Estradiol regulates the number of α1 but not β or α2 noradrenergic receptors in hypothalamus of female rats

Neurochem. Int. Vol. 16, No. I, pp. 1-9, 1990 Printed in Great Britain. All rights reserved

0197-0186/90 $3.00 + 0.00 Copyright © 1990 Pergamon Press plc

E S T R A D I O L R E G U L A T E S T H E N U M B E R O F 0~1 B U T N O T fl O R 0~2 N O R A D R E N E R G I C R E C E P T O R S IN HYPOTHALAMUS OF FEMALE RATS ANNE M. ETGEN* and GEORGE B. KARKANIAS Departments of Psychiatry and Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, U.S.A. (Received 13 April 1989; accepted 12 June 1989)

Abstract--Present experiments examined whether previously observed hormone-dependent differences in norepinephrine-stimulated cAMP accumulation in hypothalamic and preoptic area slices are attributable to differences in noradrenergic receptor number or binding affinity. When compared to ovariectomized controls, hypothalamic and preoptic area membranes from estradiol-treated rats had significantly elevated numbers of [3H]prazosin (cq) binding sites. Estradiol affected neither the number of~t I sites in frontal cortex nor the affinity of [3H]prazosin binding in any brain region sampled. Estradiol had no effect on [3H]idazoxan (a2) or [3H]dihydroalprenolol (//) binding in hypothalamus, preoptic area or cortex. Progesterone reversed estradiol elevation of prazosin binding in preoptic area but had no other measurable effects on any noradrenergic receptor binding when given alone or in combination with estradiol. Neither estradiol nor progesterone altered binding of radiolabeled antagonists when they were included in the in vitro incubation mixture. These data suggest that the increased cq receptor augmentation of cAMP accumulation seen in hypothalamic and preoptic area slices from estradiol-treated rats is correlated with increased ~tI receptor number. In contrast, estradiol attenuation of fl receptor function and progesterone depression of norepinephrine-stimulated cAMP generation in slices from estradiol-treated females is not correlated with downregulation of any noradrenergic receptor subtype.

One mechanism by which steroid hormones might regulate neural activity is to modulate neuronal responsiveness to released neurotransmitters. Work from this laboratory suggests that estradiol (E2) and progesterone (P), the ovarian steroids which coordinate female reproductive physiology and behavior, modify norepinephrine (NE)-stimulated c A M P formation in slices from the preoptic area and hypothalamus. When compared to slices from ovariectomized (OVX) rats, slices from E2-treated females exhibit decreased fl receptor stimulation and increased ~ receptor augmentation of c A M P formation (Etgen and Petitti, 1987). Cortical (Wagner and Davies, 1980) and hippocampal (Harrelson and McEwen, 1987) slices from estrogen-exposed females also show reduced c A M P responses to fl receptor activation. In contrast, slices from rats given both E2 and P show an overall depression in NE-stimulated c A M P accumulation (Etgen and Petitti, 1986;

Petitti and Etgen, 1989). This effect of P is estrogendependent and is associated with an apparent loss of ctI receptor augmentation of c A M P generation and the appearance of ct2 receptor augmentation of fl-receptor-stimulated c A M P formation (Etgen and Petitti, 1987; Petitti and Etgen, 1989). Thus ovarian steroids seem to regulate the efficacy of fl, cq, and ct2 receptors to activate the c A M P second messenger system in brain regions which control reproduction. The major question left unanswered by our previous work is the mechanism by which E 2 and P modulate N E receptor function in slices. The most obvious hypothesis is that hormone-dependent alterations in slice c A M P responses are attributable to changes in N E receptor number and/or ligand binding affinity. Other laboratories have reported that E: increases (Wilkinson et al., 1979; Vacas and Cardinali, 1980) or decreases (Wagner et al., 1979; Biegon et al., 1983)/3 receptor number in rats. In guinea-pigs, estrogen has been reported to increase ct2 receptor density in some hypothalamic regions but to decrease • 2 binding in others (Johnson et al., 1985). With

*Author to whom all reprint requests and correspondence should be addressed. N.C.L 16/I--A

I

2

ANNE M. ETGEN and GEORGEB. KARKANIAS

regard to ~ receptors, no change (Wilkinson et al., 1979; Vacas and Cardinali, 1980; Orensanz et al., 1982; Nock and Feder, 1983; A m b r o s i o et al., 1984), decreases in several h y p o t h a l a m i c regions (Weiland a n d Wise, 1987; J o h n s o n et al., 1988) or a n increase in the nucleus tractus solitarius (Shackelford et al., 1988) has been reported following E 2 treatments. N o n e of these studies reported h o r m o n e - i n d u c e d changes in ligand binding affinity. Some of the studies cited above (e.g. W a g n e r et al., 1979; Nock and Feder, 1983; Biegon et al., 1983; J o h n s o n et al., 1988) have looked at c o m b i n e d E 2 and P treatments and have reported no effect of P. Therefore, the present studies were u n d e r t a k e n to assess the effects of E2 and P on N E receptor density a n d ligand binding affinity in brain slices. To facilitate direct c o m p a r i s o n with o u r previous work, Scatchard analyses of [3H]antagonist binding to cq, ~2 and fl adrenergic receptors were conducted in membranes from slices of h y p o t h a l a m u s and preoptic area of animals given the same E 2 a n d P treatments which had been shown to alter N E receptor activation o f c A M P f o r m a t i o n in our previous studies (Etgen and Petitti, 1986, 1987; Petitti a n d Etgen, 1989). We also evaluated N E receptor binding parameters in frontal cortex from the same animals to determine whether any observed h o r m o n e effects were regionspecific. EXPERIMENTAL PROCEDURES

