0013-7227/79/1041-0189$02.00 Endocrinology Copyright © 1979 by The Endocrine Society
Vol. 104, No. 1 Printed in U.SA.
Modulation of Follicle-Stimulating Hormone-Sensitive Rat Testicular Adenylate Cyclase Activity by Guanyl Nucleotides* HUSSEIN ABOU-ISSA AND LEO E. REICHERT, JR. Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322
ABSTRACT. We have studied modulation of FSH-sensitive adenylate cyclase activity in testes of immature rats by guanyl nucleotides. Highly purified hFSH alone stimulated adenylate cyclase activity 2.2-fold over basal levels. Addition of the GTP analog, 5'-guanylyl imidodiphosphate [Gpp(NH)p], caused an additional 2.8-fold augmentation of adenylate cyclase activity to 6 times over basal levels and 3.7 times greater than that seen in the presence of Gpp(NH)p alone. GTP did not significantly stimulate basal levels of adenylate cyclase and augmented FSH stimulated activity by 1.4-fold; other nucleotides were without effect. Half-maximum activation of adenylate cyclase in each instance was produced by approximately similar concentrations of either guanyl nucleotide (about 10 /nM). The Km for hormone
I
N PREVIOUS studies, we have characterized the interaction of biologically active, radioiodinated human FSH ([125I]i.odo-hFSH) with hormone-specific receptors in membrane fractions derived from rat and beef testis (1, 2). The properties of the [125I]iodo-hFSH interaction with detergent-solubilized beef testicular receptors have also been described (1). The ability of FSH to stimulate adenylate cyclase consequent to its interaction with testicular receptor has been reported by several laboratories. However, factors regulating hormone stimulation of adenylate cyclase by FSH are not yet understood. It is known that purine nucleotides amplify the response of adenylate cyclase to a variety of hormones (3-6). Various analogs of GTP, including 5'-guanylylimidodiphosphate [Gpp(NH)p] (7-11), have proven more effective than GTP in many such systems. In this report, the role of guanyl nucleotides and their analogs in modulation of FSH stimulation of testicular adenylate cyclase activity has been studied. The results suggest a specific role of guanyl nucleoside triphosphate in this effect.
activation of adenylate cyclase was nearly the same in the presence and absence of Gpp(NH)p. Maximum adenylate cyclase stimulation in the presence of nucleotide and/or hFSH was always less than obtained by fluoride alone. Of all nucleotides tested, only GTP and its analog, Gpp(NH)p, significantly augmented FSH stimulation of testicular adenylate cyclase activity. Gpp(NH)p also markedly inhibited binding of radiolabeled hFSH to testicular receptor, but at a concentration 15-fold greater than that required for significant stimulation of testicular adenylate cyclase activity. The results suggest a specific role for guanyl nucleoside triphosphate in regulation of FSH effects on testicular adenylate cyclase activity. (Endocrinology 104: 189, 1979)
was used in this study for stimulation of adenylate cyclase activity and as the radioligand in binding studies. Iodination of LER-1801-3 was by a modified chloramine T procedure (12). GTP and other nucleotides were purchased from the Sigma Chemical Co.; GppNHp was purchased from ICN Pharmaceuticals; and (a-32P)ATP (200 Ci/mmol) and [3H]cAMP (50 Ci/mmole) were obtained from New England Nuclear. Preparation of testes membranes Testis from immature (12 day old) rats of the SpragueDawley strain were utilized in these studies. In a typical experiment, testes were excised from 20 rats after dry ice asphyxiation, the tunica albuginea was removed, and testicular tissue was homogenized in 0.25 M sucrose-0.02 M Tris-HCl buffer, pH 7.5. After filtration through four layers of cheesecloth, the homogenate was centrifuged at 32,000 X g for 20 min (4 C). The final pellet was suspended on 0.02 M Tris-HCl buffer, pH 7.5, containing 0.1% bovine serum albumin and was used immediately either for adenylate cyclase assays or for measurement of [125I]iodo-hFSH binding. Assay of adenylate cyclase
Materials and Methods Materials Highly purified hFSH fraction LER-1801-3 (4019 IU/mg) Received February 13, 1978. * This work was supported by USPHS Grant HD-08228.
The assay of adenylate cyclase was performed as described by Salomon et al. (13) and based on the formation of [32P]cAMP from [a32P]ATP in a mixture containing 40 mM TrisHCl buffer (pH 7.5), 5 mM MgCl2, 0.5 mM cAMP, 0.1% bovine serum albumin, 10 mM creatine phosphate, 0.1 mg/ml creatine phosphokinase, 1 mM [a32P]ATP (approximately 2 jnCi), 0.25
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ABOU-ISSA AND REICHERT, JR.
190
mM l-methyl-3-isobutylxanthine (MIX), and 100-250 jug membrane protein in afinalvolume of 0.2 ml. Hormones, nucleotides, and other agents were added, as detailed in individual experiments. Incubation was carried out for 15 min at 35 C, and the reaction was then stopped by immersion in a boiling water bath for 5 min. Approximately 30,000 cpm [3H]cAMP were included for recovery, and water was then added to a final volume of 1 ml. The mixture was clarified by centrifugation (7000 rpm for 10 min at 4 C). The resulting supernatant was chromatographed on Dowex AG-50 WX4 and then on neutral alumina (13). The radioactivity in the final column eluates was counted in a Beckman LS-100C liquid scintillation spectrometer. The conditions of the assay have been described in detail elsewhere (14). The rate of [a-32P]cAMP formation increases linearly with time of incubation up to about 15 min at 32 C in the absence and presence of FSH or fluoride and is also linear with enzyme protein concentration up to 500 /xg (14, 15).
