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The significance of protein disulfide and sulfhydryl groups in prostaglandin action
THE SIGNIFICANCE OF PROTEIN DISULFIDE AND
SULFHYDRYLGROUPSIN PROSTAGLANDINACTION1 Malcolm Johnson, Reginald Jessup and Peter W. Ramwell
Department of Physiology &Biophysics Georgetown University Washington, D. C.
ABSTRACT
The contraction of the rat uterus and gerbil colon to prostaglandins E1, E2, FI~ and F2,~ and the modification of ADP-induced platelet aggregation by PGE1 and PGE2 is inhibited by prior treatment with I, 4-dithiothreitol (DTT) or with p-hydroxymercuribenzoate (PHMB). The inhibition by both reagents is characterized by an increase in the concentration of prostaglandin needed to elicit half-maximal and maximal responses. The inhibition due to PHMB is reversed by subsequent treatment with thiol compounds whereas, in the case of DTT, oxidising 'agents are required. N-ethylmaleimide (NEM) prevents the reversal of DTT inhibition by the oxidising agent 5,5'-dlthiobis-(2-nitrobenzoate). The effect of DTT is attentuated by the presence of prostaglandin or 7-oxa- 13-prostynoic acid, a competitive antagonist for prostaglandins, suggesting protection of those disulfide groups specifically associated with prostaglandin action. It is concluded that dithiothreitol reduces disulfide groups and that PHMB complexes with sulfhydryl groups in the vicinity of one or more components of the prostaglandin receptor.
Accepted December II
1 Supported by the Office of Naval Research #3287-888
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INTRODUCTION
We h a v e r e p o r t e d that some m e m b r a n e - b o u n d e n z y m e s y s t e m s w h i c h c o n t a i n functional s u l f h y d r y l g r o u p s a r e r e s p o n s i v e to p r o s t a g l a n d i n s (1), and t h e r e is e v i d e n c e for the d i r e c t i n v o l v e m e n t of thio g r o u p s in p r o s t a g l a n d i n b i n d i n g (2) and action ( 3 , 3 a ) . S u l f h y d r y l g r o u p s h a v e b e e n implicated in a v a r i e t y of p h y s i o l o g i c a l l y i m p o r t a n t m e m b r a n e p h e n o m e n a (4), i n c l u d i n g active t r a n s p o r t a n d m e m b r a n e s t a b i l i t y , a n d a r e i n v o l v e d in the action of s u c h p h a r macological a g o n i s t s as p o l y p e p t i d e s ( 5 ) , . ~ - a d r e n e r g i c a g e n t s ( 6 ) . a n d a c e t y l c h o l i n e (7). In a d d i t i o n , s u l f h y d r y l r e a g e n t s have p r e v i o u s l y b e e n r e p o r t e d to i n h i b i t inflammatory r e s p o n s e s a s s o c i a t e d with p r o s t a f f l a n d i n r e l e a s e and with the action of a r a c h i d o n i c acid ( 8 ) . In t h i s communication we r e p o r t the effects of s u l f h y d r y l , d i s u l f i d e - r e d u c i n g and o x i d i s i n g a g e n t s in the p r o s t a g l a n d i n - r e s p o n s i v e s y s t e m s of p l a t e l e t a g g r e g a t i o n and smooth m u s c l e c o n t r a c tion.
MATERIALS AND METHODS
Rat U t e r u s Female v i r g i n S p r a g u e - D a w l e y r a t s (120-140 g) w e r e s a c r i f i c e d 14 d a y s a f t e r b i l a t e r a l o v a r i e c t o m y . A 1 cm. s e g m e n t of u t e r i n e h o r n was e x c i s e d and s u s p e n d e d in a lucite b a t h c o n t a i n i n g 0.5 ml. o r a e r a t e d De J a l o n ' s s o l u t i o n (mM, Na 160, K 5 . 6 , Ca 0.54, Cl 162, HCO 3 5.95, g l u c o s e 2.78) pH 7.4 at 22° C as p r e v i o u s l y d e s c r i b e d (9). T i s s u e s w e r e s u b j e c t e d to a r e s t i n g t e n s i o n of 300 rag. and c o n t r a c t i o n s w e r e m e a s u r e d i s o t o n i c a l l y u s i n g a l i n e a r motion t r a n s d u c e r ( P h i p p s and B i r d ) . U t e r i n e h o r n s w e r e c y c l e d at 5 rain. i n t e r v a l s w h i c h i n c l u d e d an a g o n i s t contact time of 90 s e e . G e r b i l Colon The a s c e n d i n g colon from m a t u r e male g e r b i l s (Meriones u n g u i c u l a t u s , 60-70 g) w a s s u s p e n d e d in 0.9 ml of a e r a t e d De J a l o n ' s s o l u t i o n m a i n t a i n e d at 30° C. A r e s t i n g t e n s i o n of 500 mg was a p p l i e d a n d c o n t r a c t i o n r e c o r d e d i s o t o n i c a l l y . A g o n i s t s , a d d e d i n a volume of 0.1 ml De J a l o n ' s s o l u t i o n , r e m a i n e d in c o n t a c t w i t h the t i s s u e for 1 rain followed b y a 2 rain w a s h a n d r e s t p e r i o d . Platelet Aggregation Blood w a s c o l l e c t e d from the d o r s a l a o r t a of male S p r a g u e - D a w l e y r a t s (500-600 g ) o r from the e u b i t a l v e i n of h u m a n v o l u n t e e r s into 1/9 volume sodium c i t r a t e (1.8% w / v ) , a n d p l a t e l e t r i c h p l a s m a (PRP) w a s p r e p a r e d b y t h e m e t h o d of Shio (10). Platelet a g g r e g a t i o n w a s followed at 600 nm in a r e c o r d i n g s p e c t r o p h o t o m e t e r (Gilford) with c o n s t a n t s t i r r i n g (900 r p m ) (11).
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Dithiothreitol, N-ethylmaleimide, 5,5'-dithiobis- (2-nitrobenzoic acid) and p-hydroxymercuribenzoic acid were obtained from Sigma Chemical Co, and 2-mercaptoethanol from Eastman Organic Chemicals. Prostaglandins were a generous flirt from the Upjohn Co. and 7-oxa-13-prostynoic acid was kindly supplied by Dr. J. Fried, University of Chicago. Solutions of thiol compounds were prepared fresh daily i n saline (0.9% w / v ) . The sodium salt of DTNB was produced by reacting the acid with an equivalent amount of NaHCO3. RESULTS
Figure I- Comparison of the effects of DTT and DTNB o n prostaglandin and serotonin induced contractions in the rat u t e r u s . Serotonin ing/ml (0) and 2 ng/ml (0) and PGE 2 0.75 ng/ml (O) and 1 ng/ml (1) were added at the points indicated. The tissue was exposed to DTT (0. imM) for 20 rain (~) and then washed for 2 rain before testing. DTNB (0.1 raM) was applied for 20 rain (~) and removed before agonists were added.
