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Calcitonin secreting property of ipriflavone in the presence of estrogen
Life Sciences, Vol. 38, pp. 1535-1541 Printed in the U.S,A.
Pergamon Press
CALCITONIN SECRETING PROPERTY OF IPRIFLAVONE IN THE PRESENCE OF ESTROGEN
lwao Yamazaki and Masako Kinoshita B i o l o g y L a b o r a t o r i e s , C e n t r a l Research D i v i s i o n , Takeda Chemical I n d u s t r i e s , Ltd., 17-85, Jusohonmachi 2-chome, Yodogawaku, Osaka 532, Japan (Received in final form February ii~ 1986) Summary C a l c i t o n i n s e c r e t i o n i s i n f l u e n c e d by e s t r o g e n . In t h e p r e s e n t s t u d y , b a s a l and c a l c i u m - s t i m u l a t e d serum c a l c i t o n i n l e v e l s were r e d u c e d i n o v a r i e c t o m i z e d r a t s , and r e p l a c e m e n t e s t r o g e n a d m i n i s t r e d f o r 3 weeks i n c r e a s e d both c a l c i t o n i n l e v e l s t o t h o s e in i n tact rats. Ipriflavone, 7-isopropoxy-3-phenyl-4H-l-benzopyran-4one, a l o n e d i d n o t i n f l u e n c e e i t h e r c a l c i t o n i n l e v e l . However, i p r i f l a v o n e and s u b e f f e c t i v e d o s e s o f e s t r o g e n a d m i n i s t e r e d s i m u l t a n e o u s l y i n c r e a s e d both l e v e l s ; t h e i n c r e a s e depended upon t h e dose of ipriflavone. F u r t h e r m o r e , p r e t r e a t m e n t w i t h e s t r o n e r e s u l t e d in g r e a t e r c a l c i t o n i n r e l e a s e in r e s p o n s e t o v a r i o u s doses of c a l c i u m and p r e t r e a t m e n t w i t h e s t r o n e and i p r i f l a v o n e caused an even g r e a t e r release. These f i n d i n g s i n d i c a t e t h a t i p r i f l a v o n e i n c r e a s e s t h e s e n s i t i v i t y o f t h e t h y r o i d g la n d t o e s t r o g e n t o s e c r e t e c a l c i t o n i n in response to calcium. I p r i f l a v o n e , 7-isopropoxy-3-phenyl-4H-l-benzopyran-4-one, is devoid of est r o g e n i c a c t i v i t y but i n c r e a s e s t h e u t e r o t r o p i c a c t i v i t y o f e s t r o g e n (1). This p r o p e r t y o f i p r i f l a v o n e i s t h o u g h t t o be produced by d i r e c t a c t i o n on t h e t a r g e t organ. C a l c i t o n i n s e c r e t i o n from t h e t h y r o i d gland i s u n d e r t h e i n f l u e n c e of estrogen (2-5). I p r i f l a v o n e a l s o i n f l u e n c e s c a l c i t o n i n s e c r e t i o n in the presence o f e s t r o g e n ( 1 ) ; however, t h e d e t a i l s o f t h i s a c t i o n a r e n o t known, and t h e p r e s e n t s t u d i e s were d e s i g n e d t o o b t a i n i n f o r m a t i o n about t h e s e d e t a i l s . Materials and Methods Animal s Female Sprague-Dawley r a t s , 9 weeks o f age, were p u r ch ased from Japan CLEA Co. They were f e d a commercial d i e t (CE-2, Japan CLEA) and d r i n k i n g w a t e r ad l i b i t u m , in an a i r c o n d i t i o n e d e n v i r o n m e n t (24 ± I°C, 50-60~ h u m i d i t y ) i l l ~ - - T h a t e d f o r 14 h / d a y from 07:30 t o 21:30 h. The c a l c i u m , p h o sp h o r u s, and v i t a m i n D c o n t e n t s o f t h e d i e t were 1.2~, 1.1~, and 2.0 I U / g , r e s p e c t i v e l y . The animals were o v a r i e c t o m i z e d a t 10 weeks o f age. Compounds used Ipriflavone was synthesized in our Chemical Development Laboratories. The compound was suspended in water containing 1% hydroxypropyl cellulose (HPC-L, Nippon Soda Co., Japan). Estrone was purchased from Merck A.G. (West Germany), 0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Press Ltd.
