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Atrial natriuretic factor stimulates testosterone production by mouse interstitial cells
European Journal of Pharmacology, 115 (1985) 125-126
125
Elsevier
Rapid communication ATRIAL NATRIURETIC FACTOR STIMULATES TESTOSTERONE PRODUCTION BY MOUSE INTERSTITIAL CELLS FREDERICKBEX * and ALANCORBIN Division of Experimental Therapeutics, Wyeth Laboratories, Inc., P.O. Box 8299, Philadelphia, PA 19101, U.S.A.
Received31 July 1985, accepted 1 August1985
Peptides have been isolated from mammalian atria that exhibit natriuretic, diuretic and vasodilatory properties. Purification, identification and synthesis have revealed that these atrial natriuretic factors (ANF) share a biologically active core amino acid sequence and that they presumably arise from a common large precursor polypeptide (for review see Needleman et al., 1984). Extensive investigation with representative bioactive synthetic atrial peptides has demonstrated that, in addition to potent vascular and renal effects, they also inhibit adrenal mineralocorticoid and glucocorticoid secretion (De Lean et al., 1984). The mechanism by which these peptides exert these diverse effects is as yet unclear. In this regard there is evidence to indicate that certain of ANF's actions may be mediated through stimulation of particulate guanylate cyclase (Waldman et al., 1984), whereas inhibition of adenylate cyclase may be involved at other target sites (De Lean et al., 1985). To further explore the possibility of a general effect of these peptides on steroidogenesis and to evaluate a potential functional correlate for the increase in particulate guanylate cyclase produced by ANF in the testis (Waldman et al., 1984), we investigated the effect of human(h)ANF-(1-28) (Ser 1-Leu-Arg-Arg-Ser-Ser-CysT-Phe-Gly-Gly-ArgMet 12-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu,GlyC~23-Asn-Ser-Phe-Arg-Tyr 28) and rat atriopeptin II (APII; [Ile12]hANF-(5-27)) on mouse interstitial (Leydig) cell testosterone production. The method for determining the testosterone response of mouse interstitial cells in vitro was
* All correspondenceshouldbe addressedto F.B. 0014-2999/85/$03.30 © 1985 ElsevierSciencePublishersB.V.
similar to that reported by Ellinwood and Resko (1980), Testes from adult male CD1 mice (Charles River) were decapsulated and then mechanically dispersed in ice cold preincubation medium: medium 199 with 0.9% BSA, 25 mM Hepes buffer (pH 7.4), penicillin/streptomycin (100 IU/ml) and fungizone (1.25 /xg/ml). The homogenate was filtered through nylon mesh (100 #m) and the filtrate was incubated for 1 h at 34°C with shaking at 90 cycles/min under 95% 02-5% CO2. The homogenate was then centrifuged at 250 × g for 15 min. The packed cells were resuspended in incubation medium (preincubation medium containing 0.25 mM 3-methyl isobutyl xanthine and sodium heparin, 100 IU/ml). The concentration was adjusted to give a final cell count of 2-4 × 106 cells/ml. To duplicate polystyrene tubes were added the luteinizing hormone (LH) standards or experimental peptides in 75/~1 incubation medium along with 25/~1 preincubation medium containing 5% BSA and 100/~1 of the cell suspension. After incubation for 3 h (34°C, 90 cycles/min, 95% 02-5% CO2), 1 ml 0.01 M phosphate buffer, pH 7.0 was added to each tube followed by centrifugation at 1500 × g for 15 min. The supernatants were decanted and stored frozen at - 2 0 ° C until analyzed for testosterone by radioimmunoassay (Bex and Corbin, 1981). The testosterone response of mouse interstitial cells in vitro is a widely used bioassay for the glycoprotein hormone, LH (fig. 1, upper panel). These results demonstrate for the first time that, at micromolar concentrations, the atrial peptide, hANF-(1-28), also can produce a dose-related increase in testosterone secretion by these cells. This steroidogenic response appears to be specific and
126 MICT RESPONSE TO LH TESTOSTERONE PERCENT BASELINE (X.01) 21
"
10 100 LH (ng) MICT RESPONSE TO ATRIAL PEPTIDES 25 hANF5-28 21 17 APII hANF1-28
13 9 5 1 .01
, .1
1
PEPTIDE CONCENTRATION (/JM)
SRIF,CRF LHRH,ADH 10 hANF 1-11 hANF 18-28
Fig. 1. Mouse interstitial cell testosterone (MICT) production in response to rat luteinizing hormone (LH-RP1, NIAMDDK; upper panel) and various atrial and hypothalamic peptides (lower panel). Human atrial natriuretic factor (hANF-(1-28)), rat atriopeptin II (APII), somatostatin (SRIF) and corticotropin releasing factor (CRF) were obtained from Bachem, Inc.; hANF-(1-11), hANF-(5-28) and hANF-(18-28) were from Peninsula Labs; and luteinizing hormone releasing hormone (LHRH, Gonadorelin) was from Ayerst. An LH standard curve was included in each assay. Values represent means _ S.E.M. of assay replicates (LH, N=10; APII, N = 6; hANF, N = 2; single assay for remaining peptides) expressed as a percentage of respective 0 dose control. Testosterone production for controls (N = 10) after 3 h was 1.46 5-0.22 ng/ml medium.
