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Endocrinology Vol. 143, No. 5 1950-1963
Copyright © 2002 by The Endocrine Society

NEUROENDOCRINOLOGY

Neuropeptide Y Inhibits the Biosynthesis of Sulfated Neurosteroids in the Hypothalamus through Activation of Y1 Receptors

Delphine Beaujean1, Jean-Luc Do-Rego, Ludovic Galas, Ayikoe G. Mensah-Nyagan, Robert Fredriksson, Dan Larhammar, Alain Fournier, Van Luu-The, Georges Pelletier and Hubert Vaudry2

European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U-413, UA Centre National de la Recherche Scientifique, University of Rouen (D.B., J.-L.D.-R., L.G., A.G.M.-N., H.V.), 76821 Mont-Saint-Aignan, France; Department of Neuroscience, Unit of Pharmacology, Uppsala University (R.F., D.L.), 75124 Uppsala, Sweden; Institut National de la Recherche Scientifique-Institut Armand Frappier, University of Quebec (A.F.), Pointe-Claire, Montréal, Canada H9R 1G6; and Medical Research Council Group in Molecular Endocrinology, Laval University Medical Center (V.L.-T., G.P.), Québec, Canada G1V 4G2

Address all correspondence and requests for reprints to: Dr. Hubert Vaudry, European Institute for Peptide Research (IFRMP 23), Laboratory of Cellular and Molecular Neuroendocrinology, INSERM U-413, UA Centre National de la Recherche Scientifique, University of Rouen, 76821 Mont Saint Aignan, France. E-mail: . hubert.vaudry{at}univ-rouen.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have recently shown that hydroxysteroid sulfotransferase (HST), the enzyme responsible for the biosynthesis of pregnenolone sulfate ({Delta}5PS) and dehydroepiandrosterone sulfate (DHEAS), is expressed in neurons located in the anterior preoptic area and the dorsal magnocellular nucleus of the frog diencephalon. As these two nuclei are richly innervated by NPY-immunoreactive fibers, we investigated the possible implication of NPY in the control of {Delta}5PS and DHEAS biosynthesis. Double labeling of frog brain sections revealed that 42% of the HST-immunoreactive perikarya in the diencephalon were contacted by NPY-containing fibers. In situ hybridization studies showed that Y1 and Y5 receptor mRNAs are expressed in the anterior preoptic area and the dorsal magnocellular nucleus. Pulse-chase experiments with 35S-labeled 3'-phosphoadenosine 5'-phosphosulfate as a sulfate donor demonstrated that frog NPY (fNPY) inhibited the conversion of [3H]{Delta}5P and [3H]dehydroepiandrosterone ([3H]DHEA) into [3H,35S]{Delta}5PS and [3H,35S]DHEAS by diencephalic explants. The inhibitory effect of fNPY on {Delta}5PS and DHEAS formation was mimicked by (pPYY) and [Leu31,Pro34]pNPY, which is an agonist for non-Y2 receptors in mammals, and was completely suppressed by the Y1 receptor antagonist BIBP3226. Conversely, the Y2 receptor agonist pNPY-(13–36) and the Y5 receptor agonist [D-Trp32]pNPY did not significantly modify the biosynthesis of [3H,35S]{Delta}5PS and [3H,35S]DHEAS. The present study provides the first evidence for the innervation of neurosteroid-producing neurons by NPY fibers. Our data also demonstrate that NPY, acting via Y1 receptors, exerts an inhibitory effect on the biosynthesis of sulfated neurosteroids.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE TERM NEUROSTEROIDS designates steroid compounds that are synthesized within the central nervous system and act locally to regulate nerve cell activity (1, 2). Among the most potent neurosteroids identified to date are the two sulfated 3-hydroxysteroids, pregnenolone sulfate ({Delta}5PS) and dehydroepiandrosterone sulfate (DHEAS) (3, 4, 5). The synthesis of these sulfated neurosteroids is catalyzed by a cytosolic enzyme termed hydroxysteroid sulfotransferase (HST), which transfers the sulfonate moiety from the donor molecule 3'-phosphoadenosine 5'-phosphosulfate (PAPS) onto the 3-hydroxy acceptor site of pregnenolone ({Delta}5P) or dehydroepiandrosterone (DHEA) (Fig. 1Go). Although early studies have demonstrated the occurrence of HST-like activity in the brains of primates (6) and rodents (7), the cellular localization of HST in the central nervous system has long remained unknown. It is only recently that the first immunohistochemical mapping of HST has been described in the brain of a vertebrate, the European green frog Rana ridibunda (8). Pulse-chase experiments using [3H]{Delta}5P and [3H]DHEA as steroid precursors and [35S]PAPS as a sulfonate donor have also demonstrated that frog diencephalic tissues are capable of synthesizing {Delta}5PS and DHEAS (8).



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  		Figure 1. Biosynthesis of the sulfoconjugated neurosteroids {Delta}5PS and DHEAS by HST. The enzyme transfers the sulfonate moiety from the donor molecule PAPS on the 3-hydroxy acceptor site of {Delta}5P and DHEA.

 
To date, the neuronal mechanisms regulating neurosteroid biosynthesis are poorly documented in mammals (9). In contrast, the roles of neurotransmitters and neuropeptides in the control of neurosteroid production have been intensely investigated in the brains of amphibians (10). In particular, the involvement of {gamma}-aminobutyric acid (GABA) (11), the octadecaneuropeptide (12), and the triakontatetraneuropeptide (13) in the regulation of 3ß-hydroxysteroid dehydrogenase has been clearly demonstrated in the brain of R. ridibunda. Concurrently, the actions of neurosteroids on GABAA receptors have been thoroughly investigated in frog pituitary melanotrope cells (14, 15). Thus, the frog brain appears to be a very suitable model in which to investigate the regulation of neurosteroid biosynthesis.

