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Synthesis of prostaglandin F by cultured human endometrial cells
PROSTAGLANDINS
S Y N T H E S I S OF P R O S T A G L A N D I N F BY C U L T U R E D HUMAN E N D O M E T R I A L CELLS S.J.M.
Skinner,
G.C.Liggins,
T . W i l s o n and G. Neale
P o s t g r a d u a t e School of O b s t e t r i c s U n i v e r s i t y of Auckland, Auckland,
and Gynaecology, New Zealand.
ABSTRACT Human e n d o m e t r i a l cells were d i s p e r s e d with c o l l a g e n a s e and m a i n t a i n e d in culture overnight. The synthesis of PGF by the d i s p e r s e d cells i n c u b a t e d at 37oc in s e r u m - f r e e m e d i u m was s t i m u l a t e d b y _ e s t r a d i o l ( 1 0 - 7 M - 10-5M). h i s t a m i n e (5x10-7M - 5xl0-bM), b r a d y k i n i n (10-6M), p h o { b o l m y r i s t a t e (PMA, 3x10-SM) and a r a c h i d o n a t e (5xl0-UM). P r e i n c u b a t i o n of the cells for 3 h w i t h c o r t i s o l (5x10-7M - 5x10-5M), p r o g e s t e r o n e (10-6M) or m e p a c r i n e (10-6M - 2x10-4M) i n h i b i t e d the r e s p o n s e to histamine, b r a d y k i n i n and PMA but not to arachidonate. P e r i f u s i o n of the c u l t u r e d cells in f i l t r a t i o n chambers y i e l d e d s i m i l a r results to those obtained in the i n c u b a t i o n system but d i f f e r e n c e s in the onset and d u r a t i o n of the r e s p o n s e s to stimuli were found. In the p e r i f u s i o n s y s t e m the r e s p o n s e s to h i s t a m i n e and b r a d y k i n i n were rapid and of short d u r a t i o n (peak r e s p o n s e in <60 min) w h i l e the r e s p o n s e s to PMA and a r a c h i d o n a t e were of longer d u r a t i o n with a slower onset. We c o n c l u d e that these o b s e r v a t i o n s u s i n g d i s p e r s e d e n d o m e t r i a l cells are c o n s i s t e n t w i t h p r e v i o u s work showing that histamine, b r a d y k i n i n and PMA act by s t i m u l a t i n g a c y l h y d r o l a s e activity, thereby l i b e r a t i n g p r e c u r s o r s such as a r a c h i d o n i c acid w h i c h are c o n v e r t e d to p r o s t a g l a n d i n s by the c y c l o - o x y g e n a s e complex. INTRODUCTION The m e c h a n i s m c o n t r o l l i n g the i n i t i a t i o n of human p a r t u r i t i o n p o s s i b l y takes the form of an i n t e r a c t i o n b e t w e e n the cells of the amnion and c h o r i o n and those of the e n d o m e t r i u m (i) . The i n t e r a c t i o n may result in the i n h i b i t i o n during p r e g n a n c y of p r o s t a g l a n d i n p r o d u c t i o n by the e n d o m e t r i u m f o l l o w e d by d e c l i n i n g i n h i b i t i o n a n d / o r i n c r e a s i n g s t i m u l a t i o n at parturition. The study of such i n t e r a c t i o n s r e q u i r e s a system that will detect the p r e s e n c e of i n h i b i t o r y or s t i m u l a t o r y m a t e r i a l s a c t i n g at any point in the b i o s y n t h e t i c p a t h w a y
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of p r o s t a g l a n d i n s . Microsomal prostaglandin synthetase, p r e p a r e d from bovine seminal vesicles, may be used to detect c y c l o - o x y g e n a s e inhibitors (2) but not inhibitors of a r a c h i d o n i c acid release. A w h o l e cell system is e s s e n t i a l if p o t e n t i a l l y i m p o r t a n t b i o l o g i c a l l y active m a t e r i a l s are not to be overlooked. Furthermore, tissue and species s p e c i f i c i t y is possible. Accordingly, we have d e v e l o p e d a d i s p e r s e d cell p r e p a r a t i o n of human endometrium. R e q u i r e m e n t s of the p r e p a r a t i o n are that the cells have a low basal p r o d u c t i o n of p r o s t a g l a n d i n s i n d i c a t i n g r e c o v e r y from trauma, that they r e s p o n d in a d e f i n e d m a n n e r to known p h y s i o l o g i c a l i n h i b i t o r s and stimuli and that s e n s i t i v i t y to these agents is not lost as a result of p r o l o n g e d culture. MATERIALS AND METHODS P r e p a r a t i o n of Cells Uteri were o b t a i n e d from p a t i e n t s u n d e r g o i n g h y s t e r e c t o m y or d i l a t a t i o n and c u r e t t a g e for benign disease (e.g. fibroids, d y s f u n c t i o n m e n o r r h a g i a ) . The patients were of r e p r o d u c t i v e age and were not r e c e i v i n g t r e a t m e n t with hormones. The p r o j e c t was a p p r o v e d by the H o s p i t a l Ethics Committee. E n d o m e t r i a were c l a s s i f i e d by m e n s t r u a l stage as d e s c r i b e d p r e v i o u s l y (3). The u t e r i n e tissues were p r o c e s s e d under a s e p t i c c o n d i t i o n s w i t h i n 30 min of o p e r a t i v e removal. The uterus was opened and the e n d o m e t r i u m was g e n t l y excised w i t h a blunt spatula and p l a c e d in HEPES b u f f e r e d saline (pH 7.3) s u p p l e m e n t e d w i t h glucose (2 mg/ml) and bovine a l b u m i n g/ml) at 37°C. The tissue was finely chopped (i - 2 mm 3 and then t r a n s f e r r e d to fresh HEPES b u f f e r e d saline c o n t a i n i n g c o l l a g e n a s e (0.5 mg/ml) and s u p p l e m e n t e d as above. This m i x t u r e was s t i r r e d at 37°C for 70 min. C u r e t t i n g s were c o l l e c t e d d i r e c t l y into culture m e d i u m (Medium 199, E a r l e ' s Salts), t r a n s f e r r e d to HEPES b u f f e r e d saline and then t r e a t e d as for e n d o m e t r i a from uteri. The c o l l a g e n a s e - d i g e s t e d m a t e r i a l was f i l t e r e d through a i00 ~m sieve to remove u n d i g e s t e d and fibrous m a t e r i a l s and the filtrate c o n t a i n i n g the d i s p e r s e d cells was c e n t r i f u g e d at 500 x g for 5 min at 20°C. The pellet was w a s h e d twice by r e s u s p e n d i n g in culture m e d i u m and centrifuging. The cell p e l l e t was r e s u s p e n d e d in culture m e d i u m s u p p l e m e n t e d with bovine fetal serum (5% v/v) in a sterile p l a s t i c jar and cultured o v e r n i g h t at 37oc with an a i r / C O 2 (95:5 v/v) gas phase. The cells were h a r v e s t e d by gently m i x i n g the s e d i m e n t e d cells and media with a sterile plastic p i p e t t e and then t r a n s f e r r e d
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to a glass tube and c e n t r i f u g e d . The cell p e l l e t was w a s h e d twice in d e f i n e d m e d i u m (Medium 199, Hank's B a l a n c e d Salts) to remove s e r u m and the p r o d u c t s of o v e r n i g h t culture. The pellet was r e s u s p e n d e d in d e f i n e d medium. A small p o r t i o n was stained w i t h T r y p a n Blue and the number of live cells (40 - 70%) was d e t e r m i n e d in a h e m o c y t o m e t e r . The r e m a i n i n g cells w e r e d i l u t e d w i t h the a p p r o p r i a t e v o l u m e of d e f i n e d m e d i u m to give a c o n c e n t r a t i o n of 10 -5 c e l l s / 1 0 0 ~i. Incubation
Experiments
The cells were m a i n t a i n e d in s u s p e n s i o n by gentle m a n u a l a g i t a t i o n w h i l e i00 ~i a l i q u o t s w e r e d i s p e n s e d into glass tubes c o n t a i n i n g 1 ml d e f i n e d m e d i u m w i t h or w i t h o u t c o n d i t i o n i n g agents (progesterone, cortisol, e s t r a d i o l or mepacrine) as a p p r o p r i a t e . Steroids and a r a c h i d o n i c acid were d i s s o l v e d in ethanol and added in a volume of 5 ~i. O t h e r agents w e r e d i s s o l v e d in water. Each t r e a t m e n t and c o n t r o l p r e i n c u b a t i o n was c a r r i e d out in t r i p l i c a t e at 37°C for 3 hr w i t h air as the gas phase. The tubes w e r e c e n t r i f u g e d and the s u p e r n a t a n t was r e p l a c e d with an equal volume of d e f i n e d m e d i u m w i t h or w i t h o u t a s t i m u l a t o r (histamine, p h o r b o l m y r i s t a t e , b r a d y k i n i n , e s t r a d i o l - 1 7 ~ or a r a c h i d o n i c acid) and the same c o n d i t i o n i n g agent. A f t e r further i n c u b a t i o n for up to 2 h the tubes were c e n t r i f u g e d (1500 x g, 5 min at 20°C) and the s u p e r n a t a n t removed. Perifusion
Experiments
D e f i n e d m e d i u m was p u m p e d fAutoanalyser P r o p o r t i o n i n g Pump i, T e c h n i c o n Instruments, Chauncey, New York, U.S.A.) from r e s e r v o i r s in six p a r a l l e l systems at a c o n s t a n t rate (0.i ml/min) t h r o u g h b u b b l e traps (i ml) and then t h r o u g h chambers (27 m m diameter, 5 ~m pore size, Acrodisc, Gelman S c i e n c e s Inc., A n n Arbor, MI, U.S.A.) c o n t a i n i n g the d i s p e r s e d cells. F r a c t i o n s w e r e c o l l e c t e d w i t h a m o d i f i e d LKB 17000 M i n i r a c F r a c t i o n C o l l e c t o r (LKB, Bromma, S w e d e n ) . Before the i n t r o d u c t i o n of the cells, the c h a m b e r s were filled w i t h d i s t i l l e d w a t e r to c o m p l e t e l y remove air b u b b l e s and the w a t e r was then d i s p l a c e d b z medium. The system, from the b u b b l e trap to the p e r i f u s i o n c h a m b e r out-flow, was i m m e r s e d in a 37oc w a t e r bath. The cells were i n t r o d u c e d into the p e r i f u s i o n chamber by u n c o u p l i n g the i n f l o w and p i p e t t i n g i00 ~i of
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s u s p e n s i o n d i r e c t l y into the chamber. The pump was then r e - s t a r t e d and 30 min f r a c t i o n s w e r e collected. A g e n t s were d i s s o l v e d in d e f i n e d m e d i u m and either added to the r e s e r v o i r s or, for 'pulsed' a d m i n i s t r a t i o n , were m a n u a l l y p i p e t t e d d i r e c t l y into the cell p e r i f u s i o n chamber. A s s a y of PGF PGF in the s u p e r n a t a n t was assayed in d u p l i c a t e as d e s c r i b e d p r e v i o u s l y (3) and the q u a n t i t i e s were e x p r e s s e d as the amount r e l e a s e d by 105 cells per hour (pmol/105 cells/h). S t a n d a r d curves from the r a d i o - i m m u n o a s s a y were g e n e r a t e d using an iterative c o m p u t e r p r o g r a m m e (4) and the mean fitted value of the d u p l i c a t e d e t e r m i n a t i o n s of each t r i p l i c a t e i n c u b a t i o n was c o m b i n e d to give a m e a n value for each e x p e r i m e n t a l data point. Chemicals P r o s t a g l a n d i n F2~ was o b t a i n e d from the Upjohn Company, Kalamazoo, MI, U.S.A. [9-3H]-Prostaglandin F2d and [ 5 , 6 , 8 , 1 1 , 1 2 , 1 4 , 1 5 - 3 H ] - a r a c h i d o n i c acid were o b t a i n e d from A m e r s h a m International, Bucks, England. A s c o r b i c acid, a r a c h i d o n i c acid, h i s t a m i n e diphosphate, mepacrine, cortisol, p r o g e s t e r o n e , e s t r a d i o l - 1 7 B , 4 B - p h o r b o l - 1 2 - m y r i s t a t e 1 3 - a c e t a t e (PMA) and c o l l a g e n a s e were o b t a i n e d from Sigma, St Louis, U.S.A. B r a d y k i n i n was o b t a i n e d from Serva, Heidelberg, FRG. M e d i u m 199 (Earle's Salts), M e d i u m 199 (Hank's B a l a n c e d Salts) and bovine fetal serum was o b t a i n e d from Gibco (New York, U.S.A.). M e d i u m 199 was p r e p a r e d by sterile f i l t r a t i o n and was s u p p l e m e n t e d w i t h p e n i c i l l i n (i00 m/ml), s t r e p t o m y c i n (i00 ~g/ml) and g e n t a m y c i n (25 ~g/ml) and was a d j u s t e d to pH 7.4. Arachidonic acid was p u r i f i e d by HPLC (Waters, Milford, Mass., U.S.A.) using a W a t e r s R a d i a l - P a k C18 c a r t r i d g e column (10 ~m p a r t i c l e size) w i t h a 5 m m internal diameter. The a r a c h i d o n i c acid was e l u t e d i s o c r a t i c a l l y with a c e t o n i t r i l e / 0 . 1 % p h o s p h o r i c acid (65:35 v/v) at a rate of 1.5 m l / m i n with a r e t e n t i o n time of 7 min and was m o n i t o r e d by its a b s o r p t i o n at 214 nm. Column fractions c o n t a i n i n g the p u r i f i e d a r a c h i d o n i c acid were n e u t r a l i s e d with IM NaOH, the a c e t o n i t r i l e e v a p o r a t e d under N 2 gas and the r e m a i n i n g aqueous s o l u t i o n d i l u t e d as appropriate. The r e c o v e r y of a r a c h i d o n i c acid from the HPLC was 90% as d e t e r m i n e d
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PROSTAGLANDINS
by the r e c o v e r y of [ 3 H ] - a r a c h i d o n i c two c o l u m n c a l i b r a t i o n e x p e r i m e n t s .
