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Endocrinology Vol. 142, No. 2 599-607
Copyright © 2001 by The Endocrine Society

ARTICLES

Insulin-Like Growth Factor-1 Regulation of {alpha}1-Adrenergic Receptor Signaling Is Estradiol Dependent in the Preoptic Area and Hypothalamus of Female Rats1

Arnulfo Quesada and Anne M. Etgen

Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461

Address all correspondence and requests for reprints to: Dr. Arnulfo Quesada, Department of Neuroscience, Albert Einstein College of Medicine, 1300 Morris Park Avenue, F113, Bronx, New York 10461. E-mail: quesada{at}aecom.yu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Recently, we demonstrated that estradiol (E2) modulates cross-talk between protein tyrosine kinases and norepinephrine (NE) receptor signaling in the hypothalamus (HYP) and preoptic area (POA), brain areas that govern female reproductive function. We are now investigating the identity of protein tyrosine kinase(s) that modify NE receptor signaling in the HYP and POA. Incubation of POA and HYP slices with insulin-like growth factor I (IGF-I), which signals via a receptor (IGF-IR) with endogenous tyrosine kinase activity, enhances NE-stimulated cAMP accumulation only in tissue derived from ovariectomized, E2-primed animals. JB-1, an antagonist for IGF-IR, prevents the IGF-I enhancement of NE-stimulated cAMP accumulation in both POA and HYP slices. IGF-I enhances NE-stimulated cAMP accumulation via modulation of {alpha}1-adrenoceptor potentiation of adenylyl cyclase. Binding studies in membranes demonstrate that ovariectomized, E2-primed animals show a significant increase in the density of [125I]IGF-I-binding sites in both POA and HYP compared with ovariectomized control animals. Neither the IC50 for [125I]IGF-I displacement by IGF-I nor the levels of IGF-I binding proteins in serum or brain tissue are affected by E2. RIA results showed that E2 does not modify serum or brain IGF-I levels. These results indicate that E2 regulation of NE receptor function in the POA and HYP involves increased expression of IGF-IR, and that after E2 treatment, IGF-IR activation augments {alpha}1-adrenoceptor signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ESTRADIOL (E2) ACTS in the hypothalamus (HYP) and preoptic area (POA) to ensure that female mammals display reproductive behavior coincident with the timing of ovulation (1, 2). Significant progress has been made in understanding how the ovarian steroids and norepinephrine (NE) participate in the neuroendocrine regulation of reproductive function. E2 action in the HYP and POA modifies several cellular and molecular components of NE neurotransmission, including adrenergic receptor (AR)-mediated signal transduction (3, 4, 5). These effects of E2 in the POA and HYP are thought to be in part responsible for hormonal regulation of reproductive function in female vertebrates.

Recently, we were able to demonstrate that inhibition of protein tyrosine kinase activity with genistein, a general tyrosine kinase inhibitor, augments NE-stimulated cAMP accumulation in the HYP of ovariectomized rats and that this effect is augmented by E2 treatment. In the POA, genistein augments NE-stimulated cAMP accumulation only if ovariectomized rats are primed with E2 (6). Thus, protein tyrosine kinases may regulate NE receptor function in these two brain regions in a hormone-dependent manner. The current study investigates the existence and possible effects of E2 on cross-talk between insulin-like growth factor I (IGF-I) and AR signaling in the HYP and POA of female rats.

IGF-I has been identified as one of the major growth factors regulating the reproductive system of female rats. IGF-I signals via its receptor, IGF-IR, which is a member of the receptor tyrosine kinase family of growth factor receptors. IGF-I regulates neuroendocrine events such as the release of GH (7) and GnRH (8), the timing of puberty (9, 10), and, perhaps, female sexual behavior (11). IGF-I, IGF-IR, and IGF-I-binding proteins (IGF-IBPs) are locally synthesized by glia and neurons of the HYP (12, 13). In various tissues and cell types, E2 can modulate IGF-I action by regulating IGF-I gene expression (14, 15), IGF-IR (16, 17), or IGF-IBPs (17, 18). Synergistic interactions between E2 and IGF-I on neurite growth have also been demonstrated in monolayer hypothalamic cultures (19).

Because IGF-I and E2 act synergistically in both the nervous system and peripheral reproductive organs, we examined the possible existence of cross-talk between ARs and IGF-I in the POA and HYP. Adrenergic agonist-induced cAMP accumulation in brain slices was used as the marker of intracellular interaction among E2, IGF-I, and ARs. In brain, the regulation of adenylyl cyclase by NE receptors is complex; ß-ARs are linked directly to adenylyl cyclase activation via the stimulatory G protein, Gs. In many cases, including HYP and POA, the {alpha}1-AR alone has no effect on adenylyl cyclase, but potentiates ß-AR activation of cAMP synthesis (20). Thus, when appropriate agonists are employed, cAMP accumulation can be used to monitor the function of both ß- and {alpha}1-ARs. The ability of protein kinase C (PKC) inhibitors to block and of PKC activators to mimic {alpha}1-AR augmentation of cAMP synthesis suggest that PKC mediates {alpha}1-AR potentiation of adenylyl cyclase activity in rat brain (20).

