Reevaluation of the Role of Cyclic Adenosine 3',5'Monophosphate and Protein Kinase in the Stimulation of Steroidogenesis by Luteinizing Hormone in Bovine Corpus Luteum Slices1 WILLIAM Y. LING 2 AND JOHN M. MARSH3 Endocrine Laboratory and the Department of Biochemistry, University of Miami School of Medicine, P.O. Box 520875 Biscayne Annex, Miami, Florida 33152 ABSTRACT. Incubation of bovine corpus luteum slices at 37 C with luteinizing hormone showed that 10.0 fig LH/ml caused a maximum rise in tissue cyclic AMP content within 15 min. Slices incubated with 1.0 or 0.1 /ig LH/ml showed a much more gradual accumulation of this nucleotide. In the absence of added LH, a marked decline in the amount of cyclic AMP was observed during the first 60 min. The possible role of cyclic AMP in the action of LH was reexamined by studying the dose-response effect of LH on the stimulation of progesterone synthesis, cyclic AMP-dependent protein kinase activation, and cyclic AMP accumulation. After a 2-h incubation, the results showed that the dose required to elicit a minimal significant stimulation

I

HAS BEEN shown that LH4 will stimulate progesterone biosynthesis in incubating slices of bovine corpora lutea (1) and several lines of evidence point to cyclic AMP as a probable mediator of this effect. First of all, exogenous cyclic AMP mimicked the action of LH on steroidogenesis (2) and the effect of LH was not additive to that produced by a maximal amount of cyclic AMP (3). LH also brought about an increase in adenylate cyclase activity (4), and the concentration of endogenous cyclic AMP (5). Both of these effects were specific for LH, T

Received December 6, 1976. 1 Supported in part by a National Institute of Health Grant HD 03142 and a Ford Foundation Grant 0338. 2 Present address, Departments of Obstetrics and Gynaecology and Physiology, Dalhousie University, Halifax, N.S. Canada. 3 To whom reprint requests should be addressed. 4 The abbreviations used are: LH, luteinizing hormone; cyclic AMP, cyclic adenosine-3',5'-monophosphatc.

of steroidogenesis was 0.01 {JLg/m\ LH. At this and higher concentrations of LH, a concomitant stimulation of protein kinase activity and progesterone synthesis was also consistently observed. However, significant accumulation ot cyclic AMP became consistently detectable only at 0.1 Atg/ml LH. This report is the first to show a positive correlation between the activation of cyclic AMP-dependent protein kinase and the stimulation of steroidogenesis in the corpus luteum at the same minimal effective level of LH. These results indicate that cyclic AMP and the cyclic AMP-dependent protein kinase probably play important intermediary roles in the stimulation of steroidogenesis by LH in the bovine corpus luteum. (Endocrinology 100: 1571, 1977)

as had been shown for the stimulation of progesterone synthesis. Furthermore, under LH stimulation, the increase in endogenous cyclic AMP preceded the increase in progesterone synthesis (5). In terms of doseresponse, there was a positive correlation between cyclic AMP accumulation and steroidogenesis at LH concentrations between 200 to 2000 ng/ml. At 20 ng of LH/ml, however, there was usually an increase in steroidogenesis without any measurable change in cyclic AMP (5). This discrepancy was not considered to be a serious one in view of the variability of the incubating slice technique and the fact that the assay method used for the cyclic AMP had sufficient experimental error to obscure small increases in the cyclic nucleotide. Since that time other investigators such as Beall and Sayers (6), Catt and Dufau (7), and Moyle and Ramachandran (8) have shown rather conclusively that steroidogenesis can be elicited in other tissues, with low con-