acid added in
Statistics The d i f f e r e n c e b e t w e e n the m e a n PGF value for each t r i p l i c a t e d e t e r m i n a t i o n was d e t e r m i n e d using S t u d e n t ' s t-test. D o s e - r e s p o n s e r e l a t i o n s h i p s were tested using linear r e g r e s s i o n analysis. Results are e x p r e s s e d in the text, figures and tables as m e a n ± SEM. RESULTS In the d e v e l o p m e n t of these e x p e r i m e n t a l p r o c e d u r e s it was found that 38% of the cell preparations from e n d o m e t r i a o b t a i n e d on days 6-27 of the m e n s t r u a l cycle failed to r e s p o n d to a d d e d PMA w i t h i n c r e a s e d PGF production. T h e r e was no c o r r e l a t i o n b e t w e e n cycle stage and s t i m u l a t e d PGF production. It was t h e r e f o r e found a d v a n t a g e o u s to c o m b i n e the cells from two or more uteri so that the p r o b a b i l i t y of o b t a i n i n g an u n r e s p o n s i v e p r e p a r a t i o n w a s minimised. All of the results p r e s e n t e d b e l o w were o b t a i n e d w i t h cells p r e p a r e d in this manner. E n d o m e t r i a l Cell
Incubations
H i s t a m i n e (5x10-7M - 5x10-5M) c a u s e d a c o n c e n t r a t i o n - d e p e n d e n t i n c r e a s e in PGF p r o d u c t i o n (r=0.863, n=15, p <0.01); Fig la). The lowest c o n c e n t r a t i o n of h i s t a m i n e w h i c h caused s i g n i f i c a n t l y i n c r e a s e d PGF p r o d u c t i o n was 10-6M (p <0.02). 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 h i s t a m i n e (10-5M) s i g n i f i c a n t l y s t i m u l a t e d PGF p r o d u c t i o n in seven cell p r e p a r a t i o n s (Table i). The a d d i t i o n of b r a d y k i n i n (10-6M) to the cell i n c u b a t i o n s s i g n i f i c a n t l y s t i m u l a t e d PGF p r o d u c t i o n in three p r e p a r a t i o n s but in one (Table i, Expt 2) the response was small in c o m p a r i s o n to o t h e r stimuli. Similarly, the a d d i t i o n of PMA or a r a c h i d o n i c acid to the cell i n c u b a t i o n s s i g n i f i c a n t l y s t i m u l a t e d PGF p r o d u c t i o n (Table i). In one experiment, the cells were p r e i n c u b a t e d w i t h e s t r a d i o l for three hours and then for a further hour w i t h the same estradiol c o n c e n t r a t i o n s . E s t r a d i o l (10-7M - 10-5M) s t i m u l a t e d a c o n c e n t r a t i o n d e p e n d e n t increase in PGF but at a h i g h e r c o n c e n t r a t i o n (5x10-5M) the r e s p o n s e d e c l i n e d (Fig 2).
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Cortisol caused a c o n c e n t r a t i o n - d e p e n d e n t fall in h i s t a m i n e - s t i m u l a t e d PGF p r o d u c t i o n to 30 ± 9% at a c o n c e n t r a t i o n of 5 x 10-5M (r=0.697, n=14, p <0.01; Fig ib). P r o g e s t e r o n e (10-6M) also inhibited the response to histamine (p <0.01). M e p a c r i n e was found to inhibit 'the responses to histamine ~(:~0-5M), b r a d y k i n i n (10-6M) and P M A (3x10-8M; Table 2). T h e lowest c o n c e n t r a t i o n of m e p a c r i n e that inhibited the response to histamine and b r a d y k i n i h w a s ' b e t w e e n }0-6M and 10-5M and for PMA was 10-5M. S t i m u l a t i o n of PGF p r o d u c t i o n by arachidonate was relatively unaffected by mepacrine (10-6M - 2x10-4M) in two of three experiments (Table 2) but in the remaining e x p e r i m e n t an inhibitory action of m e p a c r i n e was observed (Table 2, Expt 8). Endometrial
Cell Perifusions
A t o t a l of 42 experiments were performed, e a c h of which included one control and five experimental perifusions from the~ same pool of d i s p e r s e d cells. "Th~ p r o d u c t i o n of PGF in the first 60 min w a s high but decreased to control levels after 90 m i n . The introduction of histamine (10-5M), b r a d y k i n i n (10-6M), PMA (3x10 -8) or a r a c h i d o n a t e (5x10-5M) into the p e r i f u s i o n m e d i u m at 120 min or later stimulated PGF p r o d u c t i o n (Fig 3 a - d ) . A l t h o u g h the effect of each of these agents on the p r o d u c t i o n of PGF was similar, the onset and d u r a t i o n of release differed. The response to h i s t a m i n e was rapid and of short d u r a t i o n (60 min) d e s p i t e c o n t i n u e d a d m i n i s t r a t i o n (Fig 3a]. T h e - i n c l u s i o n Of p r o g e s t e r o n e (10-6M) or corti~01 (5x10-6M) in £he m e d i u m from the b e g i n n i n g of p e r i f u s i o n (i.e. 150 min before the i n t r o d u c t i o n of histamine) inhibited the response to histamine by 80-100% ~(data not shown) although neither hormone c o n s i s t e n t l y inhibited the elevated baseline ~values usually present during the f i r s t 30-60 min of perifusion. A similar p a t t e r n of response with rapid onset and short duration, was seen when £he cells were stimulated by b r a d y k i n i n (Fig 3 b ) . The response to P M A and a r a c h i d o n a t e were different (Fig 9c and d), being slightly slower in onset and p e r s i s t i n g longer (120 min or more) ~ The contrast between rapid response (histamine or
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TABLE
i
The effect of various agents on the production rates of PGF (pmol/105 cells/h) by dispersed human endometrial cells during short-term culture in defined medium.
Expt.
No. Control
0.21
1
±0.20
2
<0.04
Arachidonate (5x10-6M)
8.07+ ±0.72
~.839 ±0.40
Stimulating
agent
Histamine (10-5M)
Bradykinin (10-6M)
5.60+ ±0.91
2.97+ ~0.91
3
0,70 ~0.12
1.55 % ±0.17
4
<0.04
0.52~ ±0.Ii
5
6.83 ±1.15
12.20 ~ ±1.33
6
0.83 ~0.25
7
0.58 ±0.ii
8
1.90 ±0.12
2.03 % ±0.15
3.03 % ±1.05
0.19 t ±0.05
PMA ¸ (3xlO-8M)
2.03 %
±0.50 1.2q % ±0.03 1.76 % ±0.17
2.13 % ~±0.18 1.50 % ±0.02 3.0 % ±0'.18
Eight different cell preparations were incubated (triplicates) in defined m e d i u m (see Materials and Methods) with or w i t h o u t the agents listed. The PGF released into the m e d i u m was determined by radioimmunoassay after ether extraction,, T h e results ar e expressed as pmol PGF/105 cells/h (mean ± SEM)~ Significant differences between the PGF production rates in control incubations (using a value of 0.04 in expts. 2 and 4) and those with agents are denoted % (p <0.05) ~ PMA = phorbol myristate
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PROSTAGLANDINS
®
®
14 12
1.2 A J~
%(: \
u~
2 \
0.9
1
0
0£
E
Q.
~i,
10
U.
0.4
~ 4
O.2
o
5xlo"7 lO"e SxlO"s lO"s sxlo"s Histamine (M)
0
5~0-7 ~
~-5
5x10-5
Co~isol(M)
Figure i: PGF p r o d u c t i o n rates by d i s p e r s e d human e n d o m e t r i a l cells in s h o r t - t e r m culture; (a) S t i m u l a t i o n by h i s t a m i n e (b) I n h i b i t i o n of h i s t a m i n e (10-5M) s t i m u l a t i o n by cortisol. The cells were i n c u b a t e d (triplicates) in d e f i n e d m e d i u m (see M a t e r i a l s and Methods) in the absence or p r e s e n c e of histamine. The PGF r e l e a s e d into the m e d i u m was d e t e r m i n e d by r a d i o i m m u n o a s s a y after ether extraction. (a) In the a b s e n c e or p r e s e n c e of i n c r e a s i n g c o n c e n t r a t i o n s of histamine. (b) The cells were p r e i n c u b a t e d for 3 hours in the a b s e n c e or p r e s e n c e of c o r t i s o l (5x10-7M - 5x10-5M), the s u p e r n a t a n t removed and r e p l a c e d with m e d i u m c o n t a i n i n g the same c o r t i s o l c o n c e n t r a t i o n and h i s t a m i n e (10-5M) .