Our studies demonstrate that IGF-I enhances NE-stimulated cAMP accumulation in HYP and POA from ovariectomized rats, but only if slices are derived from E2-primed animals. This change in IGF-I function is accompanied by increased [125I]IGF-I binding density in HYP and POA membranes. Examination of AR subtypes mediating the cAMP response to NE reveals that IGF-I enhances {alpha}1-AR potentiation of cAMP synthesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue slice preparation
Female Sprague Dawley rats (Taconic Farms, Inc., Germantown, NY), weighing 150–175 g, were anesthetized with ketamine and xylazine and were bilaterally ovariohysterectomized (OVX) to remove the primary source of estrogen and progesterone. Four to 7 days later they were injected sc twice, at 24 and 48 h before decapitation, with either 0.1 cc peanut oil (control) or 2 µg E2 benzoate (EB) dissolved in oil. The animals were rapidly killed, and the brains were placed in artificial cerebral spinal fluid (aCSF) (21). In all experiments, brain slices were prepared between 1000–1100 h to eliminate potential diurnal variation in cAMP (22) and AR (23, 24) contents. The HYP and POA were dissected, and 350-µm slices were made on a McIlwain tissue chopper (Mickle Lab. Engineering, Ltd., Surrey, UK) beginning approximately 2 mm anterior to the optic chiasm and ending 1 mm anterior to the mammillary bodies. The first four slices containing the medial and lateral POA, suprachiasmatic nucleus, and supraoptic nucleus were taken and designated POA. The next slice was discarded, and the following four slices containing the anterior, lateral, ventromedial, paraventricular, arcuate, and dorsomedial nuclei of the HYP were kept and designated HYP. Individual slices were incubated at 35 C in 300 µl oxygenated aCSF, which included a phosphodiesterase (PDE) inhibitor, 1 mM 8-methoxymethyl-3-isobutyl-1-methylxanthine (IBMX). IBMX was dissolved in absolute ethanol and added to slices such that the final concentration of ethanol was 1%. All animal experimentation was carried out in accordance with guidelines recommended by the NIH Guide for the Care and Use of Laboratory Animals.

Drug treatment
Slices were incubated for 75 min to allow cAMP levels to equilibrate (25), then were exposed for 15 min to IGF-I or vehicle. After the 15-min incubation with IGF-I, slices were stimulated with vehicle, NE dissolved in 0.01 N HCl, phenylephrine (PHE) or isoproterenol (ISO) dissolved in aCSF, phorbol 12,13-dibutyrate (PDB) or UK 14304 dissolved in dimethylsulfoxide (DMSO), or forskolin dissolved in 1% ethanol. Stimulation was carried out for 20 min. In experiments using prazosin (PRAZ), the drug was dissolved in DMSO and added 5 min before NE stimulation. The IGF-I antagonist JB-1, dissolved in aCSF, was added 5 min before IGF-I. The final concentration of DMSO or ethanol on control slices was 0.1%. Each experiment used tissue from one control and one EB-treated animal, and each experiment was replicated four times.

cAMP determination
After drug treatment the slices were disrupted by sonication in 5% ice-cold trichloroacetic acid. The supernatant containing cAMP and the pellet containing tissue protein were separated by centrifugation. The supernatant was acidified with 1 N HCl and extracted four times with hydrated ether. The samples were dried by lyophilization and analyzed for cAMP content using a modified Gilman cAMP assay (26). The protein concentration was determined by resuspending the pellet in 2 N NaOH and assaying by a modified Lowry assay (27). All cAMP values were expressed as picomoles of cAMP per mg protein. The EC50 for IGF-I enhancement of NE-stimulated cAMP accumulation was calculated using the EBDA program (Elsevier-Biosoft, Cambridge, UK).

IGF-IR binding assay
A modified version of a published protocol was used (28). The HYP and POA were dissected separately and immersed in ice-cold buffer [15% (v/w) 1 mM NaHCO3, 2 mM phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, and aprotinin at 1 trypsin inhibitory unit/ml], and homogenized in a glass/Teflon homogenizer. Each experiment used combined tissue from two control and two hormone-treated animals. The homogenate was centrifuged at 1,000 x g for 10 min at 4 C. The supernatant was decanted, and the pellet was homogenized again as described above. The two resulting supernatants were pooled and centrifuged at 25,000 x g for 30 min at 4 C. The pellet was washed once and stored at -70 C until assayed.

Displacement binding assays for IGF-IR were performed as described by Pons et al. (17). Pellets were resuspended in assay buffer [Krebs-Ringer phosphate buffer (KRP), Ca2+ free, containing 150 mM NaCl, 5 mM KCl, 1.2 mM KH2PO4, 16 mM Na2HPO4, and 1.2 mM MgSO4] at a final protein concentration of 250 µg/ml. The total reaction volume of the assay was 150 µl KRP buffer containing 50 µl membranes, 0.1% BSA, 5 x 10-11 M [125I]IGF-I (SA, 2,000 Ci/mmol; Amersham Pharmacia Biotech, Piscataway, NJ), 1 mg/ml bacitracin, and increasing concentrations (10-7–10-12 M) of unlabeled human recombinant IGF-I (Sigma, St. Louis, MO) diluted in KRP. Radioactivity bound in the presence of 10-7 M cold IGF-I was considered nonspecific binding. All assays were performed in triplicate. Samples were incubated for 18 h at 4 C on a shaking platform, then centrifuged at 10,000 x g for 5 min. The supernatants were aspirated, and the pellets were washed once with ice-cold KRP containing 0.5 M sucrose. Radioactivity in the precipitates was counted in a {gamma}-spectrometer. The density of [125I]IGF-I specific binding was analyzed by subtracting nonspecific binding from total binding and was expressed as femtomoles of [125I]IGF-I per mg protein. Competition experiments were analyzed using the EBDA program to calculate IC50 for IGF-I.

IGF-IBP assay
Trunk blood samples were collected when the animal was decapitated, allowed to clot overnight at 4 C, then centrifuged at 2,000 x g for 30 min. Serum was decanted into polypropylene tubes and frozen at -20 C until analysis. For tissue analysis three POA and three HYP from control or E2-treated animals were pooled for each experiment. Tissues were frozen in liquid nitrogen and stored at -70 C. The extraction procedure for IGF-IBPs from both serum and brain tissue was adapted from that described by Funston et al. (29). Samples were homogenized in a glass/Teflon homogenizer on ice at a concentration of 100 mg tissue/ml SDS-cholate buffer (0.5% SDS, 1% cholate, and 0.1 mM phenylmethylsulfonylfluoride), sonicated for 15 sec, and centrifuged at 13,600 x g for 10 min. Aliquots of supernatant were removed to determine protein concentration by a modified Lowry assay (27). Samples of POA and HYP (200 µg protein) and 5 µl serum were subjected to SDS-PAGE under nondenaturing conditions using a 4% stacking gel and 12.5% separating gel (30). Proteins were then electrophoretically transferred to nitrocellulose filter, and IGF-IBPs were quantified by incubating the filters with [125I]IGF-I [5 x 10-6 cpm/5 ml saline, 1% BSA (A 7030, Sigma), and 0.1% Tween-20] (31). Quantitative analysis of IGF-IBPs was conducted by exposing filters to autoradiographic film (3H Hyperfilm, Amersham Pharmacia Biotech) and scanning the autoradiograms on a Kodak DC-120 digital camera with a +3 diopter lens (Eastman Kodak Co., Rochester, NY). The image was digitized and analyzed quantitatively using the Kodak 1D gel analysis program.

