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Occurrence of D-aspartic acid and N-methyl-D-asparticacid in rat neuroendocrine tissues and their role inthe modulation of luteinizing hormone and growth

hormone releaseANTIMO 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;llDepartment of Radioimmunology, Laboratorio Igea, 80027, Frattamaggiore, Napoli, Italy;**Institute of Nephrology, School of Medicine, II University of Naples, 80131 Napoli, Italy; andInstitute of Biochemistry of Macromolecules, School of Medicine, II University of Naples, 80138Napoli, Italy

ABSTRACT Using two specific and sensitive flu-orometric/HPLC methods and a GC-MS method,alone and in combination with D-aspartate oxidase,

we have demonstrated for the first time that N-methyl-D-aspartate (NMDA), in addition to D-aspar-tate (D-Asp), is endogenously present as a naturalmolecule in rat nervous system and endocrineglands. Both of these amino acids are mostly concen-trated at nmol/g levels in the adenohypophysis,hypothalamus, brain, and testis. The adenohypophy-

sis maximally showed the ability to accumulate D-Aspwhen the latter is exogenously administered. In vivoexperiments, consisting of the i.p. injection of D-

Asp, showed that D-Asp induced both growth hor-mone and luteinizing hormone (LH) release. How-ever, in vitro experiments showed that D-Asp wasable to induce LH release from adenohypophysisonly when this gland was co-incubated with thehypothalamus. This is because D-Asp also inducesthe release of GnRH from the hypothalamus, whichin turn is directly responsible for the D-Asp-inducedLH secretion from the pituitary gland. Compared toD-Asp, NMDA elicits its hormone release action atconcentrations 100-fold lower than D-Asp. D-AP5,a specific NMDA receptor antagonist, inhibited D-

Asp and NMDA hormonal activity, demonstratingthat these actions are mediated by NMDA receptors.NMDA is biosynthesized from D-Asp by an S-adeno-sylmethionine-dependent enzyme, which we tenta-tively denominated as NMDA synthase.DAniello,

A., Di Fiore, M. M., Fisher, G. H., Milone, A., Seleni,A., DAniello, S., Perna, A. F., Ingrosso, D. Occur-rence of D-aspartic acid and N-methyl-D-asparticacid in rat neuroendocrine tissues and their role in

the modulation of luteinizing hormone and growthhormone release. FASEB J. 14, 699714 (2000)

Key Words: D-Asp NMDA methyltransferase S-adenosyl-methionine NMDA synthase testosterone progesterone endocrine glands nervous system

D-Aspartic acid (D-Asp) is an endogenous aminoacid present in nervous and endocrine tissues ofinvertebrates and vertebrates. It was first found in the

brain, stellate ganglia, and the axoplasmic fluid ofthe cephalopods Octopus vulgaris, Loligo vulgaris, andSepia officinalis (12). Later this amino acid wasfound in the tissues of many other invertebrates(35) and vertebrates. In vertebrates, this amino acidoccurs in the nervous tissues of chicken (6), rat(79), and human (10, 11). In humans, it is presentin brains of embryos (10) and adults (11), as well asin the cerebrospinal fluid (12). In addition to ner-vous tissues, in mammals D-Asp is also present in thepituitary gland and the gonads (8, 13). D-Asp occursat high levels in embryos, whereas in adult animals it

nearly disappears in nervous tissues, but increases inendocrine glands, particularly in the pituitary, hypo-thalamus (8, 13), and pineal gland (14), where it hasbeen hypothesized to play an important role as anovel messenger molecule (14). We also found thatD-Asp levels increase in the testes during the twophases of life span: immediately before birth andduring sexual maturity (13), and coincidently with

1 Correspondence: Stazione Zoologica A. Dohrn, VillaComunale 1, 80121 Napoli, Italy. E-mail: [email protected]

6990892-6638/00/0014-0699/$02.25 FASEB

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testosterone synthesis. Recently, we found that D-Aspis also present in the reproductive organs of rat,where it is localized in Leydig and Sertoli cells oftestis (13) and in Octopus vulgaris (5). These datasuggest that D-Asp is implicated in hormonal pro-cesses and in steroidogenesis, since Leydig cells areknown to have a role in the synthesis of testosterone.In support to this hypothesis is the discovery thatD-Asp occurs in the ovary ofRana esculenta, where it

is involved in the control of testosterone releaseduring the sexual cycle (16), and in the spermato-genesis of the rat testis (17).

