Actions of Choleragen and Gonadotropin in Isolated Leydig Cells

THE JOURNAL OF Buxcxxx~ CHEMISTRY Vol. 253, No. 10, Issue of May 25, pp. 3’721-3729, 1978

Printed m U.S A.

Actions of Choleragen and Gonadotropin in Isolated Leydig Cells FUNCTIONAL COMPARTMENTALIZATION OF THE HORMONE-ACTIVATED CYCLIC AMP RESPONSE*

(Received for publication, October 17, 1977)

MARIA L. DUFAU, KATHLEEN A. HORNER, KEIKO HAYASHI, TSUNEO TSURUHARA, P. MICHAEL CONN, AND KEVIN J. CATT

From the Endocrinology and Reproduction Research Branch, National Institute for Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20014

The role of cyclic AMP in the stimulation of target cell responses by choleragen and gonadotropic hormone was analyzed during studies on ligand binding and activation of steroidogenesis in purified testicular Leydig cells. The dose- response curve for stimulation of intracellular and extracellular cyclic AMP by choleragen was biphasic, with an initial response over the concentration range from lo-l2 to lo-” M

for each ligand and a second response up to lo-’ M choleragen. Equilibrium binding studies showed two sets of choleragen receptor sites with affinity constants of 1O’O M-’ and lo8 M-I, corresponding to the first and second components of the dose-dependent choleragen activation curve for cyclic AMP production. During stimulation by choleragen, occupancy of protein kinase receptor sites by cyclic AMP paralleled the initial rise of intracellular cyclic AMP and showed saturation at 10-l” M choleragen. The subsequent secondary rise in cyclic AMP production at higher choleragen concentrations caused no further increase in binding of cyclic AMP to intracellular receptor sites. The actions of both human chorionic gonadotropin (hCG) and choleragen upon steroidogenesis were correlated with the initial phase of cyclic AMP stimulation by each ligand. The higher affinity choleragen site was responsible for stimulation of cyclic AMP- dependent metabolic responses in the Leydig cells and for mediating the gonadotropin-like effects of choleragen. Low concentrations of choleragen were found to increase production and receptor binding of cyclic AMP without a corresponding effect upon testosterone production. This discrepancy and the higher sensitivity of steroidogenesis to hCG (ED,, lo-l2 M) than to choleragen (ED,,, lo-‘” M) in the presence of equivalent changes in cyclic AMP production are consistent with intracellular compartmentalization of the hormonal pathway for activation of steroidogenesis.

The numerous biochemical responses elicited by choleragen in the gut and elsewhere are mediated through activation of

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “WuertisemenY in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

adenylate cyclase and the effects of raised intracellular cyclic AMP concentration (1). Such responses include the stimulation of lipolysis in fat cells (2, 31, glycogenolysis in liver cells (4, 51, and steroidogenesis in adrenal cells (6-8). In endocrine target tissues, choleragen acts in a nonspecific and prolonged manner to elicit the characteristic cellular responses that are normally evoked by more highly specific peptide hormones that act via cyclic AMP-dependent pathways. In the steroido- genie target cells for luteinizing hormone (LH) in the testis, the stimulation of testosterone synthesis by luteinizing hormone and human chorionic gonadotropin is accompanied by increased cyclic AMP production, binding of cyclic AMP to the regulatory subunit of cyclic AMP-dependent protein kinase (9), and activation of the phosphorylating enzyme (9, 10).

The role of cyclic AMP in the steroidogenic response to physiological concentrations of gonadotropin has been further analyzed by comparative studies on the actions of hCGl and choleragen upon the acute metabolic responses of purified Leydig cells. This comparison was performed to gain insight into the selective ability of low concentrations of gonadotropin to produce maximum steroidogenic responses while activating only a minute change in cyclic AMP production and to evaluate the role of functional compartmentalization of the cyclic AMP response during hormone action. For this purpose, we performed parallel studies of cyclic AMP and testosterone responses to choleragen and human chorionic gonadotropin in purified Leydig cells, analysis of Y-choleragen binding to intact Leydig cells, and correlation of choleragen binding with cyclic AMP production and binding to receptor sites of protein kinase. The results of these experiments have indicated that the gonadotropin activation pathway operates through an intracellular compartment that is functionally coupled to the high affinity receptor sites for the hormonal ligand.

MATERIALS AND METHODS

Preparation of Isolated Leydig Cells - Interstitial cells were dispersed by collagenase digestion of decapsulated tests from adult rats (Charles River Laboratory, Wilmington, Mass.) as previously de-

’ The abbreviations used are: hCG, human chorionic gonadotropin; NaCUPO, buffer, Dulbecco’s phosphate saline buffer, pH 7.5, 137 rnM NaCl, 2 rnM KCl, 8 mM Na,HPO,, 1.47 rniv KH,PO,, 0.49 rnM MgCl,, 0.6 rnM CaCl,.

