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Indomethacin blocks gonadotropin stimulation of ovarian ornithine decarboxylase activity by inhibiting protein synthesis
PROSTAGLANDINS
INDOMETHACIN BLOCKS GONADOTROPIN STIMULATION OF OVARIAN ORNITHINE DECARBOXYLASE ACTIVITY BY INHIBITING PROTEIN SYNTHESIS Juraj Osterman I, Laurence M. Demers 2, and James M. Hammond 3 Departments of Medicine 1'3 and Pathology 2 The Milton S. Hershey Medical Center The Pennsylvania State Universlty Hershey, Pennsylvania 17033 ABSTRACT Both gonadotropins and prostaglandins stimulate the ornithine decarboxylase (ODC) activity of porclne granulosa cells in vitro (1,2). To investigate a possible intermedlary role of prostaglandins in this gonadotropin aetion, the effeets of indomethacln on gonadotropininduced ODC actlvity were studled. Indomethacln had no effect at concentrations lower than 10-5 M; at higher concentrations indomethaein exerted a dose-dependent suppression of LH-stimulated ODC activity which was essentially eomplete at 5 x 10-4 M. The effects of PGE 2 and 8-Bromo-cAMP, potent stimulators of ODC, were also bloeked by indomethaein (5 x 10-4 M). This effect did not represent dlrect inhlbltlon of enzyme activlty, hut appeared to be due to inhibition of protein synthesls by the drug. Thus, ineorporation of 14C-leucine into proteins by these eells was hlocked by indomethaein with a dose-response curve similar to that for ODC suppression. This distal effect of indomethaein may complicate the interpretation of some experiments if the inhibitor is assumed to aet only at the prostaglandin synthetase step. INTRODUCTION One of the weil established effeets of lutelnizing hormone (LH) on the mammalian ovary is augmentation of the activity of ornithine deearboxylase (ODC), the rate-limiting enzyme in polyamine synthesis. The effect is evident elther after administration of the hormone in vivo (3-7) or in vitro (1,8). Prostaglandins of the E serles mimic several effects of LH in the ovary including stimulation of ODC (2,9), cAMP generation (9), protein kinase actlvity (9), luteinlzatlon of granulosa cells in culture (i0) and progesterone synthesis (ii). Prostaglandins were
Icurrent address: Department of Medicine, University of South Carolina School of Medicine, Columbia, S.C. 29201. 3To whom reprint requests should be directed. Supported by NIH Grant No. HD 10122.
FEBRUARY 1979 VOL. 17 NO. 2
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PROSTAGLANDINS
initially proposed to act as obligatory medlators of LH action in the ovary (12). However, the weight of recent experimental evidenee does not support this eoncept (9,13,14). Consequently, it was surprising when the current studles showed that Indomethacin completely inhlbited the LH-stimulated ODC activity of porelne granulosa cells in vltro. Subsequent experiments, also reported here, have indicated that this effeet is probably exerted by direct inhibltlon of protein synthesis by the drug. MATERIALS AND METHODS Materials: Ovlne luteinizing hormone (NIH-LH-SI9) was obtalned from the Hormone Distrlbution Office, National Institute of Arthritis, Metabollsm and Digestive Diseases. Highly purlfled ovlne folllcle stimulating hormone (FSH-HP-G4-150C) containing FSH aetivlty approxlmately 50 times that of NIH-FSH-SI was provided by Dr. Harold Papkoff, Unlverslty of Californla, Sah Franclsco. 8-Bromo-cyclic AMP (8 BrcAMP) was purehased from the Sigma Chemical Company. Prostaglandin E 2 (PGE2) was obtalned from the UpJohn Company. Indomethacln was a gift from Dr. Frederlck Kuehl, Merek Sharp & Dohme. DL-(l-14C)-9~nlthine monohydrochloride (speclflc actlvlty: 45 mCi/mmol) and L-[±~C(U)]-leuclne (speclfie aetivity: 294 mCi/mmol) were purchased from New England Nuclear. Tissue Preparation: Porcine ovaries were collected at a loeal slaughterhouse and the granulosa cells isolated from small (i-2 mm) follleles. Approximately 108 cells/flask were incubated for 4 h in I0 ml serum-free Medium 199 as previously descrlbed (i). LH (200 hg/ ml), FSH (50 ng/ml), PGE 2 (i000 ng/ml) and 8 BreAMP (0.5 mM) were added at the beginning of the incubation in respective experiments. PGE 2 was dissolved in ethanol and the entire dose was added in a 10 ~I volume per flask. Indomethacin was dlssolved in dlmethylsulfoxide (DMSO) and the entlre dose was added in a 25 ~I volume per flask. Controls recelved appropriate volume of ethanol, DMSO or both. Granulosa cells were preincubated with indomethacln for 30 min. Ornithine Decarboxylase Assay: Following ineubation, granulosa cells were separated from the incubatlon medium, washed, homogenized and the cytosol was prepared for the assay of ODC activlty as prevlously deseribed (i). The results are expressed as pmol/mg protein/ 30 min or as percent of control. Each sample was assayed in dupllcate. Total protein in the cytosol was determined by the method of Lowry et a_~l. (15). In each experiment shown in the Results section, granulosa cells were from a single pool of cells Isolated on a particular day from 100-200 ovaries. Each experiment was repeated at least once with a different batch of ovaries and a similar pattern of response was obtained. Incorporation of 14C-Leucine into Proteins of Granulosa Cells: The filter-paper dise method of Mans and Novelll (16) was used. Granulosa eells (approxlmately 5.0 x 107 eells) were incubated for 4 h in
270
FEBRUARY 1979 VOL. 17 NO. 2
PROSTAGLANDINS
the same medium described above (containing 0.9 mM L-leucine) which was supplemented with 200,000 cpm of 14C-leucine per flask. Cells were preincubated with indomethacin for 30 min, and LH (200 ng/ml) was added at the beginning of incubation. Following incubation the cells were separated from the medium by centrifugation, resuspended in 0.5 ml of Medium 199 and 50 ~i aliquots in triplicate were applied to filter paper discs. This was followed by a series of washing procedures as described in the method of Mans and Novelli (16). The results were expressed as cpm of 14C-leucine incorporated/mg of total protein. Total protein was determined by the method of Lowry et al. (15). RESULTS As shown in Fig. and 10-5 M caused no lated ODC activity. was a more effective
i, indomethacin in concentrations between i0 -I0 consistent change in basal or gonadotropin-stimuIn agreement with our previous findings (I) FSH stimulator of the enzyme activity than LH.
2000
~1500
>
<
~
1000
« ~
o
500
n ~ C
'7 '7 T o o o o ~NDOMETHAClN (M)
Fig. I.
LH
~
~
~
°
I N D O M E T H A C I N (M) + LH
FSH
'~ ~7 T '2o oo o INDOMETHAClN (M) + FSH
Effect of low concentrations of indomethacin on ornithine decarboxylase activity of porcine granulosa cells. C = control; LH = 200 ng/ml; FSH = 50 ng/ml. Results are means and range of determinations on 2 replicate cultures. Control activity was 350 pmol/mg/30 min.
Since other investigators (17,18) have employed higher concentrations of indomethacin in studies of its effects on the ovary, we increased the concentration of indomethacin from 10-5 to 5 x 10-4 M (ù01 to .5 mM) (Fig. 2). The addition of indomethacin in this dose
FEBRUARY 1979 VOL. 17 NO. 2
271
PROSTAGLANDINS range caused a dose-dependent activity.
inhibition of the LH effect on ODC
2400 » B
~
2000 1600
o
312oo ,~ ~ 8o0 B ~ 4O0 C
Fi$. 2.
LH
LH+ LH+ LH+ LH+ 001 0.05 025 050 INDOMETHACIN (mM)
Effect of high concentrations of indomethacin on ornithine decarboxylase activity of porcine granulosa cells stimulated with LH (200 ng/ml). C = control. Results are means and range of determinations on 2 replicate cultures.
The experiment shown in Fig. 3 indicates that 0.5 mM indomethacin also inhibits the stimulatory effect of PGE 2 and 8 BrcAMP on ODC.
2800 » 2400
~ 2000 > ~ 1600
~12oo
~
~
800 400 -
C
Fi$. 3.
LH
PGE2 8BrcAMP
INDOMETHACIN (0.5 mM) +LH +PGE2 +BrcAMP
Effect of LH (200 ng/ml), PGE 2 (I000 ng/ml), and 8 BrcAMP (0.5 mM), alone and in combination with indomethacin, on ornithine decarboxylase activity of porcine granulosa cells. Results are means and range of determinations on 2 replicate eultures.