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Rat Uterus Exposure of the uterus preparation to a disulfide-reducing agent, dithiothreitol (DTT, 0.1 mM) inhibits subsequent responses to PGE2 (Figure 1) and to PGE1, PGFI~, and PGF2~ . This treatment with DTT, however, has little effect on the response to serotonin or to aeetylcholine. Inhibition of carbamylcholine in the eleotroplax (12) was also induced by DTT, but at ImM, suggesting that prostaglandin action in uterine smooth muscle is more susceptible to modification. The DTT inhibition is both dose-dependent (Figure 2) and time-dependenl,reaching a maximum after 30 min.
25-ef
•
20--
15--
E
Z
10-
5--
PGE 2
(ng/m|)
Figure 2- D.ose-dependent DTT-induced inhibition of prostaglandin ~ n the uterus. The response of the isolated uterus to PGE2 is shown (e). This was repeated after 20 min exposure to 0,1 mM (1) and to 0.2 mM (&) DTT. DTT was removed and the preparation washed for 2 min before testing.
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The p r i n c i p l e effect is to i n c r e a s e the concentration of p r o s t a g l a n d i n r e q u i r e d to elicit a half-maximal r e s p o n s e . The DTT inhibition remains unaffected by r e p e a t e d w a s h i n g but is fully r e v e r s e d by a s u l f h y d r y l - o x i d i s i n g compound, 5 , 5 ' - d i t h i o b i s - (2-nitrobenzoic a c i d ) , (DTNB, 0.1 raM) ( F i g u r e 3). DTNB alone has no effect on the p r o s t a g l a n d i n r e s p o n s e . The cycle of D T T - i n d u c e d p r o s t a g l a n d i n inhibition followed by DTNB r e v e r s a l , may be p e r f o r m e d r e p e a t e d l y on the same p r e p a r a t i o n . In c o n t r a s t , the inhibition is unaffected by L - e y s t e i n e (1 raM) but i s , h o w e v e r , p a r t i a l l y r e v e r s e d (~,50%) by ~-mer-¢aptoethanol (1.5 raM). The s u l f h y d r y l - a l k y l a t i n g agent, N - e t h y l maleimide, (NEM, 0.1 raM), which alone does not modify r e s p o n s e s to p r o s t a g l a n d i n s , p r e v e n t s the r e v e r s i n g action of D T N B . . When added b e f o r e DTT, NEM has little effect on the s u b s e q u e n t inhibition o r on the r e v e r s a l by DTNB. The D T T - i n h i b i t i o n of u t e r i n e r e s p o n s e s to p r o s t a g l a n d i n s is modified by the p r e s e n c e of e i t h e r a s u b - m a x i m a l concentration of p r o s t a g l a n d i n (i n g / m l ) or by 7 - o x a - 1 3 - p r o s t y n o i e acid (13), a r e p o r t e d r e v e r s i b l e competitive p r o s t a g l a n d i n antagonist (14). For e x a m p l e , in the p r e s e n c e of 7 - o x a - 1 3 - p r o s t y n o i c acid (30 }IM), the inhibition due to DTT (0.1 raM) was fully r e v e r s e d on removal of the antagonist and the r e d u c i n g agent (Figure 4). This is in direct contrast to the normal DTT effect which is unaffected b y w a s h i n g , and s u g g e s t s that some critical component essential to p r o s t a g l a n d i n action has been p r o t e c t e d by the antagonist. In o r d e r to clarify the role of o t h e r thio g r o u p s in p r o s t a g l a n d i n action, we have i n v e s t i g a t e d the effect of s u l f h y d r y l - c o m p l e x i n g agents such as p - h y d r o x y m e r c u r i b e n z o a t e (PHMB). At 0.03 raM, PHMB also i n h i b i t s the r e s p o n s e of the u t e r u s to p r o s t a g l a n d i n s and this inhibition is completely r e v e r s e d by ~ - m e r c a p t o e t h a n o l ( l . 5 mM). The PHMB effect could also be r e v e r s e d b y DTNB (0.1 raM) b u t only if p r e c e d e d b y e x p o s u r e to DTT (0. l raM).
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F i g u r e 3- Effect of DTNB on the i n h i b i t i o n d u e to DTT. PGF20t (1-20 n g / m l ) w a s a p p l i e d to the u t e r u s p r e p a r a t i o n b e f o r e (A) a n d after e x p o s u r e t o D T T ( 0 . 2 r a M , O ) for 2 0 r a i n , DTNB ( 0 . 2 r a M , • ) w a s t h e n a d d e d and allowed to r e m a i n in c o n t r a c t for 20 m i n . Both DTT and DTNB w e r e r e m o v e d and the t i s s u e w a s h e d for 2 rain b e f o r e t e s t i n g .
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F i g u r e 4- Effect of a p r o s t a g l a n d i n a n t a g o n i s t on the DTT i n h i b i t i o n of p r o s t a g l a n d i n r e s p o n s e s in the u t e r u s . T h e r e s p o n s e of t h e i s o l a t e d r a t u t e r u s to PGE 2 0.5 n g / m l ( 0 ) , 0.75 n g / m l ( I ) a n d 1 n g / m l (O) i s s h o w n , 7 - o x a - 1 3 - p r o s t y n 6 i e a c i d (10 p g / m l ) was a d d e d (A) a n d the r e s p o n s e to PGE^ w a s r e t e s t e d in i t s p r e s e n c e after 20 rain e x p o s u r e , a n d t h e n a g a i n following i t s r e m o v a l ( v , ) . T h e a n t a g o n i s t w a s r e a p p l i e d (A) a n d , a f t e r 5 rain, DTT (0.1 raM) w a s a d d e d ( ~ ) . After 20 rain, t h e DTT was r e m o v e d a n d t h e r e s p o n s e a g a i n m e a s u r e d in the p r e s e n c e of the a n t a g o n i s t a l o n e , F i n a l l y , the 7 - o x a - 1 3 - p r o s t y n o i e acid w a s r e m o v e d ( V ) a n d the t i s s u e retested.