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3722 Actions of Choleragen and Gonadotropin in Isolated Leydig Cells

scribed (11, 12). The Leydig cells were then further purified by Metrizamide gradient centrifugation of the interstitial cell suspension (13). The enzyme-dispersed interstitial cells (10” cells in 2.5 ml of Medium 199, 0.1% bovine serum albumin) were applied to a 40.ml gradient of 0 to 80% Metrizamide (Nyegaard, supplied by Accurate Chemical) dissolved in Medium 199 (Microbiological Associates, Bethesda, Md.) with 0.1% bovine serum albumin (Armour Pharma- ceutical, Kankakee, Ill.) and centrifuged at 3300 x g,,, for 5 min. Density gradients were prepared with an LKB gradient former model 11300 employing a linear gradient profile and reservoirs containing Medium 199, 0.1% bovine serum albumin, and the 80% Metrizamide solution. The gradients were prepared 16 h before use or when freshly prepared were centrifuged before use for 30 min at 3300 x g,,. The latter preparation gave a slightly steeper gradient and consistently sharper separation of the cell bands. The cell fraction containing Leydig cells was usually removed by aspiration into a 10.ml polyethylene pipette controlled by a propipette. In certain experiments, individual fractions were collected for timed intervals by aspiration through a plastic capillary connected to a Buchler polystatic pump. Forty-live fractions were collected from each tube, and densities were determined by measurement of refractive index at 25°C. The band of Leydig cells located just above the red blood cell band (13~ was removed and used for the experiments described below. In a few experiments, the purified Leydig cell band was further fractionated by centrifugation through a second Metri- zamide gradient (0 to 80%). By morphological and biochemical criteria, the purified preparation contained more than 90% Leydig cells (131. The Leydig cell fraction was washed once and resuspended in Medium 199 containing 0.1% bovine serum albumin. The proportion of incubation medium to cells was usually equivalent to 3 ml/ testis or approximately 2 x 10” purified Leydig cells/ml.

Cell Incuhoiion and Sample Preparation - Incubation of 2-ml aliquots of the cell suspension was performed in 20.ml polyethylene vials (Packard) in the presence of 0.125 mM 1-methyl-3-isobutyl xanthine (Aldrich, Milwaukee, Wise.). A range of hCG concentrations was added as loo-p1 aliquots to provide each dose level in three to six sample vials, and incubations were performed at 34” under 95% O,, 5% CO, with shaking at 100 cyclesimin. After 60 min, or at selected intervals in kinetic studies, incubations were terminated by transferring the vials to an ice bath. All further steps were carried out in the cold room, and samples were maintained between 0 and 4”. The cells plus incubation medium were transferred to polyethylene tubes (15 x 90 mm) and centrifuged at 1000 rpm for 12 min. The supernatant solution was saved for assay of testosterone and cyclic AMP released into the incubation medium. For assay of cyclic AMP, 900 ~1 of medium were immediately placed in glass tubes containing 100 ~1 of 10 ITIM theophylline, transferred to a boiling water bath for 10 min, and kept frozen until analyzed. Histochemical staining for A,-3P-hydroxysteroid dehydrogenase activity was performed as previously described (11).

Electron Microscopy and Autoradiography - Cells were collected by centrifugation at 1000 x g for 15 min and then fixed and dehydrated by standard procedures. The pellet was embedded in Spurr’s resin (14) and 800-A sections were placed on 150.mesh copper grids and stained with lead citrate. The grids were coated with carbon, then with 1:2 dilution of Ilford L4 emulsion, exposed for 11 to 16 weeks, and developed in Kodak Microdol-X.

Preparation of Cell Extracts for Assay of Intracellular and Recep- tor-bound Cyclic AMP (9) -The cell pellet was washed once with 8 ml of ice-cold medium containing 0.1% bovine serum albumin and 1 mM theophylline and suspended in 1 ml of Dulbecco’s phosphate- buffered saline (NaCl/PO,) containing 1 mM theophylline. The cell suspension was sonicated for 15 s using the microtip adaptor of a Branson sonifier-cell disruptor (Heat Systems Ultrasonics, Inc., Plainview, New York). A 0.4-ml aliquot of the incubation mixture was immediately placed in a boiling bath for 10 min and saved for assay of intracellular cyclic AMP. The remaining 0.6 ml was used for assay of endogenous cyclic AMP bound to receptor protein, i.e. occupied receptor sites (9). For this purpose, the protein kinase regulatory subunit plus bound cyclic AMP was isolated by adsorp- tion to cellulose filters (15). After addition of 3 ml of ice cold buffer (20 rn~ Tris-HCl, pH 7.4, containing 10 rn~ theophylline and 1 mM

