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Inhibition by glucocorticoids of PGE2 and ACTH secretion induced by phorbol esters and EGF in rat pituitary cells
J. sreroid Biochem. Vol. 30, No. 1-6, pp. 333-336, 1988 Printed in Great Britain
0022-4731188 $3.00+0.00 Pergamon Press pit
INHIBITION BY GLUCOCORTICOIDS OF PGEz AND ACTH SECRETION INDUCED BY PHORBOL ESTERS AND EGF IN RAT PITUITARY CELLS Centre de Recherches
E. D~RTOIS and M. M. BULKON* 1 I 1 route de Noisy, 93230 Romainville, France
Roussel-U&f,
Summary-In rat pituitary cells in primary culture glucocorticoids specifically inhibit PGEr and ACTH secretions induced by TPA, a potent phorbol ester derivative (triamcinolone acetonide>dexamethasone> cortisolrcorticosterone). However, while PGEl secretion can be inhibited up to 80%, ACTH secretion can only be inhibited up to 40%. Similar inhibitory effects are observed with mepacrine, an inhibitor of phospholipase A2 (PLA,). Glucocorticoids having also been described as PLA,-inhibitors, their inhibitory effect on TPA-induced secretions could thus be reiated to their anti-PLA, activity. Their inhibitory effect on PLA, has been attributed to their ability to induce the synthesis of li~co~in, the activity of which could be regulated by activation of kinase C or EGF-receptor kinase. Since in our model, EGF-induced PGEz secretion is also inhibited by dexamethasone, these results suggest that a lipocortin-like protein couid be present in pituitary cells and involved in the effect ofTPA and EGF on PGE2, and, at least partly, on ACTH release.
We have previously shown [l] that in rat pituitary cells, dexamethasone was able to inhibit the secretions of ACTH and prostaglandins E2 (PGE,) induced by 12-0-tetradecanoylphorbol I3-acetate (TPA), a potent phorbol ester derivative which is thought to act by activation of protein kinase C [2,3]. In order to see whether this TPA-induced ACTH secretion, like CAMP-induced ACTH secretion [4,5], is specifically inhibited by ~ucoco~icoids, we have compared the ability of different hormonal steroids to inhibit the effect of TPA on ACTH as well as PGE, secretion. In various experimental systems, the inhibitory effect of glucocorticoids on PGE, secretion has been attributed to the induction of a group of phospholipase A2 (PLAJ inhibitory proteins called lipocortin [6,7]. In order to see if PLA2 activation is also involved in TPA-induced ACTH secretion we have compared the inhibitory effect of glucocorticoids on PGE, and ACTH secretion to that of mepacrine, a well-known PLAr inhibitor [8]. Moreover, it has been shown that lipocortin can be phosphorylated by activation of kinases such as kinase C [9, lo] or epidermal growth factor (EGF)-receptor associated tyrosine kinase [ Ill. Since it is assumed that phospho~lation of lipocortin leads to a decrease in the PLA*-inhibitor potency of this protein [ 121, we have examined the ability of EGF to induce PGEr and ACTH secretions as well as the effect of dexamethasone on these EGF-induced secretions.
Materials. Medium 199 and fetal calf serum were obtained from Flow Laboratories; penicillinlstreptomycin from Gibco; trypsin, 12-O-tetradecanoylphorbol 13-acetate (TPA), 4cx-phorbol 12,13-didecanoate and EGF from Sigma; forskolin from Calbiochem Hoechst; and mepacrine from Boots. Dexamethasone, triamcinolone acetonide, corticosterone, cortisol, promegestone [R 5020: ( I?/?)-17-methyl- 17-( loxopropyl)-estra-4,9-dien-3-one], 5~-dihydrotestosterone (DHT), 17&estradiol and RU 486 if1 lp, 17/Q-1 1-[(4-dimethylamino)phenyl)]-17-hydroxy17-( I-propynyl)-estra-4,9-dien-3-one} were synthesized at the Roussel-Uclaf Research Center. Preparation of solutions. Steroids, phorbol ester derivatives and forskolin were dissolved in ethanol. Subsequent dilutions were prepared in medium 199 (final concentration in ethanol: 0.1%). Mepacrine and EGF were dissolved in water. The complete culture medium consisted of medium 199 supplemented with 0.3% of bovine serum albumin (BSA) 50 U/ml of penicillin and 50 &ml of streptomycin. Preparation
of dispersed anterior pituitary cells.
