Occurrence of D-aspartic acid and N-methyl-D-aspartic acid in rat neuroendocrine tissues and their role in the modulation of luteinizing

hormone and growth hormone release

ANTIMO DANIELLO,*,1 M. MADDALENA DI FIORE,*, GEORGE H. FISHER, ALFREDO MILONE, ANGELO SELENI,ll SALVATORE DANIELLO,* ALESSANDRA F. PERNA,** AND DIEGO INGROSSO *Department of Biochemistry and Molecular Biology and Neurobiology, Zoological Station A. Dohrn, 80121, Napoli, Italy; Department of Scienze della Vita, Second University of Naples, 81100, Caserta, Italy; Department of Chemistry, Barry University, Miami Shores, Florida 33161, USA; Institute of Chemistry of Molecule of Biological Interest, CNR, 80072, Arco Felice, Napoli, Italy; ll Department of Radioimmunology, Laboratorio Igea, 80027, Frattamaggiore, Napoli, Italy; **Institute of Nephrology, School of Medicine, II University of Naples, 80131 Napoli, Italy; and Institute of Biochemistry of Macromolecules, School of Medicine, II University of Naples, 80138 Napoli, Italy
ABSTRACT Using two specic and sensitive uorometric/HPLC methods and a GC-MS method, alone and in combination with D-aspartate oxidase, we have demonstrated for the rst time that Nmethyl-D-aspartate (NMDA), in addition to D-aspartate (D-Asp), is endogenously present as a natural molecule in rat nervous system and endocrine glands. Both of these amino acids are mostly concentrated at nmol/g levels in the adenohypophysis, hypothalamus, brain, and testis. The adenohypophysis maximally showed the ability to accumulate D-Asp when the latter is exogenously administered. In vivo experiments, consisting of the i.p. injection of DAsp, showed that D-Asp induced both growth hormone and luteinizing hormone (LH) release. However, in vitro experiments showed that D-Asp was able to induce LH release from adenohypophysis only when this gland was co-incubated with the hypothalamus. This is because D-Asp also induces the release of GnRH from the hypothalamus, which in turn is directly responsible for the D-Asp-induced LH secretion from the pituitary gland. Compared to D-Asp, NMDA elicits its hormone release action at concentrations 100-fold lower than D-Asp. D-AP5, a specic NMDA receptor antagonist, inhibited DAsp and NMDA hormonal activity, demonstrating that these actions are mediated by NMDA receptors. NMDA is biosynthesized from D-Asp by an S-adenosylmethionine-dependent enzyme, which we tentatively denominated as NMDA synthase.DAniello, A., Di Fiore, M. M., Fisher, G. H., Milone, A., Seleni, A., DAniello, S., Perna, A. F., Ingrosso, D. Occurrence of D-aspartic acid and N-methyl-D-aspartic acid in rat neuroendocrine tissues and their role in

the modulation of luteinizing hormone and growth hormone release. FASEB J. 14, 699 714 (2000)
Key Words: D-Asp NMDA methyltransferase S-adenosylmethionine NMDA synthase testosterone progesterone endocrine glands nervous system

D-Aspartic acid (D-Asp) is an endogenous amino acid present in nervous and endocrine tissues of invertebrates and vertebrates. It was rst found in the brain, stellate ganglia, and the axoplasmic uid of the cephalopods Octopus vulgaris, Loligo vulgaris, and Sepia ofcinalis (12). Later this amino acid was found in the tissues of many other invertebrates (35) and vertebrates. In vertebrates, this amino acid occurs in the nervous tissues of chicken (6), rat (79), and human (10, 11). In humans, it is present in brains of embryos (10) and adults (11), as well as in the cerebrospinal uid (12). In addition to nervous tissues, in mammals D-Asp is also present in the pituitary gland and the gonads (8, 13). D-Asp occurs at high levels in embryos, whereas in adult animals it nearly disappears in nervous tissues, but increases in endocrine glands, particularly in the pituitary, hypothalamus (8, 13), and pineal gland (14), where it has been hypothesized to play an important role as a novel messenger molecule (14). We also found that D-Asp levels increase in the testes during the two phases of life span: immediately before birth and during sexual maturity (13), and coincidently with
1 Correspondence: Stazione Zoologica A. Dohrn, Villa Comunale 1, 80121 Napoli, Italy. E-mail: daniello@alpha. snz.it

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testosterone synthesis. Recently, we found that D-Asp is also present in the reproductive organs of rat, where it is localized in Leydig and Sertoli cells of testis (13) and in Octopus vulgaris (5). These data suggest that D-Asp is implicated in hormonal processes and in steroidogenesis, since Leydig cells are known to have a role in the synthesis of testosterone. In support to this hypothesis is the discovery that D-Asp occurs in the ovary of Rana esculenta, where it is involved in the control of testosterone release during the sexual cycle (16), and in the spermatogenesis of the rat testis (17). These data gave rise to the hypothesis that D-Asp in the neuroendocrine tissues could have a direct role in the regulation of hormone release and synthesis in the hypothalamus-hypophysial-gonadal axis or that D-Asp represents the precursor for the synthesis of another compound (e.g., NMDA), which is then directly responsible for hormone release. To test this hypothesis, we performed, in rats, in vivo and in vitro experiments designed to elucidate the endocrine role of this molecule, with particular attention to its involvement in the hypothalamus-adenohypophysis hormone release.

Italy). Decanoic acid was purchased from Aldrich (Milwaukee, Wis.). Cation exchange resin (AG 50W-X8, H form, 100 200 mesh, 60 150 M size) was obtained from Bio-Rad Laboratories (Hercules, Calif.). N-Methyl-N-(tert-butyldimethylsilyl) triuoroacetamide (MTBSTFA) was purchased from Pierce Chemical Co. (Rockford, Ill.). Preparation of D-aspartate oxidase D-aspartate oxidase (D-AspO; EC 1.4.3.1) was obtained in puried form from beef kidney at the concentration of 20 U/ml (2 mg/ml), according the procedure of Negri et al. (18). The purity of the enzyme was determined by SDS gel electrophoresis (PAGE), and showed only one band. One enzymatic unit was dened as the amount of the enzyme capable of oxidizing 1 mol of D-Asp in 1 min at 37C in 1 ml of assay mixture containing D-Asp at the concentration of 10 mol/ml in 0.2 M Tris-HCl, pH 8.2 (the optimum pH value). The D-AspO, as obtained above, was able to oxidize only the amino acids NMDA, D-Asp, and D-glutamic acid (D-Glu) in the following ratios: 100% for NMDA, 90% for D-Asp, and 5% for D-Glu, respectively. Other D-amino acids, L-amino acids, and methylated amino acids in D- and L-form were not oxidized by this enzyme (18 20). Animals Wistar rats were purchased from Charles River Laboratories (Como, Italy) and housed, three per cage, in a controlled environment animal facility at 24C on a 12 h light/dark cycle (lights on from 07.00 to 19.00 h). The animals were fed standard laboratory food pellets and water ad libitum. Care of animals was in accordance with institutional guidelines. Rats were killed by decapitation. Preparation of amino acid solutions Each amino acid used in this study was prepared in distilled water at a concentration of 0.5 M, and the solution was adjusted to a pH of 7.4 with 0.1 M NaOH. However, since dicarboxylic amino acids (aspartic and glutamic acids and N-Methyl-D-aspartic acid) are insoluble in their acid form but are soluble as sodium salts, the sodium form of these amino acids was used to achieve complete solubilization as follows: 13.3 g of aspartic acid (D- or L-form), 14.7 g of glutamic acid (D- or L-form), or 14.7 g of N-methyl-aspartic acid (D- or L-form) was mixed in 100 ml distilled water with stirring, and 1 M NaOH solution was added dropwise to dissolve the amino acids and obtain a nal pH of 7.4. The nal volume was brought to 200 ml with distilled water. Sample purication To determine reliably the concentration of D-Asp and NMDA in tissue extracts, the samples were subjected to four steps of purication. Step 1: Homogenization As soon as rats were killed, adenohypophysis, hypothalamus, brain, liver, kidneys, testes, and blood were homogenized with 0.2 M trichloroacetic acid (TCA) in a proportion of 1:10. For each tissue, 100 mg from each animal were pooled, except for the adenohypophysis and the hypothalamus, which were accumulated from 10 animals. The homogenate was centrifuged at 30,000 g for 30 min. Blood was collected, left to clot at 37C for 30 min, and centrifuged at 5000 g for 30 min
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MATERIALS AND METHODS

