THEJOURNAL OF BIOLOGICAL CHEMISTRY Val. 263, No. 11, Issue of April 15, pp. 5070-5074, 1988 Printed in U.S.A. Angiotensin I1 Receptors and Inhibitory Actions in Leydig Cells* (Received for publication, August 3, 1987, and in revised form, November 4, 1987) Azra Khanum and Maria L. DufauS From the Molecular EndocrinologySection, Endocrinology ReproductionResearch Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892 Rat Leydig cells possessfunctional high-affinity in swine testis showing the presence of converting enzyme in receptors for angiotensinI1 (AII). AI1 inhibits adenyl- spermatids and other stages of germinal cells (6). ate cyclase activity in Leydig cell membranes and reOn the other hand, there is strong evidence that Leydig duces basal and human chorionic gonadotropin (hCG)-cells possess a membrane system with the potential for negstimulated CAMPpools and testosterone production in ative modulation of gonadotrophic action. In this regard, we intact cells. Treatment ofcells with an inhibitory dose have demonstrated a novel, high-affinity inhibitory action of of forskolin (lo-' M) and a submaximal dose of AI1 low doses of forskolin (range 10"2-10-9 M ) upon adenylate caused additive inhibition of hCG-stimulated events. cyclase activity and cAMP generation, an effect that appears The inhibitory action of AI1 was largely prevented by to be mediated by the pertussis toxin-sensitive guanine nupertussis toxin prior to the addition ofAI1 alone or in the presence ofhCG. This study and our recent reportcleotide inhibitory unit (Ni) of adenylate cyclase (7). Since on inhibitory action of low doses of forskolin, 10-l2- AI1 has beenshown to exert an inhibitory influence on cyclase system of liver (8, 9), adrenal cortex (10, lo-' M (Khanum, A., and Dufau,M. L. (1986) J. Biol. adenylate l l ) , renal cortex (12), and smooth muscle (13), this hormone Chem. 261,11456-1 1459)are indicative of a pertussis toxin-sensitive subunit of adenylate cyclase available couldbe apotential regulator of gonadal function. It is, for acute regulation of Leydig cell function. 8-bromo- therefore, conceivable that tubular and locally produced AI1 CAMP bypasses the inhibitory effect of forskolin as could modulate the action of gonadotrophin in Leydig cells. well as AII. We have, therefore, demonstrated funcEXPERIMENTALPROCEDURES tional AI1 high-affinity receptor and an acute inhibitory effect of AI1onhCG action in Leydig cells. Our Materials results have provided evidence for a pertussis toxinMedium-199 and elutriation medium were obtained from Whitsensitive guanine nucleotide inhibitory proteinas mediator of theeffect of AIL These findings further em- taker M.A. Bioproduct, Inc., Walkersville, MD and National Instiphasized the importance of theCAMPpathway in the tutes of Health Media Supply Units, Bethesda, MD respectively. hCG (CR-121) was kindly provided by the Center of Population Research, Leydig cells, and studies also suggest that tubular andNational Institute of Child Health and Human Development, Belocally producedAI1 could negatively modulate lutein- thesda, MD.AI1 was purchased from Sigma. [Sar',Alaa]AII was izing hormone stimulation of Leydig cells. obtained from Vega Biotechnologies,Tucson, A Z , and forskolin from A number of studies have provided evidence for the presence of the renin-angiotensin system in reproductive tissues. Immunoreactive renin has been detected in Leydig cells of rat and human testes andwas found to be pituitary-dependent in the rat (1, 2). Similarly, more recent studies have shown the presence of renin, angiotensin I and I1 (AI and AII)' in normal rat Leydig cells and a murine Leydig cell line (3, 4). Furthermore, angiotensin-converting enzyme activity was demonstrated in rat testis andshown to be localized predominantly in the germinal cells, whereas only minor activity was found in the purified adult rat Leydig and Sertoli cells (5). Also, [3H]captopril bound specifically to cellular fractions enriched in germinal cells (5). Velletri et al. (5) have suggested that the pituitary gland is required for development and maintenance of the rat testicular angiotensin-converting enzyme through stimulation of steroidogenesis in the testis. The biochemical evidence (5) was consistent with immunofluorescence studies * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed Bldg. 10, Rm. 8C407, NIH, 9000 Rockville Pike, Bethesda, MD 20892. 