0013-7227/92/1303-1122$03.00/0

Endocrinology Copyright 0 1992 by The Endocrine Society

Vol. 130, No. 3

Printed

in U.S.A.

Direct Effect of Arachidonic Acid on Protein Kinase C and LH-Stimulated Steroidogenesis in Rat Leydig Cells; Evidence for Tonic Inhibitory Control of Steroidogenesis by Protein Kinase C* M. PILAR ANTHONY

LOPEZ-RUIZ, MICHAEL P. WEST, AND BRIAN

S. K. CHOI, A. COOKE

Department of Biochemistry Royal Free Hospital Street, London NW3 2PF, United Kingdom

MATTHEW

School of Medicine,

The role of arachidonic acid in the regulation of ABSTRACT. steroidogenesis in rat Leydig cells was studied. A dose- and timedependent biphasic effect on maximal and submaximal LH- and dibutyryl-CAMP-stimulated testosterone production was found. The locus of the inhibition, which occurred during 3 h incubation, was prior to the side chain cleavage of cholesterol and after CAMP production. The same inhibitory effect was found with the protein kinase C (PKC) activators, phorbol-12-myristate, 13-acetate (PMA) and oleic acid, also with no change in LHstimulated CAMP production. Arachidonic acid, PMA, and diolein, all stimulated PKC activity in a dose-dependent fashion in partially purified Leydig cell homogenates. When the cells were incubated for 5 h, arachidonic acid

I

T IS well established that steroidogenesis in Leydig cells is regulated by LH (lutropin), via the second messenger CAMP. It has also been shown that other second messenger systems may be involved, including arachidonic acid and its metabolites (l), calcium (l), and efflux of chloride ions (2). The release of arachidonic acid to the cytosol can occur by a hormone-mediated process, through the activation of phospholipasa A2 (PLA,), phospholipase D, and/or phospholipase C followed by hydrolysis of diacylglycerol by diacylglycerol lipase. PLAz and phospholipase C can be coupled to a common membrane receptor by distinct GTP-binding proteins and may be activated by the same membrane active hormone (3, 4). Arachidonic acid can be further metabolized via cyclooxygenase and lipoxygenase pathways to prostaglandins and leukotrienes, respectively (5). Our previous results have shown that LH causes a rapid release of arachidonic acid from Leydig cells, probably Received August 19,199l. Address all correspondence and requests for reprints to: Dr. Brian Cooke, Department of Biochemistry, Royal Free Hospital School of Medicine, Rowland Hill Street, London NW3 2PF, United Kingdom. l This work was supported by the Spanish Ministery of Education and Science.

P. ROSE,

University

of

London,

Rowland

Hill

potentiated LH- and dibutyryl-CAMP-stimulated testosterone production. Similarly, incubation with PMA for 5 h, potentiated subsequent basal and dibutyryl-CAMP-stimulated testosterone production. PKC was down-regulated over 5 h (but not during 3 h) by pretreating Leydig cells with PMA or arachidonic acid in the presence of LH. Lipoxygenaee and cyclooxygenase inhibitors did not alter the stimulatory effects of arachidonic acid. We conclude that the short-term inhibitory effect of arachidonic acid (and PMA) is via activation of PKC, but when protein kinase C (PKC) is down-regulated by these ligands, steroidogenesis is enhanced. These results suggest that steroidogenesis is normally under tonic inhibitory control by PKC. (Endocrindogy 130: 1122-1130, 1992)

via activation of PLAz (6) and that arachidonic acid metabolites are involved in LH-induced steroidogenesis (7, 8). Furthermore, arachidonic acid by itself has been reported to have an intermediary role in GnRH-induced testosterone secretion (9, 10). Activation of protein kinase C (PKC) using the tumorpromoting phorbol ester, phorbol-12-myristate-13-acetate (PMA), has been reported to regulate, both positively and negatively, steroidogenesis in Leydig cells (ll13). In addition to diacylglycerol, arachidonic acid has been shown to be a physiological regulator of PKC in various tissues, via activation of the PKC y-isotype (14, 15). In rat Leydig cells the y-isotype as well as the (Yand P-isotypes are present (16) and, thus, it is theoretically possible that arachidonic acid also regulates PKC in Leydig cells. In the light of these observations, the present study was carried out to determine the direct effects of arachidonic acid on LH-induced steroidogenesis in rat Leydig cells and to determine the involvement of PKC in mediating these effects. Materials