Materials The following were purchased from Boehringer Mannheim (Germany): D-amino acid oxidase (D-AAO, EC 1.4.3.3) puried from hog kidney (15 U/mg, 5 mg/ml suspension in 3.2 M ammonium sulfate); malate dehydrogenase (MDH; EC 1.1.1.37) from pig heart mitochondria (1200 U/mg, 5 mg/ml suspension in 3.2 M ammonium sulfate solution); lactate dehydrogenase (LDH; EC 1.1.1.27) from pig muscle (550 U/mg, 10 mg/ml, suspension in ammonium sulfate solution); peroxidase (POD, EC 1.11.1.7) from horseradish (250 U/mg, 10 mg/ml suspension in 3.2 M ammonium sulfate solution); -nicotinamide adenine dinucleotide reduced form (-NADH); -nicotinamide adenine dinucleotide (NAD); EDTA; Pefabloc SC; leupeptin; aprotinin; chymostatin; bestain; PMSF; TPCK, and TLCK. The following were purchased from Sigma Chemical Co (St. Louis, Mo.): Daspartic acid (D-Asp), N-methyl-D-aspartic acid (NMDA), and all other D- and L-amino acids used in this work; bovine serum albumin (BSA), bacitracin, o-phthaldialdehyde (OPA), N-acetyl-L-cysteine (NAC), -mercaptoethanol, tyramine (4[2-aminoethyl] phenol; 4-hydroxyphenethylamine; tyrosamine hydrochloride), -ketoglutaric acid disodium salt (-ketoglutarate), D-2-amino-5-phosphonopentanoic acid (DAP5), Triton X-100, methylamine (CH3-NH2), Tris (Tris (hydroxymethyl aminomethane), and luteinizing hormonereleasing hormone (GnRH) antibody. The kits for radioimmunoassay (125I) determination of luteinizing hormone (LH), follicle stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and growth hormone (GH), as well as [2,3-3H]-D-aspartic acid (50 Ci/mmol), and [3H]methylNMDA (80 Ci/mmol), were purchased from Amersham International, Inc. (Buckinghamshire, U.K.). All solvents for high-performance liquid chromatography (HPLC) were reagent grade and purchased from Merck or C. Erba (Milano,
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to obtain serum. Two milliliters of serum were homogenized with 20 ml 0.2 M TCA. Step 2. Purication on cation exchange resin The supernatant so obtained was applied to a cation exchange 1 3 cm column, AG 50 W-X8 resin, H form, 100 200 mesh, 63150 m (Bio-Rad). The resin was previously regenerated by treatment with an excess of 5 M NaOH for 30 min, followed by an extensive wash with distilled water, treated with an excess of 5 M HCl for 30 min, and nally equilibrated with 0.01 M HCl. After the sample had been absorbed by the resin, the column was washed with 10 ml 0.01 M HCl, followed by 10 ml distilled water. These eluents were discarded. Then the column was eluted with 10 ml of 4 M NH4OH, and this last eluent was collected and evaporated in small petri dishes on a hot plate at 40 50C under a hood. Using this procedure, all amino acids and NMDA were recovered by over 90%, free of the TCA, salts, and organic compounds present in the tissues, and were without any racemization of aspartic acid or NMDA. After drying, the samples were dissolved in 1 ml of water and divided into two portions: one (50 l) was used to determine D-Asp; the other was further puried and used to determine NMDA as described below. Step 3: OPA treatment and C-18 cartridge purication 950 microliters of the sample obtained from cation exchange resin purication (Step 2) was mixed with 4 ml of distilled water and with 0.1 ml of 1 M of an OPA reagent prepared by dissolving 136 mg of o-phthaldialdehyde in 1 ml of methanol. The pH of the mixture was adjusted to 9.5 with 520 l of 1 M NaOH and incubated at 37C for 30 min. The mixture was brought to pH 2.5 with 10 100 l of 1 M HCl, cooled at 0C, and centrifuged at 30,000 g for 10 min. The supernatant (OPA complexed with the amino acids) was applied onto a C-18 cartridge (Sep-Pak Plus, Waters, Milford, Mass.) containing 820 mg of octadecyl silane (previously activated with 100% methanol and then equilibrated with distilled water). The eluent was collected and the cartridge was washed with 5 ml of 0.01 M HCl. This last eluent was combined with the previous eluent and evaporated in a petri dish on a hot plate at 40 50C (or using a rotoevaporator). The residue was dissolved in 100 l of distilled water. (During this procedure, all amino acids containing the primary amino group react with OPA reagent to form a strong irreversible complex, which is retained on the C-18 resin, whereas NMDA and other amino acids, which do not have a primary amino group, do not react with OPA and are eluted immediately from the C-18 cartridge.) Step 4. Thin-layer chromatography (TLC), cation exchange resin, and C-18 cartridge purication The sample obtained as described was subjected to further purication by TLC in order to obtain NMDA completely free of traces of D-Asp or D-Glu. The sample (100 l) was applied to a TLC cellulose plate (2020 cm, 0.5 mm thickness, PSC-Fertigplatten, art. 15275, Merck) using 20 l of the sample per linear centimeter of plate. The plate was developed in a solvent consisting of phenol-H2O (100 g phenol: 40 ml H2O), using an amount of solvent in the tank to reach 1 cm high, and left to run for 16 h. After migration, the plate was dried with a hair dryer. To identify the migration position of NMDA, a sample aliquot containing labeled N-[3H]NMDA was run in parallel on the same plate. After drying, the plate was subjected to electronic autoradiography acquisition (Cyclone, Packard, Canberra, Australia) for localization of labeled NMDA. The phenol-water solvent was chosen because

provides a good separation of D-Asp or L-Asp from NMDA (Rf value for D-Asp or L-Asp was 0.17, NMDA was 0.58) (Fig. 1). Cellulose in which NMDA had migrated was scraped off and mixed vigorously with 5 ml of 0.01 M HCl, then centrifuged for 30 min at 30,000 g; the supernatant was again puried on the cation exchange column as described in Step 2. The dried residues of each sample were dissolved in 1 ml of 0.01 M HCl and further puried on a C-18 cartridge in order to eliminate any pigment contaminants if still present (from the TLC cellulose or cation exchange resin), which could interfere in the enzymatic assay for NMDA or on gas chromatographymass spectrometry (GC-MS) assay. The sample was passed through a small C-18 cartridge (SepPak Light, containing 80 mg of C-18 resin) using a syringe, followed by 1 ml of distilled water. These eluents were combined and dried as above. The residue was dissolved in 100 l of distilled water and used for NMDA determination. Determination of D-Asp by enzymatic HPLC This method was based on the diastereomeric separation of D-Asp from L-Asp and other amino acids according to Aswad (21), modied as follows: 520 l of the sample obtained after purication on cation exchange resin (Step 2, sample purication) was mixed with 210 l of 0.1 M NaOH (to bring the pH to 9.0) and 0.01 M sodium borate buffer, pH 8.0, to obtain a nal volume of 100 l. Finally, 5 l of OPA-NAC reagent

Figure 1. Separation of D/L-Asp from NMDA and other amino acids on TLC cellulose. The gure represents a separation of a mixture of standard amino acids (20 l of the standard amino acid mixture in which each amino acid was at a concentration of 5 mol/ml) on TLC cellulose, 0.5 mm thickness. The solvent was phenol:H2O (100:40), run 16 h. The amino acids were visualized with a ninhydrin solution (1% in methanol) and autoradiography of labeled NMDA.

