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Regulation of anterior pituitary and brain ß-adrenergic receptors by ovarian steroids
Life Sciences, Vol. 37, pp. 1563-1570 Printed in the U.S.A.
Pergamon Press
REGULATION OF ANTERIOR PITUITARY AND BRAIN 16-ADRENERGIC RECEPTORS BY OVARIAN STEROIDS S.L. Petrovic, J . K . McDonald*, J.C. Bedran De Castro**, G.D. Snyder and S.M. McCann Department of Physiology, The University of Texas Health Science Center, Dallas, Texas 75235 (Received in final form August 13, 1985)
Summary Ovariectomy of adult female rats (200-230g) resulted in an increase in ~-adrenergic receptors in the cerebral cortex, hypothalamus and anterior pituitary. The anterior pituitary had the largest overall increase as well as the most rapid increase in ~-adrenergic receptor density of the tissues examined. The increase in hypothalamic or cerebral cortical B-adrenergic receptors became apparent only long after ovariectomy (7-14 days). Fourteen days after ovariectomy, the density of B-adrenergic receptors was 7996, 40~, and 245 in excess of control values in crude membranes prepared from anterior pituitary, hypothalamus and cerebral cortex, respectively. Over the same interval, the plasma concentration of luteinizing hormone (LH) increased 2Q-fold, while the concentration of follicle-stimulating hormone {FSH) rose S-fold compared to control levels. Estradiol replacement (20 ]~glkglday) in these animals for four days before sacrifice concomitantly reduced plasma levels of the gonadotropins as well as the density of ~-adrenergic receptors in both the anterior pituitary and the hypothalamus. Long-term steroid replacement during the fifth and sixth week after ovariectomy, with implants of estradiol and progesterone which released the steroids in approximately physiological concentrations, significantly reduced B-adrenergic density in anterior pituitary, but not in the hypothalamic membranes. This treatment significantly reduced plasma LH, but not FSH. Beta-adrenergic receptor density was also found to fluctuate significantly during the 4-day estrous cycle. The highest values were found on proestrus, and the lowest on diestrus I. These studies indicate that changes in plasma concentrations of gonadal steroids (e.g. during the estrous cycle) influence the density of 13-adrenergic receptors in tissues involved in the control and release of anterior pituitary gonadotropins. We have recently reported an increase in ~-adrenergic receptor density in the anterior pituitary and cerebral cortex of orchidectomized rats which parallels the postcastration rise in plasma levels of anterior pituitary gonadotropins. Present address: Department of Anatomy, Emory University, Atlanta, Georgia 30322. Present address: Universidado de Estado de Sao Paulo, 16100 Aracatuba S.P., Brazil. 0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
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These effects are reversed by testosterone replacement ( I ) . Wagner et al. (2) have r e p o r t e d t h a t ovariectomy causes an increase in the d e n s i t y of ~ - a d r e n e r g i c receptors in the cerebral c o r t e x w h i c h was later shown to be suppressed by estrogen replacement (3). In c o n t r a s t , estrogen treatment has been reported to u p r e g u l a t e hypothalamic 13-adrenergic receptors ( 4 , 5 ) . These f i n d i n g s have prompted us to examine the ~ - a d r e n e r g i c d e n s i t y in b r a i n areas and a n t e r i o r p i t u i t a r y of ovariectomized rats in relation to levels of c i r c u l a t i n g g o n a d o t r o p i n s . Moreover, in view of data demonstrating changes in the d e n s i t y of both LHRH (6,7) and D2-dopamine receptors (8,9) d u r i n g the estrous cycle of the r a t , we have examined the d e n s i t y of a n t e r i o r p i t u i t a r y 13-adrenergic receptors in discrete phases of the cycle. Materials and Methods Female S p r a g u e - D a w l e y rats (200-230 gms) were ovariectomized or shamoperated u n d e r ether anesthesia, and s u b s e q u e n t l y housed with a l i g h t schedule of 14 h o u r s on (0500-1900 h o u r s ) and 10 h o u r s off. All animals used in assessing changes of ~ - r e c e p t o r d e n s i t y d u r i n g the estrous cycle were followed t h r o u g h at least two r e g u l a r 4 - d a y cycles before s a c r i f i c e . For s h o r t - t e r m estradiol replacement, at the t e n t h day following s u r g e r y , sham-operated animals and h a l f of the ovariectomized rats were injected s u b c u t a n e o u s l y ( s . c . ) w i t h 0.1 ml of corn oil. The remaining ovariectomized animals were injected w i t h 5 ]~c3 (about 20 ]J~Ikg) of estradiol-17~-benzoate (Sigma, St. Louis, MO) in 0.1 ml of corn oil. T h i s treatment was repeated for the n e x t three d a y s , and the animals were sacrificed on the f o u r t e e n t h day a f t e r s u r g e r y . Long-term ( s i x - w e e k ) ovariectomized animals were e i t