and Methods

Materials

Ovine LH (batch oLH 26, potency 2.3 IU/mg) was obtained from NIAMDD

(Bethesda, MD). [T-~‘P]ATP was purchased

1122

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ROLE OF ARACHIDONIC from Amersham International (U.K.). Dulbecco’s modified Eagle’s minimum medium was obtained from GIBCO Ltd. (Middlesex, U.K.). Collagenase (type I) was purchased from Worthington Biochemical (Freehold, NJ). Dibutyryl cyclic AMP [(Bu)~cAMP], arachidonic acid (free acid, sealed ampoule), oleic acid, elaidic acid, 1,2 diolein, 22R hydroxy-cholesterol (PPR-OH-cholesterol), BSA (essentially fatty acid free), phorbol 12-myristate, 13-acetate (PMA), Lcy-phosphatidyl-serine (PS), histone type III S, ATP, Percoll, trypsin inhibitor (1% sterile filtered solution), and nordihydroguaiaretic acid (NDGA) were purchased from Sigma Chemical Co. (Dorset, U.K.). BW 755~ [3-amino-l-(3-[trifluoromethyl]phenyl)-2 pyrazoline hydrochloride] was a gift from Wellcome Research Laboratories (Beckenham, Kent, U.K.). All other chemicals used were of Analar grade. Methods Cell isolation and purification. Leydig cells were obtained from 200-300 g Sprague-Dawley rats. The testes were decapsulated and subjected to longitudinal shaking (65 strokes/min) with collagenase (0.5 mg/ml) and trypsin inhibitor (20 rl/ml) for 40 min at 37 C. The cells were filtered through 75 pm nylon gauze to remove fragments of seminiferous tubules, and subjected to centrifugal elutriation followed by Percoll density gradient (O90% vol/vol) centrifugation (17). Leydig cell purity was routinely more than 95% as established by 3&hydroxysteroid dehydrogenase cytochemistry (18). Viability of the cells was determined by diaphorase histochemistry (18) and was not affected by any of the treatments (the viability was >90%). Cell inubation, testosterone, and CAMP determinations For measurement of testosterone and CAMP production, purified Leydig cells were resuspended in Dulbecco’s modified Eagle’s minimum medium containing 10 mM Hepes (pH 7.5) and 1% BSA-fatty acid free and plated into 24-well Costar culture plates at a density of 100,000 cells/well in a final volume of 1 ml. The cells were preincubated for 2 h at 34 C in an air incubator and then incubated for different time periods, in the presence and absence of different compounds. LH and dibutyryl cyclic AMP were dissolved in incubation medium and added as 10 ~1 concentrates to give the required final concentration. Phorbol esters and PBR-OH-cholesterol were dissolved in dimethylsulphoxide at a concentration of 1 mM. The stock solutions were diluted with incubation medium before addition to the wells. Arachidonic acid and other fatty acids were dissolved in ethanol at a concentration of 40 mM. After further dilutions in incubation medium they were added directly to the cells. All the manipulations were done under a nitrogen atmosphere. A new ampoule of arachidonic acid was used in each experiment. The final concentration of dimethylsulphoxide and ethanol were always less than 0.25% and 0.5% (vol/vol), respectively, and did not alter basal or stimulated steroid release. The reactions were stopped with perchloric acid (final concentration 0.5 mol/l) and stored at -20 C. The samples were thawed and neutralized with tripotassium phosphate (final concentration 0.23 mol/l) prior to RIA of testosterone (19) and CAMP (20, 21).