IGF-I RIA
Concentrations of IGF-I in blood and tissue were determined using a RIA kit for human IGF-I (Peninsula Laboratories, Inc., Belmont, CA). Serum samples (100 µl) were diluted in an equal volume of PBS (0.01 M Na2HPO4, 0.15 M NaCl, 1 mM EDTA, and 0.1% sodium azide) containing 0.05% Triton-X 100. Diluted serum samples (200 µl) and tissue homogenates (200 µl) were extracted with 800 µl acid-ethanol (12.5% HCl and 87.5% ethanol). Samples were incubated for 2 h at 4 C and centrifuged at 1200 x g for 30 min. A 400-µl aliquot of supernatant was then neutralized with 160 µl 0.855 M Tris base. The neutralized supernatant was incubated for 2 h at 4 C and centrifuged at 1200 x g for 30 min. A 100-µl aliquot of supernatant was removed for RIA. All samples were analyzed in a single assay, with an intrassay coefficient of variation of 4%.

Materials
EB was purchased from Steraloids, Inc. (Wilton, NH). PHE, ISO, PRAZ, IBMX, and PDB were obtained from Sigma. Forskolin was obtained from Calbiochem (La Jolla, CA). NE and UK 14304 were purchased from RBI/Sigma (Natick, MA). JB-1 was obtained from Peninsula Laboratories, Inc.

Statistics
cAMP levels were analyzed with the StatView statistical program (Abacus Concepts, Inc., Berkeley, CA), using two-way ANOVA with drug treatment and hormone as the two factors. Significant differences between means were determined for main effects by the Student-Newman-Keuls test and least square means for interactions. In experiments using drug treatment as the only factor, one-way ANOVA was used. Significant differences between means were determined by Fisher’s protected least significant difference test. Student’s t test was used to determine statistical differences between control and hormone-treated groups for IGF-IR binding, IGF-I levels, and IGF-IBP analysis. Differences were considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of IGF-I on NE-stimulated cAMP accumulation
We initially investigated whether IGF-I modifies the cAMP response to the endogenous agonist NE in HYP and POA of OVX control and E2-treated animals. Treatment of POA and HYP slices with 10 nM IGF-I enhances NE-stimulated cAMP accumulation only in OVX, E2-primed animals (Fig. 1Go). IGF-I does not affect basal cAMP accumulation in POA or HYP slices from either OVX control or E2-treated animals. A concentration-response curve from 0.1–100 nM IGF-I was performed to obtain an EC50 for IGF-I enhancement of NE-stimulated cAMP synthesis in E2-treated animals. The EC50 for POA is 0.78 ± 0.4 nM, and that for HYP is 4.50 ± 1.2 nM (Fig. 2Go). Interestingly, the EC50 for IGF-I action suggests that the POA is more sensitive to IGF-I enhancement of NE-stimulated cAMP responses than the HYP. There was a tendency for 10-7 M IGF-I to have a weaker effect on the cAMP response to NE than did 10-8 M IGF-I. At higher concentrations, IGF-I might begin to interact with other receptor tyrosine kinases that inhibit, rather than augment, NE receptor activation of adenylyl cyclase.



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  		Figure 1. Effects of IGF-I and hormonal status on NE-stimulated cAMP accumulation. POA and HYP slices from OVX control and E2-treated female rats (EB) were made as described in Materials and Methods and treated with or without 10 nM IGF-I or vehicle (VEH) for 15 min, followed by a 20-min incubation with 100 µM NE. The PDE inhibitor IBMX (1 mM) was included. The data presented are the mean ± SEM from four independent replications. *, More than VEH or IGF-I alone (P < 0.05). **, More than all other groups (P < 0.05).

 


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  		Figure 2. Concentration dependence of IGF-I enhancement of NE-stimulated cAMP accumulation. POA ({diamondsuit}) and HYP ({blacksquare}) slices from E2-treated female rats were made as described in Materials and Methods and treated with the indicated concentrations of IGF-I for 15 min, followed by a 20-min incubation with 100 µM NE. The PDE inhibitor IBMX (1 mM) was included. The data presented are the mean ± SEM from four independent replications. *, More than NE alone (P < 0.05).

 
To examine whether the IGF-I effect on the cAMP response to NE in POA and HYP slices of E2-treated animals is IGF-IR mediated, JB-1, an antagonist for IGF-IR, was used. JB-1 (1 µg/ml) prevents IGF-I enhancement of NE-stimulated cAMP accumulation in both POA and HYP slices (Fig. 3Go).



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  		Figure 3. Effect of JB-1 on IGF-I enhancement of NE-stimulated cAMP accumulation. POA and HYP slices from E2-treated female rats were made as described in Materials and Methods and treated with 1 µg/ml of the IGF-IR antagonist JB-1 or vehicle (VEH) for 5 min, followed by a 15-min incubation with 10 nM IGF-I and a 20-min incubation with 100 µM NE. The PDE inhibitor IBMX (1 mM) was included. The data presented are the mean ± SEM from four independent replications. *, More than VEH (P < 0.05). **, More than all other groups (P < 0.05).

 
To test the possibility that IGF-I enhancement of NE-stimulated cAMP accumulation in POA and HYP slices of OVX, E2-treated animals is mediated via enhanced adenylyl cyclase activity, POA and HYP slices were preincubated with IGF-I and stimulated with forskolin, a potent activator of adenylyl cyclase. IGF-I does not potentiate cAMP accumulation in response to low or high doses of forskolin in POA or HYP slices (Fig. 4Go). Thus, IGF-I regulation of NE-stimulated cAMP synthesis does not appear to be due to receptor-independent activation of adenylyl cyclase. Inhibition of PDE is not likely to underlie IGF-I enhancement of NE-stimulated cAMP accumulation, because all of our experiments were performed in the presence of the PDE inhibitor, IBMX.



