- Email: [email protected]
Prostanglandin D2 modulates human neutrophil intracellular calcium flux and inhibits superoxide release via its ring carbonyl
Life Sciences, Vol. 46, pp. 793-801 Printed in the U.S.A.
Pergamon Press
P R O S T A N G L A N D I N D2 M O D U L A T E S H U M A N N E U T R O P H I L I N T R A C E L L U L A R C A L C I U M F L U X A N D INHIBITS S U P E R O X I D E R E L E A S E V I A ITS R I N G CARBONYL C u t h b e r t O. S i m p k i n s ! * , Denise L. Mazorow*#, Sione T. A l a i l i m a * , E l i n A. Tate*, W i l l i a m Sweatt*, Mark Johnson*, K h a r e e m S h a r i f f * and D a v i d B. Millar*" *Naval M e d i c a l R e s e a r c h I n s t i t u t e Bethesda, M a r y l a n d 20814-5055 ~ D i s t r i c t of C o l u m b i a General H o s p i t a l W a s h i n g t o n , D.C. 20003 (Received in final form January 17, 1990) Summary We c o m p a r e d the effects of p r o s t a g l a n d i n D 2 (PGD2), p r o s t a g l a n d i n F 2 alpha (PGF2) and v a r i o u s k e t o n e s on s u p e r o x i d e (OX) r e l e a s e by h u m a n n e u t r o p h i l s , w h i c h h a d been stimulated by N-formyl methionyl leucyl p h e n y l a l a n i n e (FMLP). Our data s u g g e s t e d that the r i n g carbonyl of PGD2 is e s s e n t i a l to its i n h i b i t o r y e f f e c t on OX release, but the carbonyl g r o u p as a ketone, alone is not sufficient. U s i n g the f l u o r e s c e n t Ca 2* probe, Fura-2AM, we found that PGD2 i n c r e a s e d the rate of d e c l i n e of FMLP s t i m u l a t e d i n t r a c e l l u l a r free Ca z+ (Ca)i, but that PGF2 had no effect, cAMP altered FMLP stimulated (Ca)i, in a pattern similar to PGD2. F u r t h e r m o r e , the ring c a r b o n y l of PGD2 is crucial to its e f f e c t on OX as well as on (Ca)i. P r o s t a g l a n d i n D2 (PGD2) is the p r o s t a n o i d w h i c h is m o s t a b u n d a n t l y r e l e a s e d by s t i m u l a t e d a l v e o l a r m a s t cells. (I). P r e v i o u s l y , we demonstrated that the r e l e a s e of OX by FMLP s t i m u l a t e d h u m a n n e u t r o p h i l s was i n h i b i t e d by PGD2. We found that PGD2 was m o r e p o t e n t t h a n PGF2, and s u g g e s t e d that the crucial d e t e r m i n a n t of p o t e n c y was the ring carbonyl group on PGD2. We also found t h a t PGD2 did not compete w i t h FMLP at the FMLP r e c e p t o r site (2). In this p r e s e n t c a r b o n y l g r o u p as also e x t e n d e d our by PGD2 t h r o u g h s t i m u l a t e d (Ca)i.
work, we t e s t e d the h y p o t h e s i s that o n l y the a k e t o n e was n e c e s s a r y for i n h i b i t i o n of OX. We u n d e r s t a n d i n g of the m e c h a n i s m of OX i n h i b i t i o n o b s e r v i n g the e f f e c t of PGD2 and cAMP on FMLP
C o r r e s p o n d e n c e should be a d d r e s s e d to C u t h b e r t O. Simpkins, M.D., Department of Surgery, D.C. General Hospital, 19th and M a s s a c h u s e t t s Avenue, S.E., W a s h i n g t o n , D.C. P r e s e n t addresses: # D e p a r t m e n t of Pathology, U n i f o r m e d S e r v i c e s U n i v e r s i t y of the H e a l t h Sciences, Bethesda, M a r y l a n d 20814 ~ a b o r a t o r y of B i o c h e m i c a l Genetics, N a t i o n a l I n s t i t u t e of M e n t a l Health, N e u r o s c i e n c e C e n t e r at St. Elizabeths, W a s h i n g t o n , D.C. 20032 0024-3205/90 $3.00 + .00 Copyright (c) 1990 Pergamon Press plc
794
Prostaglandins and Neutrophils
Vol. 46, No. ii, 1990
Methods Isolation
of N e u t r o p h i l s
B l o o d w a s o b t a i n e d by v e n i p u n c t u r e f r o m h e a l t h y , m e d i c a t i o n - f r e e volunteers. P r e s e r v a t i v e - f r e e h e p a r i n (Gibco, G r a n d I s l a n d , N . Y . ) w a s u s e d at a c o n c e n t r a t i o n of 12.5 u n i t s p e r ml of b l o o d for a n t i c o a g u l a t i o n . E r y t h r o c y t e s w e r e r e m o v e d by g r a v i t y s e d i m e n t a t i o n through 2% d e x t r a n (Pharmacia, Upsala, Sweden) for 30 min. L y m p h o c y t e S e p a r a t i o n M e d i u m ( L i t t o n B i o n e t i c s , K e n s i n g t o n , MD) w a s l a y e r e d u n d e r the s u p e r n a t a n t a n d c e n t r i f u g e d at 300 x g, at r o o m temperature for I0 min. R e s i d u a l e r y t h r o c y t e s w e r e r e m o v e d by h y p o t o n i c l y s i s a n d the c e l l s w e r e c e n t r i f u g e d a g a i n at 150 x g. The neutrophil pellet was suspended in H a n k ' s Balanced Salt Solution (HBSS, Gibco, Grand Island, NY) to the desired concentration. T h i s p r o c e d u r e y i e l d e d 96% n e u t r o p h i l s by W r i g h t ' s stain with a viability of at l e a s t 95% by t r y p a n blue dye exclusion. Assay
of OX
The s u p e r o x i d e d i s m u t a s e - i n h i b i t a b l e r e d u c t i o n of c y t o c h r o m e C a s s a y of B a b i o r et. al, w a s e m p l o y e d w i t h o u t c y t o c h a l a s i n B. (3). Neutrophils were preincubated for 15 min. with various concentrations of t e s t reagents with HBSS as control. After preincubation 500 ml of n e u t r o p h i l s u s p e n s i o n w a s a d d e d to the r e a c t i o n m i x t u r e in 12 x 75 m m p o l y s t y r e n e t u b e s c o n t a i n i n g 1,500 ml HBSS, t e s t r e a g e n t s (250 ml) at v a r i o u s c o n c e n t r a t i o n s a n d 50 uM cytochrome C (Sigma, St. Louis, M0). The final cell c o n c e n t r a t i o n w a s Ixl0~/ml. A f t e r a d d i t i o n of 25 ul of F M L P at a final concentration of 10 .6 or 10 .7 M, or H B S S as c o n t r o l , the r e a c t a n t s w e r e m i x e d a n d a i ml a l i q u o t was p l a c e d i n t o 12 x 75 m m p l a s t i c t u b e s w h i c h c o n t a i n e d i0 ul s u p e r o x i d e d i s m u t a s e (SOD: B o e h r i n g e r M a n n h e i m , W. G e r m a n y ) at a f i n a l c o n c e n t r a t i o n of 50 mg/ml. B o t h the t u b e s to w h i c h SOD h a d b e e n a d d e d a n d t h o s e w i t h o u t S O D w e r e i n c u b a t e d at 37 ° for I0 min. At the e n d of the i n c u b a t i o n p e r i o d 15 ul of SOD, a l s o at a f i n a l c o n c e n t r a t i o n of 50 m g / m l , w a s a d d e d to the t u b e s to w h i c h S O D h a d n o t b e e n a d d e d initially. C e n t r i f u g a t i o n was at 150 x g a n d a b s o r b a n c e of the s u p e r n a t a n t at 550 nm was d e t e r m i n e d . OX r e l e a s e w a s c a l c u l a t e d as the d i f f e r e n c e in a b s o r b a n c e b e t w e e n the t u b e s w h i c h r e c e i v e d S O D at the b e g i n n i n g , d i v i d e d by an e x t i n c t i o n c o e f f i c i e n t of 2 1 , 0 0 0 M I. C e l l v i a b i l i t y at all c o n c e n t r a t i o n s of P G D 2 (SIGMA, St. L o u i s , MO) w a s 95% or g r e a t e r . A s s a y s w e r e p e r f o r m e d in d u p l i c a t e . T h e e f f e c t of P G D 2 u p o n the i n i t i a l r a t e of r e a c t i o n a n d the m a x i m u m OX r e l e a s e d w a s m e a s u r e d as f o l l o w s : The r e a c t i o n m i x t u r e c o n t a i n e d 3 x 106 c e l l s / m l a n d 50 u M c y t o c h r o m e C a n d H B S S in a t o t a l v o l u m e of 3 ml. P r e i n c u b a t i o n w a s in p l a s t i c c u v e t t e s at 37 ° for 15 min. w i t h v a r i o u s c o n c e n t r a t i o n s of t e s t r e a g e n t s . The cuvettes were then placed into a temperature-controlled (37~)5 d u a l beam spectrophotometer with a magnetic stirrer. F M L P at i0- or 10 .7 M or PMA at 4 x 10 .9 M or g r e a t e r was a d d e d to one c u v e t t e a n d c o n t r o l s o l u t i o n a d d e d to the other. The r e a c t i o n w a s m o n i t o r e d c o n t i n u o u s l y u n t i l a m a x i m u m w a s r e a c h e d , w h i c h for F M L P t o o k 5-8 min. a n d w i t h P M A t o o k as l o n g as 60 min., w h e n l o w c o n c e n t r a t i o n s (4 x 10 .9) w e r e used.