ACID IN STEROIDOGENESIS Measurement of PKC

activity;

1123 partial purification of PKC

Calcium/phospholipid-dependent protein kinase activity was determined in a partially purified homogenate of rat testis Leydig cells, using modifications of previously published methods (22,23). All the procedures were performed at 4 C. Twenty million purified Leydig cells were resuspended in 0.5 ml of cold buffer A, consisting of 20 mM Tris-HCl (pH 7.5), 2.5 mM magnesium chloride, 2.5 mM EGTA, 0.1 mM phenylmethylsulfonyfluoride, and 50 mM /3-mercaptoethanol. The cells were left to swell in this hypotonic solution for half an hour prior to sonication for 30 set on ice. Lysis was checked using trypan blue exclusion. After sonication, 125 ~1 of 1.25 M sucrose was added to restore tonicity and the nuclei separated by centrifugation 1000 X g at 4 C. The supernatant protein was solubilized by incubation (1 h, 4 C) in the presence of Triton X-100 (l%), before chromatography on 1 ml columns of DE-52 cellulose. The columns were equilibrated with buffer B, consisting of 20 mM Tris-HCl (pH 7.5), 0.5 mM EGTA, and 2 mM p-mercaptoethanol. After the samples were loaded, the columns were washed with 12 ml of buffer B. The enzyme was eluted from the columns with 0.5 ml aliquot of buffer B containing 0.2 M NaCI. Assay of PKC PKC was assayed by its ability to catalyse incorporation of radiolabel from [T-~‘P]ATP into lysine-rich histone in the presence of phosphatidylserine. Reaction buffer. PKC reaction mixture for 20 assays was prepared as follows: 125 ~1 100 mM MgClz, 50 ~1 30 mM CaCl,, 50 ~110 mM EGTA, 62 ~1100 mM HEPES (pH 7.4), 50 ~125 mg/ ml histone, and 250 ~1 HzO. Mixed micelles. Immediately prior to the assay, 15 ~1 of PS dissolved in chloroform/methanol (95:5) (10 mg/ml) was sonicated with 0.5 ml 20 mM Hepes for 3 X 15 set on ice. This sonicated mixture also contained other PKC activators; diolein (5 pg/ml, final concentration), PMA (10m7M, final concentration) or arachidonic acid at different concentrations according to the experiment. Reaction mixture. The reaction buffer was split into two parts (250 ~1 each) one receiving 50 ~1 of mixed micelles and the other 50 ~1 of 20 mM HEPES. The samples were added in 10 ~1 aliquots (2-5 pg protein) to 30 ~1 of the reaction mixture. The reaction was started by adding 5 ~1 of [Y-~*P]ATP (200 cpm/pmol) and was carried out for 5 min at 35 C. Aliquots (35 ~1) were then spotted onto 2.5 cm squares of phosphocellulose paper (Whatman P81). The papers were washed four times in a large volume of cold acetic acid (30% vol/vol the two first washes and 15% vol/vol the last two, 5 min each wash). The papers were then washed once for 1 min with methanol and then diethylether and dried. The radioactivity on the papers was measured by liquid scintillation spectrometry. The difference of phosphorylation measured in the presence and absence of PS and/or activator (mixed micelles) is regarded as PKC activity and is expressed as pmol 32P incorporated per mg protein per min. Protein assays were carried out using the BioRad protein assay, according to the Bio-Rad manual 1990 (BioRad Laboratories, Richmond, CA). All data are expressed as the mean + SD from two similar experiments or mean f SEM

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ROLE OF ARACHIDONIC

1124

ACID IN STEROIDOGENESIS

Endo. 1992 Vol13O.No3

from three similar experiments, all of them performed in triplicate. Student’s t test was used for statistical analysis.

Results Arachidonic acid had no significant effect on basal levels of testosterone during 3 h of incubation (Table 1). After 5 h, a small but statistically significant increase was found. This increase was in the range of 3-4 ng testosterone/lO’j cells and did not increase further with concentrations higher than 10 pM. When the Leydig cells were incubated with increasing concentrations of arachidonic acid (l-200 PM), a biphasic effect was found in the presence of submaximum and maximum concentrations of LH, at two different time periods (Fig. 1, A and B). After 3 h of incubation, arachidonic acid gave a dose-dependent inhibition of LHstimulated testosterone production with both LH concentrations (0.01 and 10 rig/ml) (Fig. 1A). The EDh0 for arachidonic acid inhibition was approximately 100 PM with 10 rig/ml LH. With 0.01 rig/ml LH, testosterone production was inhibited by 50% with approximately 10 pM arachidonic acid and no further inhibition was obtained with higher concentration of arachidonic acid (Fig. 1A). We also have found this inhibitory effect in shorter periods of incubation (1 and 2 h) (data not shown). In contrast, when the time of incubation was longer (5 h), a dose-dependent stimulation of LH-stimulated testosterone production was found, which reached a maximum with approximately 25-50 pM arachidonic acid and then declined to control levels with 100 pM arachidonic acid or higher. The percentage of stimulation was higher for the lower dose of LH. The average stimulation with 25 pM arachidonic acid was approximately 170% and 40% for submaximal (0.01 rig/ml) and maximal (10 ng/ ml) concentrations of LH, respectively (Fig. 1B). Similar effects were found when (Bu)+AMP was TABLE