In the frog brain, HST-positive neurons are located in the anterior preoptic area and the dorsal magnocellular nucleus (8), two diencephalic nuclei that are richly innervated by NPY-immunoreactive fibers (16, 17, 18, 19) (Table 1Go). Concurrently, there is evidence that sulfated neurosteroids and NPY are involved in the regulation of similar neurophysiological processes. For instance, {Delta}5PS and DHEAS, like NPY, are potent regulators of food intake in rodents (20, 21). Similarly, {Delta}5PS and NPY have been shown to regulate reproductive behavior (22, 23). These observations suggest that some of the central effects of NPY may be mediated at least in part through modulation of sulfated 3-hydroxysteroid biosynthesis.


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  		Table 1. Localization and relative abundance of cell bodies and fibers exhibiting HST and NPY-like immunoreactivity in the brain of the frog Rana ridibunda

 
To test this hypothesis in the present report we have investigated the anatomical relationship between NPY- and HST-immunoreactive neurons in the frog brain. We have also studied the effect of synthetic NPY on HST activity in frog diencephalic explants. Finally, we characterized the pharmacological profile of the receptor involved in the action of NPY on the biosynthesis of sulfated neurosteroids.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult male frogs (R. ridibunda; 30–40 g BW) were purchased from a commercial supplier (Couétard, St. Hilaire de Riez, France). The animals were housed in a temperature-controlled room (8 ± 0.5 C) under running water on a 12-h dark, 12-h light regimen (lights on from 0600–1800 h) for at least 1 wk before use. To limit possible variations in neurosteroid biosynthesis due to circadian rhythms (24), all animals were killed between 0930 and 1030 h. Animal manipulations were performed according to the recommendations of the French ethical committee and under the supervision of authorized investigators.

Antibodies and oligonucleotides
The antiserum against sulfotransferase was raised by immunizing rabbits with a synthetic peptide corresponding to sequence 180–192 of rat liver HST (25). The sheep antiserum against porcine NPY was purchased from Auspep (Parkville, Australia). Texas Red-conjugated donkey antirabbit {gamma}-globulins (DAR/Texas Red) were supplied by Amersham Pharmacia Biotech (Little Chalfont, UK). Alexa 488-conjugated donkey antisheep {gamma}-globulins (DAS/Alexa 488) were purchased from Molecular Probe (Leiden, The Netherlands).

Oligonucleotides were designed from partial cDNA sequences corresponding to R. ridibunda NPY receptors as follows: Y1 forward, 5'-TGG ATT TTT GGA GTT GGT ATG TGT A-3'; Y1 reverse, 5'-AAC GGC AAT GAG AAC CAG TGA GAA A-3'; Y2 forward, 5'-TAT GCG GAC GGT GAC GAA CTA-3'; Y2 reverse, 5'-CCA CCA TCA TCA CCA ACA TCT-3'; Y5 forward, 5'-CAT ATT GCC CTG TCC TGT TTA-3'; Y5 reverse, 5'-AGA CCG AAT TCA TGT TGC TCA-3'; y6 forward, 5'-ACC GTG TGC AAA CTC GCT TCC-3'; and y6 reverse, 5'-CTT GCA TTT CCT CAC TTC CTG TCT-3'.

For Southern blot analysis, the following probes were used: Y1, 5'-AGT GTG TTT CAG TGA CAG TCT C-3'; Y2, 5'-GCC CAA CAC CCA GAA GTG AAA C-3'; and Y5, 5'-AGT CGA AGG TGG AGC TAC TCC T-3'.

Chemicals and reagents
T sulfate and estrone sulfate (ES) were purchased from Steraloids (Wilson, NH). {Delta}5P, {Delta}5PS, DHEA, DHEAS, 3-aminobenzoic acid ethyl ester (MS 222), and 8-bromo-cAMP were supplied by Sigma (St. Louis, MO). Tritiated {Delta}5P (7-[3H]{Delta}5P; 21 Ci/mmol), tritiated DHEA (1,2,6,7-[3H]DHEA; 60 Ci/mmol), and 35S-labeled PAPS (3'-[35S]PAPS; 1.13 Ci/mmol) were obtained from NEN Life Science Products (Les Ulis, France). Propylene glycol, HEPES, dichloromethane, hexane, and tetrahydrofuran (THF) were obtained from Merck \|[amp ]\| Co., Inc. (Darmstadt, Germany). BSA (fraction V) was purchased from Roche (Mannheim, Germany). Synthetic frog NPY (fNPY) was prepared by solid phase methodology as previously described (26). Synthetic porcine PYY (pPYY), [Leu31,Pro34]pNPY, pNPY-(13–36), and [D-Trp32]pNPY were supplied by France-Biochem (Meudon, France). (R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl] argininamide (BIBP3226) was purchased from RBI (Natick, MA).

Western blot analysis
One brain, three telencephalons, and five hypothalamus were homogenized in 10 mM Tris-HCl, pH 7.4, containing 1 mM phenylmethylsulfonylfluoride and 0.1% Triton X-100. The tissue homogenates were centrifuged (12,000 x g, 4 C, 15 min), and the proteins contained in the supernatants were precipitated with trichloroacetic acid (10% final concentration) and analyzed by PAGE in denaturing conditions (10% SDS). Proteins were then electroblotted onto a nitrocellulose filter and immunostained with the HST antibodies diluted 1:500 in 0.1 M PBS, pH 7.4, supplemented with 1% BSA using a chemiluminescence detection kit (Amersham Pharmacia Biotech).