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Gerbil Colon
T h e isolated colon p r e p a r a t i o n was u s e d to d e t e r m i n e w h e t h e r the effects of thio r e a g e n t s o b s e r v e d i n the u t e r u s w e r e s p e c i f i c , or could be a p p l i e d to other p r o s t a g l a n d i n - r e s p o n s i v e smooth m u s c l e t y p e s . P r o s t a g l a n d i n - i n d u c e d c o n t r a c t i o n s of the colon are also i n h i b i t e d b y DTT. T h i s i n h i b i t i o n was a g a i n f u l l y - r e v e r s i b l e with DTNB. H o w e v e r , i n c o n t r a s t to the D T T - i n d u c e d i n h i b i t i o n o b s e r v e d i n the u t e r u s , it was found n e c e s s a r y to expose the colon to PHMB (0.03 raM), followed b y r e v e r s a l with ~ - m e r c a p toethanol (1.5 mM), before a c h a r a c t e r i s t i c i n h i b i t i o n was evoked b y DTT. In an attempt to d e t e r m i n e the a c c e s s i b i l i t y of the thio r e a c t i v e s i t e s , a lipophobic sulfhydryl-eomplexing reagent, p - h y d r o x y m e r c u r i p h e n y l sulfonic acid (PHMPS), was employed (15). At 0.1 mM PHMPS i n h i b i t s the r e s p o n s e of the colon to PGF2~. T h i s i n h i b i t i o n was almost completely r e v e r s e d b y - m e r c a p t o e t h a n o l (5 raM).
~
Platelets To f u r t h e r o u r h y p o t h e s i s of the i n v o l v e m e n t of thio g r o u p s i n p r o s t a g l a n d i n a c t i o n , we have e x t e n d e d o u r i n v e s t i g a t i o n s to the a g g r e g a t i n g platetet. P r e - i n c u b a t i o n of p l a t e l e t - r i c h p l a s m a (PRP) with DTT (0.1 mM) for 20 m i n b e f o r e a d d i t i o n of a s u b - m a x i m a l c o n c e n t r a t i o n of PGE 1 or PGE 2, r e s u l t e d in a s i g n i f i c a n t r e d u c t i o n i n the s u b s e q u e n t i n h i b i t i o n or p o t e n t i a t i o n r e s p e c t i v e l y , of platelet a g g r e g a t i o n due to ADP. T h i s DTT effect was also c o n c e n t r a t i o n - d e p e n d e n t ( F i g u r e 5) and e v i d e n t i n both rat and h u m a n PRP. In these e x p e r i m e n t s , we w e r e u n a b l e to o b t a i n complete r e v e r s a l of the DTT r e s p o n s e with DTNB, T h i s may have b e e n due to the fact t h a t , in c o n t r a s t to the smooth m u s c l e p r e p a r a t i o n s , DTT was p r e s e n t d u r i n g the r e v e r s a l stage.
DISCUSSION
T h e f i n d i n g that both d i s u l f i d e and s u t f h y d r y l r e a g e n t s r e v e r s i b l y i n h i b i t p r o s t a g l a n d i n action s u g g e s t s that they are r e a c t i n g with a p r o t e i n component associated with the evoked c e l l u l a r r e s p o n s e . In a d d i t i o n , u n d e r c o n d i t i o n s i n which the r e s p o n s e to p r o s t a g l a n d i n s is maximally i n h i b i t e d , the actions of o t h e r a g o n i s t s r e m a i n u n a f f e c t e d , s u g g e s t i n g that this p r o t e i n complex may be specific for p r o s t a g l a n d i n s . Dithiothreitol is an a g e n t w h i c h r e d u c e s d i s u l f i d e s with minimum formation of mixed d i s u l f i d e s (16). The following e v i d e n c e implies that DTT i n h i b i t s c e l l u l a r r e s p o n s e s to p r o s t a g l a n d i n s b y r e d u c i n g d i s u l f i d e bonds: l ) The i n h i b i t i o n c a n be completely r e v e r s e d b y an e q u i m o l a r c o n c e n t r a t i o n of a s u l f h y d r y l o x i d i s i n g a g e n t (DTNB) w h i c h alone has no affect; 2) T h i s r e v e r s a l is p r e v e n t e d b y a s u l f h y d r y l - a l k y l a t i n g a g e n t (NEM) u n d e r c o n d i t i o n s w h e r e NEM alone has no s i g n i f i c a n t effect; 3) NEM
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Figure 5- Effect of various concentrations of DTT on the inhibition of PGEI responses in the human p latelet. The effect of PGE 1 (3 n g / m l ) o n the a g g r e g a t i o n of h u m a n p l a t e l e t s i n d u c e d b y ADP (400 n g / m l ) was t e s t e d b e f o r e and after e x p o s u r e to v a r y i n g c o n c e n t r a t i o n s of DTT (0.1 raM) for 20 rain. The i n h i b i t i o n due to DTT is e x p r e s s e d as p e r e e n t a g e i n h i b i t i o n of the o r i g i n a l E 1 r e s p o n s e .