828
JUNE 1984 VOL. 27 NO. 6
PROSTAGLANDINS
TABLE The effect of mepacrine stimulated with various
Expt.
No.
2
on PGF p r o d u c t i o n agents
by endometrial
Concentration
Stimulus
10-6M
cells
of mepacrine 10-5M
2x10-4M
1
Histamine
{10-5M)
i00 ± 16
12 ± 1%
<3 %
<3 t
2
Histamine
(10-5M)
i00 ± 26
82 ± 32
<3 %
52 ± 8
3
Bradykinin
i00 ± 34
23 ± 5
4
PMA
(3x10-8M)
i00 ± 25
118 ± 32
5
PMA
(3x10-SM)
i00 ± 3
176 ± 12
6
AA
(5x10-6M)
i00 ± 9
120 ± 16
7
AA
(5x10-6M)
i00 ± 22
104 ± 32
8
AA
(5x10-6M)
i00 ± 9
(10-6M)
30 ± 5 t
i0 ± 10 % 21 ± 16 <3 t
9 ± 9t
15 ± 12 % 14 ± 12 f 126 ± i0
N.D.
70 ± 23 130 ± 50 12 ± 3 t
51 ± 21
The cells were incubated (triplicates) in defined m e d i u m (see Materials and Methods) with or w i t h o u t mepacrine for 3 h. The m e d i u m was then removed and replaced with m e d i u m containing the same concentration of m e p a c r i n e and the appropriate stimulator. The values for PGF p r o d u c t i o n obtained in the absence of mepacrine were normalized and expressed as 100% ± SEM. The PGF p r o d u c t i o n in the presence of mepacrine was normalized and expressed as mean% ± SEM of the value obtained in the absence of mepacrine. Decreased PGF p r d o c t i o n by cells in the presence of mepacrine is denoted by % (p <0.05). PMA = Phorbol m y r i s t a t e AA = Arachidonic acid
JUNE 1984 VOL. 27 NO. 6
829
PROSTAGLANDINS
6 5 r~
=
4
O
qo
3
O
E 2 o I I
"3-
0
!
10"7 5x10-7 5x10"6 10-5 5x10 -5 Estradiol-178 ( M )
10 -5
Histamine ( M )
F i g u r e 2: ~ T h e e f f e c t of e s t r a d i o l - 1 7 ~ on P G F p r o d u c t i o n by e n d o m e t r i a l c e l l s d u r i n g s h o r t term cultures. The cells were preincubated (triplicates) in d e f i n e d m e d i u m ( s e e M a t e r i a l s a n d M e t h o d s ) i n ~ t h @ p r e s e n c e o r a b s e n c e of e s t r a d i o l (10-7M - 5x10~SM) for 3 h. The medium was r e m o v e d a n d r e p l a c e d w i t h m e d i u m c o n t a i n i n g the same concentration of e s t r a d i o l . The P G F r e l e a s e d i n t o the m e d i u m d u r i n g i n c u b a t i o n for 1 h w a s d e t e r m i n e d by r a d i o i m m u n o a s s a y after ether extraction. There was a positive correlation (r=0.750, n=12, p <0.001) b e t w e e n PGF p r o d u c t i o n and estradiol concentration (10-7M - 1 0 - 5 M ) . The r e s p o n s e to h i s t a m i n e in the same p r e p a r a t i o n is s h o w n for c o m p a r i s o n .
830
JUNE 1984 VOL. 27 NO. 6
PROSTAGLANDINS
bradykinin) and slow r e s p o n s e was further d e m o n s t r a t e d by e x p o s u r e of the cells to h i s t a m i n e in the p r e s e n c e and a b s e n c e of a ~ a c h i d o n i c acid (5x10-6M). The response to h i s t a m i n e alone o c c u r r e d in the first 30 min but w h e n both h i s t a m i n e and arach:idonate were present, a t w o - c o m p o n e n t r e s p o n s e was seen i n d i c a t i n g little i n t e r a c t i o n b e t w e e n them Fig 4). The r e s p o n s e to h i s t a m i n e and b r a d y k i n i n w e r e i n v e s t i g a t e d further to d e t e r m i n e w h e t h e r the failure to m a i n t a i n a response d u r i n g their c o n t i n u o u s a d m i n i s t r a t i o n was due to d e p l e t i o n of p r e c u r s o r s or c o f a c t o r s or to some form of d e s e n s i t i z a t i o n . H i s t a m i n e (10-5M; Fig 5a) or b r a d y k i n i n (10-6M); Fig 5b) was a d m i n i s t e r e d for 2.4 min at 120 min and again at 210 min. The r e s p o n s e to each pulse was c o m p a r a b l e to that seen with c o n t i n u o u s administration. DISCUSSION H u m a n ~ e n d o m e t r i a l cells d i s p e r s e d with c o l l a g e n a s e and m a i n t a i n e d o v e r n i g h t in a c u l t u r e m e d i u m c o n t a i n i n g 5% b o v i n e fetal serum appear to m e e t the r e q u i r e m e n t s of a useful s y s t e m for d e t e c t i n g i n h i b i t o r s and s t i m u l a t o r s of p r o s t a g l a n d i n synthesis in the p r e g n a n t human uterus. Basal p r o d u c t i o n of PGF in a d e f i n e d m e d i u m was u s u a l l y low and the cells r e s p o n d e d in the e x p e c t e d way to a ~ v a r i e t y of agents k n o w n t o ' i n h i b i t or s t i m u l a t e p r o s t a g l a n d i n synthesis. D i f f e r e n t cell p r e p a r a t i o n s v a r i e d c o n s i d e r a b l y both in the basal p r o d u c t i o n rate of PGF and in the m a g n i t u d e of r e s p o n s e s to the v a r i o u s agents (see Table i). This v a r i a b i l i t y b e t w e e n p r e p a r a t i o n s p r e c l u d e d e s t i m a t i o n s of the p o t e n c y o f a p a r t i c u l a r agen t by c o m b i n i n g v a l u e s ~ o b t a i n e d with d i f f e r e n t preParations. However, the v a r i a b i l i t y w i t h i n a p r e p a r a t i o n was s u f f i c i e n t l y low to o b t a i n valid m e a s u r e m e n t s r e l a t i v e to a standard r e s p o n s e (usually histamine). Both p e r i f u s i o n a n d i n c u b a t i o n of the cells p r o v e d satisfactory. P e r i f u s i o n r e q u i r e d a more c o m p l e x s y s t e m and the n u m b e r of s i m u l t a n e o u s e x p e r i m e n t s was limited but the m e t h o d a l l o w e d the t i m e , c o u r s e of r e s p o n s e s to be studied. Static i n c u b a t i o n 0f the cells was s i m p l e r and the n u m b e r
JUNE 1984 VOL. 27 NO. 6
831
PROSTAGLANDINS
®
Histamine (10"5M)
®
1
1
Bradykinin (10"sM) V / I
.
.
.
.
I
I
I
I
/
1
1
~
O.E
~o.8
~> 0
0
,n O.E
0.6
o
0.4
~0,4
~ 0.2 60
120
180
-I
~ 0.2
240
60
120
TIME (min)
©
180
240
300
TIME (rain)
@
Phorbol Myristate (3x10"8M)
-. c- 3 -
Arachidonic Acid (5x10"6 M) .~
v / / / / J~z
zz2
0"61[
o 2-
to
o
Q. U.
60
120
180
240
TIME (min)
300
30 90 120
180
240
300
TIME (rain)
Figure 3: R e p r e s e n t a t i v e examples showing the effect of various agents on PGF release by d i s p e r s e d human e n d o m e t r i a l cells in p e r i f u s i o n culture. The d i s p e r s e d cells were placed in chambers and p e r i f u s e d with d e f i n e d m e d i u m (0.I ml/min) for 120 min or 180 min and then with the same m e d i u m c o n t a i n i n g (a) h i s t a m i n e (10-5Ml, (b) b r a d y k i n i n (10-6M), (c) PMA (3xl0-~M) and (d) arachidonic acid (5x10-6M). The duration of exposure of the cells to each agent is shown by the hatched bars. The PGF released into the m e d i u m was d e t e r m i n e d in timed fractions (30 min) by r a d i o i m m u n o a s s a y after ether extraction.
832
JUNE 1984 VOL. 27 NO. 6
PROSTAGLANDINS
Histamine (10 "5 M) alone Histamine (10 "5 M ) + Arachidonic ............... Acid ( 5 x 1 0 " 6 M ) 0.7-
"---'1
0.6O
0.5"
0.4o 0.3E o. 0.2-
"-
IJ.
0
a.
L-- 1
0.1
"--'1
60
120
180
L----I___
240
300
TIME (min) Figure 4: The effect of h i s t a m i n e in the p r e s e n c e of a r a c h i d o n i c acid on PGF release by d i s p e r s e d h u m a n e n d o m e t r i a l cells in p e r i f u s i o n culture. The d i s p e r s e d cells were p e r i f u s e d (see Fig 3) for 120 min with d e f i n e d m e d i u m and then w i t h m e d i u m containing h i s t a m i n e (10-5M) alone (o---o) or with m e d i u m c o n t a i n i n g h i s t a m i n e (10-5M) and a r a c h i d o n i c acid (5x10-6M; o. o). The d u r a t i o n of e x p o s u r e of the cells to these a g e n t s is shown by the h a t c h e d bar. With h i s t a m i n e alone there was a single peak of short d u r a t i o n while w i t h h i s t a m i n e and a r a c h i d o n i c acid there was a t w o - c o m p o n e n t response.
JUNE 1984 VOL. 27 NO. 6
833
PROSTAGLANDINS of s i m u l t a n e o u s e x p e r i m e n t s was limited only by the number of cells a v a i l a b l e but it was less suitable for d y n a m i c ~ s t u d i e s. T h e m e t h o d is a m e n a b l e to further r e f i n e m e n t by s e p a r a t i n g the e p i t h e l i a l and stromal c o m p o n e n t s (5). The p r o d u c t i o n of PGF by the d i s p e r s e d cells was s t i m u l a t e d by four agents known to s t i m u l a t e p r o s t a g l a n d i n s y n t h e s i s by a c t i v a t i n g a c y l h y d r o l a s e s . These e n z y m e s liberate a r a c h i d o n i c acid from cell m e m b r a n e lipids and are known to be s t i m u l a t e d by h i s t a m i n e (6), b r a d y k i n i n (7,8), PMA (9) and e s t r a d l o l (i). This effect in the p r e s e n t cell system was c o n f i r m e d by the i n h i b i t i o n of the response to • histamine, b r a d y k i n i n or PMA by m e p a c r i n e w h i c h inhibits p h o s p h o l i p a s e A 2 by a d i r e c t action ~I~). Both cortisol, w h i c h ~ n h i b i t s p h o s p h o l i p a s e A 2
~)
Histamine (10 "5 M )
~
Bradykinin (10 "6 M )
J¢
a
~3
tj
I
to
2 i
E Q. LI. 1 • ¢3 a. 50
120
180
240
TIME (min)
300
I
60
120
180
240 • 300
TIME ( m i n )
Figure 5: The effect of h i s t a m i n e and b r a d y k i n i n a d m i n i s t e r e d as r e p e a t e d short pulses on PGF release by d i s p e r s e d human e n d o m e t r i a l cells in p e r i f u s i o n culture. The d i s p e r s e d cells were p e r i f u s e d (see Fig 3) w i t h d e f i n e d medium. The cells were e x p o s e d to short pulses (2.4 min duration, as d e p i c t e d by the h a t c h e d bars) of (a) h i s t a m i n e (10-5M) or (b) b r a d y k i n i n (10-6M) at 120 min and again at 210 min.
