European Journal of Pharmacology, 44 (1977) 75--80 © Elsevier/North-Holland Biomedical Press

75

I N C R E A S E D P R O S T A G L A N D I N S Y N T H E T A S E A C T I V I T Y IN I N F L A M E D T I S S U E S O F THE RABBIT EYE PARIMAL BHATTACHERJEE and ANDREAS PHYLACTOS Institute of Ophthalmology, Judd Street, London WCIH 9QS, England Received 16 December 1976, revised MS received 1 March 1977, accepted 17 March 1977

P. BHATTACHERJEE and A. PHYLACTOS, Increased prostaglandin synthetase activity in inflamed tissues of the rabbit eye, European J. Pharmacol. 44 (1977) 75--80. Tissues from normal and inflamed rabbit eyes were examined for prostaglandin synthetase activity using homogenates and microsomal preparation. Ocular inflammation was induced with 10 pg Shigella endotoxin injected into the vitreous body. Homogenates of iris-ciliary processes of normal and inflamed eyes synthesized 2.3 and 5.6 //g respectively of prostaglandins per g wet weight of tissues from endogenous substrate. Intact tissues from normal and inflamed eyes produced similar amounts of prostaglandins (2.2 and 5.3 pg/g wet wt. respectively). Microsomes obtained from inflamed tissues produced 565 ng of prostaglandins per mg of protein per 30 min compared with 190 ng synthetized by normal microsomes. The apparent Km for the substrate of prostaglandin synthetase from inflamed tissues compared with that from normal was found to be lower. It is suggested that prostaglandin synthetase activity in the ocular tissues is modified during Shigella endotoxin-induced inflammation. Prostaglandin synthetase activity Ocular inflammation

Microsomes

1. I n t r o d u c t i o n Prostaglandins are released d u r i n g inflamm a t o r y r e a c t i o n s in various tissues (Willis, 1 9 6 9 ; Higgs et al., 1 9 7 4 ; Di Rosa et al., 1 9 7 1 ; Greaves et at., 1 9 7 1 ) , including the eye (Eakins et al., 1 9 7 2 a , b ; B h a t t a c h e r j e e , 1975). In m o s t tissues p r o s t a g l a n d i n s (PGs) are n o t cont i n u o u s l y f o r m e d b u t are s y n t h e s i z e d and released w h e n t h e r e is an a p p r o p r i a t e stimulus. In n o r m a l tissues p r o s t a g l a n d i n - s y n t h e s i z ing e n z y m e s a p p e a r to be p r e s e n t in an active form. In i n f l a m e d tissues, a cascade o f r e a c t i o n s o c c u r s leading t o cellular infiltration, phagoc y t o s i s and s u b s e q u e n t a c t i v a t i o n o f lysosomal a n d m e m b r a n a l p h o s p h o l i p a s e w h i c h a t t a c k the p h o s p h o l i p i d o f the cell m e m b r a n e to release f a t t y acids. It seems possible, therefore, t h a t an increased availability o f free f a t t y acids, such as dihomo-3,-linolenic o r arachid o n i c acid, the substrates for PG b i o s y n t h e s i s

Shigella endotoxin

Kinetics

and (or) s o m e changes in the characteristics o f the PG s y n t h e t a s e s y s t e m m a y be responsible for t h e synthesis and release o f PGs during i n f l a m m a t i o n . The present s t u d y was u n d e r t a k e n to examine PG s y n t h e t a s e activity o f the m i c r o s o m a l f r a c t i o n and h o m o g e n a t e s o f n o r m a l and i n f l a m e d r a b b i t eyes to see if t h e r e are a n y changes in the e n z y m e activity d u r i n g experim e n t a l l y i n d u c e d ocular i n f l a m m a t i o n .