Tissue preparation Female Sprague Dawley rats (150 175 g, Taconic Farms) were housed in groups of 2-4 in a temperature-controlled environment with food and water available ad libitum. All animals were OVX bilaterally under Metofane anesthesia (Pitman Moore Inc., Atlanta, Ga) 4~7 days prior to use and were randomly assigned to each hormone treatment group. The steroid hormones E 2, E 2 benzoate (EB) and P were purchased from Steraloids (Wilton, N.H.), dissolved in peanut oil and injected subcutaneously. Except where noted otherwise, estrogen treatment consisted of 2 #g of EB given 24 and 48 h before sacrifice. P treatment was always 500/~g given 3.5h before sacrifice. OVX control animals were injected with equivalent volumes of oil vehicle. To control for potential diurnal variations in hypothalamic adrenoreceptor levels (Krauchi et al., 1984; Jhanwar-Uniyal et al., 1986; Weiland and Wise, 1987), all animals were sacrificed between 1000 and 1300h. Animals were sacrificed by decapitation; the brain was quickly removed and placed on ice. The entire hypothalamus and preoptic area were removed as described by Hatton (1984), and slices of "'middle" hypothalamus and preoptic area (350/~m thick) were cut on a Mcllwain tissue chopper beginning approx. 2 mm anterior to the optic chiasm and ending 1 mm anterior to the mamillary bodies. Based on anatomical landmarks observed in comparable slices from fixed tissue, the first four slices were taken and designated

preoptic area. The next two slices containing the anterior hypothalamus were discarded. The following three slices, containing the ventromedial hypothalamus and much of the arcuate nucleus, were saved and designated hypothalamus. A sample of frontal cortex (250-350 mg) was also removed from each brain. Tissue from each of the three brain regions of each rat was homogenized separately in 5 ml of ice cold buffer containing 50 mM Tris HCI (pH = 7.4) and 10mMMgC12. The homogenates were centrifuged for 10 min at 20,000g, the supernatant discarded and the pellet containing the crude membrane fraction frozen at -76°C until assay. We find no loss in any of the NE receptor subtypes under these conditions with storage times of up to 3 months and with up to 3 series of thawing and refreezing. Radioligand binding assays Frozen membranes were resuspended in enough homogenizing buffer to bring the protein content to 0.5-1.0mg/ml. For Scatchard analyses, aliquots of the suspended membranes from individual animals were incubated with 0.2-5 nm [3H]prazosin (~, sites) for 20min at 25:'C, or with 0.2 5 nM [3H]idazoxan (a2 sites) or 0.2-3 nM [3H]dihydroalprenolol (DHA, fl sites) for 15 min at 25"C. Scatchards were performed on 4~7 independent samples for each hormone treatment. The radiolabeled ligands [3H]DHA and [3H]prazosin were purchased from New England Nuclear (Boston, Mass.) and [3H]idazoxan was obtained from Amersham (Arlington Heights, 111.). Unlabeled competitors used to assess nonspecific binding were present in 1000-fold excess and included prazosin (oq), yohimbine (~2) and propranolol (fl). Aliquots for both total and nonspecific binding were prepared in triplicate for each concentration of radioligand. Bound and free radioligands were separated by rapid filtration through glass fiber filters (No. 25, Schleicher and Schuell, Keene, N.H.) on Millipore filter manifolds. To reduce nonspecific adsorption of ligands, filters were soaked in 1% polyethylenimine for 1 h prior to use (Bruns et al., 1983). After addition of sample, each filter was rinsed with 2 × 5 ml of ice cold homogenizing buffer and then placed into a 7 ml plastic scintillation vial. Radioactivity was measured by adding 5 ml of Hydrofluor (National Diagnostics) and counting in a Beckman LS3801 counter for 10min at approx. 50% efficiency. Receptor affinities (Kd) and numbers (Bmax)were calculated using the LIGAND program of Munson and Rodbard (1980). To evaluate the specificity of radioligand binding, triplicate aliquots of suspended cortical membranes from OVX females were incubated with 2 nM 3H-ligand in the presence or absence of increasing concentrations of unlabeled competitors (1 nM 10/aM). Extensive competition studies were not conducted on hypothalamic or preoptic area slices due to the limited amounts of tissue available from these brain regions. The IC50 of each competitor for each radioligand was calculated using the LIGAND program of Munson and Rodbard (1980). In experiments where receptor number was estimated using one-point analysis, membranes were incubated with a single, saturating concentration of each ligand in the presence or absence of 1000-fold excess unlabeled competitor as described above for Scatchard analyses. All incubations for total and nonspecific binding were done in triplicate, and the radioligand concentrations used were 5 nM [3H]prazosin, 2 nM [3Hlidazoxan and 5 nM [3H]DHA.

