a b b i t PRP lead i n i t i a l l y to an i n c r e a s e in O.D., i n d i c a t i n g a p l a t e l e t s h a p e c h a n g e , f o l l o w e d by a d e c r e a s e in O.D., and by p l a t e l e t a g g r e g a t i o n . S a m p l e s of PRP w e r e c o l l e c t e d at d i f f e r e n t time i n t e r v a l s a f t e r a d d i t i o n of AA, and a d d e d to r e c i p i e n t c u v e t t e s , w i t h r e s u l t a n t a g g r e g a t i o n of the r e c i p i e n t p l a t e l e t s . T r a n s f e r able a c t i v i t y was not due to c o n t a m i n a t i o n by the t r a n s f e r red AA as the r e c i p i e n t c u v e t t e c o n t a i n e d e n o u g h i n d o m e t h a cin to p r e v e n t its e f f e c t s . M a x i m u m a g g r e g a t i n g a c t i v i t y was o b t a i n e d w h e n s a m p l i n g was p e r f o r m e d 2-5 min. a f t e r add i t i o n of AA to the d o n o r c u v e t t e . T r a n s f e r a b l e a c t i v i t y was o n l y p r e s e n t w h e n a g g r e g a t i o n was u n d e r way; s a m p l e s c o l l e c t e d one m i n u t e a f t e r a d d i t i o n of AA to the d o n o r cuv e t t e d i s p l a y e d f e e b l e t r a n s f e r a b l e a c t i v i t y , w h e r e a s in s a m p l e s c o l l e c t e d a f t e r IO or 20 m i n u t e s of s t i r r i n g w i t h AA the t r a n s f e r a b l e a g g r e g a t i n g a c t i v i t y d i s a p p e a r e d . W h e n ADP was a d d e d to a d o n o r PRP in p l a c e of AA and s a m p l e s w e r e t r a n s f e r r e d from it to a r e c i p i e n t c u v e t t e , in o r d e r to i m m i t a t e w i t h e x o g e n o u s ADP w h a t o c c u r s w h e n AA is used, a 1 0 0 - 2 5 0 fold h i g h e r m o l a r c o n c e n t r a t i o n of AA as c o m p a r e d to that of ADP had to be a d d e d to a d o n o r c u v e t t e , to o b t a i n , in the r e c i p i e n t one, a c o m p a r a b l e a g g r e g a t i o n ( f i g u r e I). A l t h o u g h the t e n d e n c y of d o n o r PRP s a m p l e s c h a l l e n g e d w i t h ADP was to r e t a i n the t r a n s f e r a b l e a c t i v i t y for l o n g e r p e r i o d s of time than did PRP c h a l l e n g e d w i t h AA, no c l e a r d i f f e r e n c e in t i m e - r e l a t e d s t a b i l i t y c o u l d be d e m o n s t r a t e d w i t h this p r o c e d u r e b e t w e e n t r a n s f e r a b l e agg r e g a n t a c t i v i t y o b t a i n e d from AA or A D P - a g g r e g a t e d platelets. F i g u r e l(panel B) also shows that 0 . 5 m M of AA was less p o t e n t in i n d u c i n g a g g r e g a t i o n than 0.! mM. This was p o s s i b l y c a u s e d by over p r o d u c t i o n of PGE2, l i k e l y to a n t a ~ o n l z e a g g r e g a t i o n if p r e s e n t in a p p r o p r i a t e a m o u n t s . I n t e r f e r e n c e of thiol and o t h e r a s e n t s w i t h a r a c h i d o n i c acid-induced platelet a$$resation. P y r o g a l l o l , D E D T C , TG, ME, and DTT s u p p r e s s e d A A - i n d u c e d platelet aggregation ( f i g u r e 2 and T a b l e I). At c o n c e n t r a tions e f f e c t i v e a g a i n s t AA no b l o c k a d e was o b s e r v e d t o w a r d s ADP. S o d i u m t h i o g l y c o l a t e , DL-penicillamine and GSH at 1 mM r e d u c e d ADP and AA i n d u c e d a g g r e g a t i o n , w h e r e a s no b l o c k a d e was o b s e r v e d w i t h L - c y s t e i n e and N - a c e t y l - L - c y s t e ine. W h e n c o n c e n t r a t i o n s of AA b e l o w t h r e s h o l d for a g g r e g a tion ( 0 . 0 2 - 0 . 0 5 mM) w e r e p r e c e d e d or f o l l o w e d at a short i n t e r v a l by O.5-! m M of L - c y s t e i n e , a g g r e g a t i o n was l a u n c h e d and e v e n t u a l l y was total. No such e f f e c t was o b s e r v e d for ADP. A f t e r i n c u b a t i o n of r a b b i t PRP w i t h p y r o g a l l o l , D T T , T G or D E D T C (I mM) for 10 m i n u t e s , f o l l o wed by w a s h i n g and r e s u s p e n s i o n in PPP, a g g r e g a t i o n due to
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AA was not b l o c k e d . If t h e s e t r e a t e d p l a t e l e t s w e r e s u s p e n d e d in PPP c o n t a i n i n g the p o t e n t i a l a n t a g o n i s t s ( c o l l e c t e d from the i n i t i a l i n c u b a t e of PRP b e f o r e w a s h i n g ) , a g g r e g a t i o n was c o m p l e t e l y s u p p r e s s e d . On the o t h e r hand, w h e n i n d o m e t h a c i n (0.05 mM) was i n c u b a t e d w i t h PRP, f o l l o w ed by w a s h i n g and r e s u s p e n s i o n of p l a t e l e t s in d r u g - f r e e PPP, a g g r e g a t i o n was b l o c k e d , i n d i c a t i n g an i r r e v e r s i b l e e f f e c t on the p l a t e l e t s ; no b l o c k a d e was o b s e r v e d if these p r e t r e a t e d p l a t e l e t s w e r e r e s u s p e n d e d in T y r o d e s o l u t i o n (figure 3). B l o c k a d e of a g g r e g a t i o n by s u l f h y d r y l a g e n t s or by i n d o m e t h a c i n r e s u l t e d also in b l o c k a d e of the r e l e a s e or g e n e r a t i o n of t r a n s f e r a b l e a c t i v i t y (figure 4). A d d i t i o n of s u l f h y d r y l a g e n t s to the r e c i p i e n t c u v e t t e did not prev e n t the d e t e c t i o n of the t r a n s f e r a b l e a c t i v i t y o r i g i n a t i n g from an A A - c h a l l e n g e d s a m p l e of r a b b i t PRP. W a s h e d p l a t e lets s u s p e n d e d in T y r o d e s o l u t i o n w e r e not p r o t e c t e d from AA by s u l f h y d r y l a g e n t s or i n d o m e t h a c i n ; a d d i t i o n of PPP i n s u r e d b l o c k a d e s i m i l a r to that seen on PRP ( f i g u r e 3). The i n h i b i t o r y a c t i v i t y of i n d o m e t h a c i n or of thiol a g e n t s on A A - i n d u c e d p l a t e l e t a g g r e g a t i o n is thus only u n c o v e r e d if p l a s m a (or serum) is p r e s e n t . E G T A p r e v e n t e d A A - i n d u c e d a g g r e g a t i o n and the d e t e c t i o n of t r a n s f e r a b l e a g g r e g a n t a c t i v i t y . Both e f f e c t s w e r e o v e r c o m e by e q u i m o l a r CaCI 2 (figure 4). M e t h y s e r g i d e (up to 2 ~ g / m l of PRP) did not a f f e c t A A - i n d u c e d p l a t e l e t a g g r e g a t i o n or p r e v e n t d e t e c t i o n of t r a n s f e r a b l e a c t i v i t y w h e n p r e s e n t in the r e c i p i e n t cuvette. R e l e a s e of p h a r m a c o l o g i c a l l y a c t i v e s u b s t a n c e s f r o m i n c u b a tes of a r a c h i d o n i c acid w i t h p l a t e l e t p r e p a r a t i o n s . I n c u b a t i o n of PRP w i t h AA is f o l l o w e d by the g e n e r a t i o n of RCS and of P G - l i k e s u b s t a n c e s . (I-6, 19-21). As A A - i n d u c e d a g g r e g a t i o n of p l a t e l e t s s u s p e n d e d in T y r o d e s o l u t i o n was not b l o c k e d by s u l f h y d r y l a g e n t s or by i n d o m e t h a c i n , we i n v e s t i g a t e d w h e t h e r p l a s m a was also r e q u i r e d in order to d e m o n s t r a t e the i n h i b i t o r y e f f e c t s of the thiol s u b s t a n c e s on g e n e r a t i o n of p h a r m a c o l o g i c a l l y a c t i v e s u b s t a n c e s . T h e s e e x p e r i m e n t s w e r e not e n t i r e l y l e a s a b l e , b e c a u s e p l a t e l e t s s u s p e n d e d in T y r o d e s o l u t i o n and kept at r o o m t e m p e r a t u r e or at 37°C only g e n e r a t e d RCS for the initial 10-30 m i n u tes a f t e r b e i n g s u s p e n d e d ; p r e p a r a t i o n s kept for l o n g e r p e r i o d s g e n e r a t e d the rat s t o m a c h a c t i v i t y , w h e t h e r p l a s m a was p r e s e n t or not. It was h y p o t h e s i z e d that this e f f e c t m i g h t be due to r a p i d d e g r a d a t i o n of RCS at r o o m t e m p e r a ture i m m e d i a t l y u p o n f o r m a t i o n ; in o r d e r to c h e c k it, i n c u b a t i o n s of AA w i t h PRP or w i t h p l a t e l e t s s u s p e n d e d in T y r o d e s o l u t i o n w e r e p e r f o r m e d at 4°C. R e s u l t s s h o w n on f i g u r e 5 A and 5 B d e m o n s t r a t e that p l a t e l e t s s u s p e n d e d in T y r o d e s o l u t i o n at 37°C gave a v e r y low y i e l d of c o n t r a c ting a c t i v i t i e s over the rat s t o m a c h and the r a b b i t a o r t a strips, w h e r e a s w h e n i n c u b a t i o n s w e r e c a r r i e d at 4=C the a c t i v i t y was p r e s e n t and r e l a t i v e l y stable up to t h i r t y
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m i n u t e s . T i m e - r e l a t e d o b s e r v a t i o n s s h o w n in these f i g u r e s are f u r t h e r m e n t i o n e d in the d i s c u s s i o n . S u l f h y d r y l a g e n t s or i n d o m e t h a c i n added to the i n c u b a t e s of AA w i t h p l a t e l e t s at 4°C d i s p l a y e d their e x p e c t e d i n h i b i t o r y p r o p e r t i e s . Thus, c o n t r a s t i n g w i t h p h o t o m e t r i c o b s e r v a t i o n s , w h e n the a n t a g o n i s t s o n l y are e f f e c t i v e in the p r e s e n c e of plasma, the latter is not r e q u i r e d to i n h i b i t g e n e r a t i o n of RCS and PGlike a c t i v i t i e s . W h e n i n c u b a t i o n s w e r e p e r f o r m e d in p r e s e n ce of p l a s m a the y i e l d s of c o n t r a c t i n g m a t e r i a l s w e r e h i g h e r for 37°C than for 4°C and d e g r a d a t i o n w i t h time less steep. I n t e r f e r e n c e of s u l f h y d r ~ l a g e n t s w i t h the r e l e a s e of p h a r m a c o l o ~ i c a l l y a c t i v e s u b s t a n c e s in i n c u b a t e s of r a b b i t p l a t e l e t s w i t h a r a c h i d o n i c acid. P y r o g a l l o l , DTT, DEDTC, TG, ME and TG, m i x e d at a I mM c o n c e n t r a t i o n w i t h PGE 2 or w i t h PGF2~, did not r e d u c e by m o r e than 10 % the rat s t o m a c h strip c o n t r a c t i o n s . N o t w i t h s t a n d i n g , a d d e d to PRP one m i n u t e b e f o r e AA, the i n h i b i t o r s b l o c k e d the e x p e c t e d c o n t r a c t i o n s of the r a b b i t a o r t a and of the rat s t o m a c h strips. The r e s p o n s e s of the f o r m e r w e r e p r e v e n t e d at lower c o n c e n t r a t i o n s of the inhib i t o r s as c o m p a r e d to the l a t t e r (Table I). In c o n t r a s t , the a m i n o - t h i o l s GSH, D L - p e n i c i l l a m i n e and L - c y s t e i n e , p o t e n t i a t e d the t i s s u e r e s p o n s e s . Thus, in two e x p e r i m e n t s L - c y s t e i n e (I mM) p o t e n t i a t e d the r e s p o n s e of the aorta of 233 and 246 % and the r e s p o n s e of the s t o m a c h of 48 and 47 % ; D L - p e n i c i l l a m i n e (0.5 mM) p o t e n t i a t e d the a o r t a r e s p o n s e of 26 % and 69 %, and the s t o m a c h of 18 % ; GSH (1 mM) p o t e n t i a t e d the a o r t a of 24 % and the s t o m a c h of 15 %. E G T A (up to 5 mM) did not i n h i b i t the g e n e r a t i o n of P G - l i k e or of RCS a c t i v i t i e s in r a b b i t PRP i n c u b a t e d w i t h AA. I n t e r f e r e n c e of c o p p e r w i t h the g e n e r a t i o n of p h a r m a c o l o ~ically active substances from platelets. In o r d e r to c h e c k w h e r e a s the m e c h a n i s m of a c t i o n of the thiol a g e n t s i n v o l v e d i n t e r f e r e n c e w i t h c o p p e r ions, a few i n h i b i t o r s w e r e tested in its p r e s e n c e . A d d i t i o n of C u C I 2 (I mM) to p l a t e l e t i n c u b a t e s , f o l l o w e d after one m i n u t e by a d d i t i o n of AA, and by b i o a s s a y of the r e s u l t i n g i n c u b a t e ~ showed that the r e s p o n s e s of the r a b b i t a o r t a strip i n c r e a s e d by 101,88 ~ 26,6 %, w h e r e a s the r e s p o n s e s of the rat s t o m a c h w e r e i n c r e a s e d by 36.7 + 10.5 %, as c o m p a r e d to c o n t r o l i n c u b a t e s w i t h o u t CuCI2. The i n h i b i t o r y e f f e c t s of p y r o g a l l o l and of D E D T C w e r e r e v e r t e d by e q u i m o l a r CuCI2, w h e r e a s lower c o n c e n t r a t i o n s of the l a t t e r did not p r e v e n t i n h i b i t i o n of the g e n e r a t i o n of p h a r m a c o l o g i c a l l y a c t i v e m a t e r i a l s in the i n c u b a t e s ( f i g u r e 6 A and 6 B). The i n h i b i t o r y a c t i v i t y of i n d o m e t h a c i n was not p r e v e n t e d by CuCI 2 up to ] mM.