'The abbreviations used are: AI and AII, angiotensin I and II; hCG, human chorionic gonadotrophin; Ni, guanine nucleotide inhibitory unit; HPLC, high pressure liquid chromatography. Behring Diagnostics. AI1 ( 5 4 , LHRH, AI, and [des-Asp']AII were obtained through Peninsula Laboratories, San Carlos, CA. Pertussis toxin was purchased from List Biological Laboratories, Inc., Campbell, CA.'"1-AII (2200 Ci/mmol) and succinyl cAMP '251-tyrosine methyl ester (2000pCi/pg)were prepared byMeloy Laboratories, Springfield, VA, and by Hazleton Biotechnologies,Vienna, VA, using a modification of the chloramine-T method (14), followed by purification by HPLC (15). [cY-~*P]ATP (800 Ci/mmol) were obtained from Du Pont-New England Nuclear. Methods Preparation of Leydig Cellsand Membranes-Adult male SpragueDawley rats (Charles River Breeding Laboratories, Wilmington, MA) were killed and testes were removed and placed in ice-cold PBS, pH 7.4. Interstitial cells were obtained by collagenase digestion of decapsulated testes, as described previously (16). Crude cell suspension was washed and then pelleted at 200 X g for 10 min. The cell pellet was resuspended with elutriation buffer consisting of regular medium-199 with Hanks' salts and L-glutamine containing 1.4 g/liter NaHC03, 0.5% bovine serum albumin-, 1mM EDTA, 50 units/ml heparin, 12.5 pg/ml DNase and 50 pg/ml gentamycin, pH 7.4. The purified cells were obtained by centrifugal elutriation (17). Cells were centrifuged and resuspended in medium-199 containing 0.1% bovine serum albumin and were incubated a t 34 "C with shaking at 100 cycles/min under an atmosphere of 02:C02 (955, v/v) in the presence and in the absence of various concentrations of hCG with or without AH, forskolin, and 8-bromo-CAMP.For some experiments, cells were pretreated for 60 min with pertussis toxin under the same incubation conditions prior to theaddition of hCG and/or AIL The incubations were terminated by transferring the incubation tubes to an ice bath; all further stepswere carried out at 0 "C and processed for the analysis of cAMP (intracellular and receptor-bound) and testosterone; and 5070 AII Receptors and Actions 5071 r- V 0 30 60 90 I 0 120 I I I I I 30 20 TIME [MINI 10 TIME IMIN) FIG. 1. Time course specific binding of 121-AII to Leydig cells during incubation at 4 "C, 22 "C and 37 "C for 120 min. Nonspecific binding for each time intervalwas determined by addition . point represents the mean f S.E. of of unlabeled AI1 (10 p ~ ) Each triplicate determinations. I FIG. 3. Time course association and dissociation of AI1 binding to Leydig cell receptors. The rate of association of specific binding to the cells was measured as a function of time after incubating Iz5I-AIIat 22 'C. The rate of dissociation was obtained by the addition of 10 p~ unlabeled AI1 after 14 min of incubation and by the measurement of release of'"I-AII. Each point represents the mean f S.E. of three determinations. I I 10-8 10-4 1 1.o 0 m m 0.5 7.5 BOUND A Il-pM FIG. 2. Scatchard analysis and saturation curve (ineet) of AI1 binding to Leydig cells. Cells were incubated at 22 "C for 20 min with increasing concentrations of Iz5I-AII.Each point represents the mean of triplicate determinations. The S.E. was less than 10%. their measurements were performed by radioimmunoassay as described previously (18). Purified Leydig cell plasma membranes were prepared in the presence of 5 mM EDTA (19) and stored in liquid nitrogen until use. Adenylate cyclase assay was carried out by methods previously described (9). For some experiments, membranes were also preincubated for 20 min with activated pertussis toxin. Binding Studies-Binding assays with '"I-AII were performed with intact cells which were incubated in 500 p1 of Dulbecco's PBS in the presence of 0.2% bovine serum albumin and 100 FM phenylmethylsulfonyl fluoride for various time periods: 0-120 min at different temperatures: 4 "C, 22 "C and 37 "C. For equilibrium studies, cells were incubated a t 22 'C for 20 min with increasing concentrations of ~ AI1 or with '%I'261-AIIin the presence or in theabsence of 10 g i of AI1 and increasing concentrations of unlabeled AIL In some displacement studies, AII-related and -unrelated peptides were used. The reaction was terminated by adding 2 ml of ice-cold PBS, pH 7.4, in the tube and immediately filtered under vacuum through GF/C filters (Whatman, Maidstone, Great Britain). Filters were washed twice with 3 mlof PBS and bound radioactivity was