2. Materials and methods A d u l t male and female albino rabbits weighing 2 . 5 - - 3 . 5 kg were used t h r o u g h o u t the s t u d y . 2.1. A c u t e i n f l a m m a t i o n 10 t~g o f Shigella e n d o t o x i n (Difco L a b o r a tories, U.S.A.) dissolved in 50 pl sterile saline

76 were injected into the vitreous body of one eye. In a separate group of rabbits which served as controls, one eye of each animal was injected with a similar volume of saline. 24 h later, the rabbits were killed with sodium pentobarbitone (60--70 mg/kg) and the iris-ciliary processes and cornea were dissected out. The iris-ciliary processes (2--3 tissues for each sample) were homogenized in 2 ml of phosphate buffer, pH 7.4 at 4°C. Homogenates or whole tissues were then incubated in shaking water bath at 37°C for 30 min after which an equal volume of absolute alcohol was added to stop the reaction. The preincubation PG level in the aliquots of homogenate and intact tissues was also determined.

2.2. Preparation of microsomal fraction Iris-ciliary processes of normal and inflamed eyes were pooled and homogenized in ice-cold phosphate buffer pH 7.4. After centrifugation at 10,000 g for 10 min, the supernatant was recentrifuged at 80,000 g for 60 min at 4°C. Microsomal pellets obtained after the second centrifugation were suspended in phosphate buffer and were used either immediately or the following day after overnight freezing. Protein concentration in the microsomai suspension was determined according to the m e t h o d o f Lowry et al. (1951) using bovine serum albumin as the reference protein.

2.3. Incubation Microsomal suspension equal to 400--500 pg of protein was incubated in 1 ml of phosphate buffer containing 16.5 pM arachidonic acid, 1 mM reduced glutathione and 1 mM adrenaline bitartrate (as cofactors) in a shaking water bath at 37°C for 30 min. This time of incubation was chosen so that PG synthesis measured lie in the linear region of synthesis. For the determination of Kin, concentrations of arachidonic acid ranging from 2 to 33 pM were used. The arachidonic acid (Sigma Chemical Company, England) was dissolved in absolute alcohol and diluted with phos-

P. BHATTACHERJEE, A. PHYLACTOS phate buffer to give a final concentration of 0.1% alcohol in the incubation medium. At the end of the incubation, the microsomes were denatured by immersing the tubes in boiling water for 1 min. Denaturation of the enzyme in this manner resulted in the loss of approximately 10% of PG activity as determined by comparing the heated and unheated standard PGE1. This loss of activity most likely has occurred in all the samples treated similarly. The basal PG level in the microsomes was ascertained by destroying the enzyme before incubation. The contribution of the endogenous substrate present in the microsomes and of non-enzymatic conversion of arachidonic acid towards the total PG synthesis was determined by incubating the microsomes and arachidonic acid separately in the presence of cofactors. The samples were then extracted using the method of Unger et al. {1971). Prostaglandins were assayed in terms of PGE1 using rat fundus strip (Vane, 1957) suspended in 10 ml of Krebs solution at 37°C, gassed with 5% CO2 in 02, and containing methysergide, atropine, mepyramine (all 0.1 gg/ml), phenoxybenzamine, propranolol, and indomethacin (all 1 pg/ml). Prostaglandins in the extracts of incubated homogenates and microsomes were identified colorimetricaily by thin layer chromatography according to the method of Kiefer et al. (1975). Duplicate extracts of homogenates and microsomes were dissolved in chloroform and chromatographed on silica gel plates (Eastman Chromogram, No. 6061). Pure samples of PGE (El + E2) and F2~ were treated similarly and chromatographed concurrently. Zones corresponding to standard PGs were scraped off, extracted and bioassayed as described above. More than 80% of the PGs formed during incubation was found to be E-type PGs.

3. R e s u l t s

3.1. The results of this study are summarised in table 1 (A and B) and fig. 1. Under the condi-

PROSTAGLANDIN SYNTHETASE ACTIVITY IN INFLAMED EYES

77

TABLE 1 A. Production of prostaglandin E-like material by iris-ciliary processes (C.P.) and cornea from the endogenous substrate (PGE-like material/~g/g wet wt.). Normal (mean ±S.E.M.)

Intactiris-C.P. Homogenates Intact cornea

Inflamed (mean ±S.E.M.)