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  		Figure 4. Effect of IGF-I on forskolin-stimulated cAMP accumulation. POA and HYP slices from E2-treated female rats were made as described in Materials and Methods and treated with or without 10 nM IGF-I or vehicle (VEH) for 15 min, followed by a 20-min incubation with 1 or 10 µM forskolin (FSK). The PDE inhibitor IBMX (1 mM) was included. The data presented are the mean ± SEM from four independent replications. *, More than VEH or IGF-I alone (P < 0.05). **, More than all other groups (P < 0.05).

 
IGF-I effects on ß-, {alpha}1-, and {alpha}2-AR components of cAMP accumulation in POA and HYP slices of E2-treated animals
NE stimulates adenylyl cyclase by activating Gs-coupled ß-ARs. In rat brain slices, NE further increases cAMP synthesis by activating {alpha}1-ARs, which are Gq coupled and augment adenylyl cyclase activity via PKC (20). In addition, previous studies (6, 32) suggest that under some circumstances, {alpha}2-ARs also play a role in the cAMP response to NE stimulation in HYP and POA. Therefore, we examined which ARs are regulated by IGF-I in POA and HYP slices using the ß-AR agonist ISO, the {alpha}1-AR agonist PHE, and the {alpha}2-AR agonist UK 14304.

Pretreatment with IGF-I does not significantly enhance ISO-stimulated cAMP accumulation in either brain region, although there is a trend for IGF-I to increase the cAMP response to ISO (Table 1Go). Moreover, IGF-I does not enhance the cAMP response to ISO plus UK 14304, a selective full agonist for {alpha}2-AR, in either brain region (Table 1Go). Neither PHE, the {alpha}1-AR agonist, nor UK 14304, alone or in combination with IGF-I, modifies cAMP levels (data not shown). As previously reported (20), the {alpha}1-AR agonist PHE significantly increases ISO-stimulated cAMP accumulation in slices from both brain regions. Moreover, IGF-I significantly enhances the cAMP response to PHE plus ISO in slices from both brain regions (Table 1Go). To confirm that IGF-I influences the {alpha}1-AR component of the cAMP response to NE, we also examined the ability of PRAZ, a selective antagonist for {alpha}1-AR, to suppress IGF-I enhancement of NE-stimulated cAMP accumulation. PRAZ blocks IGF-I enhancement of NE-stimulated cAMP accumulation in both brain regions. Furthermore, in the HYP, PRAZ significantly inhibits the cAMP response to NE alone; in the POA, this effect was not quite significant (P < 0.08; Table 2Go).


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  		Table 1. IGF-I effects on adrenergic agonist-stimulated cAMP accumulation in POA and HYP slices from estradiol-treated rats

 

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  		Table 2. Effects of {alpha}1-AR antagonist and PKC activation on IGF-I enhancement of agonist-stimulated cAMP accumulation in the POA and HYP slices from estradiol-treated rats

 
As shown in Tables 1Go and 2Go, NE stimulates cAMP accumulation in part by activating {alpha}1-ARs, which are Gq coupled and augment adenylyl cyclase activity via PKC. If IGF-I is enhancing the cAMP response to NE by modulating the {alpha}1-AR component, as suggested by the findings with selective {alpha}1-AR agonists and antagonists, then maximal activation of PKC by the phorbol ester PDB should occlude IGF-I potentiation of the cAMP response to the ß-AR agonist ISO. Alternatively, if IGF-I potentiation of cAMP synthesis does not involve PKC activation, then PDB and IGF-I should have additive effects on the response to ISO. The phorbol ester PDB significantly enhances ISO-stimulated cAMP accumulation in slices from both brain regions (Table 2Go). However, IGF-I fails to enhance the cAMP response to PDB plus ISO (Table 2Go).

E2 modulation of the IGF-I system
In various tissues and cell types, E2 increases IGF-I gene expression (14, 15) and modulates IGF-I action by regulating the levels of IGF-IR (17) and IGF-IBPs (17, 18). Moreover, E2 increases IGF-I immunoreactivity in the arcuate nucleus of the HYP (33). Several of these factors were investigated to examine potential mechanisms underlying the E2 dependence of IGF-I enhancement of {alpha}1-AR signaling.

To test whether E2 regulates IGF-IR in POA and HYP, [125I]IGF-I binding in membranes was quantified. E2-primed animals show a significant increase in [125I]IGF-I binding density in both POA and HYP membranes compared with OVX control animals (Table 3Go). The IC50 for [125I]IGF-I displacement is not affected by hormone treatment (Table 3Go). RIA results showed that IGF-I levels are the same independent of hormonal treatment for serum, POA, and HYP (Fig. 5Go). Furthermore, analysis of [125I]IGF-I binding to serum and to soluble tissue protein after electrophoresis indicated that IGF-IBP levels are not modulated by E2 in either serum or extracts of POA and HYP (Fig. 6Go).


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  		Table 3. E2 treatment increases [125I]IGF-I binding in both HYP and POA of female rats

 


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  		Figure 5. Hormonal effects on IGF-I levels from POA, HYP, and serum. A, Levels of IGF-I measured by RIA in POA and HYP from both OVX control (CON) and E2-treated female rats (EB). The data presented are the mean ± SEM from three independent experiments. B, IGF-I levels in serum. The data presented are the mean of ± SEM from four independent experiments.

