Inhibition of choriogonadotropin-activated steroidogenesis in cultured ...

Vol. 262, No. 13, Issue of May 5, pp. 6093-610@,19S7 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1987 by The American Society of Biological Chemists, Inc.

Inhibition of Choriogonadotropin-activatedSteroidogenesis in Cultured LeydigTumor Cells by the R, Diastereoisomer of Adenosine 3’,5’-Cyclic Phosphorothioate* (Received for publication, November 25, 1986)

Maria E. PereiraS, Deborah L. SegaloffS, Mario AscoliS, and FritzEcksteing From iThe P o d a t i o n Council, New York, New York 10021 and the $Mar-Planck-Imtitutfur Experimentelk Medizin, Abteilung Chemie, 0-3400 Gottingen, West Germany

to adenylate cyclase activThe diastereoisomers of adenosine 3’,5’-cyclic phos- LH/CG have been shown increase phorothioate, (S,)-CAMPSand (R,)-CAMPS,have been ity in isolated membranes and cAMP levels in intact cells; previously shown to act as agonists and antagonists, (ii) cAMP analogues or compounds that raiseendogenous respectively, in the activation of several mammalian cAMP levels (such as cholera toxin and forskolin) have been CAMP-dependent protein kinases. shown to stimulate steroidbiosynthesis; and (iii) phosphodiIn an effort to characterize further the involvement esterase inhibitors have been shown to potentiate the stimuof cAMP in the activationof Leydig cell steroidogene- latory effects of low concentrations of LH/CG on cAMP levels sis by Iutropin/choriogonadotropin (LH/CG), we ex- and steroid biosynthesis(see Ref. 1for a review). It is imporamined the effects of these cyclic nucleotide analogues tant to note, however, that there are discrepancies between on a clonal strain of cultured murine Leydig tumor the amounts of LH/CG required to stimulate steroidbiosyncells (designated MA-10). Our results show that (i) (S,)- thesis and cAMP accumulation (Refs. 1-5 and this paper). CAMPSactivates and(RJ-CAMPSinhibits the isolated CAMP-dependent protein kinase of the MA-10 cells; Thus, atlow concentrations of LH/CG, a substantial increase (ii) both analogues inhibit the isolated cAMP phospho- in steroid biosynthesis occurs without detectable changes in diesterase(s); (iii)@,)-CAMPSactivates steroidbiosyn- cAMP accumulation. The ability of other ligands such as luteinizing hormonethesis in intactcells, but (I$,)-CAMPSdoes not; and (iv) (&,)-CAMPSis a competitive inhibitor of the activation releasing hormone (6) and epidermalgrowth factor (7)’ to of steroidogenesis by (S,)-CAMPS,8-bromo-cAMP,hu- activate steroid biosynthesis inLeydig cells without affecting man CG, cholera toxin, and forskolin. However, (Rp)- cAMP levels suggests thatintracellularsignalingsystems CAMPSis a more effective inhibitorwhen steroidogen- other than those involving cAMP can activate the steroidoesis is activated by (S,)-CAMPSor 8-bromo-CAMPthan genic pathway of these cells. These findings, together with when it is activated by human CG, cholera toxin, or recent data showing that LH/CG can affectphospholipid forskolin. This difference appears tobe related to the metabolism and/or Ca2+ fluxes in Leydig cellsand/or granucombined effects of (R,)-CAMPS onthe CAMP-depend- losa/lutealcells (4, 8, 9), have ledseveral investigators to ent protein kinases andcAMP phosphodiesterase(s). of LH/CG with these cells results propose that the interaction We conclude that cAMP is a quantitatively important in thegeneration of additionalintracellularmessenger(s) mediator of the activation of steroidogenesis by LH/ otherthancAMPthatare involved intheactivation of CG even at low concentrations of hormone where an steroidogenesis by LH/CG (4,8-10). Moreover, it has been increase in steroid biosynthesis cannot be easily cor- postulated that this‘‘CAMP-independent pathway” is responrelated with increasedcAMP accumulation. Thus, our sible for the stimulatoryeffects of low concentrations of LH/ data indicate that if other second messengers are in- CG on steroid biosynthesis (4). volved in the activation of steroidogenesis by LH/CG, Recent studies on the activitiesof the R, and S, diastereothey must do so by acting together with, rather than isomers of CAMP phosphorothioate (CAMPS) have shown that independently of, CAMP. (S,)-CAMPS is a full agonist for the activation of both isozymes of the mammalian CAMP-dependent protein kinases, whereas (R,)-CAMPS is an antagonist for the activation of Studies on the activationof steroid biosynthesis in Leydig these enzymes by cAMP on analogues thereof (11-14). The cells and granulosa/luteal cells by LHICG’ have shown that S, isomer has also been shown to mimic the actionsof cAMP cAMP fulfills all the criteria of a second messenger. Thus, (i) in the parotid gland (15), hepatocytes (16), PC12 cells (17), and thyroid cells (18); and the actions of this isomer have * This work was supported by Grant CA-40629 from the National been shown to be antagonized by the R, isomer in some of Cancer Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore these cells (16-18). Thus, (R,)-CAMPSis a potentially useful behereby marked “aduertisement” in accordance with 18 U.SC. compound to investigate CAMP-mediated effects in intact cells. Section 1734 solely to indicate this fact. ’ The abbreviations used are: LH/CG, lutropin/choriogonadotro- Since questions have been raised about the involvement of pin; hCG, human choriogonadotropin; (S& and (R,)-cAMPS, the cAMP as an obligatory mediator of the actionsof low concendiastereoisomers of adenosine 3’,5’-cyclic phosphorothioate; EGTA, trations of LH/CG (4, 8-10), it is important to utilize novel [ethylenebis(oxyethylenenitriio)]tetraacetic acid; HEPES, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid; MES, 2-(N-morpho- approaches t o address this question. Thus, the studies pre1ino)ethanesulfonate; Br-, bromo-; Rr and Ru, regulatory subunits of sentedherein were designed todetermine if (R,)-CAMPS the type 1 and I1 CAMP-dependent protein kinases, respectively; 8N2cAMP, 8-azidoadenosine 3’,5’-cyclic monophosphate; IBMX 3isobutyl-l-methylxanthine.

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M. Ascoli, J . Euffa, and D. L. Segaloff, submitted for publication.