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PROSTAGLANDINS
Table Inhibition
of A r a c h i d o n i c
Generation
of RCS
Rabbit
I
Acid and
Platelet
Induced
PG-like Rich IC
Platelet Aggregation
Antagonist
Diethyldithiocarbamate
Aggregation
Activities
and
in
Plasma 5O
(uM) *
C o n t r a c t i o n s of Rabbit Aorta Rat S t o m a c h Strip Strip
53
76
330
Mercaptoethanol
150
130
300
Thioglycerol
280
300
560
Dithiothreitol
2]0
130
630
DL P e n i c i l l a m i n e
540
~
~
Pyrogallol Indomethacin
20
57
260
3.7
2.5
11
IC 50 : c o n c e n t r a t i o n s of d r u g s (in ~M) r e q u i r e d to i n h i b i t 50 % p l a t e l e t a g g r e g a t i o n or g e n e r a t i o n of pharmacologically a c t i v e s u b s t a n c e s o b t a i n e d by i n t e r p o l a t i o n of 3-4 r e s u l t s . z~
potentiates
generation
of RCS
and
PG-like
activities.
B l o c k a d e of a r a c h i d o n i c a c i d - i n d u c e d p l a t e l e t a $ $ r e s a t i o n and r e l e a s e of m e d i a t o r s a f t e r in v i v o a d m i n i s t r a t i o n of druss. I n d o m e t h a c i n was i n t r a v e n o u s l y a d m i n i s t e r e d to two r a b b i t s at 5 m g / k g . S a m p l e s of b l o o d w e r e c o l l e c t e d b e f o r e and a f t e r t r e a t m e n t ~ and p l a t e l e t r e s p o n s e s w e r e s t u d i e d in PRP, in w a s h e d p l a t e l e t s r e s u s p e n d e d in T y r o d e s o l u t i o n , or in p l a s m a c o l l e c t e d b e f o r e and a f t e r i n d o m e t h a c i n t r e a t m e n t . PRP c o l l e c t e d a f t e r i n d o m e t h a c i n did not respond any m o r e to 0.5 m M of AA, as did the s a m p l e s c o l l e c t ed b e f o r e t r e a t m e n t , w h e r e a s r e s p o n s e s to ADP were u n a f fected. W a s h e d p l a t e l e t s from a b l o o d s a m p l e c o l l e c t e d af t e r i n d o m e t h a c i n w e r e also u n r e s p o n s i v e to AA, even if r e s u s p e n d e d in p l a s m a f r o m s a m p l e s o b t a i n e d b e f o r e i n j e c tion of i n d o m e t h a c i n ; t h e s e same p l a t e l e t s , r e s u s p e n d e d
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in T y r o d e s o l u t i o n , a g g r e g a t e d to AA d e s p i t e p r e v i o u s indomethacin treatment. Washed platelets collected before i n d o m e t h a c i n t r e a t m e n t but r e s u s p e n d e d in p l a s m a f r o m b l o o d c o l l e c t e d a f t e r i n d o m e t h a c i n , thus c o n t a i n i n g the a n t i - i n f l a m m a t o r y agent, w e r e also b l o c k e d to AA w h e r e a s r e s p o n s e s to ADP w e r e u n a f f e c t e d . A g g r e g a t i o n and r e l e a s e of m e d i a t o r s t r i g g e r e d by AA in PRP c o l l e c t e d a f t e r i n t r a v e n o u s TG (200 m g / k g ) , D E D T C (I00 m g / k g ) and p y r o g a l l o l (20 m g / k g ) w e r e i n h i b i t e d . W a s h e d p l a t e l e t s from b l o o d s a m p l e s c o l l e c t e d a f t e r a d m i n i s t r a t i o n of i n h i b i tors and r e s u s p e n d e d in p l a s m a from b e f o r e the t r e a t m e n t r e s p o n d e d to AA, w h e r e a s the same p l a t e l e t s r e s u s p e n d e d in p l a s m a f r o m after t r e a t m e n t w e r e u n r e s p o n s i v e to AA. No r e s p o n s e of the i s o l a t e d o r g a n s w e r e o b t a i n e d f r o m i n c u b a t e s of AA w i t h PRP c o l l e c t e d f r o m a n i m a l s t r e a t e d w i t h the above m e n t i o n e d d o s e s of TG, D E D T C or p y r o g a l l o l . This b l o c k a d e was s u p p r e s s e d by w a s h i n g the p l a t e l e t s and r e s u s p e n d i n g in T y r o d e s o l u t i o n or in d r u g - f r e e p l a s m a ; w a s h e d p l a t e l e t s r e s u s p e n d e d in p l a s m a c o l l e c t e d a f t e r drug t r e a t m e n t did not r e s p o n d to AA° Two e x p e r i m e n t a l r e s u l t s are d e p i c t e d in f i g u r e 7.
DISCUSSION
S u l f h y d r y l a g e n t s and p y r o g a l l o l have n o w b e e n s h o w n to i n h i b i t p l a t e l e t a g g r e g a t i o n and g e n e r a t i o n of RCS and P G - l i k e a c t i v i t i e s , w h e n added to PRP or a d m i n i s t e r e d to r a b b i t s at d o s e s that s u p p r e s s the h y p o t e n s i v e e f f e c t s of SRS-C (11). I n d o m e t h a c i n - l i k e d r u g s e x h i b i t the same a c t i v i t i e s . It thus seems l o g i c a l to a s s u m e that r e l e a s e of P G - l i k e and RCS a c t i v i t i e s , and the p h a r m a c o l o g i c a l e f f e c t s of AA and of SRS-C are r e l a t e d : we h y p o t h e s i z e that h y p o t e n s i o n and b r o n c h o c o n s t r l c t i o n in r a b b i t s , dogs and g u i n e a - p i g s are l a r g e l y due to the i n t e r a c t i o n of AA or of SRS-C w i t h p l a t e l e t PG s y n t h e t a s e w i t h release of s u b s t a n c e s d i s p l a y i n g RCS and P G - l i k e a c t i v i t i e s . The h y p o t h e s i s of a b a s i c role for p l a t e l e t s is s u p p o r t e d by the fa~t that w h e n AA or SRS-C are i n j e c t e d d i r e c t l y into a t u b i n g c a r r y i n g b l o o d p r o v i d e d by a dog onto s u p e r fused i s o l a t e d t i s s u e s , m a r k e d c o n t r a c t i o n s are o b t a i n e d , b l o c k e d by i n t r a v e n o u s AID (19), or thiol a g e n t s (21). O n l y b l o o d is i n v o l v e d , and lungs, a k n o w n s o u r c e of PG and RCS w h e n p e r f u s e d in v i t r o w i t h AA or SRS-C (13), are e x c l u d e d . M o r e o v e r , DEDTC, that s h a r e s the in v i v o inhib i t o r y p r o p e r t i e s w i t h the other thiol a g e n t s , was sole, a m o n g them, not to b l o c k the r e l e a s e of RCS by AA from i s o l a t e d g u i n e a - p i g lungs (II); n o t w i t h s t a n d i n g , D E D T C was e f f e c t i v e on p l a t e l e t s in v i v o and in vitro, i n d i c a ting a b e t t e r c o r r e l a t i o n of in v i v o i n h i b i t i o n w i t h a p l a t e l e t than w i t h a p u l m o n a r y site of action. A l t h o u g h
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PROSTAGLANDINS t a c h y p h y l a x i s m i g h t have b e e n e x p e c t e d u p o n in v i v o a d m i n i s t r a t i o n of h i g h a m o u n t s of AA, (22) r e p r o d u c i b l e h y p o t e n s i o n can be o b t a i n e d w i t h lower a m o u n t s (23) or w i t h SRS,C (13). This f u l l y a g r e e s w i t h a p l a t e l e t site of action, b e c a u s e A A - i n d u c e d a g g r e g a t i o n of r a b b i t p l a t e l e t s is r e v e r s i b l e , s p o n t a n e o u s l y or if a p p r o p r i a t e a n t a g o n i s t s (PGE I or PGE2, d i b u t y r y l c y c l i c AMP, EGTA, a p y r a s e ) are a d d e d a f t e r c o m p l e t i o n of a g g r e g a t i o n (1,5 ; and to be p u b l i s h e d ) . A u n i f y i n g t h e o r y for the m e c h a n i s m of a c t i o n of s u l f h y d r y l and a n t i - o x i d a n t a g e n t s should a c c o u n t for : a. A A - i n d u c e d p l a t e l e t a g g r e g a t i o n and g e n e r a t i o n of RCS and P G - l i k e a c t i v i t i e s are o n l y b l o c k e d in p r e s e n c e of thiol agents, w h e r e a s i n d o m e t h a c i n p r e v e n t s a g g r e g a t i o n also after the p l a t e l e t s h a v e b e e n w a s h e d and r e s u s p e n d e d in d r u g - f r e e plasma; b.
thiol a g e n t s b l o c k the c o n t r a c t i o n s of the r a b b i t a o r t a and of the rat s t o m a c h strips to p l a t e l e t incubates w i t h AA, w i t h o u t i n t e r f e r i n g w i t h the e f f e c t s of PGE 2 or PGF2~.
F r o m a it m a y be c o n c l u d e d that i n d o m e t h a c i n does not i n t e r a c t c h e m i c a l l y w i t h AA or w i t h the e x p e c t e d l i p o p e r o xide (s), as i n h i b i t i o n is r e t a i n e d after r e m o v a l of i n d o m e t h a c i n f r o m the p l a t e l e t e n v i r o n m e n t . This is not the case for thiol agents, w h i c h m i g h t i n t e r a c t w i t h AA or w i t h its p r o d u c t s . F r o m b it m a y be c o n c l u d e d that thiol a g e n t s (and also AID) did not p r e v e n t the d i r e c t e f f e c t s of p r o s t a g l a n d i n s , as has b e e n d e s c r i b e d by J o h n s o n , J e s s u p and R a m w e l l (24) in a n o t h e r system. The h y p o t h e s i z e d include : a.
mechanisms
of a c t i o n
of
thiol
agents
r e d u c t i o n of d i s u l p h i d e b o n d s of a c o m p o n e n t of the e n z y m e c o m p l e x d e a l i n g w i t h AA or of the r e c e p t o r i n v o l v e d w i t h its e f f e c t s . This m e c h a n i s m c a n n o t be ruled out, but w o u l d not e x p l a i n the f a i l u r e of a few thiol a g e n t s to b l o c k our systems. E v e n if f a i l u r e of GSH or c y s t e i n e is a c c o u n t e d for by their r a p i d a u t o o x i d a t i o n , this is not the case for p e n i c i l l a m i n e (25). D i s u l f i r a m and its r e d u c e d form, DEDTC, w e r e e f f e c t i v e at e q u i m o l a r c o n c e n t r a t i o n s . As the f o r m e r is c o n v e r t e d into the l a t t e r in p l a s m a (26) it is p r o b a b l e that the a c t i v e a n t a g o n i s t is the free thiol c o m p o u n d .