1536
Calcitonin Secretion by Ipriflavone
Vol. 38, No. 17, 1986
and dissolved in sesame oil. Calcium gluconate monohydrate was purchased from Wako Pure Chemical Industries, Ltd. (Japan) and dissolved in saline (9 g NaCI/I) Procedures for administerin~ the compound and for serum collection Ipriflavone was administered orally and estrone subcutaneously starting one day after the ovariectomy and was continued daily for three weeks. On the day after the last dosing, blood (basal) was collected from the orbital vein under ether anesthesia. Then, calcium, usually 50 mg/kg body weight as calcium gluconate, was given in a single intraperitoneal injection. Thirty minutes afterwards, the animals were anesthetized with ether and blood (stimulated) was withdrawn from the abdominal aorta. It was confirmed beforehand that serum calcitonin reached a plateau level by i0 min after the intraperitoneal administration of this dose of calcium and maintained the level for 35 min. The separated serum was stored at -20°C until used for calcium and calcitonin determinations. Determination of serum calcium A spectrophotometric method (6) for determining serum calcium, using the metal complexing dye, orthocresolphthalein complexon, was applied; a commercial kit, Calcium C-Test Wako (Wako Pure Chemical Industries, Ltd.) was used. Assay of serum calcitonin Serum calcitonin was determined using a commercial radioimmunoassay kit for human calcitonin (Daiichi Radioisotope Labs., Japan) (7) by the double antibody technique. It was confirmed beforehand that the dilution curve of the rat thyroid extract prepared according to the method of Bell and Queener (8) paralleled the standard curve for human cal¢itonin of the radioimmunoassay kit. Values are expressed as pg equivalents of synthetic human calcitonin/ml, since a human calcitonin standard was utilized. The minimum detectable amount is 65 pg/ml, and the intra- and inter-assay coefficients of variation were 12.5% and 20.9%, respectively. Statistical analysis The statistical significance of differences between means was determined by the Student's t-test for two group comparison, and by Dunnett's test (9) for multiple group comparisons. Results Effect of ipriflavone alone on calcitonin secretion Ipriflavone, in daily doses of 50 to 400 mg/kg body weight for 3 weeks, did not influence the basal or the calcium-stimulated calcitonin level in ovarectomized rats (Table I); it did not influence uterine weight either. Effect of the combined administration of ipriflavone and estrone on calcitonin secretion As shown in Fig. i, basal and calcium-stimulated calcitonin levels were decreased in ovariectomized rats, although the effect of ovariectomy was much less on the basal level. Estrone increased both the basal and calcium-stimulated calcitonin levels dose-dependently up to the levels observed in intact rats. Estrone in daily doses of I0 ~g/kg body weight induced significant secretion but 1 ~g/kg did not. Therefore, the latter was chosen to be given combined with ipriflavone. The simultaneous administration of various doses of ipri-
8
8
8
7
50
i00
200
400
219 ± 3
217 ± 3
217 ± 3
217 ± 3
217 ± 3
start
279 ± 4
288 ± 7
285 ± 6
293 ± 5
285 ± 7
autopsy
Body weight (g) at
153 ± 8
146 ± S
156 ± 5
151 ± 4
152 ± 5
Uterine weight (mg)
10.2 ± 0.i
10.3 ± 0.3
10.4 ± 0.3
10.2 ± 0.I
10.8 ± 0.6
basal
17.6 ± 0.2
17.3 ± 0.7
17.6 ± 0.2
17.7 ± 0.5
18.4 ± 0.3
stimulated
Serum calcium (mg/dl)
486 ± 25
490 ± 20
468 ± 17
474 ± 23
395 ± 49
basal (A)
(B)
89
2,254 ± 254
2,133 ± 116
2,030 ±
2,088 ± 147
95
1,764 ± 232
1,643 ± 103
1,562 ±
1,613 ± 134
1,475 ± 181
A (B-A)
(pg/ml)
1,740 ± 135
stimulated
Serum c a l c i t o n i n
Rats were ovariectomized at i0 weeks of age and the intragastric pretreatment of ipriflavone was started one day after the operation. Calcium challenge (50 mg/kg body weight) was performed on the day after the last pretreatment. Values are expressed as means ± SEM.
8
0
Daily dose of No. ipriflavone of (mg/kg) rats
Body and uterine weights and changes in serum calcium and calcitonin concentrations in response to intraperitoneal administration of calcium in ovariectomized female rats pretreated with ipriflavone for three weeks
TABLE I
~n
< O m
c~
0
~D rt
fD r~
m
0
m
~o co
--J
Z O
OO
O
1538
Calcitonin Secretion by Ipriflavone
Vol. 38, No. 17, 1986
6,000
5,000
Et30
4,000
:_o b §
3,000
§ 2,000 E U~
1,000
,¸!!i 0 Ovariectom Ipriflavone (mg/kg/day) Eistrone (F'g/kg/day)
.