Estradiol and hypothalamic noradrenergic receptors Table 1. Displacementof 3H-ligandsby unlabeledcompetitorsin cortical membranes 3H-ligand Competitor

Dihydroalprenolol (#)

Isoproterenol Propranolol Prazosin Phenylephrine Clonidine Yohimbine Schering 23390

> > > >

52.8 9.7 10,000 10,000 10,000 10,000 ND

Prazosin (~tt) 1C~0 (nM)

Idazoxan (~t2)

> 10,000 > 10,000 11.8 > 10,000 > 10,000 > 10,000 > 20,000

> I 0,000 > I 0,000 929 > 10,000 17.6 112 923

ND = not done. Pooled membranes from frontal cortex (OVX female rats) were incubated with 2 nM 3H-ligand in the presence or absence of increasing concentrations of unlabeled competitors (1 nM 10#M). ICs0s were calculated using the L I G A N D program.

Data analysis Aliquots of each membrane suspension were analyzed for protein content by the method of Lowry et al. (1951) so that total apparent receptor (i.e. Bmax)could be expressed as fmol/mg protein. Mean Bmax and Kd values for each brain region of animals from different treatment groups were analyzed by one-way analysis of variance (ANOVA). When the overall ANOVA was significant (P < 0.05), betweengroup differences were determined using the Newman Keuls analysis. RESULTS

Ligand binding characteristics Table 1 shows the ability of various noradrenergic compounds to compete for [3H]prazosin, [3H]idazoxan and [3H]DHA binding sites in cortical membranes from OVX female rats. These data suggest that the radioligands are labeling at, ~2 and fl receptor sites, respectively. Scatchard analyses of ~t~, ct2 and fl receptor binding in cortical membranes incubated with no additions, with 10 nM added E 2 or with 100 nM added P to approximate maximal expected levels of hormone that might be present in brain tissue at the time animals were sacrificed in later Table 2. Effects of E 2 and P in vitro on binding of 3H-ligands in cortical membranes Competitor 3H-ligand Dihydroalprenolol Ka (nM) Bma~ (fmol/mg) Prazosin Kd (nM) Bma~ (fmol/mg) Idazoxan K~ (nM) Bin,~ (fmol/mg)

None

E 2 (10 nM)

P (100 nM)

0.70 45.0

0.76 49.6

0.80 43.4

0.26 355

0.34 359

0.25 390

1.45 97.5

1.14 92.9

1.64 123

Pooled membranes from frontal cortex (OVX female rats) were subjected to Scatchard analysis under 3 conditions: no additions, l0 nM E 2 in the in vitro incubation or 100 nM P in the in vitro incubation.

experiments, showed that E 2 and P in vitro affected neither receptor number nor ligand binding affinity of any NE receptor subtype (Table 2). Figure 1 shows representative protein-standardized Scatchard plots of ~ , ~2 and fl receptor binding in hypothalamus, preoptic area and cortex from OVX female rats. Each of the three ligands exhibited a single class of saturable, high affinity binding sites in all three brain regions, and Hill coefficients were not significantly different from 1.0 (data not shown). The Kds of [3H]DHA and [3H]prazosin binding were very similar in all brain areas (approx. 1.0 and 0.2 nM, respectively). The KdS for [3H]idazoxan binding were somewhat more variable (0.5-1.0 nM), but they did not vary consistently as a function of brain region. Effects of ovarian steroid treatments on NE receptor binding As shown in Table 3, neither EB, P nor combined administration of EB plus P affected any aspect of ligand binding in cortical membranes. In contrast, hypothalamic membranes from EB-treated females showed a modest (30%) but significant increase in the number of ctI sites with no change in [3H]prazosin binding affinity (Fig. 2). P had no effect on prazosin binding when given alone, and it neither enhanced nor reversed the EB-induced elevation in ~tt sites in the hypothalamus. EB did not affect either idazoxan or DHA binding in the hypothalamus (Fig. 3). In preoptic area membranes, EB also induced a small (20%) but significant increase in the number of [3H]prazosin binding sites. P alone had no effect on prazosin binding in the preoptic area, but it appeared to reverse the EB-induced elevation in prazosin binding. The total number of idazoxan binding sites was similar in preoptic area membranes from OVX, EB-, P- and EB + P-treated females, and there were no effects of hormone treatment on DHA binding.

ANNE M.

ETGENand

DHA

CORTEX

GEORGE B. KARKANIAS

IDAZOXAN

PRAZOSIN

0.I00

0080

0.500

0.080-

0.400

0.060,

0060

0.300

0 040

0.200

0 020

0.100

0.040

0 000

I

60

120

90

0.00( 110

0020

160

210

260

0.000 - -

310

25

50

75

1oo

PREOPTIC AREA 0.006-

0.025 0 020

~

0004 •

"

0.060 ~

•

o 0 015

0.0,¢O

0.010

~-) 0.002

0.020

0.000 0

20

30

0.000 4,0

40

O005 50

60

70

80

90

0 000

210

- -

4*O

60

HYPOTHALAMUS 0.005 0004 0.003

0o,o

0 002

0.040-

.'>--.. •

o02o

O.001 O00C

10

2O

30

0.000 90

0 000 115

140

165

0

I0

20

3O

40

BOUND (FMOLE/MG)

Fig. 1. Representative Scatchard plots of [I (DHA), ~L (prazosin) and ~2 (idazoxan) receptor binding in membranes of frontal cortex, preoptic area slices and hypothalamic slices from OVX female rats.

