237 Honn. Metab. Res. 10 (1978) 237-242
An Increase of Pituitary 3',5' Cyclic Adenosine Monophosphate Produced by Estradiol Benzoate In Vitro: Possible Implication of This Increase in the Secretion of Luteinizing Hormone R. KObayashi, A. Harada, M. Kotani, T, Yamada, and K. Wakabayashi Department of Medicine, Institute of Adaptation Medicine, School of Medicine, Shinshu University, Matsumoto, and Hormone Assay Center, Institute of Endocrinology, Gunma University, Maebashi, Japan
mono phosphate (cyclic AMP) was studied in vitro
Summary
Downloaded by: University of Illinois. Copyrighted material.
In an attempt to study the site and mechanism of action of in the presence of estradiol benzoate (EB) and LHestrogen in producing positive feedback control, porcine anRH. Finally, a possible effect of clomiphene citrate, terior pituitary slices were incubated in vitro in the presence progesterone and testosterone propionate on the inof estradiol benzoate (EB). EB elevated pituitary cyclic AMP crease of pituitary cyclic AMP produced hy EB was concentration within 5 min and augmented pituitary restudied in vitra. lease of luteinizing hormone (LH). The magnitude of increase of cyclic AMP and LH release was related to the doses of EB used. Also, luteinizing hormone releasing hormone Materials and Methods (LH-RH) elevated pituitary cyclic AMP concentration and Porcine pituitary glands wen: obtained from an abbatoir. stimulated pituitary release of LH. The magnitude of inOnly the anterior pituitaries from male animals were used crease of cyclic AMP and LH release was inversely related in the experiments. After gentle separation of anterior pito the doses of LH-RH used. EB and LH-RH were additive tuitaries, slices 0.5 mm thick and weighing 20- 30 mg were in increasing cyclic AMP. Progesterone and clomiphene cimade with a Stadie-Riggs microtome. For the measurement trate interfered with an increase of pituitary cyclic AMP of cold cyclic AMP, the pituitary slices were preincubated produced by EB, but did not significantly affect the basal at 37°C for 30 min in a flask containing 1 ml Krebs-Ringer level of pituitary cyclic AMP. Testosterone propionate, hubicarbonate buffer (pH 7.4) plus 50mg/l00 ml glucose man chorionic gonadotropin and hexestrol were without ef(KRBG) with 10-2 M theophylline. After this preincubation fect on either basal or stimulated level of pituitary cyclic the slices were then incubated in 0.5 ml KRBG with theoAMP. Since cyclic AMP and dibutyryl cyclic AMP (DBe) phylline (l0-2M) and test material (EB1), LH-RH, elomistimulated LH release, it is suggested that EB directly stiphene citrate 2 ), progesterone 2), testosterone propionate 1), mulates the release of LH by augmenting cyclic AMP synhuman chorionic gonadotropin and hexestrol 2) at 37°C for thesis in the anterior pituitary. 5 to 60 min. The test substances were dissolved or diluted in KRBG. The KRBG was gassed with a mixture of 5 % C02Key-Words: Estrogen-Pituitary Cyclic AMP - LH Release 95 % 02 for 20 min prior to use. After the second incubation, the slices were frozen in acetone-dry ice and weighIntroduction ed accurately. The slices were then boiled for 5 min and h.omogenized with 0.5 ml of 0.1 M Tris-HCl buffer (pH 7.4). Estrogen can be shown to induce ovulation in a vaAfter centrifugation (3,000 rpm) for 20 min, the assay was riety of experimental conditions and of different done by the protein binding assay for cyclic AMP of Gilman (Gilman 1970) with a minor modification. The binding prospecies (Everett 1964). This effect is mediated by tein was obtained from rat liver according to the method restimulation of gonadotrop in secretion (Ba"aclough ported by Kumon et al. (Ku mon, Yamamura and Nishizuko 1973). The loeation of this stimulatory feedback ac- 1970). Instead of collecting the bound cyclic AMP on millition of estrogen is not weIl established, and either pore filter, the bound cyelic AMP was separated from the the anterior pituitary (Döcke and Dörner 1965; unbound form by using charcoal suspension and the inhibitor pro tein was not used. For the measurement of pituitary Weick and Davidson 1970; Weick et al. 1971) or the hypothalamus (Westman and Jacobsohn 1938; Pressel release of LH, the second incubation was performed for f20 min in the presence of EB, LH·RH,cyclic AMP and 1961; Palka, Ramirez and Sawyer 1966) has heen con- DBe. After the second incubation, the pituitary slices were sidered as the possible site of action of estrogen. Furweighed accurately and the incubation medium was stored thermore, the exact biochemical step(s) involved in at - 20 oe until used. Porcine LH was measured by radioimmunoassay according to the method by Monroe et al. this positive feedback control has never heen shown.
