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Endocrinology Vol. 140, No. 2 652-659
Copyright © 1999 by The Endocrine Society

ARTICLES

Estradiol Coupling to Endothelial Nitric Oxide Stimulates Gonadotropin-Releasing Hormone Release from Rat Median Eminence Via a Membrane Receptor1

Vincent Prevot, Dominique Croix, Christos M. Rialas, Pierre Poulain, Gregory L. Fricchione, George B. Stefano and Jean-Claude Beauvillain

INSERM, U-422, Unité de Neuroendocrinologie et Physiopathologie Neuronale (V.P., D.C., P.P., J.-C.B.), 59045 Lille Cedex, France; Neuroscience Research Institute, State University of New York (C.M.R., G.L.F., G.B.S.), Old Westbury, New York 11568; and the Division of Psychiatry, Harvard Medical School, Brigham and Women’s Hospital (G.L.F., G.B.S.), Boston, Massachusetts 02115

Address all correspondence and requests for reprints to: Dr. Vincent Prevot, INSERM, U-422, Unité de Neuroendocrinologie et Physiopathologie Neuronale, place de Verdun, 59045 Lille Cedex, France. E-mail: prevot{at}biserte.lille.inserm.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The median eminence (ME), which is the common termination field for adenohypophysiotropic systems, has been shown to produce nitric oxide (NO), a signaling molecule involved in neuroendocrine secretion. Using an ex vivo technique, 17ß-estradiol exposure to ME fragments, including vascular tissues, stimulated NO release within seconds in a concentration-dependent manner, whereas 17{alpha}-estradiol or testosterone had no effect. 17ß-Estradiol conjugated to BSA (E2-BSA) also stimulated NO release, suggesting mediation by a membrane surface receptor. Tamoxifen, an estrogen receptor inhibitor, antagonized the action of both 17ß-estradiol and E2-BSA. Furthermore, estradiol-stimulated NO stimulates GnRH release. This was demonstrated by hemoglobin (a NO scavenger), N{omega}-nitro-L-arginine methyl ester, and L-N5-(1-iminoethyl)ornithine (nitric oxide synthase inhibitors) inhibition of estradiol stimulated NO and GnRH release. In this regard, L-N5-(1-iminoethyl)ornithine, specific for endotheliol constitutive nitric oxide synthase, was significantly more potent, suggesting that the estradiol-stimulated NO release arose from vascular endothelial cells. Additionally, the NO-stimulated GnRH release occurs via guanylyl cyclase activation in GnRH nerve terminals, as ODQ, a potent and selective inhibitor of NO-sensitive guanylyl cyclase, abolished the estradiol-stimulated GnRH release. The results suggest that at physiological concentrations, 17ß-estradiol may have immediate actions on ME endothelial cells via nongenomic signaling pathways leading to NO-stimulated GnRH release.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GnRH IS synthesized in neuronal cell bodies diffusely distributed in the preoptic area and is secreted from neuroendocrine terminals localized in the median eminence (ME). Once secreted, GnRH enters the portal vessels and is then transported to the anterior pituitary, where it modulates the synthesis and secretion of gonadotropins, which are essential to gonadal function and reproduction. The secretory activity of GnRH neurons is controlled by neural and glial regulatory processes (1, 2, 3, 4). Neuronal inputs are mediated via trans-synaptic mechanisms, and glial influences are conveyed via substances such as transforming growth factors (5, 6, 7, 8). Interestingly, endothelial-ME signaling can occur by nitric oxide (NO) coupling as recently demonstrated (9, 10). In regard to NO, it is a signaling molecule involved in the regulation of numerous neuroendocrine secretions (11) and particularly GnRH release from the neuroendocrine terminals located in the ME external zone (12). NO is also considered to be an important endothelium-derived relaxing factor and may also function to protect blood vessels against atherosclerotic development by inhibiting monocyte adhesion to the endothelium (for review, see Ref. 13). Concerning estrogen, estradiol can increase endothelial and neuronal nitric oxide synthase (NOS) activities in both males and females (14) and thus endothelial NO production by a cytosolic receptor-mediated system (15).

In this regard, the present study demonstrates that rat ME responds to acute estradiol exposure by releasing NO. We further demonstrate that a consequence of this estrogen-stimulated NO release is the release of GnRH from ME fragments. Taken together, this study represents the first study in which estrogen is demonstrated to exert acute actions that have immediate biological consequences, i.e. hormonal release. Furthermore, of equal importance is the demonstration that this signaling process can be initiated and regulated by vascular endothelial cells, demonstrating the presence of neurovascular regulatory processes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Experiments were performed on male rats, which have low estrogen levels but comparable estrogen receptor expression as females (16, 17, 18, 19, 20, 21). The Wistar rats (CERJ, St. Berthevin, France), weighing 300–350 g, were housed four per cage and given free access to food and water. All animals were subjected to fixed conditions of lighting (from 0500–1900 h). All experiments were carried out in accordance with the European Communities Council Directive of November 24, 1986 (86/609/EEC).

Dissection procedure
Animals were killed by decapitation. After rapid removal of the brain, the median eminence and associated vascular tissues were dissected under a binocular magnifying glass by cutting with Wecker’s scissors (Moria, France) the floor of the brain within the following limits: posterior border of the optic chiasm and the anterior border of the mamillary bodies. With this type of dissection, pieces containing ME were obtained with very little arcuate nucleus fragments. The total dissection time was less than 3 min from decapitation.

Incubation system
After dissection, ME fragments were washed twice in Krebs-Ringer bicarbonate/glucose buffer (pH 7.4) containing bacitracin (23 µM; Sigma Chemical Co., St. Louis, MO) in an atmosphere of 95% O2–5% CO2 and then immersed in Eppendorf tubes containing 700 µl of the same buffer. Three ME fragments were incubated in each tube.