 


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  		Figure 6. Hormonal effects on POA, HYP, and serum IGF-IBPs. Representative autoradiograms of IGF-IBPs from POA, HYP, and serum from OVX control (CON) and E2-treated female rats (EB) are shown. A, The Mr for [125I]IGF-I binding in extracts from POA and HYP revealed a doublet of 32 kDa corresponding to BP-2 and BP-1 and 29 kDa corresponding to BP-5. Two low Mr binding proteins at 16 and 14 kDa were also observed. B, In serum the darkest bands were observed at Mr 42 kDa, corresponding to BP-3, 32 kDa corresponding to BP-2 and BP-1, and 29 kDa corresponding to BP-5. Lower intensity bands were observed at Mr 24 kDa corresponding to BP-4 and at 21 kDa.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The data presented herein demonstrate the existence of an E2-dependent interaction between IGF-I and NE signaling in POA and HYP of female rats. Pretreatment with IGF-I enhances NE-stimulated cAMP synthesis in POA and HYP slices only in OVX, E2-primed rats. The concentration-response curve for IGF-I indicates that physiological levels of IGF-I are sufficient to enhance NE-stimulated cAMP synthesis. The IGF-IR can bind to IGF-I, IGF-II, and insulin, although insulin binds with a much lower affinity than IGF-I or IGF-II (34). JB-1, a highly selective antagonist for IGF-IR both in vitro (35) and in vivo (18), blocked IGF-I enhancement of the cAMP response to NE in slices from both brain regions. Thus, results with JB-1 and the IGF-I dose-response curve for augmentation of NE-stimulated cAMP accumulation are indicative that IGF-IR mediates the interaction of IGF-I with NE signaling in brain slices from E2-treated female rats.

Examination of AR subtypes with selective agonists for ß-, {alpha}2-, and {alpha}1-AR reveals that IGF-I enhances NE-stimulated cAMP accumulation via modulation of {alpha}1-AR potentiation of adenylyl cyclase. As shown previously (20), the {alpha}1-AR agonist PHE, which does not increase cellular cAMP by itself, potentiates the cAMP response to the ß-AR agonist ISO. Preincubation with IGF-I significantly increases cAMP accumulation only in slices stimulated with both ISO and PHE. Because IGF-I does not significantly augment the cAMP response to ISO alone, the growth factor must be modulating the {alpha}1-AR component of the cAMP response. This interpretation is supported by two other observations. First, the {alpha}1-AR antagonist PRAZ blocks IGF-I enhancement of NE-stimulated cAMP synthesis. Second, maximal activation of PKC, the downstream mediator of {alpha}1-AR potentiation of adenylyl cyclase, occludes IGF-I augmentation of ISO-stimulated cAMP accumulation.

The data also suggest that IGF-I modulation of {alpha}1-AR signaling probably occurs at the level of the receptor and/or the G protein. Potentiation of {alpha}1-AR signaling is unlikely to be mediated by IGF-I activation of adenylyl cyclase, because IGF-I does not enhance the cAMP response to forskolin, a direct activator of adenylyl cyclase. IGF-I enhancement of cAMP accumulation is observed in the presence of PDE inhibitors, ruling out PDE as a target of IGF-I action. It is also unlikely that IGF-I directly activates PKC. If IGF-I acted by this route, it should facilitate the cAMP response to both ISO and forskolin (20), neither of which was observed in the present experiments. These findings are consistent with reports that protein tyrosine kinases regulate G protein-coupled receptor activation of the phospholipase C pathway in Rat-1 fibroblasts (36) and Chinese hamster ovary cells expressing metabotropic glutamate receptor 1{alpha} (37). Moreover, IGF-I can modulate receptor-G protein coupling, resulting in modulation of receptor function (38).

Quantitation of {alpha}1-AR potentiation of cAMP synthesis, a PKC-mediated response, is an excellent means to monitor {alpha}1-AR function in rat brain slices. Agonist occupancy of {alpha}1-ARs can activate both phospholipase C and phospholipase A2 (39, 40), producing a variety of second messengers that activate PKC (41). Additionally, at least two {alpha}1-AR subtypes, termed {alpha}1B- and {alpha}1A-AR, are expressed in the HYP (42). Both subtypes of {alpha}1-AR can mediate potentiation of adenylyl cyclase (43). Thus, measurement of any single second messenger generated by phospholipase C and A2 activation may overlook the pathway by which {alpha}1-ARs stimulate PKC activity.

Previous studies from our laboratory demonstrated that E2 selectively increases {alpha}1b-AR messenger RNA (44) and {alpha}1B-AR binding density (45) in the POA and HYP of female rats. This E2-dependent increase in receptor expression correlates with increased {alpha}1-AR potentiation of ß-AR-stimulated cAMP accumulation (45). In view of these results, it is possible that the E2-dependence of IGF-I potentiation of {alpha}1-AR signaling is due to increased {alpha}1B-AR expression in the POA and HYP of female rats.

The present results were unexpected in view of our previous findings with genistein, a general tyrosine kinase inhibitor (6). We found that inhibition of tyrosine kinase activity with genistein enhances NE-stimulated cAMP accumulation in POA and HYP slices. This effect is dependent on in vivo E2 treatment in the POA and is amplified by E2 treatment in the HYP. Thus, it might have been predicted that IGF-I would inhibit NE-stimulated cAMP synthesis. However, we now find that exogenous IGF-I enhances, rather than inhibits, {alpha}1-AR signaling in both POA and HYP, and that this action of IGF-I is E2 dependent. Because genistein and IGF-I have similar effects on {alpha}1-AR signaling in these brain regions, an explanation may lie in the complexity of the system. Different protein tyrosine kinases might regulate {alpha}1-AR signaling in opposite directions. IGF-IR may also modulate {alpha}1-AR signaling independently of tyrosine kinase activation (46). Alternatively, genistein may not completely inhibit the tyrosine kinase activity of IGF-IR in brain slices from female rats. Indeed, preincubation of HYP and POA slices from E2-primed female rats with genistein does not block IGF-I potentiation of {alpha}1-AR signaling (Quesada, A., and A. M. Etgen, unpublished observations). Thus, partial IGF-IR autophosphorylation may be sufficient for IGF-IR modulation of {alpha}1-AR signaling.

