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
269
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
>
~ 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
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
269
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
>
~ 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