834
JUNE 1984 VOE. 27 NO. 6
PROSTAGLANDINS
through the m e d i a t i o n of m a c r o c o r t i n or l i p o m o d u l i n (12,13), a n d p r o g e s t e r o n e , w h i c h has a less welld e f i n e d action on p h o s p h o l i p a s e A 2 in r e p r o d u c t i v e tissues, i n h i b i t e d the response to histamine. S u p p o r t for a c y l h y d r o l a s e s as the m a j o r site of action of histamine, b r a d y k i n i n and P M A was o b t a i n e d by showing that m e p a c r i n e had little e f f e c t on the r e s p o n s e to a r a c h i d o n i c acid w h e r e a s it c o n s i s t e n t l y i n h i b i t e d the r e s p o n s e to the former agents. The t r a n s i e n t r e s p o n s e of e n d o m e t r i a l cells to h i s t a m i n e is c o n s i s t e n t with the report (6) that h i s t a m i n e in c o n c e n t r a t i o n s similar to those used in the p r e s e n t e x p e r i m e n t s (10-7M - 10-5M) c a u s e s only a brief (3 min) r e ~ e a s e of p r o S t a c y c l i n from human e n d o t h e l i a l cells~ ~ D e p l e t i o n o f - p r e c u r s o r s Or c o f a c t o r s is an u n l i k e l y cause of the t r a n s i e n t r e s p o n s e since two short pulses of :histamine s e p a r a t e d by 90 m i n of p e r i f u s i o n e l i c ± t e d c o m p a r a b l e r e s p o n s e s (Fig 5a) . C o n t i n u o u s exposure of the cells to h i s t a m i n e may cause d e s e n s i t i z a t i o n , p o s s i b l y due to d o w n - r e g u l a t i o n of c e l l - s u r f a c e receptors. The p r e s e n c e of h i s t a m i n e (HI) receptors on e n d o m e t r i a l cell m e m b r a n e s has p r e v i o u s l y been r e p o r t e d (14). S t i m u l a t i o n of p r o s t a g l a n d i n s y n t h e s i s by PMA is well d o c u m e n t e d (9,15). The p r e s e n t e x p e r i m e n t s c o n f i r m that this action occurs in e n d o m e t r i a l cells and that the r e s p o n s e is i n h i b i t e d b y m e p a c r i n e , s u g g e s t i n g that PMA acts ~ by s t i m u l a t i n g a c y l h y d r o l a s e activity. The c o n t r a s t i n g p a t t e r n s of r e s p o n s e to PMA and to h i s t a m i n e or b r a d y k i n i n d u r i n g c o n t i n u o u s p e r i f u s i o n points to d i f f e r e n c e s in their modes of action. ~ A similar C o n c l u s i o n is s u g g e s t e d by the g r e a t e r r e s i s t a n c e of t h e r e s p o n s e to PMA to i n h i b i t i o n by m e p a c r i n e (Table 2). The:obs~rved conCentration-dependent response to e s t r a d i o l ' 1 7 B accords with t h e ~ p h y s i o l o g i c a l action of this h o r m o n e i n vivo (16,17,18) and with its e f f e C t on PGF product--{-n by cultures of human e n d o m e t r i a l b i o p s y tissue (19) and p r i m a r y m o n o l a y e r c u l t u r e s of e p i t h e l i u m from h u m a n p r o l i f e r a t i v e e n d o m e t r i u m (20). In-the latter study, e s t r a d i o l (10"8M) ~stimulated a fourfold i~crease in c u m u l a t i v e levels of PGF d u r i n g ~ i n c u b a ~ i o n of g l a n d u l a r tissue for 24~h W h e r e a s t H e p r o d u c t i o n of 'PGF by stromal cells was either u n a f f e c t e d or inhibited.
JUNE 1984 VOE. 27 NO. 6
835
PROSTAGLANDINS
This suggests that in our short-term cultures of mixed dispersed cells the increased production rate of PGF in response to estradiol may reflect glandular cell activity. The rapid response to estradiol (within 4 h) observed in the present study has not been described previously. More detailed investigation of the time course of the response would be of interest since it raises the possibility of a direct action not mediated by synthesis of new protein. We conclude that human endometrial cells, prepared by a simple procedure, are useful for the detection of endogenous stimuli and inhibitors of PGF synthesis. The cells retain sensitivity to estradiol, histamine, bradykinin and PMA which stimulate the synthesis of prostaglandins by activating acylhydrolases, and to cortisol and progesterone which inhibit synthesis at the same point in the biosynthetic pathway. ACKNOWLEDGEMENTS We are grateful to the gynecologists and nursing staff of National Women's Hospital, Auckland, New Zealand for their willing co-operation in obtaining endometrial tissues. We thank Dr J.E.Pike, The Upjohn Company, Kalamazoo, MI, U.S.A. for the gift of authentic PGF2e. This study was supported by the New Zealand Medical Research Council. REFERENCES i.
Liggins, G.C. Initiation of spontaneous labor. Clin. Obstet. Gynecol. 26:47-55, 1983.
2.
Saeed, S.A., D.M.Strickland, D.C.Young, A.Dang and M.D.Mitchell. Inhibition of prostaglandin synthesis by human amniotic fluid: Acute reduction in inhibitory activity of amniotic fluid obtained during labor. J. Clin. Endocr. Metab. 55:801-803, 1982.
3.
Liggins, G.C., G.A.Campos, C.M.Roberts and S.J.M.Skinner. Production rates of prostaglandin F, 6-keto-PGF 1 and thromboxane-B 2 by perifused human endometrlum. Prostaglandins 19:461-477, 1980.
836
JUNE 1984 VOL. 27 NO. 6
PROSTAGLANDINS
4.
Wilkins, T.A., D.C.Chadney, J. Bryant, S.H. Palmstrom and R.L.Winder. In: Radioimmunoassay and related procedures in medicine (Vol i). International Atomic Energy Authority, Vienna, 1978.
5.
Satyaswaroop, P.G., R.S.Bressler, M.M. de la Pena and E.Gurpide. Isolation and culture of human endometrial glands. J. Clin. Endocr. Metab. 48:639-641, 1979.
6.
Baenziger, N.L., F.J. Fogerty, L.F. Mertz and L.F.Chernuta. Regulation of histamine-mediated prostacyclin synthesis in cultured human vascular endothelial cells. Cell 24:915-923, 1981.
7.
Hong, S.L. and L.Levine. Inhibition of arachidonic acid release from cells as the biochemical action of anti-inflammatory corticosteroids. Proc. Natl. Acad. Sci. U.S.A. 73: 1730-1734, 1976.
8.
Schremmer, J.M., M.L.Blank and R.L.Wykle. Bradykinin-stimulated release of [3HI arachidonic acid from phospholipids and plasmalogens as sources of prostaglandin precursors. Prostaglandins 18:491-505, 1979.
9.
Levine, tumour lipids Nature
L. and K.Ohuchi. Retinoids as well as promoters enhance deacylation of cellular and prostaglandin production in MDCK cells. 276:274-275, 1978.
i0.
Dey, S.K., R.K.Hoversland and D.C. Johnson. Phospholipase A 2 activity in the rat uterus: Maturation by steroid hormones. Prostaglandins 23:619-630, 1982.
ii.
Vargaftig, B.B. and N. Dao-Hai. Selective inhibition by mepacrine of the release of 'rabbit-aorta-contracting-substance' evoked by the administration of bradykinin. J. Pharm. Pharmac. 24:159-161, 1972.
12.
Flower, R.J. and G.J.Blackwell. Anti-inflammatory steroids induce biosynthesis of a phospholipase A 2 inhibitor which prevents prostaglandin generation. Nature 278:456-459, 1979.
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837
PROSTAGLANDINS
13.
Hirata, F., ~. $~lomon inhibitory induced by Sci. U.S.A.
E. Schiffman, K. V e n k a t a s u b r a m a n i a n , and J. Axelrod. A phospholipase A 2 protein in rabbit n e u t r o p h i l s glucocorticoids; Proc. Nat. Acad. 77:2533-2536, 1980.
14.
Dey, S.K., C . V i l l a n u e v a and N.I.Abdou. Histamine receptors on rabbit b l a s t o c y s t and endometrial cell membranes. N a t u r e 278: 648-649/ 1979.
15.
Levine, L. Effects of tumour promoters on a r a c h i d o n i c acid m e t a b o l i s m by cells i n culture, '~ Carcinog. Compr. Surv. 7:477-494, 1982.
16.
Barcikowski, B., J.C. Carlson, L . W i l s o n and J.A.McCracken. The effect of endogenous and exogenous e s t r a d i o l - 1 7 ~ on the release of p r o s t a g l a n d i n - F 2 ~ from the ovine uterus. E n d o c r f n o l o g y 5:1340-1349, 1974.
17.
Ham, E.A., V.J.Cirillo, M . E . Z a n e t t i and F.A. Kuehl. E s t r o g e n - d i r e c t e d synthesis of specifi c p r o s t a g l a n d i n s in the uterus. ~ Proc. Natl. Acad. Sci. U.S.A. 7 2 : 1 4 2 0 - 1 4 2 4 , 1975.
18.
caStracane, V.D. and V.C.JOrdan. The effect of e s t r o g e n and p r o g e s t e r o n e on uterine p r o s t a g l a n d i n b i o s y n t h e s i s in the o v a r i e c t o m i s e d rat. Biol. Reprod. 13:587-597, 1975. !
19.
Abel, M.H. and D.T.Baird. The effect of 17Bestradiol and p r o g e s t e r o n e on p r o s t a g l a n d i n p r o d u c t i o n by human e n d o m e t r i u m m a i n t a i n e d in organ culture. E n d o c r i n o l o g [ i06:1599u1606, i980.
20.
Schatz, F. and Gurpide, E. The effects of estradiol on p r o s t a g l a n d i n F2~ levels in p r i m a r y m o n o l a y e r c u l t u r e s ot e p i t h e l i a l cells from human p r o l i f e r a t i v e endometrium. E n d o c r i n o l o g y 11"3:1274-1279, 1983.