h e r injected over the last f o u r days before s a c r i f i c i n g w i t h 100 ~ g l k g l d a y of estradiol or the vehicle (Table I ) , or t h e y were implanted b i l a t e r a l l y in the flank w i t h c r y s t a l l i n e progesterone and u n i l a t e r a l l y w i t h a solution of estradiol, both contained in Silastic tubing. These implants were dimensioned to produce the levels of estradiol and progesterone found on proestrus (I0). T h e y were inserted f o u r weeks a f t e r c a s t r a t i o n and remained in animals f o r the two weeks p r i o r to sacrifice. Animals were sacrificed between 11:00 AM and 11.45 AM on each day of the cycle. The d e n s i t y of 13-adrenergic receptors was determined on c r u d e membrane p r e p a r a t i o n s (from 600 to 100,000 x g) of tissue homogenates for all ovariectomized animals and sham-operated controls as described p r e v i o u s l y (11). The s a t u r a t i o n b i n d i n g assays w i t h [125-1]-iodocyanopindolol ( I C Y P ) were done at e i g h t c o n c e n t r a t i o n s of the ligand o v e r the range of 5 to 120 pM of the ( - ) - f o r m . The assay medium contained a m i x t u r e of suppressors of the n o n - s p e c i f i c b i n d i n g (11), I - ( - ) Propranolol ( 5 x i 0 - - M) was used to define the n o n - s p e c i f i c b i n d i n g . Plasma concentrations of anterior pituitary gonadotropins were determined by radiommunoassay. FSH antibody and reference standards were gifts of N I A D D K . LH antibody was obtained from Dr. G. Niswender, while the LH reference preparation RP2 and the hormone for iodination were provided by N I A D D K anterior pituitary program. Results The density of ~3-adrenergic receptors in membranes from the anterior pituitary gland increased within the initial 24 hours after ovariectomy (Figure I). This increase was not significant by Student's t test due to a relatively high variance at this sampling time for the castrated group. By 7 days after castration, the density of ~-adrenergic receptors in anterior pituitary membranes was significantly increased. In contrast, the increase was not pronounced in the hypothalamic membranes. The density of
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13-adrenergic receptors in the cerebral cortex was found to be significantly elevated only after long periods of ovariectomy (see Table I ) . Six weeks after ovariectomy, all tissues displayed significantly higher 13-adrenergic densities in comparison with their respective controls (Figure I and Table I ) . Treatment of ovariectomized animals with 20 1~glkglday of estradiol benzoate for four days before sacrifice on the 14th day after surgery resulted in a significant decrease in ~-adrenergic receptor density in both anterior p i t u i t a r y and hypothalamic membranes when compared to values in ovariectomized controls (Figure I ) . This treatment dramatically reduced the plasma concentration of LH (by 74%), and also v e r y significantly decreased plasma FSH (by 37%) (Figure 2). Combined treatment with estradiol and progesterone during the 5th and 6th week after ovariectomy significantly reduced the number of p i t u i t a r y ~-adrenergic receptors, but was less effective in reducing hypothalamic ~-adrenergic receptor density. This treatment strongly reduced the circulating LH levels (approximately 50%), while plasma FSH decreased only 12%. This latter difference was not significant in comparison with ovariectomized control values for this hormone. (Figure 2, lower panel). Anterior - Pituitary
~
~
0`5
i
~E
Hypothalamus
o
E m 1.5
0.5
1
7 14 Days after surgery
42
FIGURE 1 Maximum binding values for [1251] iodocyanopindolol with membrane fractions from anterior pituitaries and hypothalami at 1-42 days after surgery. Four to seven groups of 4 animals each were analyzed at each point. Unshaded bars denote values for sham-operated controls, hatched bars represent animals ovariectomized only, while the filled bars correspond to ovariectomized animals receiving estradiol benzoate for 4 days before sacrifice (day 14) or estradiol plus progesterone for 2 weeks before sacrifice (day 42). For f u r t h e r details see Materials and Methods. Asterisks indicate significance levels in two-tailed Student's t test, with comparison always being made to the nearest column on the left at each point in time. (*) indicates significance at the level of 0.05, (**) at the level of 0.01, and ( * * * ) at the level of 0.001. Data are presented with standard errors of the corresponding means.
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TABLE I Density of B-adrenergic receptors in cerebral cortex a f t e r ovariectomy and estrogen treatment Kd, pM b
Bmax (fM/m 9 tissue) b
14,03_+1.1
2.526_+0.16
Ovariectomized (7)
14.08_+0.92
3.094+0.15
p<0.025
Ovariectomized plus estrogen (4)
14.71+I .54
2.61±0.13
p<0.05
Treatment a Controls
(7)
Significance for change in Bmax c Student's Dunnett's t test test p<0.05 N.S.