Anterior pituitary cells from mature male SpragueDawley rats were prepared as previously described [I]. Briefly, after disruption at 37°C with complete medium containing 0.25% trypsin, cells were plated in multiwell dishes at a density of 5 x 10’ cells/well and maintained for 4 days at 37°C under 5% C02-950h air in 1.5 ml of complete medium supplemented with 101 fetal calf serum. Effects of test-compounds on ACTH and PGE2 secretion. On day 5 of the culture, the cells were
Proceedings of the 8th International Symposium of The Journal of Steroid Biochemists “Recent Advances in Steroid Biochemistry” (Paris, 24-27 May 1987). *To whom correspondence should be addressed.
washed with medium 199, and incubated in 1 ml of fresh medium 199 under 5% CO*-95% air for 4 h in the presence of various concentrations of phorbol 333
. 3ooI il_ .
E. DARTOISand M. M. BOUTON
334
0
sc
I
2
x
B
“p
200
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s 2 I
l
100
E
l
l
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i
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w
01
. *Q
,;8
,;T
Pnorbol Ester Concentration
) I
10 EGF
Wtt
100
Concentration
x)ocl (ng/ml
f
Fig. 1. Effect of phorbol ester derivatives and EGF on PGE, and ACTH secretion. Pituitary cells were incubated for 4 h in the presence of the indicated concentrations of (A) TPA (-) or 4cr-phorbol 12, I3didecanoate (--). Control values: 0.3 tug/ml for PGI$ (X) and 5 rig/ml for ACTH (o), (B) EGF. Control values: 3.1 n&ml for PGEl (X) and 3.2 @ml for ACTH (of.
1001
1
, Id’
rf?
lcg
12
IO7 STEROID
IdO
12’ CONCENTRATE
x59
Iii8 16'
fM)
Fig. 2. Effect of different steroids on TPA-induced PGEl and ACTH secretion. Cells were preincubated for 1 h with the indicated concentrations of steroids, and then incubated for 4 h in the presence of TPA (100 nM). Results are expressed as per cent inhibition of the stimulation. (A) PGEl secretion. (B) ACTH secretion. T.A.: ttiamcinolone acetonide. Dex.: dexamethasone. Cortico.: corticosterone. P.: promegestone. E,,: 17/?-estradiol.
ester derivatives or EGF or forskolin. After incubation, media were removed and PGE, and ACTH concentrations were determined by radioimmunoassay [ 131. When steroids or mepacrine were tested they were added 1 h prior to the addition of TPA or EGF. RESULTS
Effect ofphor~ol ester derivatives and EGF an PGE, and ACTH secretion TPA caused a dose-dependent increase in both PGEr and ACTH secretions over a similar concentration range (Fig. 1A). Maximal release is achieved with a 100 nM TPA concentration, which is compatible with its af&nity for protein kinase C [2]. No stimulation could be observed with the inactive phorbol ester derivative (4a-phorbol 12,13-didecanoate) over the same concentration range. In contrast to TPA, EGF caused an increase in PGE, secretion only, ACTH secretion remaining unchanged over the range of concentrations ( 1- 1000 ndml) (Fig. 1B). The maxi-
ma1 release of PGE2 is observed with a 100 rig/ml concentration of EGF. Effect of steroids and mepacrine on TEA- and EGFinduced secretions Inhibition of PGEr and ACTH secretion induced by TPA (1OOnM) is observed in the presence of giucocorticoids only (Fig. 2). No significant inhibition of either PGE2 or ACTH secretion was observed in the presence of estradiol, promegestone or dihydrotestosterone. The inhibitory effect of glucocorticoids follows the same rank order as their binding affinity for the glucocorticoid receptor (GR) [ 141 (triamcinolone acetonide>dexamethasone>co~i~l~~~icosterone) and is observed at concentrations (l-100 nM) compatible with their affinity for GR. However, while PGEz secretion can be almost entirely inhibited (80%), ACTH secretion can only be inhibited up to 40%. Mepacrine, a well-known inhibitor of PI.A2, ex-
Corticoids on TPA- or EGF-induced ACTH and PC&
x-
reversed by increasing concentrations cocorticoid RU 486 (Fig. 4) [ 151.