These data gave rise to the hypothesis that D-Aspin the neuroendocrine tissues could have a directrole in the regulation of hormone release and syn-thesis in the hypothalamus-hypophysial-gonadal axisor that D-Asp represents the precursor for the syn-thesis of another compound (e.g., NMDA), which isthen directly responsible for hormone release. Totest this hypothesis, we performed, in rats, in vivoandin vitroexperiments designed to elucidate the endo-

crine role of this molecule, with particular attentionto its involvement in the hypothalamus-adenohy-pophysis hormone release.

MATERIALS AND METHODS

Materials

The following were purchased from Boehringer Mannheim(Germany): D-amino acid oxidase (D-AAO, EC 1.4.3.3) puri-fied from hog kidney (15 U/mg, 5 mg/ml suspension in 3.2M ammonium sulfate); malate dehydrogenase (MDH; EC

1.1.1.37) from pig heart mitochondria (1200 U/mg, 5 mg/mlsuspension in 3.2 M ammonium sulfate solution); lactatedehydrogenase (LDH; EC 1.1.1.27) from pig muscle (550U/mg, 10 mg/ml, suspension in ammonium sulfate solu-tion); peroxidase (POD, EC 1.11.1.7) from horseradish (250U/mg, 10 mg/ml suspension in 3.2 M ammonium sulfatesolution); -nicotinamide adenine dinucleotide reducedform (-NADH); -nicotinamide adenine dinucleotide(NAD); EDTA; Pefabloc SC; leupeptin; aprotinin; chymosta-tin; bestain; PMSF; TPCK, and TLCK. The following werepurchased from Sigma Chemical Co (St. Louis, Mo.): D-aspartic acid (D-Asp), N-methyl-D-aspartic acid (NMDA), andall other D- and L-amino acids used in this work; bovineserum albumin (BSA), bacitracin, o-phthaldialdehyde (OPA),N-acetyl-L-cysteine (NAC), -mercaptoethanol, tyramine (4-

[2-aminoethyl] phenol; 4-hydroxyphenethylamine; tyro-samine hydrochloride), -ketoglutaric acid disodium salt(-ketoglutarate), D-2-amino-5-phosphonopentanoic acid (D-

AP5), Triton X-100, methylamine (CH3-NH2), Tris (Tris(hydroxymethyl aminomethane), and luteinizing hormone-releasing hormone (GnRH) antibody. The kits for radioim-munoassay (125I) determination of luteinizing hormone(LH), follicle stimulating hormone (FSH), thyroid-stimulat-ing hormone (TSH), and growth hormone (GH), as well as[2,3-3H]-D-aspartic acid (50 Ci/mmol), and [3H]methyl-NMDA (80 Ci/mmol), were purchased from AmershamInternational, Inc. (Buckinghamshire, U.K.). All solvents forhigh-performance liquid chromatography (HPLC) were re-agent grade and purchased from Merck or C. Erba (Milano,

Italy). Decanoic acid was purchased from Aldrich (Milwau-kee, Wis.). Cation exchange resin (AG 50W-X8, H form,100200 mesh, 60150 M size) was obtained from Bio-RadLaboratories (Hercules, Calif.). N-Methyl-N-(tert-butyldi-methylsilyl) trifluoroacetamide (MTBSTFA) was purchasedfrom Pierce Chemical Co. (Rockford, Ill.).

Preparation of D-aspartate oxidase

D-aspartate oxidase (D-AspO; EC 1.4.3.1) was obtained inpurified form from beef kidney at the concentration of 20U/ml (2 mg/ml), according the procedure of Negri et al.(18). The purity of the enzyme was determined by SDS gelelectrophoresis (PAGE), and showed only one band. Oneenzymatic unit was defined as the amount of the enzymecapable of oxidizing 1 mol of D-Asp in 1 min at 37C in 1 mlof assay mixture containing D-Asp at the concentration of 10mol/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 theamino acids NMDA, D-Asp, and D-glutamic acid (D-Glu) inthe 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 notoxidized by this enzyme (1820).