measured in a y counter. For kinetic studies, Leydig cells were also incubated with 1261-AIIat various times: 0-30 min. The specific binding of '251-AII reached to equilibrium after 14 min of incubation. Therefore, the rate of dissociation of "'I-AII receptor complex was obtained by adding 10 p~ of unlabeled AI1 after 14 min of incubation. The binding studies were analyzed by the method of Scatchard (20) using computer 0 PEPTIDECONCENTRATION [MI FIG. 4. Displacement of la6I-AIIby angiotensin-related and -unrelated peptides and diterpene, forskolin. Bo and E , the fraction of total tracer bound in the absence and in the presence of unlabeled peptides, respectively, a t 22 "C for 20 min of incubation. Each point represents the mean f S.E. of three determinations. O-", AII; A-A, [des-Asp'IAII; X-X, [Sar',Alas]AII; H, AI; -, LHRH; 0-"0, AI1 (5-8); A, arginine vasopresin; 0, forskolin. analysis of binding data by a nonlinear model curve fitting program (21). RESULTS AND DISCUSSION In this study, we have demonstrated the presence of functional AI1 receptors in rat Leydig cells and have provided the evidence of its acute inhibitory effects. Cells were incubated with lZ5I-AIIfor various time periods at 37 "C, 22 "C, and 4 "C. The highest specific binding was observed when the cells were incubated at 22 "C, and thebinding reached to maximum within 30 min of incubation (Fig. 1).Thereafter, there was subsequent decrease in binding. At 37 "C, the specific binding increased rapidly after 5 min of incubation followed bya rapid decline. Thisis most likelydue totracer degradation by proteolytic enzymes as indicated by others (22) or may be due AII Receptors and Actions 5072 *--+ PT + hCG +All Y 0 10-10 10-8 10-6 All [MI 0 0.26 2.6 26 hCG IpMl FIG.5. I,& effect of pertussis toxin (PT) on AII-induced inhibition of intracellular CAMPproduction. Leydig cells (1 X lo6 cells/ml) were preincubated with pertussis toxin (30 ng/ml) for 60 min. Incubation was further carried out for 60 min in the presence or in the absence of hCG concentration with and without AII. Each point represents the mean f S.E. of triplicate incubations. Right, dose-dependent inhibitory effect of AI1 on hCGstimulated intracellular CAMP production. Leydig cells (1 X lo6 cells/ml) were incubated with a range of hCG concentrations (0.26-260 PM) for 60 min in the presence and in the absence of AI1 (lO-'M). Each point represents the mean f S.E. of triplicate incubations. B a d A I1 K + A 11 hCG hCG 12.6pMI + All PTihCG hCG + 1260 pMI All hCG PT+ hCG + + All AI1 FIG. 6. Effect of pertussis toxin (PT)on AII-induced inhibition of receptor-bound CAMP. Leydig cells (1 X lo6 cells/ml) were preincubated with pertussis toxin (30 ng/ml) for 60 min. Incubation was further carried out for 60 min in the presence or inabsence of the indicated hCG doses with and without AIL Each point represents themeans 2 S.E. of triplicate incubations. to internalization of receptor complex and subsequent receptor degradation. In fact, this receptor-mediated internalization has been indicated in cases of various peptide hormones (23). However at 4 "C, the specific binding was increased gradually and reached equilibrium after 90 min of incubation. In this case the specific binding was only 10% of that observed at 22 "C during 20 min of incubation. Therefore, for binding studies, the incubation was carried out at 22 "C for 20 min. Scatchard plot derived from equilibrium binding data obtained at 22 "C showed the presence of high-affinity binding sites with KOof 1.7 X 10'' M" f 0.41 ( n = 4) and number of 2018 & 408 ( n= 4)receptor sites percell (Fig. 2). The binding was saturable andreversible as indicated by saturation analy- sis of binding data and association and dissociation studies, respectively. The rates of association (kl) and dissociation ( k 1 ) of Iz5I-AIIbinding to Leydig cells were 0.885 nM" min-' and 0.04 rnin", respectively (Fig. 3). Furthermore, the calculated half-life was 17.5 min. From the kinetic data, the calculated equilibrium constant, K,, was 2.2 X 10" M-l. This value is veryclose to the value obtained by equilibrium analysis. Fig. 4 showed the specificity of AI1 binding to Leydig cell receptor sites. No inhibition in binding was observed with the neurohypophyseal peptide, arginine vasopressin and diterpene, forskolin. On the other hand, the binding-inhibition potency of various analogues and fragment was noted. [Sar1,Ala8]AII,AII, and [des-Asp']AII were potent displacers of specific '251-AIIbinding and theirKOvalues were similar to the observed K. for AI1 (1.7 X 10" M-'). AI was found to be 100 times less potent than AI1 in the binding assay, and the smaller fragment of AI1 (AII, Refs. 5-8) displayed weak inhibitory effect on '251-AIIbinding. LHRH (hypothalamic gonadotropin-releasing hormone), an unrelated decapeptide, competed quite effectively with lZ5I-AIIfor binding to Leydig cell receptor sites when present in relatively high concentrations, although it was less potent (1000 times) than the AII. The potency order of these compounds for AI1 receptors is AII> [Sar',Ala8]AII> [des-Asp']AII> AI>LHRH> AI1 (5-8). LHRH (hypothalamic gonadotropin-releasing hormone) has shown its ability to inhibit lZ5I-AIIbinding in Leydig cells. This is consistent with earlier studies by Capponi and Catt (25) in the adrenal cortex and uterus suggesting that the binding inhibition ofAI1by LHRH is probably due to a common structural feature in the COOH-terminal sequence of LHRH and AII. Although a number of studies have indicated the presence of the renin-angiotensinsystem in reproductive tissues on the basis of immunohistochemical, immunofluorescence, HPLC, and radioimmunoassay studies (1-4,6, 26), its physiological significance and possiblerole in gonadal function are still unclear. Our studies have provided direct evidence for the 5073 AII Receptors and Actions FIG.7. Left, inhibitory effect ofAI1 on testosterone production. Leydig cells (1 X 10' cells/ml) were incubated for 120 min with various concentrations of AI1 (lO"o-lO"j M) in the presence of hCG and hCG plus forskoli (39.Each point represents the mean f S.E. of triplicate incubations. Right, effect of pertussis toxin (Py)on AII-induced inhibition on testosterone production. Leydig cells (1 x 10' cell/ml) were preincubated with pertussis toxin (30 ng/ml) for 60 min. Incubation was further carried out for 60 min in the presence or inabsence of the indicated hCG doses with or without AII. Each point representsthe means f S.E. of triplicate incubations. 100 1- 1 hCG 1260 pMI t A 1 I hCG + F110-9 MI t A I1 1 Testosterone 150- .- 0 10°C Angiotensin II (MI I t A 1 I A11 I hcG + hCG hCG PT+hCG I260 OMI + + AI1 All A11 TABLE I1 Effect of pertussis toxin(PT)on AIZ-induced inhibition of adenylate cyclnse activity Adenylate cyclase activity was determined in the membranes of Leydig cells, pretreated and untreated with pertussis toxin (pg/ml). The membranes were incubated with GTP (lo-' M) and LH (lo-' M) in the presence and in the absence of AI1 (lo-' M). Mean values between groups by student's t test are as follows: b versus a and c ( p < 0.05);e versus d and f ( p < 0.01). Additions Adenylate cyclase activity pnwl/mg protein/ 15 min -I hCG cAMP 12.6 pMI hCG + cAMP hCG + A II CAMP+ hCG+ hCG hCG A II CAMP+ I260 pMI + A II cAMP hCG t hCG + CAM + A II TABLE I Effect of 8-bromo-CAMP on forskolin-induced inhibition of testosterone productionby Leydig cells Testosterone production was measured as described under "Experimental Procedures." Leydig cells (lo6cells/ml) were incubated for 120 min in the presence and in the absence of described additions. Mean values between groups by student's t test are as follows: b versus a and d ( p < 0.05);b versus c ( p < 0.01). Additions 10-9 M 8-Bromo-CAMP lo-' M hCG 260 pM Testosterone w/l@ &dig cells - + + + 29 f 2 39 f 3 71 f 7 (a) 88f7 51 f 4 ( b ) 113 f 10 ( c ) 121 f 14 ( d ) 82 f 8 ( e ) 154 f 12 (f) A II FIG. 8. Effect of 8-bromo-CAMP on An-induced inhibition of testosterone production. Leydig cells (1 X 10' cells/ml) were incubated for 60 min with 8-bromo-CAMPin the presence and in the absence of hCG and/or AII. Each point represents the mean f S.E. of triplicate incubations. Forskolin Control Control + PT GTP GTP + PT GTP + AI1 GTP + AI1 + PT LH + GTP LH + GTP + AI1 LH + GTP + AI1 + PT 28 f 2 16 t 1 319 f 24 337 -+ 28 (a) 338 f 12 246 f 11 ( b ) 308 -+ 15 ( c ) 299 f 10 ( d ) presence of functional AI1 receptors, since AI1 acutely inhibits (as early as 15 min) gonadotrophin stimulation of cyclic AMP pools and testosterone production in Leydig cells. Fig. 5, left, shows AII-induced inhibition of intracellular cAMP production and the effect of pertussis toxin on this inhibition. Cells were stimulated with a submaximal dose of hCG in the presence and in the absence of increasing concentrations of M. The inhibitory effect of AI1 was doseAII, lO-"-lO"j