NO determination
The tissue fragments were incubated as described above. NO release was measured directly using a NO-specific amperometric probe (World Precision Instruments, Sarasota, FL) as described previously (9, 22, 23, 24). The tip diameter of the probe (25 µm) permitted the use of a micromanipulator (Zeiss-Eppendorf, Hamburg, Germany) to position the sensor 5 mm above the tissue surface. Calibration of the electrochemical sensor was performed by use of different concentrations of a nitrosothiol donor S-nitroso-N-acetyl-D,L-penicillamine, as described in detail previously (25). The probe needed 15 min to stabilize after its immersion in the Krebs-Ringer bicarbonate/glucose buffer medium, and baseline levels of NO release were determined by evaluation of the NO concentration released from unstimulated ME fragments. Drugs were added to the buffer after the stabilization time, and NO release was monitored for 20 min. ME fragments were stimulated with various concentrations of 17ß-estradiol (10-13–10-7 M) or 17ß-estradiol conjugated to BSA (E2-BSA; 10-13–10-7 M 17ß-estradiol) or testosterone (10-13–10-7 M; four tubes were run for each condition). ME fragments were stimulated with 17{alpha}-estradiol (10-8 M; n = 4); tamoxifen (10-8 M), an estrogen receptor antagonist (n = 4); tamoxifen (10-8 M) plus 17ß-estradiol (10-8 M; n = 4); or tamoxifen (10-8 M) plus E2-BSA (10-8 M; n = 4). Tamoxifen was added to the milieu 5 min before 17ß-estradiol or E2-BSA. The concentration of NO gas in solution was measured in real-time with computer data acquisition (DUO 18, World Precision Instruments) at a sampling rate of 6/s (22, 23). NO release was evaluated with simultaneous measurement of untreated control ME fragments with a second probe to allow comparison of each treatment group to its own control preparation. Data acquisition was performed by the computer-interfaced DUO-18 software (World Precision Instruments). The experimental values were then transferred to Sigma-Plot and Sigma-Stat (Jandel, San Rafael, CA) for graphic representation and evaluation.

GnRH secretion determination
The tissue fragments were incubated as described for the NO determination. Drugs were added to the medium after an equilibration period of 15 min. The stimulation period was 10 min. NOS inhibitors, N{omega}-nitro-L-arginine methyl ester (L-NAME; 4 x 10-6 M) or L-N5-(1-iminoeth-yl)ornithine (L-NIO; 4 x 10-5, 4 x 10-6, 5 x 10-7, and 5 x 10-8 M), or the potent and selective inhibitor of NO-sensitive guanylyl cyclase, 1H-[1,2,4]oxadiazolo[4,3{alpha}]quinoxalin-1-one (ODQ; 2 x 10-6 M) (26, 27, 28, 29), were added to the milieu 5 min before 17ß-estradiol or E2-BSA. L-NIO is a well known inhibitor of NOS (30) and shows a better inhibition of endotheliol constitutive NOS (IC50 = 0.5 x 10-6 M) (30) than neuronal NOS (nNOS; IC50 = 3.9 x 10-6 M) (31). Four tubes were run for each experiment. At the end of each experiment, the ME fragments were immediately removed, and EDTA was added to the milieu (final concentration, 10-2 M). GnRH concentrations were measured in duplicate by RIA as described previously (9). The sensitivity for GnRH was 1.2 pg/tube, and the intraassay variability was 3.4%. GnRH antibody was a gift from Dr. Tramu of the CNRS URA 339, Université Bordeaux I (Talence, France). All drugs were purchased from Sigma Chemical Co. (St. Quentin Fallavier, France), except L-NIO and ODQ, which were purchased from Calbiochem (France Biochem, Meudon, France).

Plasma estradiol and testosterone determinations
After decapitation, trunk blood was collected into vials containing 50 µl 0.3 M EDTA and centrifuged. Plasma was stored at -20 C until estradiol and testosterone RIAs.

Plasma estradiol was measured using a RIA kit optimized for the direct quantitative determination of very low concentrations of 17ß-estradiol in human serum and plasma (e.g. in children), purchased from Sorin Biomedica (Antony, France). The assay sensitivity was 0.2 pg/tube, and intra- and interassay variances were 5.6% and 7.3%, respectively.

Testosterone levels were measured using a RIA kit optimized for the direct quantitative determination of very low concentrations of testosterone in human serum and plasma, purchased from Amersham (Les Ulis, France). The assay sensitivity was 5 pg/tube, and intra- and interassay variances were 5.1% and 11.5%, respectively.

Statistics
All experiments were repeated a minimum of four times, i.e. at least four tubes where run for each condition. The results were analyzed by one-way ANOVA with repeated measures, and the significance of differences was determined by the Student-Newman-Keuls test. The differences between the means of two groups were calculated by Student’s t test, where P < 0.05 was considered significantly different.

Morphology
To assure that tissues were able to maintain a high level of biological organization during the experiments, which may reflect their responses to stimulation, some ME fragments were immersed for 2 h in a fixative mixture of 2% paraformaldehyde, 0.2% picric acid, and 0.1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, for 2 h at 4 C after a 15-min stabilization time plus a 20-min stimulation time. Pieces of tissue were postfixed for 1 h at room temperature with 1% OsO4 in phosphate buffer and were embedded in Araldite after dehydration.

Semithin sections (1–2 µm thick) were obtained to observe the tissue conservation at the light microscopy level and ultrathin sections (80–90 nm thick) were obtained to observe the ultrastructural aspect of the ME.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Direct evaluation of NO release from ME fragments
NO release was measured in real-time using a NO-specific amperometric probe after stimulation of ME fragments with either 17ß-estradiol or E2-BSA.