1.

Effect of arachidonic

acid on basal levels of testosterone

production Arachidonic acid (PM) 0 10 25 50

100 200

Testosterone (ng/106 cells) 3h 5.38 4.60 4.52 3.80 4.71 6.01

f + f 3~ + f

5h 0.65 0.31 0.51 0.62 1.32 0.90

10.0 14.5 14.8 9.7 14.1 15.3

f 0.9 + 0.9” 2 1.2”

+ 1.0 f f

1.5” 1.4”

Leydig cells (10’ cells/well/ml) were incubated for 3 and 5 h in the absence and presence of increasing concentrations of arachidonic acid. The testosterone produced was measured in the incubation media as described in MaterMk and Methods. The values are mean f SEM for three different experiments each performed in triplicate. “P c 0.05 (by Student’s t test) us. testosterone production in the absence of arachidonic acid.

ok/

’ 1

0 200

I 10

I

I

100

1000

r

150

, apppy\bd

B

100

+P~

50

-6

,i ok/ 0

’ 1

Arachidonic

I

I

I

10

100

1000

acid

concentration

(PM)

FIG. 1. Effect of arachidonic acid on LH-stimulated testosterone production over 3 h (A) or 5 h (B) of incubation. The cells (lo6 cells/well/ ml) were incubated with increasing concentrations of arachidonic acid in the presence of submaximal 0.01 rig/ml (0) or maximal 10 rig/ml (0) stimulatory concentrations of LH. Testosterone accumulation was measured in the incubation media, as described in Materials and Methods. All data points are the mean + SEM of three experiments, each performed in triplicate. When SEM values are not shown, these are smaller than the respective symbols.

added in place of LH. After 3 h of incubation arachidonic acid concentrations higher than 25 pM had an inhibitory effect on (Bu)~cAMP (1 mM)-stimulated testosterone production (15% inhibition with 50 pM arachidonic acid and 35% inhibition with 200 PM arachidonic acid) (Fig. 2A). Testosterone produced by submaximal doses of (Bu,JcAMP (0.05 mM) was inhibited by 50% with approximately 10 PM arachidonic acid (Fig. 2A). In contrast, after 5 h, arachidonic acid was found to potentiate (Bu)BcAMP-induced testosterone production. The arachidonic acid concentration which produced the maximum effect was 25 pM, and with this arachidonic acid concentration, the stimulatory effect was approximately 90% and 50% for submaximal (0.05 mM) and maximal (1 mM) doses of (Bu)~cAMP, respectively (Fig. 2B). Further experiments were carried out to determine the locus of action of arachidonic acid. The results in Table

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ROLE

OF ARACHIDONIC

ACID

TABLE

A 100

IN STEROIDOGENESIS 2.

Effect of arachidonic

50

200

100

5h 111 f 9 100 f 9 119f8

85 + 16

81 + 20 75 f 15 68 f 14 81 + 20

25 50

i

10

3h

0 1 10

c3

t- -~.-.+o-..~ ok-

cyclic AMP

Cyclic AMP (pmol/106 cells)

Arachidonic acid (PM) Y

1

acid on LH-stimulated

production

--4+-l

0

1125

127 -c 5 123 f 17

100

104 f 35

llOf9

200

107 f 32

122 f

12

Leydig cells (10’ cells/well/ml) were incubated (3 and 5 h) with LH (10 rig/ml) and increasing concentrations of arachidonic acid. The levels of CAMP were determined as described in Materials and Methods. The values are expressed as mean f SEM of three different experiments, each performed in triplicate. There were no significant differences. The levels of CAMP produced in 3 and 5 h, by nontreated cells were 1.2 f 0.9 and 4.5 + 1.9 pmol/106 cells, respectively.