(a) 4-6 animals constituted each experimental group. The number ot groups is given in parentheses. Ovariectomy or sham operations were performed six weeks before s a c r i f i c i n g . Estrogen treatment was I00 ] z g l k g l d a y of estradiol-17B benzoate for 4 consecutive days before sacrifice_. I (b) Mean values ± I SEM. Kd is the dissociation constant, pmol. l i t . Bmax is the maximum b i n d i n g , fmollmg tissue equivalent for membrane preparations used. These data were obtained in saturation assays employing ICYP in the range of 5-120 pM, using eight d i f f e r e n t molarity inputs and employing a mixture of suppressors of non-specific binding (11). (c) In the respective two-tailed tests [12). Note that the comparisons are with the control group shown above the respective treatment group. Since the density of a n t e r i o r p i t u i t a r y B-adrenergic receptors was affected by ovarian steroids, we examined the concentration of these receptors over the 4-day estrous cycle. The average density of B-adrenergic receptors found in each cycle phase is tabulated in Table 2. These densities were lower (at a confidence level g r e a t e r than 95%) than the average density of a n t e r i o r p i t u i t a r y B-receptors in either 14-day or 42-day ovariectomized animals (Table 2). The density found in proestrus and diestrus 2 was s i g n i f i c a n t l y higher than that of diestrus I . The receptor density at proestrus, the greatest within the cycle, was also s i g n i f i c a n t l y higher than the mean B-adrenergic receptor concentration on the day of estrus. The difference between proestrus and diestrus I was also significant in one-way F-test and in the Student-Newman-Keuls test (12) (Table 2), indicating a change in receptor number independent of the d i s t r i b u t i o n of Bmax values constituting the compared grand averages. Discussion The B-adrenergic receptor complement of both the a n t e r i o r p i t u i t a r y and various regions of the brain is downregulated by gonadal steriods a f t e r castration of either male or female rats ( 1 , 3 - 4 , 1 3 - 1 4 ) , although s h o r t - t e r m effects of either castration or steroid replacement may not be pronounced in the case of cortical and hypothalamic B-adrenergic receptors ( r e f . ( I ) and present r e s u l t s ) .
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24 tG
m
LH
B-adrenergic Receptors and Ovarian Steroids
t
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FIGURE 2. Plasma levels of luteinizing hormone {LH) and follicle-stimulating hormone (FSH) at 1-42 days after sham-operation (unhatched bars), ovariectomy (hatched bars) or ovariectomy followed by treatment with estradiol-1713 benzoate (filled bars) as described in caption to Figure 1. Plasma samples from 8-12 animals were analyzed at each point. The data are presented with standard errors of the respective means. For f u r t h e r details see Figure 1 and Materials and Methods.
8OO
400
t 14 42 Days after surgery
TABLE II
Density of anterior p i t u i t a r y ~-adrenergic receptors during the rat estrous cycle Cycle Phase
Number a of groups
Mean receptor density a
Percent of overall mean
Estrus
16
0.181-+0.019
92.2
Diestrus I
22
0.164-+0.018
83.5
Diestrus 2
20
0.215-+0.020
109.4
Proestrus
24
0. 226-+0.017
114.8
Overall b mean 82 0.197_+0.01 100.0 (a) Two to four animals constituted each experimental group. The B-receptor density is expressed in femtomoles of ICYP specifically bound per/ mg of fresh tissue equivalent used, _+I S.E.M. (b) This mean is about 11 percent higher than the average for all sham-operated animals of Figure I (0.177-+0.013 fMlmg tissue), possibly due to seasonal and assay-related variations. The mean value of Bmax found at diestrous day 1 was d i f f e r e n t at the significance level of 0.05 from the mean of diestrus 2, and at the level of 0.01 from the mean of proestrus, using two-tailed Dunnett's test (12). The difference between proestrus and diestrus 1 was also significant in Student-Newman-Keuls test (12). The rapid change seen in anterior p i t u i t a r y B-adrenergic receptor density after ovariectomy (Figure 1) and after orchidectomy (1) might be due to the low overall B-adrenergic receptor density in the gland. This would permit large relative B-receptor density changes over short intervals. The variation observed during the early period after ovariectomy might have been due to the use of randomly cycling animals, since density of B-adrenergic receptors in anterior p i t u i t a r y gland differs significantly between the phases of the estrous cycle (Figure 1 and Table 2). However,
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at longer intervals after ovariectomy, influences of cycle phase upon the ~3-receptor density would presumably be small since the maximal difference between cycle phases varied only by about 15% of the mean density (Table 2). Following ovariectomy, the density of /3-adrenergic receptors in the hypothalamus, and especially in the cerebral cortex, increased at rates apparently much slower than in the anterior pituitary. The increase became significant between 7 and 14 days after the castration. Over that interval, the absolute increase of t3-adrenergic receptor number per mg tissue was not, however, appreciably different between anterior pituitary and hypothalamus (Figure I). On the 7th day, the increment was approximately 0.1 fM ICYP/mg in both tissues (Figure I). Six weeks after ovariectomy, the increment of ~3-adrenergic receptors in hypothalamus and cerebral cortex was quite similar, about 0.5 fM ICYP/mg tissue (Figure I and Table I), while in the anterior pituitary it was approximately 0.2 fM/mg (Figure I). Since the anterior pituitary appears to have exclusively ~2-receptors (I,11}, the increment in the pituitary could be considered as an increase in the ~2-adrenergic receptor population. Actually, cortical ~2-adrenergic responses, or overall density of 13-adrenergic receptors, in ovariectomized animals have been reported to be attenuated by estrogen treatment (2,13). The density of ~32-adrenergic receptors, but not of Dl-SUbtype, appears to be significantly uncreased in rat