ROLE OF ENDOGENOUS D-Asp AND NMDA IN REGULATING LH AND GH SECRETION

701

(prepared by mixing 10 mg of OPA and 10 mg of NAC in 4 ml 50% methanol) was added; after 2 min, 20 l was injected onto a C-18 Supelcosil HPLC column (0.4525 cm, Supelco, Inc., Belafonte, Pa.) using the Beckman-Gold HPLC system. The column was eluted with a gradient consisting of solvent A (5% acetonitrile in 30 mM sodium acetate buffer, pH 5.5) and solvent B (70% acetonitrile in 30 mM sodium acetate buffer, pH 5.5) as follows: 0 20% B over 20 min, then to 100% B over 5 min, staying at 100% B for 2 min, and returning to 0% of B in 2 min at a ow rate of 1.2 ml/min. Amino acid derivatives were detected uorometrically using an excitation wavelength at 330 nm excitation and 450 nm emission. A standard curve was obtained under the same conditions using 5 l of a solution consisting of 17 L-amino acids, each at 0.1 mM, plus 0.02 mM D-Asp. It was observed that D-Asp eluted at 7.7 min, L-Asp at 8.5 min, followed by the other amino acids (Fig. 2). To determine the amount of area peak due to D-Asp, a parallel sample was incubated with 2 l of puried D-AspO for 15 min at 37C and chromatographed as above. The total disappearance or the reduction of area peak corresponding to D-Asp elution peak conrmed the presence of D-Asp and gave the exact amount of the content of D-Asp. A typical analysis is represented in Fig. 2. Using this method, we can determine reliably as little as 1 pmol of D-Asp in 20 l of the derivatized sample. NMDA determination Three methods were used in combination to determine NMDA.

Method 1: Enzymatic uorometric This method was used for screening of NMDA, based on the determination of H2O2 produced from the reaction between NMDA and D-AspO as follows: COOH P CH2 P CH-NH-CH3 P COOH NMDA COOH P CH2 P CAO P COOH -oxaloacetate
POD

O2 H2O

D-AspO

H2O2 CH3-NH2

methylamine

fluorescent biphenyl derivative H2O2 tyramine O The enzymatic procedure was performed according to the

Figure 2. Typical HPLC determination of D-Asp by the OPA-NAC method. Upper left panel: HPLC separation of a standard mixture of amino acids (100 pmol each amino acid and 20 pmol of D-Asp) derivatized with OPA-NAC and uorescence detection. Dashed line represents the HPLC gradient program. Arrow shows the elution of D-Asp. Lower left panel: the same sample as in the upper panel, but after treatment with D-AspO. The peak corresponding to D-Asp disappears because of oxidation by D-AspO. Upper right panel: analysis of a rat adenohypophysis extract. The peaks in the chromatogram correspond to D-Asp, L-Asp, and the other amino acids. Lower right panel: the same sample as in the upper right panel, but after treatment with D-AspO. The peak corresponding to D-Asp disappears.

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method described by DAniello et al. (19) and modied as follows. In three Eppendorf tubes (marked sample, sample plus internal standard, and blank sample) was added 20 l of the puried sample. To the tube marked sample plus internal standard was added 5 l of NMDA standard at the concentration of 0.02 mM (100 pmol). To each tube was added 20 l of POD-tyramine assay mixture prepared by mixing 20 mg of tyramine hydrochloride (Sigma) in 5 ml of 0.2 M Tris-HCl, pH 8.2, plus 20 l of POD (peroxidase 2000 U/ml). To the tubes marked sample and sample plus internal standard was added 2 l of puried D-AspO (2 mg/ml; 20 U/ml), and all tubes were incubated at 37C for 30 min. After incubation, 1 ml of 0.1 M Tris-HCl, pH 8.5, was added to each tube. The uorescence of the sample and sample plus internal standard was read against the blank sample using the excitation wavelength of 320 nm and emission wavelength 415 nm. To calculate the concentration of NMDA in the sample, the following formula was applied: pmole of NMDA in 30 l of sample As 100 A(S1) As

Method 3: GC-MS This method was based on the direct measurement of NMDA by GC-MS and used to conrm the occurrence of NMDA in neuroendocrine tissues. The analyses were performed with a Hewlett Packard gas chromatograph 5890 Series II Plus. The gas chromatograph was linked via a direct capillary column HP5-MS (cross-linked 5% PH Me siloxane) (30 m x 0.25 mm, 0.25 M lm thickness) to a Hewlett Packard 5989B mass spectrometer. Helium ow at 1 ml/min was used as a carrier gas. For this purpose, 30 l of the sample (puried as above) was dried and mixed with 5 l of decanoic acid (as internal standard) at a concentration of 1 ng/l, 5.8 pmol/l, in chloroform. The mixture was dried under nitrogen ow. Then 20 l of MTBSTFA used as derivatizing agent for carboxylic groups, was added and heated at 80C for 45 min in a sealed vial. Finally, 6 l of the tert-butyldimethylsilyl derivatized sample was injected into the GC-MS system with a split ratio 1:6. The programmed column temperature was 120 300C, 3C/min. Injector and detector were at 240C. A GC-EIMS (electron impact mass spectrum) of a standard consisting of derivatized decanoic acid and NMDA was registered in the 50 550 amu (m/z) range in order to determine the GC retention time (R.T.) and fragmentation of these derivatized substances. Under these conditions, R.T. for the derivatized decanoic acid and NMDA was 17.89 and 23.86 min, respectively. Since previous analyses have revealed that NMDA levels present in the tissues were undetectable using the above scan conditions, the more sensitive single ion monitoring (SIM) technique was adopted. Direct introduction EIMS spectra of decanoic acid and NMDA standards were run (data not shown), and fragments for the SIM technique were chosen. The SIM fragments used for decanoic acid were m/z 229, 230, 231, obtained from 286, 287, 288 (which are MW, MW1, MW2) 57, which is the tert-butyl radical fragment, -C(CH3)3, from the derivatizing group (Fig. 4, CL) and for derivatized NMDA, m/z 318, 319, 320 obtained from 375, 376, 377 (MW, MW1, MW2) 57 (Fig. 4, DL). These SIM fragments were chosen because the 57 fragment is characteristic and a well-known radical fragment of each derivatized compound with NTBSTFA. Naturally, the retention times of the GC peaks for decanoic acid and NMDA obtained in the SIM run are the same as those obtained in the Scan run (Fig. 4, AL). Since the GC-MS technique does not distinguish between NMDA and NMLA (N-methyl-L-aspartate), each sample was also analyzed after treatment with D-AspO. Fifteen microliters of the sample was mixed with 15 l of 0.02 M borate buffer (pH 8.2) and 1 l of D-AspO. The mixture was incubated for 20 min at 37C, then ltered on a membrane with a cutoff of 30 kDa (microcon lter, Amicon) in order to separate the D-AspO from the smaller molecules. The ltrate was mixed with 5 l of decanoic acid, dried, derivatized, and analyzed by GC-MS under the same conditions as above. Since the DAspO oxidizes NMDA and not NMLA (18, 2122), the difference in abundances of the peak between before and after incubation with D-AspO indicated the amount of NMDA present in the standard (Fig. 4, BL) and the samples (Fig. 4, BR). To verify that the reduction of the NMDA peak was actually due to the action of D-AspO and not due to sample injection errors, the internal standard decanoic acid was used as a general reference point. Determination of L-amino acids and other D-amino acids The determination of each L-amino acid was performed according the method of Godel et al. (23), and determination of other D-amino acids was performed using the method of Okuma and Abe (24).
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where AS absorbance of the sample; A(S I) absorbance of the sample plus the internal standard. Method 2: Enzymatic HPLC This method is based on the measurement of the CH3-NH2 (methylamine), which is generated from the reaction between NMDA and D-AspO as shown by the reaction: NMDA O2 H2O O -oxaloacetate H2O2 CH3-NH2 The CH3-NH2 produced was determined quantitatively by HPLC after its reaction with OPA-mercaptoethanol. In two 200 l Eppendorf tubes (marked sample and blank sample) was placed 20 l of sample as puried above and 20 l of 0.1 M borate buffer, pH 8.2. Then 1 l of puried D-AspO (2 mg/ml; 20 U/ml) was added to the sample and both tubes were incubated at 37C for 20 min. The blank sample tube was then kept at 0C. Four microliters of OPA-mercaptoethanol reagent (consisting of 10 mg OPA in 1 ml methanol and 1 ml of 0.2 M borate buffer pH 9.5 and 20 l of -mercaptoethanol) was added to the sample and mixed. After 2 min (needed to obtain complete derivatization of the amino acids), 20 l of derivatized sample was injected onto a C-18 Supelcosil HPLC column (0.4525 cm, Supelco, Inc.) using the Beckman-Gold HPLC system. The column was eluted with a gradient consisting of solvent A (10% acetonitrile in 30 mM sodium acetate buffer, pH 5.5) and solvent B (70% acetonitrile in 30 mM sodium acetate buffer, pH 5.5) using the following gradient program: 040% B over 15 min; to 100% B in 4 min, staying at 100% B for 3 min and back 0% B in 1 min, at a ow rate of 1.2 ml/min. CH3-NH2 was detected uorometrically at an excitation wavelength of 330 nm and an emission wavelength of 450 nm. The CH3-NH2 eluted as a sharp peak at the retention time of 21.2 min, well separated from the other amino acids (Fig. 3). After running the sample, 5 l of OPA-mercaptoethanol was added to the blank sample and chromatographed as with the sample. The difference of the peak areas obtained between the sample and the blank sample gave the net amount of the area due to the methylamine generated by the action of the D-AspO. To quantify the concentration of NMDA in the sample, a standard curve of different concentrations of NMDA (range 0.11.0 nmol/ml) was performed under the same assay conditions as the samples. Using this method, it was possible to determine reliably NMDA in amounts as small as 1 pmol.
D-AspO