p o t e n t i a t e s the DTT i n h i b i t i o n , b u t only w h e n a d d e d after DTT, and t h e r e f o r e may be r e a c t i n g with n e w l y f o r m e d s u l f h y d r y l g r o u p s ; 4) DTT r e s u l t s in a c h a n g e in the s e n s i t i v i t y of the u t e r u s to p r o s t a g l a n d i n s and t h e i r a n a l o g u e s , PGE 2 b e i n g i n h i b i t e d w h e r e a s the effect of 7 - o x a - 1 3 - p r o s t y n o i e acid is e n h a n c e d : and 5) Both p r o s t a g l a n d i n and 7 - o x a - 1 3 - p r o s t y n o i e a c i d , a r e p o r t e d s p e c i f i c and competitive p r o s t a g l a n d i n a n t a g o n i s t , p r o t e c t a g a i n s t DTT i n h i b i tion p r e s u m a b l y b y making u n a v a i l a b l e for r e d u c t i o n only t h o s e d i s u l f i d e g r o u p s c r i t i c a l to p r o s t a g l a n d i n a c t i o n . The r e s u l t s s u g g e s t that DTT r e d u c e s the S-S g r o u p s of a m e m b r a n e p r o t e i n c o m p o n e n t e s s e n t i a l to the a c t i o n s of
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p r o s t a g l a n d i n s . The r e s u l t i n g s u l f h y d r y l g r o u p s a r e a c c e s s i b l e for alkylation or r e - o x i d a t i o n to the o r i g i n a l form. PHMB will r e a c t p r e f e r e n t i a l l y with a v a i l a b l e s u l f h y d r y l g r o u p s and r e a c t i o n w i t h d i s u l f i d e s is u n l i k e l y u n d e r the c o n d i t i o n s e m p l o y e d (17). The complex formed b y PHMB m u s t be slowly d i s s o c i a b l e s i n c e the i n h i b i t i o n is u n a f f e c t e d b y r e p e a t e d w a s h i n g . T h e c o m p l e t e r e v e r s i b i l i t y of t h i s i n h i b i tion by thiol c o m p o u n d s a g a i n s u g g e s t s that the s i t e s of r e a c t i o n a r e a c c e s s i b l e to l i p o p h o b i c m o l e c u l e s . That t h i s is i n d e e d the c a s e is s u p p o r t e d b y o u r f i n d i n g s that the s u l f o n i c acid d e r i v a t i v e of PHMB, I i p o p h o b i e in n a t u r e , is e q u i p o t e n t with the p a r e n t c o m p o u n d as an i n h i b i t o r of p r o s t a g l a n d i n a c t i o n . The i n h i b i t i o n by both d i s u l f i d e and s u l f h y d r y l r e a g e n t s may b e the r e s u l t of a c h a n g e in conformation of a r e c e p t o r p r o t e i n , as is r e a d i l y initiated b y c l e a v a g e of d i s u l f i d e b r i d g e s (18). H o w e v e r , thio r e a g e n t s may also i n d u c e i n h i b i t i o n b y i n t e r a c t i n g with f r e e d i s u l f i d e o r s u l f h y d r y l g r o u p s c r i t i c a l to the c e l l u l a r r e s p o n s e . T h e s e g r o u p s a r e t h e n r e a d i l y r e s t o r e d c h e m i c a l l y , b u t show no s i g n of s p o n t a n e o u s l y r e v e r t i n g to t h e i r n a t i v e f o r m . I n d e e d , it has b e e n r e p o r t e d that f r e e s u l f h y d r y t g r o u p s , r e s p o n s i v e to DTNB, a r e n e c e s s a r y for full e x p r e s s i o n of a d e n y l a t e c y c l a s e activity (19) • The complete reversibility of the physiological inhibition in a number of systems indicates, however, that the protein component involved is not irreversibly denatured. The native configuration of the receptor, if not fully restored, must at least return to a responsive conformation. T h e m a r k e d s e n s i t i v i t y of m e m b r a n e d i s u l f i d e and s u l f h y d r y l g r o u p s to c h e m i c a l modification, and t h e i r s p e c i f i c p r o t e c t i o n b y p r o s t a g l a n d i n s and p r o s t a g l a n d i n a n t a g o n i s t s , s u g g e s t s a d i r e c t and c r i t i c a l r o l e t o t t h e s e g r o u p s in p r o s t a g l a n d i n a c t i o n . T h e i n h i b i t i o n d u e to the r e s p e c t i v e thio r e a g e n t s may b e the r e s u l t of a d e c r e a s e in p r o s t a g l a n d i n r e c e p t o r b i n d i n g . H o w e v e r . s u l f h y d r y l r e a g e n t s have b e e n r e p o r t e d to h a v e minimal effects on p r o s t a g t a n d i n b i n d i n g (2). I n d e e d , the c r u c i a l thio g r o u p s may not be at the b i n d i n g site i t s e l f , but may i n t e r a c t with it by i n f l u e n c i n g molecular conformation as has b e e n s u g g e s t e d by Karlin (20) for the a c e t y l e h o l i n e r e c e p t o r . An action on any of the as yet u n d e f i n e d s t e p s , s u b s e q u e n t to the b i n d i n g of p r o s t a g l a n d i n to its r e c e p t o r , could also account for the data o b t a i n e d .
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REFERENCES
i.
Johnson, M. ,and P. W. Ramwell. Prostaglandin Modification of Membrane Enzyme Activity: A Possible Mechanism of Action?, Prostaglandins 3: 703, 1973.
2.
Moore, W.V., and J . Wolff. Binding of Prostaglandins E 1 to Beef Thyroid Membranes. J . Biol. Chem. 248: 5705, 1973.
3.
Mills, D . C . B . , and J . B . Smith. The Control of Platelet Responsiveness by Agents that Influence Cyclic AMP Metabolism, Ann. N.Y. Acad. Sci. 201: 391, 1972.
3a.
Johnson, M., R. J e s s u p , and P.W. Ramwell. Ultraviolet Light Modification of the Prostaglandin Receptor, Prostaglandins, 4:4 , 1973.
4.
Sutherland, R . M . , A. Rothstein, and R . I . Weed. Erythrocyte Membrane Sulfhydryl Groups and Cation Permeability, J , Cell Physiol. 69: 185, 1967.
5.
Picarelli, Z . P . , O.B. Henriques, and M.C. Oliviera. Potentiation of Bradykinin Action on Smooth Muscle by Cysteine, Experientia 18: 77, 1962.
6.
Goldman, J .M., and M.E. Hadley. Sulfhydryl Requirement for ~ - A d r e n e r g i c Receptor Activity and MSH Action in Melanophores, J . Pharm. Exp. T h e r . 182: 93, 1972.
7.
Vargaftig, B . B . and C . J . de Vos. Anti-lnflammatory Properties of Sulfhydryl and Anti-Oxidant Reagents, Br. J . Pharmaeol. 15: 601, 1960.
8.
Vargaftig, B . B . , and N. Dao. Release of Vasoactive Substances from Guinea-Pig Lungs by Slow-Reacting Substance C and Araehidonie Acid, Pharmacology 6: 99, 1971.
9.
Ramwell, P.W., J . E . Shaw, and R. J e s s u p . Spontaneous and Evoked Release of Prostaglandins from Frog Spinal Cord, Am. J . Physiol. 211: 998, 1966.
I0.
Shio, H . , A.M. Plasse, and P.W. Ramwell, Platelet Swelling and Prostaglandins, Microvascular Research 2: 294, 1970.
11.
Shio, H . , P.W. Ramwell, and S . J . J e s s u p . Prostaglandin E2: Effects on Aggregation, Shape Change and Cyclic AMP of Rat Platelets, Prostaglandins I: 29, 1972.
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12.
K a r l i n , A . , a n d E. B a r t e t s . Effects of B l o c k i n g S u l f h y d r y l G r o u p s a n d of R e d u c i n g Disulfide B o n d s on t h e A c e t y l c h o l i n e - A c t i v a t e d P e r m e a b i l i t y S y s t e m of t h e E l e c t r o p l a x . B i o c h e m . et B i o p h y s . A c t a . 126: 525, 1966.