Vol. 46, No. ii, 1990
Measurement
Prostaglandins and Neutrophils
of I n t r a c e l l u l a r
795
Calcium
5 x 106 cells w e r e l a b e l l e d w i t h the m e m b r a n e p e r m e a n t p r o b e F u r a 2AM ( C a l b i o c h e m La-Jolla, Calif.) at 37 o in a P e r k i n E l m e r M o d e l 44B s p e c t r o p h o t o f l u o r o m e t e r . The cells w e r e c o n t i n u a l l y s t i r r e d during measurement. A l t e r n a t i v e e ~ c i t a t i o n w a v e l e n g t h s of 335 nm and 362 nm (slit 4 nm) w e r e e m p l o y e d and e m i s s i o n was m e a s u r e d at 510 nm (slit 8 nm). C a l c i u m ion c o n c e n t r a t i o n s w e r e c a l c u l a t e d by the t e c h n i q u e of G r y n k i e w i c z et al. (4) u s i n g the c o n s t a n t s of M a r t e l l and S m i t h (5). FMLP control spectra w e r e r u n p r i o r to and a f t e r each e x p e r i m e n t a l scan. Since our r e s t i n g c a l c i u m l e v e l s determined with FURA-2AM were so m u c h lower, we i n v e s t i g a t e d w h e t h e r Q u i n - 2 A M gave us the same value for (Ca)i w i t h F U R A - 2 A M of 16- 20 nM, w i t h Q U I N - 2 A M we had r e s t i n g levels of (Ca)i from several i n d i v i d u a l s similar to those r e p o r t e d by o t h e r s of 100200nM. I n v e s t i g a t a t i o n w i t h o t h e r cell lines gave s i m i l a r r e s u l t s ( m a n u s c r i p t in p r e p a r a t i o n , D:L. M a z o r o w and D.B. Millar). DMSO at the c o n c e n t r a t i o n s u s e d had no e f f e c t on (Ca)i. Reagents All r e a g e n t s w e r e p r e p a r e d f r e s h l y each day. FMLP, p r o s t a g l a n d i n s , l i p o p h i l i c ketones, cAMP, cGMP, and phorbol m y r i s t a t e a c e t a t e (PMA) w e r e d i s s o l v e d in d i m e t h y l s u l f o x i d e (DMSO). Control solutions c o n t a i n e d equal amounts of DMSO. All of these r e a g e n t s w e r e o b t a i n e d from Sigma (St. Louis, MO.). The ketones, and a l c o h o l s w e r e o b t a i n e d from A l d r i c h (Milwaukee, Wis.). Statistical
Analysis
All e x p e r i m e n t s w e r e of p a i r e d design. The p a i r e d S t u d e n t ' s t - t e s t was employed. P< 0.05 was c o n s i d e r e d significant. Unstimulated O X r e l e a s e was s u b s t r a c t e d from all values. The d e r i v e d number, % of control, was u s e d b e c a u s e of the large v a r i a b i l i t y in F M L P r e s p o n s e among individuals. % of control was c a l c u l a t e d for e a c h e x p e r i m e n t as OX r e l e a s e w i t h test r e a g e n t s over OX r e l e a s e w i t h o u t test r e a g e n t times i00. S t a t i s t i c a l a n a l y s i s of data e x p r e s s e d as p e r c e n t a g e s was t r a n s f o r m e d b e c a u s e p e r c e n t a g e s are n o t n o r m a l l y distributed. The transformation used was a n g l e = A r c s i n ( p e r c e n t a g e ) 5. The rate of d e c r e a s e in i n t r a c e l l u l a r c a l c i u m was d e t e r m i n e d by m e a s u r i n g the d e c r e a s e that o c c u r e d as of one m i n u t e after the m a x i m u m F M L P - s t i m u l a t e d intracellular calcium had been attained. These data were expressed as percentages of control and transformed using the arcsin t r a n f o r m a t i o n as above. Results P r e v i o u s e x p e r i m e n t s showed no e f f e c t of PGD2 on the the r e l e a s e of OX g e n e r a t e d by a c e l l - f r e e x a n t h i n e - x a n t h i n e o x i d a s e s y s t e m (2). In six e x p e r i m e n t s performed with neutrophils from six i n d i v i d u a l s a b i p h a s i c dose r e s p o n s e curve of the i n h i b i t i o n of O X r e l e a s e by PGD2 was found (Fig. I). The first p h a s e o c c u r r e d b e t w e e n 10 .9 and I0 ~ M and showed an i n h i b i t o r y e f f e c t of up to 64%. In this c o n c e n t r a t i o n range PGD2 had no e f f e c t on FMLP binding (2). In the second phase, b e t w e e n 10 .5 and 10 .4 M, OX r e l e a s e was p r a c t i c a l l y e l i m i n a t e d . This phase c o r r e s p o n d s to the c o n c e n t r a t i o n at w h i c h PGD2 d e c r e a s e s FMLP binding.
796
Prostaglandins and Neutrophils
100
i
Vol. 46, No. Ii, 1990
I
7[
u3 90 +1 IJJ
'~ IJJ
8O 70 605o
O
30'
1,1.,
O o.e, 2 0 10I
I
I
-11 - 1 0 - 9 - 8 - 7 - 6 - 5 - 4 LOG PGD 2 CONCENTRATION (M)
-3
FIG.I C e l l s w e r e s t i m u l a t e d w i t h 10 .6 M FMLP. Results are expressed as the p e r c e n t of c o n t r o l O X r e l e a s e + S.E. in the a b s e n c e of PGD2. U n s t i m u l a t e d O X r e l e a s e is s u b t r a c t e d f r o m all v a l u e s . E a c h p o i n t is the a v e r a g e of six e x p e r i m e n t s performed with c e l l s f r o m six d i f f e r e n t i n d i v i d u a l s .