.,.x x._. . __. .. .___. . . _.. .__. - __-. x-.
x
335
of the antiglu-
If---*,_
-.
.
i.
““,f
DISCUSSION
‘.
x llx
I
‘\
In rat pituitary cells in primary steroids only glucocorticoids are
x
\
-
leg
16’
165
lnhlb~lor ConcentratmdM)
Fig. 3. Effect of mepacrine and dexamethasone on TPA- and forskolin-induced ACTH secretion. Cells were preincubated for I h with the indicated concentrations of mepacrine (--) or dexamethasone (-), and then incubated for 4 h in the presence of TPA (100 nM) (X) or forskolin (10,aM) (0). Results are expressed as per cent inhibition of the stimulation.
Dex.(5 x10-8 M) + RU486
: :
I
1001
10-1' Test compound
10-q
concentration
10-7 (M
)
Fig. 4. Effect of dexamethasone on EGF-induced PGEl secretion. Cells were preincubated for 1 h in the presence of the indicated concentrations of dexamethasone alone (-_) or dexamethasone (5 x 10-s M) and the indicated concentration of RU 486 (--), and then incubated for 4 h in the presence of EGF (100 ng/ml). Results are expressed as per cent inhibition of the stimulation.
hibits an inhibitory pattern on PGEl and ACTH secretion induced by TPA similar to that observed with glucocorticoids, i.e. a complete inhibition of PGE2 secretion and a maximum of 40% inhibition of ACTH secretion. When ACTH secretion is induced by forskolin, mepacrine does not exhibit any inhibitory effect (Fig. 3). PGE2 secretion induced by 100 ng/ml EGF can be also inhibited in the presence of dexamethasone (Fig. 4). This inhibition is dose dependent, and maximal inhibition (80%) is achieved with a 100 nM concentration of dexamethasone. Moreover, the inhibitory effect of dexamethasone (5 x lo-* M) can be totally
culture, able to
among inhibit
ACTH and PGE2 secretions induced by TPA, and the efficacy of the compounds parallels their affinity for the glucocorticoid receptor. However, while PGE2 secretion can be entirely inhibited, ACTH secretion can only be inhibited up to 40%, in contrast to the total inhibition observed with glucocorticoids on CAMP-dependent ACTH secretion. Mepacrine, a well-known inhibitor of PLA?, has an effect similar to that of glucocorticoids, i.e. a complete inhibition of PGEl release and a partial (maximum of 40%) inhibition of TPA-induced ACTH release, in contrast to its inability to inhibit CAMPdependent ACTH release. Glucocorticoids having also been described as PLA*-inhibitors their inhibitory effect on TPA-induced secretions could thus be related to their anti-PLA, activity. The inhibitory effect of glucocorticoids on PLAz has been attributed to their ability to induce the synthesis of a PLA*-inhibitor lipocortin [6, 7, 161. It has been previously shown that phosphorylated lipocortin is unable to inhibit PLA, [9] and that lipocortin phosphorylation can be induced by kinase C activation [9, 10, 171 or by EGF-receptor activation [ 1I]. In our model, release of PGEl and ACTH is observed at a TPA concentration of 100 nM, which is compatible with its affinity for a cellular phorbol ester specific binding protein [ 181 as well as with its potency as a protein kinase C activator [ 191. Moreover, no