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Inhibition of Steroidogenesis by a CAMPAnalogue

antagonizes the activationof steroid biosynthesis by LH/CG, specifically at concentrations of hCG that activate steroid biosynthesis without detectable changes incAMP accumulation.

cold buffer C containing 100 unitslml aprotinin, 20 p~ leupeptin, 1 mM phenylmethylsulfonyl fluoride, and 5 mM 2-mercaptoethanol and recovered by centrifugation. The cell pellets were then resuspended in cold buffer D (50 mM Tris-C1,5 mM 2-mercaptoethanol, pH 8) and homogenized as described above. The homogenates were centrifuged at 800 X g (4 "C) for 10 min, and the supernatant was saved. The EXPERIMENTALPROCEDURES pellets were homogenized and centrifuged again. The combined suCells-The origin and handling of the MA-10 cells have been pernatants were used as a source of cAMP phosphodiesterase since described (19). Cells were plated a t a density of3-4 X 104/cm2and they contained 75-85% of the total enzyme activity present in the used 3 days after plating. At this time, the cell density was 1.5-2 x homogenate. cAMP phosphodiesterase activity was measured in these extracts 105/cm2.The cells were plated in 6 X 35-mm clusters for the progesterone biosynthesis experiments, in 60-mm dishes for the CAMP (1 mg of protein/ml) by the method of Thompson et al. (24) as experiments, or in 100-mm dishes for the preparation of cell extracts. modified by Brothers et al. (25) during a 60-min incubation at 30 "C. Enzyme activity was linear with time and protein concentration. DEAE Chromatographyof CAMP-dependentProtein Kinases-Ten Determination of Progesterone Levels-All experiments were perto fifteen dishes of cells were placed on ice and washed twice with 3ml portions of cold buffer A (0.25 M sucrose, 10 mM Tris-C1, 10 mM formed with cells plated in 6 X 35-mm wells. The wells were washed EDTA, pH 7.4). The cells were scraped from the dishes with the help twice with 1-ml portions of warm assay medium (Waymouth's of a rubber policeman into cold buffer A supplemented with 100 MB752/1 modified to contain 1.1 g/liter NaHC03, 20 mM HEPES, 1 units/ml aprotinin, 20 p~ leupeptin, and 1mM phenylmethylsulfonyl mg/ml albumin, pH 7.4). Incubations were performed ina total fluoride. The cells were recovered by centrifugation and resuspended volumeof 1 mlof assay medium. Cholera toxin and hCGwere in cold buffer B (10 mM Tris-C1, 1 mM EDTA, 1 mM dithiothreitol, dissolved in 0.15 M NaCI, 20 mM HEPES, 1 mg/ml albumin, pH 7.4. 20 mM benzamidine, 100 units/ml aprotinin, 20 p~ leupeptin, and 1 Cyclic nucleotides were dissolved in the same buffer devoid of albumM phenylmethylsulfonyl fluoride, pH 7.4). The cell suspension was min, and forskolin was dissolvedin 95% ethanol. All compounds were homogenized (4 "C) with five strokes of a motor-driven teflon pestle added to the wells in 40-50 pl aliquots. Cells were incubated for 4 h (5,000 rpm). The homogenate was centrifuged (4 "C) at 800 X g for at 37 "C in a humidified atmosphere containing 5% CO,. At the end 10 min, and the supernatant was saved. The pellet was rehomogenized of the incubation, the medium wassaved, and progesterone (the major and centrifuged as described above. The combined supernatants were steroid produced by the MA-10 cells; see Ref. 19) was measured by then centrifuged (4 "C) at 12,000 X g for 30 min to give the cell radioimmunoassay in suitable aliquots of the unextracted medium (19). cytosol. Determination of intracellular CAMP Levels-Experiments were Two ml of cytosol (3-5 mg of protein/ml) were applied to a 1 X 8cm column of DEAE-Sephacel equilibrated with buffer B. The column performed with cells plated in 60-mm dishes. The cells were washed twice with 2-ml portions of warm assay medium and incubated as was washed with 10 ml of buffer B and then eluted with a linear gradient (0-0.3 M) of NaCl in buffer B at 4 "C. The flow rate was 0.5 described above in 2 mlof assay medium. Intracellular cAMP was ml/min, and 2-ml fractions were collected. Aliquots of the individual measured by a previously described method (26) modified as follows. fractions were assayed for protein kinase activity and [3H]cAMP At the end of the incubation, the dishes were placedon ice and washed twice with 2-ml portions of cold Hanks' balanced salt solution conbinding; they were also labeled with [32P]8-N3cAMP. Assay of CAMP-dependent Protein Kinase Activity-The activity taining 1 mM theophylline. After adding 2 ml of cold 0.5 N H3C104 of the CAMP-dependent protein kinases was determined by their containing 1 mM theophylline and 10,000 cpm of [3H]cAMP(used to ability to phosphorylate a synthetic peptide (kemptide). Briefly, 10 determine procedural losses), the cells were scraped, transferred to p1 of the column fractions (or pooled fractions) were incubated in a tubes, frozen and thawed once, and centrifuged. The supernatants total volume of 50 pl. The reaction mixture contained 40 mM Tris- were then chromatographed on a combination of alumina and Dowex C1, pH 7.4, 10 mM MgCl,, 0.2 mM [y-32P]ATP(100-200 cpm/pmol), columns as described by Lorenz and Wells (27). The fractions con1 mM EGTA, and 0.1 mg/ml kemptide. After a 5-min incubation at taining the cAMP were diluted, acetylated (28), and assayed by 30 "C, the phosphorylation of the synthetic peptide was determined radioimmunoassay (29, 30). In preliminary experiments, 50 pM solutions of CAMP, (Rp)as described by Roskoski (20). The reaction was linear with time and CAMPS, or (S,)-cAMPS were chromatographed on these columns, enzyme concentration. PHICAMPBinding Assay-Cyclic nucleotide binding was meas- and the effluents were assayed for the presence of the cyclic nucleoured by a modification of the Millipore filter technique described by tides by measuring AZm. We found that in contrast to CAMP, (Rp)Rannels and Corbin (21). Aliquots (20 21)of the column fractions CAMPSwas not adsorbed to the alumina columns. (S,)-CAMPS was were incubated overnight a t 4 "C with 50 p1 of a buffer containing 2 adsorbed to thealumina columns but was eluted during the extensive M NaCl, 25 mM potassium phosphate, pH 6.8, 0.5 mg/ml histone, 1 washing performed prior to eluting the cAMP onto the Dowex colmM EDTA, 0.4 mM IBMX, and 1.4 pM [3H]cAMP (12,000-16,000 umns. Thus, cAMP was completely separated from these analogues cpm/pmol). At the end of the incubation period, the contents of each prior to radioimmunoassay. Other Methods-Protein concentrations were measured by the tube were rapidly taken up in 1 mlof coldwash buffer (10 mM potassium phosphate, 1 mM EDTA, pH 6.8) and filtered through method of Bradford (31) using bovine serum albumin as a standard. Millipore filters. The filters were then washed three times (3 ml each lZ5I-Tyrosine methyl ester succinyl-CAMPwas prepared by the method of Richman et al. (30). The different parameters that describe time) with cold wash buffer, dried, and counted. concentration-response curves were calculated using the computer Photoaffinity Labeling of CAMP-dependent Protein Kinases-Aliquots (25 p l ) of the column fractions were incubated in a total volume program ALLFIT (32) and used to draw the lines shown. Supplies-Purified hCG (batch CR-121; 12,800 units/mg) was obof 100 pl. The final concentrations of reagents in each incubation were 50 mM sodium MES, 10 mM MgC12,2 mM EGTA, 1mM IBMX, tained from the National Institutes of Health. Cholera toxin, forsko10 units/ml aprotinin, 1 mM 2-mercaptoethanol, 1 p M [32P]8- lin, 8-Br-CAMP, theophylline, IBMX, histone (type IIAfromcalf N3cAMP, pH 6.2. After a 90-min incubation at room temperature, thymus), benzamidine, phenylmethylsulfonyl fluoride, aprotinin, the tubes were incubated on ice for 10 min and irradiated for 10 min alumina (type WN-3, neutral), bovine serum albumin, CAMP, and (on ice) with a 254-nm UV lamp placed 7.5 cm above the bottom of tyrosine methyl ester succinyl-CAMP wefe purchased from Sigma. the tubes (22). Following irradiation, the samples were resolved on DEAE-Sephacel and Dowex resins were from Pharmacia P-L Bio9% sodium dodecyl sulfate polyacrylamide gels as described previously chemicals and Bio-Rad, respectively. Kemptide (Leu-Arg-Arg-Ala(30(23). Autoradiograms of the gels were obtained after overnight expo- Ser-Leu-Gly) was from Peninsula Laboratories, Inc. [cY-~'P]ATP sure (-70 "C) to Kodak XAR-5 film. Following autoradiography, the 40 Ci/mmol), [2,8-3H]cAMP(30 Ci/mmol), [1,2,6,7-3H]progesterone radioactive bands were excised from the gels and counted in a liquid (90-100 Ci/mmol), and 5'-[U-"C]AMP (600 mCi/mmol) were obscintillation counter. The specificity of labeling was assessed by tained from New England Nuclear. [32P]8-N3cAMP(50-80 Ci/mmol) incubating parallel samples in the presence of 100 p~ CAMP. Under was from ICN, and NaIz5Iwas from Amersham Corp. P-81 cellulose these conditions, the incorporation of radioactivity was completely phosphate paper was obtained from Whatman, and cellulose ester filters (type HAWP, 0.45 pm) were from Millipore Corp. All tissue prevented. Assay of cAMP Phosphodiesterase Activity-Dishes of cells were culture supplies were from GIBCO,and tissue culture plasticware was placed on ice and washed twice with 3-ml portions of cold buffer C from Falcon Labware or Corning Glass Works. The antiserum to (0.25 M sucrose, 50 mM Tris-C1, pH 7.4). The cells were scraped into cAMP was generously provided by Dr. David Garbers (Department