1000

r

TABLE 3. Effect of arachidonic acid on the conversion of 22R-hydroxycholesterol to testosterone by Leydig cells

Testosterone (ng/lO’ cells) Arachidonic acid (NM)

ov0 Arachidonic

22R-OH-cholesterol 3h 0.25 PM

1

10 acid

100 concentration

0

1000

1 (PM)

2. Effect of arachidonic acid on (BuhcAMP-stimulated testosterone production for 3 h (A) or 5 h (B) of incubation. Leydig cells (10’ cells/well/ml) were incubated with increasing concentrations of arachidonic acid in the presence of submaximal 0.05 mM (0) or maximal 1 mM (0) stimulatory concentrations of (BuhcAMP. Testosterone production was measured in the incubation media, as described in Materials and Methods. All data points are the mean f SEM of three experiments, each performed in triplicate. When SEM values are not shown, these are smaller than the respective symbols. ETC.

2 show that, under our conditions (i.e. in the presence of 1% albumin), the levels of CAMP did not change at any of the concentrations of arachidonic acid studied. The testosterone formed in the presence of 22R-OH-cholesterol (0.25 and 2.5 FM) was unaffected by all concentrations of arachidonic acid after both 3 and 5 h of incubation (Table 3). These results indicate that arachidonic acid has its action after the formation of CAMP and before cholesterol side chain cleavage. When we studied the effect of the phorbol ester, PMA, on steroidogenesis, a dose-dependent inhibition of LHstimulated testosterone production was also found, both with maximal (10 rig/ml) and submaximal (0.01 rig/ml) doses of LH in 3 h (Fig. 3). However, when the incubations were continued for 5 h, the levels of testosterone were back to controls except at the highest PMA concentration used (3 x 10m6 M) (Fig. 4). By contrast, phorbol

3

10 25 50

100 200

69 f 66 +54 f 62 f 68+4 68-18 63 f 67 f

5h 2.5 /.tM

0.25 PM

8 9

478 f 82 457 f 37

1

481 f 88

102 f 28 97 + 15 86 + 13

2

496 512 457 470 457

125 f 13 99 f 18 97 f 15 105 f 10

7 8

f f * f f

43 53 41 53 59

91+ 12

2.5 PM 844+10 806 f 785+99 807 + 722 f 741 + 811 f 764 5

59 60 99 65 27 59

The cells (10’ cells/well/ml) were incubated (3 and 5 h) with submaximal (0.25 PM) and maximal (2.5 PM) 22R-OH-cholesterol concentrations in the presence of increasing concentrations of arachidonic acid. The amounts of testosterone were measured in the incubation media as described in Materials and Methods and are expressed as the mean + SD of two different experiments, each performed in triplicate. There were no significant differences.

12,13didecanoate, which does not activate protein kinase C, did not alter steroid production induced by LH at any of the concentrations used (data not shown). No changes in CAMP levels were found (Table 4). The effects of incubating the cells with PMA (400 nM) for 5 h on the subsequent response during 2 h to (Bu)*cAMP was also investigated. It was found (Table 5) that up to a 2fold potentiation of basal and stimulated steroidogenesis occurred. In parallel incubations with phorbol-12,13-d& decanoate no potentiation was found. Since arachidonic acid and PMA have similar inhibitory effects on steroidogenesis during 3 h of incubation, we next investigated if arachidonic acid could directly stimulate protein kinase C activity as has been shown in other systems (14-15). Figure 5 shows the effect of in-

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ROLE OF ARACHIDONIC

‘/

PMA

concentration,

nM

FIG. 3. Dose-response of PMA on LH-stimulated testosterone production in Leydig cells. The cells were incubated for 3 h with LH 0.01 ng/ ml (0) and 10 rig/ml (0) in the presence and absence of increasing concentrations of PMA. Testosterone was determined in the incubation media as described in Materida and Methods. Each point represent the mean f SD of two different experiments, each performed in triplicate. Where SD values are not shown, these are smaller than the respective symbols.