Immunofluorescence procedure
Fifteen animals were anesthetized by immersion in 0.1% MS 222 and perfused transcardially with 50 ml 0.1 M PBS (pH 7.4). The perfusion was performed with 50 ml Bouin’s fixative. The brains were rapidly dissected, postfixed overnight at 4 C, embedded in Tissue-Tek (Reichert-Jung, Nussloch, Germany), and frozen at -80 C. Brain sections were cut at 7 µm in the frontal or sagittal plane in a cryomicrotome (Frigocut 2700, Leica Corp., Nussloch, Germany). The tissue sections were incubated overnight at 4 C in a humid atmosphere with the HST antiserum (1:100) or the antibodies against NPY (1:200) diluted in PBS containing 0.3% Triton X-100 and 1% BSA. At the end of the incubation, the sections were rinsed for 1 h in PBS and incubated for 1.5 h at room temperature with DAR/Texas Red or DAS/Alexa 488 (10 µg/ml). For colocalization studies, brain sections were incubated with both the HST and NPY antisera, and the immunoreactivities were revealed with DAR/Texas Red and DAS/Alexa 488. Finally, the sections were rinsed for 1 h in PBS and mounted with PBS/glycerol (vol/vol). The preparations were examined under an Orthoplan microscope (Leica Corp., Rockleigh, NJ) or a confocal laser scanning microscope (CLSM; Leica Corp.) equipped with a Diaplan optical system and an argon/krypton ion laser (excitation wavelengths, 488, 568, and 647 nm). Dual channel CLSM analysis was performed using a long-pass filter ({lambda} > 610 nm) for detection of Texas Red and a band-pass filter ({lambda} = 535 ± 7 nm) for detection of Alexa 488. To study the specificity of the immunoreaction, the following controls were performed: 1) substitution of the HST or NPY antiserum with PBS, 2) incubation with nonimmune rabbit serum instead of the HST or NPY antiserum, and 3) preincubation of the HST antiserum (diluted 1:100) with the synthetic peptide hapten (5 x 10-6 M) or preincubation of the NPY antiserum (diluted 1:200) with 10-6 M synthetic fNPY.

Neuroanatomical nomenclature was based on the atlas of Neary and Northcutt for the bullfrog diencephalon (27).

RT-PCR analysis
Total RNA from frog diencephalon was purified by the acid guanidinium-thiocyanate-phenol-chloroform method (28) using the Tri-Reagent (Sigma). Approximately 5 µg RNA were reverse transcribed using an oligo(deoxythymidine)12–18 primer and SuperScript II reverse transcriptase RNase H- (Life Technologies, Inc., Cergy Pontoise, France) in the buffer supplied with the enzyme. PCR amplification was performed in a 50-µl volume containing 2 µl reverse transcribed RNA solution, 200 µM of each dNTP, 1 mM MgCl2, 1 U Taq DNA polymerase (Promega Corp., Charbonnières, France), and 20 pmol sense and antisense primers in 5 µl of the buffer (pH 9) supplied with the enzyme for 40 cycles (40 sec at 94 C, 60 sec at 50 C, and 90 sec at 72 C) in a Robocycler Gradient 40 (Stratagene, La Jolla, CA).

The PCR products were separated on a 2% agarose gel and transferred to Hybond-N membrane (Amersham Pharmacia Biotech). The membranes were prehybridized for 4 h at 42 C in a solution containing 5x SSC, 0.1x SDS, 10x Denhardt’s solution, and 50 µg/ml denatured salmon sperm DNA. Hybridization was performed overnight at 42 C in a solution containing 5x SSPE and 1x SDS in the presence of 32P-labeled NPY receptor probe. The membranes were washed twice in 5x SSPE/0.1% SDS at 42 C and exposed on Kodak X-OMAT films (Sigma).

In situ hybridization histochemistry
Adult male frogs were perfused transcardially with 4% paraformaldehyde as described above. Brain slices (12-µm thick) were cut on a cryostat, mounted on 0.5% gelatin/0.05% chrome alun/0.01% poly-L-lysine-coated slides, and kept at -80 C until use. The partial NPY receptor cDNA sequences were subcloned into the pGME-T vector between SpeI and NcoI sites, and sense and antisense riboprobes were generated by in vitro transcription using T7 and SP6 RNA polymerases in the presence of [35S]UTP (Combination Systems, Promega Corp.). Sections were incubated for 10 min in 0.1 M triethanolamine, 0.9% NaCl (pH 8.0), and 0.25 acetic anhydride; rinsed in 2x SSC; and covered for 60 min with prehybridization buffer (pH 7.5) containing 50% formamide, 0.6 M NaCl, 10 mM Tris-HCl (pH 7.5), 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% BSA, 1 mM EDTA (pH 8.0), 550 µg/ml denatured salmon sperm DNA, and 50 µg/ml yeast tRNA. Hybridization was performed overnight at 40 C for the Y1 receptor and at 50 C for the Y2, Y5, and y6 receptors in the same buffer (except for salmon sperm DNA, the concentration of which was lowered to 60 µg/ml) supplemented with 10 mM dithiothreitol, 10% dextran sulfate, and 1.5 x 107 cpm/ml heat-denatured RNA riboprobes. The tissue sections were then washed in 2x SSC at 50 C and treated with ribonuclease A (50 µg/ml) for 60 min at 37 C. Five final high stringency washes were performed in 0.01x SSC containing 14 mM ß-mercaptoethanol and 0.05% sodium pyrophosphate. The slices were dehydrated in ethanol and exposed on Hyperfilm-ßmax films (Amersham Pharmacia Biotech) for 1 month. Tissue slices were subsequently dipped into Kodak NTB2 liquid emulsion at 40 C, exposed for 2 months, and developed. To identify anatomical structures, slices were stained with hematoxylin and eosin.