mercaptoethanol), the contents of each tube were transferred to HAWP cellulose filters (Millipore). The tubes were washed once with 3-ml aliquots of cold buffer and then transferred to the respec- tive filters. Each filter was washed once with 2 ml of cold buffer, dried under suction, and transferred to glass tubes (12 x 75 mm). After addition of 0.6 ml of NaCl/PO, containing 1 rn~ theophylline,

the tubes were placed in a boiling water bath for 10 min. After cooling, the supernatant solution containing released endogenous cyclic AMP was stored frozen until analyzed by radioimmunoassay.

Measurement of Testosterone and Cyclic AMP- For testosterone determinations, incubation media were diluted 1:lO to 1:25 with NaCl/PO, and subjected to radioimmunoassay as previously described (16). Radioimmunoassay of cyclic AMP was performed by a modification (6) of the method of Steiner et al. (171, with addition of the acetylation step described by Harper and Brooker (18). This procedure improved the assay sensitivity by approximately 50.fold, reducing the detection limit to a few femtomoles of cyclic AMP, and permitted assay of the small quantities of cyclic AMP bound to the regulatory subunit of protein kinase. The acetylation step also improved the specificity of the assay at low cyclic AMP concentrations, allowing detection of small changes in cyclic AMP levels that were perceived as relatively constant when analyzed by the conventional assay method. This form of the assay was also employed to measure intra- and extracellular cyclic AMP concentrations. For comparative purposes, cyclic AMP was measured in selected experiments by the conventional radioimmunoassay method (6, 17) and by the more sensitive version employing acetylation prior to assay (181.

Binding of “V-hCG to Leydig Cells- The iodinated tracer used for these studies was prepared by the lactoperoxidase method and purified by agarose-Concanavalin A chromatography as previously described (19). Purified Leydig cells from individual fractions of Metrizamide gradients (5 x lo5 cells) were incubated for 3 h at 34°C with lo5 dpm of ‘““I-hCG (specific activity 40 &i/pg) with addition of 10 ng of hCG (10,000 IU/mg of protein) to approach receptor saturation. Thus, the final hCG concentration was 200 PM, approximately lo-fold higher than the K, for binding to gonadotropin receptors (6). Nonspecific binding was determined from tubes in which the labeled hormone was incubated with receptor in the presence of 20 /*g of unlabeled hCG and also in the absence of cells. After incubation, cells were washed with 3 ml of Medium 199, 0.1% bovine serum albumin and centrifuged at 120 x g for 15 min. Following aspiration of the supernatants the bound hormone in the pellet was determined by y spectrometry in an automatic counter with efficiency of 50% for lz51-hCG.

Binding Studies with “Wabeled Choleragen- rz51-choleragen was prepared by the chloramine-T method essentially as described by Cuatrecasas (20). The specific activity of the labeled toxin was 25 PCiIpg, and 60% of the tracer was bound during incubation with an excess of Leydig cells. Aliquots of the Leydig cell suspension (1 ml, 2 x lo5 cells) purified by two successive Metrizamide gradients were incubated with 0.1 ml of labeled choleragen (200,000 dpm) in the presence of increasing concentrations of choleragen (range 0.1 to 10 fig1 at 24” for 1 h. These conditions were found during initial experiments to approach equilibrium of the binding reaction. Non- specific binding of rz51-choleragen was determined by incubation in control tubes containing an excess (50 pg) of unlabeled choleragen (SchwarziMann, Orangeburg, N. J.) and did not exceed 1% of the total added radioactivity.

Equilibrium binding data were analyzed by the Scatchard method (21) or by an equation relating the concentration of bound ligand to the total cyclic AMP concentration (22). An interactive computer program with differential equation solving ability (23) was used to perform all curve fitting and calculations. Such programs were executed on a PDP-10 time sharing computer, with graphic output facilities, via a Tektronix terminal 4010-l.

