Chem -Btol. Inte~ct~ons, 76 (1990) 77--87 77 Elsewer Scmntlfic Pubhshers Ireland Ltd

SPECIFIC ROLE OF AN a,~-UNSATURATED CARBONYL GROUP IN y-GLUTAMYLCYSTEINE S Y N T H E T A S E INDUCTION BY P R O S T A G L A N D I N A 2

KOUJI OHNO, JUNKO HIGAKI, SHOUZO TAKECHI and MASAHARU HIRATA*

Sh~onog~ Research Laboratories, Sh~onog~ and Co Ltd., Fukushtma-ku, Osaka 553 (Japan]

(Received April 9th, 1990) (Rewmon received May 27th, 1990) (Accepted May 29th, 1990)

SUMMARY

The effects of prostaglandins (PGs) on cellular glutathione (GSH) status in L-1210 cells were examined. PGA 2 and J2, whlch have an a,f~-unsaturated carbonyl group in the cyclopentane ring, elevated the GSH content, but PGB 2, D 2, E 2 and F2~ did not show the effect. When L-1210 cells were incu- bated with various 2-cyclopentenone demvatives, 4-hydroxy-2-cyclopentenone and some of related compounds elevated cellular GSH levels. Subsequent study with cell-free extract of cultured L-1210 cells revealed that PGA 2 and 4-hydroxy-2-cyclopentenone induced ),-glutamylcysteine synthetase activity at the transcriptional level. This induction was also found in other cultured mammalian cells such as HeLa $3, NIH/3T3 and porcine aorta endothelial cells. When L-1210 cells were incubated with PGA 2 in the presence of 4- hydroxy-2-cyclopentenone and its analogues, they inhibited the accumulation of PGA 2 in cell nuclei. Our findings thus suggest that an a,/3-unsaturated car- bonyl mmety is responsible for enhancing the biosynthesis of ),-glutamylcy- steine synthetase in cultured cells.

Key words Prostaglandin A 2 - - ),-Glutamylcystelne synthetase - Gluta- thlone - - L-1210 cells - 4-Hydroxy-2-cyclopentenone

INTRODUCTION

Glutathione (GSH: L-y-glutamyl-L-cysteinylglycine), which is widely distrib- uted in mammalian cells, has many important roles in cellular functions [1].

*Correspondence to Masaharu H1rata, Shlonogl Research Laboratorles, Shlonogl and Co, Ltd, Fukushlmaku, Osaka, 553 Japan Abbrevlatlons BSO, buthlonme sulfoxlmme, FCS, fetal calf serum, GSH, glutathlone, PBS, phos- phate-buffered sahne, PG, prostaglandln

0009-2797/90/$03 50 © 1990 Elsevier Scientific Pubhshers Ireland Ltd Printed and Pubhshed m Ireland

78

They include: (a) protectmn of cell membranes by the maintenance of essen- tml thml groups of protein and other molecules [2], (b) ehmmatmn of hydro- gen peroxide and other peroxides [3,4], (c) translocatmn of amino acMs, small peptldes and amines across cell membranes [5], (d) detoxffmatmn of xenobmt- ms via conjugation with metabohte(s) [6,7]. Intracellular GSH synthesis pro- ceeds by two successive steps catalyzed by soluble enzymes, ),- glutamylcysteme synthetase and GSH synthetase, respectively Recently, we found that PGA 2, which has an a,/3-unsaturated carbonyl group m its cyclo- pentane ring, caused marked and sustained elevatmn of cellular GSH con- tent, as a result of mductmn of ),-glutamylcysteme synthetase [8]. PGA 2 is an enzymatmally dehydrated product of PGE 2 m serum [9,10] and inhibits the growth of varmus cultured mammahan cells [10-12] Unhke other PGs, PGA~ is actively taken up by the cells and accumulated In cell nuclei with brad- mg to nuclear proteins [13--15]. Lines of ewdence suggest that accumulation and binding of PGA 2 m nuclei could be the mltml step to the expressmn of its actmn and that the aft-unsaturated carbonyl group m the cylopentane ring of the PG structure plays an important part m the PGA 2 actmn. In this study, we examined the structure-actlwty relationships of PGs and varmus 2-cyclopentenone demvatlves and clarffmd the reqmrement of the aft-unsatu- rated carbonyl group for the mductmn of ),-glutamylcystelne synthetase by PGs.