PGD2 is i d e n t i c a l to PGF2 e x c e p t t h a t PGF2 has an h y d r o x y l g r o u p i n s t e a d of a c a r b o n y l g r o u p at the Cll p o s i t i o n . The structures a r e s h o w n in Fig.2.
Vol. 46, No. ii, 1990
Prostaglandins and Neutrophils
797
HO
~ ~
POD2
0
COOH OH
HO
PGF2~ ~ HO
COOH OH
FIG.2 Comparative
Structures
of PGD2
and P G F 2
T h e i m p o r t a n c e of the r i n g c a r b o n y l w a s d e m o n s t r a t e d by d i r e c t l y c o m p a r i n g the e f f e c t of P G D 2 to t h a t of P G F 2 on O X r e l e a s e ( T a b l e i).
TABLE C o m p a r i s o n of the E f f e c t of in R e s p o n s e to F M L P ( i 0 ~ ) .
Mean P<
Subject A B C D E + S.E.
0.005
for
1 0 7 M PGD2 a n d 1 0 7 M PGF2 on O X R e l e a s e E x p r e s s e d as % of C o n t r o l
PGD2 50.0 51.0 8.1 46.6 39.8 39.1+7.3 significant
1
difference
PGF2 86.4 98.5 91.5 94. 1 69.8 88.1+4.5 of P G D 2
Ratio 0 0 0 0 0 0
PGPD2/PGF2 58 52 089 50 57 46+0.094
from PGF2
P< 0 . 0 0 5 for d i f f e r e n c e s b e t w e e n P G F 2 and c o n t r o l a n d P G D 2 a n d control. Both PGD2 and PGF2 inhibited OX release but the i n h i b i t o r y e f f e c t of P G D 2 w a s g r e a t e r . These results suggest that a m a j o r d e t e r m i n a n t of the i n h i b i t i o n of O X r e l e a s e by P G D 2 is t h e c a r b o n y l g r o u p s i n c e the o n l y d i f f e r e n c e b e t w e e n P G D 2 a n d P G F 2 is a Cll c a r b o n y l and h y d r o x y l g r o u p , r e s p e c t i v e l y . N o n e of the k e t o n e s or a l c o h o l s c a u s e d a m a r k e d d e c r e a s e in OX r e l e a s e .
798
Prostaglandins and Neutrophils
TABLE Effect
of K e t o n e s
2
and A l c o h o l s
Reagent (i0~) Cyclopentanone Cyclopentanol 2,4 D i m e t h y l c y c l o p e n t a n o n e Methanol Acetone PGD2 PGF2
Vol. 46, No. ii, 1990
on O X R e l e a s e
% of C o n t r o l 9 8 . 4 ~ 6.0 8 8 . 5 ~ 5.1 7 4 . 6 ~ 7.8* 81.5+ 4.0* 87.7+ 6.8 54.5+ 3.3* 74.2+ 5.3*
+ S.E.
*= s i g n i f i c a n t l y d i f f e r e n t f r o m OX r e l e a s e in the a b s e n c e of a n y test reagent with p<0.05.10~ F M L P was u s e d to s t i m u l a t e the cells. A l s o i n c l u d e d w i t h e a c h e x p e r i m e n t w e r e P G D 2 a n d P G F 2 at I 0 ~ as positive controls. We c o n c l u d e f r o m t h e s e e x p e r i m e n t s t h a t the c a r b o n y l g r o u p a l o n e as a k e t o n e is n o t s u f f i c i e n t to i n h i b i t OX r e l e a s e to a l a r g e d e g r e e . 2,4 d i m e t h y l c y c l o p e n t a n o n e is m o r e lipophilic than cyclopentanone and decreased OX release s i g n i f i c a n t l y w h i l e c y c l o p e n t a n o n e d i d not. This result suggests that lipophilicity is a f a c t o r in the i n h i b i t o r y e f f e c t of a ketone. It is a l s o of i n t e r e s t t h a t in m o s t c a s e s PGD2 i n h i b i t e d OX r e l e a s e by a p p r o x i m a t e l y 50% r e g a r d l e s s of w h e t h e r P G D 2 a n d F M L P w e r e u s e d at I 0 ~ or I0 ~ . For u n k n o w n r e a s o n s s u b j e c t C f r o m T a b l e 1 w a s m u c h m o r e s e n s i t i v e to P G D 2 t h a n o t h e r i n d i v i d u a l s t e s t e d . We f o u n d t h a t P G D 2 d e c r e a s e d b o t h the i n i t i a l r a t e a n d the m a x i m u m q u a n i t y of OX r e l e a s e (Table 111). P G D 2 a l o n e c a u s e d no c h a n g e in OX o v e r 15 min. p r e i n c u b a t i o n p e r i o d (data n o t shown). TABLE Effect
of P G D 2
Release
(I0~)
on the
Stimulated
Subject A B C D E F M e a n + S.E.
by
i0~
3 Initial
FMLP
Initial Rate 85.1 74.5 73.6 77.9 66.7 55.0 72.1+4.21"
Rate
and Emax
of OX
(as % of c o n t r o l ) Emax 46.1 79.5 56.2 48.6 50.3 41.4 53.7+5.5*
* = P < 0 . 0 5 c o n t r o l . I n i t i a l r a t e was 2.82 to 9.33 n M / M i n / l . 5 x 107 cells. D a t a are p r e s e n t e d as p e r c e n t of c o n t r o l OX. Control Emax w a s f r o m 5.43 to 2 7 . 9 9 n M o l e s / l . 5 x 107 cells. These data were o b t a i n e d f r o m 6 d i f f e r e n t s u b j e c t s in six d i f f e r e n t e x p e r i m e n t s . The r e d u c t i o n of the i n i t i a l r a t e of O X r e l e a s e by P G D 2 s u g g e s t s t h a t P G D 2 a c t e d u p o n the r a t e l i m i t i n g e n z y m e N A D P H o x i d a s e . This e f f e c t m a y be d i r e c t or i n d i r e c t . In o r d e r to d i s t i n g u i s h b e t w e e n t h e s e two p o s s i b i l i t i e s , we d e t e r m i n e d t h a t e f f e c t of P G D 2 on O X r e l e a s e s t i m u l a t e d w i t h PMA. PMA activates NADPH oxidase via a
Vol. 46, No. ii, 1990
Prostaglandins and Neutrophils
799