RESULTS

Stimulation of Cyclic AMP and Testosterone Production by

hCG and Choleragen - In initial studies the total cyclic AMP production (extracellular plus intracellular nucleotide) was evaluated in Leydig cells incubated in the presence of hCG or choleragen over a wide dose of hormone or toxin concentrations. The total cyclic AMP production was measured in a preparation of collagenase-dispersed interstitial cells (containing approximately 40% Leydig cells) and in purified Leydig cells isolated by Metrizamide density gradient centrifugation (Fig. 1, A and B). In each case, the stimulation of cyclic AMP production by choleragen showed a biphasic pattern, with an initial response from 10. ‘I to lo-!’ M and a second response up to lo-’ M choleragen. Stimulation of cyclic AMP production by


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Actions of Choleragen and Gonadotropin in Isolated Leydig Cells

- ID) Purified Interstitial Cells ID) Purified Interstitial Cells

0

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CHOLERAGEN OR hCG (M) HCG OR CHOLERA TOXIN (M)

FIG. 1. Total cyclic AMP production (cells + medium) and testosterone production by testis interstitial cells (A and C) and of purified Leydig cells (B and D) following incubation for 2 h with a wide range of hCG and choleragen concentrations. In this experiment, cyclic AMP was measured by radioimmunoassay without acetylation.

hCG followed closely the initial segment of the dose-response curve elicited by choleragen. However, by contrast with the closely similar dose-response curves for cyclic AMP production evoked by hCG and choleragen, the steroid responses were widely dissociated, such that hCG was approximately loo-fold more potent than choleragen in stimulating testosterone production in both interstitial cells and purified Leydig cells (Fig. 1, C and D). These preliminary findings prompted us to perform more detailed comparative studies on the activation of purified Leydig cells by gonadotropin and choleragen, with measurements of extracellular, intracellular, and receptor- bound cyclic AMP, and testosterone production.

Choleragen Binding and Activation in Purified Leydig Cells - The characteristic dose-response curves for stimulation of cyclic AMP production by hCG and choleragen were observed in three further experiments with interstitial cell preparations that contained 25 to 40% Leydig cells and in four experiments with purified Leydig cells that contained 80 to 90% Leydig cells by morphological criteria and histochemical analysis (11). An additional criterion of purity was provided by the ‘2”I-hCG binding profile of cell fractions obtained during density gradient purification of the isolated Leydig cells (Fig. 2). Equilibration of each cell fraction with 12”I-labeled hCG showed that specific gonadotropin binding per cell was relatively constant over the peak of Leydig cells (equivalent to 20,200 2 2,850 sites/cell, mean 2 S. D., n = 8). The refractive index of the fractions (21 to 28) containing the Leydig cell layer was equivalent to density of 1.3703 to 1.3595 g/cm3, and that of the peak tube (Fraction 24) was 1.3655 g/cm”. Histo- chemical analysis of the peak fractions containing the cell band indicated that formazan-positive Leydig cells comprised 95 f 3% (S. D.) of the total cell population.

The biphasic activation curve for stimulation of cyclic AMP by choleragen suggested that heterogeneity of choleragen receptor sites was present in the Leydig cell. Direct evidence of binding site heterogeneity was provided by equilibrium binding studies during incubation of purified Leydig cells with “‘I-choleragen. Data analysis by Scatchard plots or saturation curves demonstrated two orders of receptors, a high affinity

22

18

4

12 16 20 24 28 32 36 40 44 46

TUBE NUMBER ,topi

FIG. 2. Sedimentation profile of Leydig cells on a density gradient of 0 to 80% Metrizamide. Leydig cells previously obtained by density gradient purification of dispersed interstitial cells were layered on a second Metrizamide gradient. After centrifugation, a single layer of cells was observed and the gradient was divided into l-ml fractions. Aliquots of 0.5 ml were incubated for 3 h at 34” with a saturating concentration of lz51-hCG (200,000 dpm, 10 rig/ml).

group of approximately lo5 sites/cell with K,, of 2 x 10’” M-I and a low affinity group of about 2 x 10” sites/cell with K,, of 4 x lox Mm’ (Fig. 3). By comparison, equilibrium binding studies with 1251-choleragen and rat red blood cells purified by gradient centrifugation demonstrated a single population of approximately 5.5 x lo4 sites/cell with affinity constant of 6 X lo” M-‘.

Morphological comparison of hCG and cholera toxin binding by purified Leydig cells, performed by autoradiographic anal-


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ysis of cells equilibrated with saturating concentrations of each 12”I-labeled ligand, revealed marked differences in the uptake of each ligand, with predominant localization at or near the cell membrane in each case (Fig. 4). This analysis also demonstrated the predominantly pericellular binding of choleragen under the conditions employed for uptake studies and derivation of binding constants for the choleragen receptors.