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LING AND MARSH

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centrations of trophic hormones without concomitant rise in cyclic AMP levels. These results raised the question of whether cyclic AMP was an obligatory mediator in the regulation of steroidogenesis by trophic hormones. We were thus prompted to reinvestigate the relationships of dose of LH to steroidogenic and cyclic AMP responses in this model system using a more sensitive method for cyclic AMP measurement. In addition, it has been shown that bovine corpora lutea contains a cyclic AMPdependent protein kinase5 (9,10) which is specifically stimulated by LH in vitro (11). Furthermore, a cyclic AMP-dependent protein kinase prepared from corpora lutea has been shown to stimulate cholesterol side-chain cleavage activity (12). We have, therefore, utilized the activation of cyclic AMP-dependent protein kinase as another indicator of the possible role of cyclic AMP on steroidogenesis under LH stimulation in this tissue. Materials and Methods Chemicals and animal tissues ATP, cyclic AMP, EDTA and histone Type II-A from calf thymus were purchased from Sigma Chemical Co. The triethylammonium salt of adenosine [y-32P]triphosphate (specific activity 28-30 Ci/mmol) was purchased from ICN Pharmaceuticals, Inc. [l,2-3H]Progesterone (specific activity 40-60 Ci/mmol) was purchased from New England Nuclear and [8-3H]adenosine 3', 5'-monophosphate (specific activity 27 Ci/ mmol) from Amersham Searle. Dowex 50 (AG50W X8, 100-200 mesh, hydrogen form) and Dowex 1 (AG-1-X8, 200-400 mesh, chloride form) was purchased from Bio Rad Laboratories. Beef heart cyclic AMP-dependent protein kinase and protein kinase inhibitor were obtained from Sigma Co. Cyclic nucleotide phosphodiesterase (prepared from bovine heart) was purchased from Boehringer Mannheim. Ovine LH (NIH-LHS18) was a gift of the National Institute of Arthritis, Metabolism, and Digestive Diseases and l-methyl-3-isobutylxanthine was a gift of Searle Laboratories. 5

Cyclic AMP-dependent protein kinase (EC 2.7.1.37).

Kndo • 1977 Vol 100 • No 6

Ovaries from cows in the first 6 months of pregnancy (as determined by the size of the fetus (13)) were obtained at slaughter and transported to the laboratory in 0.154M NaCl at 0 C. The coipora lutea were then dissected free from adjacent ovarian tissue in a cold room at 4 C. Preparation and incubation of tissue slices Tissue slices were prepared in the cold room from a single corpus luteum for each experiment. The corpus luteum was quartered and slices of approximately 0.5 mm in thickness were prepared using a Stadie-Riggs hand microtome. These slices were distributed so that each incubation vessel had the same number of slices from approximately equivalent areas of the corpus luteum. When protein kinase measurements were carried out, one slice in each incubation vessel was marked for protein kinase assay with a small cut in one side. Unless otherwise indicated the slices were incubated for 2 h in 10 ml of Krebs-Ringer bicarbonate buffer in an atmosphere of 95% O2 and 5% CO2 in a Dubnoff metabolic incubator. At the end of the incubation the slice for protein kinase determination was removed and was homogenized at 0 C in an appropriate volume (125 mg/ml) of 10 mM potassium phosphate, pH 6.5, containing 0.5M NaCl, 10 mM EDTA and 0.5 mM l-methyl-3-isobutylxanthine. The other slices were also removed and both the slices and the medium were kept frozen until analyzed for progesterone and cyclic AMP. Protein kinase assay The luteal tissue homogenate was assayed for protein kinase activity by a modification of the method of Corbinet al. (14). The homogenate was centrifuged at 12,000 x gfor5 min at 4 C, and the supernatant assayed for its ability to transfer 32 P from adenosine (y-32P) triphosphate to histone in the presence and absence of cyclic AMP. Aliquots of 10 /xl were added to 55 jul of a solution of 15.4 mM potassium phosphate, pH 6.8, 0.3 mM adenosine (y-32P) triphosphate (approximately 1.5 x 106 cpm/tube), 5 mM magnesium acetate, 0.5 mg histone type 11-A, 12 mM NaF, and where added, 1.8 x 10~6M cyclic AMP. This mixture was incubated for 10 min in air with shaking. The reaction was terminated by pipetting duplicate 20 /x\ aliquots of the mixture onto rectangles (1.5 cm x 2.5 cm) of Whatman 3 MM chromatograph paper and dropping the