p a t h w a y w h i c h is d i f f e r e n t from t h a t of FMLP. PMA directly a c t i v a t e s p r o t e i n k i n a s e C, t h e r e b y b y p a s s i n g the F M L P r e c e p t o r and o t h e r e a r l y signal t r a n s d u c t i o n m e c h a n i s m s (6). We r e a s o n e d t h a t if PGD2 w e r e d i r e c t l y a c t i n g u p o n N A D P H o x i d a s e t h e n its i n h i b i t o r y e f f e c t on OX r e l e a s e s h o u l d be i n d e p e n d e n t of the p a t h w a y of N A D P H oxidase activation. We found that PGD2 had no e f f e c t on P M A s t i m u l a t e d OX r e l e a s e e v e n at PMA c o n c e n t r a t i o n s as low as 4 x I0~. L o w e r PMA c o n c e n t r a t i o n s did not s t i m u l a t e OX r e l e a s e (data not shown). T h e r e f o r e , PGD2 is not a c t i n g d i r e c t l y u p o n N A D P H oxidase. N e x t we e x a m i n e d the e f f e c t of PGD2 on (Ca)i u s i n g the f l u o r e s c e n t dye Fura2AM. PGD2 had no e f f e c t on the i n i t i a l r i s e in (Ca)i, a f t e r s t i m u l a t i o n by i x i 0 ~ FMLP. However, in the PGD2 t r e a t e d cells, the rate at w h i c h (Ca)i d e c r e a s e d a f t e r the i n i t i a l r i s e w a s more rapid than in c o n t r o l cells (Fig.3A) (p<0.05). Since p r o s t a g l a n d i n s h a v e b e e n shown to i n c r e a s e cAMP l e v e l s (7), we d e t e r m i n e d w h e t h e r cAMP m a y m e d i a t e the c h a n g e s in (Ca)i c a u s e d by PGD2. I n c u b a t i o n of cells w i t h d i b u t y r y l cAMP p r o d u c e d a c h a n g e in the r a t e of (Ca)i d e c l i n e s i m i l a r to that o b t a i n e d by u s i n g PGD2 (Fig. 3B). cAMP c a u s e d no c h a n g e in the rate of r e t u r n to b a s e l i n e (data not shown). DISCUSSION F a n t o n e and K i n n e s (8) d e m o n s t r a t e d t h a t p r o s t a g l a n d i n El a n d 12 i n h i b i t e d FMLP s t i m u l a t e d OX r e l e a s e in h u m a n n e u t r o p h i l s (8). T h e y found no e f f e c t of PGF2, w h i c h c o n t r a s t s w i t h our f i n d i n g s of a small effect. It is p o s s i b l e t h a t small d i f f e r e n c e s in t e c h n i q u e a c c o u n t for this d i s c r e p a n c y . By c o m p a r i n g two p r o s t a g l a n d i n s w h i c h d i f f e r e d in o n l y one s t r u c t u r a l aspect, we s h o w e d t h a t the c a r b o n y l g r o u p of PGD 2 was e s s e n t i a l to its i n h i b i t o r y effect. The fact t h a t a c a r b o n y l as a k e t o n e alone was not s u f f i c i e n t to i n h i b i t OX r e l e a s e was shown by the i n e f f e c t i v e n e s s of the ketones, i n c l u d i n g acetone. Lipophilicity of the ketone may be important in d e t e r m i n i n g potency, as d e m o n s t r a t e d by the i n c r e a s e d e f f e c t of 24 d i m e t h y l c y c l o p e n t a n o n e o v e r that of c y c l o p e n t a n o n e . Our p r e v i o u s e x p e r i m e n t s s h o w e d that c o n c e n t r a t i o n s equal to and less t h a n i0 ~ PDG2 had no e f f e c t on FMLP r e c e p t o r binding. Togni et al. s h o w e d t h a t 10 .3 cAMP c a u s e d a more r a p i d r e c o v e r y of (Ca)i l e v e l s b a c k to b a s e l i n e (9). Our r e s u l t s s u g g e s t t h a t PGD2 m a y i n h i b i t OX r e l e a s e via cAMP and (Ca)i. We f o u n d t h a t c A M P but not c G M P c a u s e s the same c h a n g e s in (Ca)i as does PGD2. Furthermore, PGF2, w h i c h had a s i g n i f i c a n t but small e f f e c t on OX r e l e a s e , h a d no e f f e c t on (Ca)i. The e x p e r i m e n t s w i t h PMA, in w h i c h PGD2 h a d no effect, e v e n at a PMA c o n c e n t r a t i o n as low as 4 x 10 .9 s h o w e d t h a t the PGD2 a c t i o n was l o c a l i z e d at a step e a r l i e r t h a n the a c t i v a t i o n of N A D P H oxidase. T h e r e f o r e , we s u g g e s t t h a t PGD2 i n t e r a c t s w i t h the n e u t r o p h i l in a c a r b o n y l - d e p e n d e n t m a n n e r to e f f e c t an i n c r e a s e in i n t r a c e l l u l a r cAMP w h i c h e n h a n c e s the i n i t i a l r a t e of d e c r e a s e in (Ca)i. It is also p o s s i b l e h o w e v e r t h a t PGD2 and cAMP a t t a i n s i m i l a r e f f e c t s t h r o u g h d i f f e r e n t m e c h a n i s m s . S i n c e p r o t e i n k i n a s e C and o t h e r k i n a s e s are Ca 2÷ d e p e n d e n t , it is p o s s i b l e that the d e c l i n e in (Ca)i c a u s e d by PGD2 leads to the i n h i b i t i o n of OX r e l e a s e (9). The l i p o p h i l i c i t y r e n d e r e d by the side c h a i n s of PGD2 m a y a l l o w the m o l e c u l e to e n t e r a c r i t i c a l a r e a of the cell m e m b r a n e . The h y d r o x y l g r o u p s at C9 and C15 m a y be i m p o r t a n t in a n c h o r i n g the p r o s t a g l a n d i n into an i m p o r t a n t p o s i t i o n
800
Prostaglandins and Neutrophils
Vol. 46, No. ii, 1990
/i hi U,I 0
-J I,I. I.M >
p-.
I.g w-
I 0
I 5
I 10 TIME
I 15
(rain)
W 0
I I
.........
0
I
i
I
5
10
15
i~ . . . . .
j
L- ..... I
0
J 2-D|BUTYRL c A M P ,
I
I
I
5
10
15
TIME (rain)
FIG.3 Effect of PGD2 Prostaglandins
and PGF2 = I0~.
on Intracellular Calcium. FMLP=IO~. cAMP = 10r~. Preincubation = 15 m i n .
Vol. 46, No. ii, 1990
Prostaglandlns and Neutrophils
by h y d r o g e n bonding. Further r e l a t i o n s h i p s of p r o s t a n g l a n d i n s needed.
801
w o r k on the s t r u c t u r e / f u n c t i o n and l i p o p h i l i c k e t o n e s is c l e a r l y
ACKNOWLEDGEMENTS This work was supported by the Naval Medical Research and D e v e l o p m e n t Command, W o r k Unit N.O. M 0 0 9 5 . 0 0 1 - I 0 0 5 . The o p i n i o n s and a s s e r t i o n s c o n t a i n e d h e r e i n are the p r i v a t e ones of the a u t h o r s and s h o u l d not be c o n s t r u e d as r e f l e c t i n g the views of the U.S. Navy, the naval service at large, or the D e p a r t m e n t of Defense. We g r a t e f u l l y a c k n o w l e d g e of the m a n u s c r i p t .
Mrs.