cerebral cortex affer orchidectomy (I). Since 139-adrenergic receptors constitute, in intact animals, only about 20% of cortical~3-adrenergic receptor complement (11,15), and about half of the hypothalamic ~3-receptor density (11,16), even an increase comprised entirely of 13~-species will be appreciably masked in these tissues by their ~1-acrrenergic receptor complement. On the other hand, there is evidence for an activation of neurons, possibly involving an increase in synaptization between neurons in the arcuate and the mediobasal hypothalamus after ovariectomy (17,18). Such activity would likely result in increased numbers of one or both t3-adrenergic receptor subtypes. Since these changes appear to develop slowly, they might account for a significant proportion of the long-term accumulation of ~3-adrenergic receptor density in hypothalamus and cerebral cortex after ovariectomy. The physiological role of ~2-adrenergic receptors in anterior pituitary and hypothalamus is at present not sufficiently understood. These receptors are presumably involved in activation of adenylate cyclase systems and thus in secretion of protein hormones and peptides. For example, ~32-adrenergic mediation is involved in the release of ~ - M S H from posterior pituitary (19}. Positive ~-adrenergic regulation could also be shown for release of A C T H in vivo (20), for the pulsed release of growth hormone {GH) from dispersed anterior pituitary cells (21), and for the release of prolactin from such cells {22). ~-receptors, which are increased after castration in both male and female r~ts (reference (I) and the present results) might cooperate with LHRH receptors in modulating the increased output of LH following castration. L H R H receptors are also strongly upregulated by ovariectomy in the rat (7). It is of interest to note that patterns of change in ~-receptors of the anterior pituitary over the estrous cycle as found in this work are similar to those established for L H R H receptors in the cycle phases (6,7). The peak concentration of both species of receptor might occur at the beginning of proestrus, thereby providing for a maximal responsiveness of the tissue to either L H R H or to ~32-adrenergic inputs. The increase in B-receptors in pituitary on proestrus suggests that estrogen has a biphasic action on their density, first to upregulate and then to downregulate it. The upregulatory effect might correspond with estrogen enhancement of pituitary responsiveness to L H R H (e.g., ref. 22), and would probably not be seen in
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c h r o n i c estrogen replacement s t u d i e s . Our data i n d i c a t e , as in the case of orchidectomized males ( I ) , a parallelism in p a t t e r n s of change for a n t e r i o r p i t u i t a r y 132-adrenergic receptors and the levels of plasma LH a f t e r ovariectomy and replacement of ovarian steroids. T h e parallelism between b r a i n 132-receptors and LH levels remains as yet to be e l u c i d a t e d . However, the p o s s i b i l i t y t h a t increased 13-receptors in hypothalamic areas might facilitate release of g o n a d o t r o p i n - r e l e a s i n g peptides, and in t h i s way c o n t r i b u t e to elevated LH and FSH levels a f t e r c a s t r a t i o n , should be viewed w i t h c a u t i o n , since B~-agonists appear to e x e r t l a r g e l y i n h i b i t o r y i n f l u e n c e s upon LH release at tl~e level o f hypothalamus (23,24). I f a n t e r i o r p i t u i t a r y ~ - a d r e n e r g i c receptors s i g n i f i c a n t l y c o n t r i b u t e to the r e g u l a t i o n of g o n a d o t r o p i n s e c r e t i o n , i t would appear t h a t t h e i r role in LH secretion should be much more d i r e c t than in the case of FSH secretion. It has been demonstrated p r e v i o u s l y t h a t the elevated levels of FSH w h i c h r e s u l t from ovariectomy are r e f r a c t o r y to steroid replacement while LH levels are not ( 1 7 , 2 5 ) . A l s o , o u r recent r e s u l t s indicate t h a t 13-adrenergic a n t a g o n i s t s i n f l u e n c e the plasma levels of LH much more d i r e c t l y and to a much l a r g e r degree than is the case w i t h levels of FSH (26). In the p r e s e n t s t u d y , steroid replacement also had much more d i r e c t effect on plasma levels of LH. It could be t h a t the control of LH secretion is more d i r e c t l y associated w i t h p i t u i t a r y a d r e n e r g i c mechanisms. References
I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
S.L. P E T R O V I C , J.K. M C D O N A L D , G.D. S N Y D E R and S.M. M C C A N N , Life Sci. 34, 2399-2406 (1984). H.R. W A G N E R and J.N. DAVIES, Brain Res. 201, 235-239 (1980). A. BIEGON, A. R E C H E S , L. S N Y D E R and B. M C E W E N , Life Sci. 32, 2015-2021 (1983). M. V A C A S and D.P. C A R D I N A L I , Neurosci. Lett. 17, 73-77 (1980). M. WILKINSON, H. H E R D O N , M. P E A R C H and C. WILSON, Brain Res. 168, 652-655 (1979). E. H A Z U M and D. KEINAN, Biochem. Biophys. Res. Commun. 107, 695-698 (1982). J.J. REEVES, G.K. T A R N A V S K Y and T. P L A T T , Biol. Reprod. 27, 316-319 (1982). M. H E I M A N and N. B E N - J O N A T H A N , Endocrinology 111, 37 (1982). C. PASQUALINI, V. LENOIR, A. EL A B E D and B. K E R D E L H E U , Neuroendocrinology 38, 39-44 (1984). R.L. G O O D M A N , Endocrinology 102, 142-150 (1978). S.L. P E T R O V I C , J.K. M C D O N A L D , G.D. S N Y D E R and S.M. M C C A N N , Brain Res. 261, 249-259 (1983). J. ZAR, Biostatistical Analysis. Prentice-Hall Inc. Englewood Cliffs, N.J. (1974). H.R. W A G N E R , K.A. C R U T C H E R and J.N. DAVIES, Brain Res. 171, 147-151 (1979). M.I. V A C A S , R.P. L O W E N S T E I N and D.P. C A R D I N A L I , J. Neural. Transm., 53, 49-57 (1982). K.P. M I N N E M A N , A. H E D B E R G and P.B. M O L I N O F F , J. Pharmacol. Exp. Therap. 211, 502-508 (1979). T.C. R A I N B O W , B. P A R S O N S and B.B. WOLFE, Proc. Natl. Acad. Sci. U.S. 81, 1585-1589 (1984). J.R. B R A W E R and C. S O N N E N S C H E I N , Am. J. Anat. 144, 57-88 (1976). J. B R A W E R , H. SCHIPPER, and B. R O B A I R E , Endocrinology 112, 194-199 (1983).