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PROTEIN KINASE AND STEROIDOGENESIS papers into a bath of cold 10% TCA. The papers were washed according to the procedure of Reiman et al. (15) except that the wash in 5% TCA at room temperature for 30 min was done only once. The papers were dried and counted in 10 ml of toluene scintillation fluid. The cpm of 32P were converted into pmoles of 32P incorporated into histone from the specific activity of the (y-32P) ATP in the assay mixture, and the protein kinase activity expressed as pmoles 32P incorporated per min. A background activity was determined for each assay using a boiled aliquot of the supernatant. The experimental values were corrected for this background activity (about 2.55 pmol/min) and are usually expressed in terms of endogenous activity (without cyclic AMP), total activity (with cyclic AiMP) and the ratio of endogenous to total activity. Isolation and measurement of progesterone and cyclic AMP Progesterone was isolated and measured using a modification of a previously described procedure (5). The frozen tissue (about 0.25 g-0.5 g) was homogenized in 1 ml of 10% TCA containing [l,2- 3 H]progesterone (15,000 cpm) and [8-3H]adenosine-3', 5'-monophosphate (5,000 cpm). The homogenate was centrifuged at 2,000 X g for 10 min at 2 C and the supernatant removed and used for cyclic AMP measurement. The pellet was homogenized with 0.2 ml of 2% NaOH and extracted 4 times with 4 ml of ethyl ether. The pooled ether extract was backwashed with H2O and the ether evaporated using a rotary evaporator. The residue material was defatted by standing overnight at - 2 0 C in 3 ml of 70% CH3OH and then being centrifuged at 2000 x g for 30 min at - 1 0 C . The supernatant was evaporated under N2 and the residue subjected to two dimensional, thin layer chromatography on silical gel (5). The mass of progesterone was determined spectrophotometrically at 280 11111 as described previously (5). Progesterone synthesis was calculated from the amount present after incubation, less the amount present in an unincLibated portion of the corpus luteum. A 0.5 ml aliquot of the supernatant from the centrifugation of the tissue homogenate was subjected to chromatography on Dowex 50 and analyzed for cyclic AMP by a competitive protein binding assay as previously described (16). The purity of the cyclic AMP isolated in this way from corpora lutea extracts was determined by subjecting a portion of the eluate from the

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TABLE 1. A test of the purity of the cyclic AMP isolated by Dowex 50 chromatography Specific activity samples (cpm/pmol) 1

Treatment 1. After Dowex 50 2. After Dowex 1 3. After Phosphodiesterase

11.9 14.5 18.4 12.0 11.7 14.0 17.3 14.0 —' —' —' —'

1 There was no mass or radioactivity detected in the samples after phosphodiesterase treatment and rechromatography on Dowex 50. Four samples were subjected to Dowex 50 chromatography and the amount of cyclic AMP determined on aliquots of the eluate fractions as described previously (16). The amount of [8-3H]adenosine 3',5'-monophosphate tracer in the eluate was also determined and the specific activity calculated. Another portion of the eluate fractions was subjected to Dowex 1 chromatography and the specific activity of the eluted cyclic AMP determined again. Finally, another portion of the eluate fraction of the first Dowex 50 chromatography was incubated with cyclic nucleotide phosphodiesterase, rechromatographed on Dowex 50, and the final eluate analyzed for mass of cyclic AMP and 3H cpm.

Dowex 50 column to further chromatography on Dowex 1. As shown in Table 1, the specific activity (cpm of 3H-adenosine-3',5'-monophosphate/pmol of cyclic AMP measured) of the cyclic AMP remained essentially the same after this further purification step. Furthermore, it was shown that treatment of the eluate fraction from Dowex 50 chromatography with cyclic nucleotide phosphodiesterase, followed by rechromatography on the same system completely eliminated the apparent mass of cyclic AMP. These tests indicate that our method was adequate for measuring the mass of cyclic AMP in extracts of corpora lutea.