E d i t o r : Harold Behrman
838
Received: 10-28-83
Accented: 4-16-84
JUNE 1984 VOL. 27 NO. 6
S Y N T H E S I S OF P R O S T A G L A N D I N F BY C U L T U R E D HUMAN E N D O M E T R I A L CELLS S.J.M.
Skinner,
G.C.Liggins,
T . W i l s o n and G. Neale
P o s t g r a d u a t e School of O b s t e t r i c s U n i v e r s i t y of Auckland, Auckland,
and Gynaecology, New Zealand.
ABSTRACT Human e n d o m e t r i a l cells were d i s p e r s e d with c o l l a g e n a s e and m a i n t a i n e d in culture overnight. The synthesis of PGF by the d i s p e r s e d cells i n c u b a t e d at 37oc in s e r u m - f r e e m e d i u m was s t i m u l a t e d b y _ e s t r a d i o l ( 1 0 - 7 M - 10-5M). h i s t a m i n e (5x10-7M - 5xl0-bM), b r a d y k i n i n (10-6M), p h o { b o l m y r i s t a t e (PMA, 3x10-SM) and a r a c h i d o n a t e (5xl0-UM). P r e i n c u b a t i o n of the cells for 3 h w i t h c o r t i s o l (5x10-7M - 5x10-5M), p r o g e s t e r o n e (10-6M) or m e p a c r i n e (10-6M - 2x10-4M) i n h i b i t e d the r e s p o n s e to histamine, b r a d y k i n i n and PMA but not to arachidonate. P e r i f u s i o n of the c u l t u r e d cells in f i l t r a t i o n chambers y i e l d e d s i m i l a r results to those obtained in the i n c u b a t i o n system but d i f f e r e n c e s in the onset and d u r a t i o n of the r e s p o n s e s to stimuli were found. In the p e r i f u s i o n s y s t e m the r e s p o n s e s to h i s t a m i n e and b r a d y k i n i n were rapid and of short d u r a t i o n (peak r e s p o n s e in <60 min) w h i l e the r e s p o n s e s to PMA and a r a c h i d o n a t e were of longer d u r a t i o n with a slower onset. We c o n c l u d e that these o b s e r v a t i o n s u s i n g d i s p e r s e d e n d o m e t r i a l cells are c o n s i s t e n t w i t h p r e v i o u s work showing that histamine, b r a d y k i n i n and PMA act by s t i m u l a t i n g a c y l h y d r o l a s e activity, thereby l i b e r a t i n g p r e c u r s o r s such as a r a c h i d o n i c acid w h i c h are c o n v e r t e d to p r o s t a g l a n d i n s by the c y c l o - o x y g e n a s e complex. INTRODUCTION The m e c h a n i s m c o n t r o l l i n g the i n i t i a t i o n of human p a r t u r i t i o n p o s s i b l y takes the form of an i n t e r a c t i o n b e t w e e n the cells of the amnion and c h o r i o n and those of the e n d o m e t r i u m (i) . The i n t e r a c t i o n may result in the i n h i b i t i o n during p r e g n a n c y of p r o s t a g l a n d i n p r o d u c t i o n by the e n d o m e t r i u m f o l l o w e d by d e c l i n i n g i n h i b i t i o n a n d / o r i n c r e a s i n g s t i m u l a t i o n at parturition. The study of such i n t e r a c t i o n s r e q u i r e s a system that will detect the p r e s e n c e of i n h i b i t o r y or s t i m u l a t o r y m a t e r i a l s a c t i n g at any point in the b i o s y n t h e t i c p a t h w a y
JUNE 1984 VOL. 27 NO. 6
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of p r o s t a g l a n d i n s . Microsomal prostaglandin synthetase, p r e p a r e d from bovine seminal vesicles, may be used to detect c y c l o - o x y g e n a s e inhibitors (2) but not inhibitors of a r a c h i d o n i c acid release. A w h o l e cell system is e s s e n t i a l if p o t e n t i a l l y i m p o r t a n t b i o l o g i c a l l y active m a t e r i a l s are not to be overlooked. Furthermore, tissue and species s p e c i f i c i t y is possible. Accordingly, we have d e v e l o p e d a d i s p e r s e d cell p r e p a r a t i o n of human endometrium. R e q u i r e m e n t s of the p r e p a r a t i o n are that the cells have a low basal p r o d u c t i o n of p r o s t a g l a n d i n s i n d i c a t i n g r e c o v e r y from trauma, that they r e s p o n d in a d e f i n e d m a n n e r to known p h y s i o l o g i c a l i n h i b i t o r s and stimuli and that s e n s i t i v i t y to these agents is not lost as a result of p r o l o n g e d culture. MATERIALS AND METHODS P r e p a r a t i o n of Cells Uteri were o b t a i n e d from p a t i e n t s u n d e r g o i n g h y s t e r e c t o m y or d i l a t a t i o n and c u r e t t a g e for benign disease (e.g. fibroids, d y s f u n c t i o n m e n o r r h a g i a ) . The patients were of r e p r o d u c t i v e age and were not r e c e i v i n g t r e a t m e n t with hormones. The p r o j e c t was a p p r o v e d by the H o s p i t a l Ethics Committee. E n d o m e t r i a were c l a s s i f i e d by m e n s t r u a l stage as d e s c r i b e d p r e v i o u s l y (3). The u t e r i n e tissues were p r o c e s s e d under a s e p t i c c o n d i t i o n s w i t h i n 30 min of o p e r a t i v e removal. The uterus was opened and the e n d o m e t r i u m was g e n t l y excised w i t h a blunt spatula and p l a c e d in HEPES b u f f e r e d saline (pH 7.3) s u p p l e m e n t e d w i t h glucose (2 mg/ml) and bovine a l b u m i n g/ml) at 37°C. The tissue was finely chopped (i - 2 mm 3 and then t r a n s f e r r e d to fresh HEPES b u f f e r e d saline c o n t a i n i n g c o l l a g e n a s e (0.5 mg/ml) and s u p p l e m e n t e d as above. This m i x t u r e was s t i r r e d at 37°C for 70 min. C u r e t t i n g s were c o l l e c t e d d i r e c t l y into culture m e d i u m (Medium 199, E a r l e ' s Salts), t r a n s f e r r e d to HEPES b u f f e r e d saline and then t r e a t e d as for e n d o m e t r i a from uteri. The c o l l a g e n a s e - d i g e s t e d m a t e r i a l was f i l t e r e d through a i00 ~m sieve to remove u n d i g e s t e d and fibrous m a t e r i a l s and the filtrate c o n t a i n i n g the d i s p e r s e d cells was c e n t r i f u g e d at 500 x g for 5 min at 20°C. The pellet was w a s h e d twice by r e s u s p e n d i n g in culture m e d i u m and centrifuging. The cell p e l l e t was r e s u s p e n d e d in culture m e d i u m s u p p l e m e n t e d with bovine fetal serum (5% v/v) in a sterile p l a s t i c jar and cultured o v e r n i g h t at 37oc with an a i r / C O 2 (95:5 v/v) gas phase. The cells were h a r v e s t e d by gently m i x i n g the s e d i m e n t e d cells and media with a sterile plastic p i p e t t e and then t r a n s f e r r e d
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to a glass tube and c e n t r i f u g e d . The cell p e l l e t was w a s h e d twice in d e f i n e d m e d i u m (Medium 199, Hank's B a l a n c e d Salts) to remove s e r u m and the p r o d u c t s of o v e r n i g h t culture. The pellet was r e s u s p e n d e d in d e f i n e d medium. A small p o r t i o n was stained w i t h T r y p a n Blue and the number of live cells (40 - 70%) was d e t e r m i n e d in a h e m o c y t o m e t e r . The r e m a i n i n g cells w e r e d i l u t e d w i t h the a p p r o p r i a t e v o l u m e of d e f i n e d m e d i u m to give a c o n c e n t r a t i o n of 10 -5 c e l l s / 1 0 0 ~i. Incubation
Experiments
The cells were m a i n t a i n e d in s u s p e n s i o n by gentle m a n u a l a g i t a t i o n w h i l e i00 ~i a l i q u o t s w e r e d i s p e n s e d into glass tubes c o n t a i n i n g 1 ml d e f i n e d m e d i u m w i t h or w i t h o u t c o n d i t i o n i n g agents (progesterone, cortisol, e s t r a d i o l or mepacrine) as a p p r o p r i a t e . Steroids and a r a c h i d o n i c acid were d i s s o l v e d in ethanol and added in a volume of 5 ~i. O t h e r agents w e r e d i s s o l v e d in water. Each t r e a t m e n t and c o n t r o l p r e i n c u b a t i o n was c a r r i e d out in t r i p l i c a t e at 37°C for 3 hr w i t h air as the gas phase. The tubes w e r e c e n t r i f u g e d and the s u p e r n a t a n t was r e p l a c e d with an equal volume of d e f i n e d m e d i u m w i t h or w i t h o u t a s t i m u l a t o r (histamine, p h o r b o l m y r i s t a t e , b r a d y k i n i n , e s t r a d i o l - 1 7 ~ or a r a c h i d o n i c acid) and the same c o n d i t i o n i n g agent. A f t e r further i n c u b a t i o n for up to 2 h the tubes were c e n t r i f u g e d (1500 x g, 5 min at 20°C) and the s u p e r n a t a n t removed. Perifusion
Experiments
D e f i n e d m e d i u m was p u m p e d fAutoanalyser P r o p o r t i o n i n g Pump i, T e c h n i c o n Instruments, Chauncey, New York, U.S.A.) from r e s e r v o i r s in six p a r a l l e l systems at a c o n s t a n t rate (0.i ml/min) t h r o u g h b u b b l e traps (i ml) and then t h r o u g h chambers (27 m m diameter, 5 ~m pore size, Acrodisc, Gelman S c i e n c e s Inc., A n n Arbor, MI, U.S.A.) c o n t a i n i n g the d i s p e r s e d cells. F r a c t i o n s w e r e c o l l e c t e d w i t h a m o d i f i e d LKB 17000 M i n i r a c F r a c t i o n C o l l e c t o r (LKB, Bromma, S w e d e n ) . Before the i n t r o d u c t i o n of the cells, the c h a m b e r s were filled w i t h d i s t i l l e d w a t e r to c o m p l e t e l y remove air b u b b l e s and the w a t e r was then d i s p l a c e d b z medium. The system, from the b u b b l e trap to the p e r i f u s i o n c h a m b e r out-flow, was i m m e r s e d in a 37oc w a t e r bath. The cells were i n t r o d u c e d into the p e r i f u s i o n chamber by u n c o u p l i n g the i n f l o w and p i p e t t i n g i00 ~i of
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s u s p e n s i o n d i r e c t l y into the chamber. The pump was then r e - s t a r t e d and 30 min f r a c t i o n s w e r e collected. A g e n t s were d i s s o l v e d in d e f i n e d m e d i u m and either added to the r e s e r v o i r s or, for 'pulsed' a d m i n i s t r a t i o n , were m a n u a l l y p i p e t t e d d i r e c t l y into the cell p e r i f u s i o n chamber. A s s a y of PGF PGF in the s u p e r n a t a n t was assayed in d u p l i c a t e as d e s c r i b e d p r e v i o u s l y (3) and the q u a n t i t i e s were e x p r e s s e d as the amount r e l e a s e d by 105 cells per hour (pmol/105 cells/h). S t a n d a r d curves from the r a d i o - i m m u n o a s s a y were g e n e r a t e d using an iterative c o m p u t e r p r o g r a m m e (4) and the mean fitted value of the d u p l i c a t e d e t e r m i n a t i o n s of each t r i p l i c a t e i n c u b a t i o n was c o m b i n e d to give a m e a n value for each e x p e r i m e n t a l data point. Chemicals P r o s t a g l a n d i n F2~ was o b t a i n e d from the Upjohn Company, Kalamazoo, MI, U.S.A. [9-3H]-Prostaglandin F2d and [ 5 , 6 , 8 , 1 1 , 1 2 , 1 4 , 1 5 - 3 H ] - a r a c h i d o n i c acid were o b t a i n e d from A m e r s h a m International, Bucks, England. A s c o r b i c acid, a r a c h i d o n i c acid, h i s t a m i n e diphosphate, mepacrine, cortisol, p r o g e s t e r o n e , e s t r a d i o l - 1 7 B , 4 B - p h o r b o l - 1 2 - m y r i s t a t e 1 3 - a c e t a t e (PMA) and c o l l a g e n a s e were o b t a i n e d from Sigma, St Louis, U.S.A. B r a d y k i n i n was o b t a i n e d from Serva, Heidelberg, FRG. M e d i u m 199 (Earle's Salts), M e d i u m 199 (Hank's B a l a n c e d Salts) and bovine fetal serum was o b t a i n e d from Gibco (New York, U.S.A.). M e d i u m 199 was p r e p a r e d by sterile f i l t r a t i o n and was s u p p l e m e n t e d w i t h p e n i c i l l i n (i00 m/ml), s t r e p t o m y c i n (i00 ~g/ml) and g e n t a m y c i n (25 ~g/ml) and was a d j u s t e d to pH 7.4. Arachidonic acid was p u r i f i e d by HPLC (Waters, Milford, Mass., U.S.A.) using a W a t e r s R a d i a l - P a k C18 c a r t r i d g e column (10 ~m p a r t i c l e size) w i t h a 5 m m internal diameter. The a r a c h i d o n i c acid was e l u t e d i s o c r a t i c a l l y with a c e t o n i t r i l e / 0 . 1 % p h o s p h o r i c acid (65:35 v/v) at a rate of 1.5 m l / m i n with a r e t e n t i o n time of 7 min and was m o n i t o r e d by its a b s o r p t i o n at 214 nm. Column fractions c o n t a i n i n g the p u r i f i e d a r a c h i d o n i c acid were n e u t r a l i s e d with IM NaOH, the a c e t o n i t r i l e e v a p o r a t e d under N 2 gas and the r e m a i n i n g aqueous s o l u t i o n d i l u t e d as appropriate. The r e c o v e r y of a r a c h i d o n i c acid from the HPLC was 90% as d e t e r m i n e d
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PROSTAGLANDINS
by the r e c o v e r y of [ 3 H ] - a r a c h i d o n i c two c o l u m n c a l i b r a t i o n e x p e r i m e n t s .