ROLE OF ENDOGENOUS D-Asp AND NMDA IN REGULATING LH AND GH SECRETION

Figure 3. Typical HPLC determination of methylamine (CH3-NH2) coming from the oxidation of NMDA with D-AspO. Upper left panel: HPLC separation of a standard mixture of amino acids (100 pmol each amino acid and 50 pmol of NMDA) derivatized with OPA-mercaptoethanol and uorescence detection. Dashed line represents the HPLC gradient program. Note that NMDA is not seen because NMDA does not react with OPA. Middle left panel: same sample of standard amino acids as in left top panel was incubated with D-AspO before HPLC analysis (see Materials and Methods). A new peak, corresponding to methylamine coming from the oxidation of NMDA appears at 21.2 min. Lower left panel: same sample of standard amino acids as in left top panel, to which 100 pmol of authentic methylamine was added and incubated with D-AspO before HPLC analysis. Upper right panel: analysis of rat hypothalamus extract after purication (cation exchange resin, OPA treatment and TLC separation). The peaks observed are traces of amino acids still present in the sample after purication (each peak represents 0.51% of the each amino acid originally present in the tissue extract). Middle right panel: same sample as upper panel, but after treatment with D-AspO, showing the peak of methylamine coming from the D-AspO oxidation of NMDA. Biosynthesis of NMDA: in vivo and in vitro studies Since D-Asp has been well documented to be an endogenous molecule (117) and involved in neuroendocrine activity (13, 16 17), and NMDA is the methylated form of D-Asp, we hypothesized that there exists a biosynthesis for NMDA. To validate this hypothesis, in vivo and in vitro experiments were performed. The in vivo experiments consisted of injecting intraperitoneally (i.p.) into a rat a solution of 0.5 M D-Asp at a dose to obtain 2 mol/g body weight of animal. One hour after injection, the rat was killed and tissues were processed for purication and determination of NMDA, as described above. The in vitro experiments consisted of incubating at 37C for 60 min with shaking a homogenate of 200 mg of tissue with 1 ml of phosphate buffer saline (PBS) solution containing 10 mg/ml of BSA, 20 mM sodium D-aspartate, 10 mM sodium EDTA (used as metalloprotease inhibitor), 20 mM sodium and potassium tartrate (used as inhibitor for mammalian D-AspO), and 5 mM of S-adenosyl-L-methionine (AdoMet). After incubation, 1 ml of 1 M TCA was added to
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the assay mixture and centrifuged at 30,000 g. The supernatant was subjected to the purication of NMDA as described above and analyzed for NMDA. Parallel experiments were also performed in presence of S-adenosyl-L-homocysteine (AdoHcy) at the concentration of 10 mM, as an inhibitor for the methyltransferase. Effects of D-Asp on LH and GH release: in vivo and in vitro studies To study the effects of D-Asp on LH and GH release, in vivo and in vitro experiments were performed. The in vivo experiments consisted of i.p. injection of a solution of 0.5 M sodium D-aspartate, pH 7.4, into 85-day-old male rats using an appropriate volume to inject 2.0 mol/g body weight. After 1 h and 5 h, the animals were killed by decapitation. Blood was collected, incubated at 37C for 30 min, and then centrifuged for 30 min at 3000 g. Serum was separated from the red cells and used for hormonal analyses. Solid tissues were removed, homogenized, and puried as described above, then used to
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mixture also contained 0.1 mM D-AP5, used as an antagonist of NMDA receptors. Effects of D-Asp on GnRH synthesis and release This experiment was undertaken to ascertain whether D-Asp induced the release and the synthesis of GnRH from isolated hypothalamus. Hypothalami collected from four adult male rats were each cut into four portions and incubated at 37C for 2 h under gentle shaking in 2 ml of a medium consisting of PBS containing 5 mg/ml of BSA, 10 mM Na-EDTA, 20 mM sodium and potassium tartrate, and 2 mM D-aspartic acid. After incubation, the medium was centrifuged at 1000 g, and the supernatant was homogenized with 8 ml of 100% methanol and centrifuged at 30,000 g. The supernatant was dried by using a rotoevaporator at 2530C. The residue was dissolved in 1 ml of distilled water and passed through a C-18 Sep-Pak cartridge (C-18 of 1 g size, Waters). After the sample had been absorbed, the cartridge was washed with 5 ml of 15% methanol. This eluent was discarded. The cartridge was then eluted with 5 ml of 70% methanol; this last eluent (which contained the GnRH) was dried as described above. The residue was dissolved in 100 l of 0.1 M phosphate buffer and used to determine GnRH by HPLC analyses. For HPLC analyses, 50 l of the sample was injected onto a C-18 supelcosil HPLC column (0.4525 cm, Supelco, Inc.) using a Beckman-Gold HPLC system. The column was eluted with a gradient of solvent A consisting of 5% acetonitrile in 30 mM sodium acetate buffer, pH 5.5, and solvent B consisting of 70% acetonitrile in 30 mM sodium acetate buffer, pH 5.5. The gradient program consisted of 0 50% B over 22 min; then to 100% B over 3 min, staying at 100% B for 2 min, and nally back 0% B over 1 min, at a ow rate of 1.2 ml/min. The GnRH was detected using a wavelength of 215 nm. A standard curve was performed under the same conditions by injecting 50 l of synthetic GnRH (Sigma) at concentrations between 10 and 100 pg/ml. The peak of GnRH eluted at the retention time 16.8 min (Fig. 6). To verify that the peak attributed by us as GnRH in the sample was really this compound, the remaining 50 l of the sample were treated with 20 l of a solution of the antibody against mammalian GnRH and incubated overnight at 4C. The GnRHantibody complex was then centrifuged on a molecular weight lter with a cutoff of 30,000 (microcon lter, Waters) and the ltrate was injected on the HPLC. In this last case the GnRH-antibody complex does not pass through the lter because of its high molecular weight. Consequently, the peak previously observed at the elution time corresponding to the GnRH disappears (Fig. 6). Radioimmunoassays Concentrations of LH, GH, and TSH in serum and in the medium from the in vitro experiments, as well as in the pituitary homogenate, were determined by double-antibody autoimmunoassay methods using the rat reagent kits purchased from Amersham Life Science (Buckinghamshire, U.K.) with magnetic separation, according to the suggested assay procedures. The assay system uses a high specic activity [125I] tracer, together with a highly specic and sensitive antiserum. The sensitivity of the assay was in the range 0.08 to 5.0 ng per tube. The serum samples, derived from the in vivo experiments, were examined undiluted and after 1:2, 1:4, 1:8, and 1:16 dilution with PBS. Tissue incubation media from in vitro experiments of the indicated periods were diluted with PBS 1:1000, 1:10,000, and 1:100,000 and then used for the hormonal analyses. Serum testosterone, progesterone, 17estradiol, androstenedione, 17-hydroxyprogesterone, corti705