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Vol. 104, No. 1 Printed in U.SA.
Modulation of Follicle-Stimulating Hormone-Sensitive Rat Testicular Adenylate Cyclase Activity by Guanyl Nucleotides* HUSSEIN ABOU-ISSA AND LEO E. REICHERT, JR. Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322
ABSTRACT. We have studied modulation of FSH-sensitive adenylate cyclase activity in testes of immature rats by guanyl nucleotides. Highly purified hFSH alone stimulated adenylate cyclase activity 2.2-fold over basal levels. Addition of the GTP analog, 5'-guanylyl imidodiphosphate [Gpp(NH)p], caused an additional 2.8-fold augmentation of adenylate cyclase activity to 6 times over basal levels and 3.7 times greater than that seen in the presence of Gpp(NH)p alone. GTP did not significantly stimulate basal levels of adenylate cyclase and augmented FSH stimulated activity by 1.4-fold; other nucleotides were without effect. Half-maximum activation of adenylate cyclase in each instance was produced by approximately similar concentrations of either guanyl nucleotide (about 10 /nM). The Km for hormone
I
N PREVIOUS studies, we have characterized the interaction of biologically active, radioiodinated human FSH ([125I]i.odo-hFSH) with hormone-specific receptors in membrane fractions derived from rat and beef testis (1, 2). The properties of the [125I]iodo-hFSH interaction with detergent-solubilized beef testicular receptors have also been described (1). The ability of FSH to stimulate adenylate cyclase consequent to its interaction with testicular receptor has been reported by several laboratories. However, factors regulating hormone stimulation of adenylate cyclase by FSH are not yet understood. It is known that purine nucleotides amplify the response of adenylate cyclase to a variety of hormones (3-6). Various analogs of GTP, including 5'-guanylylimidodiphosphate [Gpp(NH)p] (7-11), have proven more effective than GTP in many such systems. In this report, the role of guanyl nucleotides and their analogs in modulation of FSH stimulation of testicular adenylate cyclase activity has been studied. The results suggest a specific role of guanyl nucleoside triphosphate in this effect.
activation of adenylate cyclase was nearly the same in the presence and absence of Gpp(NH)p. Maximum adenylate cyclase stimulation in the presence of nucleotide and/or hFSH was always less than obtained by fluoride alone. Of all nucleotides tested, only GTP and its analog, Gpp(NH)p, significantly augmented FSH stimulation of testicular adenylate cyclase activity. Gpp(NH)p also markedly inhibited binding of radiolabeled hFSH to testicular receptor, but at a concentration 15-fold greater than that required for significant stimulation of testicular adenylate cyclase activity. The results suggest a specific role for guanyl nucleoside triphosphate in regulation of FSH effects on testicular adenylate cyclase activity. (Endocrinology 104: 189, 1979)
was used in this study for stimulation of adenylate cyclase activity and as the radioligand in binding studies. Iodination of LER-1801-3 was by a modified chloramine T procedure (12). GTP and other nucleotides were purchased from the Sigma Chemical Co.; GppNHp was purchased from ICN Pharmaceuticals; and (a-32P)ATP (200 Ci/mmol) and [3H]cAMP (50 Ci/mmole) were obtained from New England Nuclear. Preparation of testes membranes Testis from immature (12 day old) rats of the SpragueDawley strain were utilized in these studies. In a typical experiment, testes were excised from 20 rats after dry ice asphyxiation, the tunica albuginea was removed, and testicular tissue was homogenized in 0.25 M sucrose-0.02 M Tris-HCl buffer, pH 7.5. After filtration through four layers of cheesecloth, the homogenate was centrifuged at 32,000 X g for 20 min (4 C). The final pellet was suspended on 0.02 M Tris-HCl buffer, pH 7.5, containing 0.1% bovine serum albumin and was used immediately either for adenylate cyclase assays or for measurement of [125I]iodo-hFSH binding. Assay of adenylate cyclase
Materials and Methods Materials Highly purified hFSH fraction LER-1801-3 (4019 IU/mg) Received February 13, 1978. * This work was supported by USPHS Grant HD-08228.
The assay of adenylate cyclase was performed as described by Salomon et al. (13) and based on the formation of [32P]cAMP from [a32P]ATP in a mixture containing 40 mM TrisHCl buffer (pH 7.5), 5 mM MgCl2, 0.5 mM cAMP, 0.1% bovine serum albumin, 10 mM creatine phosphate, 0.1 mg/ml creatine phosphokinase, 1 mM [a32P]ATP (approximately 2 jnCi), 0.25
189
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 15 November 2015. at 19:17 For personal use only. No other uses without permission. . All rights reserved.
ABOU-ISSA AND REICHERT, JR.
190
mM l-methyl-3-isobutylxanthine (MIX), and 100-250 jug membrane protein in afinalvolume of 0.2 ml. Hormones, nucleotides, and other agents were added, as detailed in individual experiments. Incubation was carried out for 15 min at 35 C, and the reaction was then stopped by immersion in a boiling water bath for 5 min. Approximately 30,000 cpm [3H]cAMP were included for recovery, and water was then added to a final volume of 1 ml. The mixture was clarified by centrifugation (7000 rpm for 10 min at 4 C). The resulting supernatant was chromatographed on Dowex AG-50 WX4 and then on neutral alumina (13). The radioactivity in the final column eluates was counted in a Beckman LS-100C liquid scintillation spectrometer. The conditions of the assay have been described in detail elsewhere (14). The rate of [a-32P]cAMP formation increases linearly with time of incubation up to about 15 min at 32 C in the absence and presence of FSH or fluoride and is also linear with enzyme protein concentration up to 500 /xg (14, 15).
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o 50 < -B x