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T. COTE, M. MUNEMURA, R.L. ESKAY and J.W. KEBABIAN, Endocrinology 107:108-116 (1980). E. MEZEY, T . D . REISINE, M. PALKOVlTS, M.J. BROWNSTEIN and J. AXELROD, Proc. Natl. Acad. Sci., U.S. 80:6728-6731 (1980). S,N. PERKINS, W.S. EVANS, M.O. THORNER and M.J. CRONIN, Neuroendocrinology 37, 473-475 (1983). M. BAES and C. DENEF, Life Sci. 34, 1447-1454 (1984). X . Y . HUANG and S.M. MCCANN, Proc. Soc. Exp. Biol. Med. 174, 244-248 (1983). P.C. LEUNG, G.W. ARENDASH, D.I. WHITMAYER, R.A. GORSKI and C.H. SAWYER, Neuroendocrinology 34, 207-210 (1982). S.M. MCCANN, H. MIZUNUMA, W.K. SAMSON and M.D. LUMPKIN, Psychoneuroendocrinology 8, 299-308 (1983). S.L. PETROVlC, J.C. BEDRAN DE CASTRO and S.M. MCCANN, Abstract #1749, 7th Intnl. Congress of Endocrin., Quebec (1984).
Pergamon Press
REGULATION OF ANTERIOR PITUITARY AND BRAIN 16-ADRENERGIC RECEPTORS BY OVARIAN STEROIDS S.L. Petrovic, J . K . McDonald*, J.C. Bedran De Castro**, G.D. Snyder and S.M. McCann Department of Physiology, The University of Texas Health Science Center, Dallas, Texas 75235 (Received in final form August 13, 1985)
Summary Ovariectomy of adult female rats (200-230g) resulted in an increase in ~-adrenergic receptors in the cerebral cortex, hypothalamus and anterior pituitary. The anterior pituitary had the largest overall increase as well as the most rapid increase in ~-adrenergic receptor density of the tissues examined. The increase in hypothalamic or cerebral cortical B-adrenergic receptors became apparent only long after ovariectomy (7-14 days). Fourteen days after ovariectomy, the density of B-adrenergic receptors was 7996, 40~, and 245 in excess of control values in crude membranes prepared from anterior pituitary, hypothalamus and cerebral cortex, respectively. Over the same interval, the plasma concentration of luteinizing hormone (LH) increased 2Q-fold, while the concentration of follicle-stimulating hormone {FSH) rose S-fold compared to control levels. Estradiol replacement (20 ]~glkglday) in these animals for four days before sacrifice concomitantly reduced plasma levels of the gonadotropins as well as the density of ~-adrenergic receptors in both the anterior pituitary and the hypothalamus. Long-term steroid replacement during the fifth and sixth week after ovariectomy, with implants of estradiol and progesterone which released the steroids in approximately physiological concentrations, significantly reduced B-adrenergic density in anterior pituitary, but not in the hypothalamic membranes. This treatment significantly reduced plasma LH, but not FSH. Beta-adrenergic receptor density was also found to fluctuate significantly during the 4-day estrous cycle. The highest values were found on proestrus, and the lowest on diestrus I. These studies indicate that changes in plasma concentrations of gonadal steroids (e.g. during the estrous cycle) influence the density of 13-adrenergic receptors in tissues involved in the control and release of anterior pituitary gonadotropins. We have recently reported an increase in ~-adrenergic receptor density in the anterior pituitary and cerebral cortex of orchidectomized rats which parallels the postcastration rise in plasma levels of anterior pituitary gonadotropins. Present address: Department of Anatomy, Emory University, Atlanta, Georgia 30322. Present address: Universidado de Estado de Sao Paulo, 16100 Aracatuba S.P., Brazil. 0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
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These effects are reversed by testosterone replacement ( I ) . Wagner et al. (2) have r e p o r t e d t h a t ovariectomy causes an increase in the d e n s i t y of ~ - a d r e n e r g i c receptors in the cerebral c o r t e x w h i c h was later shown to be suppressed by estrogen replacement (3). In c o n t r a s t , estrogen treatment has been reported to u p r e g u l a t e hypothalamic 13-adrenergic receptors ( 4 , 5 ) . These f i n d i n g s have prompted us to examine the ~ - a d r e n e r g i c d e n s i t y in b r a i n areas and a n t e r i o r p i t u i t a r y of ovariectomized rats in relation to levels of c i r c u l a t i n g g o n a d o t r o p i n s . Moreover, in view of data demonstrating changes in the d e n s i t y of both LHRH (6,7) and D2-dopamine receptors (8,9) d u r i n g the estrous cycle of the r a t , we have examined the d e n s i t y of a n t e r i o r p i t u i t a r y 13-adrenergic receptors in discrete phases of the cycle. Materials and Methods Female S p r a g u e - D a w l e y rats (200-230 gms) were ovariectomized or shamoperated u n d e r ether anesthesia, and s u b s e q u e n t l y housed with a l i g h t schedule of 14 h o u r s on (0500-1900 h o u r s ) and 10 h o u r s off. All animals used in assessing changes of ~ - r e c e p t o r d e n s i t y d u r i n g the estrous cycle were followed t h r o u g h at least two r e g u l a r 4 - d a y cycles before s a c r i f i c e . For s h o r t - t e r m estradiol replacement, at the t e n t h day following s u r