stimulation could be observed with the inactive phorbol ester derivative (4cr-phorbol 12,13-didecanoate). This suggests that the effect of TPA is mediated through an activation of kinase C. Moreover, PGE2 secretion induced by EGF is observed at a concentration compatible with its affinity for EGF-receptor and thus with an activation of the tyrosine kinase [20,21]. Since all these secretions are inhibited by glucocorticoids, these results suggest that a lipocortin-like protein could be present in pituitary cells and involved in the effect of TPA and EGF on PGE2 release, and at least partly on TPA-induced ACTH release. Experiments are now in progress in order to verify that a lipocortin-like protein is indeed present in pituitary cells and that it could play a role in the secretory processes at the pituitary level. REFERENCES 1. Dartois E. and Bouton M. M.: Role of Ca*+ on TPAinduced secretion of ACTH and PGEz by pituitary cells: effect of dexamethasone. Biochem. biophys. Rex Commun. 138 (1986) 323-329. 2. Castagna M., Takai Y., Kaibuchi K., Sano K., Kikkawa U. and Nishizuka Y.: Direct activation of calciumactivated, phospholipid-dependent protein kinase by
E. DARTOIS and M. M. BOUTON
336
tumor-promoting phorbol esters. J. biol. Chem. 257 (1982) 7847-785 1. 3. Uratsuji Y., Nakanishi H., Takeyama Y., Kishimoto A. and Nishizuka Y.: Activation of cellular protein kinase C and mode of inhibitory action of phospholipidinteracting compounds. Biochem. biophys. Res. Commun. 130 (1985) 654-661.
4. Bilezikjian L. M. and Vale W. W.: Glucocorticoids inhibit corticotropin-releasing factor-induced production of adenosine 3, 5’-monophosphate in cultured anterior pituitary cells. Endocrinology 113 (1983) 657-662. 5. Giguere V., Labrie F., Cote J., Coy D. H., Sueiras-Diaz J. and Schally A. V.: Stimulation of cyclic AMP accumulation and corticotropin release by synthetic ovine corticotropin-releasing factor in rat anterior pituitary cells: site of glucocorticoid action. Proc. natn. Acad. Sci. U.S.A. 79 (1982) 3466-3469.
6. Di Rosa M., Flower R. J., Hirata F., Parente L. and Russo-Marie F.: Anti-phospholipase proteins. Prostnglundins 28 (1984) 441-442.
I. Wallner B. P., Mattaliano R. J., Hession C., Cate R. L.,Tizard R., Sinclair L. K., Foeller C., Chow E. P., Browning J. L., Ramachandran K. L. and Pepinsky R. B.: Cloning and expression of human lipocortin, a phospholipase A2 inhibitor with potential anti-inflammatory activity. Nature 320 (1986) 77-8 1. 8. Vigo C., Lewis G. P. and Piper P. J.: Mechanism of inhibition of phospholipase Ar. Biochem. Pharm. 29 (1979) 623-627.
9. Khanna N. C., Tokuda M. and Waisman D. M.: Phosphorylation of lipocortins in vitro by protein kinase C. Biochem. biophys. Res. Commun. 141 (1986) 547-554. 10. Gould K. L., Woodgett J. R., Isacke C. M. and Hunter T.: The protein-tyrosine kinase substrate ~36 is also a substrate for protein kinase C in vitro and in vivo. Molec. Cell. Biol. 6 (1986) 2738-2744.
11. Pepinsky R. B. and Sinclair L. K.: Epidermal growth factor-dependent phosphorylation of lipocortin. Nature 321 (1986) 81-84.