Measurement of HST activity
For each experiment, the hypothalami of five frogs were rapidly dissected, sliced, and preincubated at 24 C for 30 min in the presence of [35S]PAPS (4.5 x 10-6 M) diluted in 1 ml Ringer’s solution consisting of 15 mM HEPES, 112 mM NaCl, 15 mM NaHCO3, 2 mM CaCl2, and 2 mM KCl, supplemented with 2 mg glucose/ml and 0.3 mg BSA/ml. The incubation medium was gassed with a 95% O2/5% CO2 mixture, and the pH was adjusted to 7.4. The diencephalic slices were incubated at 24 C for 2 h in 500 µl Ringer’s medium containing 10-6 M [3H]{Delta}5P or 10-7 M [3H]DHEA, 4.5 x 10-6 M [35S]PAPS, and 4% propylene glycol in the absence or presence of test substances. At the end of the incubation period, the medium was removed, the reaction was stopped by adding 1 ml ice-cold Ringer’s medium, and the tissues were homogenized. Sulfated and unconjugated steroids were extracted three times with 1 ml dichloromethane as previously described (29). The aqueous phase containing sulfated steroids was evaporated in a Speed-Vac concentrator (Savant, Hicksville, NY), and the dry extract was prepurified on a Sep-Pak C18 cartridge (Waters Corp., Milford, MA) equilibrated with hexane. Sulfated steroids were eluted with 3 ml of a solution of 70% hexane and 30% THF. The solvent was evaporated in a Speed-Vac concentrator, and the dry extract was kept at 4 C until HPLC analysis.

HPLC
Sep-Pak-prepurified diencephalic extracts were analyzed by reverse phase HPLC on a Gilson liquid chromatograph (Unipoint System, Villiers-le-Bel, France) equipped with a 0.39 x 30-cm Nova-Pak C18 column (Waters Corp.) equilibrated with 100% hexane. Radioactive steroids were eluted at a flow rate of 1 ml/min using a gradient of THF (0–100% over 45 min) including three isocratic steps at 0% (0–10 min), 2% (15–20 min), and 100% (35–45 min). Fractions were collected at 0.5-min intervals. A 400-µl aliquot of each HPLC fraction was mixed with 4 ml liquid scintillator (Aquasafe 300 Plus, Zimer Analytic, Frankfurt, Germany) and counted in a double channel liquid scintillation counter (1211 Minibeta, LKB Wallac, Inc. Gaithersburg, MD) using the specific windows for 3H and 35S. Synthetic steroids used as reference standards were chromatographed under the same conditions as the tissue extracts, and their elution positions were determined by UV absorption using a UV-VIS 119 detector (Gilson).

Quantification of steroid biosynthesis and statistical analysis
The amounts of radioactive sulfated steroids formed by conversion of [3H]{Delta}5P or [3H]DHEA in the presence of [35S]PAPS were expressed as a percentage of the total radioactivity contained in all peaks resolved by HPLC, including [3H]{Delta}5P or [3H]DHEA for the 3H-labeled neosynthesized steroids or including [35S]PAPS for the 35S-labeled neosynthesized steroids. Each value is the mean of three independent experiments. Statistical significance for comparisons among groups was determined by ANOVA with post-hoc Tukey’s test using the Instat version 3.01 program (GraphPad Software, Inc., San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Western blot analysis
Western blot analysis of whole frog brain, telencephalon, and hypothalamus extracts revealed that the HST antiserum stained two major bands and four minor bands with apparent molecular masses ranging from 30–46 kDa (Fig. 2Go).



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  		Figure 2. Western blot analysis of HST-immunoreactive proteins in frog brain (Br), telencephalon (Tel), and hypothalamus (HT) extracts. The tissues were homogenized, and 30-µg protein samples from 12,000 x g supernatants were analyzed by SDS-PAGE, followed by immunoblotting. HST antibodies were used at a 1:1000 dilution. Molecular mass markers (in kilodaltons) are indicated on the left.

 
Immunocytochemistry
A comparison of the cellular distribution of HST to that of NPY in the frog brain revealed that the two diencephalic nuclei that contain HST-immunoreactive neurons (i.e. the anterior preoptic area and the dorsal magnocellular nucleus) are innervated by NPY-positive fibers (Table 1Go). Labeling of consecutive sections of the frog diencephalon with the antisera against HST or against NPY showed that the HST-positive cell bodies are located in territories traversed by beaded NPY-immunoreactive nerve fibers in the anterior preoptic area (Fig. 3Go, A and B) and the dorsal magnocellular nucleus (Fig. 3Go, C and D).