Carolyn
McCoy
for her
preparation
REFERENCES CC. HARDY, C. ROBINSON, A.E. T A T T E R S F I E L D , and S.J. HOLGATE, N. E n g l a n d J. of Medicine, 311 209-212 (1984) C.O. SIMPKINS, S.T. ALAILIMA, E.A. TATE, and M. JOHNSON. J. Surg. Res. 4__1 645-652 (1986) B.M. BABIOR, R.S. KIGNES, J.T. CURNETTE. J. Clin. Invest. 52 741-744 (1973) G. G R Y N K I E W I C Z , M. POENIE, and R.Y. TSIEN. J. Biol. Chem. 260 3 4 4 0 - 3 4 5 0 (1985) A.E. M A R T E L L and R.M. SMITH. C r i t i c a l S t a b i l i t y C o n s t a n t s , i, P l e n u m Press, NY (1974) J.E. NIEDEL, L.J. KUHN, G.R. V A N D E R B A R K . Proc. Nat'l. Acad. Sci. USA 75 3838-3822 (1978) T. T A K E N A W A G Q , J. ISHITOYA, Y. NAGIA. J. Biol. Chem. 261 10921098 (1986) J.C. F A N T O N E and D.A. KINNES. Biochem. Biophys. Res. Commun. 113 506-512 (1983) P. TOGNI, G. CARBRINI, F. DIVIRGILIO. Biochem. J. 224 629-635 (1984)
Pergamon Press
P R O S T A N G L A N D I N D2 M O D U L A T E S H U M A N N E U T R O P H I L I N T R A C E L L U L A R C A L C I U M F L U X A N D INHIBITS S U P E R O X I D E R E L E A S E V I A ITS R I N G CARBONYL C u t h b e r t O. S i m p k i n s ! * , Denise L. Mazorow*#, Sione T. A l a i l i m a * , E l i n A. Tate*, W i l l i a m Sweatt*, Mark Johnson*, K h a r e e m S h a r i f f * and D a v i d B. Millar*" *Naval M e d i c a l R e s e a r c h I n s t i t u t e Bethesda, M a r y l a n d 20814-5055 ~ D i s t r i c t of C o l u m b i a General H o s p i t a l W a s h i n g t o n , D.C. 20003 (Received in final form January 17, 1990) Summary We c o m p a r e d the effects of p r o s t a g l a n d i n D 2 (PGD2), p r o s t a g l a n d i n F 2 alpha (PGF2) and v a r i o u s k e t o n e s on s u p e r o x i d e (OX) r e l e a s e by h u m a n n e u t r o p h i l s , w h i c h h a d been stimulated by N-formyl methionyl leucyl p h e n y l a l a n i n e (FMLP). Our data s u g g e s t e d that the r i n g carbonyl of PGD2 is e s s e n t i a l to its i n h i b i t o r y e f f e c t on OX release, but the carbonyl g r o u p as a ketone, alone is not sufficient. U s i n g the f l u o r e s c e n t Ca 2* probe, Fura-2AM, we found that PGD2 i n c r e a s e d the rate of d e c l i n e of FMLP s t i m u l a t e d i n t r a c e l l u l a r free Ca z+ (Ca)i, but that PGF2 had no effect, cAMP altered FMLP stimulated (Ca)i, in a pattern similar to PGD2. F u r t h e r m o r e , the ring c a r b o n y l of PGD2 is crucial to its e f f e c t on OX as well as on (Ca)i. P r o s t a g l a n d i n D2 (PGD2) is the p r o s t a n o i d w h i c h is m o s t a b u n d a n t l y r e l e a s e d by s t i m u l a t e d a l v e o l a r m a s t cells. (I). P r e v i o u s l y , we demonstrated that the r e l e a s e of OX by FMLP s t i m u l a t e d h u m a n n e u t r o p h i l s was i n h i b i t e d by PGD2. We found that PGD2 was m o r e p o t e n t t h a n PGF2, and s u g g e s t e d that the crucial d e t e r m i n a n t of p o t e n c y was the ring carbonyl group on PGD2. We also found t h a t PGD2 did not compete w i t h FMLP at the FMLP r e c e p t o r site (2). In this p r e s e n t c a r b o n y l g r o u p as also e x t e n d e d our by PGD2 t h r o u g h s t i m u l a t e d (Ca)i.
work, we t e s t e d the h y p o t h e s i s that o n l y the a k e t o n e was n e c e s s a r y for i n h i b i t i o n of OX. We u n d e r s t a n d i n g of the m e c h a n i s m of OX i n h i b i t i o n o b s e r v i n g the e f f e c t of PGD2 and cAMP on FMLP
C o r r e s p o n d e n c e should be a d d r e s s e d to C u t h b e r t O. Simpkins, M.D., Department of Surgery, D.C. General Hospital, 19th and M a s s a c h u s e t t s Avenue, S.E., W a s h i n g t o n , D.C. P r e s e n t addresses: # D e p a r t m e n t of Pathology, U n i f o r m e d S e r v i c e s U n i v e r s i t y of the H e a l t h Sciences, Bethesda, M a r y l a n d 20814 ~ a b o r a t o r y of B i o c h e m i c a l Genetics, N a t i o n a l I n s t i t u t e of M e n t a l Health, N e u r o s c i e n c e C e n t e r at St. Elizabeths, W a s h i n g t o n , D.C. 20032 0024-3205/90 $3.00 + .00 Copyright (c) 1990 Pergamon Press plc
794
Prostaglandins and Neutrophils
Vol. 46, No. ii, 1990
Methods Isolation
of N e u t r o p h i l s
B l o o d w a s o b t a i n e d by v e n i p u n c t u r e f r o m h e a l t h y , m e d i c a t i o n - f r e e volunteers. P r e s e r v a t i v e - f r e e h e p a r i n (Gibco, G r a n d I s l a n d , N . Y . ) w a s u s e d at a c o n c e n t r a t i o n of 12.5 u n i t s p e r ml of b l o o d for a n t i c o a g u l a t i o n . E r y t h r o c y t e s w e r e r e m o v e d by g r a v i t y s e d i m e n t a t i o n through 2% d e x t r a n (Pharmacia, Upsala, Sweden) for 30 min. L y m p h o c y t e S e p a r a t i o n M e d i u m ( L i t t o n B i o n e t i c s , K e n s i n g t o n , MD) w a s l a y e r e d u n d e r the s u p e r n a t a n t a n d c e n t r i f u g e d at 300 x g, at r o o m temperature for I0 min. R e s i d u a l e r y t h r o c y t e s w e r e r e m o v e d by h y p o t o n i c l y s i s a n d the c e l l s w e r e c e n t r i f u g e d a g a i n at 150 x g. The neutrophil pellet was suspended in H a n k ' s Balanced Salt Solution (HBSS, Gibco, Grand Island, NY) to the desired concentration. T h i s p r o c e d u r e y i e l d e d 96% n e u t r o p h i l s by W r i g h t ' s stain with a viability of at l e a s t 95% by t r y p a n blue dye exclusion. Assay
of OX
The s u p e r o x i d e d i s m u t a s e - i n h i b i t a b l e r e d u c t i o n of c y t o c h r o m e C a s s a y of B a b i o r et. al, w a s e m p l o y e d w i t h o u t c y t o c h a l a s i n B. (3). Neutrophils were preincubated for 15 min. with various concentrations of t e s t reagents with HBSS as control. After preincubation 500 ml of n e u t r o p h i l s u s p e n s i o n w a s a d d e d to the r e a c t i o n m i x t u r e in 12 x 75 m m p o l y s t y r e n e t u b e s c o n t a i n i n g 1,500 ml HBSS, t e s t r e a g e n t s (250 ml) at v a r i o u s c o n c e n t r a t i o n s a n d 50 uM cytochrome C (Sigma, St. Louis, M0). The final cell c o n c e n t r a t i o n w a s Ixl0~/ml. A f t e r a d d i t i o n of 25 ul of F M L P at a final concentration of 10 .6 or 10 .7 M, or H B S S as c o n t r o l , the r e a c t a n t s w e r e m i x e d a n d a i ml a l i q u o t was p l a c e d i n t o 12 x 75 m m p l a s t i c t u b e s w h i c h c o n t a i n e d i0 ul s u p e r o x i d e d i s m u t a s e (SOD: B o e h r i n g e r M a n n h e i m , W. G e r m a n y ) at a f i n a l c o n c e n t r a t i o n of 50 mg/ml. B o t h the t u b e s to w h i c h SOD h a d b e e n a d d e d a n d t h o s e w i t h o u t S O D w e r e i n c u b a t e d at 37 ° for I0 min. At the e n d of the i n c u b a t i o n p e r i o d 15 ul of SOD, a l s o at a f i n a l c o n c e n t r a t i o n of 50 m g / m l , w a s a d d e d to the t u b e s to w h i c h S O D h a d n o t b e e n a d d e d initially. C e n t r i f u g a t i o n was at 150 x g a n d a b s o r b a n c e of the s u p e r n a t a n t at 550 nm was d e t e r m i n e d . OX r e l e a s e w a s c a l c u l a t e d as the d i f f e r e n c e in a b s o r b a n c e b e t w e e n the t u b e s w h i c h r e c e i v e d S O D at the b e g i n n i n g , d i v i d e d by an e x t i n c t i o n c o e f f i c i e n t of 2 1 , 0 0 0 M I. C e l l v i a b i l i t y at all c o n c e n t r a t i o n s of P G D 2 (SIGMA, St. L o u i s , MO) w a s 95% or g r e a t e r . A s s a y s w e r e p e r f o r m e d in d u p l i c a t e . T h e e f f e c t of P G D 2 u p o n the i n i t i a l r a t e of r e a c t i o n a n d the m a x i m u m OX r e l e a s e d w a s m e a s u r e d as f o l l o w s : The r e a c t i o n m i x t u r e c o n t a i n e d 3 x 106 c e l l s / m l a n d 50 u M c y t o c h r o m e C a n d H B S S in a t o t a l v o l u m e of 3 ml. P r e i n c u b a t i o n w a s in p l a s t i c c u v e t t e s at 37 ° for 15 min. w i t h v a r i o u s c o n c e n t r a t i o n s of t e s t r e a g e n t s . The cuvettes were then placed into a temperature-controlled (37~)5 d u a l beam spectrophotometer with a magnetic stirrer. F M L P at i0- or 10 .7 M or PMA at 4 x 10 .9 M or g r e a t e r was a d d e d to one c u v e t t e a n d c o n t r o l s o l u t i o n a d d e d to the other. The r e a c t i o n w a s m o n i t o r e d c o n t i n u o u s l y u n t i l a m a x i m u m w a s r e a c h e d , w h i c h for F M L P t o o k 5-8 min. a n d w i t h P M A t o o k as l o n g as 60 min., w h e n l o w c o n c e n t r a t i o n s (4 x 10 .9) w e r e used.