Results

Cyclic AMP concentration and steroidogenesis in incubating luteal slices In our previous work (5) where a discrepancy between the effect of LH on cyclic AMP accumulation and steroidogenesis had been discovered, we measured steroidogenesis after 2 h of incubation and cyclic AMP concentrations after 15 min of incubation of the corpora lutea slices. This was done because it had been shown that these times represented the optimum times to detect maximum changes in cyclic AMP

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FIG. 1. Time course of cyclic AMP accumulation in corpora lutea slices incubated with various concentrations of LH. Corpora lutea slices (0.25-0.5 g) were incubated in 10 ml of Krebs-Ringer bicarbonate buffer for intervals of 15, 30, 60 or 120 min at which times they were analyzed for cyclic AMP (cAMP). The circles and brackets for the incubations carried out with 1,000 ng LH/ml and 10,000 ng LH/ml represent the mean c-AMP concentrations and range for 2 experiments, respectively. The circles and brackets for the incubations carried out with 10 or 100 ng LH/ml represent the mean and standard error, respectively, of 4 experiments. The dots and brackets for the controls represent the mean and standard error of 12 experiments.

and progesterone synthesis when high concentrations of LH were used (5). It occurred to us that part of the discrepancy between cyclic AMP accumulation and steroidogenesis could have been due to the possibility that 15 min did not represent an optimum time to measure cyclic AMP when lower concentrations of LH were used. Incubations of corpora lutea slices were then carried out for 15, 30, 60 and 120 min, under control conditions and with several concentrations of LH. The results are shown in Fig. 1. It was found that there was an initial marked decrease in the cyclic AMP content of control tissues which leveled off after about 60 min. This drop could not be attributed simply to a release of cyclic

1977 No 6

AMP into the medium because no cyclic AMP or progesterone could ever be measured in the medium of these tissue slices. The time course of the changes in cyclic AMP in tissue slices incubated with 10,000 ng LH/ml were very similar to those previously reported when high concentrations of LH were used (5). There was a rapid increase in cyclic AMP accumulation which reached a maximum at about 30 min. At lower concentrations of LH, however, the time courses were quite different. There was an almost linear increase in cyclic AMP throughout the 2 h period when 1000 ng LH/ml was used. At 100 ng LH/ml or 10 ng LH/ml there was a fall in cyclic AMP concentrations like those in control tissues, and the level in the tissues treated with 100 ng LH/ml significantly (P < 0.05) exceeded the control values only after 2 h of incubation. The levels of cyclic AMP in the

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FIG. 2. Effect of several concentrations of LH on cyclic AMP accumulation and progesterone synthesis in corpora lutea slices incubated for 2 h. Corpora lutea slices were incubated for 2 h in Krebs-Ringer bicarbonate buffer and then they were analyzed for cyclic AMP (cAMP, A A) and progesterone ( • D). The symbols and the brackets for the control tissues, incubated without LH, represent the mean and standard error, respectively, of 36 experiments. The symbols and the brackets for the tissues incubated with 1 to 10,000 ng LH/ml represent the means and standard errors of 12 paired experiments. The * indicates the lowest concentration of LH which had a significant (P < 0.01) stimulatory effect on either progesterone or cyclic AMP accumulation.

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PROTEIN KINASE AND STEROIDOGENESIS tissue slices treated with 10 ng LH/ml were not different from those in the control tissues in these experiments. From the foregoing data, the optimum time to detect maximum changes in cyclic AMP in luteal tissue slices incubated with different concentrations of LH appeared to be at 2 h. A comparison of the effects of different doses of LH on cyclic AMP accumulation and progesterone synthesis was, therefore, undertaken again, only this time both parameters were measured after 2 h of incubation. The results are shown in Fig. 2. Again, however, even after 12 experiments were carried out at each of several doses of LH, there was still a discrepancy between the minimum effective doses of LH which would stimulate progesterone synthesis and that which would stimulate cyclic AMP accumulation. Progesterone synthesis was significantly (P < 0.01) increased at 10 ng LH/ml while the cyclic AMP accumulation was not significantly increased until 100 ng LH/ml was used. There was a tendancy for the level of cyclic AMP accumulation to be greater in the slices incubated with 10 ng LH/ml (272 pmol/g ± 53.1, mean ± SE) than in the control incubations (177 ± 10.4) but this was not significant (P > 0.05). Protein kinase activity in luteal tissue It is possible that the inability to detect a consistent increase in cyclic AMP at lower concentrations of LH might be due to the confinement of the effective cyclic AMP to a small compartment of the cell. One component of the cell which could be considered as a compartment is the regulatory subunit of the enzyme protein kinase. We attempted then to detect an interaction of endogenous cyclic AMP with this subunit indirectly by measuring the changes in protein kinase activity. The protein kinase activity was measured in a 12,000 x g supernatant of corpora lutea homogenates prepared in a high concentration of NaCl according to the procedure of Corbin et al. (14). The only major modification that we carried out was