The possibility that indomethacin directly inhibited the ornithine decarboxylase enzyme or interferred in its assay was tested next. The results are shown in Table I. The addition of indomethacin (0.05-0.5 mM) and appropriate vehicle to the assay flasks caused at most, 10%
272
FEBRUARY 1979 VOL. 17 NO. 2
PROSTAGLANDINS
inhibition of ornithine decarboxylase activity. This was insignificant compared to the profound inhibition observed during incubation of granulosa cells with the drug. Table I. Effect of Various Concentrations of Indomethacin on the Assäy Of Ornithine Decarboxylase Aetivity Ornithine Decarboxylase Activity (% of LH-stimulated activity)
Treatment a LH LH LH LH LH
100 b
+ + + +
DMSO Indomethacin (0.05 mM) Indomethacin (0.25 mM) Indomethaein (0.5 mM)
88 92 90 93
aIndomethacin and DMSO were added to the enzyme preparatlon (20,000 x g supernatant from granulosa cells previously treated with LH) at the beginning of the enzyme assay. bValues are means of duplicate assays. Since hormonal stimulation of ODC activity is blocked by inhibitors of protein synthesis (4,5), the possibility that the indomethacin effect was exerted at this level was considered. In the experiment shown in Fig. 4, LH and increasing doses of indomethacin were added to granulosa cells, and the incorporation of 14C-leucine into TCA-precipitable material was measured. I000
« õ ~
800
o
~ 600 L E
~~~°° ~
2oo CONTROL
Fi$. 4.
LH
LH~ 001
LH+ LH+ LH+ 005 025 050 INDOMETHACIN (mM)
Effect of LH (200 ng/ml) and various concentrations of indomethacin on 14C-leucine incorporation into proteins of granulosa cells. Results are mean ± SEM of 3 replicate cultures.
LH caused a small but significant (p <.05) increase of 14C-leucine incorporation into acid precipitable proteins. Indomethacin caused a dose-dependent inhibition of 14C-leucine incorporation. At the highest dose, there was virtually complete inhibition of protein synthesis.
FEBRUARY 1979 VOL. 17 NO. 2
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PROSTAGLANDINS DISCUSSION Since the effects of LH and prostaglandins on ehe ovary show significant overlap, and since LH raises ovarian prostaglandin levels (19-21), numerous investigators have tested the plausability of prostaglandin mediation of LH effects. In approaching this question, many workers have employed the prostaglandin synthetase inhibitor, indomethacin. Indomethacin evidently does not block LH-induced generation of cAMP (13,18) or LH stimulation of steroidogenesis in vivo (21). However, it appears to block ovulation (21). We wished to assess the role of prostaglandins in gonadotropin stimulation of ODC. The studies with indomethacin reported here have failed to confirm or deny an intermediary role for prostaglandins in this gonaßotropin action. They have, however, revealed an effect of indomethacin which may complicate its use as a molecular probe of some prostaglandin actions. This effect appears to be distal to prostaglandin synthetase and adenylyl cyclase since ODC stimulation by PGE 2 and 8 BrcAMP was blocked by indomethacin. The demonstrated inhibitiön of 14C-leucine incorporation indicates that the drug interferes with protein synthesis but does not define the mechanism; the results could be accounted for by blockade of RNA synthesis or translation, decreased amino acid transport, or a nonspecific cytotoxic effect. To our knowledge, this action of indomethacin has not been previously recognized. Young Lai has suggested that indomethacin inhibition of steroidogenesis by rabbit follicles at very high concentrations of the drug (i mg/ml) might be due to toxic effects (22). With isolated porcine granulosa cells "toxic" effects appear to be demonstrable at 10-fold lower concentrations as w e l l - concentrations which are widely used in studies of prostaglandin action in vitro. ACKNOWLEDGEMENTS The authors wish to thank Dr. Harold Papkoff for ovine FSH, Dr. Frederick Kuehl of Merck Sharp & Dohme for indomethacin, the Upjohn Company for PGE^, and the Hormone Distribution Program, NIAMDD z for ovine LH. We are also grateful for the technical assistance of Ms. Elizabeth Krall and the secretarial help of Mrs. Marlene Thompson. REFERENCES i)
2)
3)
274
Osterman, J., and J.M. Hammond. FSH and LH Stimulation of Ornithine Decarboxylase Activity: Studies with Porcine Granulosa Cells in vitro. Endocrinology 101:1335, 1977 Osterman, J., and J.M. Hammond. Prostaglandin Stimulation of Ovarian Ornithine Decarboxylase in vitro. Biochem. Biophys. Res. Commun. 83:794, 1978. Kobayashi, Y., T. Kupelian, and D.V° Maudsley. Ornithine Decarboxylase Stimulation in Rat Ovary by Luteinizing Hormone. Science 172:379, 1971.