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  		Figure 3. CLSM photomicrographs comparing the distribution of HST- and NPY-like immunoreactivities in the frog diencephalon. A and B, Consecutive frontal sections through the anterior preoptic area (Poa) labeled with the antiserum against HST (A) or the antiserum against NPY (B). III, Third ventricle. C and D, Consecutive frontal sections through the dorsal magnocellular nucleus (Mgd) labeled with the antiserum against HST (C) or the antiserum against NPY (D). E and F, Specificity control of the HST immunoreaction on two consecutive sections in the Mgd. E, Positive control with nonabsorbed HST antiserum. F, Negative control with HST antiserum preincubated with the synthetic peptide hapten (5 x 10-6 M). The contours of HST-immunoreactive neurons appearing in A and C, which are schematically represented in B and D, respectively, show that NPY-immunoreactive elements are found in the vicinity of HST-positive neurons (asterisks). Scale bars, 10 µm.

 
Preincubation of the HST antiserum with the synthetic peptide hapten (5 x 10-6 M) resulted in total loss of the immunoreaction (Fig. 3Go, E and F). Similarly, preincubation of the NPY antiserum with synthetic fNPY (10-6 M) suppressed immunolabeling (data not shown). No fluorescence was observed when the HST and NPY antisera were substituted by nonimmune rabbit serum or PBS (data not shown).

Double labeling of diencephalic slices with the HST and NPY antisera combined with dual channel CLSM analysis revealed the existence of NPY-positive varicosities in close vicinity to HST-immunoreactive cell bodies in the dorsal magnocellular nucleus (Fig. 4Go, A and B). Similarly, in the anterior preoptic area, HST-positive perikarya were surrounded by a network of beaded NPY-immunoreactive processes (Fig. 4Go, C and D). Quantitative analysis performed on 72 HST-immunoreactive neurons revealed that 42% of them were contacted by NPY-containing fibers.



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  		Figure 4. Dual channel CLSM analysis of frontal sections of the frog brain, illustrating the distribution of HST- and NPY-like immunoreactivity in the dorsal magnocellular nucleus (Mgd; A and B) and the anterior preoptic area (Poa; C and D). Each brain section was labeled with the rabbit antiserum against HST revealed with DAR/Texas Red and the sheep antiserum against NPY revealed with DAS/Alexa 488. The Texas Red and Alexa 488 fluorescence signals were analyzed separately using a long-pass filter ({lambda} > 610 nm) and a band-pass filter ({lambda} = 535 nm), respectively, and the two acquisitions were superimposed on the same image. Asterisks show that beaded NPY-immunoreactive nerve fibers are located close to HST-positive neurons. III, Third ventricle. Scale bars, 20 µm.

 
Expression of NPY receptors in the frog diencephalon
RT-PCR analysis was carried out to determine which NPY receptor subtypes are expressed in the frog diencephalon. Oligonucleotides were designed to amplify cDNA fragments of 93, 581, 422, and 434 bp corresponding, respectively, to Y1, Y2, Y5, and y6 receptor mRNAs. Y1 and Y5 receptor mRNAs were amplified by RT-PCR, and the identities of the cDNA sequences were confirmed by Southern blot analysis using specific Y1 and Y5 cDNA riboprobes (Fig. 5Go, A and B). In contrast, no Y2 or y6 mRNA signal could be detected in the frog diencephalon (data not shown). When reverse transcriptase was omitted in the RT-PCR mixture, no amplification product was observed (Fig. 5Go, A and B).



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  		Figure 5. RT-PCR analysis of Y1 and Y5 receptor mRNA in the frog diencephalon. Total RNA was incubated in the presence (+) or absence (-) of reverse transcriptase, and the cDNAs were amplified by PCR using specific Y1 (A) or Y5 (B) receptor primers. A DNA mass ladder (kilobases) is shown between the two gels. The identity of the bands was confirmed by Southern blot analysis with specific frog 32P-labeled Y1 (A) or Y5 (B) receptor riboprobes, as shown under the ethidium bromide-stained gels.

 
The tissue distribution of Y1 and Y5 receptor mRNAs was determined by in situ hybridization histochemistry (Figs. 6Go and 7Go). Receptor mRNA expression was considered significant when the density of silver grains was 5 times higher than that in the respective control. A high concentration of Y1 receptor mRNA was found notably in the anterior preoptic area and the lateral amygdala (Fig. 6AGo) as well as in the dorsal magnocellular nucleus, the ventromedial thalamic nucleus, and the habenular nuclei (Fig. 6BGo). A moderate concentration of Y5 receptor mRNA was detected in the anterior preoptic area (Fig. 7AGo) and the dorsal magnocellular nucleus (Fig. 7BGo). Control sections incubated with the sense Y1 and Y5 probes exhibited only weak background staining (Fig. 6Go, A and B, and Fig. 7Go, A and B).



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  		Figure 6. In situ hybridization of Y1 receptor mRNA in the frog diencephalon. Frontal brain sections, at the level of the anterior preoptic area (A) and the dorsal magnocellular nucleus (B), were hybridized with the antisense Y1 receptor riboprobe (upper panels). Consecutive sections were hybridized with the sense Y1 receptor riboprobe (lower panels). The anatomical structures are designated on the left hemisections according to Neary and Northcutt (20 ). For abbreviations, see Table 1Go. Brain sections were exposed onto Hyperfilm-ßmax for 1 month (right hemisections). The tissue slices were then dipped into Kodak liquid emulsion for 2 months, and photomicrographs at the level of the anterior preoptic area (A) and the dorsal magnocellular nucleus (B) are shown on the right. The mean densities of silver grains in positive neurons of the preoptic area and the magnocellular nucleus were, respectively, 4.6 and 6.1 times higher than the background in the same sections. III, Third ventricle. Scale bars, 20 µm.