n o t artifactual since a variety of p e p t i d e h o r m o n e s including somatostatin, CRF, LHRH and ADH h a d no effect over a wide range of concentrations. Initial a t t e m p t s at elucidating a structure-activity relationship have revealed that r e m o v a l of the first 4 N - t e r m i n a l a m i n o acids, ( h A N F - ( 5 - 2 8 ) ) or Ct e r m i n a l tyrosine d e l e t i o n a n d 12 p o s i t i o n lie substitution ( A P I I ; [Ilet2]hANF-(5-27)), does n o t affect the testosterone s t i m u l a t i n g p r o p e r t y . H o w -
ever m o r e extensively altered fragments, such as h A N F - ( 1 - 1 1 ) a n d h A N F - ( 1 8 - 2 8 ) , were inactive. This might reflect a r e q u i r e m e n t for the presence of the disulfide b o n d (Cys7-Cys 23) for testosterone s t i m u l a t i o n as has been shown for natriuretic a n d diuretic activity. W h i l e s t i m u l a t i o n of g u a n y l a t e cyclase has been i m p l i c a t e d in the renal a n d vascular actions of the atrial peptides, c o n c u r r e n t inc u b a t i o n with 10 - 5 M m e t h y l e n e blue, a cyclase inhibitor, d i d n o t m o d i f y the interstitial cell doseresponse to A P I I ( u n p u b l i s h e d observations). Since a n increase in c A M P is involved in the steroidogenic s t i m u l a t i o n b y L H , it is possible that this cyclic n u c l e o t i d e might also b e involved in the testicular action of A N F . However, it should b e n o t e d that a n i n h i b i t i o n rather t h a n s t i m u l a t i o n of a d e n y l a t e cyclase in the p i t u i t a r y b y [Ile~2]hANF (3-28) has been r e p o r t e d ( D e L e a n et al., 1985). T h e m e c h a n i s m for the s t i m u l a t i o n of testosterone p r o d u c t i o n b y the atrial p e p t i d e s a n d its physiological significance is the subject of o n g o i n g investigation.
References Bex, F.J. and A. Corbin, 1981, In vivo and in vitro investigation of the extrapituitary antireproductive effects of a potent LHRH agonist in immature and adult male rats, J. Androl. 3, 152. De Lean, A., J. Gutkowska, N. McNicoll, P.W. Schiller, M. Cantin and J. Genest, 1984, Characterization of specific receptors for atrial natriuretic factor in bovine adrenal zona glomerulosa, Life Sci. 35, 2311. Ellinwood, W.E. and J.A. Resko, 1980, Sex differences in biologically active and immunoactive gonadotropins in the fetal circulation of Rhesus monkeys, Endocrinology 107, 902. Needleman, P., M.G. Currie, D.M. Geller, B.R. Cole and S.P. Adams, 1984, Atriopeptins: potential mediators of an endocrine relationship between heart and kidney, Trends Pharmacol. Sci., December, 506. Waldman, S.A., R.M. Rapoport and F. Murad, 1984, Atrial natriuretic factor selectively activates particulate guanylate cyclase and elevates cyclic GMP in rat tissues, J. Biol. Chem. 250, 14332.