0 min

30 rain

Net

0 min

30 rain I

Net

0.20+ 0.05(5) 0.30± 0.10(6) 0.02+ 0.01(4)

2.40± 0 . 2 0 ( 5 ) 2.60± 0.50(12) 0.06± 0 . 0 2 ( 5 )

2.20 2.30 0.04

0.50± 0.20(6) 0.40+_ 0.25(9) 0.03+_ 0.01(4)

5.80 + 0 . 8 0 ( 8 ) ( p < 0.001) 5.30 6.0 + 0 . 7 0 ( 9 ) ( p < 0.001) 5.60 0.27± 0 . 0 6 ( 6 ) ( p < 0.001) 0.24

B. Synthesis of PGE-like material by microsomal fraction prepared from normal and inflamed-ciliary processes from exogenous substrate (ng of PGE-like material/mg of protein). Normal (mean ±S.E.M.)

Inflamed (mean ±S.E.M.)

0 rain

30 rain

Net

0 rain

30 rain I

Net

20 +- 5(9)

210 + 20(9)

190

35 ± 10

600 +- 60(6) (p < 0.001)

565

' All p values were calculated by Student's t-test from the difference between the means of normal and inflamed at 30 rain. S.E.M. = standard error of the mean. Figures in parentheses are the number of experiments. tions of the experiments, the homogenates of the normal iris-ciliary processes of the rabbit eyes produced a net amount of 2.3 pg of PGE-like material/g wet weight, compared with 5.6 pg PGE-like material by the homo-

3s T 3O

genates of the inflamed iris-ciliary processes. Similar amounts (2.2 and 5.3 pg PGE-like material/g wet weight) were produced by intact tissues indicating that homogenization d o e s n o t m a k e a n y d i f f e r e n c e in t h e a m o u n t of PGE-like material formed. The cornea from inflamed eyes also produced PGE-like material in q u a n t i t i e s m u c h h i g h e r t h a n d i d t h e n o r mal tissues (table 1A).

25

3.2. 20-

15-I> I0-

-0"4

-0'3

-0"2

-0'1

0

J 01

I 0"2

I 03

1

0.4

I 0"5

I 06

5

Fig. 1. Lineweaver--Burk plots relating arachidonic acid concentration and velocity. Abscissa, reciprocal of arachidonic acid concentration; ordinate, reciprocal of velocity expressed as nmoles of PGs per mg of protein per 30 rain. e, Normal PG synthetase K m = 6.8 X 10 -6 M; A, inflamed PG synthetase K m = 3.3 X 10 -6 M.

Microsomal fraction prepared from normal and inflamed iris-ciliary processes synthesized 190 and 565 ng of PGE-like material/mg of protein/30 min respectively using 16.5 pM a r a c h i d o n i c a c i d as t h e s u b s t r a t e ( t a b l e 1 B ) . T h e fig. 1 s h o w s t h e L i n e w e a v e r - - B u r k p l o t s o f a r a c h i d o n i c a c i d in 10 -6 M c o n c e n t r a t i o n (abscissa) and of the PGE-like material forme d e x p r e s s e d as n m o l e / m g o f p r o t e i n i n 3 0 m i n . T h e a p p a r e n t Km f o r a r a c h i d o n i c a c i d of the PG synthetase from normal and i n f l a m e d t i s s u e s a r e 6 . 8 × 1 0 -6 M a n d 3 . 3 × 1 0 -6 M r e s p e c t i v e l y . T h e s a t u r a t i o n c o n c e n t r a tion of the substrate for the enzyme from b o t h s o u r c e s lies b e t w e e n 25 X 10 -6 M a n d 3 3 X 1 0 -6 M.

78 Prostaglandin E-like material formed during incubation of the microsomal fraction and arachidonic acid separately were 20--30 ng PGE-like material per mg of protein and 10 ng respectively. Also direct addition of 5 pg of arachidonic acid (conc. used in incubations) to the organ bath produced a response of the rat fundus equal to 15 ng of PGE1 approximately. Hence the contribution of the enzyme and substrate blanks towards the synthesis of total PGE-like material is insignificant. We would like to point out that although PGEI not PGE~ was used as a reference standard in the present investigation, it seems unlikely t h a t this would alter the differences or changes in the PG synthesis observed in view of the fact that PGE2 has been reported to be either equiactive (Horton and Main, 1963; Main, 1973) or slightly more active than E~ (not more than 1.5 times; Ian Stamford, personal communication). 4. Discussion