13.
F r i e d , J ° , T . S. S a n t h a n a K r i s h n a n , J . Himizu, C. H. Lin, S. H. F o r d , B. R u b i n , a n d E. O. G r i g a s . P r o s t a g l a n d i n A n t a g o n i s t s : S y n t h e s i s a n d Smooth Muscle A c t i v i t y , N a t u r e 223: 208, 1969.
14.
K u e h l , F. A . , a n d J . L. H u m e s . D i r e c t E v i d e n c e for a P r o s t a g l a n d i n R e c e p t o r a n d its A p p l i c a t i o n to P r o s t a g l a n d i n M e a s u r e m e n t s , P r o c . Nat. A c a d . Sci. U. S. A. 69: 480, 1972.
15.
A l e d o r t , L. M . , S. B. T r o u p , a n d R. I. Weed. I n h i b i t i o n of S u l f h y d r y l Dependent Platelet Functions by Penetrating and Non-Penetrating A n a l o g u e s of P a r a c h l o r o m e r c u r i b e n z e n e , Blood 31: 471, 1968.
16.
C l e l a n d , W. W. D i t h i o t h r e i t o l , A New P r o t e c t i v e R e a g e n t for SH G r o u p s , B i o c h e m i s t r y 3: 480, 1964.
17.
C u n n i n g h a m , L. W . , B. J . Nuenke~ a n d W. D. S t r a y h o r n . S u l f h y d r y l C o n t e n t a n d T r y p t i c S u s c e p t i b i l i t y of T h e r m a l l y D e n a t u r e d O v a l b u m i n , J . Biol. C h e m . 228: 835, 1957.
18.
S c h a c h m a n , H. K. C o n s i d e r a t i o n s on the T e r t i a r y S t r u c t u r e of P r o t e i n s , Cold S p r i n g H a r b o r Syrup. Q u a n t . Biol. 28: 409, 1963.
19.
K a r l i n , A. T h e A c e t y l c h o l i n e R e c e p t o r , N e u r o s c i e n c e R e s . P r o g . B u l l . 8: 393, 1972.
20.
F e r r e n d e l l i , J . A . , E. M. J o h n s o n , M. M. C h a n g , a n d P. N e e d l e m a n . I n h i b i t i o n of B r a i n A d e n y l a t e C y c l a s e b y E t h a c r y n i c Acid a n d D i t h i o b i s n i t r o b e n z o i c A c i d , B i o c h e m . P h a r m a c o l . 22: 3313, 1973
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VOL. 5 NO. 2
SULFHYDRYLGROUPSIN PROSTAGLANDINACTION1 Malcolm Johnson, Reginald Jessup and Peter W. Ramwell
Department of Physiology &Biophysics Georgetown University Washington, D. C.
ABSTRACT
The contraction of the rat uterus and gerbil colon to prostaglandins E1, E2, FI~ and F2,~ and the modification of ADP-induced platelet aggregation by PGE1 and PGE2 is inhibited by prior treatment with I, 4-dithiothreitol (DTT) or with p-hydroxymercuribenzoate (PHMB). The inhibition by both reagents is characterized by an increase in the concentration of prostaglandin needed to elicit half-maximal and maximal responses. The inhibition due to PHMB is reversed by subsequent treatment with thiol compounds whereas, in the case of DTT, oxidising 'agents are required. N-ethylmaleimide (NEM) prevents the reversal of DTT inhibition by the oxidising agent 5,5'-dlthiobis-(2-nitrobenzoate). The effect of DTT is attentuated by the presence of prostaglandin or 7-oxa- 13-prostynoic acid, a competitive antagonist for prostaglandins, suggesting protection of those disulfide groups specifically associated with prostaglandin action. It is concluded that dithiothreitol reduces disulfide groups and that PHMB complexes with sulfhydryl groups in the vicinity of one or more components of the prostaglandin receptor.
Accepted December II
1 Supported by the Office of Naval Research #3287-888
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INTRODUCTION
We h a v e r e p o r t e d that some m e m b r a n e - b o u n d e n z y m e s y s t e m s w h i c h c o n t a i n functional s u l f h y d r y l g r o u p s a r e r e s p o n s i v e to p r o s t a g l a n d i n s (1), and t h e r e is e v i d e n c e for the d i r e c t i n v o l v e m e n t of thio g r o u p s in p r o s t a g l a n d i n b i n d i n g (2) and action ( 3 , 3 a ) . S u l f h y d r y l g r o u p s h a v e b e e n implicated in a v a r i e t y of p h y s i o l o g i c a l l y i m p o r t a n t m e m b r a n e p h e n o m e n a (4), i n c l u d i n g active t r a n s p o r t a n d m e m b r a n e s t a b i l i t y , a n d a r e i n v o l v e d in the action of s u c h p h a r macological a g o n i s t s as p o l y p e p t i d e s ( 5 ) , . ~ - a d r e n e r g i c a g e n t s ( 6 ) . a n d a c e t y l c h o l i n e (7). In a d d i t i o n , s u l f h y d r y l r e a g e n t s have p r e v i o u s l y b e e n r e p o r t e d to i n h i b i t inflammatory r e s p o n s e s a s s o c i a t e d with p r o s t a f f l a n d i n r e l e a s e and with the action of a r a c h i d o n i c acid ( 8 ) . In t h i s communication we r e p o r t the effects of s u l f h y d r y l , d i s u l f i d e - r e d u c i n g and o x i d i s i n g a g e n t s in the p r o s t a g l a n d i n - r e s p o n s i v e s y s t e m s of p l a t e l e t a g g r e g a t i o n and smooth m u s c l e c o n t r a c tion.