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FIG. 3. Effect of NaF on protein kinase activity of corpora lutea extracts. Assays were performed in the absence ( • • ) (endogenous activity) and presence of 1.5 x 10-6M cyclic AMP ( • • ) (total activity). A 10 fi\ aliquot of the enzyme preparation (equivalent to 1.25 mg wet weight of tissue) was added to 55 fi\ of reaction mixture (prepared as described in Materials and Methods) containing different concentrations of NaF. The assay was carried out for 10 min as described in Materials and Methods. The symbols represent the means and the brackets the standard errors for 3 experiments. Activity is expressed as pmoles of 32P incorporated into substrate per min per weight of tissue equivalent.

to decrease the concentration of NaF to 10 mM in the final reaction mixture. Corbin et al. (14) had shown that NaF markedly stimulated protein kinase activity of adipose tissue and had an optimum effect at 70 mM. As shown in Fig. 3, NaF also stimulated total and endogenous corpora luteal protein kinase activity to some extent, but the optimal concentration seemed to be at about 10 mM NaF in the samples assayed in the presence of 1.5 x 10"6M cyclic AMP. The effect of NaF on endogenous protein kinase activity was somewhat smaller and the maximum ratio of total to endogenous activity was achieved at 10 mM NaF. In all subsequent assays, therefore, NaF was present at 10 mM. The time course of protein kinase activity was linear up to 5 min and then its rate decreased slightly (Fig. 4). The amount of

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LING AND MARSH

corpora lutea slices was also examined in this study and the results are shown in Figs. 5A and B. There was no apparent change in the cyclic AMP dependency of the protein kinase upon dilution of the extracts in the absence (Figs. 5A) or presence (Fig. 5B) of NaCl, as can be seen by the relative constancy of the ratio of endogenous (-cAMP) to total protein kinase activities ( + cAMP). There was, however, no apparent deleterious effect of NaCl, so it was included in the homogenization medium of all subsequent experiments.

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INCUBATION TIME (min) FIG. 4. The time course of the protein kinase assay of corpora lutea extracts. The measurement of endogenous and total protein kinase activity was determined as described in Fig. 3, but using 4 different assay incubation times. The symbols represent the means of three determinations.

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activity at 5 min or less was, however, difficult to estimate accurately because the total accumulated counts of 32P-histone were relatively close to the background. A 10 min assay time was therefore chosen for all subsequent assays. Using this assay time, we found that the assay was proportional to the amount of enzyme, up to an amount equivalent to 2.5 mg of wet weight of tissue (Figs. 5A and B). In all subsequent assays the amount of enzyme assayed was equivalent to 1.25 mg of wet weight tissue. Corbin et al. (14) also reported that the inclusion of 0.5M NaCl in the homogenizing buffer served to prevent the reassociation of the subunits of protein kinase prepared from adipose tissue. In the absence of 0.5M NaCl there was a marked increase in the cyclic AMP dependence of protein kinase after several dilutions of the crude extiact ofadipose tissue (14). The effect of including 0.5M NaCl in the homogenizing medium of

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FIG. 5. Effect of dilution on the endogenous and total protein kinase activity of corpora lutea extracts. 1.25 g of corpora lutea slices were homogenized in 2.5 ml of 10 mM potassium phosphate, pH 6.5, containing 10 mMEDTAand0.5 mM l-methyl-3-isobutylxanthine. The homogenate was centrifuged at 12,000 x g for 5 min, at 4 C. Ten /JL\ aliquots of the supernatant and several dilutions of the supernatant, in the same homogenizing medium (no NaCl), were assayed for protein kinase activity in the absence (endogenous) and presence (total) of 1.5 x 10~6M cyclic AMP, as described in Materials and Methods. The symbols represent the mean of duplicate samples. The ratio values (O O) represent the ratio of the endogenous to total activities.