Figure 4. GC-MS analyses of NMDA. Left panels (standards): AL shows the gas chromatographic prole of the derivatized internal standard decanoic acid (1.16 pmol; R.T.17.89 min) and NMDA (27.7 pmol; R.T.23.86 min). BL is the same standard mixture as AL, but after treatment with D-AspO (note the disappearance of the peak at R.T. 23.86 corresponding to NMDA). CL and DL show the SIM mass spectrum of the derivatized standard decanoic acid peak at R.T. 17.89 (m/z 229, 230, 231) and the NMDA peak at R.T. 23.86 (m/z 318, 319, 320). Right panels (tissue sample): analyses of a puried sample from rat hypothalamus plus decanoic acid under the conditions depicted in the left panel. AR shows the gas chromatographic prole of decanoic acid and other peaks from the tissue extract (the peak corresponding to NMDA is well shown). BR is the same sample after the treatment with D-AspO, showing the disappearance of the NMDA peak. CR and DR show the SIM mass spectrum of the GC peak of the internal standard (decanoic acid) and of the peak corresponding to NMDA, respectively. determine D-Asp and NMDA. Parallel experiments were also conducted using other D- and L- amino acids instead of D-Asp. In vitro experiments were conducted: as soon as the animal was killed, the pituitary gland was separated from the neurohypophysis and cut into four portions by making longitudinal and vertical cuts. The four specimens were incubated at 37C under gentle shaking in 2 ml of a medium consisting of PBS containing 5 mg/ml BSA and D-Asp at concentrations from 0.2 to 2 mM. After 1 h, the medium was diluted with PBS 1:10; 1:100; 1:1000, and 1:10000, and these solutions were used for RIA determination of LH and GH. Other experiments were conducted under these same conditions in which the assay

ROLE OF ENDOGENOUS D-Asp AND NMDA IN REGULATING LH AND GH SECRETION

sol, 3,5,3-triiodothyronine (T3), and 3,5,3,5-tetraiodothyronine (T4) were also assayed by radioimmunoassay using the reagent kits for human blood purchased from Biochemical Immunosystem Company (Milan, Italy). Statistical analysis The results described in the text are expressed as the mean sd. Statistical analyses were performed using the SPSS software package (v 8.0), running on a Pentium II computer equipped with Windows 95 operating system.

RESULTS Endogenous occurrence of D-Asp in rat tissues and its accumulation in response to acute treatment Using a specic and sensitive HPLC method to determine D-Asp based on the diastereomeric separation of D-Asp from the other amino acids (Fig. 2), associated with the use of D-aspartate oxidase, we have determined the endogenous occurrence of D-Asp in the neuroendocrine and other tissues of rat. The results obtained conrmed that rat tissues possess D-Asp, as we and others had previously reported (6 14), and that among various tissues this amino acid is concentrated mostly in the endocrine glands: adenohypophysis, testes, and adrenal, where its concentration corresponds to a mean value of 114 18, 90 14, and 78 8 nmol/g tissue, respectively (Table 1). The nervous tissues possess an amount of D-Asp four- to vefold lower than endocrine glands (between 1526 nmol/g) except for the hypothalamus, where this amino acid reaches a

mean value of 53 9 nmol/g tissue. The other tissues (liver, kidney, muscle, and blood) show very low amounts of D-Asp (2 8 nmol/g tissue). When exogenous D-Asp was administered to rats via i.p. injection (2.0 mol/g body weight), it accumulated predominantly in the endocrine glands and especially in the adenohypophysis. One hour after injection of D-Asp, the adenohypophysis accumulated 3200 nmol/g tissue (28-fold higher than the basal value); 5 h after treatment, this gland had accumulated 5250 nmol (46-fold higher than the basal value) (Table 1). The testes and adrenal also presented this phenomenon, but to a much lower extent than the adenohypophysis. D-Asp accumulation in the brain was low compared to the adenohypophysis, because the brain barrier actively prevents a large amount of D-Asp from entering (25). However, it is interesting to observe that the hypothalamus is a brain region capable of binding exogenous D-Asp (13424 nmol/g tissue 1 h after D-Asp injection and 19539 nmol/g after 5 h) (Table 1). These last data are of particular interest in that the hypothalamus and the adenohypophysis are directly connected in the control of the pituitary hormone secretion. The results also showed that 24 h after the injection of D-Asp, the adenohypophysis still strongly bound D-aspartate at an elevated concentration (1500250 nmol/g tissue), whereas in other tissues the D-Asp level decreased to near basal values. Other D-amino acids investigated (D-Ala, D-Glu, and DMet) were not signicantly taken up by the adenohypophysis or other neuroendocrine tissues (data not shown), indicating that D-Asp is the only D-

TABLE 1. Endogenous occurrence of free D-Asp in rat tissues and its accumulation in response to acute treatment
D-Asp accumulation after i.p. injectiona (nmol/g tissue) Time post treatment 1h 5h 24 h

Endogenous occurrence of D-Asp in rat tissues (nmol/g tissue)

Endocrine glands Adenohypophysis Testes Adrenal Brain Hypothalamus Hippocampus Cerebellum Frontal lobe Parietal lobe Occipital lobe Other tissues Blood Liver Kidney Hind leg muscle

114 18 90 14 78 8 53 9 26 5 18 4 15 3 18 4 21 5 21 74 86 32

3200 460 210 65 220 58 134 24 60 10 62 9 54 8 45 6 44 7 252 48 224 44 212 38 15 5

5250 690 370 85 345 67 195 39 76 18 70 15 64 12 58 11 63 14 80 15 90 21 120 32 14 4

1500 250 120 24 91 25 60 12 35 8 28 6 18 4 17 4 20 3 73 94 12 3 42

a This consisted of an i.p. injection of sodium D-aspartate solution, 0.5 M, pH 7.4, using an appropriate volume to administer 2.0 mol/g body weight. The rats were injected at about 10 a.m. and killed after the times indicated. Data were obtained using the specic HPLC method for determination of D-Asp (see Materials and Methods). The results represent the mean sd, obtained from 5 adult male rats.

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amino acid that is actively taken up by the adenohypophysis. Effects of D-Asp on hormone release: in vivo and in vitro studies Since D-Asp occurs at high concentrations in the adenohypophysis as an endogenous natural compound; since this gland responds positively by accumulating this amino acid, we hypothesized it could be involved in the endocrine activity. To verify this, rats were injected with a solution of 0.5 M D-Asp (i.p. injection) to obtain a concentration of 2 mol/g body weight. After 1 and 5 h, the levels of some adenohypophysial and gonadal hormones were measured in the blood. The results indicated that GH, LH, and the gonadal hormones testosterone and progesterone were signicantly increased in the blood in response to D-Asp injection (Table 2). One hour after the injection of D-Asp, GH concentration in plasma increased 1.96-fold over basal levels (P0.01), and after 5 h reached 2.6-fold the basal level (P0.01) (Table 2). Similarly, LH increased signicantly (2.1- and 2.5-fold over basal level 1 and 5 h after injection, respectively) (Table 2). Testosterone and progesterone also increased in the blood, but only 5 h after D-Asp treatment was this increase signicant. Testosterone rose 3.36-fold higher than the basal serum levels (P0.01), whereas progesterone increased 2.72-fold (P0.01). Other hormones17 -estradiol, androstenedione, 17 hydroxyprogesterone, cortisol, thyroid-stimulating hormone (TSH), 3,5,3-Triiodothyronine (T3), and 3,5,3,5-tetraiodothyronine (T4,)were not affected by D-Asp injection (Table 2). The free amino acids L-Asp, D- and L-Glu, D-, and L-Ala, and D- and