Dose dependence of EB effects on NE receptor binding Since the experiments above indicated that E 2 and P have no effect on antagonist affinity for u~ NE receptors, one-point assays were used to examine the dose dependence of estrogen elevation of [3H]prazosin binding. When the EB dose was varied from 0.5-5.0 #g given 24 and 48 h before sacrifice, we found that all EB doses significantly increased the number of u~ sites in hypothalamus from 150 (OVX) to about 200 fmol/mg (Fig. 3). Additional animals were injected with: (1) a single dose of 3/~ g of EB 48 h before sacrifice to mimic the estrogen priming dose we normally use to facilitate female sexual behavior (e.g. Etgen, 1987), or (2) 2 #g of EB every other day for 10 days prior to sacrifice since this treatment has been reported to increase u~ sites in cortex (Favit et al., 1988). These EB treatments also produced approx. 30% elevations in the level of [3H]prazosin

binding in hypothalamus (Table 4). Moreover, as suggested by the Scatchard analysis, injection of 500 pg of P in animals given 2 y g of EB 24 and 48 h before sacrifice did not reduce the elevated level of [3H]prazosin binding. None of the EB treatments altered the number of DHA or idazoxan binding sites in hypothalamus (Fig. 3), nor were there any significant EB-induced changes in ~,, ~2 or fl binding sites in cortical or preoptic area membranes (Table 5). Based on the Scatchard results, we had expected to see modest EB-dependent increases in [3H]prazosin binding in preoptic area. The failure to see statistically significant effects of EB in preoptic area analyzed with one-point assays probably results from increased variability and reduced accuracy in estimates of receptor density using this method. The receptor density values reported for estrogen-exposed rats in Table 5 are very close to those found using Scatchard

Estradiol and hypothalamic noradrenergic receptors Table 3. Effects of/n vivo treatments with ovarian steroids on NE receptor binding parameters Treatment EB

OVX

P

EB + P

0.96_+0.19 95 _+6.1

1.1 +0.10 96 + 3.1

Dihydroalprenolol (fl) Cortex Kd (nM) Bm~x (fmol/mg) Preoptic area Kd (nM) B,,x (fmol/mg) Hypothalamus Kd (nM) Bma~ (fmol/mg)

0.92+0.13 95 + 12

0.98_+0.10 92 _+7.3

1.0_+0.16 47 _+7.7

1.1 _+0.14 51 _+3.6

1.1 _ + 0 . 1 6 33 + 3.2

1.3_+0.55 37 + 2.4

0.95_+0.10 34 _+4.4

0.97_+0.18 35 _+5.7

0.28_+0.07 274 -+ 41

0.19+0.02 284 + 31

0.18+0.03 255 -+ 30

0.29+_0.14 226 -+ 36

0.20 _+0.02 87+3.1

0.24 _+0.02 108+6.1"

0.22 _+0.04 94-+5.2

0.19 + 0.02 91_+4.6

0.19_+0.03 152+21

0.15_+0.02 195+4.3"*

0.17_+0.01 154_+3.4

0.19_+0.02 181_+11

1.0+0.16 105_+11

0.79_+0.06 112_+4.1

0.78+_0.02 101_+5.5

0.85_+0.05 110_+25

0.88 _+0.10"** 64 _+ 10

0.50 _+0.08 54 _+7.8

0.57 _+0.07 62 _+6.3

0.52 _+0.10 54 _+6.8

0.49_+0.13 41 _+2.9

0.69_+0.11 51 _+6.5

0.55_+0.04 49 _+ 1.0

0.57_+0.07 44 _+ 5.0

1.1 _+0.09 46 _+5.4

1.0+0.13 45 + 2.6

Prazosin (~1) Cortex Kd (nM) B~x (fmol/mg) Preoptic area Kd (nM) Bm~ (fmol/mg) Hypothalamus Kd (nM) Bm,~ (fmol/mg)

ldazoxan (~z) Cortex Kd (riM) Bmax(fmol/mg) Preoptic area Kd (nM) Bm~~ (fmol/mg) Hypothalamus Kd (nM) Bin,~ (fmol/mg)

Each value represents the mean of 4-7 independent replications. Data from the 4 different hormone treatment groups were analyzed by 1-way analysis of variance for each ligand and each brain region. *EB ~- OVX = P = EB + P (P < 0.05, Newman-Keuls). **EB > OVX = P (P < 0.05, Newman-Keuls). ***OVX > EB = P = EB + P (P < 0.05, Newman-Keuls).

a n a l y s i s in T a b l e 3. H o w e v e r , t w o u n u s u a l l y h i g h r e c e p t o r d e n s i t y v a l u e s in t h e O V X c o n t r o l s p r e v e n t e d t h e E B effect f r o m r e a c h i n g s t a t i s t i c a l significance.