125
Elsevier
Rapid communication ATRIAL NATRIURETIC FACTOR STIMULATES TESTOSTERONE PRODUCTION BY MOUSE INTERSTITIAL CELLS FREDERICKBEX * and ALANCORBIN Division of Experimental Therapeutics, Wyeth Laboratories, Inc., P.O. Box 8299, Philadelphia, PA 19101, U.S.A.
Received31 July 1985, accepted 1 August1985
Peptides have been isolated from mammalian atria that exhibit natriuretic, diuretic and vasodilatory properties. Purification, identification and synthesis have revealed that these atrial natriuretic factors (ANF) share a biologically active core amino acid sequence and that they presumably arise from a common large precursor polypeptide (for review see Needleman et al., 1984). Extensive investigation with representative bioactive synthetic atrial peptides has demonstrated that, in addition to potent vascular and renal effects, they also inhibit adrenal mineralocorticoid and glucocorticoid secretion (De Lean et al., 1984). The mechanism by which these peptides exert these diverse effects is as yet unclear. In this regard there is evidence to indicate that certain of ANF's actions may be mediated through stimulation of particulate guanylate cyclase (Waldman et al., 1984), whereas inhibition of adenylate cyclase may be involved at other target sites (De Lean et al., 1985). To further explore the possibility of a general effect of these peptides on steroidogenesis and to evaluate a potential functional correlate for the increase in particulate guanylate cyclase produced by ANF in the testis (Waldman et al., 1984), we investigated the effect of human(h)ANF-(1-28) (Ser 1-Leu-Arg-Arg-Ser-Ser-CysT-Phe-Gly-Gly-ArgMet 12-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu,GlyC~23-Asn-Ser-Phe-Arg-Tyr 28) and rat atriopeptin II (APII; [Ile12]hANF-(5-27)) on mouse interstitial (Leydig) cell testosterone production. The method for determining the testosterone response of mouse interstitial cells in vitro was
* All correspondenceshouldbe addressedto F.B. 0014-2999/85/$03.30 © 1985 ElsevierSciencePublishersB.V.
similar to that reported by Ellinwood and Resko (1980), Testes from adult male CD1 mice (Charles River) were decapsulated and then mechanically dispersed in ice cold preincubation medium: medium 199 with 0.9% BSA, 25 mM Hepes buffer (pH 7.4), penicillin/streptomycin (100 IU/ml) and fungizone (1.25 /xg/ml). The homogenate was filtered through nylon mesh (100 #m) and the filtrate was incubated for 1 h at 34°C with shaking at 90 cycles/min under 95% 02-5% CO2. The homogenate was then centrifuged at 250 × g for 15 min. The packed cells were resuspended in incubation medium (preincubation medium containing 0.25 mM 3-methyl isobutyl xanthine and sodium heparin, 100 IU/ml). The concentration was adjusted to give a final cell count of 2-4 × 106 cells/ml. To duplicate polystyrene tubes were added the luteinizing hormone (LH) standards or experimental peptides in 75/~1 incubation medium along with 25/~1 preincubation medium containing 5% BSA and 100/~1 of the cell suspension. After incubation for 3 h (34°C, 90 cycles/min, 95% 02-5% CO2), 1 ml 0.01 M phosphate buffer, pH 7.0 was added to each tube followed by centrifugation at 1500 × g for 15 min. The supernatants were decanted and stored frozen at - 2 0 ° C until analyzed for testosterone by radioimmunoassay (Bex and Corbin, 1981). The testosterone response of mouse interstitial cells in vitro is a widely used bioassay for the glycoprotein hormone, LH (fig. 1, upper panel). These results demonstrate for the first time that, at micromolar concentrations, the atrial peptide, hANF-(1-28), also can produce a dose-related increase in testosterone secretion by these cells. This steroidogenic response appears to be specific and
126 MICT RESPONSE TO LH TESTOSTERONE PERCENT BASELINE (X.01) 21
"
10 100 LH (ng) MICT RESPONSE TO ATRIAL PEPTIDES 25 hANF5-28 21 17 APII hANF1-28
13 9 5 1 .01
, .1
1
PEPTIDE CONCENTRATION (/JM)
SRIF,CRF LHRH,ADH 10 hANF 1-11 hANF 18-28
Fig. 1. Mouse interstitial cell testosterone (MICT) production in response to rat luteinizing hormone (LH-RP1, NIAMDDK; upper panel) and various atrial and hypothalamic peptides (lower panel). Human atrial natriuretic factor (hANF-(1-28)), rat atriopeptin II (APII), somatostatin (SRIF) and corticotropin releasing factor (CRF) were obtained from Bachem, Inc.; hANF-(1-11), hANF-(5-28) and hANF-(18-28) were from Peninsula Labs; and luteinizing hormone releasing hormone (LHRH, Gonadorelin) was from Ayerst. An LH standard curve was included in each assay. Values represent means _ S.E.M. of assay replicates (LH, N=10; APII, N = 6; hANF, N = 2; single assay for remaining peptides) expressed as a percentage of respective 0 dose control. Testosterone production for controls (N = 10) after 3 h was 1.46 5-0.22 ng/ml medium.