 


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  		Figure 7. In situ hybridization of Y5 receptor mRNA in the frog diencephalon. Frontal brain sections, at the level of the anterior preoptic area (A) and the dorsal magnocellular nucleus (B), were hybridized with the antisense Y5 receptor riboprobe (upper panels). Consecutive sections were hybridized with the sense Y5 receptor riboprobe (lower panels). See Fig. 6Go for other designations. The mean densities of silver grains in positive neurons of the preoptic area and the magnocellular nucleus were, respectively, 5.6 and 5 times higher than the background in the same sections. III, Third ventricle. Scale bars, 20 µm.

 
Effect of NPY on biosynthesis of sulfated neurosteroids
Frog hypothalamic explants were incubated with both [3H]{Delta}5P as a steroid precursor and [35S]PAPS as a sulfate donor, and the tissue extracts were analyzed by reverse phase HPLC. The HPLC gradient used made it possible to resolve several double labeled metabolites (sulfated steroids) including [3H,35S]{Delta}5PS, [3H,35S]T sulfate and [3H,35S]ES (Fig. 8AGo). When [3H]DHEA was used as a precursor in the presence of [35S]PAPS, the formation of [3H,35S]DHEAS and [3H,35S]ES was observed (Fig. 8BGo). Addition of fNPY (10-7 M) to the incubation medium markedly reduced the conversion of [3H]{Delta}5P or [3H]DHEA to sulfated steroids (Fig. 8Go, C and D). Incubation of frog diencephalic slices with graded concentrations of fNPY (10-9–10-5 M) induced a dose-dependent decrease in the conversion of [3H]{Delta}5P to [3H,35S]{Delta}5PS (Fig. 9AGo) as well as the conversion of [3H]DHEA to [3H,35S]DHEAS (Fig. 9BGo).



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  		Figure 8. HPLC analysis of radioactive metabolites formed during a 2-h incubation of frog hypothalamic explants with a 3H-labeled steroid precursor ({Delta}5P or DHEA) and the 35S-labeled sulfate donor PAPS. Frog hypothalamic slices were incubated with 4.5 x 10-6 M [35S]PAPS together with 10-6 M [3H]{Delta}5P (A and C) or 10-7 M [3H]DHEA (B and D), in the absence (A and B) or presence (C and D) of 10-7 M synthetic frog NPY. The tissues were homogenized, and the radioactive metabolites were analyzed using a hexane/THF gradient. The ordinates indicate the radioactivity (3H and 35S) measured in the HPLC fractions (0.4 ml each). The dashed line represents the gradient of secondary solvent (percent THF). The arrows indicate the elution position of standards. TS, T sulfate.

 


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  		Figure 9. Effects of graded concentrations of fNPY on the conversion of [3H]{Delta}5P to [3H,35S]{Delta}5PS (A) and of [3H]DHEA to [3H,35S]DHEAS (B) by frog hypothalamic explants. The values were obtained from experiments similar to those presented in Fig. 8Go. {bullet}, Relative amounts of [3H]{Delta}5PS or [3H]DHEAS compared with the total amount of 3H-labeled compounds resolved by HPLC analysis (x100). {circ}, Relative amounts of [35S]{Delta}5PS or [35S]DHEAS compared with the total amount of 35S-labeled compounds resolved by HPLC analysis (x100). Each value is the mean ± SEM of three independent experiments.

 
Incubation of frog hypothalamic explants with a low concentration of 8-bromo-cAMP (10-4 M) did not affect the spontaneous biosynthesis of sulfated neurosteroids, but suppressed the inhibitory effect of NPY (10-7 M) on the conversion of [3H]{Delta}5P to [3H,35S]{Delta}5PS (Fig. 10AGo) and of [3H]DHEA to [3H,35S]DHEAS (Fig. 10BGo).



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  		Figure 10. Effect of 8-bromo-cAMP on NPY-induced inhibition of the conversion of [3H]{Delta}5P to [3H,35S]{Delta}5PS (A) and of [3H]DHEA to [3H,35S]DHEAS (B) by frog hypothalamic explants. The values were obtained from experiments similar to those presented in Fig. 8Go. The ordinates represent the relative amount of [3H]{Delta}5PS or [3H]DHEAS compared with the total amount of 3H-labeled compounds resolved by HPLC analysis (x100). Values are the mean ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01; NS, not statistically different from control. f, Frog.

 
Effects of NPY receptor agonists and antagonists on the biosynthesis of sulfated neurosteroids
The pharmacological profile of the receptors involved in the action of NPY was studied using various NPY receptor agonists and antagonists. pPYY (10-6 M), which is a nonselective NPY receptor agonist in mammals, and [Leu31,Pro34]pNPY (10-6 M), which is an agonist for Y1, Y4, Y5, and y6, but not for Y2 receptors, both mimicked the inhibitory effect of fNPY on the conversion of [3H]{Delta}5P to [3H,35S]{Delta}5PS (Fig. 11AGo) and the conversion of [3H]DHEA to [3H,35S]DHEAS (Fig. 11BGo). In addition, compound BIBP3226 (10-6 M), which is a selective Y1 receptor antagonist in mammals, completely suppressed the inhibitory effect of fNPY on {Delta}5PS (Fig. 11AGo) and DHEAS formation (Fig. 11BGo). Conversely, the selective Y2 receptor agonist pNPY-(13–36) (10-6 M) and the Y5 receptor agonist [D-Trp32]pNPY (10-6 M) did not significantly modify the biosynthesis of [3H,35S]{Delta}5PS (Fig. 11AGo) and [3H,35S]DHEAS (Fig. 11BGo) by diencephalic explants.