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pools (3), they have often been interpreted to mean that cAMP is not involved in the activation of steroid biosynthesis bylow concentrations of hCG (4, 10). In the experiments summarized below, we have examined this possibility by testing the effects of an antagonist of cAMP on the activation of steroidogenesis by low concentrations of hCG. RESULTS Characterization of CAMP-dependent Protein Kinases and Effects of LH/CG on CAMP Accumulation and Steroid Bio- CAMPPhosphodiesterases of MA-10 Cells-Before performing synthesis in MA-10 Cells-Several investigators have previ- any experiments on the cyclic nucleotide analogues, it was ously shown that thereare discrepancies between the amounts important todetermine some of the properties of the CAMPof LH/CG required to activate steroidbiosynthesis and cAMP dependent protein kinases and cAMP phosphodiesterases of accumulation in normal rat and mouse Leydig cells (2, 3, 5). the MA-10 cells. This phenomenon is illustrated with the MA-10 cells in Fig. The CAMP-dependent protein kinases were characterized 1. In these experiments, we incubated the MA-10 cells with by DEAE chromatography of the MA-10 cytosol. As shown increasing concentrations of hCG and measured the accu- in Fig. 2 (upper), all of the CAMP-dependent protein kinase mulation of intracellular cAMP and extracellular progester- activity was adsorbed to DEAE and subsequently eluted with one. These parameters were measured during a 45-min and a NaCl gradient. Two peaks of activity were resolved the first 4-h incubation, respectively, since we have previously shown peak contained 80-85% of the kinase activity and eluted at that maximal levels are attained under these conditions (19, 0.01-0.075 M NaC1; the second peak contained 15-20% of the 26). The results presented in Fig. 1 show that (i) hCG stimu- kinaseactivity and eluted at 0.13-0.2 M NaCl. When the lated progesterone biosynthesis about 1000-foldand increased fractions were assayed for [3H]cAMP binding (center), we the levels of intracellular cAMP about 7-fold; (ii) the EC,, for detected a major peak (containing about 60% of the total cAMP accumulation (11ng/ml) was about 5-fold higher than binding activity recovered) that eluted at 0.01-0.075 M NaCl the EC50for progesterone biosynthesis (2 ng/ml); (iii) maxi- and a broad peak (or peaks) that eluted at 0.075-0.2 M NaCI. mal progesterone biosynthesis occurred at concentrations of hCG that elicited 30-50%of the maximal cAMP response; and (iv) at low concentrations of hCG (up to about 0.4 ng/ ml), asubstantial increase in progesterone biosynthesis (about 100-fold above basal) occurred with no detectable change in the level of intracellular CAMP. Similar results (not shown) were obtained when extracellular cAMP was measured. The resultspresentedin Fig. 1 aresimilar to those previously described for normal rat and mouse Leydig cells (2, 4,5). Although results such as thesemay be explained by lack of sensitivity of the methods used to measure CAMP, signal amplification (34, 35), or by compartmentalization of cAMP 15 I

of Pharmacology and Howard Hughes Medical Institute, Vanderbilt University). (Rp)-CAMPSand (&)-CAMPS were synthesized and purified as described previously (33). (Sp)-CAMPSwas also obtained from Boehringer Mannheim.