.
. +
.
.
;/
.
.
.
.
.
.
.
+
' +
0 50 100200400
0
0 0
0 0
0
0
0 0
~
0
FIG.
0 0
1 10100
+
' +
+
25 50 100200 1 1
1 1
1
Dose-related effect of estrone alone or ipriflavone combined with a given dose of estrone administered for 3 weeks on basal (shadowed column) and calcium (50 mg/kg body weight)-stimulated (open column) calcitonin release in ovariectomized rats. Each bar represents the mean ± SEM of 6-8 rats. Significantly different from ovariectomized control : * 0.01 < p < 0.05, ~ p < 0.01 (Dunnett's test).
flavone with 1 ~g/kg body weight of estrone caused a dose-dependent increase in the level of calcium-stimulated calcitonin up to the level observed in the intact rats. Fig. 2 shows the effect of the simultaneous administration of various subeffective doses of estrone with a constant dose of ipriflavone, i00 mg/kg body weight. Neither the basal nor the calcium-stimulated calcitonin levels were influenced by estrone alone. However, both levels were increased significantly when ipriflavone was combined with estrone. Dose-related effect of calcium on calcitonin release The effect of various doses of calcium, 6.25 to 25 mg/kg body weight, on calcitonin release was compared among ovariectomized rats pretreated with vehi-
Vol. 38, No. 17, 1986
Calcitonin Secretion by Ipriflavone
4,000
Stimulated
I
1539
*
3,500
3,000
t~
2,500
2,00(] c o
u_
1,500 u
Basal m
50(
1
Daily dose of
estrone (yg/kg)
FIG. 2 Dose-related effect of estrone with ( • ) or without ( Q ) ipriflavone (100 mg/ kg b o d y w e i g h t / d a y ) a d m i n i s t e r e d f o r 3 weeks on b a s a l a n d c a l c i u m (50 mg/kg body weight)-stimulated calcitonin release in ovariectomized rats. Each p o i n t r e p r e s e n t s t h e mean ± SEM o f 7 r a t s . Significantly different from estrone treated g r o u p : * 0 . 0 0 1 < p < 0 . 0 1 , ** p < 0 . 0 0 1 ( S t u d e n t ' s t - t e s t ) .
cle, estrone (0.5 ~g/kg body weight/day) alone, and estrone (0.5 ~g/kg body weight/day) in combination with ipriflavone (100 mg/kg body w e i g h t / d a y ) f o r 3 weeks ( F i g . 3 ) . Estrone pretreatment appeared to cause greater calcitonin rel e a s e t h a n was o b s e r v e d i n t h e c o n t r o l i n r e s p o n s e t o a g i v e n d o s e o f c a l c i u m . Simultaneous pretreatment of estrone with ipriflavone appeared to cause a further increase in the level of calcium-stimulated calcitonin.
1540
Calcitonin Secretion by Ipriflavone
Vol. 38, No. 17, 1986
2,000
E O.
1,500 t'-
._o
i "~
1,000
I 500
0
L 0
/!
,
6.25 Dose of
, 12.5
, 25
calcium (mg/kg)
FIG. 3 Dose-related effect of calcium on calcitonin release in ovariectomized rats treated with vehicle ( 1 ) , estrone (0.5 pg/kg body weight/day) alone ( • ) or ipriflavone (I0~ mg/kg body weight/day) in combination with estrone (0.5 ~g/kg body weight/day) ( • ) for 3 weeks. Significantly different from vehicle treated control : ** p < 0.01 (Dunnett's test).