Time dependence o f estrogen elevation o f prazosin binding OVX female rats were injected with 3 #g of EB and sacrificed 4, 12, 24 o r 48 h l a t e r to e v a l u a t e t h e t i m e

Cortex

0.60

c o u r s e o f e s t r o g e n e l e v a t i o n o f ~ b i n d i n g sites in h y p o t h a l a m u s a n d p r e o p t i c area. C o n t r o l a n i m a l s received oil v e h i c l e 48 h b e f o r e sacrifice. A s s h o w n in Fig. 4, E B d i d n o t s i g n i f i c a n t l y i n c r e a s e [3H]prazosin b i n d i n g in h y p o t h a l a m u s u n t i l 48 h p o s t - i n j e c t i o n . O n e - p o i n t e s t i m a t e s o f [3H]prazosin b i n d i n g in p r e o p t i c a r e a b e t w e e n 4 a n d 48 h a f t e r E B were somewhat variable (data not shown). However, mean levels o f p r a z o s i n b i n d i n g in p r e o p t i c a r e a i n c r e a s e d

HypothoLomus

P r e o p t i c area

0.06

o. 12

o,or

oo, F

o

o2o _

o o2 F

o o,

i ol

100

•

, 200

o, 300

40

, 60

, \o, 80

100

o •

ol 120

80

,

,

100

120

o 140

160

180

Bound (fmol.e/mg) Fig. 2. Representative Scatchard plots of prazosin binding in cortical, preoptic area and hypothalamic m e m b r a n e s from OVX (solid circles) and EB-treated (open circles) female rats.

200

6

ANNE M. ETGEN and GEORGE B. KARKANIAS

"•250 T

Prozosin

*

E 15o+: "1o E 100-

~

:::}

Idozoxon

50-

0

o

0

DHA

e

~

t-n

1.0

o

2.0

5.0 EB, /Jg

Fig. 3. Effects of EB dose on NE receptor number in hypothalamic slices. OVX female rats received the indicated doses of EB 24 and 48 h prior to sacrifice. The 0 dose represents OVX controls which received injections of oil vehicle 24 and 48 h prior to sacrifice. Each point represents the mean ( + SEM) of at least 4 independent determinations. Error bars are not visible on many of the D H A points because the magnitude of the SEM was smaller than the symbol size. *Significantly different from control (P < 0.05, Newman Keuls).

from 79.5 + 10.5 fmol/mg (n---2) in OVX rats to 116 + 41 fmol/mg (n = 4) in rats given EB 48 h previously. These numbers agree closely with the OVX and EB values reported for preoptic area using Scatchard analysis (Table 3). Elevations in [3H]prazosin binding in preoptic area were not seen at 4, 12 or 24h post-EB. DISCUSSION

These experiments demonstrate that E 2, even in very low doses, moderately (30%) but significantly increases the number of ~ binding sites in female rat hypothalamus. The Scatchard analyses suggest further that cq receptors in the preoptic area are also elevated by E2, but the hormonal effect is less robust than in the hypothalamus. Present data thus suggest

that the enhanced ~t1 receptor augmentation of cAMP accumulation seen in hypothalamic and preoptic area slices from estrogen-treated rats (Etgen and Petitti, 1987) is attributable to increased numbers of cq binding sites in these brain regions. Because of the multiple levels of signal amplification in second messenger systems, it is possible to produce marked changes in second messenger levels with relatively small changes in receptor number. Our data are also consistent with the hypothesis that estrogendependent elevations in cq binding sites are mediated by classical E2 receptors. Three observations support this hypothesis: (!) the antiestrogen tamoxifen, which prevents E2-receptor interaction with chromatin binding sites (Etgen, 1979; Etgen and Robisch, 1988), blocks E 2 effects on ~q augmentation of the cAMP response (Etgen and Petitti, 1987); (2) increased levels of cq sites are measurable only at relatively long times after estrogen exposure (48 h); (3) in agreement with Biegon et al. (1983), we found that EB treatments had no effect on prazosin binding in frontal cortex, a brain region which is almost devoid of E 2 receptors in adult female rats. Estrogen treatment had no effect on any NE receptor subtype in frontal cortex, nor did EB alter ct2 or fl adrenergic receptor binding in any of the three brain regions examined. Thus our results suggest that E2 regulation of NE receptors is both region- and subtype-specific. Furthermore, the finding that E 2 elevates cq binding sites in preoptic area and hypothalamus agrees well with reported effects of E 2 and NE on female reproductive physiology and behavior. In estrogen-primed female rats, NE acts via a~ receptors to facilitate release of luteinizing hormone-releasing hormone (LHRH). This ~t~ receptor-mediated release of L H R H is not seen in OVX females in the absence of E 2 priming (see Taleisnik and Sawyer, 1986), except perhaps in the first day or two following ovariectomy (Leipheimer et al., 1984). It has also

Table 4. Effects of different EB priming schedules on NE receptor binding in hypothalamus EB priming schedule Ligand DHA Prazosin Idazoxan

OVX 3/ag, 48h 2~g+P 2/~g, 10days Specific binding [fmol/mg protein (X _+SEM)I 19 + 4.0 149_+22 33 + 5.9

27 + 3.0 212_+11" ND

27 +_2.2 223_+10" ND

23 + 1.9 189+17 44 _+5.4

Values represent the mean of 3-6 independent determinations. All animals were OVX; some received a single injection of 3 ~g of EB 48 h prior to sacrifice, some received 2#g of EB 24 and 48h before sacrifice plus 500 #g of P 3.5 h before sacrifice, and some received 2 #g of EB every other day for 10 days before sacrifice. *Significantly different from OVX control (P < 0.05, Newman-Keuls). ND = not done.