12. Hirata F.: The regulation of lipomodulin, a phospholipase inhibitory protein, in rabbit neutrophils by phosphorylation. J. biol. Chem. 256 (1981) 7730-7733. 13. Dray F., Charbonnel B. and Maclouf J.: Radioimmunoassay of prostaglandins F2, E, and E2 in human plasma. Eur. J. clin. Invest. 5 (1975) 31 l-318. 14. Raynaud J. P., Ojasoo T., Bouton M. M. and Philibert D.: Receptor binding as a tool in the development of new bioactive steroids. In Drug Design. Academic Press, New York (1979) Vol. 8, p. 169. 15. Baulieu E. E. and Segal S. J.: In The Antiprogestin Steroid RU 486 and Human Fertility Control. Plenum Press, New York (1985). 16. Touqui L., Rothhut B., Shaw A. M., Fradin A., Vargaftig B. B. and Russo-Marie F.: Platelet activation: a role for a 40K anti-phospholipase A, protein indistinguishable from lipocortin. Nature 321 (1986) 177- 180. 17. Hirata F., Matsuda K., Notsu Y., Hattory T. and Del Carmine R.: Phosphorylation at a tyrosine residue of lipomodulin in mitogen-stimulated murine thymocytes. Proc. natn. Acud. Sci. U.S.A. 81 (1984) 4717-4721. 18. Salomon D. S.: Inhibition of epidermal growth factor binding to mouse embryonal carcinoma cells by phorbol esters mediated by specific phorbol ester receptors. J. biol. Chem. 256 (1981) 7958-7966. 19. Hermon J., Azrad A., Reiss N. and Naor Z.: Phospholipid-dependent Ca*+-activated protein kinase (C-kinase) in the pituitary: further characterization and endogenous redistribution. Molec. Cell. Endocr. 47 (1986) 201-208. 20. Aoyagi T., Suya H., Kato N., Nemoto O., Kobayashi H. and Miura Y.: Epidermal growth factor stimulates release of arachidonic acid in pig epidermis. J. invest. Derm 84 (1985) 168-171. 21. King L. E.: What does epidermal growth factor do and how does it do it? J. invest. Derm. 84 (1985) 165-167.
0022-4731188 $3.00+0.00 Pergamon Press pit
INHIBITION BY GLUCOCORTICOIDS OF PGEz AND ACTH SECRETION INDUCED BY PHORBOL ESTERS AND EGF IN RAT PITUITARY CELLS Centre de Recherches
E. D~RTOIS and M. M. BULKON* 1 I 1 route de Noisy, 93230 Romainville, France
Roussel-U&f,
Summary-In rat pituitary cells in primary culture glucocorticoids specifically inhibit PGEr and ACTH secretions induced by TPA, a potent phorbol ester derivative (triamcinolone acetonide>dexamethasone> cortisolrcorticosterone). However, while PGEl secretion can be inhibited up to 80%, ACTH secretion can only be inhibited up to 40%. Similar inhibitory effects are observed with mepacrine, an inhibitor of phospholipase A2 (PLA,). Glucocorticoids having also been described as PLA,-inhibitors, their inhibitory effect on TPA-induced secretions could thus be reiated to their anti-PLA, activity. Their inhibitory effect on PLA, has been attributed to their ability to induce the synthesis of li~co~in, the activity of which could be regulated by activation of kinase C or EGF-receptor kinase. Since in our model, EGF-induced PGEz secretion is also inhibited by dexamethasone, these results suggest that a lipocortin-like protein couid be present in pituitary cells and involved in the effect ofTPA and EGF on PGE2, and, at least partly, on ACTH release.
We have previously shown [l] that in rat pituitary cells, dexamethasone was able to inhibit the secretions of ACTH and prostaglandins E2 (PGE,) induced by 12-0-tetradecanoylphorbol I3-acetate (TPA), a potent phorbol ester derivative which is thought to act by activation of protein kinase C [2,3]. In order to see whether this TPA-induced ACTH secretion, like CAMP-induced ACTH secretion [4,5], is specifically inhibited by ~ucoco~icoids, we have compared the ability of different hormonal steroids to inhibit the effect of TPA on ACTH as well as PGE, secretion. In various experimental systems, the inhibitory effect of glucocorticoids on PGE, secretion has been attributed to the induction of a group of phospholipase A2 (PLAJ inhibitory proteins called lipocortin [6,7]. In order to see if PLA2 activation is also involved in TPA-induced ACTH secretion we have compared the inhibitory effect of glucocorticoids on PGE, and ACTH secretion to that of mepacrine, a well-known PLAr inhibitor [8]. Moreover, it has been shown that lipocortin can be phosphorylated by activation of kinases such as kinase C [9, lo] or epidermal growth factor (EGF)-receptor associated tyrosine kinase [ Ill. Since it is assumed that phospho~lation of lipocortin leads to a decrease in the PLA*-inhibitor potency of this protein [ 121, we have examined the ability of EGF to induce PGEr and ACTH secretions as well as the effect of dexamethasone on these EGF-induced secretions.