MATERIALS AND METHODS

Matemals L-[G-SH]Glutamm acid (46 C1/mmol) and L-[14C]glycme (108 mC1/mmol) were

obtained from Amersham International plc. [5,6,8,11,14,15(n)-3H]PGA2 (80 C1! retool) was purchased from Hungaman Academy of Scmnces (Budapest, Hun- gary). PGA~, PGB2, PGD 2, PGE 2, PGF2o and PGJ 2 were from Funakoshl Yaku- hm Co. (Tokyo, Japan) Drmd powder of RPMI-1640, Ham's F-12, 199, Dulbecco's MEM and Dulbecco's PBS were from Nlssm Selyaku Ltd. (Tokyo, Japan), fetal calf serum (FCS) from M A. Bloproducts (Walkersvllle, MD) GSH and ATP from Sigma Chemmals Co. (St Lores, MO), L-glutamm acid, L- glycme, L-cysteme and ),-glutamylcysteme from Nakaral Tesque (Kyoto, Japan), 2-cyclopentenone, 2-methyl-2-cyclopentenone, 3-methyl-2-cyclopenten- one, 2-cyclopenten-l,4-dlol and 2-cyclopenten-l,4-dlone from Aldrmh Chemi- cals Co (Milwaukee, WI). (R)-4-Hydroxy-2-cyclopentenone, (S)-4-hydroxy-2- cyclopentenone and 4-methoxy-2-cyclopentenone were prepared m the con- ventlonal manner from 2-cyclopenten-l,4-dlol (_)-trans-5-Ethyl-4-hydroxy-2- cyclopentenone was synthemzed as descmbed by Plancatelh et al [16] All other chemicals used were of reagent grade

Cells and culture cond~twns L-1210 murme leukemia cells, NIH/3T3 mouse flbroblasts and HeLa $3

human eplthehal carcinoma cells were grown m RPMI-1640, Dulbecco's MEM and Ham's F-12 medmm supplemented with 10% FCS and antlblotms,

79

respectively. Porcine aorta endothelial cells were prepared as described by Wey et al. [17] and cultured in 199 Medium containing 20% FCS. The aorta endothelial cells at passage 4--6 were used for all experiments.

Incubation procedures L-1210 cells were suspended at a density of 5 x 105 cells/ml m culture

flasks (Nunc, Roshlde, Denmark} with 4 ml of culture medium. The cells were incubated at 37°C with 10 wM PGJ e, 20/~M PG (PGA 2, B 2, D 2, E e or Feo) or 20 wM of a 2-cyclopentenone demvative After 0, 12 and 24 h of mcubatmn, the number of viable cells was counted and the cells were washed twine with PBS, then resuspended m 50 ~l of PBS to determme the cellular GSH con- tent. Determination of GSH content was performed by the fluorometrm method as descmbed by Hissm and Hflf [18] and the results were confirmed by HPLC analysis as descmbed below To measure the activity of the enzymes related to GSH biosynthesis, L-1210 cells, HeLa $3 cells, NIH/3T3 cells and porcine aorta endothelial cells grown at the confluent phase were incubated for 12 h w~th 20 ~M of each type of PG or of the 2-cyclopentenone demvative. After the incubatmn, the cells were collected and were pelleted by centmfugatmn at 100 x g for 10 mm The cell pellets were lysed by brief homogemzatmn w~th Potter homogemser m 500 ~l of 50 mM Tins--C1 buffer (pH 7.4) containing 25 mM KC1, 10 mM MgC12, 2 mM CaCl 2, 1 mM NaHCO s and 1 mM EDTA. The lysates were then centrifuged at 15 000 × g for 20 min at 4°C. The 15 000 × g supernatants were used as the enzyme source

Determination of the enzyme act~wty related to GSH b~osynthes~s y-Glutamylcysteine synthetase and GSH synthetase actlvltms were deter-