b. D i r e c t i n t e r a c t i o n of the i n h i b i t o r s w i t h the e n z y m e or w i t h a c o - f a c t o r . D i e t h y l d i t h i o c a r b a m a t e b l o c k s the v e s i c u l a r g l a n d e n z y m e r e s p o n s i b l e for o x y g e n a t i o n of AA (27). R e v e r s i b i l i t y was o b t a i n e d w i t h Cu ++, w h i c h could be due to r e m o v a l of D E D T C by the m e t a l and not to r e p l a c e m e n t of f u n c t i o n a l l y a c t i v e Cu ++ (see h y p o thesis c). Lee and L a n d s (28) h a v e also d e m o n s t r a t e d that in--the a b s e n c e of Cu ++, DTT i n h i b i t s the o x i d a t i o n of AA by PG s y n t h e t a s e , w h e r e a s the c o m p l e x of Cu ++
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w i t h D T T i n c r e a s e s the e x t e n t of s u b s t r a t e o x i d a t i o n and s h i f t s the s y n t h e s i s of P G E p to PGF2e. This is s i m i l a r to our r e s u l t s . E f f e c t i v e n e S s of th~ non c h e l a t o r mp h e n a n t h r o l i n e in i n h i b i t i n g the d i o x y g e n a s e (28) indic a t e s that o t h e r m e c h a n i s m than Cu ++ c o m p l e x a t i o n m a y be involved. c. All a g e n t s we u s e d are c o p p e r c h e l a t o r s , but no d i r e c t r e l a t i o n s h i p was found b e t w e e n the a b i l i t y to c o m p l e x Cu ++ and to b l o c k the e f f e c t s of AA. Thus p e n i c i l l a m i n e , E D T A and E G T A are e x p e c t e d to c o m p l e x all free copper, as their K 1 ( l o g a r i t h m of the s t a b i l i t y c o n s t a n t for Cu ++) is r e s p e c t i v e l y of 16,5, 18.8 and 17.7, but did not p r e v e n t g e n e r a t i o n of R C S - l i k e a c t i v i t y ; p y r o g a l l o l has lower c h e l a t i n g p r o p e r t i e s (K I = 12.4), and was the s t r o n g e s t a n t a g o n i s t of A A - i n d u c e d e f f e c t s . As 2,2d i p y r i d y l and 8 - h y d r o x y q u i n o l i n e , w h i c h h a v e no SH groups, also i n h i b i t e d the e f f e c t s of AA (to be p u b l i shed), it is p o s s i b l e that e n z y m e b o u n d c o p p e r d i s p l a y s a role in PG s y n t h e t a s e , b e i n g e i t h e r n e u t r a l i z e d by c h e l a t o r s as D E D T C or t u r n e d m o r e c h e m i c a l l y r e a c t i v e by a c o o p e r a t i v e e f f e c t (29), as m i g h t be the case for the a m i n o - t h i o l s . The role of Cu ++ is f u r t h e r m o r e supp o r t e d by the fact that it s t i m u l a t e s the p r o d u c t i o n of P G F 2 ~ in a n o t h e r s y s t e m (30); d. A n o t h e r p o s s i b l e m e c h a n i s m of a c t i o n of thiol and a n t i o x i d a n t s u b s t a n c e s c o n s i s t s in the r e d u c t i o n of the i n t e r m e d i a t e a c t i v e l i p o p e r o x i d e . This w o u l d e x p l a i n i n t e r f e r e n c e w i t h a g g r e g a t i o n , a t t r i b u t e d to the s h o r t l a s t i n g c y c l i c e n d o p e r o x i d e (1,6) and w i t h r a b b i t a o r t a c o n t r a c t i n g a c t i v i t y , if the l a t t e r is also due to l i p o p e r o x i d e s (31). Rabbit aorta contracting activity obtained from platelets, is not PGE 2 or PGF2~, w h i c h do not a f f e c t the aorta. P G F 2 ~ was p r o b a b l y o n l y p r e s e n t in low a m o u n t s in our AA i n c u b a tes, as the rat c o l o n c o n t r a c t e d f e e b l y w h e n c h a l l e n g e d w i t h them, a l t h o u g h it r e s p o n d e d to c o n c e n t r a t i o n s of P G F 2 e e q u i a c t i v e w i t h PGE 2 and w i t h the p l a t e l e t i n c u b a t e s w i t h r e s p e c t to the s t o m a c h strip. M o r e o v e r , PGE 2 r a t h e r than P G F 9 ~ is g e n e r a t e d by p l a t e l e t s d u r i n g a g g r e g a t i o n (20). F i ~ a l l y , the drop in rat s t o m a c h strip c o n t r a c t i n g a c t i v i t y in p r e s e n c e of thiol a g e n t s is c e r t a i n l y not a c c o u n t e d for by a shift f r o m PGE 2 to P G F g ~ p r o d u c t i o n due to a d d e d r e d u c i n g e q u i v a l e n t s , as the c o n t r a c t i o n s of the rat c o l o n w e r e not i n c r e a s e d , as c o u l d o c c u r in p r e s e n c e of h i g h e r a m o u n t s of P G F g ~ , but w e r e d e c r e a s e d . D i s a p p e a r a n c e w i t h time of the a ~ t i v i t y of i n c u b a t e s of AA w i t h p l a t e l e t s s u s p e n d e d in T y r o d e , b o t h w i t h r e s p e c t to rat s t o m a c h and r a b b i t a o r t a strip, i n d i c a t e s that the role of PGE 2 is small, if any, in these c o n d i t i o n s . If the rat s t o m a c h a c t i v i t y w o u l d be due to PGEg, it s h o u l d o n l y s l i g h t l y d e c a y d u r i n g t i m e - r e l a t e d i ~ c u b a t i o n s , or w h e n
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i n c u b a t e s at 4 ° C are t r a n s f e r r e d to 37°C~ It is thus p r o b a ble that p l a t e l e t s in T y r o d e s o l u t i o n p r e d o m i n a n t l y s y n t h e size s o m e t h i n g else, w h i c h a f f e c t s b o t h a s s a y t i s s u e s . H i g h e r y i e l d s at 4°C, as c o m p a r e d to 37°C, i n d i c a t e that the g e n e r a t e d m a t e r i a l is h i g h l y u n s t a b l e in T y r o d e s o l u t i o n , w h e r e a s s t a b i l i t y is h i g h e r in plasma, as are the y i e l d s at 37°C c o m p a r e d to 4°C. W h e n s a m p l e s are a l l o w e d to stand at r o o m t e m p e r a t u r e , the r a b b i t a o K t a a c t i v i t y d i s a p p e a r s t o g e t h e r w i t h part of the rat s t o m a c h a c t i v i t y , w h e r e a s a r e s i d u a l t i m e - r e s i s t a n t e f f e c t on the rat s t o m a c h is o b s e r ved, p r o b a b l y a c c o u n t e d for by PGE 2. That part of the rat s t o m a c h a c t i v i t y w h i c h d i s a p p e a r s c o n c u r r e n t l y w i t h loss of a c t i v i t y over the r a b b i t a o r t a strip, is thus p r o b a b l y due to RCS. If the r a b b i t a o r t a a c t i v i t y w o u l d p u r e l y be due to a P G - p r e c u r s o r , such as the c y c l i c e n d o p e r o x i d e , and the rat s t o m a c h a c t i v i t y due to PGE 2 o r i g i n a t i n g from it, as i n i t i a l l y t h o u g h t , a drop in the r a b b i t a o r t a a c t i v i t y should be a c c o m p a n i e d by i n c r e a s e d rat s t o m a c h strip a c t i vity. A s i m i l a r o b j e c t i o n a p p l i e s to the a c t i v i t i e s found in p l a t e l e t s s u s p e n d e d in T y r o d e s o l u t i o n and c h a l l e n g e d w i t h AA at r o o m t e m p e r a t u r e : d e g r a d a t i o n of RCS should be f o l l o w e d by i n c r e a s e d P G - l i k e g e n e r a t i o n , w h i c h was not the case. The h a l f - l i v e s of the c y c l i c e n d o p e r o x i d e s are l o n g e r (7) than those r e p o r t e d by us (13) for lung RCS ; our r e s u l t s now show that the a g g r e g a n t and t r a n s f e r a b l e m a t e rials g e n e r a t e d in p l a t e l e t i n c u b a t e s w i t h AA have a shorter life than RCS, w h e n p l a s m a is p r e s e n t , w h e r e a s its a c t i v i t y d e c a y s p r e c i p i t o u s l y if p l a s m a is o m i t t e d . A " l a b i l e a g g r e g a t i o n s t i m u l a t i n g s u b s t a n c e " , formed from AA by p l a t e l e t m i c r o s o m a l p r e p a r a t i o n s is m a x i m a l l y a v a i l a b l e a f t e r 45 s e c o n d s (6), w h i c h c o n f l i c t s w i t h our data that the a g g r e g a n t a c t i v i t y r e q u i r e s a r o u n d two m i n u t e s to appear. D i f f e r e n t p r o c e d u r e s m a y e x p l a i n the d i s c r e p a n c y : we used r a b b i t PRP as d o n o r and r e c i p i e n t m a t e r i a l s , the l a t t e r b e i n g p r i m e d w i t h i n d o m e t h a c i n to p r e v e n t the " p a r a s i t i c " effect of AA. W i l l i s (6) added h u m a n PRP to i n c u b a t e s of m i c r o s o m e s in b u f f e r and AA, after b i o t r a n s f o r m a t i o n of the latter had b e e n i n i t i a t e d , p o s s i b l y m a k i n g the a g g r e g a n t a c t i v i t y m o r e r e a d i l y a v a i l a b l e . M o r e o v e r , as in his e x p e r i m e n t s h u m a n p l a t e l e t s in p l a s m a did not r e s p o n d to AA, the " p a r a s i t i c " a c t i v i t y of the l a t t e r was not feared and i n d o m e t h a c i n was o m i t t e d from the r e c i p i e n t system, w h i c h m i g h t thus have r e s p o n d e d m o r e r e a d i l y than in our case to the joint e f f e c t s of the t r a n s f e r r e d AA and of the g e n e r a t e d l i p o p e r o x l d e . ADP is p r e s u m a b l y r e l e a s e d w h e n a g g r e g a t i o n is e v o k e d by AA, and may p a r t l y a c c o u n t for the e f f e c t i v e n e s s of EGTA, w h i c h did not p r e v e n t g e n e r a t i o n of RCS and P G - l i k e m a t e r i a l s , b u t p r e v e n t e d g e n e r a t i o n or d e t e c t i o n of a g g r e g a t i n g a c t i v i t y . This a c t i v i t y of E G T A may also be e x p l a i n e d by a Ca ++ r e q u i r e m e n t for a g g r e g a t i o n or for b i n d i n g of the lipop e r o x i d e to the m e m b r a n e , as occurs for PGs (32). As our
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PROSTAGLANDINS a i m was to s t u d y the e f f e c t of AA on i n t a c t p l a t e l e t s , A D P free PG s y n t h e t a s e p r e p a r a t i o n s w e r e not used. M o r e o v e r , as a n t a g o n i s t s of A D P - i n d u c e d a g g r e g a t i o n (PGEI, a p y r a s e or a d e n o s i n e ) also i n h i b i t the e f f e c t s of AA on r a b b i t p l a t e lets (1,5), it was u s e l e s s to try to d i s c r i m i n a t e w i t h them b e t w e e n ADP and o t h e r p o t e n t i a l m e d i a t o r s . Such a d i s c r i m i n a t i o n has r e c e n t l y b e e n p e r f o r m e d on dog p l a t e l e t s , w h i c h are c o m p l e t e l y r e f r a c t o r y to the a g g r e g a t i n g a c t i v i t y of AA, but g e n e r a t e t r a n s f e r a b l e a g g r e g a t i n g a c t i v i t y to recipient rabbit platelets (33, and to be p u b l i s h e d ) , acc o m p a n i e d by RCS and P G - l i k e s u b s t a n c e s (19). This t r a n s f e r a b l e a g g r e g a n t a c t i v i t y c a n n o t be a c c o u n t e d for by ADP, w h i c h i n d u c e s on dog p l a t e l e t s , as do t h r o m b i n or c o l l a g e n , the e x p e c t e d a g g r e g a t i o n . An i m p o r t a n t role for 5HT to i n d u c e a g g r e g a t i o n or a c c o u n t for t r a n s f e r a b l e a c t i v i t y was d i s c a r d e d w i t h m e t h y s e r g i d e , show n to be i n a c t i v e in p r e v e n t i n g e i t h e r of these e f f e c t s . Moreover, in p i l o t e x p e r i m e n t s , p l a t e l e t s c o l l e c t e d from r e s e r p i n i z e d r a b b i t s w e r e as a g g r e g a t e d by AA as c o n t r o l unreserpinized s a m p l e s . Our r e s u l t s , p a r t i c u l a r l y w i t h washed platelets, those of W i l l i s (6) and of H a m b e r g et al (7) w o u l d thus i n d i c a t e that the s p e c i f i c c y c l i c e n d o p e r o xide (s), w i t h a short h a l f - l i f e in a p l a s m a m e d i u m , m a y be i n v o l v e d w i t h p l a t e l e t a g g r e g a t i o n . The m a t e r i a l s that ~ a c c o u n t for r a b b i t a o r t a c o n t r a c t i n g a c t i v i t y m a y c o n t a i n the c y c l i c e n d o p e r o x i d e (s) but o t h e r s u b s t a n c e s as well, w i t h a l o n g e r h a l f - l i f e . T h e s e a g e n t s are p o s s i b l y l i a b l e to c h e m i c a l i n t e r a c t i o n w i t h thiol r e d u c i n g a g e n t s , a l t h o u g h an i n h i b i t o r y e f f e c t over a c o m p o n e n t of the PG s y n t h e t a s e c o m p l e x c a n n o t be e x c l u d e d . A s u m m a r y of the r e s u l t s (figure 8), h i g h l i g h t s the fact that all a g e n t s e f f e c t i v e in v i t r o in our s y s t e m i n h i b i t the in v i v o effects of AA, $RS-C (11) and b r a d y k i n i n (34), thus r e i n f o r cing the t h e o r y that the in v i v o e f f e c t s of the a g o n i s t s l i a b l e to b l o c k a d e by AID or by s u l f h y d r y l a g e n t s are due to a c o m m o n m e d i a t o r , r e l e a s e d t h r o u g h a c t i v a t i o n of PG s y n t h e t a s e or o t h e r l i p o p e r o x i d i z i n g e n z y m e s \ An i m p o r t a n t role of 5HT for a g g r e g a t i o n or t r a n s f e r of a g g r e g a t i n g a c t i v i t y was d i s c a r d e d w i t h m e t h y s e r g i d e , shown to be i n a c t i v e a g a i n s t b o t h A A - i n d u c e d a g g r e g a t i o n and d e t e c t i o n of t r a n s f e r a b l e a g g r e g a t i n g a c t i v i t y .
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Experientia
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PROSTAGLANDINS
A
t 1 I
B
C
IM 2O
0.1
I 0.5 0.1
6O
8o1
0.5
~
20
Figure 1 : Arachidonic acid and ADP-induced platelet aggregation and 6eneration of transferable activity Panel A:
Concentration-related aggregation of rabbit platelets by ADP (concentrations in ~M indicated next to each curve).
Panel B:
Aggregation of rabbit platelets by arachidonic acid; curve labelled A was obtained with a platelet rich plasma kept for the overnight; (concentrations in mM indicated next to each curve). Observe that an optimal amount of AA (O.| mM) is more aggregant than 0.5 mM.
Panel C:
Aggregation of rabbit PRP contained in recipient cuvettes induced by 0.2 ml of PRP transferred from donor euvettes of panel B; (concentrations refer to those used to induce aggregation in the donor PRP); indomethacin (0.5 mM) was added to prevent effects of arachidonic acid.