As mentioned previously, E2 can modulate IGF-I action by stimulating IGF-I gene expression or by increasing IGF-IR and/or IGF-IBPs in several tissues and cell cultures. Our studies demonstrate that E2 modestly, but significantly, increases [125I]IGF-I binding density in the POA and HYP, indicating an increase in IGF-IR in these two brain regions. Toran-Allerand et al. (19) suggested that an increase in IGF-IR in neurons exposed to E2 might explain the synergistic interaction between E2 and insulin/IGF-I on neurite outgrowth in hypothalamic explants. Thus, induction of IGF-IR by E2 may increase the sensitivity of cells to IGF-I action, thereby enhancing the biological effects of the peptide. Other important molecules involved in the regulation of IGF-I action are its binding proteins. E2 can increase IGF-IBP expression in the HYP. Incubation of cultured HYP neurons with 0.1 nM E2 for 5 days increases IGF-IR and IGF-IBP-2 levels (17). Likewise, a single injection of E2 to OVX female rats, which does not modulate IGF-IR protein levels quantified by immunoblotting in the HYP after 24 h, increases IGF-BP-2 immunostaining in the arcuate nucleus (47). We found increases in IGF-IR, but no change in IGF-IBPs, when quantified with [125I]IGF-I binding methods after a 48-h exposure to E2. The difference between doses and duration of hormone treatment and experimental methods may explain why we did not observe E2-induced increases in IGF-IBPs in the HYP.

In addition to regulation of IGF-I function in neurons, E2 can modulate IGF-IR, IGF-IBPs, and the morphology of hypothalamic astrocytes (48). Tanycytes are specialized astroglia that modulate the secretion of GnRH and other neurohormones from nerve terminals in the median eminence (49). Glial cells may modulate neuroendocrine events at least in part by synthesizing and/or releasing trophic factors that, in turn, act on neurons or other glial cells (17). Duenas et al. (33) demonstrated that levels of IGF-I immunoreactivity increase in hypothalamic tanycytes on the morning and afternoon of proestrus, when the preovulatory LH surge and sexual receptivity are initiated in female rats. IGF-I immunoreactivity decreases to basal levels by the morning of metestrus, when LH levels and sexual receptivity are low. Furthermore, IGF-I immunoreactivity in the median eminence decreases when gonadal steroid levels are reduced by OVX and increases in a dose-dependent manner when OVX rats are injected with E2. Unlike Duenas et al. (33), we did not find that E2 increases IGF-I levels in the POA, HYP, or serum of OVX rats. However, our data on IGF-I levels in whole HYP and POA do not rule out the possibility that E2-dependent increases in IGF-I levels might be limited to specific hypothalamic regions.

Behavioral data from this laboratory suggest that activation of hypothalamic {alpha}1-AR by endogenously released NE participates in hormonal facilitation of female rat sexual behavior (50). Pharmacological blockade of {alpha}1B-AR blocks both the facilitation of sexual behavior and the electrophysiological responses of ventral medial hypothalamic neurons to {alpha}1-AR agonists in E2-primed female rats (51). In addition, NE acts via {alpha}1-ARs to facilitate the release of GnRH in an E2-dependent manner (52). The facilitatory effect of E2 on GnRH release may be exerted at the median eminence, where the GnRH axons converge to release the neuropeptide into the portal vasculature. Arcuate neurons and tanycytes express IGF-IRs (53), and immunocytochemical studies from our laboratory show {alpha}1B-AR immunoreactivity in arcuate fibers and possibly in tanycytes of the median eminence (54). Thus, it can be hypothesized that IGF-IRs and {alpha}1-ARs, particularly the {alpha}1B-AR subtype, interact in these two brain regions to modulate female reproductive function.

In summary, our results demonstrate that IGF-I enhances NE-stimulated cAMP accumulation in POA and HYP only if slices are derived from E2-primed rats. Pharmacological studies reveal that IGF-I enhances NE-stimulated cAMP accumulation by modulating {alpha}1-AR potentiation of adenylyl cyclase. Thus, E2 regulation of NE receptor function in the POA and HYP might involve increased expression of IGF-IR, which, in turn, augments NE signaling via E2-induced {alpha}1B-AR. Our finding that both {alpha}1B-AR expression and IGF-I modulation of the {alpha}1-AR signaling are E2-regulated events suggests that interactions between IGF-I and NE receptor signaling pathways may participate in E2 regulation of GnRH release and female sexual behavior. Future research in our laboratory will examine this possibility in more detail.


   Acknowledgments
 
We thank Dr. Michael Ansonoff for technical assistance, and Drs. Maynard Makman, Jorge Larocca, and George Karkanias for critical review of the manuscript.


   Footnotes
 
1 This work was supported by Grants MH-41414, HD-29856, and T32-DK-07513. The data in this paper are from a thesis to be submitted by A.Q. in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Sue Golding Division of Medical Sciences, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY. Back