17ß-Estradiol stimulates NO release
17ß-Estradiol induced a concentration-dependent increase in NO release from ME fragments (Figs. 1Go and 2Go). Increasing concentrations of 17ß-estradiol (10-11–10-7 M) resulted in a dose-dependent increase in NO release, with a maximal effect observed after 10-8 M 17ß-estradiol treatment (Fig. 1Go). NO release peaked after 2 min of 17ß-estradiol treatment of the ME fragments (Fig. 2Go). Addition of 10-12 M 17ß-estradiol failed to induce a significant increase in NO release. The median effective concentration (EC50) value for 17ß-estra-diol-induced NO release was approximately 10-10 M.



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  		Figure 1. Dose-dependent release of NO after in vitro stimulation of median eminence fragments by 17ß-estradiol and E2-BSA. Testosterone has no effect on NO release. The graphed values represent peak values obtained 2 min after drug exposure. The ME fragments are exposed to the agents for the entire observation period (15 min; see Fig. 2Go, inset). Each experiment was repeated four times, and the resulting means ± SEM are graphed.

 


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  		Figure 2. Steroid-specific stimulation of NO release by 17ß-estradiol via a membrane receptor. 17ß-Estradiol stimulates NO release from ME fragments within 2 min of its application. 17{alpha}-Estradiol had no effect on NO release (P < 0.001). 17ß-Estradiol coupled to BSA stimulates NO release at the same time as 17ß-estradiol (P < 0.01), indicating that estradiol acts at the membrane surface. The 17ß-estradiol- and E2-BSA-stimulated NO releases were antagonized by tamoxifen, an antiestrogen compound. 17ß-Estradiol, E2-BSA, and 17{alpha}-estradiol were added to the milieu at 2 min. Inset, Representative amperometric NO release after in vitro stimulation of NO production by 17ß-estradiol (arrow, time of drug application). Each experiment was repeated four times, and the resulting means ± SEM are graphed.

 
Testosterone does not stimulate NO release
Increasing concentrations of testosterone (10-13–10-7 M) had no effect on NO release from ME fragments (Fig. 1Go).

The action of estradiol is steroid specific
17{alpha}-Estradiol (10-8 M) did not stimulate NO release (Fig. 2Go). Tamoxifen (10-8 M), an estradiol receptor inhibitor, significantly diminished (Fig. 2Go; P < 0.01) 17ß-estradiol-stimulated NO release.

17ß-Estradiol acts at a surface receptor
17ß-Estradiol appears to stimulate NO release by acting at the membrane surface, not on an intracellular receptor. E2-BSA (10-8 M), which does not penetrate the cellular membrane due to its size, also stimulates NO release from ME fragments within 2 min of its application in a tamoxifen-sensitive manner (Fig. 2Go). As for 17ß-estradiol, E2-BSA-stimulated NO release is dose dependent (Fig. 1Go). Stimulation of the ME with 10-11 M E2-BSA failed to stimulate a significant increase in NO release. The median effective concentration (EC50) for E2-BSA-stimulated NO release is approximately 3 x 10-10 M.

17ß-Estradiol and 17ß-estradiol coupled to BSA stimulate a rapid GnRH secretion from ME nerve terminals
17ß-Estradiol (10-10 and 10-8 M), like E2-BSA (10-8 M), induced a release of GnRH from ME fragments (Figs. 3Go and 4Go) within 10 min of their application. GnRH levels were significantly increased in the medium after 17ß-estradiol or E2-BSA addition (Fig. 4Go; P < 0.05) compared with those in the untreated groups. E2-BSA was as potent as 17ß-estradiol in inducing GnRH release from ME fragments, whereas 17{alpha}-estradiol had no effect on GnRH release (Fig. 4Go).



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  		Figure 3. In vitro stimulation of GnRH release from ME fragments by 17ß-estradiol (10-10 and 10-8 M; black columns). In this figure, the height of the column represents the mean, and the vertical line represents SEM. *, Significantly different from the white columns, P < 0.05. In the following experiments, 17ß-estradiol was used at 10-8 M because this concentration induces maximal NO release (see Fig. 1Go).

 


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  		Figure 4. In vitro stimulation of GnRH release from ME fragments by 17ß-estradiol (10-8 M) and E2-BSA (10-8 M; black columns). Estradiol-stimulated GnRH release is steroid specific, as 17{alpha}-estradiol (10-8 M) had no effect (white columns). In this figure, the height of the column represents the mean, and the vertical line represents the SEM. *, Significantly different from the white columns, P < 0.05; **, significantly different from the white columns, P < 0.01.

 
Estradiol-stimulated GnRH release occurs via NO release
E2-BSA-stimulated GnRH release was significantly inhibited by hemoglobin (2 µg/ml), a NO scavenger, and L-NAME (4 x 10-6 M), a NOS inhibitor (Fig. 5Go). The same results were obtained for 17ß-estradiol.



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  		Figure 5. In vitro stimulation of GnRH release from ME fragments by E2-BSA (10-8 M; black column) and its inhibition by hemoglobin (Hb; 2 µg/ml) and L-NAME (4 x 10-6 M; white columns), indicating that the E2-BSA-stimulated GnRH release occurs via NO release. In this figure, the height of the column represents the mean, and the vertical line represents the SEM. *, Significantly different from the white columns, P < 0.05.

 
Estradiol-NO-mediated GnRH release occurs via ecNOS activation
L-NIO, a more selective inhibitor of ecNOS than of nNOS, succeeded in inhibiting E2-BSA-stimulated GnRH release when added at 5 x 10-7 M (Fig. 6Go). The same results were obtained for 17ß-estradiol.