Estradiol and hypothalamic noradrenergic receptors Table 5. Effectsof EB dose on NE receptor bindingin cortex and preoptic area EB dose (#g) Lignnd/tissue DHA Cortex Preoptic area Prazosin Cortex Preoptic area Idazoxan Cortex Preoptic area

0

0.5

1.0

2.0

5.0

76 _+4.2 42 + 5.1

78 + 4.7 48 _+ 3.5

81 + 3.8 52 + 6.5

88 _+ 5.0 48 + 4.2

80 + 4.7 39 + 4.8

294 + 27 91 + 13.4

301 + 38 101 +5.1

280 + 36 99-+8.5

299 + 27 103-+9.2

272 + 22 111 -+ 11

101 + 32 87 -+ 17

131 -+ 27 94 -+ 15

105 -+ 28 85 -+ 8.9

141 + 36 92 + 11

124 -+ 32 92 -+ 11

Data represent the number of binding sites (fmol/mg tissue protein) estimated by one-point assays (see Experimental Procedures). Each value reports the mean (_+SEM) of 4 - 6 independent replications. Animals were injected with the indicated dose of EB twice at 24 and 48 h before sacrifice. The " 0 " dose represents OVX animals injected with oil vehicle 24 and 48 h before sacrifice. One-way ANOVAs revealed no statistically significant differences as a function of EB dose for any lignnd in either brain region.

been shown that the ~ antagonist prazosin can inhibit E2 + P induction of female sexual behavior when given systemically (Nock and Feder, 1984; Blaustein, 1987; Vincent and Feder, 1988) or directly into the ventromedial hypothalamus (Etgen, in preparation). Taken together, these data are consistent with the notion that E~ regulation of female reproductive function involves enhanced ~ NE receptor activity in the preoptic area and hypothalamus, mediated in part by estrogen-induced increases in ~ receptor number. A number of factors may account for discrepancies in the reported effects of estrogen on :q receptor number. Many of the earlier studies utilized ligands which do not discriminate between ~ and a2 receptors (Wilkinson et al., 1979; Vacas and Cardinali, 1980; Orensanz et al., 1982). Species differences in E2 action or the use of one-point estimates of prazosin binding might explain the failure of Nock and Feder (1983)

E

200

-~ 180 ,4-"~c 160 m c "~ o N ~ n

140 120 10C

, '2

2',

J8

&

Hours After EB

Fig. 4. Time course of EB-induced increase in ~1 receptor number in hypothalamic membranes. OVX females received 3 gg of EB at the indicated times before sacrifice. Each point represents the mean of 2 (0 control and 48 h) or 4 (4, 12, 24 h) independent determinations.

to observe estrogen-dependent changes in hypothalamic or preoptic area membranes of guinea-pigs. Differences in the method of estrogen priming might also be important (Shackelford et al., 1988). It is more difficult to reconcile the present findings with those of Weiland and Wise (1987). Using receptor autoradiography to quantify [3H]prazosin binding in hypothalamic regions, they found that 2-day exposure of OVX females to low E2 levels via Silastic capsules decreased the density of a~ binding sites in several hypothalamic regions involved in the coordination of female reproductive physiology and behavior (e.g. medial preoptic nucleus, median eminence, ventromedial nucleus, suprachiasmatic nucleus) without affecting a~ binding in the lateral septum. Major methodological differences between the present and former experiments could account for the divergent observations. Our studies utilized EB injections and measured 0~1 receptors in la,-ge blocks of hypothalamus and preoptic area via Scatchard and one-point analyses of [3H]prazosin binding to membranes. Had we assayed ~m receptors in micropunched subregions of the hypothalamus, our results might have been more comparable to those of Weiland and Wise (1987). However, the purpose of the present studies was to make direct comparisons between our previously reported effects of steroids on NE-stimulated cAMP accumulation in hypothalamic and preoptic area slices and NE receptor binding in comparably dissected tissue. With regard to ~ receptors, acute estrogen exposure has been shown to elevate ~ NE receptors in hypothalamus but not cortex (Wilkinson et al., 1979; Vacas and Cardinali, 1980), whereas chronic (2 week) estrogen treatment depressed D H A binding in cortical membranes (Biegon et al., 1983). In view of the estrogen-dependent inhibition of ~ receptor-