creasing concentrations of arachidonic acid on Ca/phospholipid dependent protein kinase. Arachidonic acid directly stimulated PKC activity in a dose-dependent fashion. The maximum stimulatory effect was 200% above basal and was similar to that obtained with diolein (5 rg/ml) or PMA (lo-’ M). The basal activity was measured in the presence of PS. In further experiments, Leydig cells were incubated over 3 and 5 h with arachidonic acid (25 PM) or PMA (lo-’ M) in the presence of 10 rig/ml LH or media alone, before measurement of PKC activity. No significant loss of PKC activity was found after 3 h incubation but, after 5 h PKC was down-regulated by both PMA (84% loss) and arachidonic acid (70% loss) (Fig. 6). The specificity of the arachidonic acid action was also studied by using two other fatty acids, oleic acid (l&l cis) which has been shown to stimulate PKC activity and elaidic acid (18:l truns) which has no effect on PKC activity. During 3 h of incubation, oleic acid inhibited LH-stimulated testosterone production, whereas elaidic acid had no effect. The levels of LH-stimulated testosterone production during 5 h did not change with either

Endo. 1992 Voll30. No 3

ACID IN STEROIDOGENESIS

10

100

PMA

1000

concentration

(nM)

4. Comparative effects of PMA on LH-stimulated testosterone production in Leydig cells incubated for 3 and 5 h. Leydig cells (l@ cells/well/ml) were incubated with LH (0.01 rig/ml) in the presence and absence of increasing concentrations of PMA. The testosterone production was measured in the incubation media after 3 h (0) or 5 h (0) of incubation. Data represent the mean f SD of two different experiments, each performed in triplicates. Where SD values are not shown, these are smaller than the respective symbols.

FIG.

TABLE

4.

Lack of effect of PMA on LH-stimulated

PMA (M)

Cyclic AMP (pmol/106 cells) 3h

0 lo+ 10-I lo4 1o-6

CAMP production

102 101 104 107 108

f f f f f

5h 10 14 9 8 10

151 121 140 138 147

f f f f f

33 14 22 17 18

Leydig cells (10’ cells/well/ml) were incubated with LH (10 rig/ml) in the absence and presence of increasing concentrations of PMA. The CAMP produced was measured in the incubation media as described in Materiuls and Methods. Data represent mean f SD of two different experiments, each performed in triplicate. There were no significant differences.

oleic or elaidic acid (Table 6). These fatty acid had no effect on basal levels of testosterone (data not shown). To investigate whether the stimulatory effect of arachidonic acid on steroidogenesis is brought about by arachidonic acid itself or its metabolites, we used inhibitors of the various pathways of arachidonic acid metabolism. In agreement with our previous studies (7), both

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ROLE OF ARACHIDONIC

ACID IN STEROIDOGENESIS

1127

TABLE 5. The effects of incubating Leydig cells for 5 h on subsequent (BuhcAMP-stimulated steroidogenesis

(Bu),cAMP

Testosterone (ng/lO’ cells/2 h)

(mM)

Control

0 0.001

20.0 f 20.7 f 22.2 f 24.2 f 64.2 f

0.005 0.01

0.05 0.1 1.0

PMA

2.2 1.4 0.4 1.2 5.8

108.1 + 12.0 176.8 f 20.5

31.6 2 33.8 + 37.7 + 40.2 + 116.8 +

PDD

0.9 2.3 3.1 2.6 11.5

21.7 + 0.4 22.1 f 2.9 22.5 -t 0.8 25.0 f 1.4 66.9 f 6.0

192.9 + 21.1 286.0 + 17.0

109.6 f 6.7 166.7 + 12.5

LER

Leydig cells were preincubated for 5 h with either the active phorbol ester PMA (400 nM) or the inactive ester PDD (400 nM) prior to stimulation with different concentrations of (Bu)gAMP for 2 h. The results are typical of one of two similar experiments and are the means f SD for triplicate incubations.