MATERIALS AND METHODS
Rat U t e r u s Female v i r g i n S p r a g u e - D a w l e y r a t s (120-140 g) w e r e s a c r i f i c e d 14 d a y s a f t e r b i l a t e r a l o v a r i e c t o m y . A 1 cm. s e g m e n t of u t e r i n e h o r n was e x c i s e d and s u s p e n d e d in a lucite b a t h c o n t a i n i n g 0.5 ml. o r a e r a t e d De J a l o n ' s s o l u t i o n (mM, Na 160, K 5 . 6 , Ca 0.54, Cl 162, HCO 3 5.95, g l u c o s e 2.78) pH 7.4 at 22° C as p r e v i o u s l y d e s c r i b e d (9). T i s s u e s w e r e s u b j e c t e d to a r e s t i n g t e n s i o n of 300 rag. and c o n t r a c t i o n s w e r e m e a s u r e d i s o t o n i c a l l y u s i n g a l i n e a r motion t r a n s d u c e r ( P h i p p s and B i r d ) . U t e r i n e h o r n s w e r e c y c l e d at 5 rain. i n t e r v a l s w h i c h i n c l u d e d an a g o n i s t contact time of 90 s e e . G e r b i l Colon The a s c e n d i n g colon from m a t u r e male g e r b i l s (Meriones u n g u i c u l a t u s , 60-70 g) w a s s u s p e n d e d in 0.9 ml of a e r a t e d De J a l o n ' s s o l u t i o n m a i n t a i n e d at 30° C. A r e s t i n g t e n s i o n of 500 mg was a p p l i e d a n d c o n t r a c t i o n r e c o r d e d i s o t o n i c a l l y . A g o n i s t s , a d d e d i n a volume of 0.1 ml De J a l o n ' s s o l u t i o n , r e m a i n e d in c o n t a c t w i t h the t i s s u e for 1 rain followed b y a 2 rain w a s h a n d r e s t p e r i o d . Platelet Aggregation Blood w a s c o l l e c t e d from the d o r s a l a o r t a of male S p r a g u e - D a w l e y r a t s (500-600 g ) o r from the e u b i t a l v e i n of h u m a n v o l u n t e e r s into 1/9 volume sodium c i t r a t e (1.8% w / v ) , a n d p l a t e l e t r i c h p l a s m a (PRP) w a s p r e p a r e d b y t h e m e t h o d of Shio (10). Platelet a g g r e g a t i o n w a s followed at 600 nm in a r e c o r d i n g s p e c t r o p h o t o m e t e r (Gilford) with c o n s t a n t s t i r r i n g (900 r p m ) (11).
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Dithiothreitol, N-ethylmaleimide, 5,5'-dithiobis- (2-nitrobenzoic acid) and p-hydroxymercuribenzoic acid were obtained from Sigma Chemical Co, and 2-mercaptoethanol from Eastman Organic Chemicals. Prostaglandins were a generous flirt from the Upjohn Co. and 7-oxa-13-prostynoic acid was kindly supplied by Dr. J. Fried, University of Chicago. Solutions of thiol compounds were prepared fresh daily i n saline (0.9% w / v ) . The sodium salt of DTNB was produced by reacting the acid with an equivalent amount of NaHCO3. RESULTS
Figure I- Comparison of the effects of DTT and DTNB o n prostaglandin and serotonin induced contractions in the rat u t e r u s . Serotonin ing/ml (0) and 2 ng/ml (0) and PGE 2 0.75 ng/ml (O) and 1 ng/ml (1) were added at the points indicated. The tissue was exposed to DTT (0. imM) for 20 rain (~) and then washed for 2 rain before testing. DTNB (0.1 raM) was applied for 20 rain (~) and removed before agonists were added.
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Rat Uterus Exposure of the uterus preparation to a disulfide-reducing agent, dithiothreitol (DTT, 0.1 mM) inhibits subsequent responses to PGE2 (Figure 1) and to PGE1, PGFI~, and PGF2~ . This treatment with DTT, however, has little effect on the response to serotonin or to aeetylcholine. Inhibition of carbamylcholine in the eleotroplax (12) was also induced by DTT, but at ImM, suggesting that prostaglandin action in uterine smooth muscle is more susceptible to modification. The DTT inhibition is both dose-dependent (Figure 2) and time-dependenl,reaching a maximum after 30 min.
25-ef
•
20--
15--
E
Z
10-
5--
PGE 2
(ng/m|)
Figure 2- D.ose-dependent DTT-induced inhibition of prostaglandin ~ n the uterus. The response of the isolated uterus to PGE2 is shown (e). This was repeated after 20 min exposure to 0,1 mM (1) and to 0.2 mM (&) DTT. DTT was removed and the preparation washed for 2 min before testing.
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The p r i n c i p l e effect is to i n c r e a s e the concentration of p r o s t a g l a n d i n r e q u i r e d to elicit a half-maximal r e s p o n s e . The DTT inhibition remains unaffected by r e p e a t e d w a s h i n g but is fully r e v e r s e d by a s u l f h y d r y l - o x i d i s i n g compound, 5 , 5 ' - d i t h i o b i s - (2-nitrobenzoic a c i d ) , (DTNB, 0.1 raM) ( F i g u r e 3). DTNB alone has no effect on the p r o s t a g l a n d i n r e s p o n s e . The cycle of D T T - i n d u c e d p r o s t a g l a n d i n inhibition followed by DTNB r e v e r s a l , may be p e r f o r m e d r e p e a t e d l y on the same p r e p a r a t i o n . In c o n t r a s t , the inhibition is unaffected by L - e y s t e i n e (1 raM) but i s , h o w e v e r , p a r t i a l l y r e v e r s e d (~,50%) by ~-mer-¢aptoethanol (1.5 raM). The s u l f h y d r y l - a l k y l a t i n g agent, N - e t h y l maleimide, (NEM, 0.1 raM), which alone does not modify r e s p o n s e s to p r o s t a g l a n d i n s , p r e v e n t s the r e v e r s i n g action of D T N B . . When added b e f o r e DTT, NEM has little effect on the s u b s e q u e n t inhibition o r on the r e v e r s a l by DTNB. The D T T - i n h i b i t i o n of u t e r i n e r e s p o n s e s to p r o s t a g l a n d i n s is modified by the p r e s e n c e of e i t h e r a s u b - m a x i m a l concentration of p r o s t a g l a n d i n (i n g / m l ) or by 7 - o x a - 1 3 - p r o s t y n o i e acid (13), a r e p o r t e d r e v e r s i b l e competitive p r o s t a g l a n d i n antagonist (14). For e x a m p l e , in the p r e s e n c e of 7 - o x a - 1 3 - p r o s t y n o i c acid (30 }IM), the inhibition due to DTT (0.1 raM) was fully r e v e r s e d on removal of the antagonist and the r e d u c i n g agent (Figure 4). This is in direct contrast to the normal DTT effect which is unaffected b y w a s h i n g , and s u g g e s t s that some critical component essential to p r o s t a g l a n d i n action has been p r o t e c t e d by the antagonist. In o r d e r to clarify the role of o t h e r thio g r o u p s in p r o s t a g l a n d i n action, we have i n v e s t i g a t e d the effect of s u l f h y d r y l - c o m p l e x i n g agents such as p - h y d r o x y m e r c u r i b e n z o a t e (PHMB). At 0.03 raM, PHMB also i n h i b i t s the r e s p o n s e of the u t e r u s to p r o s t a g l a n d i n s and this inhibition is completely r e v e r s e d by ~ - m e r c a p t o e t h a n o l ( l . 5 mM). The PHMB effect could also be r e v e r s e d b y DTNB (0.1 raM) b u t only if p r e c e d e d b y e x p o s u r e to DTT (0. l raM).