Because of these uncertainties, we have attempted 1) These compounds were obtained from the Company in to study the effect of estrogen, LH-RH, cyclic AMP the form of solution, and were diluted with KRBG. Solvents of the compounds were also obtained from the and DBC on in vitra release of LH from the porcine Company, and were similarly diluted with KRBG. The anterior pituitary. To study a possible hiochemical diluted solution was used in the control experiment. step involved in positive feedback control, an increase 2) These compounds were dissolved in the absolute alcohol of pituitary concentration of 3',5' -cyclic adenosine and then diluted with KRBG. Absolute a\cohol was also diluted with KRBG to use in the control experiment. Received: 14 Jan. 1976 0018-5043/78
0332-0237
Accepted: 15 May 1977 S 05.00
© 1978 Gerog
Thieme Publishers
238
R. Kobayashi, A. Harada, M. Kotani, T. Yamada, and K. Wakabayashi
Q)
~2
B
g'
----+-------------C~~rOI
~
E a. ........
Results
a. ~
u 0'
20'
10'
30'
TIME
IN
40'
50
60'
MINUTES
Fig. 1 Effect of estradiol benzoate (EB) on pituitary cyclic AMP concentration was indicated. Circles and vertical Iines indicated mean ± SE calculated from 5 determinations. Statistical analysis: Control 0 min vs EB 5 min p < 0.001 Control 15 min vs EB 15 min P < 0.001 Control 15 min vs EB 30 min p< 0.05 Control 60 min vs EB 30 min p < 0.05
6
P
An Increase of Pituitary 3',5' Cyclic Adenosine Monophosphate Produced by Estradiol Benzoate In Vitro: Possible Implication of This Increase in the Secretion of Luteinizing Hormone R. KObayashi, A. Harada, M. Kotani, T, Yamada, and K. Wakabayashi Department of Medicine, Institute of Adaptation Medicine, School of Medicine, Shinshu University, Matsumoto, and Hormone Assay Center, Institute of Endocrinology, Gunma University, Maebashi, Japan
mono phosphate (cyclic AMP) was studied in vitro
Summary
Downloaded by: University of Illinois. Copyrighted material.
In an attempt to study the site and mechanism of action of in the presence of estradiol benzoate (EB) and LHestrogen in producing positive feedback control, porcine anRH. Finally, a possible effect of clomiphene citrate, terior pituitary slices were incubated in vitro in the presence progesterone and testosterone propionate on the inof estradiol benzoate (EB). EB elevated pituitary cyclic AMP crease of pituitary cyclic AMP produced hy EB was concentration within 5 min and augmented pituitary restudied in vitra. lease of luteinizing hormone (LH). The magnitude of increase of cyclic AMP and LH release was related to the doses of EB used. Also, luteinizing hormone releasing hormone Materials and Methods (LH-RH) elevated pituitary cyclic AMP concentration and Porcine pituitary glands wen: obtained from an abbatoir. stimulated pituitary release of LH. The magnitude of inOnly the anterior pituitaries from male animals were used crease of cyclic AMP and LH release was inversely related in the experiments. After gentle separation of anterior pito the doses of LH-RH used. EB and LH-RH were additive tuitaries, slices 0.5 mm thick and weighing 20- 30 mg were in increasing cyclic AMP. Progesterone and clomiphene cimade with a Stadie-Riggs microtome. For the measurement trate interfered with an increase of pituitary cyclic AMP of cold cyclic AMP, the pituitary slices were preincubated produced by EB, but did not significantly affect the basal at 37°C for 30 min in a flask containing 1 ml Krebs-Ringer level of pituitary cyclic AMP. Testosterone propionate, hubicarbonate buffer (pH 7.4) plus 50mg/l00 ml glucose man chorionic gonadotropin and hexestrol were without ef(KRBG) with 10-2 M theophylline. After this preincubation fect on either basal or stimulated level of pituitary cyclic the slices were then incubated in 0.5 ml KRBG with theoAMP. Since cyclic AMP and dibutyryl cyclic AMP (DBe) phylline (l0-2M) and test material (EB1), LH-RH, elomistimulated LH release, it is suggested that EB directly stiphene citrate 2 ), progesterone 2), testosterone propionate 1), mulates the release of LH by augmenting cyclic AMP synhuman chorionic gonadotropin and hexestrol 2) at 37°C for thesis in the anterior pituitary. 