25 0

3h

5h

FIG. 6. Arachidonic acid- and PMA-induced protein kinase C depletion in Leydig cells. Cells (20 million) were treated for 3 and 5 h with 25 pM arachidonic acid (4 or lo-’ M PMA (8) in the presence of LH (10 rig/ml) or with media alone (Cl). At the end of this treatment, cells were washed twice and processed to determine PKC activity, as described in Materials and Methods. The values are the mean f SD of two different experiment. PKC activity was determined in triplicate. TABLE 6. Specificity of fatty acid-effects on LH-stimulated one production

Fatty acid (PM)

testoster-

Testosterone (ng/106 cells) Arachidonic acid

Oleic

Elaidic

3 h of incubation 0

25 50 100 I

0

I I

1

50

100

AA concentration

(PM)

hG. 5. Dose-response of arachidonic acid-induced activation of PKC. Crude soluble enzyme preparation from 20 million of untreated Leydig cells, was partially purified on DE-52 columns and samples were assayed as described in Materials and Methods, in the presence of increasing concentrations of arachidonic acid (0) or 5 pg/ml diolein (Cl) or lo-’ M PMA (m). Values represent the mean f SEM of three different experiments PKC activity was measured in triplicate.

NDGA (10 PM) and BW 755~ (100 PM) inhibited LHstimulated testosterone production. However, when exogenous arachidonic acid was present, NDGA and BW 755~ did not modify the arachidonic acid stimulation, because a similar increase above LH stimulated testosterone was observed in the presence of either inhibitor (Fig. 7).

90 f 3.3 73 f 2.3" 60 f 4.4b 45 -c5.1b

96 + 2.0

98 + 4.0

100 + 2.4

105 f 8.0

79 f 4.5" 62 f 7.0"

96 f 2.8 93 + 4.1

5 h of incubation 0

171 f 12 175 + 6 162 It 8 132 f 24 238 f 8" 158 f 10 155 f 57 100 182 f 12 151 f 9 169 f 14 Leydig cells (10’ cells/well/ml) were incubated with LH (10 rig/ml) in the absence or presence of arachidonic acid, oleic or elaidic acid for 3 and 5 h. The levels of testosterone were determined as described in Materials and Methods. The values are the mean f SD of two different experiments, each performed in triplicate. a P < 0.05. b P c 0.01 (by Student’s t test) us. levels of testosterone in the presence of LH alone.

25 50

169 f 13 205 3~ 10”

Discussion Arachidonic acid has previously been shown to modulate hormone secretion in a variety of tissues (24-27) and its role as a potential second messenger has recently been reviewed (28). Its role in the control of steroidogenesis, however, is unclear. In this study we have concentrated

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ROLE

OF ARACHIDONIC

NDGA lOjAM

BW

755c

IOO~M

FIG. 7. Bat Leydig cells (lo6 cells/well/ml) were incubated for 5 h with LH (10 rig/ml) in the absence (m) and presence (0) of arachidonic acid (25 PM) and with and without NDGA (10 FM) or BW 755~ (100 PM). Testosterone production was determined in the incubation media as described in Materials and Methods. Values are mean zk SD of two different experiments, each performed in triplicate.

on its direct effects on steroidogenesis and compared them with the actions of other stimulators of PKC. We have demonstrated that arachidonic acid and phorbol esters have two different effects on the regulation of steroidogenesis in rat Leydig cells; an initial inhibition followed by a stimulation of LH/(B&cAMP-regulated testosterone production. Phorbol esters (e.g. PMA) have previously been reported to have a stimulatory (29, 30) or an inhibitory (10-13) effect on steroidogenesis in various steroidogenic cell types. Similar results have been found for the effects of arachidonic acid (9, 10, 27, 31). The present studies differ from those in which an inhibition was observed because PMA and arachidonic acid had no effect on LHstimulated CAMP production. However, it should be noted that this lack of effect on CAMP production, is due to the high concentration (1%) of albumin used. In the presence of lower concentrations (O.l%), inhibition of LH-stimulated CAMP production by arachidonic acid (our unpublished observations) and desensitization of LH-mediated CAMP production by PMA (32) does occur. Arachidonic acid was equipotent with phorbol ester and diacylglycerol in the stimulation of PKC activity in Leydig cell homogenate preparations, which suggests a role for PKC activation in mediating the effects of arachidonic acid on LH-stimulated testosterone production. It has also been reported that in Leydig cells the GnRHinduced testosterone formation is mediated by arachi-