L-Met were tested under the same conditions as D-Asp; they did not induce any signicant increase of the above mentioned hormones (results not shown). To verify whether D-Asp has a direct effect on the adenohypophysis, experiments in vitro were performed on this isolated gland (see Materials and Methods). These experiments indicate that D-Asp is able to increase the in vitro release of GH, but not of LH (data not shown); the results show that D-Asp is not the direct effector of LH release in vivo, but D-Asp could be the precursor of another molecule, which in turn is directly involved in LH release. Endogenous occurrence of NMDA It has been reported that NMDA stimulates the release of hypothalamus and adenohypophysis hormones (26 50). Since NMDA is a molecule biochemically very similar to D-Asp, which is the methylated form of D-Asp, we hypothesized that NMDA could be present in neuroendocrine tissues as an endogenous compound and D-Asp as its natural precursor. Previous attempts to determine this amino acid in rat tissues indicated that NMDA was present in very low amounts, and it was necessary to accurately purify the sample by cation exchange resin, OPA treatment, purication on C-18 cartridge, and TLC (Fig. 1) in order to detect the small amount of NMDA without interference by other compounds present in the sample. To determine the NMDA concentration, we set up an enzymatic uorometric method based on the determination of the hydrogen peroxide generated from the reaction between NMDA and D-AspO, and an enzymatic HPLC method based on determination of methylamine generated by the oxidation of NMDA by D-AspO (Fig. 3). We found that with both

TABLE 2. Effects of D-Asp on hormone release in the blood of adult male rats
Hormone levels after i.p. D-Asp injectiona (ng/ml serum) Control (ip injection 2 mM NaCl) Time post treatment 1h 5h

Growth hormone (GH) Luteinizing hormone (LH) Testosterone Progesterone 17-Estradiol 17-Hydroxyprogesterone Androstenedione Cortisol Thyroid-stimulating hormone (TSH) 3,5,3-Triiodothyronine (T3) 3,5,3,5-Tetraiodothyronine (T4)

31.6 6.5 3.7 0.6 5.5 1.3 10.8 3.3 2.5 0.5 20.1 5.2 1.0 0.2 7.0 2.3 6.6 1.9 1.0 0.2 50.5 9.4

61.6 9.6* 7.8 1.1* 7.5 2.6 14.5 6.2 2.7 0.6 20.9 5.2 1.2 0.4 7.3 1.1 6.4 2.9 1.1 0.3 61.3 10.5

82.5 17.8* 9.1 1.3* 18.5 5.4* 29.4 6.7* 2.8 0.7 22.3 4.8 1.4 0.3 7.8 1.2 8.5 3.5 1.4 0.5 62.4 11.5

a The experiment consisted of an i.p. injection of sodium D-aspartate solution, 0.5 M, pH 7.4, using an appropriate volume to administer 2.0 mol/g body weight; after 1 and 5 h, the blood was taken from animal and analyzed for hormone release. The results represent the mean sd as ng/ml serum as obtained from 5 adult male rats (85 days old). Statistical analyses were performed using the unpaired Students t test; *P 0.01 compared with control (NaCl 2 mol/g body weight animal).

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methods, the highest concentration of endogenous NMDA occurred in the adenohypophysis (6.81.1 and 6.90.8 nmol/g tissue by the enzymatic uorometric and HPLC methods, respectively), followed by the hypothalamus (4.40.6 and 4.30.4 nmol/g tissue), testis (3.50.5 and 3.40.4 nmol/g tissue) and frontal cortex (1.50.3 and 1.40.3 nmol/g tissue) (Table 3). In the kidney, muscle, and serum, NMDA was either very low or almost undetectable. To further validate the results obtained by these two methods, analysis was conducted on the same samples by GC-MS. Results obtained by this technique conrmed that NMDA is present in the same tissues tested above (Table 3). In this case, since the GC-MS technique does not provide the opportunity to quantify the exact amount of NMDA, we have only reported the presence or the absence of this amino acid as indicated by the symbol or (Table 3). Figure 4 shows typical examples of the GC-EIMS analyses conducted on a standard of NMDA plus a standard of decanoic acid (Fig. 4, AL, CL, DL) and on a sample of rat hypothalamus plus standard decanoic acid (Fig. 4, AR, CR, DR). A GC peak at R.T. of 23.86 min, corresponding to NMDA, was observed in both the standard and the tissue samples. In each case, this GC peak shows the same SIM abundance at m/z 318, 319, 320, characteristic of NMDA. In addition, when the standard and/or sample was treated with D-AspO and subjected to GC-MS analysis, the GC peak at 23.86 min (corresponding to NMDA) disappeared or was signicantly reduced (Fig. 4, BL, BR). Biosynthesis of NMDA: in vivo and in vitro studies When D-Asp was acutely administrated to adult male rats (i.p. 2 mol/g body weight), a signicant increase of NMDA in the endocrine glands and brain was found 60 min after D-Asp injection (Fig. 5).
TABLE 3. Endogenous occurrence of NMDA in rat tissuesa
Enzymatic uorometric method

Figure 5. In vivo biosynthesis of NMDA. The experiment consisted of an i.p. injection of sodium D-aspartate solution (0.5 M, pH 7.4) to adult male rat (85 days old) using an appropriate volume to administer 2.0 mol/g body weight. After 1 h tissues were taken from the animals, processed as detailed under Materials and Methods, and subjected to HPLC analysis for NMDA through the determination of methylamine. The values represent the mean sd obtained from 6 determinations. *P0.01 **P0.05.

Enzymatic HPLC method

GC-MS method

nmol/g tissue Adenohypophysis Hypothalamus Testis Brain (frontal cortex) Liver Kidney Muscle Blood 6.8 1.1 4.4 0.6 3.5 0.5 1.5 0.3 0.2 0.1 0.1 0.1 0.1 6.9 0.8 4.3 0.4 3.4 0.4 1.4 0.3 0.2 0.1 0.1 0.1 0.1

a The results are the mean sd obtained from the tissues of 5 adult male rats (85 days old) on sample puried by cation exchange resin, OPA-mercaptoethanol, and TLC according the procedure described in Materials and Methods. () NMDA was well detected by GC-MS. () NMDA was slightly detected. () not detected.

Compared to other tissues, the hypothalamus registered the largest increase in NMDA, corresponding to 11.2-fold over the basal value (48.25.8 nmol/g vs. 4.30.8 nmol/g basal level; P0.01). In the adenohypophysis, the increase was 2.6-fold (15.42.2 nmol/g vs. the 5.91.5 nmol/g basal value; P0.01), in the brain 2.6-fold (3.40.8 nmol/g vs. the 1.30.4 nmol/g basal value; P0.05), and in the testis the increase was 2.5-fold (8.51.9 nmol/g vs. the 3.40.8 nmol/g the basal value; P0.05) (Fig. 5). These data as a whole led us to hypothesize that the NMDA increase in these tissues was due to the transformation of D-Asp into NMDA, supporting the hypothesis that an enzyme capable of synthesizing NMDA from D-Asp could exist. Therefore, we conducted in vitro experiments in which rat tissue homogenates were incubated with D-Asp and S-adenosyl-L-methionine (AdoMet, the universal methyl donor in transmethylation reactions), and NMDA in the medium was determined. The results show that a synthesis of NMDA occurs, with the highest NMDA production observed in the hypothalamus (31.45.9 nmol/ml assay mixture) followed by brain (17.33.3), adenohypophysis (15.33.5), and liver (13.83.4) (Table 4). Both D-Asp and AdoMet were necessary to obtain NMDA synthesis. In fact, if the assay mixture contained only D-Asp, the amount of NMDA synthesized was very low (30- to 40-fold lower) compared with previous conditions. In addition, incubation with only AdoMet or without D-Asp showed no NMDA biosynthesis. The specicity of this reaction was conrmed by the addition of the transmethylation inhibitor S-AdoHcy to the incubation medium. In this case, addition of AdoHcy to the assay mixture
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TABLE 4. In vitro biosynthesis of NMDA from rat tissues homogenatea

Amount of NMDA (nmol/ml assay mixture) produced in presence of D-Asp AdoMet Only AdoMet Without D-Asp and AdoMet D-Asp AdoMet AdoHcy