Kinetic Analysis of Leydig Cell Activation by Gonadotro- pin - The kinetics of cyclic AMP production during incubation of Leydig cells with the trophic hormone were determined at two gonadotropin concentrations, a near maximal steroido- genie level (50 pg/ml; 10W2 M) and a supramaximal steroido- genie level (10 rig/ml; 2 x lo-l0 M) of hCG. The higher hCG concentration caused a 2-fold increase in extracellular cyclic AMP levels at the earliest time measured (1 min), followed by a rise to almost 200-fold stimulation at 20 min, and a progres- sive increase to nearly lO,OOO-fold at 180 min (Fig. 5). The smaller hCG dose caused significant elevation of extracellular cyclic AMP between 30 and 60 min, rising to a 5-fold increase at 180 min (not shown). When the extracellular cyclic AMP production evoked by 2 x 1OW’ hCG in purified Ieydig cells was compared with that of an interstitial cell preparation containing 25% Leydig cells, in the presence and absence of 25% Metrizamide, a similar pattern of cyclic AMP production was observed (Fig. 5). The quantitative differences in cyclic AMP response were proportional to the concentration of Ley-

dig cells of the individual preparations, i.e. 95% versus 25% as determined by histochemical criteria. In addition, it was apparent that Metrizamide did not alter the kinetics and magnitude of cyclic AMP responses stimulated by hCG.

The intracellular cyclic AMP content followed a time course similar to that exhibited by the changing extracellular cyclic AMP concentration during hormone stimulation. Thus, the high hCG dose caused an immediate increase of intracellular cyclic AMP, which rose from the basal value of 0.7 pmol/lOe cells to 2.8 pmol/W cells at 2 min, with a maximum of 3.3 pmol/lO” cells at 15 min, and remained near 2.5 pmol/lO” cells at subsequent times during the incubation period. The low dose of hCG caused a more gradual increase in intracellular cyclic AMP, such that significant increases were not observed until 30 min and the maximum increase was reached between 120 and 180 min. The maximum intracellular content of cyclic AMP was relatively small when compared with the extracellular levels that accumulated in the incubation medium (Fig. 6). The cyclic AMP bound to receptor protein rose in parallel with the hormone-induced elevation of intracellular cyclic AMP. The higher hCG dose caused almost immediate saturation of cyclic AMP receptor to levels (1.4 pmol/lO” cells) that revealed approximately one-half of the total intracellular cyclic AMP to be bound to receptor protein. In contrast, the low dose of hCG caused a gradual rise in receptor-bound cyclic AMP, again in parallel with the intracellular levels, with a significant increase from 30 min to reach the maximum at 120

FIG. 3. Right, Scatchard plot of binding data obtained by incubation of 8 N, = 2.60 x lOa sites/cell

Leydig cells with ?-choleragen and ,g s increasing concentrations of cholera- 0 gen for 1 h at 24”. Left, semilogarithmic plot of specific binding of choleragen to

8 i 2

Leydig cells obtained by computer analysis employing a two-site weighted fit analysis (22, 23) of “total” and “spe- citically bound” toxin.

$ u 4 1

z 0

10” lo- lo-’

TOTAL CHOLERAGEN IM)

FIG. 4. Electron microscopic auto- radiograph of Leydig cell labeled with lz51-hCG (left) and ‘“I-choleragen (right). Metrizamide-purified cells were incubated with ‘Z51-hCG or 9. choleragen as described under “Mate- rials and Methods” and subjected to autoradiography and electron microscopy. The bar represents 1 p.

0.6 ., I I0 I K, = 1.68 Y lO’OM-’ I K, 3.80 x lPM-’ =

N, = 1.99 x lo6 sites/cell

kli

8

“8 N, = 2.66 x 1W sites/cell

I:

2 0.3 3

- b,

B ?h:

q., ‘a0

ha 0 0 .‘Q._

260 500

BOUND CHOLERAGEN-picomolar


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choleragen dose (1 rig/ml), significant elevation of cyclic AMP was observed at 45 min, with a lo-fold rise at 90 min and a rise up to 300-fold at 180 min (Fig. 9). Intracellular cyclic AMP levels during incubation with the higher choleragen dose were

INC”BATION THE mln”te*

FIG. 9. Extracellular cyclic AMP production by Leydig cells after 2 to 180 min with 1 ng or 10 pg of choleragen/ml. Each point represents the mean + S. E. of quadruplicate incubations.

T -7-- 7

- Basal

- Choleragen - 1 rig/ml - Choleragen - 10 pg/ml

Y ..-.

0 30 60 90 120 150 180

INCUBATION TIME - minutes

FIG. 10. Intracellular cyclic AMP formation in Leydig cells stimulated with 1 ng or 10 pg of choleragenlml for 2 to 180 min. Each

point represents the mean 5 S. E. of quadruplicate incubations.