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  		Figure 6. In vitro stimulation of GnRH release from ME fragments by E2-BSA (10-8 M; black column) and its inhibition by L-NIO. The inhibition of the E2-BSA-stimulated GnRH release initiated by L-NIO is significant at 5 x 10-7 M, indicating that the major source of NO might be endothelial in origin (see Discussion). L-NIO is unable to significantly inhibit E2-BSA-stimulated GnRH release when applied at 5 x 10-8 M. In this figure, the height of the column represents the mean, and the vertical line represents SEM. **, Significantly different from the columns with *, P < 0.05.

 
NO stimulation of GnRH release occurs via guanylyl cyclase activation
The NO-mediated GnRH release after estradiol stimulation (17ß-estradiol or E2-BSA 10-8 M) was inhibited by ODQ (2 x 10-6 M), an inhibitor of NO-sensitive guanylyl cyclase (Fig. 7Go).



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  		Figure 7. In vitro stimulation of GnRH release from ME fragments by E2-BSA (10-8 M; black column) and its inhibition by ODQ (2 x 10-6 M), indicating that the NO-mediated GnRH release occurs via the stimulation of the soluble guanylyl cyclase in GnRH nerve terminals. In this figure, the height of the column represents the mean, and the vertical line represents the SEM. **, Significantly different from the white columns, P < 0.01.

 
Testosterone and estradiol plasmatic levels at the time of death
The average testosterone level was 5.63 ± 0.45 nM (n = 6), and the average estradiol level was 0.12 ± 0.01 nM (n = 6). These results are in accordance with those of Chowen et al. (32).

Tissue preservation
Structural examination of the ME fragments with light (Fig. 8AGo) and electronic (Fig. 8BGo) microscopies demonstrated that the tissues were in an excellent state of preservation after a 35-min incubation period. The parenchyme of the ME external zone was well preserved (Fig. 8Go), the nerve endings were in close contact with ependymal processes that reached the portal capillary bed. Inside the nerve endings and tanycytic end feet, the different organelles were also well preserved. The parenchymatous and endothelial basement membranes were well visible (Fig. 8bGo). Together, these observations proved that even after a 35-min incubation period, the ME structure was comparable to that observed upon placing the tissues immediately, without delay, in the fixative solution (33), and was compatible with the occurrence of physiological processes throughout the experiment.



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  		Figure 8. Light microphotograph obtained from coronal section of ME fragments (A). Electron microphotograph of ultrathin section showing the ME external zone (B). The observation of the structure (A) and the ultrastructure (B) of the treated ME fragments demonstrates that the conservation of the tissues was excellent after the 35-min incubation in the survival milieu. A: Asterisk, Third ventricle; arrowhead, capillaries. Scale bar, 760 µm. B: Big arrow, Nerve terminal; big arrowhead, parenchymatous basal lamina; little arrowhead, endothelial basal lamina; little arrow, fenestrated endothelium. Cap., Capillary; p.s., pericapillary space; Tan., tanycytes. Scale bar, 0.65 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrates that at physiological concentrations, 17ß-estradiol rapidly stimulates NO release from ME fragments, and this, in turn, stimulates GnRH release. This process appears to be mediated by a specific estradiol receptor, as noted by its antagonism by tamoxifen and the lack of 17{alpha}-estradiol and testosterone action. The fact that E2-BSA, an impermeable cell membrane estradiol analog, also stimulates NO release indicates that this receptor is located on the surface of the NOS-containing cells. Furthermore, 17ß-estradiol- and E2-BSA-stimulated GnRH release is inhibited by hemoglobin (a NO scavenger), and by L-NAME and L-NIO, NOS inhibitors, indicating that the effects of estradiol on GnRH secretion are mediated by coupling to NO. Moreover, according to Rees et al. (30) and Moore et al. (31), L-NIO is much more potent in inhibiting ecNOS than nNOS at the concentrations used in this study, suggesting that the major source of NO after E2-BSA or 17ß-estradiol stimulation is endothelial in origin. Furthermore, the NO stimulation of GnRH release occurs via the soluble guanylyl cyclase activation in the GnRH nerve terminals, as estradiol-stimulated GnRH release was inhibited by ODQ, a NO-sensitive guanylyl cyclase inhibitor (26, 27, 28, 29). Taken together, the rapid action of estradiol on GnRH release from ME fragments appears to be mediated by a NOS-coupled estrogen receptor located on the surface of endothelial cells found in the ME fragments.

NO release has been shown to be crucial for the occurrence of basal LH release in males (12) and the LH surge in ovariectomized females treated with estradiol plus progesterone (34, 35). Further, NO donors have been shown to be able to induce LH surge in estradiol-treated ovariectomized females (36) and thus to have a progesterone-like effect. Very recent findings show that estradiol stimulates nNOS expression in the preoptic area and in that way exerts a facilitatory influence on NO-producing neurons (29). The NO released appears to be able to modulate the activity of GnRH neurons (29). These observations implicate neuronal NO in the regulation of GnRH cell activity in the preoptic area. Contrary to these results, the present study, in accordance with our previous work (9), suggests that at the ME level, the NO implicated in the modulation of GnRH release is endothelial in origin rather than neuronal. This is consistent with the fact that unlike in the preoptic area where GnRH perikarya are surrounded by nNOS-containing cells, nNOS fibers and GnRH fibers in the ME are distributed separately in the internal and external zones, respectively (37). Further, in the ME, ecNOS immunoreactivity is observed in endothelial cells of the pituitary portal blood vessels (38), located at the immediate proximity of the GnRH terminals (33). The endothelial origin of NO secreted from ME fragments is strengthened by the results of a previous study which showed that central administration of ecNOS antisense is more efficacious than nNOS antisense administration in suppressing the estradiol-/progesterone-induced LH surge in ovariectomized females (10).