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12-
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11-
I0-
98-0
7-
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C, 5z 0 Q.
432-
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0
1
2
4
10
20
PGF2~ ( n g / m l )
F i g u r e 3- Effect of DTNB on the i n h i b i t i o n d u e to DTT. PGF20t (1-20 n g / m l ) w a s a p p l i e d to the u t e r u s p r e p a r a t i o n b e f o r e (A) a n d after e x p o s u r e t o D T T ( 0 . 2 r a M , O ) for 2 0 r a i n , DTNB ( 0 . 2 r a M , • ) w a s t h e n a d d e d and allowed to r e m a i n in c o n t r a c t for 20 m i n . Both DTT and DTNB w e r e r e m o v e d and the t i s s u e w a s h e d for 2 rain b e f o r e t e s t i n g .
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o
•
•
A
o
•
•
V
o
•
•
~o
~
•
o
•
•
F i g u r e 4- Effect of a p r o s t a g l a n d i n a n t a g o n i s t on the DTT i n h i b i t i o n of p r o s t a g l a n d i n r e s p o n s e s in the u t e r u s . T h e r e s p o n s e of t h e i s o l a t e d r a t u t e r u s to PGE 2 0.5 n g / m l ( 0 ) , 0.75 n g / m l ( I ) a n d 1 n g / m l (O) i s s h o w n , 7 - o x a - 1 3 - p r o s t y n 6 i e a c i d (10 p g / m l ) was a d d e d (A) a n d the r e s p o n s e to PGE^ w a s r e t e s t e d in i t s p r e s e n c e after 20 rain e x p o s u r e , a n d t h e n a g a i n following i t s r e m o v a l ( v , ) . T h e a n t a g o n i s t w a s r e a p p l i e d (A) a n d , a f t e r 5 rain, DTT (0.1 raM) w a s a d d e d ( ~ ) . After 20 rain, t h e DTT was r e m o v e d a n d t h e r e s p o n s e a g a i n m e a s u r e d in the p r e s e n c e of the a n t a g o n i s t a l o n e , F i n a l l y , the 7 - o x a - 1 3 - p r o s t y n o i e acid w a s r e m o v e d ( V ) a n d the t i s s u e retested.
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Gerbil Colon
T h e isolated colon p r e p a r a t i o n was u s e d to d e t e r m i n e w h e t h e r the effects of thio r e a g e n t s o b s e r v e d i n the u t e r u s w e r e s p e c i f i c , or could be a p p l i e d to other p r o s t a g l a n d i n - r e s p o n s i v e smooth m u s c l e t y p e s . P r o s t a g l a n d i n - i n d u c e d c o n t r a c t i o n s of the colon are also i n h i b i t e d b y DTT. T h i s i n h i b i t i o n was a g a i n f u l l y - r e v e r s i b l e with DTNB. H o w e v e r , i n c o n t r a s t to the D T T - i n d u c e d i n h i b i t i o n o b s e r v e d i n the u t e r u s , it was found n e c e s s a r y to expose the colon to PHMB (0.03 raM), followed b y r e v e r s a l with ~ - m e r c a p toethanol (1.5 mM), before a c h a r a c t e r i s t i c i n h i b i t i o n was evoked b y DTT. In an attempt to d e t e r m i n e the a c c e s s i b i l i t y of the thio r e a c t i v e s i t e s , a lipophobic sulfhydryl-eomplexing reagent, p - h y d r o x y m e r c u r i p h e n y l sulfonic acid (PHMPS), was employed (15). At 0.1 mM PHMPS i n h i b i t s the r e s p o n s e of the colon to PGF2~. T h i s i n h i b i t i o n was almost completely r e v e r s e d b y - m e r c a p t o e t h a n o l (5 raM).
~
Platelets To f u r t h e r o u r h y p o t h e s i s of the i n v o l v e m e n t of thio g r o u p s i n p r o s t a g l a n d i n a c t i o n , we have e x t e n d e d o u r i n v e s t i g a t i o n s to the a g g r e g a t i n g platetet. P r e - i n c u b a t i o n of p l a t e l e t - r i c h p l a s m a (PRP) with DTT (0.1 mM) for 20 m i n b e f o r e a d d i t i o n of a s u b - m a x i m a l c o n c e n t r a t i o n of PGE 1 or PGE 2, r e s u l t e d in a s i g n i f i c a n t r e d u c t i o n i n the s u b s e q u e n t i n h i b i t i o n or p o t e n t i a t i o n r e s p e c t i v e l y , of platelet a g g r e g a t i o n due to ADP. T h i s DTT effect was also c o n c e n t r a t i o n - d e p e n d e n t ( F i g u r e 5) and e v i d e n t i n both rat and h u m a n PRP. In these e x p e r i m e n t s , we w e r e u n a b l e to o b t a i n complete r e v e r s a l of the DTT r e s p o n s e with DTNB, T h i s may have b e e n due to the fact t h a t , in c o n t r a s t to the smooth m u s c l e p r e p a r a t i o n s , DTT was p r e s e n t d u r i n g the r e v e r s a l stage.