mined by measuring the rate of formation of radlolabeled y-glutamylcysteme and GSH as descmbed by Davis et al. [19] and Oppenheimer et al [20]. The ),- glutamylcysteme formed was measured m the reachon mixture (final vol- ume, 50 ~l) containing 100 mM Tins--C1 buffer (pH 8.0}, 50 mM KCI, 20 mM MgCl 2, 2 mM EDTA, 10 mM ATP, 5 mM cysteme, 5 mM [3H]glutamm acid (0.8 mCi/mmol) and the cell extracts (100 ~g of soluble proteins). After incu- bation for 10 mm at 37°C, the reactmn was terminated by adding 50 ~l of acehc acid and 800 ~l of acetone, then the mixtures were centrifuged at 15 000 x g for 10 mm to separate the labeled products and cellular proteins. The supernatants were evaporated to dryness and the residual labeled prod- ucts were subjected to HPLC analysis. HPLC was performed on a Nucleosll 5NH 2 column (4.6 x 250 mm) with 50 mM KH2PO4-acetomtrlle (3:1) acldlfmd by phosphoric acid to pH 3.5 as a mobile phase at a flow rate of 1.0 ml/mm and monitored at 230 mm using a Jasco TWINCLE hqmd chromatograph The fractmns corresponding to ),-glutamylcysteme and GSH were collected and then the radioachvlty m these fractmns were determined. For the assay of GSH synthetase achvlty, 5 mM [:4C]glycme (0.8 mC1/mmol), 5 mM gluta- mylcysteine and 10 mM ATP were incubated with the cell extracts (100 ~g of soluble proteins) for 10 mm at 37 °C m 50/~l of the same buffer as descmbed above. The procedures for termmatmn of the reaction and preparatmn of

80

s a m p l e s for H P L C a n a l y s i s w e r e t h e s a m e as t h o s e d e s c r i b e d for l a b e l e d 7- g l u t a m y l c y s t e m e .

Effect of 2-cyclopentenone demvat~ves on accumulatmn of PGA 2 ~n nuclez L-1210 ce l l s (1 5 - - 2 . 0 x 107 cel ls /ml) w e r e i n c u b a t e d m 1 ml of P B S w i t h

10 ~M [3H]PGA2 (20 mC1/mmol) in t h e p r e s e n c e of 0, 50, 100 a n d 200 /aM va r -

ious 2 - c y c l o p e n t e n o n e d e r i v a t i v e s for 10 min a t 37°C A f t e r t h e i n c u b a t i o n , t h e cel ls w e r e w a s h e d t w i c e w i t h ice-cold P B S a n d l y s e d b y h o m o g e n i z a t i o n m 500 ~l of h y p o t o n l c s o l u t m n c o n t a i n i n g 5 m M MgC12, 2 m M CaCl 2 and 1 m M N a H C O 3 A p o r t m n of t h e l y s a t e s w a s t a k e n in to t h e c o u n t i n g v ia l to m e a s u r e t o t a l P G u p t a k e . T h e r e s t of t h e l y s a t e s w e r e c e n t r i f u g e d a t 15 000 x g for 20 m m a t 4 °C and t h e s u p e r n a t a n t s w e r e t r a n s f e r r e d to c o u n t i n g

v ia l s . T h e p e l l e t s w e r e s u s p e n d e d m 500 ~l of h y p o t o n i c so lu t ion , t h e n t r a n s - f e r r e d to c o u n t i n g v ia l s T h e r a d m a c t i v i t y in t h e s u p e r n a t a n t s and t h e pel- l e t s w e r e d e t e r m i n e d to m e a s u r e t h e P G a c c u m u l a t i o n in c y t o s o l and nucle i ,

r e s p e c t i v e l y [14,15]

RESULTS

Effect of PGs on cellular GSH level W h e n L-1210 ce l l s w e r e t r e a t e d w i t h 20 jaM P G A 2, B 2, D 2, E 2, Fe~ or 10 jaM

P G J 2 for 12 h, e l e v a t i o n of t h e G S H c o n t e n t w a s d i s t r a c t in cel ls t r e a t e d w i th

P G A 2 and P G J 2 w h i c h h a v e an a , / 3 -unsa tu r a t ed c a r b o n y l g r o u p a t t h e 10,11- p o s i t i o n a n d t h e 9 ,10-pos i t ion , r e s p e c t i v e l y (F ig 1) T h e n u m b e r of t h e con-

PGA2

PGB2

PGD2

PGE2

PGF2

PGJ 2

GSH content (% control)

0 100 200 3OO i

h

F b

I I I

Fig 1 Effect of PGs on cellular GSH content in L-1210 cells L-1210 cells (5 x 105 cells/ml) sus- pended in 4 ml of culture medmm were incubated with 10 ~M PGJ 2 or with 20 ~M PGA 2, B 2, D 2, E 2, or F2° for 12 h at 37°C After the incubation, the cells were washed and subjected to the determination of cellular GSH content as described under Materials and Methods Results are expressed as the means _+ S E of three separate experiments The amounts of GSH m control cells was 2 4 _+ 0 3 nmol/106 cells