Vertical scale: percent transmission Horizontal scale: time (one minute)
148
OCTOBER 25, 1974 VOL. 8 NO. 2
PROSTAGLANDINS
1
I
100
I00
20-
l 50
40-
10
60,
5
80
Figure 2: Inhibition of platelet aggresation due to arachidonic acid by 2~merca~toethanol and sodium diethyldithiocarbamate.
Superposed tracings of aggregation due to arachidonic acid (added at the arrow 0.5 mM) in rabbit platelet rich plasma. Concentrations of the inhibitors added one minute before arachidonic acid are indicated in ~M. Left panel: 2-mercaptoethanol; Right panel: sodium diethyldithiocarbamate. Scales as in figure I
OCTOBER 25, 1974
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PROSTAGLANDINS
!
4
20
dO
80
Figure 3-: Comparison between the effects of indomethacin and thio~l~cerol in platelets suspended in plasma or in Tyrode solution.
Superposed tracings of platelet aggregation induced by arachidonic acid (0.5 mM), as follows: a: control aggregation in rabbit PRP; b and c: aggregation blocked respectively by 0.5 mM of indomethacin and 5 mM of thioglycerol. d and e: responses of platelets suspended in Tyrode solution are not blocked by respectively 0.5 mM of indome~hacin and 5 mM of thioglycerol. Scales as in Figure I.
150
OCTOBER 25, 1974
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PROSTAGLANDINS 1 O
,I
C'
20 ME
40
60
80
Figure 4: Inhibition of a~regation and of ~eneration of transferable activity in rabbit platelet rich plasma Left Panel: superposed tracings of platelet aggregation A = control response to 0.5 D~M of AA; B = protection against aggregation by 0.05 ~M of indomethacin added at the arrow one minute before AA. C = protection against aggregation by 0.5 mM of 2-mercaptoethanol added at the arrow, one minute before AA. Right Panel:superposed tracings of platelet aggregation, obtained after transfer of 0.2 ml of the samples from the left panel. A', B' and C' correspond to A, B and C respectively, collected after three minutes allowed for aggregation. Recipient cuvettes contained 0.5 mM of indomethacin. No transferable activity was obtained from cuvettes where aggregation was inhibited.
OCTOBER 25, 1974
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PROSTAGLANDINS
F' 20
E
40
60
80
Figure 4 Cont'd
Left Panel
: superposed tracings of aggregation. D = control responses to 0.5 mM of AA ; E = equimolar CaCI 2 partially overcomes the inhibition by EGTA (5 mM) of platelet aggregation due to AA; F = inhibition by EGTA of platelet aggregation due to AA.
Right Panel : superposed tracings of platelet aggregation obtained after transfer of samples from the right panel. E' and F' correspond to E and F, respectively, collected after three minutes allowed for aggregation. Recipient cuvette contained 0.5 mM of indomethacin. For simplicity, transfer of material from D is not shown, as it was equivalent to A', for the first part of the figure. Scales as in Figure 1.
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mm
A- PLATELETS
SUSPENDED IN
TYRODE []
SOLUTION
RABBIT AORTk STRIP
[~RAT
STOMACH STRIP
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5 : Time-related degradation of rabbit aorta and rat stomach strip activities generated in platelets suspended in plasma and in Tyrode solution.
Columns indicate the height of contraction of the assay tissues at different intervals and incubation temperatures due to incubates of arachidonic acid with platelets suspended in Tyrode solution (panel A) or in plasma (panel B).
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PROSTAGLANDINS
B RAIIIT AORTA =TRIP % POT[ NTIATION
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Figure 6: Interference by CuCI 2 with inhibition by diethyldithiocarbamate and by pyrogallol of the seneration of pharmacological activity in incubates of arachidonic acid with platelet rich plasma. Vertical columns indicate, for each assay tissue, the percent inhibition of the contractions due to incubates of AA with PRP in the presence of inhibitors, and the percent potentiation of these contractions when CuCI 2 was added.
154
OCTOBER 25, 1974
VOL. 8 NO. 2
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OCTOBER 25, 1974
VOL. 8 NO. 2
155
PROSTAGLANDINS
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156
O C T O B E R 25, 1974
s,3
VOL. 8 NO. 2
V a r g a f t i g , Y. the t e c h n i c a l MERRELL
T r a n i e r and M. C h i g n a r d c o l l a b o r a t i o n of J. K i n t z
ACID-INDUCED INFLAMMATORY
and M.L.
Part
INTERNATIONAL RESEARCH CENTER 16, rue d ' A n k a r a 67000 S T R A S B O U R G , F r a n c e
ABSTRACT Sulfhydryl agents (mercaptoethanol, thioglycerol, dithiot r e i t o l , and s o d i u m d i e t h y l d l t h i o c a r b a m a t e ) prevented a g g r e g a t i o n of r a b b i t p l a t e l e t s and the a c c o m p a n y i n g g e n e r a t i o n of p h a r m a c o l o g i c a l l y a c t i v e s u b s t a n c e s due to arac h i d o n i c acid. I n h i b i t i o n was also found a f t e r in vivo a d m i n i s t r a t i o n of the a n t a g o n i s t s . This a n t a g o n i s m was s u p p r e s s e d if the i n h i b i t o r s w e r e r e m o v e d f r o m the p l a t e l e t s u s p e n s i o n by w a s h i n g p r o c e d u r e s , w h e r e a s i n h i b i t i o n by i n d o m e t h a c i n was i r r e v e r s i b l e . A m i n o - t h i o l r e a g e n t s e i t h e r f a i l e d to a n t a g o n i z e the e f f e c t s of AA or p o t e n t i a t e d them. CuCI 2 i n c r e a s e d the a m o u n t s of p h a r m a c o l o g i c a l l y a c t i v e s u b s t a n c e s g e n e r a t e d in i n c u b a t e s of intact p l a t e lets w i t h a r a c h i d o n i c acid, and r e v e r s e d the i n h i b i t i o n due to thiol agents, but did not i n t e r f e r e w i t h i n h i b i t i o n by i n d o m e t h a c i n . P l a t e l e t s s u s p e n d e d in T y r o d e s o l u t i o n generated unstable pharmacologically active substances u p o n i n c u b a t i o n w i t h a r a c h i d o n i c acid ; s t a b i l i t y of these s u b s t a n c e s c o u l d be m a i n t a i n e d at 4°C. G e n e r a t i o n of this t e m p e r a t u r e - s e n s i t i v e m a t e r i a l was i n h i b i t e d by i n d o m e t h a c i n and by thiol agents. I n t e r f e r e n c e w i t h a c o p p e r c o n t a i n i n g c o m p o n e n t of PG s y n t h e t a s e or r e d u c t i o n of an i n t e r m e d i a t e l i p o p e r o x i d e a p p e a r as two p o s s i b l e m e c h a n i s m s of a c t i o n of thiol agents.
~ccepted
September
7,
1974.
PROSTAGLANDINS OCTOBER 25, 1974
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133
PROSTAGLANDINS
INTRODUCTION Platelet a g g r e g a t i o n and the a c c o m p a n y i n g release of p h a r m a c o l o g i c a l l y active substances due to a r a c h i d o n i c acid +(AA) are blocked by n o n s t e r o i d a n t i i n f l a m m a t o r y drugs (AID) (],2,3). A r a c h l d o n i c acid is the p r e c u r s o r of prostaglandins E 2 and F2~ (PGE 2 and PGF2~) ; as p r o s t a g l a n d i n s do not a g g r e g a t e p l a t e l e t s (4), the h y p o t h e s i s was raised that an i n t e r m e d i a t e is r e s p o n s i b l e for the platelet effect; the cyclic e n d o p e r o x i d e i n t e r m e d i a t e s formed during bioc o n v e r s i o n of AA into PG have in fact been suggested to be a g g r e g a n t a g e n t s ( 1 , 3 , 5 ~ 6 , 7 ) . These i n t e r m e d i a t e s are supposed to account at least in part for the c o n t r a c t i o n s of the isolated rabbit aorta strip, induced by incubates of blood or of platelet s u s p e n s i o n s with AA or with slow reacting substance C (SRS-C), an incubate of egg yolk with p h o s p h o lipase A 2 (8,9) that shares in vivo p h a r m a c o l o g i c a l activities with AA (9,|0,11). The m a t e r i a l g e n e r a t e d in incubates of platelets with AA or with SRS-C was named rabbit aorta c o n t r a c t i n g substance (RCS) and can be also obtained from g u i n e a - p i g lungs during a n a p h y l a c t i c shock (12), or injected with AA or SRS-C (13). Blockade by sulfhydryl agents of the in vivo effects a t t r i b u t e d to a c t i v a t i o n of PG s y n t h e t a s e that f o l l o w the a d m i n i s t r a t i o n of AA and of SRS-C (11) lead us now to study the i n t e r a c t i o n of those sulfhydryl agents with the g e n e r a t i o n of p h a r m a c o l o g i c a l l y active substances in incubates of p l a t e l e t s and AA. It was d e m o n s t r a t e d that those thlol agents that prevent the in vivo effects of AA and of SRS-C interfere with the g e n e r a t i o n of p h a r m a c o l o g i c a l l y active substances in incubates of PRP with AA and prevent AA induced platelet aggregation. The m e c h a n i s m of such an i n h i b i t i o n may involve r e d u c t i o n of the l l p o p e r o x i d e i n t e r m e d i a t e to inactive h y d r o x y - f a t t y acids, or i n t e r a c t i o n with a metal c o m p o n e n t of the PG s y n t h e t a s e complex.
+ A b b r e v i a t i o n s used : AA, a r a c h i d o n i c acid; PGE 2 and PGF2~ , r e s p e c t i v e l y prostaglandins E 2 and F2~; RCS, rabbit aorta c o n t r a c t i n g substance; SRS-C, slow reacting substance C; 5HT, 5 - h y d r o x y tryptamine c r e a t i n i n e sulfate (serotonin); AID, non steroid acidic a n t i i n f l a m m a t o r y drugs; ME, 2 - m e r e a p t o e t h a n o l ; TG, 2 - t h i o g l y c e r o l ; DTT, d i t h i o t h r e i t o l ; DEDTC, sodium d i e t h y l d i t h i o c a r b a m a t e ; GSH, reduced g l u t a t h i o n e ; EGTA, ethyleneglycol bis (amino-2 ethylether) N, N - t e t r a c e t a t e , d i s o d i u m salt; PRP and PPP, r e s p e c t i v e l y platelet rich and platelet poor plasma; O.D., optical d e n s i t y
134
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MATERIALS
AND M E T H O D S
Platelet a~re~ation B l o o d was c o l l e c t e d f r o m the c e n t r a l ear a r t e r y of u n a n a e s t h e t i z e d r a b b i t s d i r e c t l y into p l a s t i c tubes c o n t a i n i n g a 4 % w/v s o d i u m c i t r a t e s o l u t i o n (9 ml of b l o o d and | ml of s o d i u m c i t r a t e ) . A f t e r c e n t r i f u g a t i o n at r o o m t e m p e r a t u r e (220 g for |2 mln.) the tubes w e r e left for one h o u r on the bench. P l a t e l e t r i c h p l a s m a (PRP) was c o l l e c t e d and k e p t at r o o m t e m p e r a t u r e for the r e m a i n d e r of the day. A g g r e g a t i o n was s t u d i e d on two B r y s t o n a g g r e g o m e t e r s , w i t h t e m p e r a t u r e k e p t at 37°C. P l a t e l e t c o u n t s w e r e p e r f o r m e ~ m i c r o s c o p i c a l ly and c o n c e n t r a t i o n a d j u s t e d to 3 O O , O O O / m m with equal v o l u m e s of p l a s m a and n o n - p y r o g e n i c 0.9 % NaCI. S t i r r i n g was of I|00 rpm. A f t e r ]-3 m i n u t e s a l l o w e d to a d j u s t the t e m p e r a t u r e of the sample, AA (0.02 to 0.5 mM) was added. P o t e n t i a l a n t a g o n i s t s w e r e i n c u b a t e d w i t h PRP for one m i n u t e b e f o r e a d d i t i o n of AA. All a d d i t i o n s w e r e of |0-50 ~I. Washed platelets PRP was c e n t r i f u g e d for 15 m i n u t e s at 720 g. The p e l l e t was w a s h e d twice w i t h T y r o d e s o l u t i o n w i t h the f o l l o w i n g c o m p o s i t i o n ( g / l ) : N a C i , 8 ; KCI, 0.2; KH 9 PO&, 0.05; Mg Clp 6 H20, 0.]; g l u c o s e , I; N a H C O ~ , 0.035. F i r s t - w a s h i n g s o l u t i o n ( T y r o d e A) c o n t a i n e d also EGTA, 0 . 0 7 6 g/l, and b o v i n e s e r u m a l b u m i n , 3.5 g/l; s e c o n d w a s h i n g s o l u t i o n (Tyrode B) c o n tained no EGTA, w h e r e a s third w a s h i n g s o l u t i o n (Tyrode C) c o n t a i n e d no E G T A or a l b u m i n , CaCI 2 (0.2 g/l) b e i n g added. The p e l l e t o b t a i n e d f r o m PRP was r e s u s p e n d e d g e n t l y in T y r o d e A; a new p e l l e t was r e c o v e r e d a f t e r c e n t r l f u g a t i o n (15 m i n u t e s at 580 g), and r e s u s p e n d e d in T y r o d e B. A new c e n t r i f u g a t i o n (15 m i n u t e s at 470 g) a l l o w e d to r e c o v e r a final p e l l e t w h i c h was r e s u s p e n d e d in T y r o d e C in h a l f the o r i g i n a l v o l u m e of PRP ( 1 4 , ] 5 , ] 6 ) . W a s h e d p l a t e l e t s w e r e u s u a l l y k e p t at 37°C and gave r e p r o d u c i b l e r e s u l t s w h e n t e s t e d f r o m one to five a f t e r final s u s p e n s i o n . D o n o r and r e c i p i e n t c u v e t t e s One h y p o t h e s i s we a i m e d to c h e c k was that a p r o - a g g r e g a n t m a t e r i a l was f o r m e d from AA, w h e n a d d e d to p l a t e l e t s . A s y s t e m was thus d e v i s e d to d i s c r i m i n a t e b e t w e e n the d i r e c t e f f e c t of AA, and that of r e l e a s e d a g g r e g a n t m a t e r i a l s . In this system, p l a t e l e t s a m p l e s w e r e c o l l e c t e d f r o m the c u v e t t e s d u r i n g a g g r e g a t i o n due to ADP or to AA, w i t h and w i t h o u t a n t a g o n i s t s , and w e r e added (0.05 to 0.2 ml) to a n o t h e r s a m p l e of PRP, p r e p a r e d in a s e c o n d a g g r e g o m e t e r and c o n t a i n i n g e n o u g h i n d o m e t h a c i n (0.5 mM) to b l o c k c o m p l e t e l y any e f f e c t of AA. It was e x p e c t e d that a g g r e g a t i o n in the r e c i p i e n t c u v e t t e w o u l d thus not be due to a " p a r a s i t i c " e f f e c t of AA, t r a n s f e r r e d f r o m the d o n o r cuvette.