Materials. Medium 199 and fetal calf serum were obtained from Flow Laboratories; penicillinlstreptomycin from Gibco; trypsin, 12-O-tetradecanoylphorbol 13-acetate (TPA), 4cx-phorbol 12,13-didecanoate and EGF from Sigma; forskolin from Calbiochem Hoechst; and mepacrine from Boots. Dexamethasone, triamcinolone acetonide, corticosterone, cortisol, promegestone [R 5020: ( I?/?)-17-methyl- 17-( loxopropyl)-estra-4,9-dien-3-one], 5~-dihydrotestosterone (DHT), 17&estradiol and RU 486 if1 lp, 17/Q-1 1-[(4-dimethylamino)phenyl)]-17-hydroxy17-( I-propynyl)-estra-4,9-dien-3-one} were synthesized at the Roussel-Uclaf Research Center. Preparation of solutions. Steroids, phorbol ester derivatives and forskolin were dissolved in ethanol. Subsequent dilutions were prepared in medium 199 (final concentration in ethanol: 0.1%). Mepacrine and EGF were dissolved in water. The complete culture medium consisted of medium 199 supplemented with 0.3% of bovine serum albumin (BSA) 50 U/ml of penicillin and 50 &ml of streptomycin. Preparation
of dispersed anterior pituitary cells.
Anterior pituitary cells from mature male SpragueDawley rats were prepared as previously described [I]. Briefly, after disruption at 37°C with complete medium containing 0.25% trypsin, cells were plated in multiwell dishes at a density of 5 x 10’ cells/well and maintained for 4 days at 37°C under 5% C02-950h air in 1.5 ml of complete medium supplemented with 101 fetal calf serum. Effects of test-compounds on ACTH and PGE2 secretion. On day 5 of the culture, the cells were
Proceedings of the 8th International Symposium of The Journal of Steroid Biochemists “Recent Advances in Steroid Biochemistry” (Paris, 24-27 May 1987). *To whom correspondence should be addressed.
washed with medium 199, and incubated in 1 ml of fresh medium 199 under 5% CO*-95% air for 4 h in the presence of various concentrations of phorbol 333
. 3ooI il_ .
E. DARTOISand M. M. BOUTON
334
0
sc
I
2
x
B
“p
200
”
s 2 I
l
100
E
l
l
Y
i
-10
w
01
. *Q
,;8
,;T
Pnorbol Ester Concentration
) I
10 EGF
Wtt
100
Concentration
x)ocl (ng/ml
f
Fig. 1. Effect of phorbol ester derivatives and EGF on PGE, and ACTH secretion. Pituitary cells were incubated for 4 h in the presence of the indicated concentrations of (A) TPA (-) or 4cr-phorbol 12, I3didecanoate (--). Control values: 0.3 tug/ml for PGI$ (X) and 5 rig/ml for ACTH (o), (B) EGF. Control values: 3.1 n&ml for PGEl (X) and 3.2 @ml for ACTH (of.
1001
1
, Id’
rf?
lcg
12
IO7 STEROID
IdO
12’ CONCENTRATE
x59
Iii8 16'
fM)
Fig. 2. Effect of different steroids on TPA-induced PGEl and ACTH secretion. Cells were preincubated for 1 h with the indicated concentrations of steroids, and then incubated for 4 h in the presence of TPA (100 nM). Results are expressed as per cent inhibition of the stimulation. (A) PGEl secretion. (B) ACTH secretion. T.A.: ttiamcinolone acetonide. Dex.: dexamethasone. Cortico.: corticosterone. P.: promegestone. E,,: 17/?-estradiol.