FEBRUARY 1979 VOL. 17 NO. 2
PROSTAGLANDINS
4)
5)
6)
7)
8)
9)
I0)
Ii)
12)
13)
14)
15)
16)
Kaye, A.M., I. Icekson, S.A. Lamprecht, R. Gruss, A. Tsafriri, and H.R. Lindner. Stimulation of Ornlthine Decarboxylase Activity by Luteinizing Hormone in Immature and Mature Rat Ovarles. Biochemistry 12:3072, 1973. Maudsley, D.V. and Y. Kobayashi. Induetion of Ornithine Decarboxylase in Rat Ovary after Administration of Luteinizlng Hormone or Human Chorionic Gonadotropin. Biochem. Pharmacol, 23:2697, 1974. leekson, I., A.M. Kaye, M.E. Lieberman, S.A. Lamprecht, M. Lahav, and H.R. Lindner. Stimulation by Luteinizing Hormone of Ornlthine Deearboxylase in Rat: Preferential Response by Follicular Tissue. J. Endocr, 63: 417, 1974. Nureddin, A. Ovarian Ornithlne Decarboxylase Induction: A Specific and Rapid in vivo Bioassay of LH. Biochem. Med. l_~7:67, 1977. Osterman, J., L.M. Demers, and J.M. Hammond. Gonadotropin Stimulation of Poreine Ovarlan Ornlthine Decarboxylase in vitro: The Role of Cyelic AMP. Endocrlnology, 1978, in press. Lampreeht, S.A., U. Zor, A. Tsafriri and H.R. Lindner. Act±on of Prostaglandin E2 and of Luteinizing Hormone on Ovarian Adenylate Cyclase, Protein Kinase and Ornithine Deearboxylase Actlvity during Postnatal Development and Maturity in the Rat. J. Endocr. 57:217, 1973. Ch---anning,C.P. Influenee of the in vivo and invitro Hormonal Envlronment upon Luteinization of Granulosa Cells in Tissue Culture. Rec. Prog. Horm. Res. 26:589, 1970. Speroff, L., and P.W. Ramwell. Prostaglandin Stimulat±on of i__n_n vitro Progesterone Synthesls. J. Clin. Endocrinol. Metab. 30: 345, 1970. Kuehl, I.A., J.L. Humes, J. Tarnoff, V.J. C$rilo, and E.A. Ham. Prostagland±n Receptor Site: Evldence for an Essential Role in the Action of Luteinizing Hormone. Science 169:883, 1970. Lindner, H.R., U. Zor, S. Bauminger, A. Tsafriri, S.A. Lamprecht, Y. Koch, S. Antebi, and A. Schwartz. Use of Prostaglandin Synthetase Inhibitors in Analyzing the Role of Prostaglandins in Reproductive Phys±ology. In: Prostaglandin Synthetase Inhibitors (H.J. Robinson and J.R. Vane, eds.) Raven Press, New York, 1974, p. 271. Rigler, G.L., G.T. Peake, and A. Ratner. Effect of FollicleStlmulating Hormone and Luteinizlng Hormone on Ovarian Cyclic AMP and Prostaglandin E in vlvo in Rats Treated with Indomethaein. J. Endocr. 70:285, 1976. Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall. Protein Measurement with the Folln Phenol Reagent. J. Biol. Chem. 193:365, 1951. Mans, R.J. and G,D. Novelli. Measurement of the Ineorporation of Radioactive Amino Acids into Protein by a Filter-Paper Disk Method. Arch. Bioehem. Biophys. 9_~4:48, 1961.
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18)
19)
20)
21)
22)
22)
Zor, U., S. Bauminger, S,A. Lamprecht, Y. Koch, P. Chobsieng, and H.R. Lindner. Stimulatlon of Cyclic AMP Produetlon in the Rat Ovary by Luteinizing Hormone: Independence of Prostaglandin Mediation. Prostaglandins ~:499, 1973. Goff, A.K., and D.T. Armstrong. Stimulatory Action of Gonadotroplns and Prostaglandins on Adenoslne-3~,5"-Monophosphate Production by Isolated Rat Granulosa Cells. Endocrlnology i01: 1461, 1977. Marsh, J.M., N.S.T. Yang, and W.J. LeMalre. Prostagland±n Synthesls in Rabbit Graafian Follicles in vltro: Effect of Luteinizlng Hormone and CycllcAMP. Prostaglandins !:269, 1974. Clark, M.R., J.M. Marsh, and W.J. LeMaire. The Role of Protein Synthesls in the Stimulatlon by LH of Prostaglandin Accumulation in Rat Preovulatory Folllcleslnvitro. Prostaglandins 12:209, 1976. Clark, M.R., J.M. Marsh, and W.J. LeMaire. Stimulatlon of Prostaglandin Accumulation in Preovulatory Rat Folllcles by Adenosine 3%5~-Monophosphate. Endocrinology 102:39, 1978. Grinwich, D.L., T.G. Kennedy, and D.T. Armstrong. Dissoc±ation of Ovulatory and Steroidogenlc Actions of Lutenlzing Hormone in Rabbits with Indomethacin, an Inhibitor of Prostaglandin Biosynthesis. Prostaglandlns ~:89, 1972. Young Lai, E.V. Prostaglandlns and Steroldogenesis by Isolated Rabbit Ovarian Follicles. Horm. Res. ~:31, 1978.