81

trol cells and PGA 2- and PGJ2-treated cells increased from 5 × l0 s cells/ml (initial) to 7.0, 6.5 and 6.4 × 10 s cells/ml, respectively, during 12 h of incuba- tion without any effects on cell viability The cells treated wlth other PGs were grown at the same rate as the control cells. Therefore, GSH contents in the cells treated wlth PGs were standardlzed based on cell number and were expressed as % control. In the cells treated wlth PGD 2 or PGE 2, the GSH content had not increased at 12 h after the Incubatmn, but rose to about 2-fold during further incubation for 12 h Since PGD 2 and PGE2 were time-dependently dehydrated to PGJ z and PGA 2 in culture medium supple- mented with serum [10,21,22], the dehydration products of PGD 2 and PGE 2 could be responsible for the elevatmn of GSH content. On the other hand, PGB 2, which has an unsaturated carbonyl group at the 8,12-posltion, did not affect on the GSH content. These results suggest that the carbonyl group and the non-substltuted double bond in the cyclopentane ring of the PG structure is important for the elevation of cellular GSH

Stimulation of GSH productwn by 2-cyclopentenone demvat~ves To investigate the role of the a,~-unsaturated carbonyl group, we incu-

bated L-1210 cells for 12 and 24 h in the presence of various 2-cyclopenten- one derivatives (Table I). 2-Cyclopentenone reduced the GSH content in the cells, while its derivatives, substituted at the 4-position by a hydroxy or methoxy group, elevated cellular GSH levels. (_+)-trans-5-Ethyl-4-hydroxy-2- cyclopentenone raised the GSH content, while 2-cyclopenten-l,4-diol and 2- cyclopenten-l,4-dlone had no effect. It seems that introduction of a substituent to a 2-cyclopentenone at the 4-position is critical for the elevation of cellular the GSH level and steric configuration at the 4-position is not important for their action. Furthermore, simultaneous addltmn of 1 mM buthionlne sulfoximine (BSO), an inhibitor of ),-glutamylcysteine synthetase and these 4-substituted derivatives to the cell culture medium completely abohshed the rise of GSH concentration (data not shown), suggesting that the elevation of GSH level caused by these derivatives is due to the acceler- ation of GSH bmsynthesis

Induction of y-glutamylcysteme synthetase We next examined if 4-substituted 2-cyclopentenone derivatives have the

potencies to induce the enzymes involved in GSH biosynthesis. The cell extracts prepared from L-1210 cells, which had been pretreated with 20 ~M PGA 2 or 20 ~M (R)-4-hydroxy-2-cyclopentenone, were used to determine the activities of y-glutamylcystelne synthetase and GSH synthetase. ),-Glutamyl- cysteine synthetase activity in control cells was 2.26 _+ 0.62 nmol/min/mg protein, while the activities In cells treated with PGA 2 and (R)-4-hydroxy-2- cyclopentenone for 12 h were 5.35 _+ 0.11 and 4.53 _+ 0.20 nmol/mln/mg pro- teln, respectively. Other 4-substituted derivatives such as (S)-4-hydroxy- and 4-methoxy-2-cyclopentenone also enhanced ),-glutamylcystelne synthetase activity to the same level as PGA 2, but 2-cyclopentenone did not elevate the enzyme activity (data not shown). The activity of GSH synthetase, which

82

TABLE I

EFFECT OF 2-CYCLOPENTENONE DERIVATIVES ON CELLULAR GSH CONTENT IN L-1210 CELLS

L-1210 cells (5 x 105 cells/ml) suspended in 4 ml of culture medmm were incubated with 20 ~M of each 2-cyclopentenone derlvahve for 12 and 24 h at 37°C After the incubation, the cells were washed and subjected to determmahon of the GSH content GSH content m the control cells at 12 and 24 h was 2 5 ± 0 1 and 2 4 ± 0 2 nmol/106 cells, respectively Values expressed as means ± S E (n = 3)

Compound GSH content (% control)

12 h 24 h

0

0 @ CH3

0

CH3

O

O

HO¢ O

;5 CH30

0

O

OH

HO

59 ± 3 54 ± 3

118 __ 4 124 ± 2

132 ± 6 100 ± 2

263 ± 10 362 ± 23

244 ± 10 372 ± 28

291 _+ 17 320 ± 7

136 _+ 6 277 ± 1

135 _+ 2 85 ± 1

108 _+ 7 84 -+ 1

83

catalyzes the formation of GSH from glutamylcysteme and glycme was not affected by these compounds; the actlvitms m control cells, cells treated with PGA 2 and (R)-4-hydroxy-2-cyclopentenone were 3.97 _+ 0.07, 3.98 _+ 0.01 and 4.01 +_ 0.20 nmol/mm/mg protein, respectively. We further examined the effect of 2-cyclopentenone demvatlves on y-glutamylcysteme synthetase activ- ity in other mammahan cell hnes such as NIH/3T3 hbroblasts, HeLa $3 and porcine aorta endothehal cells. The enzyme actiwty m each cell hnes were elevated to about 2--2.5-fold by PGA 2 and (R)-4-hydroxy-2-cyclopentenone was as active as PGA 2 {Table II).