5 to 60 min. The test substances were dissolved or diluted in KRBG. The KRBG was gassed with a mixture of 5 % C02Key-Words: Estrogen-Pituitary Cyclic AMP - LH Release 95 % 02 for 20 min prior to use. After the second incubation, the slices were frozen in acetone-dry ice and weighIntroduction ed accurately. The slices were then boiled for 5 min and h.omogenized with 0.5 ml of 0.1 M Tris-HCl buffer (pH 7.4). Estrogen can be shown to induce ovulation in a vaAfter centrifugation (3,000 rpm) for 20 min, the assay was riety of experimental conditions and of different done by the protein binding assay for cyclic AMP of Gilman (Gilman 1970) with a minor modification. The binding prospecies (Everett 1964). This effect is mediated by tein was obtained from rat liver according to the method restimulation of gonadotrop in secretion (Ba"aclough ported by Kumon et al. (Ku mon, Yamamura and Nishizuko 1973). The loeation of this stimulatory feedback ac- 1970). Instead of collecting the bound cyclic AMP on millition of estrogen is not weIl established, and either pore filter, the bound cyelic AMP was separated from the the anterior pituitary (Döcke and Dörner 1965; unbound form by using charcoal suspension and the inhibitor pro tein was not used. For the measurement of pituitary Weick and Davidson 1970; Weick et al. 1971) or the hypothalamus (Westman and Jacobsohn 1938; Pressel release of LH, the second incubation was performed for f20 min in the presence of EB, LH·RH,cyclic AMP and 1961; Palka, Ramirez and Sawyer 1966) has heen con- DBe. After the second incubation, the pituitary slices were sidered as the possible site of action of estrogen. Furweighed accurately and the incubation medium was stored thermore, the exact biochemical step(s) involved in at - 20 oe until used. Porcine LH was measured by radioimmunoassay according to the method by Monroe et al. this positive feedback control has never heen shown.
Because of these uncertainties, we have attempted 1) These compounds were obtained from the Company in to study the effect of estrogen, LH-RH, cyclic AMP the form of solution, and were diluted with KRBG. Solvents of the compounds were also obtained from the and DBC on in vitra release of LH from the porcine Company, and were similarly diluted with KRBG. The anterior pituitary. To study a possible hiochemical diluted solution was used in the control experiment. step involved in positive feedback control, an increase 2) These compounds were dissolved in the absolute alcohol of pituitary concentration of 3',5' -cyclic adenosine and then diluted with KRBG. Absolute a\cohol was also diluted with KRBG to use in the control experiment. Received: 14 Jan. 1976 0018-5043/78
0332-0237
Accepted: 15 May 1977 S 05.00
© 1978 Gerog
Thieme Publishers
238
R. Kobayashi, A. Harada, M. Kotani, T. Yamada, and K. Wakabayashi
Q)
~2
B
g'
----+-------------C~~rOI
~
E a. ........
Results
a. ~
u 0'
20'
10'
30'
TIME
IN
40'
50
60'
MINUTES
Fig. 1 Effect of estradiol benzoate (EB) on pituitary cyclic AMP concentration was indicated. Circles and vertical Iines indicated mean ± SE calculated from 5 determinations. Statistical analysis: Control 0 min vs EB 5 min p < 0.001 Control 15 min vs EB 15 min P < 0.001 Control 15 min vs EB 30 min p< 0.05 Control 60 min vs EB 30 min p < 0.05
6
P