n o t artifactual since a variety of p e p t i d e h o r m o n e s including somatostatin, CRF, LHRH and ADH h a d no effect over a wide range of concentrations. Initial a t t e m p t s at elucidating a structure-activity relationship have revealed that r e m o v a l of the first 4 N - t e r m i n a l a m i n o acids, ( h A N F - ( 5 - 2 8 ) ) or Ct e r m i n a l tyrosine d e l e t i o n a n d 12 p o s i t i o n lie substitution ( A P I I ; [Ilet2]hANF-(5-27)), does n o t affect the testosterone s t i m u l a t i n g p r o p e r t y . H o w -
ever m o r e extensively altered fragments, such as h A N F - ( 1 - 1 1 ) a n d h A N F - ( 1 8 - 2 8 ) , were inactive. This might reflect a r e q u i r e m e n t for the presence of the disulfide b o n d (Cys7-Cys 23) for testosterone s t i m u l a t i o n as has been shown for natriuretic a n d diuretic activity. W h i l e s t i m u l a t i o n of g u a n y l a t e cyclase has been i m p l i c a t e d in the renal a n d vascular actions of the atrial peptides, c o n c u r r e n t inc u b a t i o n with 10 - 5 M m e t h y l e n e blue, a cyclase inhibitor, d i d n o t m o d i f y the interstitial cell doseresponse to A P I I ( u n p u b l i s h e d observations). Since a n increase in c A M P is involved in the steroidogenic s t i m u l a t i o n b y L H , it is possible that this cyclic n u c l e o t i d e might also b e involved in the testicular action of A N F . However, it should b e n o t e d that a n i n h i b i t i o n rather t h a n s t i m u l a t i o n of a d e n y l a t e cyclase in the p i t u i t a r y b y [Ile~2]hANF (3-28) has been r e p o r t e d ( D e L e a n et al., 1985). T h e m e c h a n i s m for the s t i m u l a t i o n of testosterone p r o d u c t i o n b y the atrial p e p t i d e s a n d its physiological significance is the subject of o n g o i n g investigation.
References Bex, F.J. and A. Corbin, 1981, In vivo and in vitro investigation of the extrapituitary antireproductive effects of a potent LHRH agonist in immature and adult male rats, J. Androl. 3, 152. De Lean, A., J. Gutkowska, N. McNicoll, P.W. Schiller, M. Cantin and J. Genest, 1984, Characterization of specific receptors for atrial natriuretic factor in bovine adrenal zona glomerulosa, Life Sci. 35, 2311. Ellinwood, W.E. and J.A. Resko, 1980, Sex differences in biologically active and immunoactive gonadotropins in the fetal circulation of Rhesus monkeys, Endocrinology 107, 902. Needleman, P., M.G. Currie, D.M. Geller, B.R. Cole and S.P. Adams, 1984, Atriopeptins: potential mediators of an endocrine relationship between heart and kidney, Trends Pharmacol. Sci., December, 506. Waldman, S.A., R.M. Rapoport and F. Murad, 1984, Atrial natriuretic factor selectively activates particulate guanylate cyclase and elevates cyclic GMP in rat tissues, J. Biol. Chem. 250, 14332.