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FIG. 1. Concentration-response curves for activation of cAMP and steroid biosynthesis in MA-10cells. Cells were incubated in 2 ml of assay medium containing the indicated concentrations of hCG. Intracellular cAMP and extracellular progesterone were measured after a 45-min and 4-h incubation (37 "C), respectively, as described under "Experimental Procedures." Each point shows the mean +. S.E. of three independent experiments (duplicate points/experiment).

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FIG. 2. DEAE chromatography of CAMP-dependent protein kinases of MA-10 cells. Two ml of cytosol (10.9 mgof protein) were applied to a 1 X 8-cm column of DEAE-Sephacel equilibrated (at 4 "C) in buffer B (see "Experimental Procedures"). The column was washed with 10 ml of buffer B and then eluted with a 0-0.3 M NaCl gradient in the same buffer (the gradient profile is shown (upper)).Fractions of 1 ml were collected and assayed for protein kinase activity in the presence of 5 g M cAMP (upper), [3H]cAMP binding (center),or incorporation of [32P]8-N3cAMPinto RI and Rii (lower). Protein kinase activity (upper) is shown only when assayed in the presence of CAMP. In the absence of CAMP, kinase activity was detectable only in fractions 18-30, and it was less than 10% of the activity detected in the presence of CAMP.Lower, only the data for RI is plotted because RII was undetectable (see text for details). The results of a representative experiment are shown. RiC,, = CAMPdependent protein kinase type I; Ri'C,, = CAMP-dependent protein kinase type 11.

Inhibition of Steroidogenesis by a cAMP Analogue

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When the fractions were labeled with [32P]8-N3cAMPand resolved on sodium dodecyl sulfate gels, we only detected labeling of a protein whose M, (49,000) corresponds to RI, the regulatory subunit of the type I CAMP-dependent protein kinase (36). We were not able to detect by this method any proteins whose M, corresponded to that of the regulatory subunit of the type I1 CAMP-dependent protein kinase (€ill). The results presented in Fig. 2 (lower) show that two peaks of RI could be resolved on the DEAE column. The first peak (containing about 70% of the total RI)eluted at 0.01-0.075 M NaCl and coincided with the major peak of kinase activity(cf. upper) and [3H]cAMP binding (cf. center). The second peak eluted at 0.075-0.13 M NaCl and coincided with an area of [3H]cAMP binding (cf. center), but did not coincide with an area of protein kinase activity (cf. upper). Based on these results,we conclude that most of the CAMPdependent protein kinase activitypresent in the MA-10 cells corresponds to the type I isozyme and that these cells also have free RI. The second peak of CAMP-dependent protein kinase activity shown in Fig. 2 (upper) eluted with a salt concentration equivalent to that required to elute the type I1 isozyme of CAMP-dependent protein kinase. Although we were unable to detect RIIin these column fractions (see above), we have been able to identify a 52-54-kDa protein by immunoprecipitation of the [32P]8-N3cAMP-labeledfractions with an antibody to the bovine brain RrI (kindly provided by Dr. Charles Rubin, Albert Einstein Medical College). Thus, we conclude that thispeak represents the CAMP-dependent protein kinase type 11. The datapresented in Fig. 3 show an Eadie-Hofstee plotof cAMP phosphodiesterase activity in extracts of the MA-10 cells. As expected from results obtained in a numberof other cell types (37), this plot was biphasic, indicating that theMA10 cells have at least two types of cAMP phosphodiesterases. Effects of (S,)-cAMPS and (R,)-CAMPS on CAMP-dependent Protein Kinase and CAMPPhosphodiesterase Activities of MA-IO Cells-The res Its presented in Fig. 4 show that (Sp)CAMPS was capable f activating the type I kinase to the same extentas CAM . The concentration of @,)-CAMPS required to give half- aximal activation of the CAMP-dependent protein kinase type I (1.7 PM) was, however, about 8-fold higher than the equivalent concentration of cAMP (0.21 PM). The data presented in Fig. 4 also show that (Rp)CAMPSproduced a partial activation of the enzyme at concentrations higher than 10 pM. We next tested if (R,)-CAMPS was capable of inhibiting the CAMP- or (S,)-CAMPS-induced activation of the CAMP-

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v / \ s ~(rnln-ll FIG. 3. Eadie-Hofstee plot of cAMP phosphodiesterase activity in extracts of MA-10 cells. An extract of the MA-10 cells was prepared and assayed for hydrolysis of different concentrations of 13H]cAMPas described under “Experimental Procedures.” The results of a representative experiment are shown. Each point represents the average of duplicate samples. The arrow indicates the concentration of substrate used in subsequent experiments.

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Cyclic Nucleotide ( M I

FIG. 4. Effects of CAMP, (S,)-CAMPS,and (22,)-CAMPSon activity of CAMP-dependent protein kinase type I. The CAMPdependent protein kinase type I of the MA-10 cella was isolated by chromatography on DEAE-Sephacel as shown for Fig. 2. Fractions 21-24were pooled, and 10-rl aliquots of the pooled enzyme were assayed for protein kinase activity in the presence of the indicated concentrations of cyclic nucleotides as described under “Experimental Procedures.” The results presented are the average of three experiments performed with three different preparations of the enzyme. Error bars are not shown because we used different amounts of cytosol to isolate the enzyme, and the activity was not corrected for protein concentration in each experiment. Thus, the absolute numbers are somewhat variable. Within a given experiment, however, all determinations (done in duplicate) varied by, a t most, 10%.