8

ANNEM. ETGENand GEORGEB. KARKANIAS

stimulated cAMP accumulation seen in cortex (Wagner and Davies, 1980), hippocampus (Harrelson and McEwen, 1987), hypothalamus and preoptic area (Etgen and Petitti, 1987), we had expected that E2 might decrease DHA binding in all three brain regions examined. However, this was clearly not the case. Thus it is unlikely that E2 reduces isoproterenolinduced cAMP formation by fl receptor downregulation. Future studies will assess the hypothesis that E 2 acts by interfering with fl receptor coupling to the adenylate cyclase/cAMP generating system. A recent report that chronic estrogen exposure uncouples dopamine D 2 receptors from their effector systems in pituitary membranes (Munemura et al., 1989) provides some precedent for proposing such a mechanism of estrogen action in brain. Similar mechanisms might account for the poor correlation between other steroid-dependent changes in NE receptor-mediated cAMP formation and changes in NE receptor number or binding affinity. For example, slices from OVX and E2-treated females demonstrate ~tI receptor augmentation of fl receptorstimulated cAMP accumulation (Etgen and Petitti, 1987) whereas slices from E 2 + P-treated animals do not (Petitti and Etgen, 1989). Nonetheless, we find no evidence that P downregulates ~t, receptors. Thus P may promote uncoupling of Ctl receptors from G proteins in the hypothalamus and preoptic area. An additional possibility is that, as has been proposed for other brain regions (e.g. Han et al., 1987; Minneman et al., 1988), two aq receptor subtypes exist in hypothalamus and preoptic area. Perhaps only one of the ~, receptor subtypes is regulated by ovarian steroids and involved in mediating augmentation of adenylate cyclase activity. The issue of multiple ~t receptor subtypes must also be considered for ~2 receptors where the existence of as many as four distinct genes has been reported (see Bylund, 1988). In our cAMP studies, we find predominantly inhibitory effects of ct2 receptors on cAMP formation in slices from OVX rats but ct2 augmentation of adenylate cyclase in E2 + P-treated slices (Etgen and Petitti, 1987; Petitti and Etgen, 1989). These changes in ~t2 linkage to the cAMP generating system were not found to correlate with measurable changes in [3H]idazoxan binding in the present experiments. In summary, these results demonstrate that physiological doses of E2 selectively increase ~ adrenergic receptor number in the hypothalamus and preoptic area but not frontal cortex. Thus the increased ~q receptor augmentation of adenylate cyclase activity in hypothalamic and preoptic area slices from E2-

primed female rats could result from elevated numbers of ~t, receptors. However, the reduced fl receptor stimulation of cAMP accumulation observed in slices from E2-exposed females, the overall depression of the cAMP response to NE in E2 + P-treated rats, and steroid-dependent changes in ct2 receptor coupling to adenylate cyclase are not correlated with measurable changes in antagonist binding to any of the NE receptor subtypes examined. Future experiments will explore the hypotheses that ovarian steroids modulate NE receptor coupling to G proteins and selectively regulate subtypes of the cq and ct2 receptors. work was supported by NSF Grant BNS-8607247, by DHHS Grant MH41414 and by an ADAMHA RSDA MH00636. The authors express their appreciation for technical assistance to Dr Maynard Makman, Bea Dworkin and Nicolas Petitti. Acknowledgements--This

REFERENCES

Ambrosio E., Montero M. T., Fernandez I., Azuara M. C. and Orensanz M. L. (1984) [3H]Prazosin binding to central nervous system regions of male and female rats. Neurosci. Lett. 49, 193-197. Biegon A., Reches A., Snyder L. and McEwen B. S. (1983) Serotonergic and noradrenergic receptors in the rat brain: modulation by chronic exposure to ovarian hormones. Life Sci. 32, 2015-2021. BlausteinJ. D. (1987) The alpha-1 noradrenergic antagonist prazosin decreases the concentration of estrogen receptors in female rat hypothalamus. Brain Res. 404, 39 50. Bruns R. F., Lawson-WendlingK. and PugsleyT. A. (1983) A rapid filtration assay for soluble receptors using polyethylenimine-treated filters. Analyt. Biochem. 132, 74-81. Bylund D. B. (1988) Subtypes of ~2-adrenoreceptors: pharmacological and molecular biological evidence converge. TIPS 9, 356-361. Etgen A. M. (1979) Antiestrogens: effects of tamoxifen, nafoxidine, and C1-628 on sexual behavior, cytoplasmic receptors, and nuclear binding of estrogen. Horm. Behav. 13, 97-112. Etgen A. M. (1987) Inhibition of estrous behaviour in rats by intrahypothalamic application of agents that disrupt nuclear binding of estrogen-receptor complexes. Horm. Behav. 21, 528-535. Etgen A. M. and Petitti N. (1986) Norepinephrinestimulated cAMP accumulation in rat hypothalamic slices: effects of estrous cycle and ovarian steroids. Brain Res. 375, 385-390. Etgen A. M. and Petitti N. (1987) Mediation of norepinephrine-stimulated cAMP accumulation by adrenergic receptors in hypothalamic and preoptic area slices: effects of estradiol. J. Neurochem. 49, 1732-1739. Etgen A. M. and Robisch D. R. (1988) Differential extraction of estradiol- and tamoxifen-receptor complexes from hypothalamic cell nuclei. Brain Res. 452, 1-10. Favit A., Fiore L. and Canonico P. L. (1988) Estrogens modulate stimulation of phosphoinositide hydrolysis by norepinephrine (NE) in rat brain slices. Soc. Neurosci. Abstr. 14, 1305.