Only D-Asp

Hypothalamus Brain (frontal cortex) Adenohypophysis Liver Kidney

31.4 5.9 17.3 3.2 15.3 3.5 13.8 3.4 0.1

0.9 0.3 0.8 0.3 0.7 0.3 0.5 0.2 0.1

0.1 0.1 0.1 0.1 0.1

0.1 0.1 0.1 0.1 0.1

5.8 1.5 3.5 1.1 3.2 0.9 2.3 0.5 0.1

a After the experiment, the sample was puried as described in Materials and Methods and synthesized NMDA was determined by HPLC. The results are the mean sd obtained from 3 individual experiments. AdoMet S-adenosyl-L-methionine; AdoHcy S-adenosyl-Lhomocysteine.

caused a strong inhibition of NMDA synthesis (Table 4). To verify that the NMDA found really came from the biosynthesis and was not a contaminant in the D-Asp used, the D-Asp was analyzed for the presence of NMDA; no NMDA was found in the D-Asp. Molecular mechanism by which D-Asp and NMDA affect GH and LH release Previous in vitro experiments indicated that D-Asp alone elicits GH release but not LH. On the other hand, since in the in vivo experiments we observed that LH was released in response to D-Asp injection, we deduced that D-Asp could play a role in promoting the synthesis of another compound (i.e., NMDA), which is the molecule responsible for LH release through GnRH. Therefore, we tested the action of D-Asp and NMDA, individually or together, on the isolated adenohypophysis or adenohypophysis plus hypothalamus in hormone release. Results indicated that using 1 mM D-Asp (the concentration at which D-Asp exhibits the maximum activity in inducing hormone release), GH rose 2.61-fold compared to the control (82.59.8 ng/ml medium vs. 31.65.5 ng/ml, P0.01) (Table 5). LH release also was found to increase, but not

signicantly. However, using NMDA alone (0.1 mM), LH release becomes signicant over basal levels (6.81.2 ng/ml medium vs. 3.70.6 ng/ml of the control). In addition, when D-Asp and NMDA were used together, a more signicant increase in the release of LH was observed compared to the action of NMDA alone (8.81.9 ng/ml medium vs. 3.70.6 ng/ml of its control). However, when the adenohypophysis was coincubated together with the hypothalamus and D-Asp, a more signicant increase of LH was registered (14.53.5 ng/ml medium vs. 8.42.3 ng/ml of its control) (Table 5). In addition, when D-Asp is incubated with the adenohypophysis and the hypothalamus together with NMDA, then LH release in the medium was further increased (25.44.2 ng/ml medium). Our interpretation of these results is that D-Asp is converted to NMDA (see Table 4), which in turn induces release of the GnRH from the hypothalamus, as previously inferred (see refs 34, 37, 47, 50). GnRH then induces the release of LH from the pituitary gland. This interpretation is further conrmed by the fact that when the hypothalamus is incubated with D-Asp, both NMDA and GnRH are increased (Table 4; Fig. 6). To know whether the action of these two amino acids is mediated by the NMDA receptors, additional

TABLE 5. In vitro effects of D-Asp and NMDA on GH and LH release from isolated adenohypophysis and adenohypophysis plus the hypothalamus of rata
Adenohypophysis GH Incubation medium (ng/ml medium) LH Adenohypophysis hypothalamus GH (ng/ml medium) LH

Control (1 mM NaCl) D-Asp 1 mM NMDA 0.1 mM D-Asp 1 mM NMDA 0.1 mM D-Asp 1 mM D-AP5 0.1 mM NMDA 0.1 mM D-AP5 0.1 mM D-Asp 1 mM NMDA 0.1 mM D-AP5 0.1 mM

31.6 5.5 82.5 9.8* 90.3 10.2* 110.3 14.6* 42.3 6.1 42.4 9.5 50.3 7.5

3.7 0.6 4.3 1.0 6.8 1.2* 8.8 1.9* 4.0 0.9 4.0 0.7 4.1 0.6

80.2 11.4* 120.2 20.3* 150.4 20.4* 189.6 22.5* 88.2 13.7 110.5 15.7 90.3 10.5

8.4 2.1* 14.5 3.5* 25.4 4.2* 25.3 4.7* 9.2 2.2 11.5 2.2 12.3 2.4

a The experiment consisted of incubating the adenohypophysis alone or together with the hypothalamus in 2 ml of the medium indicated at 37C for 60 min under moderate agitation. Hormone concentrations in the medium were measured using the RIA method. Values represent the mean sd from 5 experiments. Statistical analyses were performed using the Students unpaired t test. *P 0.01 vs. control (1 mM NaCl). P 0.01 vs. its control (the respective same components, but without D-AP5).

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Figure 6. Effect of D-Asp on the release of gonadotropin-releasing hormone (GnRH). A) HPLC analysis of standard mammalian GnRH (10 pmol). B) Same sample as in panel A, but after treatment with mammalian GnRH antibody. C) Medium of rat hypothalamus incubated with D-Asp (2 mM). D) Same sample as panel C, but after treatment with the mammalian GnRH antibody.

experiments were conducted using D-AP5, a specic NMDA receptor antagonist. The results indicate that D-AP5 at a minimal concentration of 0.1 mM produced a signicant inhibition in the release of the abovementioned hormones (Table 5), indicating that the actions of D-Asp and NMDA are mediated by the NMDA receptors. Effects of D-Asp on the release of gonadotropinreleasing hormone The experiments so far described showed that D-Asp represents the precursor for NMDA synthesis. In addition, NMDA elicits release of LH (26 35, 3739, 41 43, 4550) and GH (36, 39, 41, 44 49) in male rats, as previously inferred by other authors. It appears that D-Asp and/or NMDA are involved in pituitary LH release through hypothalamic GnRH. To verify this hypothesis, we tested the effects of D-Asp on the hypothalamus in the in vitro release of GnRH. Results showed a signicant increase of GnRH (Fig. 6). In fact, a signicant amount of GnRH was detected in response to D-Asp treatment (Fig. 6C, peak at retention time 16.8 min). In the hypothalamus incubated with NaCl instead of D-Asp, this peak was almost undetectable (data not shown). To unequivocally identify GnRH, the sample was treated with the antibody anti mGnRH. With the sample (Fig. 6D) and in the case of standard GnRH (Fig. 6B), the peak corresponding to elution time of GnRH disappeared.
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Relationship between the endogenous occurrence of D-Asp in rat adenohypophysis and GH and LH release and synthesis during the day-night circadian cycle It is widely recognized that rats and many other animals show a predominantly nocturnal activity (51). We, therefore, investigated the possibility that D-Asp in the rat pituitary gland varied during the day-night circadian cycle and examined whether a relationship exists between the endogenous occurrence of D-Asp and GH release. Six adult male rats (85 days old) were killed at 10 a.m. and six adult male rats of the same age were killed at midnight. Blood and pituitaries were collected and treated as above for D-Asp, GH, and LH determination. Hormone release was measured directly in the blood. The results are shown in Fig. 7. A signicant difference in D-Asp content in the blood between morning and nocturnal levels is present. During the night, D-Asp concentration in the adenohypophysis was found to be increased nearly twofold compared to that at 10 a.m. (22030 nmol/g adenohypophysis in the night vs. 11415 nmol/g during the morning; P0.01). These events were paralleled by a signicant increase in blood of GH and LH concentrations. In fact, as shown in Fig. 7, blood GH increased 1.76-fold (6012 ng/ml serum at night vs. 345 ng/ml at 10 a.m., P0.01); LH increased 1.75-fold (5.60.8 ng/ml of serum at night vs. 3.20.4 ng/ml at 10 a.m., P0.01) (Fig. 7).
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Figure 7. Relationship between adenohypophysis D-Asp occurrence and GH and LH release during day-night circadian cycle. The results represent mean sd obtained from 6 adult male rats (85 days old). The determination of the hormone release was performed on serum. *P 0.01 compared with rats killed at 10 a.m.