ester derivatives or EGF or forskolin. After incubation, media were removed and PGE, and ACTH concentrations were determined by radioimmunoassay [ 131. When steroids or mepacrine were tested they were added 1 h prior to the addition of TPA or EGF. RESULTS
Effect ofphor~ol ester derivatives and EGF an PGE, and ACTH secretion TPA caused a dose-dependent increase in both PGEr and ACTH secretions over a similar concentration range (Fig. 1A). Maximal release is achieved with a 100 nM TPA concentration, which is compatible with its af&nity for protein kinase C [2]. No stimulation could be observed with the inactive phorbol ester derivative (4a-phorbol 12,13-didecanoate) over the same concentration range. In contrast to TPA, EGF caused an increase in PGE, secretion only, ACTH secretion remaining unchanged over the range of concentrations ( 1- 1000 ndml) (Fig. 1B). The maxi-
ma1 release of PGE2 is observed with a 100 rig/ml concentration of EGF. Effect of steroids and mepacrine on TEA- and EGFinduced secretions Inhibition of PGEr and ACTH secretion induced by TPA (1OOnM) is observed in the presence of giucocorticoids only (Fig. 2). No significant inhibition of either PGE2 or ACTH secretion was observed in the presence of estradiol, promegestone or dihydrotestosterone. The inhibitory effect of glucocorticoids follows the same rank order as their binding affinity for the glucocorticoid receptor (GR) [ 141 (triamcinolone acetonide>dexamethasone>co~i~l~~~icosterone) and is observed at concentrations (l-100 nM) compatible with their affinity for GR. However, while PGEz secretion can be almost entirely inhibited (80%), ACTH secretion can only be inhibited up to 40%. Mepacrine, a well-known inhibitor of PI.A2, ex-
Corticoids on TPA- or EGF-induced ACTH and PC&
x-
reversed by increasing concentrations cocorticoid RU 486 (Fig. 4) [ 151.
.,.x x._. . __. .. .___. . . _.. .__. - __-. x-.
x
335
of the antiglu-
If---*,_
-.
.
i.
““,f
DISCUSSION
‘.
x llx
I
‘\
In rat pituitary cells in primary steroids only glucocorticoids are
x
\
-
leg
16’
165
lnhlb~lor ConcentratmdM)
Fig. 3. Effect of mepacrine and dexamethasone on TPA- and forskolin-induced ACTH secretion. Cells were preincubated for I h with the indicated concentrations of mepacrine (--) or dexamethasone (-), and then incubated for 4 h in the presence of TPA (100 nM) (X) or forskolin (10,aM) (0). Results are expressed as per cent inhibition of the stimulation.
Dex.(5 x10-8 M) + RU486
: :
I
1001
10-1' Test compound
10-q
concentration
10-7 (M
)
Fig. 4. Effect of dexamethasone on EGF-induced PGEl secretion. Cells were preincubated for 1 h in the presence of the indicated concentrations of dexamethasone alone (-_) or dexamethasone (5 x 10-s M) and the indicated concentration of RU 486 (--), and then incubated for 4 h in the presence of EGF (100 ng/ml). Results are expressed as per cent inhibition of the stimulation.