dependent with an ID5, of 0.5 X 10"' M. However, this effect was found to be largely prevented when cells were incubated with pertussis toxin prior to the addition of AI1 and/or hCG. The inhibitory effect of AI1 affected the hormonal stimulus over the entire dose-response range of hCG concentrations. As a result of addition of a submaximal dose of AI1 to the Leydig cells stimulated by various doses of hCG, the doseresponse curve showed a significant increase in EDso(2-fold), Fig. 5, right. Our studies have also provided evidence for an inhibitory effect of AI1 on receptor-bound cAMP (Fig. 6). Both basal and hCG-stimulated cAMP production were inhibited by the addition ofAI1 to the cells.However, this inhibition was prevented when cells were incubated with pertussis toxin prior to the addition of AI1 and/or hCG. As a result of reduced production of CAMP,AI1 (10"o-10-6 M), in a dose-dependent manner, inhibited testosterone production by Leydig cells stimulated by hCG (ID50l x 10"' M). This was commensurate with its binding affinity. Further addition 5074 AII Receptors and Actions of forskolin (lo-’ M) to a submaximal inhibitory dose of AI1 caused an additive inhibition of testosterone production (Fig. 7, left),and this finding, therefore, indicated that both substances are exerting their inhibitory effects through a common pathway. This inhibitory action of AI1 was found to be prevented when cells were pretreated with pertussis toxin prior to theaddition of hCG and/or AI1 (Fig. 7, right).This finding resembles our previous studies, demonstrating the involvement of a pertussis toxin-sensitive regulatory protein in the inhibitory action of low doses of forskolin (7). Moreover, our results are consistent with early studies on the adrenal (22) and more recent studies in liver (27) and kidney (28) which showed that AI1 receptors are functionally linked to the inhibitory unit of the adenylate cyclase system. M) bypassed the inhibFurthermore, 8-bromo-CAMP( itory effect of AI1in hCG-stimulatedcells (Fig. 8).This cAMP derivative was also shown to exert a similar effect on the inhibitory action of a low dose of forskolin (Table I). Also, AI1 significantly decreased GTP and luteinizing hormone plus GTP-stimulated adenylate cyclase activities in membranes, and this inhibition was prevented by pretreatment of membranes with pertussis toxin (Table 11). Thus, thereduction in cAMP levels by AI1 is attributable to the inhibition of adenylate cyclase activity in the plasma membrane. It is, therefore, proposed on the basis of our studies that the Ni unit of adenylate cyclase is involved in the inhibitory action of AI1 on adenylate cyclase and consequently on cAMP pools and testosterone production. However, we cannot rule out the participation of other additional signal-transducing systems in mediating the inhibitory action of AI1 in the Leydig cells, since the role of calcium as a second messenger in the action ofAI1 is well documented in liver (29), adrenals (30,31), smooth muscle (32), and kidney (28). Also, the existence of a pertussis toxin-independentguanine nucleotide inhibition has been postulated for several systems (27,33,34). Furthermore, in liver (27) and in kidney (28), two different kinds of receptor-dependent mechanisms were delineated for the cellular responses of AI1 including phospholipase C/increase in intracellular calcium and adenylate cyclase system. However, in the present study, the finding that 8-bromo-CAMPbypassed the inhibitory effect of AI1 on hCG-stimulated testosterone production is suggestive that the pertussis-sensitive regulatory protein (Ni) mainly mediates the inhibitory effect of AI1 and modulates LH stimulationof Leydig cells.Because of the predominant localization of angiotensin-converting enzyme in the testicular tubular elements, it is likely that AI1 possesses a physiological paracrine regulatory function and the locally produced hormone would also effectively exert homologous negative modulatory influence on hormonal-stimulated events in the Leydig cells. REFERENCES 1. Parmentier, M., Inagami, T., Pochet, R., and Desclin, J. C. (1983) Endocrinology 112,1318-1323 2. Naruse, K., Murakoshi, M., Osamura, R. Y., Naruse, M., Toma, H., Watanabe, K., Demura, H., Inagami, T., and Shizume, K. View publication stats (1985) J. Clin. Endocrinol. Metab. 61, 172-177 3. Pandey, K. N., Misono, K. S., and Inagami, T. 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