Received July 24, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Barfield RJ, Chen JJ 1977 Activation of estrous behavior in ovariectomized rats by intracerebral implants of estradiol benzoate. Endocrinology 101:1716–1725[Abstract/Free Full Text]
  2. Chappel SC 1985 Neuroendocrine regulation of luteinizing hormone and follicle stimulating hormone: a review. Life Sci 36:97–103[CrossRef][Medline]
  3. Etgen AM, Karkanias GB 1994 Estrogen regulation of noradrenergic signaling in the hypothalamus. Psychoneuroendocrinology 19:603–610[CrossRef][Medline]
  4. Vathy I, Etgen AM 1988 Ovarian steroids and hypothalamic norepinephrine release: studies using in vivo brain microdialysis. Life Sci 43:1493–1499[CrossRef][Medline]
  5. Ungar S, Makman MH, Morris SA, Etgen AM 1993 Estrogen uncouples ß-adrenergic receptor from the stimulatory guanine nucleotide-binding protein in female rat hypothalamus. Endocrinology 133:2818–2826[Abstract/Free Full Text]
  6. Quesada A, Etgen AM 2000 Tyrosine kinase effects on adrenoceptor-stimulated cyclic AMP accumulation in preoptic area and hypothalamus of female rats: modulation by estradiol. Brain Res 861:117–125[CrossRef][Medline]
  7. Tannenbaum GS, Guyda HJ, Posner BI 1983 Insulin-like growth factors: a role in growth hormone negative feedback and body weight regulation via brain. Science 220:77–79[Abstract/Free Full Text]
  8. Hiney JK, Ojeda SR, Dees WL 1991 Insulin-like growth factor I: a possible metabolic signal involved in the regulation of female puberty. Neuroendocrinology 54:420–423[Medline]
  9. Wilson VJ, Rattray M, Thomas CR, Moreland BH, Schulster D 1995 Growth hormone increases IGF-I, collagen I and collagen III gene expression in dwarf rat skeletal muscle. Mol Cell Endocrinol 115:187–197[CrossRef][Medline]
  10. Hiney JK, Srivastava V, Nyberg CL, Ojeda SR, Dees WL 1996 Insulin-like growth factor I of peripheral origin acts centrally to accelerate the initiation of female puberty. Endocrinology 137:3717–3728[Abstract]
  11. Apostolakis EM, Garai J, Lohmann JE, Clark JH, O’Malley BW 2000 Epidermal growth factor activates reproductive behavior independent of ovarian steroids in female rodents. Mol Endocrinol 14:1086–1098[Abstract/Free Full Text]
  12. Bondy C, Werner H, Roberts Jr CT, LeRoith D 1992 Cellular pattern of type-I insulin- like growth factor receptor gene expression during maturation of the rat brain: comparison with insulin-like growth factors I and II. Neuroscience 46:909–923[CrossRef][Medline]
  13. Garcia-Segura LM, Perez J, Pons S, Rejas MT, Torres-Aleman I 1991 Localization of insulin-like growth factor I (IGF-I)-like immunoreactivity in the developing and adult rat brain. Brain Res 560:167–174[CrossRef][Medline]
  14. Dickson RB, McManaway ME, Lippman ME 1986 Estrogen-induced factors of breast cancer cells partially replace estrogen to promote tumor growth. Science 232:1540–1543[Abstract/Free Full Text]
  15. Sahlin L, Norstedt G, Eriksson H 1994 Estrogen regulation of the estrogen receptor and insulin like growth factor-I in the rat uterus: a potential coupling between effects of estrogen and IGF-I. Steroids 59:421–430[CrossRef][Medline]
  16. Wimalasena J, Meehan D, Dostal R, Foster JS, Cameron M, Smith M 1993 Growth factors interact with estradiol and gonadotropins in the regulation of ovarian cancer cell growth and growth factor receptors. Oncol Res 5:325–337[Medline]
  17. Pons S, Torres-Aleman I 1993 Estradiol modulates insulin-like growth factor I receptors and binding proteins in neurons from the hypothalamus. J Neuroendocrinol 5:267–271[CrossRef][Medline]
  18. Cardona-Gomez GP, Trejo JL, Fernandez AM, Garcia-Segura LM 2000 Estrogen receptors and insulin-like growth factor-I receptors mediate estrogen-dependent synaptic plasticity. NeuroReport 11:1735–1738[Medline]
  19. Toran-Allerand CD, Ellis L, Pfenninger KH 1988 Estrogen and insulin synergism in neurite growth enhancement in vitro: mediation of steroid effects by interactions with growth factors? Brain Res 469:87–100[Medline]
  20. Petitti N, Etgen AM 1991 Protein kinase C and phospholipase C mediate {alpha}1- and ß-adrenoceptor intercommunication in rat hypothalamic slices. J Neurochem 56:628–635[CrossRef][Medline]
  21. Yamamoto C 1972 Activation of hippocampal neurons by mossy fiber stimulation in thin brain sections in vitro. Exp Brain Res 14:423–435[Medline]
  22. Kant GJ, Sessions GR, Lenox RH, Meyerhoff JL 1981 The effects of hormonal and circadian cycles, stress, and activity on levels of cyclic AMP and cyclic GMP in pituitary, hypothalamus, pineal and cerebellum of female rats. Life Sci 29:2491–2499[CrossRef][Medline]
  23. Jhanwar-Uniyal M, Roland CR, Leibowitz SF 1986 Diurnal rhythm of {alpha}2-noradrenergic receptors in the paraventricular nucleus and other brain areas: relation to circulating corticosterone and feeding behavior. Life Sci 38:473–482[CrossRef][Medline]
  24. Krauchi K, Wirz-Justice A, Morimasa T, Willener R, Feer H 1984 Hypothalamic {alpha}2- and ß-adrenoceptor rhythms are correlated with circadian feeding: evidence from chronic methamphetamine treatment and withdrawal. Brain Res 321:83–90[CrossRef][Medline]
  25. Fredholm BB, Dunwiddie TV, Bergman B, Lindstrom K 1984 Levels of adenosine and adenine nucleotides in slices of rat hippocampus. Brain Res 295:127–136[CrossRef][Medline]
  26. Brostrom CO, Kon C 1974 An improved protein binding assay for cyclic AMP. Anal Biochem 58:459–468[CrossRef][Medline]
  27. Larson E, Howlett B, Jagendorf A 1986 Artificial reductant enhancement of the Lowry method for protein determination. Anal Biochem 155:243–248[CrossRef][Medline]
  28. Torres-Aleman I, Pons S, Garcia-Segura LM 1991 Climbing fiber deafferentation reduces insulin-like growth factor I (IGF-I) content in cerebellum. Brain Res 564:348–351[CrossRef][Medline]
  29. Funston RN, Moss GE, Roberts AJ 1995 Insulin-like growth factor-I (IGF-I) and IGF-binding proteins in bovine sera and pituitaries at different stages of the estrous cycle. Endocrinology 136:62–68[Abstract]