hibits an inhibitory pattern on PGEl and ACTH secretion induced by TPA similar to that observed with glucocorticoids, i.e. a complete inhibition of PGE2 secretion and a maximum of 40% inhibition of ACTH secretion. When ACTH secretion is induced by forskolin, mepacrine does not exhibit any inhibitory effect (Fig. 3). PGE2 secretion induced by 100 ng/ml EGF can be also inhibited in the presence of dexamethasone (Fig. 4). This inhibition is dose dependent, and maximal inhibition (80%) is achieved with a 100 nM concentration of dexamethasone. Moreover, the inhibitory effect of dexamethasone (5 x lo-* M) can be totally
culture, able to
among inhibit
ACTH and PGE2 secretions induced by TPA, and the efficacy of the compounds parallels their affinity for the glucocorticoid receptor. However, while PGE2 secretion can be entirely inhibited, ACTH secretion can only be inhibited up to 40%, in contrast to the total inhibition observed with glucocorticoids on CAMP-dependent ACTH secretion. Mepacrine, a well-known inhibitor of PLA?, has an effect similar to that of glucocorticoids, i.e. a complete inhibition of PGEl release and a partial (maximum of 40%) inhibition of TPA-induced ACTH release, in contrast to its inability to inhibit CAMPdependent ACTH release. Glucocorticoids having also been described as PLA*-inhibitors their inhibitory effect on TPA-induced secretions could thus be related to their anti-PLA, activity. The inhibitory effect of glucocorticoids on PLAz has been attributed to their ability to induce the synthesis of a PLA*-inhibitor lipocortin [6, 7, 161. It has been previously shown that phosphorylated lipocortin is unable to inhibit PLA, [9] and that lipocortin phosphorylation can be induced by kinase C activation [9, 10, 171 or by EGF-receptor activation [ 1I]. In our model, release of PGEl and ACTH is observed at a TPA concentration of 100 nM, which is compatible with its affinity for a cellular phorbol ester specific binding protein [ 181 as well as with its potency as a protein kinase C activator [ 191. Moreover, no stimulation could be observed with the inactive phorbol ester derivative (4cr-phorbol 12,13-didecanoate). This suggests that the effect of TPA is mediated through an activation of kinase C. Moreover, PGE2 secretion induced by EGF is observed at a concentration compatible with its affinity for EGF-receptor and thus with an activation of the tyrosine kinase [20,21]. Since all these secretions are inhibited by glucocorticoids, these results suggest that a lipocortin-like protein could be present in pituitary cells and involved in the effect of TPA and EGF on PGE2 release, and at least partly on TPA-induced ACTH release. Experiments are now in progress in order to verify that a lipocortin-like protein is indeed present in pituitary cells and that it could play a role in the secretory processes at the pituitary level. REFERENCES 1. Dartois E. and Bouton M. M.: Role of Ca*+ on TPAinduced secretion of ACTH and PGEz by pituitary cells: effect of dexamethasone. Biochem. biophys. Rex Commun. 138 (1986) 323-329. 2. Castagna M., Takai Y., Kaibuchi K., Sano K., Kikkawa U. and Nishizuka Y.: Direct activation of calciumactivated, phospholipid-dependent protein kinase by
E. DARTOIS and M. M. BOUTON
336
tumor-promoting phorbol esters. J. biol. Chem. 257 (1982) 7847-785 1. 3. Uratsuji Y., Nakanishi H., Takeyama Y., Kishimoto A. and Nishizuka Y.: Activation of cellular protein kinase C and mode of inhibitory action of phospholipidinteracting compounds. Biochem. biophys. Res. Commun. 130 (1985) 654-661.
4. Bilezikjian L. M. and Vale W. W.: Glucocorticoids inhibit corticotropin-releasing factor-induced production of adenosine 3, 5’-monophosphate in cultured anterior pituitary cells. Endocrinology 113 (1983) 657-662. 5. Giguere V., Labrie F., Cote J., Coy D. H., Sueiras-Diaz J. and Schally A. V.: Stimulation of cyclic AMP accumulation and corticotropin release by synthetic ovine corticotropin-releasing factor in rat anterior pituitary cells: site of glucocorticoid action. Proc. natn. Acad. Sci. U.S.A. 79 (1982) 3466-3469.
6. Di Rosa M., Flower R. J., Hirata F., Parente L. and Russo-Marie F.: Anti-phospholipase proteins. Prostnglundins 28 (1984) 441-442.
I. Wallner B. P., Mattaliano R. J., Hession C., Cate R. L.,Tizard R., Sinclair L. K., Foeller C., Chow E. P., Browning J. L., Ramachandran K. L. and Pepinsky R. B.: Cloning and expression of human lipocortin, a phospholipase A2 inhibitor with potential anti-inflammatory activity. Nature 320 (1986) 77-8 1. 8. Vigo C., Lewis G. P. and Piper P. J.: Mechanism of inhibition of phospholipase Ar. Biochem. Pharm. 29 (1979) 623-627.
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