Vol. 46, No. ii, 1990
Measurement
Prostaglandins and Neutrophils
of I n t r a c e l l u l a r
795
Calcium
5 x 106 cells w e r e l a b e l l e d w i t h the m e m b r a n e p e r m e a n t p r o b e F u r a 2AM ( C a l b i o c h e m La-Jolla, Calif.) at 37 o in a P e r k i n E l m e r M o d e l 44B s p e c t r o p h o t o f l u o r o m e t e r . The cells w e r e c o n t i n u a l l y s t i r r e d during measurement. A l t e r n a t i v e e ~ c i t a t i o n w a v e l e n g t h s of 335 nm and 362 nm (slit 4 nm) w e r e e m p l o y e d and e m i s s i o n was m e a s u r e d at 510 nm (slit 8 nm). C a l c i u m ion c o n c e n t r a t i o n s w e r e c a l c u l a t e d by the t e c h n i q u e of G r y n k i e w i c z et al. (4) u s i n g the c o n s t a n t s of M a r t e l l and S m i t h (5). FMLP control spectra w e r e r u n p r i o r to and a f t e r each e x p e r i m e n t a l scan. Since our r e s t i n g c a l c i u m l e v e l s determined with FURA-2AM were so m u c h lower, we i n v e s t i g a t e d w h e t h e r Q u i n - 2 A M gave us the same value for (Ca)i w i t h F U R A - 2 A M of 16- 20 nM, w i t h Q U I N - 2 A M we had r e s t i n g levels of (Ca)i from several i n d i v i d u a l s similar to those r e p o r t e d by o t h e r s of 100200nM. I n v e s t i g a t a t i o n w i t h o t h e r cell lines gave s i m i l a r r e s u l t s ( m a n u s c r i p t in p r e p a r a t i o n , D:L. M a z o r o w and D.B. Millar). DMSO at the c o n c e n t r a t i o n s u s e d had no e f f e c t on (Ca)i. Reagents All r e a g e n t s w e r e p r e p a r e d f r e s h l y each day. FMLP, p r o s t a g l a n d i n s , l i p o p h i l i c ketones, cAMP, cGMP, and phorbol m y r i s t a t e a c e t a t e (PMA) w e r e d i s s o l v e d in d i m e t h y l s u l f o x i d e (DMSO). Control solutions c o n t a i n e d equal amounts of DMSO. All of these r e a g e n t s w e r e o b t a i n e d from Sigma (St. Louis, MO.). The ketones, and a l c o h o l s w e r e o b t a i n e d from A l d r i c h (Milwaukee, Wis.). Statistical
Analysis
All e x p e r i m e n t s w e r e of p a i r e d design. The p a i r e d S t u d e n t ' s t - t e s t was employed. P< 0.05 was c o n s i d e r e d significant. Unstimulated O X r e l e a s e was s u b s t r a c t e d from all values. The d e r i v e d number, % of control, was u s e d b e c a u s e of the large v a r i a b i l i t y in F M L P r e s p o n s e among individuals. % of control was c a l c u l a t e d for e a c h e x p e r i m e n t as OX r e l e a s e w i t h test r e a g e n t s over OX r e l e a s e w i t h o u t test r e a g e n t times i00. S t a t i s t i c a l a n a l y s i s of data e x p r e s s e d as p e r c e n t a g e s was t r a n s f o r m e d b e c a u s e p e r c e n t a g e s are n o t n o r m a l l y distributed. The transformation used was a n g l e = A r c s i n ( p e r c e n t a g e ) 5. The rate of d e c r e a s e in i n t r a c e l l u l a r c a l c i u m was d e t e r m i n e d by m e a s u r i n g the d e c r e a s e that o c c u r e d as of one m i n u t e after the m a x i m u m F M L P - s t i m u l a t e d intracellular calcium had been attained. These data were expressed as percentages of control and transformed using the arcsin t r a n f o r m a t i o n as above. Results P r e v i o u s e x p e r i m e n t s showed no e f f e c t of PGD2 on the the r e l e a s e of OX g e n e r a t e d by a c e l l - f r e e x a n t h i n e - x a n t h i n e o x i d a s e s y s t e m (2). In six e x p e r i m e n t s performed with neutrophils from six i n d i v i d u a l s a b i p h a s i c dose r e s p o n s e curve of the i n h i b i t i o n of O X r e l e a s e by PGD2 was found (Fig. I). The first p h a s e o c c u r r e d b e t w e e n 10 .9 and I0 ~ M and showed an i n h i b i t o r y e f f e c t of up to 64%. In this c o n c e n t r a t i o n range PGD2 had no e f f e c t on FMLP binding (2). In the second phase, b e t w e e n 10 .5 and 10 .4 M, OX r e l e a s e was p r a c t i c a l l y e l i m i n a t e d . This phase c o r r e s p o n d s to the c o n c e n t r a t i o n at w h i c h PGD2 d e c r e a s e s FMLP binding.
796
Prostaglandins and Neutrophils
100
i
Vol. 46, No. Ii, 1990
I
7[
u3 90 +1 IJJ
'~ IJJ
8O 70 605o
O
30'
1,1.,
O o.e, 2 0 10I
I
I
-11 - 1 0 - 9 - 8 - 7 - 6 - 5 - 4 LOG PGD 2 CONCENTRATION (M)
-3
FIG.I C e l l s w e r e s t i m u l a t e d w i t h 10 .6 M FMLP. Results are expressed as the p e r c e n t of c o n t r o l O X r e l e a s e + S.E. in the a b s e n c e of PGD2. U n s t i m u l a t e d O X r e l e a s e is s u b t r a c t e d f r o m all v a l u e s . E a c h p o i n t is the a v e r a g e of six e x p e r i m e n t s performed with c e l l s f r o m six d i f f e r e n t i n d i v i d u a l s .