Animals

Wistar rats were purchased from Charles River Laboratories(Como, Italy) and housed, three per cage, in a controlledenvironment animal facility at 24C on a 12 h light/dark cycle(lights on from 07.00 to 19.00 h). The animals were fedstandard laboratory food pellets and water ad libitum. Care ofanimals 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 distilledwater at a concentration of 0.5 M, and the solution was

adjusted to a pH of 7.4 with 0.1 M NaOH. However, sincedicarboxylic amino acids (aspartic and glutamic acids andN-Methyl-D-aspartic acid) are insoluble in their acid form butare soluble as sodium salts, the sodium form of these aminoacids 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- orL-form) was mixed in 100 ml distilled water with stirring, and1 M NaOH solution was added dropwise to dissolve the aminoacids and obtain a final pH of 7.4. The final volume wasbrought to 200 ml with distilled water.

Sample purification

To determine reliably the concentration of D-Asp and NMDAin tissue extracts, the samples were subjected to four steps ofpurification.

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 wascentrifuged at 30,000 gfor 30 min. Blood was collected, left toclot at 37C for 30 min, and centrifuged at 5000 g for 30 min

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to obtain serum. Two milliliters of serum were homogenizedwith 20 ml 0.2 M TCA.

Step 2. Purification on cation exchange resin

The supernatant so obtained was applied to a cation exchange1 3 cm column, AG 50 W-X8 resin, H form, 100200 mesh,63150 m (Bio-Rad). The resin was previously regenerated bytreatment with an excess of 5 M NaOH for 30 min, followed byan extensive wash with distilled water, treated with an excess of5 M HCl for 30 min, and finally equilibrated with 0.01 M HCl.

After the sample had been absorbed by the resin, the columnwas washed with 10 ml 0.01 M HCl, followed by 10 ml distilledwater. These eluents were discarded. Then the column waseluted with 10 ml of 4 M NH4OH, and this last eluent wascollected and evaporated in small petri dishes on a hot plate at4050C under a hood. Using this procedure, all amino acidsand NMDA were recovered by over 90%, free of the TCA, salts,and organic compounds present in the tissues, and were withoutany racemization of aspartic acid or NMDA. After drying, thesamples were dissolved in 1 ml of water and divided into twoportions: one (50 l) was used to determine D-Asp; the other

was further purified and used to determine NMDA as describedbelow.

Step 3: OPA treatment and C-18 cartridge purification

950 microliters of the sample obtained from cation exchangeresin purification (Step 2) was mixed with 4 ml of distilled waterand with 0.1 ml of 1 M of an OPA reagent prepared by dissolving136 mg ofo-phthaldialdehyde in 1 ml of methanol. The pH ofthe mixture was adjusted to9.5 with 520l of 1 M NaOH andincubated at 37C for 30 min. The mixture was brought to pH2.5 with 10100 l of 1 M HCl, cooled at 0C, and centrifugedat 30,000 g for 10 min. The supernatant (OPA complexed withthe amino acids) was applied onto a C-18 cartridge (Sep-PakPlus, Waters, Milford, Mass.) containing 820 mg of octadecylsilane (previously activated with 100% methanol and thenequilibrated with distilled water). The eluent was collected andthe cartridge was washed with 5 ml of 0.01 M HCl. This lasteluent was combined with the previous eluent and evaporated ina petri dish on a hot plate at 4050C (or using a rotoevapora-tor). The residue was dissolved in 100 l of distilled water.(During this procedure, all amino acids containing the primaryamino group react with OPA reagent to form a strong irrevers-ible complex, which is retained on the C-18 resin, whereasNMDA and other amino acids, which do not have a primaryamino group, do not react with OPA and are eluted immediatelyfrom the C-18 cartridge.)

Step 4. Thin-layer chromatography (TLC), cation exchange resin,and C-18 cartridge purification