ACID

IN STEROIDOGENESIS

Endo. 1992 Voll30. No 3

donic acid through PKC activation (10). In contrast, Johnson and Tilly (32) have shown that the inhibitory effect of arachidonic acid on granulosa cell function was not mediated by PKC. It is important to note, however, that in granulosa cells the P-isoenzyme of PKC is the predominant isotype, with no detectable (Y, or y (31). However, Leydig cells express the three isotypes cr, p, and y at similar levels (16) and y-isotype is the most sensitive to activation by arachidonic acid (28, 33,34). After 5 h of incubation, the inhibitory effect on LHstimulated testosterone production by PMA was lost. It is well known that prolonged incubation with PMA causes an increase in PKC degradation without a concomitant increase in synthesis (35, 36). This was also shown to occur in Leydig cells because a dramatic decrease in PKC activity occurred following prolonged treatment (5 h) of Leydig cells with PMA. This was consistent with data reported for large cells from the corpus luteum (37), pituitary cells (38), granulosa cells (31) and Leydig cells (39). Arachidonic acid also caused enzyme depletion in a similar manner to PMA. Oleic acid, a fatty acid which has been shown to stimulate PKC (14, 15) had a similar inhibitory effect on LHstimulated testosterone production. Elaidic acid which does not stimulate PKC (15) had no effect on steroidogenesis produced by LH. All of these data further indicate the intermediary role for PKC in the short-term action of arachidonic acid. It has been postulated, that PKC activates the Ca2+transport adenosine triphosphatase and the Na+/Ca2+ exchange protein, both of which remove Ca2+ from the cytosol (40, 41). In Leydig cells intracellular calcium influences testosterone production in response to LH by affecting the transport of cholesterol to the mitochondria (42, 43). Therefore, we can postulate that the effect of arachidonic acid through PKC may be exerted by modulating Ca2+ and hence the transport of cholesterol. PKC could also phosphorylate the sterol carrier protein 2, which is involved in cholesterol transport, either to activate or deactivate this protein (44). In long-term incubations (5 h), when PKC was downregulated, arachidonic acid was no longer inhibitory, but instead showed a potentiation of LH and (Bu)~cAMPstimulated testosterone production. This effect was specific for arachidonic acid since other fatty acids did not have such an effect. In agreement with previous findings (7), both NDGA and BW 755C, two arachidonic acid metabolism inhibitors, inhibited LH-stimulated testosterone production. However, when arachidonic acid (25 pM) was present in the incubation, the same stimulatory effect was found with and without the inhibitors. These results indicate that the conversion of arachidonic acid to either leukotrienes or prostaglandins is not required for this effect on Leydig cells.

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ROLE OF ARACHIDONIC In conclusion, the results obtained in this study have demonstrated the regulatory role that PKC exerts on steroidogenesis. They suggest that steroidogenesis is under a continuous inhibitory control by PKC which may be further modulated by trophic hormones, and/or paracine and autocrine factors via diacylglycerol or arachidonic acid. The observed long-term stimulatory effects of arachidonic acid and PMA on basal and (Bu)*cAMPstimulated steroidogensis can be explained, therefore, by removal (via down-regulation of PKC) of the tonic inhibitory effect of PKC. This may also explain the conflicting reports for the effects of arachidonic acid and phorbol esters, because the effects obtained will depend on the incubation times and doses of ligands used. The activation of PKC by arachidonic acid presumably will also depend on the presence of the y-isotype of PKC, although the other isotypes can be activated but higher concentrations of arachidonic acid are required (28). From the studies carried out on the release of arachidonic acid it is apparent that this is very transitory. It is probable, therefore, that the main action of arachidonic acid itself is to exert a negative effect on steroidogenesis via PKC activation, whereas its metabolites formed via the lipoxygenase pathway, exert a stimulatory action. Acknowledgments We are grateful to Dr. E. Podesta for advice on the incubation conditions required for studying the effects of arachidonic acid and to Dr. R. G. Wickremasinghe for the technical advice about the protein kinase C assay.

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