acid added in
Statistics The d i f f e r e n c e b e t w e e n the m e a n PGF value for each t r i p l i c a t e d e t e r m i n a t i o n was d e t e r m i n e d using S t u d e n t ' s t-test. D o s e - r e s p o n s e r e l a t i o n s h i p s were tested using linear r e g r e s s i o n analysis. Results are e x p r e s s e d in the text, figures and tables as m e a n ± SEM. RESULTS In the d e v e l o p m e n t of these e x p e r i m e n t a l p r o c e d u r e s it was found that 38% of the cell preparations from e n d o m e t r i a o b t a i n e d on days 6-27 of the m e n s t r u a l cycle failed to r e s p o n d to a d d e d PMA w i t h i n c r e a s e d PGF production. T h e r e was no c o r r e l a t i o n b e t w e e n cycle stage and s t i m u l a t e d PGF production. It was t h e r e f o r e found a d v a n t a g e o u s to c o m b i n e the cells from two or more uteri so that the p r o b a b i l i t y of o b t a i n i n g an u n r e s p o n s i v e p r e p a r a t i o n w a s minimised. All of the results p r e s e n t e d b e l o w were o b t a i n e d w i t h cells p r e p a r e d in this manner. E n d o m e t r i a l Cell
Incubations
H i s t a m i n e (5x10-7M - 5x10-5M) c a u s e d a c o n c e n t r a t i o n - d e p e n d e n t i n c r e a s e in PGF p r o d u c t i o n (r=0.863, n=15, p <0.01); Fig la). The lowest c o n c e n t r a t i o n of h i s t a m i n e w h i c h caused s i g n i f i c a n t l y i n c r e a s e d PGF p r o d u c t i o n was 10-6M (p <0.02). 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 h i s t a m i n e (10-5M) s i g n i f i c a n t l y s t i m u l a t e d PGF p r o d u c t i o n in seven cell p r e p a r a t i o n s (Table i). The a d d i t i o n of b r a d y k i n i n (10-6M) to the cell i n c u b a t i o n s s i g n i f i c a n t l y s t i m u l a t e d PGF p r o d u c t i o n in three p r e p a r a t i o n s but in one (Table i, Expt 2) the response was small in c o m p a r i s o n to o t h e r stimuli. Similarly, the a d d i t i o n of PMA or a r a c h i d o n i c acid to the cell i n c u b a t i o n s s i g n i f i c a n t l y s t i m u l a t e d PGF p r o d u c t i o n (Table i). In one experiment, the cells were p r e i n c u b a t e d w i t h e s t r a d i o l for three hours and then for a further hour w i t h the same estradiol c o n c e n t r a t i o n s . E s t r a d i o l (10-7M - 10-5M) s t i m u l a t e d a c o n c e n t r a t i o n d e p e n d e n t increase in PGF but at a h i g h e r c o n c e n t r a t i o n (5x10-5M) the r e s p o n s e d e c l i n e d (Fig 2).
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PROSTAGLANDINS
Cortisol caused a c o n c e n t r a t i o n - d e p e n d e n t fall in h i s t a m i n e - s t i m u l a t e d PGF p r o d u c t i o n to 30 ± 9% at a c o n c e n t r a t i o n of 5 x 10-5M (r=0.697, n=14, p <0.01; Fig ib). P r o g e s t e r o n e (10-6M) also inhibited the response to histamine (p <0.01). M e p a c r i n e was found to inhibit 'the responses to histamine ~(:~0-5M), b r a d y k i n i n (10-6M) and P M A (3x10-8M; Table 2). T h e lowest c o n c e n t r a t i o n of m e p a c r i n e that inhibited the response to histamine and b r a d y k i n i h w a s ' b e t w e e n }0-6M and 10-5M and for PMA was 10-5M. S t i m u l a t i o n of PGF p r o d u c t i o n by arachidonate was relatively unaffected by mepacrine (10-6M - 2x10-4M) in two of three experiments (Table 2) but in the remaining e x p e r i m e n t an inhibitory action of m e p a c r i n e was observed (Table 2, Expt 8). Endometrial
Cell Perifusions
A t o t a l of 42 experiments were performed, e a c h of which included one control and five experimental perifusions from the~ same pool of d i s p e r s e d cells. "Th~ p r o d u c t i o n of PGF in the first 60 min w a s high but decreased to control levels after 90 m i n . The introduction of histamine (10-5M), b r a d y k i n i n (10-6M), PMA (3x10 -8) or a r a c h i d o n a t e (5x10-5M) into the p e r i f u s i o n m e d i u m at 120 min or later stimulated PGF p r o d u c t i o n (Fig 3 a - d ) . A l t h o u g h the effect of each of these agents on the p r o d u c t i o n of PGF was similar, the onset and d u r a t i o n of release differed. The response to h i s t a m i n e was rapid and of short d u r a t i o n (60 min) d e s p i t e c o n t i n u e d a d m i n i s t r a t i o n (Fig 3a]. T h e - i n c l u s i o n Of p r o g e s t e r o n e (10-6M) or corti~01 (5x10-6M) in £he m e d i u m from the b e g i n n i n g of p e r i f u s i o n (i.e. 150 min before the i n t r o d u c t i o n of histamine) inhibited the response to histamine by 80-100% ~(data not shown) although neither hormone c o n s i s t e n t l y inhibited the elevated baseline ~values usually present during the f i r s t 30-60 min of perifusion. A similar p a t t e r n of response with rapid onset and short duration, was seen when £he cells were stimulated by b r a d y k i n i n (Fig 3 b ) . The response to P M A and a r a c h i d o n a t e were different (Fig 9c and d), being slightly slower in onset and p e r s i s t i n g longer (120 min or more) ~ The contrast between rapid response (histamine or
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TABLE
i
The effect of various agents on the production rates of PGF (pmol/105 cells/h) by dispersed human endometrial cells during short-term culture in defined medium.
Expt.
No. Control
0.21
1
±0.20
2
<0.04
Arachidonate (5x10-6M)
8.07+ ±0.72
~.839 ±0.40
Stimulating
agent
Histamine (10-5M)
Bradykinin (10-6M)
5.60+ ±0.91
2.97+ ~0.91
3
0,70 ~0.12
1.55 % ±0.17
4
<0.04
0.52~ ±0.Ii
5
6.83 ±1.15
12.20 ~ ±1.33
6
0.83 ~0.25
7
0.58 ±0.ii
8
1.90 ±0.12
2.03 % ±0.15
3.03 % ±1.05
0.19 t ±0.05
PMA ¸ (3xlO-8M)
2.03 %
±0.50 1.2q % ±0.03 1.76 % ±0.17
2.13 % ~±0.18 1.50 % ±0.02 3.0 % ±0'.18
Eight different cell preparations were incubated (triplicates) in defined m e d i u m (see Materials and Methods) with or w i t h o u t the agents listed. The PGF released into the m e d i u m was determined by radioimmunoassay after ether extraction,, T h e results ar e expressed as pmol PGF/105 cells/h (mean ± SEM)~ Significant differences between the PGF production rates in control incubations (using a value of 0.04 in expts. 2 and 4) and those with agents are denoted % (p <0.05) ~ PMA = phorbol myristate
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PROSTAGLANDINS
®
®
14 12
1.2 A J~
%(: \
u~
2 \
0.9
1
0
0£
E
Q.
~i,
10
U.
0.4
~ 4
O.2
o
5xlo"7 lO"e SxlO"s lO"s sxlo"s Histamine (M)
0
5~0-7 ~
~-5
5x10-5
Co~isol(M)
Figure i: PGF p r o d u c t i o n rates by d i s p e r s e d human e n d o m e t r i a l cells in s h o r t - t e r m culture; (a) S t i m u l a t i o n by h i s t a m i n e (b) I n h i b i t i o n of h i s t a m i n e (10-5M) s t i m u l a t i o n by cortisol. The cells were i n c u b a t e d (triplicates) in d e f i n e d m e d i u m (see M a t e r i a l s and Methods) in the absence or p r e s e n c e of histamine. The PGF r e l e a s e d into the m e d i u m was d e t e r m i n e d by r a d i o i m m u n o a s s a y after ether extraction. (a) In the a b s e n c e or p r e s e n c e of i n c r e a s i n g c o n c e n t r a t i o n s of histamine. (b) The cells were p r e i n c u b a t e d for 3 hours in the a b s e n c e or p r e s e n c e of c o r t i s o l (5x10-7M - 5x10-5M), the s u p e r n a t a n t removed and r e p l a c e d with m e d i u m c o n t a i n i n g the same c o r t i s o l c o n c e n t r a t i o n and h i s t a m i n e (10-5M) .