DISCUSSION
T h e f i n d i n g that both d i s u l f i d e and s u t f h y d r y l r e a g e n t s r e v e r s i b l y i n h i b i t p r o s t a g l a n d i n action s u g g e s t s that they are r e a c t i n g with a p r o t e i n component associated with the evoked c e l l u l a r r e s p o n s e . In a d d i t i o n , u n d e r c o n d i t i o n s i n which the r e s p o n s e to p r o s t a g l a n d i n s is maximally i n h i b i t e d , the actions of o t h e r a g o n i s t s r e m a i n u n a f f e c t e d , s u g g e s t i n g that this p r o t e i n complex may be specific for p r o s t a g l a n d i n s . Dithiothreitol is an a g e n t w h i c h r e d u c e s d i s u l f i d e s with minimum formation of mixed d i s u l f i d e s (16). The following e v i d e n c e implies that DTT i n h i b i t s c e l l u l a r r e s p o n s e s to p r o s t a g l a n d i n s b y r e d u c i n g d i s u l f i d e bonds: l ) The i n h i b i t i o n c a n be completely r e v e r s e d b y an e q u i m o l a r c o n c e n t r a t i o n of a s u l f h y d r y l o x i d i s i n g a g e n t (DTNB) w h i c h alone has no affect; 2) T h i s r e v e r s a l is p r e v e n t e d b y a s u l f h y d r y l - a l k y l a t i n g a g e n t (NEM) u n d e r c o n d i t i o n s w h e r e NEM alone has no s i g n i f i c a n t effect; 3) NEM
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I00"
J
80Z o t.u ct:
z o
60-
40-
-7 Z
20"
f
.,
'2
~
'4
E°-] mM
Figure 5- Effect of various concentrations of DTT on the inhibition of PGEI responses in the human p latelet. The effect of PGE 1 (3 n g / m l ) o n the a g g r e g a t i o n of h u m a n p l a t e l e t s i n d u c e d b y ADP (400 n g / m l ) was t e s t e d b e f o r e and after e x p o s u r e to v a r y i n g c o n c e n t r a t i o n s of DTT (0.1 raM) for 20 rain. The i n h i b i t i o n due to DTT is e x p r e s s e d as p e r e e n t a g e i n h i b i t i o n of the o r i g i n a l E 1 r e s p o n s e .
p o t e n t i a t e s the DTT i n h i b i t i o n , b u t only w h e n a d d e d after DTT, and t h e r e f o r e may be r e a c t i n g with n e w l y f o r m e d s u l f h y d r y l g r o u p s ; 4) DTT r e s u l t s in a c h a n g e in the s e n s i t i v i t y of the u t e r u s to p r o s t a g l a n d i n s and t h e i r a n a l o g u e s , PGE 2 b e i n g i n h i b i t e d w h e r e a s the effect of 7 - o x a - 1 3 - p r o s t y n o i e acid is e n h a n c e d : and 5) Both p r o s t a g l a n d i n and 7 - o x a - 1 3 - p r o s t y n o i e a c i d , a r e p o r t e d s p e c i f i c and competitive p r o s t a g l a n d i n a n t a g o n i s t , p r o t e c t a g a i n s t DTT i n h i b i tion p r e s u m a b l y b y making u n a v a i l a b l e for r e d u c t i o n only t h o s e d i s u l f i d e g r o u p s c r i t i c a l to p r o s t a g l a n d i n a c t i o n . The r e s u l t s s u g g e s t that DTT r e d u c e s the S-S g r o u p s of a m e m b r a n e p r o t e i n c o m p o n e n t e s s e n t i a l to the a c t i o n s of
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p r o s t a g l a n d i n s . The r e s u l t i n g s u l f h y d r y l g r o u p s a r e a c c e s s i b l e for alkylation or r e - o x i d a t i o n to the o r i g i n a l form. PHMB will r e a c t p r e f e r e n t i a l l y with a v a i l a b l e s u l f h y d r y l g r o u p s and r e a c t i o n w i t h d i s u l f i d e s is u n l i k e l y u n d e r the c o n d i t i o n s e m p l o y e d (17). The complex formed b y PHMB m u s t be slowly d i s s o c i a b l e s i n c e the i n h i b i t i o n is u n a f f e c t e d b y r e p e a t e d w a s h i n g . T h e c o m p l e t e r e v e r s i b i l i t y of t h i s i n h i b i tion by thiol c o m p o u n d s a g a i n s u g g e s t s that the s i t e s of r e a c t i o n a r e a c c e s s i b l e to l i p o p h o b i c m o l e c u l e s . That t h i s is i n d e e d the c a s e is s u p p o r t e d b y o u r f i n d i n g s that the s u l f o n i c acid d e r i v a t i v e of PHMB, I i p o p h o b i e in n a t u r e , is e q u i p o t e n t with the p a r e n t c o m p o u n d as an i n h i b i t o r of p r o s t a g l a n d i n a c t i o n . The i n h i b i t i o n by both d i s u l f i d e and s u l f h y d r y l r e a g e n t s may b e the r e s u l t of a c h a n g e in conformation of a r e c e p t o r p r o t e i n , as is r e a d i l y initiated b y c l e a v a g e of d i s u l f i d e b r i d g e s (18). H o w e v e r , thio r e a g e n t s may also i n d u c e i n h i b i t i o n b y i n t e r a c t i n g with f r e e d i s u l f i d e o r s u l f h y d r y l g r o u p s c r i t i c a l to the c e l l u l a r r e s p o n s e . T h e s e g r o u p s a r e t h e n r e a d i l y r e s t o r e d c h e m i c a l l y , b u t show no s i g n of s p o n t a n e o u s l y r e v e r t i n g to t h e i r n a t i v e f o r m . I n d e e d , it has b e e n r e p o r t e d that f r e e s u l f h y d r y t g r o u p s , r e s p o n s i v e to DTNB, a r e n e c e s s a r y for full e x p r e s s i o n of a d e n y l a t e c y c l a s e activity (19) • The complete reversibility of the physiological inhibition in a number of systems indicates, however, that the protein component involved is not irreversibly denatured. The native configuration of the receptor, if not fully restored, must at least return to a responsive conformation. T h e m a r k e d s e n s i t i v i t y of m e m b r a n e d i s u l f i d e and s u l f h y d r y l g r o u p s to c h e m i c a l modification, and t h e i r s p e c i f i c p r o t e c t i o n b y p r o s t a g l a n d i n s and p r o s t a g l a n d i n a n t a g o n i s t s , s u g g e s t s a d i r e c t and c r i t i c a l r o l e t o t t h e s e g r o u p s in p r o s t a g l a n d i n a c t i o n . T h e i n h i b i t i o n d u e to the r e s p e c t i v e thio r e a g e n t s may b e the r e s u l t of a d e c r e a s e in p r o s t a g l a n d i n r e c e p t o r b i n d i n g . H o w e v e r . s u l f h y d r y l r e a g e n t s have b e e n r e p o r t e d to h a v e minimal effects on p r o s t a g t a n d i n b i n d i n g (2). I n d e e d , the c r u c i a l thio g r o u p s may not be at the b i n d i n g site i t s e l f , but may i n t e r a c t with it by i n f l u e n c i n g molecular conformation as has b e e n s u g g e s t e d by Karlin (20) for the a c e t y l e h o l i n e r e c e p t o r . An action on any of the as yet u n d e f i n e d s t e p s , s u b s e q u e n t to the b i n d i n g of p r o s t a g l a n d i n to its r e c e p t o r , could also account for the data o b t a i n e d .
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