dependent protein kinase type I. In these experiments, we preincubated the enzyme preparation with increasing concentrations of (R,)-CAMPS for 5 min and then added concentrations of cAMP (1 PM) or (S,)-cAMPS (10 PM) that fully activate the enzyme. As can be seen in Fig. 5A, (R,)-CAMPS antagonized the effects of both agonists to the same extent. The maximal level of inhibition (about 50%) was observed with 1-2 mM (R,)-CAMPS. We also tested the effects of (R,)-CAMPSand (S,)-CAMPS on the cAMP phosphodiesterase activity of MA-10 cell extracts. Phosphodiesterase activity was measured in the presence of 1 p~ (3H]cAMP since this was the concentration of CAMP used to activate the CAMP-dependent protein kinase type I (cf. Fig. 5A).The results presentedin Fig. 5B show that (R,)-CAMPS was also an effective inhibitor of the cAMP phosphodiesterase at the same range of concentrations that antagonize the activation of the CAMP-dependent protein kinase type I. At 1mM (R,)-CAMPS,phosphodiesterase activity was inhibited by 30%, and the kinase activitywas inhibited by 50% (cf. Fig. 5A).The datapresented in Fig. 5B also show that (S,)-CAMPS was about 10 times more effective than (R,)-CAMPS in inhibiting phosphodiesterase activity. At a concentration of 100 p ~ (S,)-CAMPS , was as effective as IBMX in inhibiting phosphodiesterase activity. Taken together, these results show that (S,)-CAMPS activates the CAMP-dependent protein kinase type I and inhibits the cAMP phosphodiesterase(s)inextracts of the MA-10 cells. The concentrations required to inhibit phosphodiesterase activity, however, appear to be higher than those required to stimulate the kinase. On the other hand, (R,)-CAMPS has little or no effect on the activity of the CAMP-dependent protein kinase type I, but antagonizes the activation of this

Inhibition of Steroidogenesis by a cAMP Analogue I

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(Rp)-cAMPS ( m M )

FIG.6. Effects of@,)-CAMPS,(R,)-CAMPS,and 8-BrcAMP on steroid biosynthesis in intact MA-10 cells. Cells were incubated withthe indicated concentrationsof cyclic nucleotidesfor 4 h at 37 "C. Progesterone was measured by radioimmunoassay in aliquots of the incubation medium. The results of a representative experimentareshown.Each bar shows the average f range of duplicate incubations. Note the different scales in the upper and lower panels.

FIG.5. Effects of (R,)-CAMPSand (S,)-CAMPSon activity of cAMP phosphodiesterase and CAMP-dependentprotein kinase type I of MA- 10 cells. A , the CAMP-dependent protein kinase type I of the MA-10 cells was isolated by chromatography on DEAE- maximal activation of steroidogenesis was about 0.2 mM. At Sephacel as shown for Fig. 2. Fractions 21-24 were pooled, and 10-rl this concentration, 8-Br-CAMP and (S,)-CAMPS increased aliquots of the enzyme were preincubated withthe indicated concen1000-fold. In contrast,(It,)-CAMPS trations of (E,)-CAMPS for 5 min at 30 "C. After addition of 1 pM steroid biosynthesis about CAMPor10 p~ (SJ-CAMPS, the mixtures were incubated for an has only a minor effect on basal steroid biosynthesis. At a additional 5 min at 30 'C and assayed for protein kinase activity as concentration of 1.0 mM, this analogueincreased steroid described under "Experimental Procedures." Each point shows the biosynthesis only 1.5-fold (Fig. 6, lower). Thus, the effects of average f S.E. of three experiments performed with three different the two isomers on progesterone biosynthesis in the intact preparations of the CAMP-dependent protein kinase. In each experi- MA-10 cells correlate well with their effects on the CAMPment, incubations were performed induplicate. Results are expressed dependent protein kinase (see above). as percent of the enzyme activity detected in the absence of (Ep)Effects of (R,)-CAMPS on Steroidogenesis Activated by CAMPS.B , phosphodiesterase activitywas measured in MA-10 cell extracts that were preincubated with the indicated concentrationsof cAMPAnalogues, hCG, Cholera Toxin, or Forskolin-We next (S,)-CAMPS, (R,)-CAMPS,or IBMX for 5 min at 30 "C. After addi- tested the abilityof (R,)-CAMPS to antagonize the activation tion of 1p~ [3H]cAMP,the mixtures were incubated for an additional of steroid biosynthesis by (S,)-CAMPS and hCG. In these 60 min at 30 "C and assayed for cAMP phosphodiesterase activity as experiments, we preincubated theMA-10 cells withor without described under "Experimental Procedures." Each point shows the average f S.E. oftwo experimentsperformedwith two different (R,)-CAMPS for 30 min at 37 "C and then added increasing preparations of MA-10 cell extract. In each experiment incubations concentrations of (S,)-CAMPS or hCG. After a further 4-h were performed in duplicate. Results are expressedas percent of the incubation at 37 "C,progesterone was measured. The results enzyme activity detected with [3H]cAMP only. are shown in Fig. 7 . As expected for a competitive inhibitor, we found that thedegree of inhibition of steroidogenesis was enzyme by cAMP or(S,)-CAMPS. It is important to note thatdependent on the concentrations of agonist (hCG or (Sp)of CAMPS) and antagonist((R,)-CAMPS) used. At any concenconcentrations of (R,)-CAMPSthat prevent the activation the CAMP-dependent protein kinase type I also inhibit cAMP tration of either agonist, thedegree of inhibition increased as phosphodiesterases. the antagonist concentration increased. On the other hand, Effects of (R,)-CAMPS and (S,)-CAMPS on Steroid Bwsyn- at a given antagonist concentration, the degree of inhibition thesis in IntactMA-10 Cells-Previous studies froma number decreased as theconcentration of agonist increased. It is of laboratories have shown that the stimulatory effects of LH/ important to note, however, that (&,)-CAMPSwas an effective CG on steroid biosynthesis in Leydig cells can be mimicked inhibitor of hCG-activated steroidogenesis even at low conwith cAMP analogues (reviewed in Ref. 1).This is also true centrations of hCG (i.e. 0.4 ng/ml) that activate steroid bioin the MA-10 cells, where we have shown that hCG, 8-Br- synthesiswithout inducing detectableincreases in cAMP CAMP, and dibutyryl cAMP activate progesterone biosyn- accumulation (cf. Fig. 1). thesis to thesame maximal extent(reviewed in Ref. 38). Because we wanted to compare the effectiveness of (Rp)The results presented in Fig. 6 (upper) show that (S,)- CAMPS to inhibitsteroidogenesis when it was stimulated to or the concentrations CAMPS was as effective as 8-Br-CAMP in activatingproges- the same extent with (S,)-CAMPShCG, terone biosynthesis in theMA-10 cells. Progesterone biosyn- of hCG and (S,)-CAMPS used in the experiment presented in thesis was activated to 30-50% of maximal when either ana- Fig. 6 were chosen t o give graded and equivalent degrees of logue was added at a concentration of 0.05 mM. The minimal stimulation of steroid biosynthesis. The data presented indiconcentration of either cyclic nucleotide required to provoke catethat 0.4 ng/mlhCG gave aboutthesame degree of