828
JUNE 1984 VOL. 27 NO. 6
PROSTAGLANDINS
TABLE The effect of mepacrine stimulated with various
Expt.
No.
2
on PGF p r o d u c t i o n agents
by endometrial
Concentration
Stimulus
10-6M
cells
of mepacrine 10-5M
2x10-4M
1
Histamine
{10-5M)
i00 ± 16
12 ± 1%
<3 %
<3 t
2
Histamine
(10-5M)
i00 ± 26
82 ± 32
<3 %
52 ± 8
3
Bradykinin
i00 ± 34
23 ± 5
4
PMA
(3x10-8M)
i00 ± 25
118 ± 32
5
PMA
(3x10-SM)
i00 ± 3
176 ± 12
6
AA
(5x10-6M)
i00 ± 9
120 ± 16
7
AA
(5x10-6M)
i00 ± 22
104 ± 32
8
AA
(5x10-6M)
i00 ± 9
(10-6M)
30 ± 5 t
i0 ± 10 % 21 ± 16 <3 t
9 ± 9t
15 ± 12 % 14 ± 12 f 126 ± i0
N.D.
70 ± 23 130 ± 50 12 ± 3 t
51 ± 21
The cells were incubated (triplicates) in defined m e d i u m (see Materials and Methods) with or w i t h o u t mepacrine for 3 h. The m e d i u m was then removed and replaced with m e d i u m containing the same concentration of m e p a c r i n e and the appropriate stimulator. The values for PGF p r o d u c t i o n obtained in the absence of mepacrine were normalized and expressed as 100% ± SEM. The PGF p r o d u c t i o n in the presence of mepacrine was normalized and expressed as mean% ± SEM of the value obtained in the absence of mepacrine. Decreased PGF p r d o c t i o n by cells in the presence of mepacrine is denoted by % (p <0.05). PMA = Phorbol m y r i s t a t e AA = Arachidonic acid
JUNE 1984 VOL. 27 NO. 6
829
PROSTAGLANDINS
6 5 r~
=
4
O
qo
3
O
E 2 o I I
"3-
0
!
10"7 5x10-7 5x10"6 10-5 5x10 -5 Estradiol-178 ( M )
10 -5
Histamine ( M )
F i g u r e 2: ~ T h e e f f e c t of e s t r a d i o l - 1 7 ~ on P G F p r o d u c t i o n by e n d o m e t r i a l c e l l s d u r i n g s h o r t term cultures. The cells were preincubated (triplicates) in d e f i n e d m e d i u m ( s e e M a t e r i a l s a n d M e t h o d s ) i n ~ t h @ p r e s e n c e o r a b s e n c e of e s t r a d i o l (10-7M - 5x10~SM) for 3 h. The medium was r e m o v e d a n d r e p l a c e d w i t h m e d i u m c o n t a i n i n g the same concentration of e s t r a d i o l . The P G F r e l e a s e d i n t o the m e d i u m d u r i n g i n c u b a t i o n for 1 h w a s d e t e r m i n e d by r a d i o i m m u n o a s s a y after ether extraction. There was a positive correlation (r=0.750, n=12, p <0.001) b e t w e e n PGF p r o d u c t i o n and estradiol concentration (10-7M - 1 0 - 5 M ) . The r e s p o n s e to h i s t a m i n e in the same p r e p a r a t i o n is s h o w n for c o m p a r i s o n .
830
JUNE 1984 VOL. 27 NO. 6
PROSTAGLANDINS
bradykinin) and slow r e s p o n s e was further d e m o n s t r a t e d by e x p o s u r e of the cells to h i s t a m i n e in the p r e s e n c e and a b s e n c e of a ~ a c h i d o n i c acid (5x10-6M). The response to h i s t a m i n e alone o c c u r r e d in the first 30 min but w h e n both h i s t a m i n e and arach:idonate were present, a t w o - c o m p o n e n t r e s p o n s e was seen i n d i c a t i n g little i n t e r a c t i o n b e t w e e n them Fig 4). The r e s p o n s e to h i s t a m i n e and b r a d y k i n i n w e r e i n v e s t i g a t e d further to d e t e r m i n e w h e t h e r the failure to m a i n t a i n a response d u r i n g their c o n t i n u o u s a d m i n i s t r a t i o n was due to d e p l e t i o n of p r e c u r s o r s or c o f a c t o r s or to some form of d e s e n s i t i z a t i o n . H i s t a m i n e (10-5M; Fig 5a) or b r a d y k i n i n (10-6M); Fig 5b) was a d m i n i s t e r e d for 2.4 min at 120 min and again at 210 min. The r e s p o n s e to each pulse was c o m p a r a b l e to that seen with c o n t i n u o u s administration. DISCUSSION H u m a n ~ e n d o m e t r i a l cells d i s p e r s e d with c o l l a g e n a s e and m a i n t a i n e d o v e r n i g h t in a c u l t u r e m e d i u m c o n t a i n i n g 5% b o v i n e fetal serum appear to m e e t the r e q u i r e m e n t s of a useful s y s t e m for d e t e c t i n g i n h i b i t o r s and s t i m u l a t o r s of p r o s t a g l a n d i n synthesis in the p r e g n a n t human uterus. Basal p r o d u c t i o n of PGF in a d e f i n e d m e d i u m was u s u a l l y low and the cells r e s p o n d e d in the e x p e c t e d way to a ~ v a r i e t y of agents k n o w n t o ' i n h i b i t or s t i m u l a t e p r o s t a g l a n d i n synthesis. D i f f e r e n t cell p r e p a r a t i o n s v a r i e d c o n s i d e r a b l y both in the basal p r o d u c t i o n rate of PGF and in the m a g n i t u d e of r e s p o n s e s to the v a r i o u s agents (see Table i). This v a r i a b i l i t y b e t w e e n p r e p a r a t i o n s p r e c l u d e d e s t i m a t i o n s of the p o t e n c y o f a p a r t i c u l a r agen t by c o m b i n i n g v a l u e s ~ o b t a i n e d with d i f f e r e n t preParations. However, the v a r i a b i l i t y w i t h i n a p r e p a r a t i o n was s u f f i c i e n t l y low to o b t a i n valid m e a s u r e m e n t s r e l a t i v e to a standard r e s p o n s e (usually histamine). Both p e r i f u s i o n a n d i n c u b a t i o n of the cells p r o v e d satisfactory. P e r i f u s i o n r e q u i r e d a more c o m p l e x s y s t e m and the n u m b e r of s i m u l t a n e o u s e x p e r i m e n t s was limited but the m e t h o d a l l o w e d the t i m e , c o u r s e of r e s p o n s e s to be studied. Static i n c u b a t i o n 0f the cells was s i m p l e r and the n u m b e r
JUNE 1984 VOL. 27 NO. 6
831
PROSTAGLANDINS
®
Histamine (10"5M)
®
1
1
Bradykinin (10"sM) V / I
.
.
.
.
I
I
I
I
/
1
1
~
O.E
~o.8
~> 0
0
,n O.E
0.6
o
0.4
~0,4
~ 0.2 60
120
180
-I
~ 0.2
240
60
120
TIME (min)
©
180
240
300
TIME (rain)
@
Phorbol Myristate (3x10"8M)
-. c- 3 -
Arachidonic Acid (5x10"6 M) .~
v / / / / J~z
zz2
0"61[
o 2-
to
o
Q. U.
60
120
180
240
TIME (min)
300
30 90 120
180
240
300
TIME (rain)
Figure 3: R e p r e s e n t a t i v e examples showing the effect of various agents on PGF release by d i s p e r s e d human e n d o m e t r i a l cells in p e r i f u s i o n culture. The d i s p e r s e d cells were placed in chambers and p e r i f u s e d with d e f i n e d m e d i u m (0.I ml/min) for 120 min or 180 min and then with the same m e d i u m c o n t a i n i n g (a) h i s t a m i n e (10-5Ml, (b) b r a d y k i n i n (10-6M), (c) PMA (3xl0-~M) and (d) arachidonic acid (5x10-6M). The duration of exposure of the cells to each agent is shown by the hatched bars. The PGF released into the m e d i u m was d e t e r m i n e d in timed fractions (30 min) by r a d i o i m m u n o a s s a y after ether extraction.
832
JUNE 1984 VOL. 27 NO. 6
PROSTAGLANDINS
Histamine (10 "5 M) alone Histamine (10 "5 M ) + Arachidonic ............... Acid ( 5 x 1 0 " 6 M ) 0.7-
"---'1
0.6O
0.5"
0.4o 0.3E o. 0.2-
"-
IJ.
0
a.
L-- 1
0.1
"--'1
60
120
180
L----I___
240
300
TIME (min) Figure 4: The effect of h i s t a m i n e in the p r e s e n c e of a r a c h i d o n i c acid on PGF release by d i s p e r s e d h u m a n e n d o m e t r i a l cells in p e r i f u s i o n culture. The d i s p e r s e d cells were p e r i f u s e d (see Fig 3) for 120 min with d e f i n e d m e d i u m and then w i t h m e d i u m containing h i s t a m i n e (10-5M) alone (o---o) or with m e d i u m c o n t a i n i n g h i s t a m i n e (10-5M) and a r a c h i d o n i c acid (5x10-6M; o. o). The d u r a t i o n of e x p o s u r e of the cells to these a g e n t s is shown by the h a t c h e d bar. With h i s t a m i n e alone there was a single peak of short d u r a t i o n while w i t h h i s t a m i n e and a r a c h i d o n i c acid there was a t w o - c o m p o n e n t response.
JUNE 1984 VOL. 27 NO. 6
833
PROSTAGLANDINS of s i m u l t a n e o u s e x p e r i m e n t s was limited only by the number of cells a v a i l a b l e but it was less suitable for d y n a m i c ~ s t u d i e s. T h e m e t h o d is a m e n a b l e to further r e f i n e m e n t by s e p a r a t i n g the e p i t h e l i a l and stromal c o m p o n e n t s (5). The p r o d u c t i o n of PGF by the d i s p e r s e d cells was s t i m u l a t e d by four agents known to s t i m u l a t e p r o s t a g l a n d i n s y n t h e s i s by a c t i v a t i n g a c y l h y d r o l a s e s . These e n z y m e s liberate a r a c h i d o n i c acid from cell m e m b r a n e lipids and are known to be s t i m u l a t e d by h i s t a m i n e (6), b r a d y k i n i n (7,8), PMA (9) and e s t r a d l o l (i). This effect in the p r e s e n t cell system was c o n f i r m e d by the i n h i b i t i o n of the response to • histamine, b r a d y k i n i n or PMA by m e p a c r i n e w h i c h inhibits p h o s p h o l i p a s e A 2 by a d i r e c t action ~I~). Both cortisol, w h i c h ~ n h i b i t s p h o s p h o l i p a s e A 2
~)
Histamine (10 "5 M )
~
Bradykinin (10 "6 M )
J¢
a
~3
tj
I
to
2 i
E Q. LI. 1 • ¢3 a. 50
120
180
240
TIME (min)
300
I
60
120
180
240 • 300
TIME ( m i n )
Figure 5: The effect of h i s t a m i n e and b r a d y k i n i n a d m i n i s t e r e d as r e p e a t e d short pulses on PGF release by d i s p e r s e d human e n d o m e t r i a l cells in p e r i f u s i o n culture. The d i s p e r s e d cells were p e r i f u s e d (see Fig 3) w i t h d e f i n e d medium. The cells were e x p o s e d to short pulses (2.4 min duration, as d e p i c t e d by the h a t c h e d bars) of (a) h i s t a m i n e (10-5M) or (b) b r a d y k i n i n (10-6M) at 120 min and again at 210 min.