FIG. 11. Occupancy of cyclic AMP receptor protein, measured by radioimmunoassay of bound cyclic AMP after Leydig cell stimulation with 1 ng or 10 pglml of choleragen for 2 to 180 min. Eachpoint represents the mean + S. E. of quadruplicate incubations.

FIG. 12. Testosterone production in Leydig cells after 2- to 180- min incubation with 1 ng or 10 pg of choleragen. Each point represents the mean + S. E. of quadruplicate incubations.

7 INTRACELLULAR cAMP

s-

CAMP SOUND TO RECEPTOR

TESTOSTERONE v - - --._____.

I PO13 10 l2 to ”

CHOLERAGEN OR hCG IM,

FIG. 13. Correlation between intracellular cyclic AMP (top), receptor-bound cyclic AMP (middle), and testosterone production (bottom) during stimulation of Leydig cells with a wide range of choleragen concentrations, from lOWI3 to 1O-7 M. Incubation time for top and middle was 60 min and for bottom was 180 min. Stimulation of cyclic AMP by a maximally effective hCG dose (10-l’ M) and a supramaximal dose (W8 M) is also shown (0). Testosterone production was determined during incubation of Leydig cells with choleragen (Al or hCG (lo-l3 to lo-* M) (0). Each point represents the mean k S. E. of quadruplicate incubations.

significantly increased at 15 min, with a 4-fold increase at 60 min and a g-fold increase at 180 min. The low choleragen dose caused a significant increase at 30 min, to almost double the control values (Fig. 10). Bound intracellular cyclic AMP was significantly increased by the higher choleragen dose at 15 to 20 min, with a 2.5-fold increase at 60 min and a maximum increase of 3-fold at 90 min, followed by a slight fall thereafter. With the low dose, significant increases were observed at 30 min, with a 1.75fold increase above control at 60 min and no further increase at later times (Fig. 11). The corresponding testosterone responses were of particular interest because steroidogenesis did not increase above the basal value (2 pmol/ 106 cells) until 4.5 to 60 min with the higher dose (10 pg/ml)


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Actions of Choleragen and Gonadotropin in Isolated Leydig Cells 3727

B 86Kl

I CAMP BOUND TO RECEPTOR

I I I-- ~~~~

- Choleragen -4 hCG 5

E . hCG

8

km . s FIG. 14. Occupancy of cyclic AMP

‘c ! receptor protein by endogenous cyclic

% AMP in cells treated with 1Om’3 to lo-~’

/’ M choleragen (A---A) and with maximal hCG doses (*) (lo-“’ and 10~’ M) in the same experiment, and with 10-l” to 10m7 M hCG in a parallel experiment (0).

2 I ls

1 1 10

'*

10

”

10

‘O

109 CHOLERAGEN OR hCG (Ml

FIG. 15. Effects of individual and combined treatment of Leydig cells with hCG and choleragen upon cyclic AMP responses (abone) and testosterone production <below). Cells were incubated for 1 h in the presence or absence of choleragen (2 x 10-l’ M, 1 rig/ml) followed by 2 h in the presence of hCG (3 x lo-‘” M). Cyclic AMP production is plotted as the intracellular (hatched), receptor-bound (solid), and extracellular (open) levels determined by radioimmunoassay as described under “Materials and Methods.” The intracellular cyclic AMP level includes the receptor-bound cyclic AMP value shown in the lower solid portion of each column. Bars represent the mean 2 S. D. of quadruplicate incubation vessels, each assayed in duplicate.

and then rose between 2 and 3 h to the maximum value of 300 pmol/lO” cells. In contrast, no significant stimulation of testosterone production was observed at any time during incubation with the low choleragen dose (1 ngiml) (Fig. 121, although this dose significantly increased cyclic AMP formation.

Comparison of Cyclic AMP and Testosterone Production during Stimulation of Purified Leydig Cells by Choleragen and hCG - Further studies were performed to investigate the relationships between intracellular cyclic AMP, receptor- bound cyclic AMP, and testosterone production during stimulation of purified Leydig cells with a wide dose range of choleragen concentrations, from lo-‘” M to 10e7 M (Fig. 13). The biphasic cyclic AMP response to choleragen was again

lo8 10’

apparent, as noted previously for total cyclic AMP concentration (Fig. l), with an initial rise from 10. I2 to lo-“I M

choleragen and a secondary rise up to 10mx M choleragen. In the same cell preparation, stimulation of intracellular cyclic AMP by hCG did not exceed the maximal levels of the initial stimulation curve to choleragen (Fig. 13, top). The rise in occupancy of the cyclic AMP receptor protein by endogenous nucleotide paralleled the initial rise of intracellular cyclic AMP described above, and complete occupancy of the cyclic AMP receptor protein was achieved at 10-l” M choleragen

(Fig. 13, middle). Saturation of the cyclic AMP receptors during stimulation with hCG was observed in this experiment at the same concentration as with the toxin.