Discussion It is well known that calcitonin secretion is influenced by estrogen. In postmenopausal women, plasma calcitonin levels, especially one that is calciumstimulated, decrease in parallel with decreased ovarian estrogen secretion (lO, ii). Estrogen administration, for a period, to postmenopausal women causes a rise in the plasma calcitonin level (2, 3). In rats plasma calcitonin levels both basal and calcium-stimulated, are decreased by ovariectomy and the replacement administration of estrogen for 3 weeks restores these levels (4). In the present study, the calcium-stimulated calcitonin level was also significantly reduced by ovariectomy and estrone replacement administration for 3 weeks produced an increase in both basal and calcium-stimulated calcitonin levels dosedependently. Ipriflavone alone did not influence either level or uterine growth in ovariectomized rats; this confirms our earlier findings (I). On the other hand, the simultaneous administration of ipriflavone with subeffective doses of estrogen produced increases in both basal and calcium-stimulated serum calcitonin
Vol. 38, No. 17, 1986
Calcitonin Secretion by Ipriflavone
1541
levels. The i n c r e a s e i n serum c a l c i t o n i n s e c r e t i o n depended upon b o t h t h e dose o f i p r i f l a v o n e and t h a t o f e s t r o g e n , and r e a c h e d t h e maximal l e v e l . As r e p o r t e d e a r l i e r , i p r i f l a v o n e has no e f f e c t on c a l c i t o n i n s e c r e t i o n i n i n t a c t r a t s (13). T h e r e f o r e , t h e e f f e c t o f endogenous e s t r o g e n on c a l c i t o n i n s e c r e t i o n a p p e a r s t o be maximal. This maximal l e v e l o f c a l c i t o n i n s e c r e t i o n was a c h i e v e d by d a i l y doses o f 10 p g / k g body w e i g h t o f e s t r o n e and by combined d o ses o f 1 p g / k g body weight of estrone with i00 mg/kg body weight of ipriflavone in ovariectomized rats. The dose response curve of calcitonin secretion to calcium loading appeared to be shifted leftwards by estrogen pretreatment. The combined estrogen and ipriflavone pretreatment led to a marked left shift of the curve (Fig. 3). These data suggest that estrogen increases the sensitivity of the thyroid gland to secrete calcitonin in response to calcium, and ipriflavone augments the activity of estrogen. The precise mechanism of these phenomena remains to be elucidated. It might involve the estrogen receptor in the target organs. Calcitonin is known to suppress bone resorption directly (14). It was reported that although ipriflavone alone has no effect on the decreases in calci tonin secretion and bone density in ovariectomized rats, a daily dose of estrone suppressed the reduction of these variables and ipriflavone administered simultaneously with estrone exerted a more marked suppression (15). These results accord well with those of the present study and suggest that ipriflavone could suppress bone resorption, at least in part, by augmenting the action of estrogen on calcitonin secretion. Acknowledgements We are grateful to Mr. Yoshihoro Shirakawa and Mrs. Masako Suzuki for technical assistance; and to Drs. Shintaro Kikuchi, Hisashi lwatsuka, and J.R. Miller for comments on the manuscript. References 1. 2. 3.
I. YAMAZAKI, Program o f t h e 7th I n t e r n a t i o n a l Congress o f E n d o c r i n o l o g y , Quebec C i t y , Canada, p. 1628 ( A b s t r a c t ) , E x c e r p t a Medica, Amsterdam (1984). S. MORIMOTO, M. TSUJ}, Y. OKADA, T. GNISHI and Y. KU~a~tARA, C l i n . Endocr. (Japan) 13 135-143 (1980). J.C. STEVENSON, G.A. ABEYASEKARA, C.J. HILLYARD, K.G. PHANfi, I. MACINTYRE, S. CAMPBELL, P.T. TOWNSEND, O. YOUNG and M.I. ~HITEHEAD, Lancet I 693-695
(1981), 4.
B.P. FIRST, M. MILLER and L.J. DEFTOS, Calcified Tissue Internat.
33 304
(1981). S.
B.D. CATHERWOOD, T. ONISHI and L . J . DEPTOS, C a l c i f . T i s s u e I n t .
35 502-507
(1983). 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
H.V. CONNERTY and A.R. BRIGGS, Am. J. Clin. Path. 45 290-296 (1966). S. MORIHOTO, Y. OKADA, T. ONISHI, M. TSUJI and Y. Kumahara, Clin. Endocr. 28 313-319 (1980), N.H. BELL and S. QUEENER, Nature 248 343-344 (1974). C.W. DUNNETT, B i o m e t r i c s 20 482-491 (1964). S. MORIMOTO, T. ONISHI, Y. OKADA, K. TANAKA, M. TSUJI and Y. KUMAHARA, E n d o c r i n o l . Japon. 26 207-211 (1979). L . J . DEFTOS, M.H. WEISMAN, G.W. WILLIAMS, D.B. KARPE, A.M. FRUMAR, B . J . DAVIDSON, J.G. PARTHEMORE and H.L. JUDD, N. Engl. J. Med. 302 1351-1353 (1980). D.L. SILVA and K.L. BECKER, Immunoreactive C a l c i t o n i n i n E x t r a t h y r o i d a l Tissue. I n : A. PECILE ( e d ) , C a ' l c i t o n i n 1980, 144-153, E x c e r p t a Medica, Amsterdam (1981). I . YAMAZAKI, L i f e S c i . (In p r e s s ) . D. ATKINS and M. PEACOCK, J . E n d o c r i n o l . 64 573-583 (1975). I . YAMAZAKI, A. SHINO and R. TSUKUDA, J . Bone M i n e r a l Metab. (In p r e s s ) .