The preceding results clearly showed that the a m o u n t of PGE-like material synthesized by normal and inflamed tissues differ significantly. Production of PGE-like material by the homogenates and microsomes of inflamed iris-ciliary processes was 2.3 and 2.9 times higher than that formed by normal tissue homogenates and microsomes respectively. It is known that during phagocytosis, hydrolytic enzymes such as phospholipases, ~-glucuronidase, elastase, etc., are released from lysosomes (Anderson and Irwin, 1973). Phospholipases, by their hydrolytic action on phospholipids of the cell membrane release free f a t t y acids such as arachidonic and dihomo-7-1inolenic acid which are substrates for prostaglandin biosynthesis and are implicated in PG biosynthesis in inflamed tissues (Anderson et al., 1971). Although free fatty acid levels in the tissues were n o t determined in the present study, higher level of substrates for PG synthesis in inflamed tissues is to be expected as phospholipases released during phagocytosis are

P. BHATTACHERJEE, A. PHYLACTOS known to act on phospholipids (Anderson et al., 1971; Flower and Blackwell, 1976) to liberate free fatty acids which may be enough to ensure PG synthesis during endotoxin-induced inflammation. Therefore, the increased formation of PGE-like material by inflamed tissues might have been partly due to the availability of greater a m o u n t of free fatty acids because of phospholipase action. However, the same explanation does not account for the increased synthetic activity of microsomes which utilised exogenous substrate. As discussed below the enhancement of the microsomal PG synthetase activity may be responsible for such an increased synthesis. The important feature of the present experiments is the finding that the apparent Km of the PG synthetase from normal and inflamed tissues differ considerably. In fact, the enzyme from the latter source has lower K m value which implies that the affinity of the enzyme for the substrate has gone up. The alteration of this kinetic parameter of the enzyme suggests that the inflammatory reactions induced by Shigella endotoxin modulated the activity of the PG synthetase system. Whether this modulation of the synthetic activity is due to the activation or induction of the enzyme is difficult to distinguish, particularly if PG synthetase system constitutes only a small part of the microsomal protein. Any significant change in the concentration of the enzyme protein might therefore produce only undetectable changes in total protein concentration. Previous studies also reported several fold increase in PG synthesis when leucocytes and intestinal tissues were incubated in the presence of dead bacteria and E. coli endotoxin respectively (Higgs and Youlten, 1972; McCall and Youlten, 1973; Higgs et al., 1975). Herman and Vane (1975) reported that PGs formed by jejuna taken from rabbits injected intravenously with endotoxin was increased several fold compared with that by normal jejuna. Recently Floman and Zor (1976) demonstrated an eight-fold increase in PG production by inflamed synovial tissues

PROSTAGLANDIN SYNTHETASE ACTIVITY IN INFLAMED EYES

compared with that by normal tissues. The increased prostaglandin production reported in these studies could have been due to the stimulation of PG synthetase system. Endotoxins (Lipopolysaccharides of bacterial cell membrane) cause inflammation by activating complement system which is known to produce chemotactic factors for neutrophils and to promote phagocytosis (Gewurz et al., 1971). McCall and Youlten (1973) suggested that the activation of prostaglandin synthetase during phagocytosis may have a controlling influence in the acute inflammatory process. Precisely how the endotoxin-induced cellular changes stimulate the PG synthetase system and whether only one or all the components of this multi-enzyme system (cyclo oxygenase, peroxidase and endoperoxide isomerase) is affected is not known. On the basis of the evidence presented above, it is concluded that during experimentally induced inflammation of the rabbit eye PG synthetase system is stimulated and increased PG synthesis is probably the consequence of such a modulation of the enzyme activity.

Acknowledgements We wish to thank the Medical Research Council for financial help, Mr. C. Higgins for technical assistance, Mrs. F.H. Dolamore and Mrs. C.M. Dawson for secretarial help. Prostaglandins were generously supplied by Dr. John E. Pike of the Upjohn Co., Kalamazoo, Michigan, U.S.A.

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