Inhibition of Steroidogenesis a by

6098

cAMP Analogue

hCG

ISPI-CAMPS

8-Br~cAMP 0.05mM

ilOOIiLilll8l

ilM1159111LI

hCG

Forsko1,n

Choler0 Tom" 10 ngiml

llO011931i791

1100118911681

0.05mM 08 ngiml 03mM _ _ __ 1100118016L1

5 300

100

50

dd o

iRp)bcAMPS

0 0.2 10

0 02 1.0

0 021.0

0 0210

0 0.21.0

0 0.2 10

ImMI

FIG. 7. Effects of (R,)-CAMPS on hCG- and (&',)-CAMPSactivated steroidogenesis. Cells were preincubated with the indicated concentrations of (I?,)-CAMPS for 30 min at 37 "C. Increasing concentrations ofhCG or (S,)-CAMPS were then added, and the incubation was continued for 4 h at 37 "C prior to the assay of progesterone. Each bar represents the mean k S.E. of two independent experiments (duplicate points/experiment). The numbers in parentheses above each set of bars indicate the amount of progesterone produced in the presence of the indicated concentrations of ( R J CAMPS expressed as percentage of that produced in the absence of (I?,)-CAMPS.The amount of progesterone produced in cells incubated with buffer only was about 1 ng/106 cells X 4 h. Increasing the concentration of hCG or (S,)-CAMPS beyond the highest concentrations shown does not result in additional increases in steroid biosynthesis.

stimulation as 0.04 mM (S,)-CAMPS (10-20% of the maximal response), 0.8 ng/ml hCG gave aboutthesame degree of stimulation as 0.05 mM (S,)-CAMPS (30-50% of the maximal response), and 20 ng/ml hCG gave about the same degree of stimulation as0.2 mM (S,)-CAMPS (90-100% of the maximal response). By comparing the ability of (I?,)-CAMPS to inhibit steroidogenesis in these pairs, it is apparent that(I?,)-CAMPS was more effective against submaximal concentrations of (S,)CAMPS than againstsubmaximal concentrations of hCG. in order to examine this phenomenon more precisely, we performed several experiments in which two concentrations of (I?,)-CAMPS were tested against single concentrations of (S,)-CAMPS, 8-Br-cAMP, hCG, forskolin, or cholera toxin. We chose these compounds because we wanted to compare the effectiveness of (I?,)-CAMPS against (S,)-CAMPS and other exogenously supplied cAMP analogues, as well as against compounds that raise endogenous cAMP levels. We used 8-Br-CAMP and (S,)-CAMPS at 0.05 mM because, as shown above (cf. Fig. 5 ) , these agonists activate steroid biosynthesis to about the same extent ( i e . 30-50% of the maximal response) at this concentration.Likewise, the concentrations of hCG, cholera toxin, and forskolin used were those that also give 30-50% of the maximal activation of steroidogenesis. The results presentedin Fig. 8 show that (I?,)-CAMPS was equally effective in inhibiting the stimulatoryeffects of (Sp)CAMPS or 8-Br-CAMP. A t a concentration of 1.0 mM, (&,)CAMPS inhibitedsteroidogenesis by about 80%.On the other hand, the same concentrationof (I?,)-CAMPS was less effective in inhibiting steroidogenesis activated by hCG, cholera toxin, or forskolin. A t a concentration of 1.0 mM, (I?,)-CAMPS inhibited steroidogenesis activated by these agents only 2040%. Effects of (I?,)-CAMPS and (S,)-CAMPS on Intracellular CAMPLeuek-The results presentedabove clearly show that (&)-CAMPS is a more effective inhibitor of Steroidogenesis

(Rp).c~Mw irnM1

0 02 O I

0 0.2 1.0

a2

IO

I

0 02 1.0

0 02 1.0

FIG. 8. Effects of (R,)-CAMPSon steroidogenesis activated by cAMP analogues and compounds that increase endogenous CAMP.Cells werepreincubated with the indicated concentrations of (It,)-CAMPS for 30 min at 37 "C. Single concentrations of (S,)CAMPS, 8-Br-cAMP, hCG, cholera toxin, or forskolin were then added, and theincubation was continued for 4 h at 37 "C prior to the assay of progesterone. Each bar shows the mean 5 S.E. of three to five independent experiments. The numbers in parentheses above each set of bars indicate the amount of progesterone produced in the presence of the indicated concentrations of (I?,)-CAMPSexpressed as percentage of that produced in cells incubated in the absence of (It,)-CAMPS. At the concentrations used, (SJ-CAMPS, 8-Br-cAMP, cholera toxin, forskolin, or hCG activated steroidbiosynthesis to 3050% of maximal. The amount of progesterone produced by cells incubated with buffer only was about 1 ng/106 cells X 4 h.

when this process is stimulated with cAMP analogues than when it is stimulated with compounds that increase endogenous cAMP levels (such as hCG, cholera toxin, or forskolin). This difference does not appear t o be due to a differential inhibition of CAMP-dependent protein kinase activity when activated by cAMP or its analogues since, as shown above, (I?,)-CAMPS is equally effective in inhibiting this enzyme when it is activated with cAMP or (S,)-CAMPS (cf. Fig. 5A). On the other hand, the inhibition of cAMP phosphodiesterase activity by (I?,)-CAMPS (cf. Fig. 5B and Refs. 18 and 39-41) may explain the observed difference since endogenous cAMP is more sensitive to the action of cAMP phosphodiesterase than (S,)-CAMPS or 8-Br-CAMP (40-43). It appeared to us that when endogenous cAMP levels are increased by incubating the cells in the presence of cholera toxin, forskolin, or hCG, the inhibition of cAMP phosphodiesterase activity by (I?,)-CAMPS would lead to a further increase in the levels of endogenous cAMP and that this increase could partially overcome the inhibitory effects of (I?,)-CAMPS on the activity of the CAMP-dependent protein kinase. On the other hand, in the presence of (S,)-CAMPSor 8-Br-cAMP, the inhibition of cAMP phosphodiesteraseactivity by (R,)-CAMPS (or the added (S,)-CAMPS; see Fig. 5B) would have little effect on the levels of intracellular cyclic nucleotides since (S,)-CAMPS and 8-Br-CAMP bind to this enzyme with a lower affinity than cAMP and are hydrolyzed much more slowly than cAMP (39-42). This hypothesis was tested by measuring intracellular cAMP levels in cells preincubatedwith or without (I?,)CAMPS for 30 min at 37 "C and then stimulated with (S,)CAMPSor hCG for 45 min at 37 "C. The effects of IBMX (a widely used phosphodiesterase inhibitor) were also tested for comparison. These results are presented in Table I and show that at the concentrations tested, hCG increased the levels of intracellular cAMP1.7-fold. (R,)-CAMPS increasedthe levels of intracellular cAMP in control cells and produced a further increase in the cAMP content of cells incubated with hCG,