Pergamon Press
CALCITONIN SECRETING PROPERTY OF IPRIFLAVONE IN THE PRESENCE OF ESTROGEN
lwao Yamazaki and Masako Kinoshita B i o l o g y L a b o r a t o r i e s , C e n t r a l Research D i v i s i o n , Takeda Chemical I n d u s t r i e s , Ltd., 17-85, Jusohonmachi 2-chome, Yodogawaku, Osaka 532, Japan (Received in final form February ii~ 1986) Summary C a l c i t o n i n s e c r e t i o n i s i n f l u e n c e d by e s t r o g e n . In t h e p r e s e n t s t u d y , b a s a l and c a l c i u m - s t i m u l a t e d serum c a l c i t o n i n l e v e l s were r e d u c e d i n o v a r i e c t o m i z e d r a t s , and r e p l a c e m e n t e s t r o g e n a d m i n i s t r e d f o r 3 weeks i n c r e a s e d both c a l c i t o n i n l e v e l s t o t h o s e in i n tact rats. Ipriflavone, 7-isopropoxy-3-phenyl-4H-l-benzopyran-4one, a l o n e d i d n o t i n f l u e n c e e i t h e r c a l c i t o n i n l e v e l . However, i p r i f l a v o n e and s u b e f f e c t i v e d o s e s o f e s t r o g e n a d m i n i s t e r e d s i m u l t a n e o u s l y i n c r e a s e d both l e v e l s ; t h e i n c r e a s e depended upon t h e dose of ipriflavone. F u r t h e r m o r e , p r e t r e a t m e n t w i t h e s t r o n e r e s u l t e d in g r e a t e r c a l c i t o n i n r e l e a s e in r e s p o n s e t o v a r i o u s doses of c a l c i u m and p r e t r e a t m e n t w i t h e s t r o n e and i p r i f l a v o n e caused an even g r e a t e r release. These f i n d i n g s i n d i c a t e t h a t i p r i f l a v o n e i n c r e a s e s t h e s e n s i t i v i t y o f t h e t h y r o i d g la n d t o e s t r o g e n t o s e c r e t e c a l c i t o n i n in response to calcium. I p r i f l a v o n e , 7-isopropoxy-3-phenyl-4H-l-benzopyran-4-one, is devoid of est r o g e n i c a c t i v i t y but i n c r e a s e s t h e u t e r o t r o p i c a c t i v i t y o f e s t r o g e n (1). This p r o p e r t y o f i p r i f l a v o n e i s t h o u g h t t o be produced by d i r e c t a c t i o n on t h e t a r g e t organ. C a l c i t o n i n s e c r e t i o n from t h e t h y r o i d gland i s u n d e r t h e i n f l u e n c e of estrogen (2-5). I p r i f l a v o n e a l s o i n f l u e n c e s c a l c i t o n i n s e c r e t i o n in the presence o f e s t r o g e n ( 1 ) ; however, t h e d e t a i l s o f t h i s a c t i o n a r e n o t known, and t h e p r e s e n t s t u d i e s were d e s i g n e d t o o b t a i n i n f o r m a t i o n about t h e s e d e t a i l s . Materials and Methods Animal s Female Sprague-Dawley r a t s , 9 weeks o f age, were p u r ch ased from Japan CLEA Co. They were f e d a commercial d i e t (CE-2, Japan CLEA) and d r i n k i n g w a t e r ad l i b i t u m , in an a i r c o n d i t i o n e d e n v i r o n m e n t (24 ± I°C, 50-60~ h u m i d i t y ) i l l ~ - - T h a t e d f o r 14 h / d a y from 07:30 t o 21:30 h. The c a l c i u m , p h o sp h o r u s, and v i t a m i n D c o n t e n t s o f t h e d i e t were 1.2~, 1.1~, and 2.0 I U / g , r e s p e c t i v e l y . The animals were o v a r i e c t o m i z e d a t 10 weeks o f age. Compounds used Ipriflavone was synthesized in our Chemical Development Laboratories. The compound was suspended in water containing 1% hydroxypropyl cellulose (HPC-L, Nippon Soda Co., Japan). Estrone was purchased from Merck A.G. (West Germany), 0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Press Ltd.