PGD2 is i d e n t i c a l to PGF2 e x c e p t t h a t PGF2 has an h y d r o x y l g r o u p i n s t e a d of a c a r b o n y l g r o u p at the Cll p o s i t i o n . The structures a r e s h o w n in Fig.2.
Vol. 46, No. ii, 1990
Prostaglandins and Neutrophils
797
HO
~ ~
POD2
0
COOH OH
HO
PGF2~ ~ HO
COOH OH
FIG.2 Comparative
Structures
of PGD2
and P G F 2
T h e i m p o r t a n c e of the r i n g c a r b o n y l w a s d e m o n s t r a t e d by d i r e c t l y c o m p a r i n g the e f f e c t of P G D 2 to t h a t of P G F 2 on O X r e l e a s e ( T a b l e i).
TABLE C o m p a r i s o n of the E f f e c t of in R e s p o n s e to F M L P ( i 0 ~ ) .
Mean P<
Subject A B C D E + S.E.
0.005
for
1 0 7 M PGD2 a n d 1 0 7 M PGF2 on O X R e l e a s e E x p r e s s e d as % of C o n t r o l
PGD2 50.0 51.0 8.1 46.6 39.8 39.1+7.3 significant
1
difference
PGF2 86.4 98.5 91.5 94. 1 69.8 88.1+4.5 of P G D 2
Ratio 0 0 0 0 0 0
PGPD2/PGF2 58 52 089 50 57 46+0.094
from PGF2
P< 0 . 0 0 5 for d i f f e r e n c e s b e t w e e n P G F 2 and c o n t r o l a n d P G D 2 a n d control. Both PGD2 and PGF2 inhibited OX release but the i n h i b i t o r y e f f e c t of P G D 2 w a s g r e a t e r . These results suggest that a m a j o r d e t e r m i n a n t of the i n h i b i t i o n of O X r e l e a s e by P G D 2 is t h e c a r b o n y l g r o u p s i n c e the o n l y d i f f e r e n c e b e t w e e n P G D 2 a n d P G F 2 is a Cll c a r b o n y l and h y d r o x y l g r o u p , r e s p e c t i v e l y . N o n e of the k e t o n e s or a l c o h o l s c a u s e d a m a r k e d d e c r e a s e in OX r e l e a s e .
798
Prostaglandins and Neutrophils
TABLE Effect
of K e t o n e s
2
and A l c o h o l s
Reagent (i0~) Cyclopentanone Cyclopentanol 2,4 D i m e t h y l c y c l o p e n t a n o n e Methanol Acetone PGD2 PGF2
Vol. 46, No. ii, 1990
on O X R e l e a s e
% of C o n t r o l 9 8 . 4 ~ 6.0 8 8 . 5 ~ 5.1 7 4 . 6 ~ 7.8* 81.5+ 4.0* 87.7+ 6.8 54.5+ 3.3* 74.2+ 5.3*
+ S.E.
*= s i g n i f i c a n t l y d i f f e r e n t f r o m OX r e l e a s e in the a b s e n c e of a n y test reagent with p<0.05.10~ F M L P was u s e d to s t i m u l a t e the cells. A l s o i n c l u d e d w i t h e a c h e x p e r i m e n t w e r e P G D 2 a n d P G F 2 at I 0 ~ as positive controls. We c o n c l u d e f r o m t h e s e e x p e r i m e n t s t h a t the c a r b o n y l g r o u p a l o n e as a k e t o n e is n o t s u f f i c i e n t to i n h i b i t OX r e l e a s e to a l a r g e d e g r e e . 2,4 d i m e t h y l c y c l o p e n t a n o n e is m o r e lipophilic than cyclopentanone and decreased OX release s i g n i f i c a n t l y w h i l e c y c l o p e n t a n o n e d i d not. This result suggests that lipophilicity is a f a c t o r in the i n h i b i t o r y e f f e c t of a ketone. It is a l s o of i n t e r e s t t h a t in m o s t c a s e s PGD2 i n h i b i t e d OX r e l e a s e by a p p r o x i m a t e l y 50% r e g a r d l e s s of w h e t h e r P G D 2 a n d F M L P w e r e u s e d at I 0 ~ or I0 ~ . For u n k n o w n r e a s o n s s u b j e c t C f r o m T a b l e 1 w a s m u c h m o r e s e n s i t i v e to P G D 2 t h a n o t h e r i n d i v i d u a l s t e s t e d . We f o u n d t h a t P G D 2 d e c r e a s e d b o t h the i n i t i a l r a t e a n d the m a x i m u m q u a n i t y of OX r e l e a s e (Table 111). P G D 2 a l o n e c a u s e d no c h a n g e in OX o v e r 15 min. p r e i n c u b a t i o n p e r i o d (data n o t shown). TABLE Effect
of P G D 2
Release
(I0~)
on the
Stimulated
Subject A B C D E F M e a n + S.E.
by
i0~
3 Initial
FMLP
Initial Rate 85.1 74.5 73.6 77.9 66.7 55.0 72.1+4.21"
Rate
and Emax
of OX
(as % of c o n t r o l ) Emax 46.1 79.5 56.2 48.6 50.3 41.4 53.7+5.5*
* = P < 0 . 0 5 c o n t r o l . I n i t i a l r a t e was 2.82 to 9.33 n M / M i n / l . 5 x 107 cells. D a t a are p r e s e n t e d as p e r c e n t of c o n t r o l OX. Control Emax w a s f r o m 5.43 to 2 7 . 9 9 n M o l e s / l . 5 x 107 cells. These data were o b t a i n e d f r o m 6 d i f f e r e n t s u b j e c t s in six d i f f e r e n t e x p e r i m e n t s . The r e d u c t i o n of the i n i t i a l r a t e of O X r e l e a s e by P G D 2 s u g g e s t s t h a t P G D 2 a c t e d u p o n the r a t e l i m i t i n g e n z y m e N A D P H o x i d a s e . This e f f e c t m a y be d i r e c t or i n d i r e c t . In o r d e r to d i s t i n g u i s h b e t w e e n t h e s e two p o s s i b i l i t i e s , we d e t e r m i n e d t h a t e f f e c t of P G D 2 on O X r e l e a s e s t i m u l a t e d w i t h PMA. PMA activates NADPH oxidase via a
Vol. 46, No. ii, 1990
Prostaglandins and Neutrophils
799
p a t h w a y w h i c h is d i f f e r e n t from t h a t of FMLP. PMA directly a c t i v a t e s p r o t e i n k i n a s e C, t h e r e b y b y p a s s i n g the F M L P r e c e p t o r and o t h e r e a r l y signal t r a n s d u c t i o n m e c h a n i s m s (6). We r e a s o n e d t h a t if PGD2 w e r e d i r e c t l y a c t i n g u p o n N A D P H o x i d a s e t h e n its i n h i b i t o r y e f f e c t on OX r e l e a s e s h o u l d be i n d e p e n d e n t of the p a t h w a y of N A D P H oxidase activation. We found that PGD2 had no e f f e c t on P M A s t i m u l a t e d OX r e l e a s e e v e n at PMA c o n c e n t r a t i o n s as low as 4 x I0~. L o w e r PMA c o n c e n t r a t i o n s did not s t i m u l a t e OX r e l e a s e (data not shown). T h e r e f o r e , PGD2 is not a c t i n g d i r e c t l y u p o n N A D P H oxidase. N e x t we e x a m i n e d the e f f e c t of PGD2 on (Ca)i u s i n g the f l u o r e s c e n t dye Fura2AM. PGD2 had no e f f e c t on the i n i t i a l r i s e in (Ca)i, a f t e r s t i m u l a t i o n by i x i 0 ~ FMLP. However, in the PGD2 t r e a t e d cells, the