Regarding estradiol, it has been implicated in vascular reactivity (for review, see Refs. 39, 40) and more particularly in the stimulation of NO synthesis (40, 41). The long term stimulatory effects of estradiol on NO activity may also take place by increasing ecNOS synthase production (14, 42, 43). Estradiol can increase ecNOS expression within 8 h after its application on human vein endothelial cells via a cytosolic receptor-mediated system, and this action can be inhibited by the estrogen receptor antagonist, tamoxifen (15). The presence of two left-half palindromic sites of an estrogen receptor-binding element on the human ecNOS gene supports a potential receptor-mediated effect of estrogen on gene expression (44). Our study clearly shows that beside its long term action via a nuclear receptor on NO release from endothelia (15, 44), estradiol can have a short term stimulating action, i.e. NO release, via a specific membrane estrogen receptor on ME fragments. Tamoxifen is considered to be an antagonist at the nuclear estrogen receptor, but our results, in accordance with those of others (45, 46), suggest that tamoxifen antagonizes the effect of estradiol on its membrane receptor. In this way, tamoxifen has been shown to block the internalization of the nonactivated membrane estrogen receptor in the goat uterus (47).

This acute effect of estrogen on GnRH release from mediobasal hypothalamus has been poorly investigated compared with processes involving progesterone (48). Only one study showed that 17ß-estradiol had a receptor-mediated effect on GnRH release within 30 min in response to depolarization evoked by elevating K+ concentration (46). These researchers concluded that a membrane effect of 17ß-estradiol is present (46). In the present study we substantiate this observation and provide a mechanism, i.e. via NO, for the acute stimulatory effect of 17ß-estradiol on GnRH release. We surmise that estradiol-stimulated NO causes activation of the soluble guanylyl cyclase in GnRH terminals and results in cGMP production and, thus, in a depolarization involving a cationic conductance (49) that leads to GnRH release (46). Our findings are in accordance with recent evidence showing that NO stimulates GnRH release by an intracellular signal transduction process involving cGMP (34, 50, 51). As suggested by others, NO may also activate cyclooxygenase in GnRH terminals and/or glia that results in the production of PGE2 (52). PGE2 would then lead to the mobilization of calcium from intracellular stores (53) and cAMP formation (54), which finally induces the exocytosis of GnRH secretory granules (52). In this regard, a recent study showed PGE2 receptor gene expression in GnRH neurons (55).

In the male rat, estrogen plasma levels, as shown by our results, are comparable to those found in early proestrus female rats (33). Part of this plasma estradiol appear to arise from testosterone aromatization (32). In the present study we show that testosterone, in contrast to estradiol, is unable to stimulate NO release from male rat ME fragments. Our results suggest that on ME fragments, testosterone does not act on membrane receptors coupled to NOS activity and is not metabolized into estradiol. The latest observation is consistent with the fact that aromatase is not expressed in the ME (56). In male rats the effect of estrogen on GnRH release has not been investigated. One study on the adult male quail showed that basal GnRH release was increased by short term exposure of hypothalamic slices to 17ß-estradiol, whereas testosterone had no effect (57). In female rats estrogen has both stimulatory and inhibitory effects on GnRH release depending on their plasma levels (for review, see Refs. 58, 59). Kalra and Kalra (60) and Smith et al. (61) have shown that on diestrous day 1 and proestrus, GnRH output increases markedly, coincident with the rise in serum 17ß-estradiol. Moreover, we have shown in a previous work (33) that the peak level of estradiol obtained at 0900 h on proestrus was associated with GnRH release between 0900–1000 h on proestrus. In this same study (33) a new peak of estradiol appeared at 1700 h on proestrus, which might be associated with the GnRH surge that might occur at the same time, as shown by Sarkar and Minami (62).

The local and acute effect of estradiol on ME endothelium might have physiological implications, the major being the modulation of pulsatile GnRH secretion (63, 64) leading to LH release. It could also be implied in circumstances where a role for estrogen has been demonstrated, such as in mood disturbance (65, 66), mental stress (67), and premenstrual syndrome (65). In addition, extrapolation from the present neuroendocrinological model appears to identify the existence of another important mode of communication between the brain and the blood. Indeed, as shown by the present study and supported by our previous work (9), various signaling molecules, such as estradiol, morphine, and anandamide, conveyed in the blood can communicate with the brain without penetrating it, via NO production originating from the endothelium. We surmise that in other brain areas, molecules that cannot penetrate the blood-brain barrier could also modulate neuronal activity via endothelial NO signaling.

In summary, these observations are in direct line with our previous study demonstrating that rat ME produces NO in response to stimulatory molecules acting on ME endothelial cells (9). In the present study, 17ß-estradiol coupling to endothelial NO stimulates GnRH release from ME fragments via a membrane receptor. These effects occur at physiological concentrations and may play an important role in the modulation of pulsatile GnRH secretion from GnRH neuroendocrine terminals of the ME and thus represent a synchronizing link to anatomically scattered GnRH neurons (63, 64, 68). Additionally, this work demonstrates that the vascular endothelium in the ME is involved in the modulation of neurosecretions.


   Acknowledgments
 
The authors thank Dr. L. Buée for support and advice, and Mrs. G. Mortreux for excellent technical assistance with the RIAs.