Inh~b~twn of PGA~ nuclear accumulatwn Our prevmus study showed that PGA 2 was incorporated into the cells,

accumulated m nuclei and became bound to nuclear proteins, whmh caused the inductmn of specific genes [23]. It was also found that PGA 2 reduced y- glutamylcysteine synthetase at the transcmptmnal level [8]. We, therefore, examined whether a common mechamsm is revolved m the actmn of PGA 2 and 4-substituted 2-cyclopentenone demvatlves. L-1210 cells were incubated with 10 pM [3H]PGA 2 m the presence of 2-cyclopentenone demvatlves and the

TABLE II

EFFECT OF PGA 2 AND (R~4-HYDROXY-2-CYCLOPENTENONE ON y-GLUTAMYLCY- STEINE SYNTHETASE AND GSH SYNTHETASE IN VARIOUS CULTURED MAMMALIAN CELLS

L-1210, NIH/3T3, HeLa $3 and porcine aorta endothelial cells (PAEC) were each incubated wlth 20 /~M PGA 2 or (R) 4-hydroxy-2-cyclopentenone for 12 h After the incubation, the cells were washed and lysed by homogemzatlon The cell lysates were centrifuged at 15 000 × g for 20 mm at 4°C, and the supernatants were used as the enzyme source y-GlutamylcysteIne synthetase (A)

and GSH synthetase (B) activity were assayed as described under Materials and Methods Mean ± S E (n = 3) *Slgmfmantly ddferent from control, P < 0 05

Cell line Activity (nmol/mln/mg protein)

Control PGA 2 (R)-4-OH-2- cyclopentenone

(A) y-Glutamylcyste~ne synthetase L-1210 226 _+ 062 535 ± 0 11" 4 53 ± 0 20* NIH/3T3 0 78 ± 0 12 1 81 ± 0 06* 1 89 -+ 009" H e L a S 3 052 ± 006 129 -+ 012" 125 ± 009" PAEC 055 _+ 006 102 ± 004" 104 ± 006"

(B] GSH synthetase L-1210 397 _ 007 398 _+ 001 401 -+ 020 NIH/3T3 449 ± 015 469 ± 029 449 _ 030 H e L a S 3 916 ± 011 939 _ 009 922 ± 009 PAEC 371 ± 009 387 ± 011 364 _+ 011

Mean -e S E (n -- 3)

84

cellular uptake and accumulation of PGA 2 in nuclm were measured. When the cells were incubated with PGA 2 alone, the total uptake was 219 0 _+ 6.3 pmol/106 cells (n = 4) and the amounts of PGA~ accumulated m nuclear and soluble fractions were 109.5 _+ 2 8 and 108.2 ± 2.3 pmol/106 cells (n = 4), respectively. (R)-4-Hydroxy-2-cyclopentenone inhibited the nuclear accumula- tion of PGA 2 in a concentration-dependent manner (Fig 2B). The amount of PGA 2 in the nuclear fraction was reduced to about 60% by 100 ~M (R)-4- hydroxy-2-cyclopentenone and to <50% when the concentration was more than 200 ~M, though 100 ~M (R)-4-hydroxy-2-cyclopentenone did not affect the amount of PGA 2 in soluble fraction (Fig. 2A) Other 4-substituted 2-cyclo- pentenone derivatives, such as (S)-4-hydroxy-, 4-methoxy- and 5-ethyl-4- hydroxy-2-cyclopentenone, also inhibited the accumulation of PGA 2 m the nuclei (data not shown). On the other hand, 200 ~M 2-cyclopentenone only shghtly inhibited the accumulatmn of the PG m the nuclear fractmn (Fig. 2B). No significant effect on the nuclear accumulation of PGA 2 was found with PGB 2 or other 2-cyclopentenone derivatives substi tuted with a methyl group at the 2- or 3-positron (data not shown) These results suggest that 2-cyclo- pentenone derivatives, which can cause the Induction of ),-glutamylcysteine synthetase actwity, inhibit the accumulation of PGA 2 in cell nuclei.