rate at w h i c h (Ca)i d e c r e a s e d a f t e r the i n i t i a l r i s e w a s more rapid than in c o n t r o l cells (Fig.3A) (p<0.05). Since p r o s t a g l a n d i n s h a v e b e e n shown to i n c r e a s e cAMP l e v e l s (7), we d e t e r m i n e d w h e t h e r cAMP m a y m e d i a t e the c h a n g e s in (Ca)i c a u s e d by PGD2. I n c u b a t i o n of cells w i t h d i b u t y r y l cAMP p r o d u c e d a c h a n g e in the r a t e of (Ca)i d e c l i n e s i m i l a r to that o b t a i n e d by u s i n g PGD2 (Fig. 3B). cAMP c a u s e d no c h a n g e in the rate of r e t u r n to b a s e l i n e (data not shown). DISCUSSION F a n t o n e and K i n n e s (8) d e m o n s t r a t e d t h a t p r o s t a g l a n d i n El a n d 12 i n h i b i t e d FMLP s t i m u l a t e d OX r e l e a s e in h u m a n n e u t r o p h i l s (8). T h e y found no e f f e c t of PGF2, w h i c h c o n t r a s t s w i t h our f i n d i n g s of a small effect. It is p o s s i b l e t h a t small d i f f e r e n c e s in t e c h n i q u e a c c o u n t for this d i s c r e p a n c y . By c o m p a r i n g two p r o s t a g l a n d i n s w h i c h d i f f e r e d in o n l y one s t r u c t u r a l aspect, we s h o w e d t h a t the c a r b o n y l g r o u p of PGD 2 was e s s e n t i a l to its i n h i b i t o r y effect. The fact t h a t a c a r b o n y l as a k e t o n e alone was not s u f f i c i e n t to i n h i b i t OX r e l e a s e was shown by the i n e f f e c t i v e n e s s of the ketones, i n c l u d i n g acetone. Lipophilicity of the ketone may be important in d e t e r m i n i n g potency, as d e m o n s t r a t e d by the i n c r e a s e d e f f e c t of 24 d i m e t h y l c y c l o p e n t a n o n e o v e r that of c y c l o p e n t a n o n e . Our p r e v i o u s e x p e r i m e n t s s h o w e d that c o n c e n t r a t i o n s equal to and less t h a n i0 ~ PDG2 had no e f f e c t on FMLP r e c e p t o r binding. Togni et al. s h o w e d t h a t 10 .3 cAMP c a u s e d a more r a p i d r e c o v e r y of (Ca)i l e v e l s b a c k to b a s e l i n e (9). Our r e s u l t s s u g g e s t t h a t PGD2 m a y i n h i b i t OX r e l e a s e via cAMP and (Ca)i. We f o u n d t h a t c A M P but not c G M P c a u s e s the same c h a n g e s in (Ca)i as does PGD2. Furthermore, PGF2, w h i c h had a s i g n i f i c a n t but small e f f e c t on OX r e l e a s e , h a d no e f f e c t on (Ca)i. The e x p e r i m e n t s w i t h PMA, in w h i c h PGD2 h a d no effect, e v e n at a PMA c o n c e n t r a t i o n as low as 4 x 10 .9 s h o w e d t h a t the PGD2 a c t i o n was l o c a l i z e d at a step e a r l i e r t h a n the a c t i v a t i o n of N A D P H oxidase. T h e r e f o r e , we s u g g e s t t h a t PGD2 i n t e r a c t s w i t h the n e u t r o p h i l in a c a r b o n y l - d e p e n d e n t m a n n e r to e f f e c t an i n c r e a s e in i n t r a c e l l u l a r cAMP w h i c h e n h a n c e s the i n i t i a l r a t e of d e c r e a s e in (Ca)i. It is also p o s s i b l e h o w e v e r t h a t PGD2 and cAMP a t t a i n s i m i l a r e f f e c t s t h r o u g h d i f f e r e n t m e c h a n i s m s . S i n c e p r o t e i n k i n a s e C and o t h e r k i n a s e s are Ca 2÷ d e p e n d e n t , it is p o s s i b l e that the d e c l i n e in (Ca)i c a u s e d by PGD2 leads to the i n h i b i t i o n of OX r e l e a s e (9). The l i p o p h i l i c i t y r e n d e r e d by the side c h a i n s of PGD2 m a y a l l o w the m o l e c u l e to e n t e r a c r i t i c a l a r e a of the cell m e m b r a n e . The h y d r o x y l g r o u p s at C9 and C15 m a y be i m p o r t a n t in a n c h o r i n g the p r o s t a g l a n d i n into an i m p o r t a n t p o s i t i o n
800
Prostaglandins and Neutrophils
Vol. 46, No. ii, 1990
/i hi U,I 0
-J I,I. I.M >
p-.
I.g w-
I 0
I 5
I 10 TIME
I 15
(rain)
W 0
I I
.........
0
I
i
I
5
10
15
i~ . . . . .
j
L- ..... I
0
J 2-D|BUTYRL c A M P ,
I
I
I
5
10
15
TIME (rain)
FIG.3 Effect of PGD2 Prostaglandins
and PGF2 = I0~.
on Intracellular Calcium. FMLP=IO~. cAMP = 10r~. Preincubation = 15 m i n .
Vol. 46, No. ii, 1990
Prostaglandlns and Neutrophils
by h y d r o g e n bonding. Further r e l a t i o n s h i p s of p r o s t a n g l a n d i n s needed.
801
w o r k on the s t r u c t u r e / f u n c t i o n and l i p o p h i l i c k e t o n e s is c l e a r l y
ACKNOWLEDGEMENTS This work was supported by the Naval Medical Research and D e v e l o p m e n t Command, W o r k Unit N.O. M 0 0 9 5 . 0 0 1 - I 0 0 5 . The o p i n i o n s and a s s e r t i o n s c o n t a i n e d h e r e i n are the p r i v a t e ones of the a u t h o r s and s h o u l d not be c o n s t r u e d as r e f l e c t i n g the views of the U.S. Navy, the naval service at large, or the D e p a r t m e n t of Defense. We g r a t e f u l l y a c k n o w l e d g e of the m a n u s c r i p t .
Mrs.
Carolyn
McCoy
for her
preparation
REFERENCES CC. HARDY, C. ROBINSON, A.E. T A T T E R S F I E L D , and S.J. HOLGATE, N. E n g l a n d J. of Medicine, 311 209-212 (1984) C.O. SIMPKINS, S.T. ALAILIMA, E.A. TATE, and M. JOHNSON. J. Surg. Res. 4__1 645-652 (1986) B.M. BABIOR, R.S. KIGNES, J.T. CURNETTE. J. Clin. Invest. 52 741-744 (1973) G. G R Y N K I E W I C Z , M. POENIE, and R.Y. TSIEN. J. Biol. Chem. 260 3 4 4 0 - 3 4 5 0 (1985) A.E. M A R T E L L and R.M. SMITH. C r i t i c a l S t a b i l i t y C o n s t a n t s , i, P l e n u m Press, NY (1974) J.E. NIEDEL, L.J. KUHN, G.R. V A N D E R B A R K . Proc. Nat'l. Acad. Sci. USA 75 3838-3822 (1978) T. T A K E N A W A G Q , J. ISHITOYA, Y. NAGIA. J. Biol. Chem. 261 10921098 (1986) J.C. F A N T O N E and D.A. KINNES. Biochem. Biophys. Res. Commun. 113 506-512 (1983) P. TOGNI, G. CARBRINI, F. DIVIRGILIO. Biochem. J. 224 629-635 (1984)