1536
Calcitonin Secretion by Ipriflavone
Vol. 38, No. 17, 1986
and dissolved in sesame oil. Calcium gluconate monohydrate was purchased from Wako Pure Chemical Industries, Ltd. (Japan) and dissolved in saline (9 g NaCI/I) Procedures for administerin~ the compound and for serum collection Ipriflavone was administered orally and estrone subcutaneously starting one day after the ovariectomy and was continued daily for three weeks. On the day after the last dosing, blood (basal) was collected from the orbital vein under ether anesthesia. Then, calcium, usually 50 mg/kg body weight as calcium gluconate, was given in a single intraperitoneal injection. Thirty minutes afterwards, the animals were anesthetized with ether and blood (stimulated) was withdrawn from the abdominal aorta. It was confirmed beforehand that serum calcitonin reached a plateau level by i0 min after the intraperitoneal administration of this dose of calcium and maintained the level for 35 min. The separated serum was stored at -20°C until used for calcium and calcitonin determinations. Determination of serum calcium A spectrophotometric method (6) for determining serum calcium, using the metal complexing dye, orthocresolphthalein complexon, was applied; a commercial kit, Calcium C-Test Wako (Wako Pure Chemical Industries, Ltd.) was used. Assay of serum calcitonin Serum calcitonin was determined using a commercial radioimmunoassay kit for human calcitonin (Daiichi Radioisotope Labs., Japan) (7) by the double antibody technique. It was confirmed beforehand that the dilution curve of the rat thyroid extract prepared according to the method of Bell and Queener (8) paralleled the standard curve for human cal¢itonin of the radioimmunoassay kit. Values are expressed as pg equivalents of synthetic human calcitonin/ml, since a human calcitonin standard was utilized. The minimum detectable amount is 65 pg/ml, and the intra- and inter-assay coefficients of variation were 12.5% and 20.9%, respectively. Statistical analysis The statistical significance of differences between means was determined by the Student's t-test for two group comparison, and by Dunnett's test (9) for multiple group comparisons. Results Effect of ipriflavone alone on calcitonin secretion Ipriflavone, in daily doses of 50 to 400 mg/kg body weight for 3 weeks, did not influence the basal or the calcium-stimulated calcitonin level in ovarectomized rats (Table I); it did not influence uterine weight either. Effect of the combined administration of ipriflavone and estrone on calcitonin secretion As shown in Fig. i, basal and calcium-stimulated calcitonin levels were decreased in ovariectomized rats, although the effect of ovariectomy was much less on the basal level. Estrone increased both the basal and calcium-stimulated calcitonin levels dose-dependently up to the levels observed in intact rats. Estrone in daily doses of I0 ~g/kg body weight induced significant secretion but 1 ~g/kg did not. Therefore, the latter was chosen to be given combined with ipriflavone. The simultaneous administration of various doses of ipri-
8
8
8
7
50
i00
200
400
219 ± 3
217 ± 3
217 ± 3
217 ± 3
217 ± 3
start
279 ± 4
288 ± 7
285 ± 6
293 ± 5
285 ± 7
autopsy
Body weight (g) at
153 ± 8
146 ± S
156 ± 5
151 ± 4
152 ± 5
Uterine weight (mg)
10.2 ± 0.i
10.3 ± 0.3
10.4 ± 0.3
10.2 ± 0.I
10.8 ± 0.6
basal
17.6 ± 0.2
17.3 ± 0.7
17.6 ± 0.2
17.7 ± 0.5
18.4 ± 0.3
stimulated
Serum calcium (mg/dl)
486 ± 25
490 ± 20
468 ± 17
474 ± 23
395 ± 49
basal (A)
(B)
89
2,254 ± 254
2,133 ± 116
2,030 ±
2,088 ± 147
95
1,764 ± 232
1,643 ± 103
1,562 ±
1,613 ± 134
1,475 ± 181
A (B-A)
(pg/ml)
1,740 ± 135
stimulated
Serum c a l c i t o n i n
Rats were ovariectomized at i0 weeks of age and the intragastric pretreatment of ipriflavone was started one day after the operation. Calcium challenge (50 mg/kg body weight) was performed on the day after the last pretreatment. Values are expressed as means ± SEM.
8
0
Daily dose of No. ipriflavone of (mg/kg) rats
Body and uterine weights and changes in serum calcium and calcitonin concentrations in response to intraperitoneal administration of calcium in ovariectomized female rats pretreated with ipriflavone for three weeks
TABLE I
~n
< O m
c~
0
~D rt
fD r~
m
0
m
~o co
--J
Z O
OO
O
1538
Calcitonin Secretion by Ipriflavone
Vol. 38, No. 17, 1986
6,000
5,000
Et30
4,000
:_o b §
3,000
§ 2,000 E U~
1,000
,¸!!i 0 Ovariectom Ipriflavone (mg/kg/day) Eistrone (F'g/kg/day)
.
.