1 0 0

0

-~ 5o

L9 n

so

(.9 D.

B A

0 , , 0 ~ ' 0 1 0 0 2 0 0 0 100 2 0 0

2-cyclopentenone denvatrve ~JM) Fig 2 Effect of 2-cyclopentenone derlvatlves on subcellular dlstrlbutlon of PGA 2 in L-1210 cells L-1210 cells (15--20 × 107 cells/ml) suspended in 1 ml PBS were incubated wlth 10 pM [3H]PGAz (20 mCl/mmol) for 10 mm at 37°C m the presence of 0, 50, 100 or 200 pM 2-cyclopen- tenone (O) or (R)-4-hydroxy-2-cyclopentenone (o) After the mcubatlon, the amounts of PGA 2 m

soluble fractmn (A) and in nuclear fractlon (B) were determined as described under Materials and Methods The results represent the means _+ S E of four experiments

85

DISCUSSION

We have previously shown that PGA 2 induced ),-glutamylcystelne synthe- tase in cultured L-1210 cells and elevated cellular GSH content [8]. In this study, we proved the carbonyl group and the non-substituted double bond in the cyclopentane ring of the PG structure are essential for the elevation of the GSH content; PGA 2 and J2 were observed to elevate the GSH content, while PGB 2, which did not possess the non-substituted double bond, had no stimulatory activity. This structure reqmrement is supported by the finding that 2-methyl- and 3-methyl-2-cyclopentenone, which have a cyclopentenone structure similar to that of PGB 2, did not elevate the GSH level. We also found that PGB 2, which did not effect on the cellular GSH content, did not inhibit the accumulation of PGA 2 in nuclei. In the case of 2-cyclopentenone derivatives, 4-substltuted 2-cyclopentenone inducted y-glutamylcysteine syn- thetase activity and inhibited the accumulation of PGA 2 in nuclei (Table II and Fig. 2). Thus, the activity for inhibiting the nuclear accumulation of PGA 2 is closely correlated with the ability to induce the ),-glutamylcysteine synthetase activity. PGA 2 and 4-substltuted 2-cyclopentenone probably share the same binding site(s) in the nuclei. According to Narumiya et al. [13-15], PGA 2 and J2 were incorporated into cultured mammalian cells, accumulated in nuclei and modified gene expression [23]. We assume the accumulation of PGA 2 in nuclei is the first key step toward ehcitlng the induction of y-gluta- mylcystelne synthetase. Recently, Cromlish and Roeder reported that N- ethylmalelmide, which is reactive to thiol, Inactivated human transcription factor IIIC through modification of its thiol group [24]. From consideration of their finding, we speculate that PGA 2 may modify the thiol group and regu- late the functions of nuclear proteins which are required for the transcrip- tional process. Among other PGs tested, PGD 2 and PGE 2 elevated the GSH levels when L-1210 cells were incubated for a longer period. However, this apparent stimulation of GSH biosynthesis was due to their dehydrated prod- ucts, PGJ 2 and PGA 2, formed during the culture [10,21,22]. While PGD 2 and PGE 2 have many biological activities such as inhibition of platelet aggrega- tion, Induction of vasoconstriction, modulation of neurotransmitter release and induction of sleep [25,26], PGJ 2 and PGA 2 lack these activities [22,27]. Instead, these cyclopentenone PGs, bear a,~-unsaturated carbonyl group, act as lnhIbitors of cell growth and inducers of heat shock protein [10-12,23]. Thus, our recent findings described in this report disclosed a new profile of their biological functions. Note that the enhancement of y-glutamylcysteine synthetase activity by PGA 2 and PGJ 2 was observed at a concentration with- out affecting the cell growth or cell viability

One aspect of present study is the finding that PGA e induced y-glutamyl- cysteine synthetase in 4 cultured cells of different origins, suggesting a same regulatory mechanism of GSH biosynthesis by PGA 2 (or PGJ 2) may operate in a wide variety of mammalian cells. Physiological significance of the enzyme induction by PGA 2 and its effect on cell functions will be a stimulat- ing future subject to be explored.

86

ACKNOWLEDGEMENTS

The authors thank Dr. M. Fujlmoto and Dr K. Suglta of Shmnogl Res Lab. for supplying porcine aorta endothehal cells and NIH/3T3 fibroblasts and Dr. S. Morn of Shionogi Res. Lab. for his valuable advice on the syn- thesis of 2-cyclopentenone demvatlves.

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