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PROTEIN KINASE AND STEROIDOGENESIS LH, the slice was homogenized, and an extract prepared and assayed for endogenous and total protein kinase activity. The effects of the different concentrations of LH on these enzyme activities are shown in Fig. 6. There was a significant increase in the endogenous protein kinase activity with as little as 10 ng LH/ml and this continued to increase until it reached a maximum in the tissue slices incubated with 1000 ng LH/ml. The total activity appeared to decrease slightly in the tissue slices incubated with 1 and 10 ng LH/ml, but this decrease was only statistically significant (P < 0.03) at the 1 ng LH/ml. The meaning of this decrease is unknown at this time. It appeared then, that although we could not detect a consistent rise in the concentration of cyclic AMP at the lowest level of LH which would stimulate progesterone synthesis, we

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FIG. 6. The effect of several concentrations of LH on protein kinase activity in corpora lutea slices incubated for 2 h. Corpora lutea slices were incubated for 2 h in Krebs-Ringer bicarbonate buffer and then they were homogenized (125mg/ml) in the homogenizing medium described in Fig. 5B (with NaCI). After centrifugation at 12,000 x g for 5 min, at 4 C, 10 (x\ aliquots were assayed for endogenous and total protein kinase activity as described in Materials and Methods. The ratio values represent the ratio of the endogenous to total activities. The circles and the brackets for the control tissues represent the means and standard errors, respectively, for 36 experiments. The circles and the brackets for the tissues incubated with 1 to 10,000 ng LH/ml represent the means and standard errors of 12 experiments. The * indicates the lowest concentration of LH which had a significant (P < 0.05) stimulatory effect on endogenous protein kinase.

could detect an increase in cyclic AMPdependent protein kinase activity. Discussion

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FIG. 5B. 1.25 g of slices of another corpus luteum was homogenized in 2.5 ml of 10 mM potassium phosphate, pH 6.5, containing 0.5M NaCI, 10 mM EDTA and 0.5M l-methyl-3-isobutylxanthine. After centrifugation at 12,000 x g for 5 min, at 4 C, 10 fi\ aliquots of this supernatant or dilutions of the supernatant in the homogenizing medium (plus NaCI), were assayed for endogenous and total protein kinase activities as described in Materials and Methods. The symbols represent the mean of duplicate samples. See legend to Fig. 5A.

It has been confirmed in this study that progesterone synthesis can be stimulated in incubating corpora lutea slices by a low dose of LH (10 ng/ml) without a consistent increase in endogenous cyclic AMP. On the other hand, we have demonstrated a good correlation of the entire dose-response curve of the cyclic AMP-dependent protein kinase to LH with the steroidogenic response. Both the minimum and the maximum effective doses of LH in terms of protein kinase activation and steroidogenesis appear to be the same. The stimulation of the enzyme at low doses of LH could be

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Endo • 1977 Vol 100 • No 6

LING AND MARSH

interpreted as a stimulation of a protein kinase which is independent of cyclic AMP changes. This is unlikely to be the case, because LH did not increase the total protein kinase activity, but decreased only that proportion which could be stimulated in the assay by exogenous cyclic AMP. We prefer to believe that this increase in endogenous protein kinase by LH at 10 ng/ml is due to an increase in endogenous cyclic AMP, but that this increase is too small to be consistently detected by our present methodology. Richardson and Schulster (17) have carried out a study similar to that reported here with isolated adrenal cells, and Podesta and Dufau (18) have reported on a comparison of the effect of hCG on steroidogenesis and protein kinase using isolated Leydig cells. Both of these studies found, however, that low concentrations of the appropriate trophic hormone would still stimulate steroidogenesis without any detectable increase in protein kinase activity. On the other hand, it has been reported in abstract form (19) that physiological concentrations of hCG (10~ 12 M) will activate protein kinase in Leydig cells and cause a translocation of the catalytic subunit to the nuclear fraction. Furthermore, Azhar et at. (20) have recently reported that both endogenous cyclic AMP and protein kinase activity were increased at the lowest concentration of hCG (2.5 ng/ml) which they found would increase progesterone production in a suspension of isolated cells from a whole ovary of an immature rat. The reasons for the differences between our results and these other reports are not clear at this time. It may be that only a portion of the total cyclic AMP-dependent protein kinase participates in the acceleration of steroidogenesis and that the portion is a larger percentage of the total in a tissue such as the corpus luteum and therefore easier to detect that in adrenal or Leydig cells of the testis. At any rate, this study indicates that a cyclic AMP-dependent protein kinase and probably cyclic AMP play important intermediary roles in the stimulation of steroidogenesis in the corpus luteum even at very low concentrations of LH.