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I n t e r f e r e n c e of 5 - h y d r o x y t r y p t a m i n e (5 HT), k n o w n to be r e l e a s e d by AA f r o m p l a t e l e t s (17), was d i s c a r d e d by the use of m e t h y s e r g i d e , a r e c o g n i z e d i n h i b i t o r of 5 H T - i n d u c e d a g g r e g a t i o n (18), a d d e d e i t h e r to d o n o r or to r e c i p i e n t c u v e t t e s b e f o r e AA. Superfusion experiments I n c u b a t e s of AA and of p l a t e l e t p r e p a r a t i o n s were t e s t e d w i t h the s u p e r f u s i o n t e c h n i q u e (12,13). A r a b b i t a o r t a strip, to d e t e c t RCS, and a rat s t o m a c h strip to d e t e c t PGlike a c t i v i t y , w e r e s u p e r f u s e d w i t h K r e b s s o l u t i o n c o n t a i n ing a m i x t u r e of a n t a g o n i s t s to p r e v e n t any e f f e c t of c a t e c h o l a m i n e s , a c e t y l c h o l i n e , s e r o t o n i n and h i s t a m i n e . In a few e x p e r i m e n t s the rat c o l o n was a d d e d as a third tissue. The a s s a y o r g a n s w e r e s t i m u l a t e d w i t h PGE 2 and P G F 2 e (|OIOOng) to c o n t r a c t the g a s t r o - i n t e s t i n a l t i s s u e s , and w i t h n o r e p i n e p h r i n e (20-250 ng) to c o n t r a c t the r a b b i t a o r t a strip. The c o m p o s i t i o n of the i n c u b a t e s was i d e n t i c a l to those used for a g g r e g a t i o n . E x p e r i m e n t s w e r e run at r o o m t e m p e r a t u r e w i t h s t i r r i n g at I]OO rpm. In vivo e x p e r i m e n t s The i n h i b i t o r s of A A - i n d u c e d p l a t e l e t a g g r e g a t i o n w e r e inj e c t e d by the i n t r a v e n o u s r o u t e to r a b b i t s . S a m p l e s of b l o o d w e r e c o l l e c t e d b e f o r e and five m i n u t e s after t r e a t ment; p l a t e l e t rich p l a s m a s a m p l e s w e r e used as such or as w a s h e d p l a t e l e t s s u s p e n d e d in T y r o d e s o l u t i o n or in p l a s m a o b t a i n e d f r o m b l o o d c o l l e c t e d r e s p e c t i v e l y b e f o r e or a f t e r i n t r a v e n o u s t r e a t m e n t . T h e s e m a t e r i a l s were s t u d i e d in the a g g r e g o m e t e r , a f t e r w h i c h the c u v e t t e s w e r e t e s t e d on the isolated tissues. Druss The f o l l o w i n g r e a g e n t s w e r e p u r c h a s e d : p h e n o x y b e n z a m i n e h y d r o c h l o r i d e (Smith, K l i n e and F r e n c h , U . S . A . ) ; m e p y r a m i ne m a l e a t e ( R h S n e - P o u l e n c , F r a n c e ) ; a t r o p i n e s u l f a t e (Laboratolres Bruneau, France); propranolol hydrochloride ( A v l o c a r d y l R, L a b o r a t o i r e s A v l o n , F r a n c e ) ; 2 m e r c a p t o e t h a nol, t h i o g l y c e r o l , and r e d u c e d g l u t a t h i o n e (Fluka, S w i t z e r land); s o d i u m diethyldithiocarbamate ( S c h u c h a r d t , G e r m a n y ) ; d i t h i o t h r e i t o l ( C a l b i o c h e m . U . S . A . ) ; D L - p e n i c i l l a m i n e , tetraethylthiuram disulfide (disulflram), N-acetyl-L-cysteine, n o r e p i n e p h r i n e and 5 - h y d r o x y t r y p t a m i n e (serotonin) (Sigma, U . S . A . ) ; i n d o m e t h a c i n (Merck, Sharp and Dohme, U . S . A . ) ; and m e t h y s e r g i d e m a l e a t e (Sandoz, S w i t z e r l a n d ) ; salts for s o l u t i o n s w e r e p u r c h a s e d from u s u a l c o m m e r c i a l s o u r c e s . D r u g s i n s o l u b l e in 0.9 % NaCI w e r e s o l u b i l i z e d by a d d i n g g r a d e d a m o u n t s of 0.2 N N a O H or HCI. T e t r a e t h y l t h i u r a m d i s u l f i d e was s o l u b i l i z e d in p o l y e t h y l e n e g l y c o l 300 (Carl R o t h OHG), w h i c h had no p h a r m a c o l o g i c a l e f f e c t of its own on r a b b i t PRP. A r a c h i d o n i c acid (Sigma) was p r e p a red as d e s c r i b e d (|3).
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VOL. 8 NO. 2
PROSTAGLANDINS
RESULTS
E f f e c t of a r a c h i d o n i c acid on p l a t e l e t s A d d i t i o n of AA ( 0 . 0 5 - 0 . 5 mM) to r a b b i t PRP lead i n i t i a l l y to an i n c r e a s e in O.D., i n d i c a t i n g a p l a t e l e t s h a p e c h a n g e , f o l l o w e d by a d e c r e a s e in O.D., and by p l a t e l e t a g g r e g a t i o n . S a m p l e s of PRP w e r e c o l l e c t e d at d i f f e r e n t time i n t e r v a l s a f t e r a d d i t i o n of AA, and a d d e d to r e c i p i e n t c u v e t t e s , w i t h r e s u l t a n t a g g r e g a t i o n of the r e c i p i e n t p l a t e l e t s . T r a n s f e r able a c t i v i t y was not due to c o n t a m i n a t i o n by the t r a n s f e r red AA as the r e c i p i e n t c u v e t t e c o n t a i n e d e n o u g h i n d o m e t h a cin to p r e v e n t its e f f e c t s . M a x i m u m a g g r e g a t i n g a c t i v i t y was o b t a i n e d w h e n s a m p l i n g was p e r f o r m e d 2-5 min. a f t e r add i t i o n of AA to the d o n o r c u v e t t e . T r a n s f e r a b l e a c t i v i t y was o n l y p r e s e n t w h e n a g g r e g a t i o n was u n d e r way; s a m p l e s c o l l e c t e d one m i n u t e a f t e r a d d i t i o n of AA to the d o n o r cuv e t t e d i s p l a y e d f e e b l e t r a n s f e r a b l e a c t i v i t y , w h e r e a s in s a m p l e s c o l l e c t e d a f t e r IO or 20 m i n u t e s of s t i r r i n g w i t h AA the t r a n s f e r a b l e a g g r e g a t i n g a c t i v i t y d i s a p p e a r e d . W h e n ADP was a d d e d to a d o n o r PRP in p l a c e of AA and s a m p l e s w e r e t r a n s f e r r e d from it to a r e c i p i e n t c u v e t t e , in o r d e r to i m m i t a t e w i t h e x o g e n o u s ADP w h a t o c c u r s w h e n AA is used, a 1 0 0 - 2 5 0 fold h i g h e r m o l a r c o n c e n t r a t i o n of AA as c o m p a r e d to that of ADP had to be a d d e d to a d o n o r c u v e t t e , to o b t a i n , in the r e c i p i e n t one, a c o m p a r a b l e a g g r e g a t i o n ( f i g u r e I). A l t h o u g h the t e n d e n c y of d o n o r PRP s a m p l e s c h a l l e n g e d w i t h ADP was to r e t a i n the t r a n s f e r a b l e a c t i v i t y for l o n g e r p e r i o d s of time than did PRP c h a l l e n g e d w i t h AA, no c l e a r d i f f e r e n c e in t i m e - r e l a t e d s t a b i l i t y c o u l d be d e m o n s t r a t e d w i t h this p r o c e d u r e b e t w e e n t r a n s f e r a b l e agg r e g a n t a c t i v i t y o b t a i n e d from AA or A D P - a g g r e g a t e d platelets. F i g u r e l(panel B) also shows that 0 . 5 m M of AA was less p o t e n t in i n d u c i n g a g g r e g a t i o n than 0.! mM. This was p o s s i b l y c a u s e d by over p r o d u c t i o n of PGE2, l i k e l y to a n t a ~ o n l z e a g g r e g a t i o n if p r e s e n t in a p p r o p r i a t e a m o u n t s . I n t e r f e r e n c e of thiol and o t h e r a s e n t s w i t h a r a c h i d o n i c acid-induced platelet a$$resation. P y r o g a l l o l , D E D T C , TG, ME, and DTT s u p p r e s s e d A A - i n d u c e d platelet aggregation ( f i g u r e 2 and T a b l e I). At c o n c e n t r a tions e f f e c t i v e a g a i n s t AA no b l o c k a d e was o b s e r v e d t o w a r d s ADP. S o d i u m t h i o g l y c o l a t e , DL-penicillamine and GSH at 1 mM r e d u c e d ADP and AA i n d u c e d a g g r e g a t i o n , w h e r e a s no b l o c k a d e was o b s e r v e d w i t h L - c y s t e i n e and N - a c e t y l - L - c y s t e ine. W h e n c o n c e n t r a t i o n s of AA b e l o w t h r e s h o l d for a g g r e g a tion ( 0 . 0 2 - 0 . 0 5 mM) w e r e p r e c e d e d or f o l l o w e d at a short i n t e r v a l by O.5-! m M of L - c y s t e i n e , a g g r e g a t i o n was l a u n c h e d and e v e n t u a l l y was total. No such e f f e c t was o b s e r v e d for ADP. A f t e r i n c u b a t i o n of r a b b i t PRP w i t h p y r o g a l l o l , D T T , T G or D E D T C (I mM) for 10 m i n u t e s , f o l l o wed by w a s h i n g and r e s u s p e n s i o n in PPP, a g g r e g a t i o n due to
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AA was not b l o c k e d . If t h e s e t r e a t e d p l a t e l e t s w e r e s u s p e n d e d in PPP c o n t a i n i n g the p o t e n t i a l a n t a g o n i s t s ( c o l l e c t e d from the i n i t i a l i n c u b a t e of PRP b e f o r e w a s h i n g ) , a g g r e g a t i o n was c o m p l e t e l y s u p p r e s s e d . On the o t h e r hand, w h e n i n d o m e t h a c i n (0.05 mM) was i n c u b a t e d w i t h PRP, f o l l o w ed by w a s h i n g and r e s u s p e n s i o n of p l a t e l e t s in d r u g - f r e e PPP, a g g r e g a t i o n was b l o c k e d , i n d i c a t i n g an i r r e v e r s i b l e e f f e c t on the p l a t e l e t s ; no b l o c k a d e was o b s e r v e d if these p r e t r e a t e d p l a t e l e t s w e r e r e s u s p e n d e d in T y r o d e s o l u t i o n (figure 3). B l o c k a d e of a g g r e g a t i o n by s u l f h y d r y l a g e n t s or by i n d o m e t h a c i n r e s u l t e d also in b l o c k a d e of the r e l e a s e or g e n e r a t i o n of t r a n s f e r a b l e a c t i v i t y (figure 4). A d d i t i o n of s u l f h y d r y l a g e n t s to the r e c i p i e n t c u v e t t e did not prev e n t the d e t e c t i o n of the t r a n s f e r a b l e a c t i v i t y o r i g i n a t i n g from an A A - c h a l l e n g e d s a m p l e of r a b b i t PRP. W a s h e d p l a t e lets s u s p e n d e d in T y r o d e s o l u t i o n w e r e not p r o t e c t e d from AA by s u l f h y d r y l a g e n t s or i n d o m e t h a c i n ; a d d i t i o n of PPP i n s u r e d b l o c k a d e s i m i l a r to that seen on PRP ( f i g u r e 3). The i n h i b i t o r y a c t i v i t y of i n d o m e t h a c i n or of thiol a g e n t s on A A - i n d u c e d p l a t e l e t a g g r e g a t i o n is thus only u n c o v e r e d if p l a s m a (or serum) is p r e s e n t . E G T A p r e v e n t e d A A - i n d u c e d a g g r e g a t i o n and the d e t e c t i o n of t r a n s f e r a b l e a g g r e g a n t a c t i v i t y . Both e f f e c t s w e r e o v e r c o m e by e q u i m o l a r CaCI 2 (figure 4). M e t h y s e r g i d e (up to 2 ~ g / m l of PRP) did not a f f e c t A A - i n d u c e d p l a t e l e t a g g r e g a t i o n or p r e v e n t d e t e c t i o n of t r a n s f e r a b l e a c t i v i t y w h e n p r e s e n t in the r e c i p i e n t cuvette. R e l e a s e of p h a r m a c o l o g i c a l l y a c t i v e s u b s t a n c e s f r o m i n c u b a tes of a r a c h i d o n i c acid w i t h p l a t e l e t p r e p a r a t i o n s . I n c u b a t i o n of PRP w i t h AA is f o l l o w e d by the g e n e r a t i o n of RCS and of P G - l i k e s u b s t a n c e s . (I-6, 19-21). As A A - i n d u c e d a g g r e g a t i o n of p l a t e l e t s s u s p e n d e d in T y r o d e s o l u t i o n was not b l o c k e d by s u l f h y d r y l a g e n t s or by i n d o m e t h a c i n , we i n v e s t i g a t e d w h e t h e r p l a s m a was also r e q u i r e d in order to d e m o n s t r a t e the i n h i b i t o r y e f f e c t s of the thiol s u b s t a n c e s on g e n e r a t i o n of p h a r m a c o l o g i c a l l y a c t i v e s u b s t a n c e s . T h e s e e x p e r i m e n t s w e r e not e n t i r e l y l e a s a b l e , b e c a u s e p l a t e l e t s s u s p e n d e d in T y r o d e s o l u t i o n and kept at r o o m t e m p e r a t u r e or at 37°C only g e n e r a t e d RCS for the initial 10-30 m i n u tes a f t e r b e i n g s u s p e n d e d ; p r e p a r a t i o n s kept for l o n g e r p e r i o d s g e n e r a t e d the rat s t o m a c h a c t i v i t y , w h e t h e r p l a s m a was p r e s e n t or not. It was h y p o t h e s i z e d that this e f f e c t m i g h t be due to r a p i d d e g r a d a t i o n of RCS at r o o m t e m p e r a ture i m m e d i a t l y u p o n f o r m a t i o n ; in o r d e r to c h e c k it, i n c u b a t i o n s of AA w i t h PRP or w i t h p l a t e l e t s s u s p e n d e d in T y r o d e s o l u t i o n w e r e p e r f o r m e d at 4°C. R e s u l t s s h o w n on f i g u r e 5 A and 5 B d e m o n s t r a t e that p l a t e l e t s s u s p e n d e d in T y r o d e s o l u t i o n at 37°C gave a v e r y low y i e l d of c o n t r a c ting a c t i v i t i e s over the rat s t o m a c h and the r a b b i t a o r t a strips, w h e r e a s w h e n i n c u b a t i o n s w e r e c a r r i e d at 4=C the a c t i v i t y was p r e s e n t and r e l a t i v e l y stable up to t h i r t y
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m i n u t e s . T i m e - r e l a t e d o b s e r v a t i o n s s h o w n in these f i g u r e s are f u r t h e r m e n t i o n e d in the d i s c u s s i o n . S u l f h y d r y l a g e n t s or i n d o m e t h a c i n added to the i n c u b a t e s of AA w i t h p l a t e l e t s at 4°C d i s p l a y e d their e x p e c t e d i n h i b i t o r y p r o p e r t i e s . Thus, c o n t r a s t i n g w i t h p h o t o m e t r i c o b s e r v a t i o n s , w h e n the a n t a g o n i s t s o n l y are e f f e c t i v e in the p r e s e n c e of plasma, the latter is not r e q u i r e d to i n h i b i t g e n e r a t i o n of RCS and PGlike a c t i v i t i e s . W h e n i n c u b a t i o n s w e r e p e r f o r m e d in p r e s e n ce of p l a s m a the y i e l d s of c o n t r a c t i n g m a t e r i a l s w e r e h i g h e r for 37°C than for 4°C and d e g r a d a t i o n w i t h time less steep. I n t e r f e r e n c e of s u l f h y d r ~ l a g e n t s w i t h the r e l e a s e of p h a r m a c o l o ~ i c a l l y a c t i v e s u b s t a n c e s in i n c u b a t e s of r a b b i t p l a t e l e t s w i t h a r a c h i d o n i c acid. P y r o g a l l o l , DTT, DEDTC, TG, ME and TG, m i x e d at a I mM c o n c e n t r a t i o n w i t h PGE 2 or w i t h PGF2~, did not r e d u c e by m o r e than 10 % the rat s t o m a c h strip c o n t r a c t i o n s . N o t w i t h s t a n d i n g , a d d e d to PRP one m i n u t e b e f o r e AA, the i n h i b i t o r s b l o c k e d the e x p e c t e d c o n t r a c t i o n s of the r a b b i t a o r t a and of the rat s t o m a c h strips. The r e s p o n s e s of the f o r m e r w e r e p r e v e n t e d at lower c o n c e n t r a t i o n s of the inhib i t o r s as c o m p a r e d to the l a t t e r (Table I). In c o n t r a s t , the a m i n o - t h i o l s GSH, D L - p e n i c i l l a m i n e and L - c y s t e i n e , p o t e n t i a t e d the t i s s u e r e s p o n s e s . Thus, in two e x p e r i m e n t s L - c y s t e i n e (I mM) p o t e n t i a t e d the r e s p o n s e of the aorta of 233 and 246 % and the r e s p o n s e of the s t o m a c h of 48 and 47 % ; D L - p e n i c i l l a m i n e (0.5 mM) p o t e n t i a t e d the a o r t a r e s p o n s e of 26 % and 69 %, and the s t o m a c h of 18 % ; GSH (1 mM) p o t e n t i a t e d the a o r t a of 24 % and the s t o m a c h of 15 %. E G T A (up to 5 mM) did not i n h i b i t the g e n e r a t i o n of P G - l i k e or of RCS a c t i v i t i e s in r a b b i t PRP i n c u b a t e d w i t h AA. I n t e r f e r e n c e of c o p p e r w i t h the g e n e r a t i o n of p h a r m a c o l o ~ically active substances from platelets. In o r d e r to c h e c k w h e r e a s the m e c h a n i s m of a c t i o n of the thiol a g e n t s i n v o l v e d i n t e r f e r e n c e w i t h c o p p e r ions, a few i n h i b i t o r s w e r e tested in its p r e s e n c e . A d d i t i o n of C u C I 2 (I mM) to p l a t e l e t i n c u b a t e s , f o l l o w e d after one m i n u t e by a d d i t i o n of AA, and by b i o a s s a y of the r e s u l t i n g i n c u b a t e ~ showed that the r e s p o n s e s of the r a b b i t a o r t a strip i n c r e a s e d by 101,88 ~ 26,6 %, w h e r e a s the r e s p o n s e s of the rat s t o m a c h w e r e i n c r e a s e d by 36.7 + 10.5 %, as c o m p a r e d to c o n t r o l i n c u b a t e s w i t h o u t CuCI2. The i n h i b i t o r y e f f e c t s of p y r o g a l l o l and of D E D T C w e r e r e v e r t e d by e q u i m o l a r CuCI2, w h e r e a s lower c o n c e n t r a t i o n s of the l a t t e r did not p r e v e n t i n h i b i t i o n of the g e n e r a t i o n of p h a r m a c o l o g i c a l l y a c t i v e m a t e r i a l s in the i n c u b a t e s ( f i g u r e 6 A and 6 B). The i n h i b i t o r y a c t i v i t y of i n d o m e t h a c i n was not p r e v e n t e d by CuCI 2 up to ] mM.
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Table Inhibition
of A r a c h i d o n i c
Generation
of RCS
Rabbit
I
Acid and
Platelet
Induced
PG-like Rich IC
Platelet Aggregation
Antagonist
Diethyldithiocarbamate
Aggregation
Activities
and
in
Plasma 5O
(uM) *
C o n t r a c t i o n s of Rabbit Aorta Rat S t o m a c h Strip Strip
53
76
330
Mercaptoethanol
150
130
300
Thioglycerol
280
300
560
Dithiothreitol
2]0
130
630
DL P e n i c i l l a m i n e
540
~
~
Pyrogallol Indomethacin
20
57
260
3.7
2.5
11
IC 50 : c o n c e n t r a t i o n s of d r u g s (in ~M) r e q u i r e d to i n h i b i t 50 % p l a t e l e t a g g r e g a t i o n or g e n e r a t i o n of pharmacologically a c t i v e s u b s t a n c e s o b t a i n e d by i n t e r p o l a t i o n of 3-4 r e s u l t s . z~
potentiates
generation
of RCS
and
PG-like
activities.
B l o c k a d e of a r a c h i d o n i c a c i d - i n d u c e d p l a t e l e t a $ $ r e s a t i o n and r e l e a s e of m e d i a t o r s a f t e r in v i v o a d m i n i s t r a t i o n of druss. I n d o m e t h a c i n was i n t r a v e n o u s l y a d m i n i s t e r e d to two r a b b i t s at 5 m g / k g . S a m p l e s of b l o o d w e r e c o l l e c t e d b e f o r e and a f t e r t r e a t m e n t ~ and p l a t e l e t r e s p o n s e s w e r e s t u d i e d in PRP, in w a s h e d p l a t e l e t s r e s u s p e n d e d in T y r o d e s o l u t i o n , or in p l a s m a c o l l e c t e d b e f o r e and a f t e r i n d o m e t h a c i n t r e a t m e n t . PRP c o l l e c t e d a f t e r i n d o m e t h a c i n did not respond any m o r e to 0.5 m M of AA, as did the s a m p l e s c o l l e c t ed b e f o r e t r e a t m e n t , w h e r e a s r e s p o n s e s to ADP were u n a f fected. W a s h e d p l a t e l e t s from a b l o o d s a m p l e c o l l e c t e d af t e r i n d o m e t h a c i n w e r e also u n r e s p o n s i v e to AA, even if r e s u s p e n d e d in p l a s m a f r o m s a m p l e s o b t a i n e d b e f o r e i n j e c tion of i n d o m e t h a c i n ; t h e s e same p l a t e l e t s , r e s u s p e n d e d
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in T y r o d e s o l u t i o n , a g g r e g a t e d to AA d e s p i t e p r e v i o u s indomethacin treatment. Washed platelets collected before i n d o m e t h a c i n t r e a t m e n t but r e s u s p e n d e d in p l a s m a f r o m b l o o d c o l l e c t e d a f t e r i n d o m e t h a c i n , thus c o n t a i n i n g the a n t i - i n f l a m m a t o r y agent, w e r e also b l o c k e d to AA w h e r e a s r e s p o n s e s to ADP w e r e u n a f f e c t e d . A g g r e g a t i o n and r e l e a s e of m e d i a t o r s t r i g g e r e d by AA in PRP c o l l e c t e d a f t e r i n t r a v e n o u s TG (200 m g / k g ) , D E D T C (I00 m g / k g ) and p y r o g a l l o l (20 m g / k g ) w e r e i n h i b i t e d . W a s h e d p l a t e l e t s from b l o o d s a m p l e s c o l l e c t e d a f t e r a d m i n i s t r a t i o n of i n h i b i tors and r e s u s p e n d e d in p l a s m a from b e f o r e the t r e a t m e n t r e s p o n d e d to AA, w h e r e a s the same p l a t e l e t s r e s u s p e n d e d in p l a s m a f r o m after t r e a t m e n t w e r e u n r e s p o n s i v e to AA. No r e s p o n s e of the i s o l a t e d o r g a n s w e r e o b t a i n e d f r o m i n c u b a t e s of AA w i t h PRP c o l l e c t e d f r o m a n i m a l s t r e a t e d w i t h the above m e n t i o n e d d o s e s of TG, D E D T C or p y r o g a l l o l . This b l o c k a d e was s u p p r e s s e d by w a s h i n g the p l a t e l e t s and r e s u s p e n d i n g in T y r o d e s o l u t i o n or in d r u g - f r e e p l a s m a ; w a s h e d p l a t e l e t s r e s u s p e n d e d in p l a s m a c o l l e c t e d a f t e r drug t r e a t m e n t did not r e s p o n d to AA° Two e x p e r i m e n t a l r e s u l t s are d e p i c t e d in f i g u r e 7.