g e r y , sham-operated animals and h a l f of the ovariectomized rats were injected s u b c u t a n e o u s l y ( s . c . ) w i t h 0.1 ml of corn oil. The remaining ovariectomized animals were injected w i t h 5 ]~c3 (about 20 ]J~Ikg) of estradiol-17~-benzoate (Sigma, St. Louis, MO) in 0.1 ml of corn oil. T h i s treatment was repeated for the n e x t three d a y s , and the animals were sacrificed on the f o u r t e e n t h day a f t e r s u r g e r y . Long-term ( s i x - w e e k ) ovariectomized animals were e i t h e r injected over the last f o u r days before s a c r i f i c i n g w i t h 100 ~ g l k g l d a y of estradiol or the vehicle (Table I ) , or t h e y were implanted b i l a t e r a l l y in the flank w i t h c r y s t a l l i n e progesterone and u n i l a t e r a l l y w i t h a solution of estradiol, both contained in Silastic tubing. These implants were dimensioned to produce the levels of estradiol and progesterone found on proestrus (I0). T h e y were inserted f o u r weeks a f t e r c a s t r a t i o n and remained in animals f o r the two weeks p r i o r to sacrifice. Animals were sacrificed between 11:00 AM and 11.45 AM on each day of the cycle. The d e n s i t y of 13-adrenergic receptors was determined on c r u d e membrane p r e p a r a t i o n s (from 600 to 100,000 x g) of tissue homogenates for all ovariectomized animals and sham-operated controls as described p r e v i o u s l y (11). The s a t u r a t i o n b i n d i n g assays w i t h [125-1]-iodocyanopindolol ( I C Y P ) were done at e i g h t c o n c e n t r a t i o n s of the ligand o v e r the range of 5 to 120 pM of the ( - ) - f o r m . The assay medium contained a m i x t u r e of suppressors of the n o n - s p e c i f i c b i n d i n g (11), I - ( - ) Propranolol ( 5 x i 0 - - M) was used to define the n o n - s p e c i f i c b i n d i n g . Plasma concentrations of anterior pituitary gonadotropins were determined by radiommunoassay. FSH antibody and reference standards were gifts of N I A D D K . LH antibody was obtained from Dr. G. Niswender, while the LH reference preparation RP2 and the hormone for iodination were provided by N I A D D K anterior pituitary program. Results The density of ~3-adrenergic receptors in membranes from the anterior pituitary gland increased within the initial 24 hours after ovariectomy (Figure I). This increase was not significant by Student's t test due to a relatively high variance at this sampling time for the castrated group. By 7 days after castration, the density of ~-adrenergic receptors in anterior pituitary membranes was significantly increased. In contrast, the increase was not pronounced in the hypothalamic membranes. The density of
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13-adrenergic receptors in the cerebral cortex was found to be significantly elevated only after long periods of ovariectomy (see Table I ) . Six weeks after ovariectomy, all tissues displayed significantly higher 13-adrenergic densities in comparison with their respective controls (Figure I and Table I ) . Treatment of ovariectomized animals with 20 1~glkglday of estradiol benzoate for four days before sacrifice on the 14th day after surgery resulted in a significant decrease in ~-adrenergic receptor density in both anterior p i t u i t a r y and hypothalamic membranes when compared to values in ovariectomized controls (Figure I ) . This treatment dramatically reduced the plasma concentration of LH (by 74%), and also v e r y significantly decreased plasma FSH (by 37%) (Figure 2). Combined treatment with estradiol and progesterone during the 5th and 6th week after ovariectomy significantly reduced the number of p i t u i t a r y ~-adrenergic receptors, but was less effective in reducing hypothalamic ~-adrenergic receptor density. This treatment strongly reduced the circulating LH levels (approximately 50%), while plasma FSH decreased only 12%. This latter difference was not significant in comparison with ovariectomized control values for this hormone. (Figure 2, lower panel). Anterior - Pituitary
~
~
0`5
i
~E
Hypothalamus
o
E m 1.5
0.5
1
7 14 Days after surgery
42
FIGURE 1 Maximum binding values for [1251] iodocyanopindolol with membrane fractions from anterior pituitaries and hypothalami at 1-42 days after surgery. Four to seven groups of 4 animals each were analyzed at each point. Unshaded bars denote values for sham-operated controls, hatched bars represent animals ovariectomized only, while the filled bars correspond to ovariectomized animals receiving estradiol benzoate for 4 days before sacrifice (day 14) or estradiol plus progesterone for 2 weeks before sacrifice (day 42). For f u r t h e r details see Materials and Methods. Asterisks indicate significance levels in two-tailed Student's t test, with comparison always being made to the nearest column on the left at each point in time. (*) indicates significance at the level of 0.05, (**) at the level of 0.01, and ( * * * ) at the level of 0.001. Data are presented with standard errors of the corresponding means.