   Footnotes
 
1 This work was supported by NIMH Grant COR 17138, NIDA Grant 09010, the Research Foundation and Central Administration of the State University of New York (to G.B.S.), NIH Fogarty Grant INT-00045 (to G.B.S.), the University of Lille II, and the FEDER (LARC network). Back

Received May 5, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ramirez VD, Feder HH, Sawyer CH 1984 The role of brain catecholamines in the regulation of LH secretion: a critical inquiry. In: Martini L, Ganong WF (eds) Frontiers in Neuroendocrinology. Raven Press, New York, vol 8:27–84
  2. Kalra SP 1986 Neural circuitry involved in the control of LHRH secretion: a model for preovulatory LH release. In: Martini L, Ganong WF, (eds) Frontiers in Neuroendocrinology. Raven Press, New York, vol 9:31–75
  3. Ojeda SR 1994 The neurobiology of mammalian puberty: has the contribution of glial cells been underestimated? J NIH Res 6:51–56
  4. Terasawa E 1995 Mechanisms controlling the onset of puberty in primates: the role of GABAergic neurons. In: Plant TM, Lee PA (eds) The Neurobiology of Puberty. Journal of Endocrinology, Bristol, pp 139–151
  5. Ojeda SR, Urbanski HF, Costa ME, Hill DF, Moholt-Siebert M 1990 Involvement of transforming growth factor {alpha} in the release of luteinizing hormone-releasing hormone from the developing female hypothalamus. Proc Natl Acad Sci USA 87:9698–9702[Abstract/Free Full Text]
  6. Melcangi RC, Galbiati M, Messi E, Magnaghi V, Cavarretta I, Riva MA, Zanisi M 1997 Astrocyte-neuron interactions in vitro: role of growth factors and steroids on LHRH dynamics. Brain Res Bull 44:465–469[CrossRef][Medline]
  7. Melcangi RC, Galbiati M, Messi E, Piva F, Martini L, Motta M 1995 Type 1 astrocytes influence luteinizing hormone-releasing hormone release from the hypothalamic cell line GT1–1: is transforming growth factor-ß the principle involved? Endocrinology 136:679–686[Abstract]
  8. Ma YJ, Berg-von der Emde K, Rage F, Wetsel WC, Ojeda SR 1997 Hypothalamic astrocytes respond to transforming growth factor-{alpha} with the secretion of neuroactive substances that stimulate the release of luteinizing hormone-releasing hormone. Endocrinology 138:19–25[Abstract/Free Full Text]
  9. Prevot V, Rialas CM, Croix D, Salzet M, Dupouy JP, Poulain P, Beauvillain JC, Stefano GB 1998 Morphine and anandamide coupling to nitric oxide stimulates GnRH and CRF release from rat median eminence: neurovascular regulation. Brain Res 790:236–244[CrossRef][Medline]
  10. Aguan K, Malesh VB, Ping L, Bhat G, Brann DW 1996 Evidence for physiological role for nitric oxide in the regulation of the LH surge: effect of central administration of antisense oligonucleotides to nitric oxide synthase. Neuroendocrinology 64:449–455[Medline]
  11. Brann W, Bhat GK, Lamar CA, Malesh VB 1997 Gaseous transmitters and neuroendocrine regulation. Neuroendocrinology 65:385–395[Medline]
  12. Rettori V, Belova N, Dees WL, Nyberg CL, Gimeno M, McCann SM 1993 Role of nitric oxide in the control of luteinizing hormone-releasing hormone release in vivo and in vitro. Proc Natl Acad Sci USA 90:10130–10134[Abstract/Free Full Text]
  13. Fricchione GL, Bilfinger TV, Hartman A, Liu Y, Stefano GB 1996 Neuroimmunologic implications in coronary artery disease. Adv Neuroimmunol 6:131–142[CrossRef][Medline]
  14. Weiner CP, Lizasoain I, Baylis SA, Knowles RG, Charles IG, Moncada S 1994 Induction of calcium-dependent nitric oxide synthases by sex hormones. Proc Natl Acad Sci USA 91:5212–5216[Abstract/Free Full Text]
  15. Hayashi T, Yamada K, Esaki T, Kuzuya M, Satake S, Ishikawa T, Hidaka H, Iguchi A 1995 Estrogen increases endothelial nitric oxide by a receptor- mediated system. Biochem Biophys Res Commun 214:847–855[CrossRef][Medline]
  16. Brown TJ, Clark AS, MacLusky NJ 1987 Regional sex differences in progestin receptor induction in the rat hypothalamus: effects of various doses of estradiol benzoate. J Neurosci 7:2529–2536[Abstract]
  17. Brown TJ, MacLusky NJ, Shanabrough M, Naftolin F 1990 Comparison of age- and sex-related changes in cell nuclear estrogen-binding capacity and progestin receptor induction in the rat brain. Endocrinology 126:2965–2972[Abstract]
  18. Simerly RB, Chang C, Muramatsu M, Swanson LW 1990 Distribution of androgen and estrogen receptor mRNA-containing cells in the rat brain: an in situ hybridization study. J Comp Neurol 294:76–95[CrossRef][Medline]
  19. Shughrue PJ, Bushnell CD, Dorsa DM 1992 Estrogen receptor messenger ribonucleic acid in female rat brain during the estrous cycle: a comparison with ovariectomized females and intact males. Endocrinology 131:381–388[Abstract]
  20. Couse JF, Lindzey J, Grandien K, Gustafsson JA, Korach KS 1997 Tissue distribution and quantitative analysis of estrogen receptor-{alpha} (ER{alpha}) and estrogen receptor-ß (ERß) messenger ribonucleic acid in the wild-type and ER{alpha}-knockout mouse. Endocrinology 138:4613–4621[Abstract/Free Full Text]
  21. Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J, Nilsson S, Gustafsson JA 1997 Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors {alpha} and ß. Endocrinology 138:863–870[Abstract/Free Full Text]