Acknowledgments The authors gratefully acknowledge the excellent technical assistance of Mrs. Adalgisa Rojo, Mrs. Sara Morales, Mr. Jorge Cidre, Mr. Jorge Safille and Mrs. Nieves Cerver for help in the preparation of the manuscript. We are also indebted to the National Institute of Arthritis, Metabolism, and Digestive Disease for the gift of LH, the Searle Laboratories for the gift of l-methyl-3-isobutylxanthine, and the Gotham Provisions Co. Inc. for the gift of cow

References 1. Mason, N. R., J. M. Marsh, and K. Savard, j . Biol Chem 237: 1801, 1962. 2. Marsh, J. M., and K. Savard, Steroids 8: 133, 1966. 3. Marsh, J. M., and W. J. LeMaire, In Moudgal, N. R. (ed.), Gonadotropins and Gonadal Function, Academic Press, New York, 1974, p. 376. 4. Marsh, J. M.J Biol Chem 245: 1596, 1970. 5. Marsh, J. M. R. W. Butcher, K. Savard, and E.W. Sutherland,/ Biol Chem 241: 5436, 1966. 6. Beall, R. J., and G. Sayers, Arch Biochem Biophys 148: 70, 1972. 7. Catt, K. J., and M. L. Dufau, Nature [Neiu Biol] 244: 219, 1973. 8. Moyle, W. R., and J. Ramachandran, Endocrinology 93: 127, 1973. 9. Menon, K. M. J . J Biol Chem 248: 494, 1973. 10. Goldstein, S., and J. M. Marsh, In Huijing, F., and E. Y. C. Lee, (eds.), Protein Phosphorylation in Control Mechanisms, Academic Press, New York, p. 123. 11. Darbon, J. M., J. Ursely, P. Leymarie, FEBS Lett 63: 159, 1976. 12. Caron, M. G., S. Goldstein, K. Savard, and J. M. Marsh, J Biol Chem 250: 5137, 1975. 13. Kristoffersen, J., Ada Endocrinol (Kbh) 33: 417, 1960. 14. Corbin, J. D., T. R. Soderling, and C.R. Park, J Biol Chem 248: 1813, 1973. 15. Reiman, E. M., D. A. Walsh, and E. G. Krebs, J Biol Chem 246: 1986, 1971. 16. Ling, W. Y., J. M. Marsh, W. N. Spellacy, A. J. Thresher, and W. J. LeMaire, J Clin Endocrinol Metab 39: 479, 1974. 17. Richardson, M. C , and D. Schulster, Biochem J 136: 993, 1973. 18. Podesta, E., and M. L. Dufau, Program of the 57th Annual Meeting of the Endocrine Society New York, 1975, Abstract No. 215. 19. Simpson, R. J., M. L. Dufau, E. Podesta, and K. J. Catt, Abstracts of the V International Congress of Endocrinology, Hamburg, 1976, p. 69. 20. Azhar, S., M. R. Clark, and K. M. J. Menon, Endocr Res Commun 3: 93, 1976.

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Reevaluation of the role of cyclic adenosine 3',5'-monophosphate and protein kinase in the stimulation of steroidogenesis by luteinizing hormone in bovine corpus luteum slices.

Reevaluation of the Role of Cyclic Adenosine 3',5'Monophosphate and Protein Kinase in the Stimulation of Steroidogenesis by Luteinizing Hormone in Bov...
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