DISCUSSION
S u l f h y d r y l a g e n t s and p y r o g a l l o l have n o w b e e n s h o w n to i n h i b i t p l a t e l e t a g g r e g a t i o n and g e n e r a t i o n of RCS and P G - l i k e a c t i v i t i e s , w h e n added to PRP or a d m i n i s t e r e d to r a b b i t s at d o s e s that s u p p r e s s the h y p o t e n s i v e e f f e c t s of SRS-C (11). I n d o m e t h a c i n - l i k e d r u g s e x h i b i t the same a c t i v i t i e s . It thus seems l o g i c a l to a s s u m e that r e l e a s e of P G - l i k e and RCS a c t i v i t i e s , and the p h a r m a c o l o g i c a l e f f e c t s of AA and of SRS-C are r e l a t e d : we h y p o t h e s i z e that h y p o t e n s i o n and b r o n c h o c o n s t r l c t i o n in r a b b i t s , dogs and g u i n e a - p i g s are l a r g e l y due to the i n t e r a c t i o n of AA or of SRS-C w i t h p l a t e l e t PG s y n t h e t a s e w i t h release of s u b s t a n c e s d i s p l a y i n g RCS and P G - l i k e a c t i v i t i e s . The h y p o t h e s i s of a b a s i c role for p l a t e l e t s is s u p p o r t e d by the fa~t that w h e n AA or SRS-C are i n j e c t e d d i r e c t l y into a t u b i n g c a r r y i n g b l o o d p r o v i d e d by a dog onto s u p e r fused i s o l a t e d t i s s u e s , m a r k e d c o n t r a c t i o n s are o b t a i n e d , b l o c k e d by i n t r a v e n o u s AID (19), or thiol a g e n t s (21). O n l y b l o o d is i n v o l v e d , and lungs, a k n o w n s o u r c e of PG and RCS w h e n p e r f u s e d in v i t r o w i t h AA or SRS-C (13), are e x c l u d e d . M o r e o v e r , DEDTC, that s h a r e s the in v i v o inhib i t o r y p r o p e r t i e s w i t h the other thiol a g e n t s , was sole, a m o n g them, not to b l o c k the r e l e a s e of RCS by AA from i s o l a t e d g u i n e a - p i g lungs (II); n o t w i t h s t a n d i n g , D E D T C was e f f e c t i v e on p l a t e l e t s in v i v o and in vitro, i n d i c a ting a b e t t e r c o r r e l a t i o n of in v i v o i n h i b i t i o n w i t h a p l a t e l e t than w i t h a p u l m o n a r y site of action. A l t h o u g h
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PROSTAGLANDINS t a c h y p h y l a x i s m i g h t have b e e n e x p e c t e d u p o n in v i v o a d m i n i s t r a t i o n of h i g h a m o u n t s of AA, (22) r e p r o d u c i b l e h y p o t e n s i o n can be o b t a i n e d w i t h lower a m o u n t s (23) or w i t h SRS,C (13). This f u l l y a g r e e s w i t h a p l a t e l e t site of action, b e c a u s e A A - i n d u c e d a g g r e g a t i o n of r a b b i t p l a t e l e t s is r e v e r s i b l e , s p o n t a n e o u s l y or if a p p r o p r i a t e a n t a g o n i s t s (PGE I or PGE2, d i b u t y r y l c y c l i c AMP, EGTA, a p y r a s e ) are a d d e d a f t e r c o m p l e t i o n of a g g r e g a t i o n (1,5 ; and to be p u b l i s h e d ) . A u n i f y i n g t h e o r y for the m e c h a n i s m of a c t i o n of s u l f h y d r y l and a n t i - o x i d a n t a g e n t s should a c c o u n t for : a. A A - i n d u c e d p l a t e l e t a g g r e g a t i o n and g e n e r a t i o n of RCS and P G - l i k e a c t i v i t i e s are o n l y b l o c k e d in p r e s e n c e of thiol agents, w h e r e a s i n d o m e t h a c i n p r e v e n t s a g g r e g a t i o n also after the p l a t e l e t s h a v e b e e n w a s h e d and r e s u s p e n d e d in d r u g - f r e e plasma; b.
thiol a g e n t s b l o c k the c o n t r a c t i o n s of the r a b b i t a o r t a and of the rat s t o m a c h strips to p l a t e l e t incubates w i t h AA, w i t h o u t i n t e r f e r i n g w i t h the e f f e c t s of PGE 2 or PGF2~.
F r o m a it m a y be c o n c l u d e d that i n d o m e t h a c i n does not i n t e r a c t c h e m i c a l l y w i t h AA or w i t h the e x p e c t e d l i p o p e r o xide (s), as i n h i b i t i o n is r e t a i n e d after r e m o v a l of i n d o m e t h a c i n f r o m the p l a t e l e t e n v i r o n m e n t . This is not the case for thiol agents, w h i c h m i g h t i n t e r a c t w i t h AA or w i t h its p r o d u c t s . F r o m b it m a y be c o n c l u d e d that thiol a g e n t s (and also AID) did not p r e v e n t the d i r e c t e f f e c t s of p r o s t a g l a n d i n s , as has b e e n d e s c r i b e d by J o h n s o n , J e s s u p and R a m w e l l (24) in a n o t h e r system. The h y p o t h e s i z e d include : a.
mechanisms
of a c t i o n
of
thiol
agents
r e d u c t i o n of d i s u l p h i d e b o n d s of a c o m p o n e n t of the e n z y m e c o m p l e x d e a l i n g w i t h AA or of the r e c e p t o r i n v o l v e d w i t h its e f f e c t s . This m e c h a n i s m c a n n o t be ruled out, but w o u l d not e x p l a i n the f a i l u r e of a few thiol a g e n t s to b l o c k our systems. E v e n if f a i l u r e of GSH or c y s t e i n e is a c c o u n t e d for by their r a p i d a u t o o x i d a t i o n , this is not the case for p e n i c i l l a m i n e (25). D i s u l f i r a m and its r e d u c e d form, DEDTC, w e r e e f f e c t i v e at e q u i m o l a r c o n c e n t r a t i o n s . As the f o r m e r is c o n v e r t e d into the l a t t e r in p l a s m a (26) it is p r o b a b l e that the a c t i v e a n t a g o n i s t is the free thiol c o m p o u n d .
b. D i r e c t i n t e r a c t i o n of the i n h i b i t o r s w i t h the e n z y m e or w i t h a c o - f a c t o r . D i e t h y l d i t h i o c a r b a m a t e b l o c k s the v e s i c u l a r g l a n d e n z y m e r e s p o n s i b l e for o x y g e n a t i o n of AA (27). R e v e r s i b i l i t y was o b t a i n e d w i t h Cu ++, w h i c h could be due to r e m o v a l of D E D T C by the m e t a l and not to r e p l a c e m e n t of f u n c t i o n a l l y a c t i v e Cu ++ (see h y p o thesis c). Lee and L a n d s (28) h a v e also d e m o n s t r a t e d that in--the a b s e n c e of Cu ++, DTT i n h i b i t s the o x i d a t i o n of AA by PG s y n t h e t a s e , w h e r e a s the c o m p l e x of Cu ++
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w i t h D T T i n c r e a s e s the e x t e n t of s u b s t r a t e o x i d a t i o n and s h i f t s the s y n t h e s i s of P G E p to PGF2e. This is s i m i l a r to our r e s u l t s . E f f e c t i v e n e S s of th~ non c h e l a t o r mp h e n a n t h r o l i n e in i n h i b i t i n g the d i o x y g e n a s e (28) indic a t e s that o t h e r m e c h a n i s m than Cu ++ c o m p l e x a t i o n m a y be involved. c. All a g e n t s we u s e d are c o p p e r c h e l a t o r s , but no d i r e c t r e l a t i o n s h i p was found b e t w e e n the a b i l i t y to c o m p l e x Cu ++ and to b l o c k the e f f e c t s of AA. Thus p e n i c i l l a m i n e , E D T A and E G T A are e x p e c t e d to c o m p l e x all free copper, as their K 1 ( l o g a r i t h m of the s t a b i l i t y c o n s t a n t for Cu ++) is r e s p e c t i v e l y of 16,5, 18.8 and 17.7, but did not p r e v e n t g e n e r a t i o n of R C S - l i k e a c t i v i t y ; p y r o g a l l o l has lower c h e l a t i n g p r o p e r t i e s (K I = 12.4), and was the s t r o n g e s t a n t a g o n i s t of A A - i n d u c e d e f f e c t s . As 2,2d i p y r i d y l and 8 - h y d r o x y q u i n o l i n e , w h i c h h a v e no SH groups, also i n h i b i t e d the e f f e c t s of AA (to be p u b l i shed), it is p o s s i b l e that e n z y m e b o u n d c o p p e r d i s p l a y s a role in PG s y n t h e t a s e , b e i n g e i t h e r n e u t r a l i z e d by c h e l a t o r s as D E D T C or t u r n e d m o r e c h e m i c a l l y r e a c t i v e by a c o o p e r a t i v e e f f e c t (29), as m i g h t be the case for the a m i n o - t h i o l s . The role of Cu ++ is f u r t h e r m o r e supp o r t e d by the fact that it s t i m u l a t e s the p r o d u c t i o n of P G F 2 ~ in a n o t h e r s y s t e m (30); d. A n o t h e r p o s s i b l e m e c h a n i s m of a c t i o n of thiol and a n t i o x i d a n t s u b s t a n c e s c o n s i s t s in the r e d u c t i o n of the i n t e r m e d i a t e a c t i v e l i p o p e r o x i d e . This w o u l d e x p l a i n i n t e r f e r e n c e w i t h a g g r e g a t i o n , a t t r i b u t e d to the s h o r t l a s t i n g c y c l i c e n d o p e r o x i d e (1,6) and w i t h r a b b i t a o r t a c o n t r a c t i n g a c t i v i t y , if the l a t t e r is also due to l i p o p e r o x i d e s (31). Rabbit aorta contracting activity obtained from platelets, is not PGE 2 or PGF2~, w h i c h do not a f f e c t the aorta. P G F 2 ~ was p r o b a b l y o n l y p r e s e n t in low a m o u n t s in our AA i n c u b a tes, as the rat c o l o n c o n t r a c t e d f e e b l y w h e n c h a l l e n g e d w i t h them, a l t h o u g h it r e s p o n d e d to c o n c e n t r a t i o n s of P G F 2 e e q u i a c t i v e w i t h PGE 2 and w i t h the p l a t e l e t i n c u b a t e s w i t h r e s p e c t to the s t o m a c h strip. M o r e o v e r , PGE 2 r a t h e r than P G F 9 ~ is g e n e r a t e d by p l a t e l e t s d u r i n g a g g r e g a t i o n (20). F i ~ a l l y , the drop in rat s t o m a c h strip c o n t r a c t i n g a c t i v i t y in p r e s e n c e of thiol a g e n t s is c e r t a i n l y not a c c o u n t e d for by a shift f r o m PGE 2 to P G F g ~ p r o d u c t i o n due to a d d e d r e d u c i n g e q u i v a l e n t s , as the c o n t r a c t i o n s of the rat c o l o n w e r e not i n c r e a s e d , as c o u l d o c c u r in p r e s e n c e of h i g h e r a m o u n t s of P G F g ~ , but w e r e d e c r e a s e d . D i s a p p e a r a n c e w i t h time of the a ~ t i v i t y of i n c u b a t e s of AA w i t h p l a t e l e t s s u s p e n d e d in T y r o d e , b o t h w i t h r e s p e c t to rat s t o m a c h and r a b b i t a o r t a strip, i n d i c a t e s that the role of PGE 2 is small, if any, in these c o n d i t i o n s . If the rat s t o m a c h a c t i v i t y w o u l d be due to PGEg, it s h o u l d o n l y s l i g h t l y d e c a y d u r i n g t i m e - r e l a t e d i ~ c u b a t i o n s , or w h e n
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i n c u b a t e s at 4 ° C are t r a n s f e r r e d to 37°C~ It is thus p r o b a ble that p l a t e l e t s in T y r o d e s o l u t i o n p r e d o m i n a n t l y s y n t h e size s o m e t h i n g else, w h i c h a f f e c t s b o t h a s s a y t i s s u e s . H i g h e r y i e l d s at 4°C, as c o m p a r e d to 37°C, i n d i c a t e that the g e n e r a t e d m a t e r i a l is h i g h l y u n s t a b l e in T y r o d e s o l u t i o n , w h e r e a s s t a b i l i t y is h i g h e r in plasma, as are the y i e l d s at 37°C c o m p a r e d to 4°C. W h e n s a m p l e s are a l l o w e d to stand at r o o m t e m p e r a t u r e , the r a b b i t a o K t a a c t i v i t y d i s a p p e a r s t o g e t h e r w i t h part of the rat s t o m a c h a c t i v i t y , w h e r e a s a r e s i d u a l t i m e - r e s i s t a n t e f f e c t on the rat s t o m a c h is o b s e r ved, p r o b a b l y a c c o u n t e d for by PGE 2. That part of the rat s t o m a c h a c t i v i t y w h i c h d i s a p p e a r s c o n c u r r e n t l y w i t h loss of a c t i v i t y over the r a b b i t a o r t a strip, is thus p r o b a b l y due to RCS. If the r a b b i t a o r t a a c t i v i t y w o u l d p u r e l y be due to a P G - p r e c u r s o r , such as the c y c l i c e n d o p e r o x i d e , and the rat s t o m a c h a c t i v i t y due to PGE 2 o r i g i n a t i n g from it, as i n i t i a l l y t h o u g h t , a drop in the r a b b i t a o r t a a c t i v i t y should be a c c o m p a n i e d by i n c r e a s e d rat s t o m a c h strip a c t i vity. A s i m i l a r o b j e c t i o n a p p l i e s to the a c t i v i t i e s found in p l a t e l e t s s u s p e n d e d in T y r o d e s o l u t i o n and c h a l l e n g e d w i t h AA at r o o m t e m p e r a t u r e : d e g r a d a t i o n of RCS should be f o l l o w e d by i n c r e a s e d P G - l i k e g e n e r a t i o n , w h i c h was not the case. The h a l f - l i v e s of the c y c l i c e n d o p e r o x i d e s are l o n g e r (7) than those r e p o r t e d by us (13) for lung RCS ; our r e s u l t s now show that the a g g r e g a n t and t r a n s f e r a b l e m a t e rials g e n e r a t e d in p l a t e l e t i n c u b a t e s w i t h AA have a shorter life than RCS, w h e n p l a s m a is p r e s e n t , w h e r e a s its a c t i v i t y d e c a y s p r e c i p i t o u s l y if p l a s m a is o m i t t e d . A " l a b i l e a g g r e g a t i o n s t i m u l a t i n g s u b s t a n c e " , formed from AA by p l a t e l e t m i c r o s o m a l p r e p a r a t i o n s is m a x i m a l l y a v a i l a b l e a f t e r 45 s e c o n d s (6), w h i c h c o n f l i c t s w i t h our data that the a g g r e g a n t a c t i v i t y r e q u i r e s a r o u n d two m i n u t e s to appear. D i f f e r e n t p r o c e d u r e s m a y e x p l a i n the d i s c r e p a n c y : we used r a b b i t PRP as d o n o r and r e c i p i e n t m a t e r i a l s , the l a t t e r b e i n g p r i m e d w i t h i n d o m e t h a c i n to p r e v e n t the " p a r a s i t i c " effect of AA. W i l l i s (6) added h u m a n PRP to i n c u b a t e s of m i c r o s o m e s in b u f f e r and AA, after b i o t r a n s f o r m a t i o n of the latter had b e e n i n i t i a t e d , p o s s i b l y m a k i n g the a g g r e g a n t a c t i v i t y m o r e r e a d i l y a v a i l a b l e . M o r e o v e r , as in his e x p e r i m e n t s h u m a n p l a t e l e t s in p l a s m a did not r e s p o n d to AA, the " p a r a s i t i c " a c t i v i t y of the l a t t e r was not feared and i n d o m e t h a c i n was o m i t t e d from the r e c i p i e n t system, w h i c h m i g h t thus have r e s p o n d e d m o r e r e a d i l y than in our case to the joint e f f e c t s of the t r a n s f e r r e d AA and of the g e n e r a t e d l i p o p e r o x l d e . ADP is p r e s u m a b l y r e l e a s e d w h e n a g g r e g a t i o n is e v o k e d by AA, and may p a r t l y a c c o u n t for the e f f e c t i v e n e s s of EGTA, w h i c h did not p r e v e n t g e n e r a t i o n of RCS and P G - l i k e m a t e r i a l s , b u t p r e v e n t e d g e n e r a t i o n or d e t e c t i o n of a g g r e g a t i n g a c t i v i t y . This a c t i v i t y of E G T A may also be e x p l a i n e d by a Ca ++ r e q u i r e m e n t for a g g r e g a t i o n or for b i n d i n g of the lipop e r o x i d e to the m e m b r a n e , as occurs for PGs (32). As our