  22. Magazine I 1995 Detection of endothelial cell-derived nitric oxide: current trends and future directions. Adv Neuroimmunol 5:479–490[CrossRef][Medline]
  23. Stefano GB, Hartman A, Bilfinger TV, Magazine HI, Liu Y, Casares F, Goligorsky MS 1995 Presence of the µ3 opiate receptor in endothelial cells. J Biol Chem 270:30290–30293[Abstract/Free Full Text]
  24. Tsukahara H, Gordienko DV, Goligorsky MS 1993 Continuous monitoring of nitric oxide release from human umbilical vein endothelial cells. Biochem Biophys Res Commun 193:722–729[CrossRef][Medline]
  25. Stefano GB, Christensen VB, Tonnesen E, Liu Y, Hughes TK, Bilfinger TV 1996 Interleukin 10 stimulation of endogenous nitric oxide release from human saphenous veins diminishes immunocyte adherence. J Cardiovasc Pharmacol 30:90–95
  26. Southam E, Charles SL, Garthwaite J 1996 The nitric oxide-cyclic GMP pathway and synaptic plasticity in the rat superior cervical ganglion. Br J Pharmacol 119:527–532[Medline]
  27. Brunner F, Stessel H, Kukovetz WR 1995 Novel guanylyl cyclase inhibitor, ODQ reveals role of nitric oxide, but not of cyclic GMP in endothelin-1 secretion. FEBS Lett 376:262–266[CrossRef][Medline]
  28. Garthwaite J, Southam E, Boulton CL, Nielsen EB, Schmidt K, Mayer B 1995 Potent and selective inhibition of nitric oxide-sensitive guanylyl cyclase by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Mol Pharmacol 48:184–188[Abstract]
  29. Pu S, Kalra PS, Kalra SP 1998 Ovarian steroid-independent diurnal rhythm in cyclic GMP/nitric oxide efflux in the medial preoptic area: possible role in preovulatory and ovarian steroid-induced LH surge. J Neuroendocrinol 10:617–625[CrossRef][Medline]
  30. Rees DD, Palmer RM, Schulz R, Hodson HF, Moncada S 1990 Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo. Br J Pharmacol 101:746–752[Medline]
  31. Moore WM, Webber RK, Jerome GM, Tjoeng FS, Misk TP, Currie MG 1994 L-N6-(1-Iminoethyl)lysine: a selective inhibitor of inducible nitric oxide synthase. J Med Chem 37:3886–3888[CrossRef][Medline]
  32. Chowen JA, Argente J, Vician L, Clifton DK, Steiner RA 1990 Pro-opiomelanocortin messenger RNA in hypothalamic neurons is increased by testosterone through aromatization to estradiol. Neuroendocrinology 52:581–588[Medline]
  33. Prevot V, Dutoit S, Croix D, Tramu G, Beauvillain JC 1998 Semi-quantitative ultrastructural analysis of the localization and neuropeptide content of gonadotropin releasing hormone nerve terminals in the median eminence throughout the estrous cycle of the rat. Neuroscience 84:177–191[CrossRef][Medline]
  34. Bonavera JJ, Sahu A, Kalra PS, Kalra SP 1993 Evidence that nitric oxide may mediate the ovarian steroid-induced luteinizing hormone surge: involvement of excitatory amino acids. Endocrinology 133:2481–2487[Abstract]
  35. Bonavera JJ, Sahu A, Kalra PS, Kalra SP 1994 Evidence in support of nitric oxide (NO) involvement in the cyclic release of prolactin and LH surges. Brain Res 660:175–179[CrossRef][Medline]
  36. Bonavera JJ, Kalra PS, Kalra SP 1996 L-Arginine/nitric oxide amplifies the magnitude and duration of the luteinizing hormone surge induced by estrogen: involvement of neuropeptide Y. Endocrinology 137:1956–1962[Abstract]
  37. Herbison AE, Simonian SX, Norris PJ, Emson PC 1996 Relationship of neuronal nitric oxide synthase immunoreactivity to GnRH neurons in the ovariectomized and intact female rat. J Neuroendocrinol 8:73–82[CrossRef][Medline]
  38. Yamada K, Emson P, Hokfelt T 1996 Immunohistochemical mapping of nitric oxide synthase in the rat hypothalamus and colocalization with neuropeptides. J Chem Neuroanat 10:295–316[CrossRef][Medline]
  39. White MM, Zamudio S, Stevens T, Tyler R, Lindenfeld J, Leslie K, Moore LG 1995 Estrogen, progesterone, and vascular reactivity: potential cellular mechanisms. Endocr Rev 16:739–751[CrossRef][Medline]
  40. Wellman GC, Bonev AD, Nelson MT, Brayden JE 1996 Gender differences in coronary artery diameter involve estrogen, nitric oxide, and Ca2+-dependent K+ channels. Circ Res 79:1024–1030[Abstract/Free Full Text]
  41. Van Buren GA, Yang DS, Clark KE 1992 Estrogen-induced uterine vasodilatation is antagonized by L-nitroarginine methyl ester, an inhibitor of nitric oxide synthesis. Am J Obstet Gynecol 167:828–833[Medline]
  42. Goetz RM, Morano I, Calovini T, Studer R, Holtz J 1994 Increased expression of endothelial constitutive nitric oxide synthase in rat aorta during pregnancy. Biochem Biophys Res Commun 205:905–910[CrossRef][Medline]
  43. Hishikawa K, Nakaki T, Marumo T, Suzuki H, Kato R, Saruta T 1995 Up-regulation of nitric oxide synthase by estradiol in human aortic endothelial cells. FEBS Lett 360:291–293[CrossRef][Medline]
  44. Miyahara K, Kawamoto T, Sase K, Yui Y, Toda K, Yang LX, Hattori R, Aoyama T, Yamamoto Y, Doi Y 1994 Cloning and structural characterization of the human endothelial nitric-oxide-synthase gene. Eur J Biochem 223:719–726[Medline]