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  		Figure 11. Effect of NPY receptor agonists and antagonist on the conversion of [3H]{Delta}5P to [3H,35S]{Delta}5PS (A) and of [3H]DHEA to [3H,35S]DHEAS (B) by frog hypothalamic explants. pPYY, Nonselective agonist; [Leu31,Pro34]pNPY, non-Y2 agonist; pNPY-(13–36), Y2 agonist; [D-Trp32]pNPY, Y5 agonist; BIBP3226, Y1 antagonist. See Fig. 10Go for other designations. Values are the mean ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not statistically different from control. f, Frog; p, porcine.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although sulfated neurosteroids are potent regulators of neuronal activity, the mechanisms of control of their biosynthesis remain unknown. The present study describes the neuroanatomical relationship between the NPYergic neuronal system and HST-containing cell bodies in the frog diencephalon. Our data demonstrate that NPY, acting through Y1 receptors, exerts an inhibitory effect on the biosynthesis of sulfated 3-hydroxysteroids.

Anatomical relationship between NPY-containing nerve fibers and HST-immunoreactive neurons
Western blot analysis revealed the existence of two groups of immunoreactive bands in whole frog brain, telencephalon, and hypothalamus, with apparent molecular masses of about 30–32 kDa and 40–42-46 kDa. In mammals, all HST isoforms characterized to date possess a conserved Asp residue (position 230 in human DHEA-sulfotransferase), which represents a potential N-glycosylation site (29). Thus, the two groups of bands detected by Western blot may correspond to two distinct isoforms of HSTs, each of which exhibits different degrees of N-glycosylation.

The mapping of NPY- and HST-immunoreactive elements has been previously studied in detail in the brain of the European green frog R. ridibunda (8, 16). A comparison of the distribution of these two neuronal systems has revealed that in the frog diencephalon, dense networks of NPY-immunoreactive fibers are present in the anterior preoptic area and the dorsal magnocellular nucleus where HST-positive neurons are located. Immunohistochemical labeling of consecutive sections confirmed the occurrence of numerous NPY-immunoreactive fibers and HST-expressing cell bodies in the anterior preoptic area and dorsal magnocellular nucleus. After double labeling of frog brain sections, CLSM analysis revealed that in these two diencephalic nuclei, many NPY-positive fibers are found in the close vicinity of HST-containing perikarya. These observations provided a neuroanatomical clue suggesting that NPY could be involved in regulation of the biosynthesis of sulfated 3-hydroxysteroids in the frog diencephalon.

Expression of NPY receptors in the diencephalic nuclei containing HST-immunoreactive neurons
At least five NPY receptor subtypes, termed Y1, Y2, Y4, Y5, and y6, have been cloned in vertebrates (for review, see Refs. 30 and 31). A Y1 receptor has been cloned in the frog Xenopus laevis, and to date this is the only NPY receptor isoform that has been characterized in amphibians (32). The partial sequences of the Y1, Y2, Y5, and y6 receptors in the frog R. ridibunda were obtained using a degenerate PCR approach on frog genomic DNA (our unpublished observations). In the present study we have taken advantage of the availability of these nucleotide sequences to investigate which NPY receptor isoforms are expressed in the frog diencephalon.

RT-PCR and Southern blot analysis revealed the presence of both Y1 and Y5 receptor mRNAs in the diencephalon, and the precise localization of each receptor was determined by in situ hybridization. Expression of Y1 and Y5 receptor mRNAs was seen in the anterior preoptic area and the dorsal magnocellular nucleus, i.e. in the two diencephalic nuclei where HST-immunoreactive neurons are located. Consistent with the RT-PCR data, neither Y2 nor y6 transcripts could be detected in the frog diencephalon by in situ hybridization histochemistry. These observations suggested that NPY might regulate the activity of HST neurons through activation of Y1 and/or Y5 receptors.

Effect of NPY on sulfated neurosteroid biosynthesis
Because the sulfate donor molecule PAPS slowly diffuses through the plasma membrane (for review, see Ref. 33), we previously used tissue homogenates to demonstrate that the frog brain has the capability of synthesizing sulfated neurosteroids, including {Delta}5PS and DHEAS (8). In the present study we had to modify this pulse-chase technique to investigate the biosynthesis of sulfated steroids by intact nerve cells. We found that a 30-min preincubation of hypothalamic explants with [35S]PAPS markedly increased the conversion of [3H]{Delta}5P and [3H]DHEA to {Delta}5PS and DHEAS, respectively, making it possible to study the effect of NPY on sulfated neurosteroid biosynthesis.

Incubation of frog hypothalamic slices with graded concentrations of synthetic fNPY (26) provoked a dose-dependent inhibition of the formation of newly synthesized sulfated steroids [3H,35S]{Delta}5PS and [3H,35S]DHEAS, with EC50 of 15 and 4 nM, respectively. The differential potencies of NPY on {Delta}5PS and DHEAS biosynthesis may be ascribed to the existence of two HST isoforms in the frog brain, as suggested by Western blot analysis. Thus, one isoform would be more specific for {Delta}5P and moderately sensitive to NPY, whereas the other isoform would be more specific for DHEA and highly sensitive to NPY.

To our knowledge, this is the first report demonstrating that NPY inhibits the biosynthesis of steroids in the central nervous system. It has been previously shown that NPY modulates the secretion of unconjugated steroid hormones from adrenocortical cells (for reviews, see Refs. 34 and 35) and ovary granulosa and luteal cells (36, 37, 38). Whether NPY can also regulate the activity of HST and thus the biosynthesis of DHEAS in the adrenal gland remains to be determined.