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TABLE I Density of B-adrenergic receptors in cerebral cortex a f t e r ovariectomy and estrogen treatment Kd, pM b
Bmax (fM/m 9 tissue) b
14,03_+1.1
2.526_+0.16
Ovariectomized (7)
14.08_+0.92
3.094+0.15
p<0.025
Ovariectomized plus estrogen (4)
14.71+I .54
2.61±0.13
p<0.05
Treatment a Controls
(7)
Significance for change in Bmax c Student's Dunnett's t test test p<0.05 N.S.
(a) 4-6 animals constituted each experimental group. The number ot groups is given in parentheses. Ovariectomy or sham operations were performed six weeks before s a c r i f i c i n g . Estrogen treatment was I00 ] z g l k g l d a y of estradiol-17B benzoate for 4 consecutive days before sacrifice_. I (b) Mean values ± I SEM. Kd is the dissociation constant, pmol. l i t . Bmax is the maximum b i n d i n g , fmollmg tissue equivalent for membrane preparations used. These data were obtained in saturation assays employing ICYP in the range of 5-120 pM, using eight d i f f e r e n t molarity inputs and employing a mixture of suppressors of non-specific binding (11). (c) In the respective two-tailed tests [12). Note that the comparisons are with the control group shown above the respective treatment group. Since the density of a n t e r i o r p i t u i t a r y B-adrenergic receptors was affected by ovarian steroids, we examined the concentration of these receptors over the 4-day estrous cycle. The average density of B-adrenergic receptors found in each cycle phase is tabulated in Table 2. These densities were lower (at a confidence level g r e a t e r than 95%) than the average density of a n t e r i o r p i t u i t a r y B-receptors in either 14-day or 42-day ovariectomized animals (Table 2). The density found in proestrus and diestrus 2 was s i g n i f i c a n t l y higher than that of diestrus I . The receptor density at proestrus, the greatest within the cycle, was also s i g n i f i c a n t l y higher than the mean B-adrenergic receptor concentration on the day of estrus. The difference between proestrus and diestrus I was also significant in one-way F-test and in the Student-Newman-Keuls test (12) (Table 2), indicating a change in receptor number independent of the d i s t r i b u t i o n of Bmax values constituting the compared grand averages. Discussion The B-adrenergic receptor complement of both the a n t e r i o r p i t u i t a r y and various regions of the brain is downregulated by gonadal steriods a f t e r castration of either male or female rats ( 1 , 3 - 4 , 1 3 - 1 4 ) , although s h o r t - t e r m effects of either castration or steroid replacement may not be pronounced in the case of cortical and hypothalamic B-adrenergic receptors ( r e f . ( I ) and present r e s u l t s ) .
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24 tG
m
LH
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t
1567
FIGURE 2. Plasma levels of luteinizing hormone {LH) and follicle-stimulating hormone (FSH) at 1-42 days after sham-operation (unhatched bars), ovariectomy (hatched bars) or ovariectomy followed by treatment with estradiol-1713 benzoate (filled bars) as described in caption to Figure 1. Plasma samples from 8-12 animals were analyzed at each point. The data are presented with standard errors of the respective means. For f u r t h e r details see Figure 1 and Materials and Methods.
8OO
400
t 14 42 Days after surgery
TABLE II
Density of anterior p i t u i t a r y ~-adrenergic receptors during the rat estrous cycle Cycle Phase
Number a of groups
Mean receptor density a
Percent of overall mean
Estrus
16
0.181-+0.019
92.2
Diestrus I
22
0.164-+0.018
83.5
Diestrus 2
20
0.215-+0.020
109.4
Proestrus
24
0. 226-+0.017
114.8
Overall b mean 82 0.197_+0.01 100.0 (a) Two to four animals constituted each experimental group. The B-receptor density is expressed in femtomoles of ICYP specifically bound per/ mg of fresh tissue equivalent used, _+I S.E.M. (b) This mean is about 11 percent higher than the average for all sham-operated animals of Figure I (0.177-+0.013 fMlmg tissue), possibly due to seasonal and assay-related variations. The mean value of Bmax found at diestrous day 1 was d i f f e r e n t at the significance level of 0.05 from the mean of diestrus 2, and at the level of 0.01 from the mean of proestrus, using two-tailed Dunnett's test (12). The difference between proestrus and diestrus 1 was also significant in Student-Newman-Keuls test (12). The rapid change seen in anterior p i t u i t a r y B-adrenergic receptor density after ovariectomy (Figure 1) and after orchidectomy (1) might be due to the low overall B-adrenergic receptor density in the gland. This would permit large relative B-receptor density changes over short intervals. The variation observed during the early period after ovariectomy might have been due to the use of randomly cycling animals, since density of B-adrenergic receptors in anterior p i t u i t a r y gland differs significantly between the phases of the estrous cycle (Figure 1 and Table 2). However,