834
JUNE 1984 VOE. 27 NO. 6
PROSTAGLANDINS
through the m e d i a t i o n of m a c r o c o r t i n or l i p o m o d u l i n (12,13), a n d p r o g e s t e r o n e , w h i c h has a less welld e f i n e d action on p h o s p h o l i p a s e A 2 in r e p r o d u c t i v e tissues, i n h i b i t e d the response to histamine. S u p p o r t for a c y l h y d r o l a s e s as the m a j o r site of action of histamine, b r a d y k i n i n and P M A was o b t a i n e d by showing that m e p a c r i n e had little e f f e c t on the r e s p o n s e to a r a c h i d o n i c acid w h e r e a s it c o n s i s t e n t l y i n h i b i t e d the r e s p o n s e to the former agents. The t r a n s i e n t r e s p o n s e of e n d o m e t r i a l cells to h i s t a m i n e is c o n s i s t e n t with the report (6) that h i s t a m i n e in c o n c e n t r a t i o n s similar to those used in the p r e s e n t e x p e r i m e n t s (10-7M - 10-5M) c a u s e s only a brief (3 min) r e ~ e a s e of p r o S t a c y c l i n from human e n d o t h e l i a l cells~ ~ D e p l e t i o n o f - p r e c u r s o r s Or c o f a c t o r s is an u n l i k e l y cause of the t r a n s i e n t r e s p o n s e since two short pulses of :histamine s e p a r a t e d by 90 m i n of p e r i f u s i o n e l i c ± t e d c o m p a r a b l e r e s p o n s e s (Fig 5a) . C o n t i n u o u s exposure of the cells to h i s t a m i n e may cause d e s e n s i t i z a t i o n , p o s s i b l y due to d o w n - r e g u l a t i o n of c e l l - s u r f a c e receptors. The p r e s e n c e of h i s t a m i n e (HI) receptors on e n d o m e t r i a l cell m e m b r a n e s has p r e v i o u s l y been r e p o r t e d (14). S t i m u l a t i o n of p r o s t a g l a n d i n s y n t h e s i s by PMA is well d o c u m e n t e d (9,15). The p r e s e n t e x p e r i m e n t s c o n f i r m that this action occurs in e n d o m e t r i a l cells and that the r e s p o n s e is i n h i b i t e d b y m e p a c r i n e , s u g g e s t i n g that PMA acts ~ by s t i m u l a t i n g a c y l h y d r o l a s e activity. The c o n t r a s t i n g p a t t e r n s of r e s p o n s e to PMA and to h i s t a m i n e or b r a d y k i n i n d u r i n g c o n t i n u o u s p e r i f u s i o n points to d i f f e r e n c e s in their modes of action. ~ A similar C o n c l u s i o n is s u g g e s t e d by the g r e a t e r r e s i s t a n c e of t h e r e s p o n s e to PMA to i n h i b i t i o n by m e p a c r i n e (Table 2). The:obs~rved conCentration-dependent response to e s t r a d i o l ' 1 7 B accords with t h e ~ p h y s i o l o g i c a l action of this h o r m o n e i n vivo (16,17,18) and with its e f f e C t on PGF product--{-n by cultures of human e n d o m e t r i a l b i o p s y tissue (19) and p r i m a r y m o n o l a y e r c u l t u r e s of e p i t h e l i u m from h u m a n p r o l i f e r a t i v e e n d o m e t r i u m (20). In-the latter study, e s t r a d i o l (10"8M) ~stimulated a fourfold i~crease in c u m u l a t i v e levels of PGF d u r i n g ~ i n c u b a ~ i o n of g l a n d u l a r tissue for 24~h W h e r e a s t H e p r o d u c t i o n of 'PGF by stromal cells was either u n a f f e c t e d or inhibited.
JUNE 1984 VOE. 27 NO. 6
835
PROSTAGLANDINS
This suggests that in our short-term cultures of mixed dispersed cells the increased production rate of PGF in response to estradiol may reflect glandular cell activity. The rapid response to estradiol (within 4 h) observed in the present study has not been described previously. More detailed investigation of the time course of the response would be of interest since it raises the possibility of a direct action not mediated by synthesis of new protein. We conclude that human endometrial cells, prepared by a simple procedure, are useful for the detection of endogenous stimuli and inhibitors of PGF synthesis. The cells retain sensitivity to estradiol, histamine, bradykinin and PMA which stimulate the synthesis of prostaglandins by activating acylhydrolases, and to cortisol and progesterone which inhibit synthesis at the same point in the biosynthetic pathway. ACKNOWLEDGEMENTS We are grateful to the gynecologists and nursing staff of National Women's Hospital, Auckland, New Zealand for their willing co-operation in obtaining endometrial tissues. We thank Dr J.E.Pike, The Upjohn Company, Kalamazoo, MI, U.S.A. for the gift of authentic PGF2e. This study was supported by the New Zealand Medical Research Council. REFERENCES i.
Liggins, G.C. Initiation of spontaneous labor. Clin. Obstet. Gynecol. 26:47-55, 1983.
2.
Saeed, S.A., D.M.Strickland, D.C.Young, A.Dang and M.D.Mitchell. Inhibition of prostaglandin synthesis by human amniotic fluid: Acute reduction in inhibitory activity of amniotic fluid obtained during labor. J. Clin. Endocr. Metab. 55:801-803, 1982.
3.
Liggins, G.C., G.A.Campos, C.M.Roberts and S.J.M.Skinner. Production rates of prostaglandin F, 6-keto-PGF 1 and thromboxane-B 2 by perifused human endometrlum. Prostaglandins 19:461-477, 1980.
836
JUNE 1984 VOL. 27 NO. 6
PROSTAGLANDINS
4.
Wilkins, T.A., D.C.Chadney, J. Bryant, S.H. Palmstrom and R.L.Winder. In: Radioimmunoassay and related procedures in medicine (Vol i). International Atomic Energy Authority, Vienna, 1978.
5.
Satyaswaroop, P.G., R.S.Bressler, M.M. de la Pena and E.Gurpide. Isolation and culture of human endometrial glands. J. Clin. Endocr. Metab. 48:639-641, 1979.
6.
Baenziger, N.L., F.J. Fogerty, L.F. Mertz and L.F.Chernuta. Regulation of histamine-mediated prostacyclin synthesis in cultured human vascular endothelial cells. Cell 24:915-923, 1981.
7.
Hong, S.L. and L.Levine. Inhibition of arachidonic acid release from cells as the biochemical action of anti-inflammatory corticosteroids. Proc. Natl. Acad. Sci. U.S.A. 73: 1730-1734, 1976.
8.
Schremmer, J.M., M.L.Blank and R.L.Wykle. Bradykinin-stimulated release of [3HI arachidonic acid from phospholipids and plasmalogens as sources of prostaglandin precursors. Prostaglandins 18:491-505, 1979.
9.
Levine, tumour lipids Nature
L. and K.Ohuchi. Retinoids as well as promoters enhance deacylation of cellular and prostaglandin production in MDCK cells. 276:274-275, 1978.
i0.
Dey, S.K., R.K.Hoversland and D.C. Johnson. Phospholipase A 2 activity in the rat uterus: Maturation by steroid hormones. Prostaglandins 23:619-630, 1982.
ii.
Vargaftig, B.B. and N. Dao-Hai. Selective inhibition by mepacrine of the release of 'rabbit-aorta-contracting-substance' evoked by the administration of bradykinin. J. Pharm. Pharmac. 24:159-161, 1972.
12.
Flower, R.J. and G.J.Blackwell. Anti-inflammatory steroids induce biosynthesis of a phospholipase A 2 inhibitor which prevents prostaglandin generation. Nature 278:456-459, 1979.
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837
PROSTAGLANDINS
13.
Hirata, F., ~. $~lomon inhibitory induced by Sci. U.S.A.
E. Schiffman, K. V e n k a t a s u b r a m a n i a n , and J. Axelrod. A phospholipase A 2 protein in rabbit n e u t r o p h i l s glucocorticoids; Proc. Nat. Acad. 77:2533-2536, 1980.
14.
Dey, S.K., C . V i l l a n u e v a and N.I.Abdou. Histamine receptors on rabbit b l a s t o c y s t and endometrial cell membranes. N a t u r e 278: 648-649/ 1979.
15.
Levine, L. Effects of tumour promoters on a r a c h i d o n i c acid m e t a b o l i s m by cells i n culture, '~ Carcinog. Compr. Surv. 7:477-494, 1982.
16.
Barcikowski, B., J.C. Carlson, L . W i l s o n and J.A.McCracken. The effect of endogenous and exogenous e s t r a d i o l - 1 7 ~ on the release of p r o s t a g l a n d i n - F 2 ~ from the ovine uterus. E n d o c r f n o l o g y 5:1340-1349, 1974.
17.
Ham, E.A., V.J.Cirillo, M . E . Z a n e t t i and F.A. Kuehl. E s t r o g e n - d i r e c t e d synthesis of specifi c p r o s t a g l a n d i n s in the uterus. ~ Proc. Natl. Acad. Sci. U.S.A. 7 2 : 1 4 2 0 - 1 4 2 4 , 1975.
18.
caStracane, V.D. and V.C.JOrdan. The effect of e s t r o g e n and p r o g e s t e r o n e on uterine p r o s t a g l a n d i n b i o s y n t h e s i s in the o v a r i e c t o m i s e d rat. Biol. Reprod. 13:587-597, 1975. !
19.
Abel, M.H. and D.T.Baird. The effect of 17Bestradiol and p r o g e s t e r o n e on p r o s t a g l a n d i n p r o d u c t i o n by human e n d o m e t r i u m m a i n t a i n e d in organ culture. E n d o c r i n o l o g [ i06:1599u1606, i980.
20.
Schatz, F. and Gurpide, E. The effects of estradiol on p r o s t a g l a n d i n F2~ levels in p r i m a r y m o n o l a y e r c u l t u r e s ot e p i t h e l i a l cells from human p r o l i f e r a t i v e endometrium. E n d o c r i n o l o g y 11"3:1274-1279, 1983.
E d i t o r : Harold Behrman
838
Received: 10-28-83
Accented: 4-16-84
JUNE 1984 VOL. 27 NO. 6