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PROSTAGLANDINS a i m was to s t u d y the e f f e c t of AA on i n t a c t p l a t e l e t s , A D P free PG s y n t h e t a s e p r e p a r a t i o n s w e r e not used. M o r e o v e r , as a n t a g o n i s t s of A D P - i n d u c e d a g g r e g a t i o n (PGEI, a p y r a s e or a d e n o s i n e ) also i n h i b i t the e f f e c t s of AA on r a b b i t p l a t e lets (1,5), it was u s e l e s s to try to d i s c r i m i n a t e w i t h them b e t w e e n ADP and o t h e r p o t e n t i a l m e d i a t o r s . Such a d i s c r i m i n a t i o n has r e c e n t l y b e e n p e r f o r m e d on dog p l a t e l e t s , w h i c h are c o m p l e t e l y r e f r a c t o r y to the a g g r e g a t i n g a c t i v i t y of AA, but g e n e r a t e t r a n s f e r a b l e a g g r e g a t i n g a c t i v i t y to recipient rabbit platelets (33, and to be p u b l i s h e d ) , acc o m p a n i e d by RCS and P G - l i k e s u b s t a n c e s (19). This t r a n s f e r a b l e a g g r e g a n t a c t i v i t y c a n n o t be a c c o u n t e d for by ADP, w h i c h i n d u c e s on dog p l a t e l e t s , as do t h r o m b i n or c o l l a g e n , the e x p e c t e d a g g r e g a t i o n . An i m p o r t a n t role for 5HT to i n d u c e a g g r e g a t i o n or a c c o u n t for t r a n s f e r a b l e a c t i v i t y was d i s c a r d e d w i t h m e t h y s e r g i d e , show n to be i n a c t i v e in p r e v e n t i n g e i t h e r of these e f f e c t s . Moreover, in p i l o t e x p e r i m e n t s , p l a t e l e t s c o l l e c t e d from r e s e r p i n i z e d r a b b i t s w e r e as a g g r e g a t e d by AA as c o n t r o l unreserpinized s a m p l e s . Our r e s u l t s , p a r t i c u l a r l y w i t h washed platelets, those of W i l l i s (6) and of H a m b e r g et al (7) w o u l d thus i n d i c a t e that the s p e c i f i c c y c l i c e n d o p e r o xide (s), w i t h a short h a l f - l i f e in a p l a s m a m e d i u m , m a y be i n v o l v e d w i t h p l a t e l e t a g g r e g a t i o n . The m a t e r i a l s that ~ a c c o u n t for r a b b i t a o r t a c o n t r a c t i n g a c t i v i t y m a y c o n t a i n the c y c l i c e n d o p e r o x i d e (s) but o t h e r s u b s t a n c e s as well, w i t h a l o n g e r h a l f - l i f e . T h e s e a g e n t s are p o s s i b l y l i a b l e to c h e m i c a l i n t e r a c t i o n w i t h thiol r e d u c i n g a g e n t s , a l t h o u g h an i n h i b i t o r y e f f e c t over a c o m p o n e n t of the PG s y n t h e t a s e c o m p l e x c a n n o t be e x c l u d e d . A s u m m a r y of the r e s u l t s (figure 8), h i g h l i g h t s the fact that all a g e n t s e f f e c t i v e in v i t r o in our s y s t e m i n h i b i t the in v i v o effects of AA, $RS-C (11) and b r a d y k i n i n (34), thus r e i n f o r cing the t h e o r y that the in v i v o e f f e c t s of the a g o n i s t s l i a b l e to b l o c k a d e by AID or by s u l f h y d r y l a g e n t s are due to a c o m m o n m e d i a t o r , r e l e a s e d t h r o u g h a c t i v a t i o n of PG s y n t h e t a s e or o t h e r l i p o p e r o x i d i z i n g e n z y m e s \ An i m p o r t a n t role of 5HT for a g g r e g a t i o n or t r a n s f e r of a g g r e g a t i n g a c t i v i t y was d i s c a r d e d w i t h m e t h y s e r g i d e , shown to be i n a c t i v e a g a i n s t b o t h A A - i n d u c e d a g g r e g a t i o n and d e t e c t i o n of t r a n s f e r a b l e a g g r e g a t i n g a c t i v i t y .
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PROSTAGLANDINS
REFERENCES
1. Vargaftig, 244:114,
B.B., 1973.
and P. Zirinis.
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2. Ingerman, C., J.B. Smith, J.J. Kocsis Fed. Proc. 32 (I), Abstract 45, 1973. 3. Willis,
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P.J.
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M.A., E.S. Warrior, M.F. Glynn, A.S. Mustard. J. Exp. Med. 126:171, 1967.
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15. Packham, M.A., M.A. Guccione, D.W. Perry, R.L. K i n l o u g h - R a t h b o n e , and J.F. Mustard. Am. J. Physiol. 223:419, 1972. 16. Ardlie, N.G,, M.A. Packham, J. Haemat. 19:7, |970.
and J.F. Mustard.
17. Leonardi, R.G., B. Alexander, Fed. Proc., 33:1480, 1974. 18. Cumings, J.N., 42:6|I, 1971. 19. Ferreira, S.H., 50:543, 1974.
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J. Pharmac.
O C T O B E R 25, 1974
VOL. 8 NO. 2
PROSTAGLANDINS
20.
Silver, Kocsis.
M.J., J.B. Smith, C. Ingerman, P r o s t a g l a n d i n s 4:863, 1973.
and J.J.
21. Vargaftig, B.B°, and N. Dao Hal. In V a s o p e p t i d e s , N. Back and F. Sicuteri, Editors, p. 155, Plenum Press, N. York, 1971. 22.
Silver, M.J., W. Hoch, J.J. Kocsis, C.M. J.B. Smith, Science, 183:1085, 1974.
23.
Larsson, C. and 25:654, 1973.
24.
Johnson, M°, R° Jessup, dins 4:593, 1973.
25. C h r i s t o p h e r s e n , 26.
Stromme,
J.H.
E. Anggard,
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28. Lee, R.E. and W.E.M. 260:203, 1972.
and P,W.
Biochem.
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27. Letellier, P.R., W.L, P r o s t a g l a n d i n s 4:837,
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Smith 1973. Lands.
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and W.E.M.
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29. Albert, London,
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30. Maddox,
J.S.
Biochim.
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31.
Gryglewski, R. and J.R. 46:449, 1972.
32.
Gorman,
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O C T O B E R 25, 1974
and N. Dao Hal.
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1973.
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33. Vargaftig, B.B., in P r o s t a g l a n d i n s , Editor) Plenum Press, N. York, vol. 34. Vargaftig, B.B. 28:59, 1972.
306:74,
1974. (P.W. Ramwell, 2, 1974.
Experientia
147
PROSTAGLANDINS
A
t 1 I
B
C
IM 2O
0.1
I 0.5 0.1
6O
8o1
0.5
~
20
Figure 1 : Arachidonic acid and ADP-induced platelet aggregation and 6eneration of transferable activity Panel A:
Concentration-related aggregation of rabbit platelets by ADP (concentrations in ~M indicated next to each curve).
Panel B:
Aggregation of rabbit platelets by arachidonic acid; curve labelled A was obtained with a platelet rich plasma kept for the overnight; (concentrations in mM indicated next to each curve). Observe that an optimal amount of AA (O.| mM) is more aggregant than 0.5 mM.
Panel C:
Aggregation of rabbit PRP contained in recipient cuvettes induced by 0.2 ml of PRP transferred from donor euvettes of panel B; (concentrations refer to those used to induce aggregation in the donor PRP); indomethacin (0.5 mM) was added to prevent effects of arachidonic acid.
Vertical scale: percent transmission Horizontal scale: time (one minute)
148
OCTOBER 25, 1974 VOL. 8 NO. 2
PROSTAGLANDINS
1
I
100
I00
20-
l 50
40-
10
60,
5
80
Figure 2: Inhibition of platelet aggresation due to arachidonic acid by 2~merca~toethanol and sodium diethyldithiocarbamate.
Superposed tracings of aggregation due to arachidonic acid (added at the arrow 0.5 mM) in rabbit platelet rich plasma. Concentrations of the inhibitors added one minute before arachidonic acid are indicated in ~M. Left panel: 2-mercaptoethanol; Right panel: sodium diethyldithiocarbamate. Scales as in figure I
OCTOBER 25, 1974
VOL. 8 NO. 2
149
PROSTAGLANDINS
!
4
20
dO
80
Figure 3-: Comparison between the effects of indomethacin and thio~l~cerol in platelets suspended in plasma or in Tyrode solution.
Superposed tracings of platelet aggregation induced by arachidonic acid (0.5 mM), as follows: a: control aggregation in rabbit PRP; b and c: aggregation blocked respectively by 0.5 mM of indomethacin and 5 mM of thioglycerol. d and e: responses of platelets suspended in Tyrode solution are not blocked by respectively 0.5 mM of indome~hacin and 5 mM of thioglycerol. Scales as in Figure I.
150
OCTOBER 25, 1974
VOL. 8 NO. 2
PROSTAGLANDINS 1 O
,I
C'
20 ME
40
60
80
Figure 4: Inhibition of a~regation and of ~eneration of transferable activity in rabbit platelet rich plasma Left Panel: superposed tracings of platelet aggregation A = control response to 0.5 D~M of AA; B = protection against aggregation by 0.05 ~M of indomethacin added at the arrow one minute before AA. C = protection against aggregation by 0.5 mM of 2-mercaptoethanol added at the arrow, one minute before AA. Right Panel:superposed tracings of platelet aggregation, obtained after transfer of 0.2 ml of the samples from the left panel. A', B' and C' correspond to A, B and C respectively, collected after three minutes allowed for aggregation. Recipient cuvettes contained 0.5 mM of indomethacin. No transferable activity was obtained from cuvettes where aggregation was inhibited.
OCTOBER 25, 1974
VOL. 8 NO. 2
151
PROSTAGLANDINS
F' 20
E
40
60
80
Figure 4 Cont'd
Left Panel
: superposed tracings of aggregation. D = control responses to 0.5 mM of AA ; E = equimolar CaCI 2 partially overcomes the inhibition by EGTA (5 mM) of platelet aggregation due to AA; F = inhibition by EGTA of platelet aggregation due to AA.
Right Panel : superposed tracings of platelet aggregation obtained after transfer of samples from the right panel. E' and F' correspond to E and F, respectively, collected after three minutes allowed for aggregation. Recipient cuvette contained 0.5 mM of indomethacin. For simplicity, transfer of material from D is not shown, as it was equivalent to A', for the first part of the figure. Scales as in Figure 1.
152
O C T O B E R 25, 1974
VOL. 8 NO. 2
PROSTAGLANDINS
mm
A- PLATELETS
SUSPENDED IN
TYRODE []
SOLUTION
RABBIT AORTk STRIP
[~RAT
STOMACH STRIP
H mm
B-
PLATELETS
SUSPENDED
IN
PLASMA
?o 60 50 40
2O
T[MPERATURE 37"C ~f INCUBATION4"C ~}VR~TION OF, IN~UI~&TION 2 (MtNUTES)
Fisure
4"13
37"C S
4°¢
3?% 15
4%
S?'C
30
5 : Time-related degradation of rabbit aorta and rat stomach strip activities generated in platelets suspended in plasma and in Tyrode solution.
Columns indicate the height of contraction of the assay tissues at different intervals and incubation temperatures due to incubates of arachidonic acid with platelets suspended in Tyrode solution (panel A) or in plasma (panel B).
OCTOBER 25, 1974
VOL. 8 NO. 2
153
PROSTAGLANDINS
B RAIIIT AORTA =TRIP % POT[ NTIATION
F~ RAT STOMACHSTRIP
m
1
140 I
L
20
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DEDTC
mM
Cu CIt
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0.5
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4
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( ~ RAT STOMACH STRIP
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20 0 20
60
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PYROGALLOL
mM
CuCl=
I o.o;
_
o.ol
0.01
0.01
0.1
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0.5
0.1
0.5
I
--
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Figure 6: Interference by CuCI 2 with inhibition by diethyldithiocarbamate and by pyrogallol of the seneration of pharmacological activity in incubates of arachidonic acid with platelet rich plasma. Vertical columns indicate, for each assay tissue, the percent inhibition of the contractions due to incubates of AA with PRP in the presence of inhibitors, and the percent potentiation of these contractions when CuCI 2 was added.
154
OCTOBER 25, 1974
VOL. 8 NO. 2
PROSTAGLANDINS
0
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OCTOBER 25, 1974
VOL. 8 NO. 2
155
PROSTAGLANDINS
u
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156
O C T O B E R 25, 1974
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VOL. 8 NO. 2