. +
.
.
;/
.
.
.
.
.
.
.
+
' +
0 50 100200400
0
0 0
0 0
0
0
0 0
~
0
FIG.
0 0
1 10100
+
' +
+
25 50 100200 1 1
1 1
1
Dose-related effect of estrone alone or ipriflavone combined with a given dose of estrone administered for 3 weeks on basal (shadowed column) and calcium (50 mg/kg body weight)-stimulated (open column) calcitonin release in ovariectomized rats. Each bar represents the mean ± SEM of 6-8 rats. Significantly different from ovariectomized control : * 0.01 < p < 0.05, ~ p < 0.01 (Dunnett's test).
flavone with 1 ~g/kg body weight of estrone caused a dose-dependent increase in the level of calcium-stimulated calcitonin up to the level observed in the intact rats. Fig. 2 shows the effect of the simultaneous administration of various subeffective doses of estrone with a constant dose of ipriflavone, i00 mg/kg body weight. Neither the basal nor the calcium-stimulated calcitonin levels were influenced by estrone alone. However, both levels were increased significantly when ipriflavone was combined with estrone. Dose-related effect of calcium on calcitonin release The effect of various doses of calcium, 6.25 to 25 mg/kg body weight, on calcitonin release was compared among ovariectomized rats pretreated with vehi-
Vol. 38, No. 17, 1986
Calcitonin Secretion by Ipriflavone
4,000
Stimulated
I
1539
*
3,500
3,000
t~
2,500
2,00(] c o
u_
1,500 u
Basal m
50(
1
Daily dose of
estrone (yg/kg)
FIG. 2 Dose-related effect of estrone with ( • ) or without ( Q ) ipriflavone (100 mg/ kg b o d y w e i g h t / d a y ) a d m i n i s t e r e d f o r 3 weeks on b a s a l a n d c a l c i u m (50 mg/kg body weight)-stimulated calcitonin release in ovariectomized rats. Each p o i n t r e p r e s e n t s t h e mean ± SEM o f 7 r a t s . Significantly different from estrone treated g r o u p : * 0 . 0 0 1 < p < 0 . 0 1 , ** p < 0 . 0 0 1 ( S t u d e n t ' s t - t e s t ) .
cle, estrone (0.5 ~g/kg body weight/day) alone, and estrone (0.5 ~g/kg body weight/day) in combination with ipriflavone (100 mg/kg body w e i g h t / d a y ) f o r 3 weeks ( F i g . 3 ) . Estrone pretreatment appeared to cause greater calcitonin rel e a s e t h a n was o b s e r v e d i n t h e c o n t r o l i n r e s p o n s e t o a g i v e n d o s e o f c a l c i u m . Simultaneous pretreatment of estrone with ipriflavone appeared to cause a further increase in the level of calcium-stimulated calcitonin.
1540
Calcitonin Secretion by Ipriflavone
Vol. 38, No. 17, 1986
2,000
E O.
1,500 t'-
._o
i "~
1,000
I 500
0
L 0
/!
,
6.25 Dose of
, 12.5
, 25
calcium (mg/kg)
FIG. 3 Dose-related effect of calcium on calcitonin release in ovariectomized rats treated with vehicle ( 1 ) , estrone (0.5 pg/kg body weight/day) alone ( • ) or ipriflavone (I0~ mg/kg body weight/day) in combination with estrone (0.5 ~g/kg body weight/day) ( • ) for 3 weeks. Significantly different from vehicle treated control : ** p < 0.01 (Dunnett's test).