Estradiol and hypothalamic noradrenergic receptors Han C., Abel P. W. and Minneman K. P. (1987) Heterogeneity of ~t~-adrenergic receptors revealed by chlorethylclonidine. Molec. Pharmac. 32, 505-510. Harrelson A. and McEwen B. (1987) Gonadal steroid modulation of neurotransmitter-stimulated cAMP accumulation in the hippocampus of the rat. Brain Res. 404, 89-94. Hatton G. I. (1984) Hypothalamic neurobiology. In: Brain Slices (Dingledine R., ed.), pp. 341-374. Plenum Press, New York. Jhanwar-Uniyal M., Roland C. R. and Leibowitz S. F. (1986) Diurnal rhythm of ct2-noradrenergic receptors in the paraventricular nucleus and other brain areas: relation to circulating corticosterone and feeding behavior. Life Sci. 38, 473-482. Johnson A. E., Nock B., McEwen B. S. and Feder H. H. (1985) Estradiol modulation of ctz-noradrenergic receptors in guinea pig brain assessed by tritium-sensitive film autoradiography. Brain Res. 336, 153-159. Johnson A. E., Nock B., McEwen B. S. and Feder H. H. (1988) ~tt- and ct2-noradrenergic receptor binding in guinea pig brain: sex differences and effects of ovarian steroids. Brain Res. 442, 205-213. Krauchi K., Wirz-Justice A., Morimasa T., Willener R. and Feer H. (1984) Hypothalamic ct2- and fl-adrenoreceptor rhythms are correlated with circadian feeding: evidence from chronic methamphetamine treatment and withdrawal. Brain Res. 321, 83-90. Leipheimer R. E., Alper R. H. and Gallo R. V. (1984) Effect of dopamine receptor blockade or norepinephrine synthesis inhibition on acute, ovariectomy-induced increases in pulsatile luteneizing hormone release in rat. Brain Res. Bull. 13, 235-240. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Minneman K. P., Han C. and Abel P. W. (1988) Comparison of cq-adrenergic receptor subtypes distinguished by chlorethylclonidine and WB 4101. Molec. Pharmac. 33, 309 514. Munemura M., Agui T. and Sibley D. R. (1989) Chronic estrogen treatment promotes a functional uncoupling of the D 2 dopamine receptor in rat anterior pituitary gland. Endocrinology 124, 346-355. Munson P. J. and Rodbard D. (1980) LIGAND: a versatile computerized approach for characterization of ligandbinding systems. Analyt. Biochem. 107, 220-239.

Nock B. and Feder H. H. (1983) Characteristics of [~H]prazosin binding to guinea pig brain ct-adrenergic receptors: effects of estradiol and progesterone. Brain Res. 262, 163-167. Nock B. L. and Feder H. H. (1984) ~q-Noradrenergic regulation of hypothalamic progestin receptors and guinea pig lordosis behavior. Brain Res. 310, 77-85. Orensanz L. M., Guillamon A., Ambrosio E., Segovia S. and Azuara M. C. (1982) Sex differences in alpha-adrenergic receptors in the rat brain. Neurosci. Lett. 30, 265278. Petitti N. and Etgen A. M. (1989) Progesterone depression of norepinephrine-stimulated cAMP accumulation in hypothalamic slices. Molec. Brain Res. 5, 109-119. Shackelford D. P. Jr, McConnaughey M. M. and Iams S. G. (1988) The effects of estradiol and mestranol on alpha-adrenoreceptors in select regions of the rat brain. Brain Res. Bull. 21, 329-333. Taleisnik S. and Sawyer C. H. (1986) Activation of the CNS noradrenergic system may inhibit as well as facilitate pituitary luteinizing hormone release. Neuroendocrinology 44, 265-268. Vacas M. I. and Cardinali D. P. (1980) Effect of estradiol on ~- and fl-adrenoreceptor density in medial basal hypothalamus, cerebral cortex and pineal gland of ovariectomized rats. Neurosci. Lett. 17, 73 77. Vincent P. A. and Feder H. H. (1988) Alpa-l- and alpha-2noradrenergic receptors modulate lordosis behavior in female guinea pigs. Neuroendocrinology 48, 477-481. Wagner H. R. and Davies J. N. (1980) Decreased fl-adrenergic responses in the female rat brain are eliminated by ovariectomy: correlation of [3H]dihydroalprenolol binding and catecholamine stimulated cAMP levels. Brain Res. 201, 235-239. Wagner H. R., Crutcher K. A. and Davies J. N. (1979) Chronic estrogen treatment decreases fl-adrenergic responses in rat cerebral cortex. Brain Res. 171, 147-151. Weiland N. G. and Wise P. M. (1987) Estrogen alters the diurnal rhythm of ~q-adrenergic receptor densities in selected brain regions. Endocrinology 121, 1751 1758. Wilkinson M., Herdon H., Pearce M. and Wilson C. (1979) Radioligand binding studies on hypothalamic noradrenergic receptors during the estrous cycle or after steroid injection in ovariectomized rats. Brain Res. 168, 652~555.