  45. Wong M, Moss RL 1991 Electrophysiological evidence for a rapid membrane action of the gonadal steroid, 17ß-estradiol, on CA1 pyramidal neurons of the rat hippocampus. Brain Res 543:148–152[CrossRef][Medline]
  46. Drouva SV, Laplante E, Gautron JP, Kordon C 1984 Effects of 17ß-estradiol on LH-RH release from rat mediobasal hypothalamic slices. Neuroendocrinology 38:152–157[Medline]
  47. Karthikeyan N, Thampan RV 1996 Plasma membrane is the primary site of localization of the nonactivated estrogen receptor in the goat uterus: hormone binding causes receptor internalization. Arch Biochem Biophys 325:47–57[CrossRef][Medline]
  48. Ramirez VD, Zheng J 1996 Membrane sex-steroid receptors in the brain. Front Neuroendocrinol 17:402–439[CrossRef][Medline]
  49. Fesenko EE, Kolesnikov SS, Lyubarsky AL 1985 Induction by cyclic GMP of cationic conductance in plasma membrane of retinal rod outer segment. Nature 313:310–313[CrossRef][Medline]
  50. Pu S, Xu B, Kalra SP, Kalra PS 1996 Evidence that gonadal steroids modulate nitric oxide efflux in the medial preoptic area: effects of N-methyl-D-aspartate and correlation with luteinizing hormone secretion. Endocrinology 137:1949–1955[Abstract]
  51. Moretto M, Lopez FJ, Negro-Vilar A 1993 Nitric oxide regulates luteinizing hormone-releasing hormone secretion. Endocrinology 133:2399–2402[Abstract]
  52. Rettori V, Gimeno M, Lyson K, McCann SM 1992 Nitric oxide mediates norepinephrine-induced prostaglandin E2 release from the hypothalamus. Proc Natl Acad Sci USA 89:11543–11546[Abstract/Free Full Text]
  53. Ojeda SR, Negro-Vilar A 1985 Prostaglandin E2-induced luteinizing hormone-releasing hormone release involves mobilization of intracellular Ca+2. Endocrinology 116:1763–1770[Abstract]
  54. Ojeda SR, Urbanski HF, Katz KH, Costa ME 1985 Stimulation of cyclic adenosine 3',5'-monophosphate production enhances hypothalamic luteinizing hormone-releasing hormone release without increasing prostaglandin E2 synthesis: studies in prepubertal female rats. Endocrinology 117:1175–1178[Abstract]
  55. Rage F, Lee BJ, Ma YJ, Ojeda SR 1997 Estradiol enhances prostaglandin E2 receptor gene expression in luteinizing hormone-releasing hormone (LHRH) neurons and facilitates the LHRH response to PGE2 by activating a glia-to-neuron signaling pathway. J Neurosci 17:9145–9156[Abstract/Free Full Text]
  56. Sanghera MK, Simpson ER, McPhaul MJ, Kozlowski G, Conley AJ, Lephart ED 1991 Immunocytochemical distribution of aromatase cytochrome P450 in the rat brain using peptide-generated polyclonal antibodies. Endocrinology 129:2834–2844[Abstract]
  57. Li Q, Tamarkin L, Levantine P, Ottinger MA 1994 Estradiol and androgen modulate chicken luteinizing hormone-releasing hormone-I release in vitro. Biol Reprod 51:896–903[Abstract]
  58. Kalra SP, Kalra PS 1989 Do testosterone and estradiol-17ß enforce inhibition or stimulation of luteinizing hormone-releasing hormone secretion? Biol Reprod 41:559–570[Abstract]
  59. Herbison AE 1998 Multimodal influence of estrogen upon gonadotropin-releasing hormone neurons. Endocr Rev 19:302–330[Abstract/Free Full Text]
  60. Kalra SP, Kalra PS 1974 Temporal interrelationships among circulating levels of estradiol, progesterone and LH during the rat estrous cycle: effects of exogenous progesterone. Endocrinology 95:1711–1718[Medline]
  61. Smith MS, Freeman ME, Neill JD 1975 The control of progesterone secretion during the estrous cycle and early pseudopregnancy in the rat: prolactin, gonadotropin and steroid levels associated with rescue of the corpus luteum of pseudopregnancy. Endocrinology 96:219–226[Abstract]
  62. Sarkar DK, Minami S 1995 Diurnal variation in luteinizing hormone-releasing hormone and ß-endorphin release in pituitary portal plasma during the rat estrous cycle. Biol Reprod 53:38–45[Abstract]
  63. Purnelle G, Gerard A, Czajkowski V, Bourguignon JP 1997 Pulsatile secretion of gonadotropin-releasing hormone by rat hypothalamic explants of GnRH neurons without cell bodies. Neuroendocrinology 66:305–312[Medline]
  64. Lopez FJ, Moretto M, Merchenthaler I, Negro-Vilar A 1997 Nitric oxide is involved in the genesis of pulsatile LHRH secretion from immortalized LHRH neurons. J Neuroendocrinol 9:647–654[CrossRef][Medline]
  65. Schmidt PJ, Nieman LK, Danaceau MA, Adams LF, Rubinow DR 1998 Differential behavioral effects of gonadal steroids in women with and in those without premenstrual syndrome. N Engl J Med 338:209–216[Abstract/Free Full Text]
  66. Elmquist JK, Scammell TE, Saper CB 1997 Mechanisms of CNS response to systemic immune challenge: the febrile response. Trends Neurosci 20:565–570[CrossRef][Medline]
  67. Del Rio G, Velardo A, Menozzi R, Zizzo G, Tavernari V, Grazia Venneri M, Marrama P, Petraglia F 1998 Acute estradiol and progesterone administration reduced cardiovascular and catecholamine responses to mental stress in menopausal women. Neuroendocrinology 67:269–274[CrossRef][Medline]
  68. Lopez FJ, Merchenthaler I, Moretto M, Negro-Vilar A 1998 Modulating mechanisms of neuroendocrine cell activity: the LHRH pulse generator. Cell Mol Neurobiol 18:125–146




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