Received
276
9/5/78
- Approved
12/4/78
FEBRUARY 1979 VOL. 17 NQ. 2
INDOMETHACIN BLOCKS GONADOTROPIN STIMULATION OF OVARIAN ORNITHINE DECARBOXYLASE ACTIVITY BY INHIBITING PROTEIN SYNTHESIS Juraj Osterman I, Laurence M. Demers 2, and James M. Hammond 3 Departments of Medicine 1'3 and Pathology 2 The Milton S. Hershey Medical Center The Pennsylvania State Universlty Hershey, Pennsylvania 17033 ABSTRACT Both gonadotropins and prostaglandins stimulate the ornithine decarboxylase (ODC) activity of porclne granulosa cells in vitro (1,2). To investigate a possible intermedlary role of prostaglandins in this gonadotropin aetion, the effeets of indomethacln on gonadotropininduced ODC actlvity were studled. Indomethacln had no effect at concentrations lower than 10-5 M; at higher concentrations indomethaein exerted a dose-dependent suppression of LH-stimulated ODC activity which was essentially eomplete at 5 x 10-4 M. The effects of PGE 2 and 8-Bromo-cAMP, potent stimulators of ODC, were also bloeked by indomethaein (5 x 10-4 M). This effect did not represent dlrect inhlbltlon of enzyme activlty, hut appeared to be due to inhibition of protein synthesls by the drug. Thus, ineorporation of 14C-leucine into proteins by these eells was hlocked by indomethaein with a dose-response curve similar to that for ODC suppression. This distal effect of indomethaein may complicate the interpretation of some experiments if the inhibitor is assumed to aet only at the prostaglandin synthetase step. INTRODUCTION One of the weil established effeets of lutelnizing hormone (LH) on the mammalian ovary is augmentation of the activity of ornithine deearboxylase (ODC), the rate-limiting enzyme in polyamine synthesis. The effect is evident elther after administration of the hormone in vivo (3-7) or in vitro (1,8). Prostaglandins of the E serles mimic several effects of LH in the ovary including stimulation of ODC (2,9), cAMP generation (9), protein kinase actlvity (9), luteinlzatlon of granulosa cells in culture (i0) and progesterone synthesis (ii). Prostaglandins were
Icurrent address: Department of Medicine, University of South Carolina School of Medicine, Columbia, S.C. 29201. 3To whom reprint requests should be directed. Supported by NIH Grant No. HD 10122.
FEBRUARY 1979 VOL. 17 NO. 2
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PROSTAGLANDINS
initially proposed to act as obligatory medlators of LH action in the ovary (12). However, the weight of recent experimental evidenee does not support this eoncept (9,13,14). Consequently, it was surprising when the current studles showed that Indomethacin completely inhlbited the LH-stimulated ODC activity of porelne granulosa cells in vltro. Subsequent experiments, also reported here, have indicated that this effeet is probably exerted by direct inhibltlon of protein synthesis by the drug. MATERIALS AND METHODS Materials: Ovlne luteinizing hormone (NIH-LH-SI9) was obtalned from the Hormone Distrlbution Office, National Institute of Arthritis, Metabollsm and Digestive Diseases. Highly purlfled ovlne folllcle stimulating hormone (FSH-HP-G4-150C) containing FSH aetivlty approxlmately 50 times that of NIH-FSH-SI was provided by Dr. Harold Papkoff, Unlverslty of Californla, Sah Franclsco. 8-Bromo-cyclic AMP (8 BrcAMP) was purehased from the Sigma Chemical Company. Prostaglandin E 2 (PGE2) was obtalned from the UpJohn Company. Indomethacln was a gift from Dr. Frederlck Kuehl, Merek Sharp & Dohme. DL-(l-14C)-9~nlthine monohydrochloride (speclflc actlvlty: 45 mCi/mmol) and L-[±~C(U)]-leuclne (speclfie aetivity: 294 mCi/mmol) were purchased from New England Nuclear. Tissue Preparation: Porcine ovaries were collected at a loeal slaughterhouse and the granulosa cells isolated from small (i-2 mm) follleles. Approximately 108 cells/flask were incubated for 4 h in I0 ml serum-free Medium 199 as previously descrlbed (i). LH (200 hg/ ml), FSH (50 ng/ml), PGE 2 (i000 ng/ml) and 8 BreAMP (0.5 mM) were added at the beginning of the incubation in respective experiments. PGE 2 was dissolved in ethanol and the entire dose was added in a 10 ~I volume per flask. Indomethacin was dlssolved in dlmethylsulfoxide (DMSO) and the entlre dose was added in a 25 ~I volume per flask. Controls recelved appropriate volume of ethanol, DMSO or both. Granulosa cells were preincubated with indomethacln for 30 min. Ornithine Decarboxylase Assay: Following ineubation, granulosa cells were separated from the incubatlon medium, washed, homogenized and the cytosol was prepared for the assay of ODC activlty as prevlously deseribed (i). The results are expressed as pmol/mg protein/ 30 min or as percent of control. Each sample was assayed in dupllcate. Total protein in the cytosol was determined by the method of Lowry et a_~l. (15). In each experiment shown in the Results section, granulosa cells were from a single pool of cells Isolated on a particular day from 100-200 ovaries. Each experiment was repeated at least once with a different batch of ovaries and a similar pattern of response was obtained. Incorporation of 14C-Leucine into Proteins of Granulosa Cells: The filter-paper dise method of Mans and Novelll (16) was used. Granulosa eells (approxlmately 5.0 x 107 eells) were incubated for 4 h in
270
FEBRUARY 1979 VOL. 17 NO. 2
PROSTAGLANDINS
the same medium described above (containing 0.9 mM L-leucine) which was supplemented with 200,000 cpm of 14C-leucine per flask. Cells were preincubated with indomethacin for 30 min, and LH (200 ng/ml) was added at the beginning of incubation. Following incubation the cells were separated from the medium by centrifugation, resuspended in 0.5 ml of Medium 199 and 50 ~i aliquots in triplicate were applied to filter paper discs. This was followed by a series of washing procedures as described in the method of Mans and Novelli (16). The results were expressed as cpm of 14C-leucine incorporated/mg of total protein. Total protein was determined by the method of Lowry et al. (15). RESULTS As shown in Fig. and 10-5 M caused no lated ODC activity. was a more effective
i, indomethacin in concentrations between i0 -I0 consistent change in basal or gonadotropin-stimuIn agreement with our previous findings (I) FSH stimulator of the enzyme activity than LH.
2000
~1500
>
<
~
1000
« ~
o
500
n ~ C
'7 '7 T o o o o ~NDOMETHAClN (M)
Fig. I.
LH
~
~
~
°
I N D O M E T H A C I N (M) + LH
FSH
'~ ~7 T '2o oo o INDOMETHAClN (M) + FSH
Effect of low concentrations of indomethacin on ornithine decarboxylase activity of porcine granulosa cells. C = control; LH = 200 ng/ml; FSH = 50 ng/ml. Results are means and range of determinations on 2 replicate cultures. Control activity was 350 pmol/mg/30 min.
Since other investigators (17,18) have employed higher concentrations of indomethacin in studies of its effects on the ovary, we increased the concentration of indomethacin from 10-5 to 5 x 10-4 M (ù01 to .5 mM) (Fig. 2). The addition of indomethacin in this dose
FEBRUARY 1979 VOL. 17 NO. 2
271
PROSTAGLANDINS range caused a dose-dependent activity.
inhibition of the LH effect on ODC
2400 » B
~
2000 1600
o
312oo ,~ ~ 8o0 B ~ 4O0 C
Fi$. 2.
LH
LH+ LH+ LH+ LH+ 001 0.05 025 050 INDOMETHACIN (mM)
Effect of high concentrations of indomethacin on ornithine decarboxylase activity of porcine granulosa cells stimulated with LH (200 ng/ml). C = control. Results are means and range of determinations on 2 replicate cultures.
The experiment shown in Fig. 3 indicates that 0.5 mM indomethacin also inhibits the stimulatory effect of PGE 2 and 8 BrcAMP on ODC.
2800 » 2400
~ 2000 > ~ 1600
~12oo
~
~
800 400 -
C
Fi$. 3.
LH
PGE2 8BrcAMP
INDOMETHACIN (0.5 mM) +LH +PGE2 +BrcAMP
Effect of LH (200 ng/ml), PGE 2 (I000 ng/ml), and 8 BrcAMP (0.5 mM), alone and in combination with indomethacin, on ornithine decarboxylase activity of porcine granulosa cells. Results are means and range of determinations on 2 replicate eultures.