DISCUSSION Here we report the occurrence of endogenous D-Asp and NMDA in the nervous system and endocrine glands of the rat and provide the rst evidence for their neuroendocrine role. D-Asp occurs at levels of 1526 nmol/g wet tissue in the brain with the exception of the hypothalamus, where D-Asp concentration is higher with respect to other brain regions (539 nmol/g wet tissue). In the endocrine glands, D-Asp occurs at a much higher concentration than in the brain. In other tissues, D-Asp is found at very low levels compared to the above tissues. The adenohypophysis is the gland in which this amino acid is present at the highest concentration (11418 nmol/g wet tissue). In addition, this gland possesses the capacity to take up and accumulate exogenously administered D-Asp (Table 1). The occurrence of D-Asp in the endocrine tissue led us to propose that this amino acid could be endowed with a neuroendocrine role. We investigated the in vivo effects of D-Asp on the release of some hypophysial hormones in the rat. The results show a signicant rise in the plasma levels of GH and LH between 1 and 5 h after i.p. injection of D-Asp, and of testosterone and progesterone after 5 h (Table 2). Since the release of testosterone and progesterone occurs only after 5 h, it is reasonable to hypothesize that their release is stimulated by the LH increase. To identify the mechanism(s) by which D-Asp elicits hormone release, we were intrigued by the structural similarities between D-Asp and its methylated derivative NMDA, hypothesizing that the latter excitatory amino acid could be present in neuroendocrine animal tissues arising from the endogenous D-Asp. This hypothesis was suggested by the widely documented ability of NMDA to elicit the release of GnRH from the hypothalamus (34, 37, 43, 50) as well as the secretion of adenohypophysial hormones in rat (26 28, 3133, 35, 38, 42 43, 49), rhesus monkey

(29 30), sheep (36), pig (40), rainbow trout (47), barrow (44), ewe (45), mares (46), gilts (48), ovine fetus (50), and pig cultured cells (41). Using two sensitive and specic uorometric methods devised in this work, we were able to demonstrate that NMDA is actually present in the rat neuroendocrine system at levels comparable to those of many known hormones of the hypothalamus-hypophysis axis. The highest concentration of this excitatory amino acid occurs in the adenohypophysis (6.8 6.9 nmol/g wet tissue), followed by hypothalamus, testis, and brain (Table 3). To further conrm the above results and ascertain whether NMLA (N-methyl-L-aspartic acid) was also present (in addition to NMDA) in the rat tissues investigated, the samples were analyzed by GC-MS in combination with D-AspO treatment. Results show that in all samples analyzed, the peak corresponding to elution time of NMDA (which also has the same elution time as NMLA) almost completely disappeared after D-AspO treatment (Fig. 4). Since D-AspO is able to oxidize NMDA and not NMLA, these results not only conrmed the presence of NMDA in rat neuroendocrine tissues, but also indicated that probably no NMLA is present or is at very low concentrations compared to NMDA. This is the rst evidence for the occurrence of endogenous NMDA in neuroendocrine tissue. Only one example of the occurrence of NMDA in living organisms has previously been reported (52). It describes the nding of NMDA in muscle extract of the blood shell, Scapharca broughtonii. In this mollusk, however, NMDA maximally occurs in muscle tissues and not in the neuroendocrine tissues. Consequently, it appears that in this animal NMDA could have an osmotic function rather than a neuroendocrine activity. The presence of NMDA in rat nervous tissues and endocrine glands and its increase in response to D-Asp administration led us to hypothesize that NMDA is biosynthesized from its parent compound, D-Asp. To conrm our hypothesis, rats were injected with D-Asp, and after 60 min tissues were analyzed for NMDA concentration. Results indicated that in vivo biosynthesis of NMDA occurs and that the hypothalamus is the site in which the synthesis occurs to the greatest extent as expressed by the amount of NMDA/g tissue (Fig. 5). In fact, in the hypothalamus NMDA was found to be increased 11.2-fold compared to basal levels, whereas in the adenohypophysis, brain, and testis NMDA was increased by 2.6-, 2.6-, and 2.5-fold, respectively. In vitro experiments conducted by incubating a tissue homogenate with D-Asp and AdoMet (the putative methyl group donor) demonstrated that the conversion of D-Asp into NMDA effectively occurs, and that in this case the hypothalamus is the tissue in which the maximum synthesis of NMDA occurs (Table 4). Thus, these results indicate that an enzyme capable of synthe711

ROLE OF ENDOGENOUS D-Asp AND NMDA IN REGULATING LH AND GH SECRETION

sizing NMDA is present in animals, and it is a methyltransferase. That this enzymatic reaction involves a methyltransferase is evidenced by the fact that the in vitro synthesis for NMDA is inhibited by AdoHcy, a compound widely known to be an inhibitor for methyltransferases (53). Therefore, we propose to call this enzyme NMDA synthase or, alternatively, S-adenosylmethionine: D-Asp-N-methyltransferase. There appears to be some discrepancy concerning the endogenous presence and the biosynthesis of NMDA. In fact, we found the maximum concentration of endogenous NMDA in the adenohypophysis (Table 3), whereas the maximum biosynthesis occurs in the hypothalamus (Table 4, Fig. 5). Our explanation is simply that more NMDA was found endogenously in the adenohypophysis, because the highest concentration of D-Asp also occurs in this gland (Table 1), from which NMDA is biosynthesized; on the other hand, the hypothalamus is the tissue where the enzyme NMDA synthase is more concentrated, so that the most biosynthesis of NMDA occurs in the hypothalamus. To understand how D-Asp is implicated in the hormonal action, in vitro experiments were performed by incubating isolated adenohypophysis or adenohypophysis plus hypothalamus with D-Asp or NMDA. The results obtained indicated that D-Asp has a direct action in inducing GH release, but not LH release. However, whether adenohypophysis is incubated together with hypothalamus and D-Asp, the release of LH in the medium becomes higher than that which occurs when only the hypothalamus and adenohypophysis are incubated together, but without D-Asp. This occurs because D-Asp exerts a double action: it has a direct action on the hypothalamus in inducing the synthesis and release of GnRH from the hypothalamus, and at the same time is a substrate for the biosynthesis of NMDA, which more actively induces the GnRH release, as previously demonstrated by others (34, 37, 43, 47, 50). Previous reports indicated that NMDA involvement in hypothalamus and adenohypophysial hormones is mediated by NMDA receptors (30, 33, 39). We also performed in vitro experiments in which D-Asp and NMDA were tested in the presence of D-AP5, an NMDA receptor antagonist. The observation was that D-AP5 blocked D-Asp-induced release of GH from the isolated adenohypophysis, as well as the action of D-Asp and/or NMDA in the release of GH and LH from isolated adenohypophysis alone or incubated with the hypothalamus. These data thus suggest that an NMDA receptor-mediated event occurs, presumably at the level of the adenohypophysis and the hypothalamus. It is common knowledge that rodents display an increase in activity during the night, including sexual activity (51). Based on this concept, we conducted a
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series of experiments designed to clarify the role of D-Asp in hormone biorhythms, both in secretion and synthesis. We found that in the basal, unstimulated state, D-Asp in the adenohypophysis occurs at a signicantly higher concentration during nighttime than at daytime (Fig. 7), and a direct relationship between the natural D-Asp occurrence in the adenohypophysis and GH and LH secretion was observed. These results strengthen the notion that D-Asp is implicated in hormone activity and agrees well with those obtained by Snyder et al. (14), who found D-Asp to be present at very high concentrations in the pineal gland (1.2 mol/g tissue), a gland that regulates the day-night circadian biorhythm. In conclusion, we provide evidence that D-Asp and NMDA are present as endogenous compounds in the mammalian neuroendocrine system, where NMDA is synthesized from D-Asp by a methyltransferase. Both of these amino acids are involved in the regulation of GH and LH release from the adenohypophysis and of GnRH from the hypothalamus. Compared with D-Asp, NMDA elicits the same action, but at a concentration 100-fold lower than D-Asp. Therefore, we believe that D-Asp has a minor role as an effector molecule for hormone release, but that it mainly acts as the precursor for NMDA synthesis, which in turn is directly involved in the regulation of GnRH secretion from the hypothalamus and of GH from the adenohypophysis.
We gratefully acknowledge Drs. Margherita Branno, Francesco Aniello, and Francesco Errico, Department of Biochemistry and Molecular Biology of the Zoological Station, Naples, Italy, for their help in the preparation of D-aspartate oxidase obtained by molecular biology using mRNA from beef kidney. This work was principally supported by and carried out at the Zoological Station, Naples, Italy. G.H.F. gratefully acknowledges support from NIH grants NIGMS-MBRS/SCORE SO6GM45455 and FIC-MIRT GMTW00033.

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