6099

kinases, on steroid biosynthesis activated by cAMP analogues or compounds that increase endogenous cAMP levels. Our results on the actions of @,)-CAMPS and (E,)-CAMPS on the isolated CAMP-dependent protein kinase type I and cAMP phosphodiesterase(s) of the MA-10 cells show that (S,)-CAMPS is (i) a full agonist for the activation of the CAMP-dependent protein kinase type I, but it is about eight times less potent than cAMP (Fig. 4); and (ii) an inhibitor of the cAMP phosphodiesterase(s) (Fig. 5B). On the other hand, (I?,)-CAMPS is (i) a weak agonist for the activation of the CAMP-dependentprotein kinase type I (Fig. 4); (ii) an antagonist for the activation of the CAMP-dependentprotein kinase type I by cAMP or @,)-CAMPS(Fig. 5A);and (iii) an inhibitor of the cAMP phosphodiesterase(s) (Fig. 5B). These results None 5.6 r+- 0.9 None are in agreement with previous results obtained with other hCG (0.8 ng/ml) 9.7 r+- 2.1 None mammalian CAMP-dependent protein kinases and cAMP (S,)-CAMPS(0.05 mM) 2.4 & 0.3 None phosphodiesterases. Thus, other investigators (13, 14) have shown that (S,)-CAMPS is a full agonist for the CAMPdependent protein kinases I and I1 from other mammalian tissues but that it is about 10 times less potent than CAMP. Likewise, (It,)-CAMPS was found to be completely inactive 8.6 & 2.0 None IBMX (1 mM) (14) or partially active (13) when tested for activation of other 26.4 +. 6.6 hCG (0.8 ng/ml) IBMX (1 mM) mammalian CAMP-dependent protein kinases. With regard to the cAMP phosphodiesterase(s), it has beenpreviously shown that both isomers of CAMPS bind to the bovine heart such that the cAMP content of cells incubated with (I?,)phosphodiesterase with a lower affinity than CAMP,but only CAMPSand hCG washigher than thatof cells incubated with the S, isomer seems to be hydrolyzed to a significant extent (It,)-CAMPS only or hCG only (Table I). These results are (40-42). similar to those obtained when cells were incubated with The results presented on the actions of these analogues on IBMX (Table I) and are consistent with the ability of (Rp)- intact MA-10 cells are consistent with their effects on the CAMPS to inhibit the isolated cAMP phosphodiesterasefs) isolated CAMP-dependent protein kinase and with the re(cf. Fig. 5B). Thus, it appears that when steroidogenesis is ported effects of these analogues on other mammalian cells. activated by increasing endogenous CAMP,the ability of (I?,)Thus, like other active cAMP analogues, (S,)-CAMPS is caCAMPS to block this process by inhibiting the CAMP-depable of stimulating steroidogenesis in intact cells, whereas pendent protein kinase is attenuated by its concomitant ability to inhibit cAMP phosphodiesterase activity. Further sup- (R,)-CAMPS is not (Fig. 6). Moreover, (I?,)-CAMPS is a port for this hypothesis was obtained in experiments in which competitive inhibitor of (S,)-CAMPS-stimulated steroidogenthe effects of (R,)-CAMPS on hCG-activated steroidogenesis esis in intact MA-10 cells (Fig. 7). Our results on the antagwere tested in cells incubated with or without IBMX. We onism of the actions of (S,)-CAMPS by (I?,)-CAMPSin intact found that IBMX reduced the ability of (R,)-CAMPS to cells are in good agreement with previous results obtained in rat hepatocytes (16, 45). In this cell type, the molar ratios of inhibit hCG-activated steroidogenesis (data not shown). The effects of (SJ-CAMPS on intracellular cAMP levels (I?,)-CAMPS to @,)-CAMPS needed toinhibit the (Sp)CAMPS-stimulated glucose production by 50 and 98% were are somewhat surprising. This isomer is more potent than (R,)-CAMPS in inhibiting the cAMP phosphodiesterase(s) calculated to be 3:l and 30:1, respectively. In our experiments, molar ratios of 4:1 and 20:l gave about 50 and 80% inhibition, (cf. Fig. 5B), yet it lowers intracellular cAMP instead of increasing it (Table I). Although we did not seek an expla- respectively (cf. Fig. 8). The absolute concentrations of diasnation for these results, it is worth noting that Corbin et al. tereoisomers used, however, are vastly different. This is pre(44) have recently shown that activation of the hepatocyte sumably due to differences in the cell types since hepatocytes CAMP-dependent protein kinase with cAMP analogues that appear to be specially sensitive to cAMP analogues (43). The ability of (R,)-CAMPS to competitively inhibit hCGare poor substrates for the phosphodiesterases leads to a reduction in the concentration of intracellular CAMP. Thus, activated and (S,)-CAMPS-activated steroidogenesis is conit is likely that a similar phenomenon occurs in the MA-10 sistent with the view that cAMP is a mediator of the actions cells when the CAMP-dependent protein kinase is activated of hCG. It is important to note, however, that (I?,)-CAMPSis with (S,)-CAMPS and that this effect attenuates theexpected a more effective inhibitor when steroidogenesis is activated inhibitory effect of @,)-CAMPS on the cAMP phosphodies- with cAMP analogues than when it is activated with hCG or terase(s). Theresults presented in TableI also show that the other compounds that activate adenylate cyclase (Fig. 7 and with previous observations made intracellular cAMP content of cells incubated with (I?,)- 8).These data are consistent CAMPS plus (S,)-CAMPS is higher than that of cells incu- in other cell types. Thus, in rat hepatocytes, (I?,)-CAMPS bated with (S,)-CAMPS only, but lower than that detected in inhibits (S,)-CAMPS-stimulated glucose production almost completely, whereas it inhibits glucagon-stimulated glucose cells incubated with (R,)-CAMPS only. production by, at most, 60% (16,45). Likewise, in dog thyroid DISCUSSION slices, (I?,)-CAMPSinhibits the actions of thyroid-stimulating The experiments presented herein were designed to inves- hormone by, at most, 50% (18). At first glance, the differential effectiveness of (R,)-CAMPS tigate further the role of cAMP as a mediator of the stimulatory effects of LH/CG on steroid biosynthesis in Leydig cells. against hCG and (SJ-CAMPS could be interpreted to mean To accomplish this goal, we tested theeffects of (R,)-CAMPS, that cAMP is not the only second messenger involved in the an antagonist of the activation of CAMP-dependent protein activation of steroidogenesis by hCG. The finding that (I?,)-