The possibility that indomethacin directly inhibited the ornithine decarboxylase enzyme or interferred in its assay was tested next. The results are shown in Table I. The addition of indomethacin (0.05-0.5 mM) and appropriate vehicle to the assay flasks caused at most, 10%
272
FEBRUARY 1979 VOL. 17 NO. 2
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inhibition of ornithine decarboxylase activity. This was insignificant compared to the profound inhibition observed during incubation of granulosa cells with the drug. Table I. Effect of Various Concentrations of Indomethacin on the Assäy Of Ornithine Decarboxylase Aetivity Ornithine Decarboxylase Activity (% of LH-stimulated activity)
Treatment a LH LH LH LH LH
100 b
+ + + +
DMSO Indomethacin (0.05 mM) Indomethacin (0.25 mM) Indomethaein (0.5 mM)
88 92 90 93
aIndomethacin and DMSO were added to the enzyme preparatlon (20,000 x g supernatant from granulosa cells previously treated with LH) at the beginning of the enzyme assay. bValues are means of duplicate assays. Since hormonal stimulation of ODC activity is blocked by inhibitors of protein synthesis (4,5), the possibility that the indomethacin effect was exerted at this level was considered. In the experiment shown in Fig. 4, LH and increasing doses of indomethacin were added to granulosa cells, and the incorporation of 14C-leucine into TCA-precipitable material was measured. I000
« õ ~
800
o
~ 600 L E
~~~°° ~
2oo CONTROL
Fi$. 4.
LH
LH~ 001
LH+ LH+ LH+ 005 025 050 INDOMETHACIN (mM)
Effect of LH (200 ng/ml) and various concentrations of indomethacin on 14C-leucine incorporation into proteins of granulosa cells. Results are mean ± SEM of 3 replicate cultures.
LH caused a small but significant (p <.05) increase of 14C-leucine incorporation into acid precipitable proteins. Indomethacin caused a dose-dependent inhibition of 14C-leucine incorporation. At the highest dose, there was virtually complete inhibition of protein synthesis.
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PROSTAGLANDINS DISCUSSION Since the effects of LH and prostaglandins on ehe ovary show significant overlap, and since LH raises ovarian prostaglandin levels (19-21), numerous investigators have tested the plausability of prostaglandin mediation of LH effects. In approaching this question, many workers have employed the prostaglandin synthetase inhibitor, indomethacin. Indomethacin evidently does not block LH-induced generation of cAMP (13,18) or LH stimulation of steroidogenesis in vivo (21). However, it appears to block ovulation (21). We wished to assess the role of prostaglandins in gonadotropin stimulation of ODC. The studies with indomethacin reported here have failed to confirm or deny an intermediary role for prostaglandins in this gonaßotropin action. They have, however, revealed an effect of indomethacin which may complicate its use as a molecular probe of some prostaglandin actions. This effect appears to be distal to prostaglandin synthetase and adenylyl cyclase since ODC stimulation by PGE 2 and 8 BrcAMP was blocked by indomethacin. The demonstrated inhibitiön of 14C-leucine incorporation indicates that the drug interferes with protein synthesis but does not define the mechanism; the results could be accounted for by blockade of RNA synthesis or translation, decreased amino acid transport, or a nonspecific cytotoxic effect. To our knowledge, this action of indomethacin has not been previously recognized. Young Lai has suggested that indomethacin inhibition of steroidogenesis by rabbit follicles at very high concentrations of the drug (i mg/ml) might be due to toxic effects (22). With isolated porcine granulosa cells "toxic" effects appear to be demonstrable at 10-fold lower concentrations as w e l l - concentrations which are widely used in studies of prostaglandin action in vitro. ACKNOWLEDGEMENTS The authors wish to thank Dr. Harold Papkoff for ovine FSH, Dr. Frederick Kuehl of Merck Sharp & Dohme for indomethacin, the Upjohn Company for PGE^, and the Hormone Distribution Program, NIAMDD z for ovine LH. We are also grateful for the technical assistance of Ms. Elizabeth Krall and the secretarial help of Mrs. Marlene Thompson. REFERENCES i)
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Osterman, J., and J.M. Hammond. FSH and LH Stimulation of Ornithine Decarboxylase Activity: Studies with Porcine Granulosa Cells in vitro. Endocrinology 101:1335, 1977 Osterman, J., and J.M. Hammond. Prostaglandin Stimulation of Ovarian Ornithine Decarboxylase in vitro. Biochem. Biophys. Res. Commun. 83:794, 1978. Kobayashi, Y., T. Kupelian, and D.V° Maudsley. Ornithine Decarboxylase Stimulation in Rat Ovary by Luteinizing Hormone. Science 172:379, 1971.