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at longer intervals after ovariectomy, influences of cycle phase upon the ~3-receptor density would presumably be small since the maximal difference between cycle phases varied only by about 15% of the mean density (Table 2). Following ovariectomy, the density of /3-adrenergic receptors in the hypothalamus, and especially in the cerebral cortex, increased at rates apparently much slower than in the anterior pituitary. The increase became significant between 7 and 14 days after the castration. Over that interval, the absolute increase of t3-adrenergic receptor number per mg tissue was not, however, appreciably different between anterior pituitary and hypothalamus (Figure I). On the 7th day, the increment was approximately 0.1 fM ICYP/mg in both tissues (Figure I). Six weeks after ovariectomy, the increment of ~3-adrenergic receptors in hypothalamus and cerebral cortex was quite similar, about 0.5 fM ICYP/mg tissue (Figure I and Table I), while in the anterior pituitary it was approximately 0.2 fM/mg (Figure I). Since the anterior pituitary appears to have exclusively ~2-receptors (I,11}, the increment in the pituitary could be considered as an increase in the ~2-adrenergic receptor population. Actually, cortical ~2-adrenergic responses, or overall density of 13-adrenergic receptors, in ovariectomized animals have been reported to be attenuated by estrogen treatment (2,13). The density of ~32-adrenergic receptors, but not of Dl-SUbtype, appears to be significantly uncreased in rat cerebral cortex affer orchidectomy (I). Since 139-adrenergic receptors constitute, in intact animals, only about 20% of cortical~3-adrenergic receptor complement (11,15), and about half of the hypothalamic ~3-receptor density (11,16), even an increase comprised entirely of 13~-species will be appreciably masked in these tissues by their ~1-acrrenergic receptor complement. On the other hand, there is evidence for an activation of neurons, possibly involving an increase in synaptization between neurons in the arcuate and the mediobasal hypothalamus after ovariectomy (17,18). Such activity would likely result in increased numbers of one or both t3-adrenergic receptor subtypes. Since these changes appear to develop slowly, they might account for a significant proportion of the long-term accumulation of ~3-adrenergic receptor density in hypothalamus and cerebral cortex after ovariectomy. The physiological role of ~2-adrenergic receptors in anterior pituitary and hypothalamus is at present not sufficiently understood. These receptors are presumably involved in activation of adenylate cyclase systems and thus in secretion of protein hormones and peptides. For example, ~32-adrenergic mediation is involved in the release of ~ - M S H from posterior pituitary (19}. Positive ~-adrenergic regulation could also be shown for release of A C T H in vivo (20), for the pulsed release of growth hormone {GH) from dispersed anterior pituitary cells (21), and for the release of prolactin from such cells {22). ~-receptors, which are increased after castration in both male and female r~ts (reference (I) and the present results) might cooperate with LHRH receptors in modulating the increased output of LH following castration. L H R H receptors are also strongly upregulated by ovariectomy in the rat (7). It is of interest to note that patterns of change in ~-receptors of the anterior pituitary over the estrous cycle as found in this work are similar to those established for L H R H receptors in the cycle phases (6,7). The peak concentration of both species of receptor might occur at the beginning of proestrus, thereby providing for a maximal responsiveness of the tissue to either L H R H or to ~32-adrenergic inputs. The increase in B-receptors in pituitary on proestrus suggests that estrogen has a biphasic action on their density, first to upregulate and then to downregulate it. The upregulatory effect might correspond with estrogen enhancement of pituitary responsiveness to L H R H (e.g., ref. 22), and would probably not be seen in
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c h r o n i c estrogen replacement s t u d i e s . Our data i n d i c a t e , as in the case of orchidectomized males ( I ) , a parallelism in p a t t e r n s of change for a n t e r i o r p i t u i t a r y 132-adrenergic receptors and the levels of plasma LH a f t e r ovariectomy and replacement of ovarian steroids. T h e parallelism between b r a i n 132-receptors and LH levels remains as yet to be e l u c i d a t e d . However, the p o s s i b i l i t y t h a t increased 13-receptors in hypothalamic areas might facilitate release of g o n a d o t r o p i n - r e l e a s i n g peptides, and in t h i s way c o n t r i b u t e to elevated LH and FSH levels a f t e r c a s t r a t i o n , should be viewed w i t h c a u t i o n , since B~-agonists appear to e x e r t l a r g e l y i n h i b i t o r y i n f l u e n c e s upon LH release at tl~e level o f hypothalamus (23,24). I f a n t e r i o r p i t u i t a r y ~ - a d r e n e r g i c receptors s i g n i f i c a n t l y c o n t r i b u t e to the r e g u l a t i o n of g o n a d o t r o p i n s e c r e t i o n , i t would appear t h a t t h e i r role in LH secretion should be much more d i r e c t than in the case of FSH secretion. It has been demonstrated p r e v i o u s l y t h a t the elevated levels of FSH w h i c h r e s u l t from ovariectomy are r e f r a c t o r y to steroid replacement while LH levels are not ( 1 7 , 2 5 ) . A l s o , o u r recent r e s u l t s indicate t h a t 13-adrenergic a n t a g o n i s t s i n f l u e n c e the plasma levels of LH much more d i r e c t l y and to a much l a r g e r degree than is the case w i t h levels of FSH (26). In the p r e s e n t s t u d y , steroid replacement also had much more d i r e c t effect on plasma levels of LH. It could be t h a t the control of LH secretion is more d i r e c t l y associated w i t h p i t u i t a r y a d r e n e r g i c mechanisms. References
I. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
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