Discussion It is well known that calcitonin secretion is influenced by estrogen. In postmenopausal women, plasma calcitonin levels, especially one that is calciumstimulated, decrease in parallel with decreased ovarian estrogen secretion (lO, ii). Estrogen administration, for a period, to postmenopausal women causes a rise in the plasma calcitonin level (2, 3). In rats plasma calcitonin levels both basal and calcium-stimulated, are decreased by ovariectomy and the replacement administration of estrogen for 3 weeks restores these levels (4). In the present study, the calcium-stimulated calcitonin level was also significantly reduced by ovariectomy and estrone replacement administration for 3 weeks produced an increase in both basal and calcium-stimulated calcitonin levels dosedependently. Ipriflavone alone did not influence either level or uterine growth in ovariectomized rats; this confirms our earlier findings (I). On the other hand, the simultaneous administration of ipriflavone with subeffective doses of estrogen produced increases in both basal and calcium-stimulated serum calcitonin
Vol. 38, No. 17, 1986
Calcitonin Secretion by Ipriflavone
1541
levels. The i n c r e a s e i n serum c a l c i t o n i n s e c r e t i o n depended upon b o t h t h e dose o f i p r i f l a v o n e and t h a t o f e s t r o g e n , and r e a c h e d t h e maximal l e v e l . As r e p o r t e d e a r l i e r , i p r i f l a v o n e has no e f f e c t on c a l c i t o n i n s e c r e t i o n i n i n t a c t r a t s (13). T h e r e f o r e , t h e e f f e c t o f endogenous e s t r o g e n on c a l c i t o n i n s e c r e t i o n a p p e a r s t o be maximal. This maximal l e v e l o f c a l c i t o n i n s e c r e t i o n was a c h i e v e d by d a i l y doses o f 10 p g / k g body w e i g h t o f e s t r o n e and by combined d o ses o f 1 p g / k g body weight of estrone with i00 mg/kg body weight of ipriflavone in ovariectomized rats. The dose response curve of calcitonin secretion to calcium loading appeared to be shifted leftwards by estrogen pretreatment. The combined estrogen and ipriflavone pretreatment led to a marked left shift of the curve (Fig. 3). These data suggest that estrogen increases the sensitivity of the thyroid gland to secrete calcitonin in response to calcium, and ipriflavone augments the activity of estrogen. The precise mechanism of these phenomena remains to be elucidated. It might involve the estrogen receptor in the target organs. Calcitonin is known to suppress bone resorption directly (14). It was reported that although ipriflavone alone has no effect on the decreases in calci tonin secretion and bone density in ovariectomized rats, a daily dose of estrone suppressed the reduction of these variables and ipriflavone administered simultaneously with estrone exerted a more marked suppression (15). These results accord well with those of the present study and suggest that ipriflavone could suppress bone resorption, at least in part, by augmenting the action of estrogen on calcitonin secretion. Acknowledgements We are grateful to Mr. Yoshihoro Shirakawa and Mrs. Masako Suzuki for technical assistance; and to Drs. Shintaro Kikuchi, Hisashi lwatsuka, and J.R. Miller for comments on the manuscript. References 1. 2. 3.
I. YAMAZAKI, Program o f t h e 7th I n t e r n a t i o n a l Congress o f E n d o c r i n o l o g y , Quebec C i t y , Canada, p. 1628 ( A b s t r a c t ) , E x c e r p t a Medica, Amsterdam (1984). S. MORIMOTO, M. TSUJ}, Y. OKADA, T. GNISHI and Y. KU~a~tARA, C l i n . Endocr. (Japan) 13 135-143 (1980). J.C. STEVENSON, G.A. ABEYASEKARA, C.J. HILLYARD, K.G. PHANfi, I. MACINTYRE, S. CAMPBELL, P.T. TOWNSEND, O. YOUNG and M.I. ~HITEHEAD, Lancet I 693-695
(1981), 4.
B.P. FIRST, M. MILLER and L.J. DEFTOS, Calcified Tissue Internat.
33 304
(1981). S.
B.D. CATHERWOOD, T. ONISHI and L . J . DEPTOS, C a l c i f . T i s s u e I n t .
35 502-507
(1983). 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
H.V. CONNERTY and A.R. BRIGGS, Am. J. Clin. Path. 45 290-296 (1966). S. MORIHOTO, Y. OKADA, T. ONISHI, M. TSUJI and Y. Kumahara, Clin. Endocr. 28 313-319 (1980), N.H. BELL and S. QUEENER, Nature 248 343-344 (1974). C.W. DUNNETT, B i o m e t r i c s 20 482-491 (1964). S. MORIMOTO, T. ONISHI, Y. OKADA, K. TANAKA, M. TSUJI and Y. KUMAHARA, E n d o c r i n o l . Japon. 26 207-211 (1979). L . J . DEFTOS, M.H. WEISMAN, G.W. WILLIAMS, D.B. KARPE, A.M. FRUMAR, B . J . DAVIDSON, J.G. PARTHEMORE and H.L. JUDD, N. Engl. J. Med. 302 1351-1353 (1980). D.L. SILVA and K.L. BECKER, Immunoreactive C a l c i t o n i n i n E x t r a t h y r o i d a l Tissue. I n : A. PECILE ( e d ) , C a ' l c i t o n i n 1980, 144-153, E x c e r p t a Medica, Amsterdam (1981). I . YAMAZAKI, L i f e S c i . (In p r e s s ) . D. ATKINS and M. PEACOCK, J . E n d o c r i n o l . 64 573-583 (1975). I . YAMAZAKI, A. SHINO and R. TSUKUDA, J . Bone M i n e r a l Metab. (In p r e s s ) .