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Kaye, A.M., I. Icekson, S.A. Lamprecht, R. Gruss, A. Tsafriri, and H.R. Lindner. Stimulation of Ornlthine Decarboxylase Activity by Luteinizing Hormone in Immature and Mature Rat Ovarles. Biochemistry 12:3072, 1973. Maudsley, D.V. and Y. Kobayashi. Induetion of Ornithine Decarboxylase in Rat Ovary after Administration of Luteinizlng Hormone or Human Chorionic Gonadotropin. Biochem. Pharmacol, 23:2697, 1974. leekson, I., A.M. Kaye, M.E. Lieberman, S.A. Lamprecht, M. Lahav, and H.R. Lindner. Stimulation by Luteinizing Hormone of Ornlthine Deearboxylase in Rat: Preferential Response by Follicular Tissue. J. Endocr, 63: 417, 1974. Nureddin, A. Ovarian Ornithlne Decarboxylase Induction: A Specific and Rapid in vivo Bioassay of LH. Biochem. Med. l_~7:67, 1977. Osterman, J., L.M. Demers, and J.M. Hammond. Gonadotropin Stimulation of Poreine Ovarlan Ornlthine Decarboxylase in vitro: The Role of Cyelic AMP. Endocrlnology, 1978, in press. Lampreeht, S.A., U. Zor, A. Tsafriri and H.R. Lindner. Act±on of Prostaglandin E2 and of Luteinizing Hormone on Ovarian Adenylate Cyclase, Protein Kinase and Ornithine Deearboxylase Actlvity during Postnatal Development and Maturity in the Rat. J. Endocr. 57:217, 1973. Ch---anning,C.P. Influenee of the in vivo and invitro Hormonal Envlronment upon Luteinization of Granulosa Cells in Tissue Culture. Rec. Prog. Horm. Res. 26:589, 1970. Speroff, L., and P.W. Ramwell. Prostaglandin Stimulat±on of i__n_n vitro Progesterone Synthesls. J. Clin. Endocrinol. Metab. 30: 345, 1970. Kuehl, I.A., J.L. Humes, J. Tarnoff, V.J. C$rilo, and E.A. Ham. Prostagland±n Receptor Site: Evldence for an Essential Role in the Action of Luteinizing Hormone. Science 169:883, 1970. Lindner, H.R., U. Zor, S. Bauminger, A. Tsafriri, S.A. Lamprecht, Y. Koch, S. Antebi, and A. Schwartz. Use of Prostaglandin Synthetase Inhibitors in Analyzing the Role of Prostaglandins in Reproductive Phys±ology. In: Prostaglandin Synthetase Inhibitors (H.J. Robinson and J.R. Vane, eds.) Raven Press, New York, 1974, p. 271. Rigler, G.L., G.T. Peake, and A. Ratner. Effect of FollicleStlmulating Hormone and Luteinizlng Hormone on Ovarian Cyclic AMP and Prostaglandin E in vlvo in Rats Treated with Indomethaein. J. Endocr. 70:285, 1976. Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall. Protein Measurement with the Folln Phenol Reagent. J. Biol. Chem. 193:365, 1951. Mans, R.J. and G,D. Novelli. Measurement of the Ineorporation of Radioactive Amino Acids into Protein by a Filter-Paper Disk Method. Arch. Bioehem. Biophys. 9_~4:48, 1961.
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Zor, U., S. Bauminger, S,A. Lamprecht, Y. Koch, P. Chobsieng, and H.R. Lindner. Stimulatlon of Cyclic AMP Produetlon in the Rat Ovary by Luteinizing Hormone: Independence of Prostaglandin Mediation. Prostaglandins ~:499, 1973. Goff, A.K., and D.T. Armstrong. Stimulatory Action of Gonadotroplns and Prostaglandins on Adenoslne-3~,5"-Monophosphate Production by Isolated Rat Granulosa Cells. Endocrlnology i01: 1461, 1977. Marsh, J.M., N.S.T. Yang, and W.J. LeMalre. Prostagland±n Synthesls in Rabbit Graafian Follicles in vltro: Effect of Luteinizlng Hormone and CycllcAMP. Prostaglandins !:269, 1974. Clark, M.R., J.M. Marsh, and W.J. LeMaire. The Role of Protein Synthesls in the Stimulatlon by LH of Prostaglandin Accumulation in Rat Preovulatory Folllcleslnvitro. Prostaglandins 12:209, 1976. Clark, M.R., J.M. Marsh, and W.J. LeMaire. Stimulatlon of Prostaglandin Accumulation in Preovulatory Rat Folllcles by Adenosine 3%5~-Monophosphate. Endocrinology 102:39, 1978. Grinwich, D.L., T.G. Kennedy, and D.T. Armstrong. Dissoc±ation of Ovulatory and Steroidogenlc Actions of Lutenlzing Hormone in Rabbits with Indomethacin, an Inhibitor of Prostaglandin Biosynthesis. Prostaglandlns ~:89, 1972. Young Lai, E.V. Prostaglandlns and Steroldogenesis by Isolated Rabbit Ovarian Follicles. Horm. Res. ~:31, 1978.
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9/5/78
- Approved
12/4/78
FEBRUARY 1979 VOL. 17 NQ. 2