Pharmacological characterization of NPY receptors
To determine the type of receptor mediating the action of NPY on HST activity in the frog diencephalon, we investigated the effects of various NPY receptor agonists and antagonists on the conversion of [3H]{Delta}5P or [3H]DHEA to [3H,35S]{Delta}5PS or [3H,35S]DHEAS by hypothalamic explants. Our data revealed that the nonselective NPY receptor agonist pPYY (30) and [Leu31,Pro34]pNPY, which is an agonist for non-Y2 receptors in mammals (39), mimic the inhibitory action of fNPY on the biosynthesis of sulfated neurosteroids. In contrast, the selective Y2 agonist pNPY-(13–36) (40) and the specific Y5 agonist [D-Trp32]pNPY (41) did not significantly affect the production of {Delta}5PS and DHEAS. In addition, compound BIBP3226, which is a selective Y1 receptor antagonist in mammals (42), totally abolished the decrease in sulfated neurosteroid formation evoked by fNPY in the frog brain. The fact that high concentrations of Y1 mRNA are present in the anterior preoptic area and dorsal magnocellular nucleus, which contain HST-immunoreactive cell bodies (8), provides additional support for the involvement of Y1 receptors in the inhibitory effect of NPY on HST activity in the frog diencephalon. Concurrently, the observation that BIBP3226 alone provoked a modest increase in the biosynthesis of {Delta}5PS and DHEAS suggests that endogenous NPY actually exerts a tonic inhibitory action on sulfated neurosteroid biosynthesis. Collectively, these data indicate that in the frog brain NPY modulates the production of {Delta}5PS and DHEAS through activation of Y1 receptors.

Physiological significance
In contrast to the lipophilic nonconjugated steroids, steroid ester sulfates, which are hydrophilic compounds, cannot easily cross the blood-brain barrier. Therefore, although sulfated steroids are produced by the adrenal gland and testis, it is likely that {Delta}5PS and DHEAS present in the brain are mainly synthesized locally. In support of this hypothesis, substantial amounts of {Delta}5PS and DHEAS are found in the brain of adrenalectomized and gonadectomized animals (3, 4). Due to the modulatory actions of sulfated neurosteroids on various membrane receptors, including GABAA, N-methyl-D-aspartate (NMDA), and {varsigma} receptors (43), it is of crucial importance to determine the neuronal mechanisms controlling their biosynthesis.

In mammals, NPY, acting through its different receptors, has been implicated in a large array of neurophysiological processes (for reviews, see Refs. 44 and 45). In particular, activation of Y1 receptors by NPY modulates behavioral response to novelty (46), food consumption (47, 48), anxiety (49, 50), depression (51), body temperature (48), blood pressure (52, 53), and neuronal proliferation (54). Similarly, {Delta}5PS and DHEAS appear to be involved in the control of food intake (20), behavioral response to a novel environment (55), and anxiety (56, 57), suggesting that some of the effects of NPY could be mediated through modulation of HST activity.

It is clearly established that activation of Y1 receptors induces mobilization of intracellular calcium and inhibition of adenylyl cyclase activity (58, 59). To our knowledge, the effect of the cytosolic calcium concentration on HST activity has never been investigated. In contrast, it has been reported that in human fetal adrenocortical cells cAMP enhances the biological activity of HST (60, 61). It has also been shown that cAMP stimulates the biosynthesis of {Delta}5P, which is one of the main substrates of HST (62). These observations strongly suggest that the decrease in {Delta}5PS and DHEAS biosynthesis induced by NPY in the hypothalamus is mediated through inhibition of adenylyl cyclase activity. In support of this hypothesis, we found that incubation of hypothalamic slices with a low concentration of 8-bromo-cAMP (to clamp intracellular cAMP at a fixed level) suppressed the inhibitory effect of NPY on the biosynthesis of sulfated neurosteroids.

In conclusion, the present report has provided the first evidence for a neuroanatomical relationship between the NPYergic system and sulfated neurosteroid-secreting cells in the central nervous system. Our data show that NPY inhibits {Delta}5PS and DHEAS biosynthesis through activation of Y1 receptors, suggesting that some of the neurobiological effects of NPY can be accounted for by inhibition of HST bioactivity.


   Footnotes
 
This work was supported by grants from INSERM (U-413), a FRSQ-INSERM exchange program (to A.F., G.P., and H.V.), and the Conseil Régional de Haute-Normandie.

1 Recipient of a fellowship from the Ministère de la Recherche. Back

2 Affiliated Professor at the INRS-Institut Armand Frappier (Montréal, Canada). Back

Abbreviations: BIBP3226, (R)-N2-(Diphenylacetyl)-N-[(4-hydroxyphenyl)methyl] argininamide; CLSM, confocal laser scanning microscope; DAR, donkey antirabbit {gamma}-globulins; DAS, donkey antisheep {gamma}-globulins; DHEA, dehydroepiandrosterone; DHEAS, dehydroepiandrosterone sulfate; ES, estrone sulfate; fNPY, frog NPY; GABA, {gamma}-aminobutyric acid; HST, hydroxysteroid sulfotransferase; PAPS, 3'-phosphoadenosine 5'-phosphosulfate; pNPY, porcine NPY; {Delta}5PS, pregnenolone sulfate; THF, tetrahydrofuran.

Received August 2, 2001.

Accepted for publication January 9, 2002.


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