In contrast to the similar cyclic AMP responses evoked by hCG and choleragen, the two ligands differed markedly in their potency as stimuli of testosterone production. The dose- response curve for hCG was elicited with hormone concentrations of lo-‘” to lo-” M, and the ED,,, for testosterone production was lo--l2 M hCG. The lower sensitivity of the dose- response curve of testosterone to choleragen was clearly apparent, with stimulation over the range of lo-” to lo-!’ M and ED,,, of 10-l” M toxin (Fig. 13, bottom). By comparison with the middle panel of Fig. 13, it is apparent that the steroid response to hCG was elicited when but a small fraction of the cyclic AMP binding sites were occupied, whereas the testosterone response to choleragen was observed only during more extensive occupancy of the cyclic AMP receptors. A more detailed study of the occupancy of cyclic AMP receptor protein by endogenous cyclic AMP in choleragen-stimulated and hCG- stimulated Leydig cells is shown in Fig. 14. Occupancy of cyclic AMP receptor protein by endogenous cyclic AMP in cells treated with choleragen was progressively increased between 10-l” M and lo-lo M toxin, and the levels of occupancy stimulated by hCG in the same experiments attained similar maximal values as in cells stimulated by the toxin. Also, increases in cyclic AMP bound to receptor protein during hCG stimulation in a parallel experiment showed that receptor occupancy occurred over the same dose range with each ligand, reaching saturation at comparable concentrations of hCG or choleragen.

An examination of the effects of choleragen plus hCG on receptor-bound cyclic AMP and testosterone responses was performed to test the possibility that low choleragen doses could exert a direct inhibitory effect on the steroidogenic activity of the Leydig cells. In this study, Leydig cells were incubated for 1 h with a choleragen concentration (2 x lo-” M) that would produce almost maximal occupancy of cyclic AMP receptors and only a slight increase (<20%) in testoster-


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one production. A near maximum steroidogenic dose of hCG (100 pg, 2 x lo-l2 M) was then added and incubation was continued for 2 h (Fig. 15). The testosterone responses of cells incubated with choleragen alone were barely augmented over those of control cells, despite the nearly maximal occupancy of cyclic AMP receptor. Cells incubated with hCG alone showed near maximal testosterone responses, and, as expected, only about 20 to 25% increase in occupancy of cyclic AMP receptors. In contrast, cells incubated in the presence of both stimuli showed additive effects on testosterone response and on cyclic AMP receptor occupancy (Fig. 15).

DISCUSSION

The presence of a biphasic activation pattern for cyclic AMP during incubation of collagenase-dispersed interstitial cells with choleragen suggested heterogeneity of toxin binding sites in the Leydig cell. Further experiments on purified Leydig cells confirmed the presence of a biphasic stimulation pattern and excluded cellular heterogeneity as the cause of this phenomenon. The homogeneity of the Leydig cell preparation has been shown by several criteria in a previous report (13) and was further confirmed in these studies during Metriza- mide gradient analysis of the Leydig cell fraction. Also, the production of cyclic AMP and testosterone showed quantitative differences in response that were consistent with the proportion of Leydig cells in the unfractionated and purified cell preparations. Direct evidence for heterogeneity of the choleragen binding sites was provided by binding studies with labeled toxin. In addition, there was a close correlation between the dose-response curves for choleragen activation of intracellular and receptor-bound cyclic AMP and the dose- related choleragen inhibition of ‘2”I-choleragen binding to isolated Leydig cells.

The cyclic AMP activation curve elicited through interaction of choleragen with high affinity sites (K,Z - lOJo M-‘)

closely followed the hormonal activation pattern obtained by stimulation with chorionic gonadotropin. Intracellular cyclic AMP receptors were fully saturated at the cyclic AMP levels attained by association of choleragen with the high affinity toxin receptors and of hCG with the specific gonadotropin receptors. Further interaction of choleragen with the low affinity toxin binding sites caused additional increases in intracellular and extracellular cyclic AMP levels, with no further change in cyclic AMP bound to intracellular receptors. The intracellular cyclic AMP content rose approximately 5- fold during activation of high affinity choleragen sites and during hCG stimulation via specific gonadotropin receptors. At the same time, there was a doubling of the receptor-bound cyclic AMP to reach the saturation level associated with maximum stimulation of steroidogenesis.