  30. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685[CrossRef][Medline]
  31. Hossenlopp P, Seurin D, Segovia-Quinson B, Hardouin S, Binoux M 1986 Analysis of serum insulin-like growth factor binding proteins using western blotting: use of the method for titration of the binding proteins and competitive binding studies. Anal Biochem 154:138–143[CrossRef][Medline]
  32. Etgen AM, Petitti N 1987 Mediation of norepinephrine-stimulated cyclic AMP accumulation by adrenergic receptors in hypothalamic and preoptic area slices: effects of estradiol. J Neurochem 49:1732–1739[CrossRef][Medline]
  33. Duenas M, Luquin S, Chowen JA, Torres-Aleman I, Naftolin F, Garcia-Segura LM 1994 Gonadal hormone regulation of insulin-like growth factor-I-like immunoreactivity in hypothalamic astroglia of developing and adult rats. Neuroendocrinology 59:528–538[Medline]
  34. Nissley SP, Haskell HJ, Sasaki N, De Vroede MA, Rechler MM 1985 Insulin-like growth factor receptors. J Cell Sci [Suppl] 3:39–51[Medline]
  35. Pietrzkowski Z, Wernicke D, Porcu P, Jameson BA, Baserga R 1992 Inhibition of cellular proliferation by peptide analogues of insulin-like growth factor 1. Cancer Res 52:6447–6451[Abstract/Free Full Text]
  36. Liu WW, Mattingly RR, Garrison JC 1996 Transformation of Rat-1 fibroblasts with the v-src oncogene increases the tyrosine phosphorylation state and activity of the {alpha} subunit of Gq/G11. Proc Natl Acad Sci USA 93:8258–8263[Abstract/Free Full Text]
  37. Umemori H, Inoue T, Kume S, Sekiyama N, Nagao M, Itoh H, Nakanishi S, Mikoshiba K, Yamamoto T 1997 Activation of the G protein Gq/11 through tyrosine phosphorylation of the {alpha} subunit. Science 276:1878–1881[Abstract/Free Full Text]
  38. Goad DL, Tashjian Jr AH 1993 Insulin-like growth factor-I inhibits parathyroid hormone-stimulated and enhances prostaglandin E2-stimulated adenosine 3',5'-monophosphate production by human osteoblast-like SaOS-2 cells. Endocrinology 133:1585–1592[Abstract/Free Full Text]
  39. Nanberg E, Putney Jr J 1986 {alpha}1-Adrenergic activation of brown adipocytes leads to an increased formation of inositol polyphosphates. FEBS Lett 195:319–322[CrossRef][Medline]
  40. Duman RS, Karbon EW, Harrington C, Enna SJ 1986 An examination of the involvement of phospholipases A2 and C in the {alpha}-adrenergic and {lambda}-aminobutyric acid receptor modulation of cyclic AMP accumulation in rat brain slices. J Neurochem 47:800–810[Medline]
  41. Schaad NC, Schorderet M, Magistretti PJ 1989 Accumulation of cyclic AMP elicited by vasoactive intestinal peptide is potentiated by noradrenaline, histamine, adenosine, baclofen, phorbol esters, and ouabain in mouse cerebral cortical slices: studies on the role of arachidonic acid metabolites and protein kinase C. J Neurochem 53:1941–1951[CrossRef][Medline]
  42. Blendy JA, Grimm LJ, Perry DC, West-Johnsrud L, Kellar KJ 1990 Electroconvulsive shock differentially increases binding to {alpha}-1 adrenergic receptor subtypes in discrete regions of rat brain. J Neurosci 10:2580–2586[Abstract]
  43. Robinson JP, Kendall DA 1989 Inositol phospholipid hydrolysis and potentiation of cyclic AMP formation by noradrenaline in rat cerebral cortex slices are not mediated by the same {alpha}-adrenoceptor subtypes. J Neurochem 52:690–698[CrossRef][Medline]
  44. Karkanias GB, Ansonoff MA, Etgen AM 1996 Estradiol regulation of {alpha}1b-adrenoceptor mRNA in female rat hypothalamus-preoptic area. J Neuroendocrinol 8:449–455[CrossRef][Medline]
  45. Petitti N, Karkanias GB, Etgen AM 1992 Estradiol selectively regulates {alpha}1B-noradrenergic receptors in the hypothalamus and preoptic area. J Neurosci 12:3869–3876[Abstract]
  46. Blakesley VA, Kato H, Roberts Jr CT, LeRoith D 1995 Mutation of a conserved amino acid residue (tryptophan 1173) in the tyrosine kinase domain of the IGF-I receptor abolishes autophosphorylation but does not eliminate biologic function. J Biol Chem 270:2764–2769[Abstract/Free Full Text]
  47. Cardona-Gomez GP, Chowen JA, Garcia-Segura LM 2000 Estradiol and progesterone regulate the expression of insulin-like growth factor-I receptor and insulin-like growth factor binding protein-2 in the hypothalamus of adult female rats. J Neurobiol 43:269–281[CrossRef][Medline]
  48. Torres-Aleman I, Rejas MT, Pons S, Garcia-Segura LM 1992 Estradiol promotes cell shape changes and glial fibrillary acidic protein redistribution in hypothalamic astrocytes in vitro: a neuronal-mediated effect. Glia 6:180–187[CrossRef][Medline]
  49. Garcia-Segura LM, Luquin S, Parducz A, Naftolin F 1994 Gonadal hormone regulation of glial fibrillary acidic protein immunoreactivity and glial ultrastructure in the rat neuroendocrine hypothalamus. Glia 10:59–69[CrossRef][Medline]
  50. Etgen AM 1990 Intrahypothalamic implants of noradrenergic antagonists disrupt lordosis behavior in female rats. Physiol Behav 48:31–36[CrossRef][Medline]
  51. Kow LM, Weesner GD, Pfaff DW 1992 {alpha}1-adrenergic agonists act on the ventromedial hypothalamus to cause neuronal excitation and lordosis facilitation: electrophysiological and behavioral evidence. Brain Res 588:237–245[CrossRef][Medline]
  52. Weesner GD, Krey LC, Pfaff DW 1993 {alpha}1-Adrenergic regulation of estrogen-induced increases in luteinizing hormone-releasing hormone mRNA levels and release. Brain Res Mol Brain Res 17:77–82[Medline]
  53. Garcia-Segura LM, Rodriguez JR, Torres-Aleman I 1997 Localization of the insulin-like growth factor I receptor in the cerebellum and hypothalamus of adult rats: an electron microscopic study. J Neurocytol 26:479–490[CrossRef][Medline]
  54. Acosta-Martinez M, Fiber JM, Brown RD, Etgen AM 1999 Localization of {alpha}1B-adrenergic receptor in female rat brain regions involved in stress and neuroendocrine function. Neurochem Int 35:383–391[CrossRef][Medline]



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