6100


CAMPS is equally effective against hCG, cholera toxin, or David Garbers for providing us with the cAMP antibody and Dr. forskolin, however, shows that this phenomenon is not re- Charles Rubin for providing us with the antibody to bovine brain RII. stricted to hCG. Thus, to explain these results, one must assume that (i) cAMP is also not the only second messenger REFERENCES involved in the activation of steroidogenesis by cholera toxin 1. Hunzicker-Dunn, M., and Birnhaumer, L. (1985) in Luteinizing Hormone Action and Receptors (Ascoli, M., ed) pp. 57-132, CRC Press, Inc., Boca or forskolin; or (ii) (I?,)-CAMPSis less effective against enRaton, FL dogenous cAMP than against exogenously supplied cAMP 2. Mendelson, C., Dufau, M., and Catt, K. (1975) J. Eiol. Chem. 260,8818M7R analogues. Our data (Table I) indicate that the second possiL., Homer, K.,A.,Hayashi, K., Tsuruhara, T., Conn, P. M., and bility is likely to be correct and that thedifferential effective- 3. Dufau,M. Catt, K. J. (1978) J. Eml. Chem. 253,3721-3729 4. Sullivan, M. H. F., and Cooke, B. A. (1986) Eiochem. J. 236.45-51 ness of (I?,)-CAMPS against endogenous cAMP and cAMP 5. Schumacher. M.. Schafer. G.. Lichtenbere. V.. and Hilz. H. (1979) FEES analogues is due to its dual effects on the CAMP-dependent 6. protein kinases and cAMP phosphodiesterases. When cells 7. are stimulatedwith (S,)-CAMPS, the dominant effect of (I?,)- 8. CAMPS is the antagonism of the CAMP-dependent protein 9. kinase; the (I?,)-CAMPS-induced inhibition of phosphodiesterase activity increases the levels of endogenous CAMP,but 10. it probably has no effect on the concentration of (S,)-CAMPS 11. since this compound is fairly resistant to hydrolysis (39-42). 12. On the other hand, when cells are stimulated by increasing 13. endogenous cAMP levels, the inhibition of cAMP phospho- 14. diesterase(s) by (I?,)-CAMPS results in a further increase in 15. endogenous cAMP (over that obtained with the stimulus 16. only). This would in turn lead to a decrease in the (I?,)17. CAMPS-induced inhibition of the CAMP-dependent protein 18. kinase because of an increase in the agonist to antagonist 19. ratio. Thus,the decreased effectiveness of (I?,)-CAMPS 20. 21. against hCG cannot be interpreted to mean that cAMP is not 22. the only mediator of the activation of steroidogenesis by hCG. 23. Taken together, the data presented here show that with 24. some precautions, (&,)-CAMPS can be safely utilized to study 25. the role of cAMP as a second messenger in hormone action. 26. In fact, our data show that (I?,)-CAMPSis an effective inhib- 21. itor of hCG-activated steroidogenesis at low concentrations 28. of hCG, where changes in intracellular cAMP are not easily 29. correlated with increased steroid biosynthesis (cf.Figs. 1 and 30. 7). Thus, an important conclusion derived from our data is 31. that cAMP is an obligatory mediator of the activation of 32. steroidogenesis by all concentrations of hCG. The data pre- 33. sented here do not, of course, rule out the possibility of the 34. involvement of other signaling systems in the activation of 35. Strickland, S., and Loeh, J. N. (1981) Proc. Noti. Acad. Sei. U.S. A. 78, 1366-1370 steroidogenesis by LH/CG. From the datapresented, however, 36. Rannels, S. R., Beasley, A., and Corbin, J. D. (1983) Methods Enzymol. 99, 55-62 it appears to us that if other signaling systems are activated 37. Strada, S.. and Thompson, W. J. (1978) Adu. Cyclic Nucleotide Res. 9,265by hCG, (i) they would participate in the activation of ste283. roidogenesis by acting together with, rather than independ- 38. Ascoll, M. (1985) in The Receptors (Conn, P. M., ed) Vol. 2, pp. 368-400, Academic Press, New York ently of, CAMP;and (ii) thiscombination of second messenger 39. Jarvest, R. L., Lowe, G., Baraniak, J., and Stec, W. J. (1982) Eiochem. J. 203,461-470 systems would be involvedin the activation of steroidogenesis 40. Van Haastert P. J. M. Di'k aaf P. A. M., Konijn, T. M., Abbad, E. G., by all concentrations of hCG, rather than each signaling Petridis, G.: and Jastkfd f ( 1 6 8 3 ) Eur. J. Eiochem. 131,659-666 Eckstein, F.,Simonson, L. P., and Bar, H.-P. (1974) Emchemistry 13, 41. system being involved at different concentrations of hCG. 3806-3810 42. B u r r s , P. M. J., Eckstein, F., Hunneman, D. H., Baraniak, J., Kinas, R. Although additional experiments are needed to resolve these K., and Stec, W. J. (1979) J. Biol. Chem. 254,9959-9961 issues, the results presented in this paper provide the neces- 43. Beebe,,Leslak, S. J., Redmon, J. B., Blackmore, P. F., and Corbin, J. D. (1985) J. Ewl. Chem. 260,15781-15788 sary foundation for further work in this area. Acknowledgments-We thank Florence Kaczorowski and Jeri Euffa for expert technical assistance. We also wish to thank Dr.

44. Corbln, J. D.. Beebe. S. J., and Blackmore, P.F.(1985) J.Eiol. Chem. 2 6 0 , 8731-8735. 45. Rothermel, J. D., Jastorff, B., and Botelho, L. H.P. (1984) J . Bid. Chern. 269,8151-8155