Kinetic studies on ligand-induced responses revealed a lag of 45 to 60 min in the steroidogenic response to choleragen, even at supramaximal doses for cyclic AMP stimulation (10 pg/ml). This was in contrast with the shorter period (10 to 15 min) required for stimulation of testosterone production by submaximal and saturating doses of trophic hormone. A lag period in nucleotide and steroid responses has often been observed during incubation of adrenal cells with choleragen (8, 24-26). This delay was attributed to the time required for initial interaction of choleragen with the receptor and the release and/or transmembrane passage of the toxin’s active subunit prior to stimulation of adenylate cyclase (26, 27). Cyclic AMP production showed no lag period during stimulation with a high concentration of trophic hormone, being

almost instantaneously elevated at supramaximal hCG doses, with immediate saturation of intracellular cyclic AMP receptors. In contrast, a delay of 15 to 30 min in cyclic AMP production was observed with choleragen concentrations of 1 rig/ml and 10 pg/ml.

These and previous studies (9) have demonstrated that formation of cyclic AMP as the second messenger for the Leydig cells precedes the testosterone responses by 10 to 15 min at all effective doses of hCG or choleragen. However, it is of interest that significant elevations of intracellular and receptor-bound cyclic AMP occurred without subsequent stimulation of steroidogenesis during incubation of cells with the low concentration of toxin (1 rig/ml). This observation, with the higher ED,,, for choleragen stimulation of testosterone production, suggests the presence of functional compartmentalization in the cyclic AMP-protein kinase response to specific hormonal stimulation. During toxin stimulation, testosterone dose-response curves occurred when 75 to 100% of cyclic AMP receptors were filled, in contrast to the 25% occupancy needed during hormonal stimulation to evoke maximum steroido- genie levels (9). The possibility that the absence of a steroido- genie response to low choleragen concentrations that elevate cyclic AMP levels could be due to an inhibitory effect on steroid production was excluded by the data obtained during combined stimulation with the two ligands. As shown in Fig. 15, addition of hCG to choleragen-treated cells evoked a full testosterone response while producing a small but significant increase in receptor-bound cyclic AMP over the elevated level produced by choleragen alone.

It is probable that cyclic AMP produced during hormonal stimulation has more ready access to cyclic AMP receptors in the vicinity of key steroidogenic enzymes which depend upon activation of protein kinase to effect increased testosterone production. After binding of choleragen and nonspecific stimulation of adenylate cyclase at multiple sites in the cell membrane, the cyclic AMP produced could also occupy cyclic AMP receptors not directly related to the protein kinase involved in the activation of steroidogenesis. This would explain the finding that a major proportion of the available cyclic AMP receptors needs to be occupied to mediate the maximal steroidogenic response to choleragen, whereas a much smaller degree of cyclic AMP receptor occupancy is adequate for maximum responses to hormonal stimulation.

In addition to the interpretation of functional compartmentalization as an explanation of these observations, other possible mechanisms should be noted. The results could also be regarded as evidence that cyclic AMP is not necessarily involved in the activation of steroidogenesis, and that the primary effects of gonadotropin could be on mechanisms other than adenylate cyclase. This would suppose a membrane effect that can be exerted selectively by gonadotropin and only at much higher concentrations by choleragen, possibly through major perturbations of the cell membrane. There is at present no evidence for the nature of alternative plasma membrane responses that are activated by hormonal ligands and cholera toxin, but the possibility cannot be excluded. However, the close correlation between receptor-bound cyclic AMP and steroidogenesis during gonadotropin action (9) strongly favors the probability that cyclic AMP production mediates the steroidogenic response of the Leydig cells to gonadotropic hormones. Another possibility, that choleragen has an inhibitory effect on steroidogenesis at low concentrations that evoke cyclic AMP responses without increasing testosterone production, has been rendered unlikely by the experimental find-


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Actions of Choleragen and Gonadotropin in Isolated Leydig Cells 3729

ings. In the absence of alternative mechanisms of hormone 12.

activation that can also be simulated by cholera toxin, it is reasonable to conclude that functional compartmentalization

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of cyclic AMP and protein kinase exists in Leydig cells and is 14,

responsible for the differential actions of hCG and choleragen 15. on cyclic AMP and steroidogenesis.

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M L Dufau, K A Horner, K Hayashi, T Tsuruhara, P M Conn and K J Cattcompartmentalization of the hormone-activated cyclic AMP response.

Actions of choleragen and gonadotropin in isolated Leydig cells. Functional

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Actions of Choleragen and Gonadotropin in Isolated Leydig Cells