The sample obtained as described was subjected to further

purification by TLC in order to obtain NMDA completely freeof traces of D-Asp or D-Glu. The sample (100 l) was appliedto a TLC cellulose plate (2020 cm, 0.5 mm thickness,PSC-Fertigplatten, art. 15275, Merck) using 20 l of thesample per linear centimeter of plate. The plate was devel-oped in a solvent consisting of phenol-H2O (100 g phenol: 40ml H2O), using an amount of solvent in the tank to reach 1cm high, and left to run for 16 h. After migration, the plate

was dried with a hair dryer. To identify the migration positionof NMDA, a sample aliquot containing labeled N-[3H]NMDA

was run in parallel on the same plate. After drying, the platewas subjected to electronic autoradiography acquisition (Cy-clone, Packard, Canberra, Australia) for localization of la-beled NMDA. The phenol-water solvent was chosen because

provides a good separation of D-Asp or L-Asp from NMDA (Rfvalue for D-Asp or L-Asp was 0.17, NMDA was 0.58) (Fig. 1).Cellulose in which NMDA had migrated was scraped off andmixed vigorously with 5 ml of 0.01 M HCl, then centrifugedfor 30 min at 30,000 g; the supernatant was again purified onthe cation exchange column as described in Step 2. The driedresidues of each sample were dissolved in 1 ml of 0.01 M HCland further purified on a C-18 cartridge in order to eliminateany pigment contaminants if still present (from the TLCcellulose or cation exchange resin), which could interfere inthe enzymatic assay for NMDA or on gas chromatography-

mass spectrometry (GC-MS) assay. The sample was passedthrough a small C-18 cartridge (SepPak Light, containing80 mg of C-18 resin) using a syringe, followed by 1 ml ofdistilled water. These eluents were combined and dried asabove. The residue was dissolved in 100 l of distilled waterand used for NMDA determination.

Determination of D-Asp by enzymatic HPLC

This method was based on the diastereomeric separation ofD-Asp from L-Asp and other amino acids according to Aswad(21), modified as follows: 520 l of the sample obtained afterpurification on cation exchange resin (Step 2, sample purifica-tion) 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 afinal volume of 100 l. Finally, 5 l of OPA-NAC reagent

Figure 1. Separation of D/L-Asp from NMDA and otheramino acids on TLC cellulose. The figure represents aseparation of a mixture of standard amino acids (20 l of thestandard amino acid mixture in which each amino acid was ata concentration of 5 mol/ml) on TLC cellulose, 0.5 mmthickness. 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.

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(prepared by mixing 10 mg of OPA and 10 mg of NAC in 4 ml50% methanol) was added; after 2 min, 20 l was injected ontoa C-18 Supelcosil HPLC column (0.4525 cm, Supelco, Inc.,Belafonte, Pa.) using the Beckman-Gold HPLC system. Thecolumn was eluted with a gradient consisting of solvent A (5%acetonitrile in 30 mM sodium acetate buffer, pH 5.5) andsolvent B (70% acetonitrile in 30 mM sodium acetate buffer, pH5.5) as follows: 020% B over 20 min, then to 100% B over 5min, staying at 100% B for 2 min, and returning to 0% of B in2 min at a flow rate of 1.2 ml/min. Amino acid derivatives weredetected fluorometrically using an excitation wavelength at 330

nm excitation and 450 nm emission. A standard curve wasobtained under the same conditions using 5 l of a solutionconsisting of 17 L-amino acids, each at 0.1 mM, plus 0.02 mMD-Asp. It was observed that D-Asp eluted at 7.7 min, L-Asp at 8.5min, followed by the other amino acids (Fig. 2). To determinethe amount of area peak due to D-Asp, a parallel sample wasincubated with 2 l of purified D-AspO for 15 min at 37C andchromatographed as above. The total disappearance or thereduction of area peak corresponding to D-Asp elution peakconfirmed the presence of D-Asp and gave the exact amount ofthe content of D-Asp. A typical analysis is represented in Fig. 2.Using this method, we can determine reliably as little as 1 pmolof D-Asp in 20 l of the derivatized sample.

NMDA determination

Three methods were used in combination to determineNMDA.

Method 1: Enzymatic fluorometric

This method was used for screening of NMDA, based on thedetermination of H2O2 produced from the reaction betweenNMDA and D-AspO as follows:

COOHPCH2

P O2 H2O 3D-AspO

CH-NH-CH3

PCOOH

NMDA

COOHPCH2P H2O2 CH3-NH2CAOPCOOH

-oxaloacetate methylamine

H2O2 tyramineO

POD

fluorescent biphenyl derivative

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 standardmixture of amino acids (100 pmol each amino acid and 20 pmol of D-Asp) derivatized with OPA-NAC and fluorescencedetection. Dashed line represents the HPLC gradient program. Arrow shows the elution of D-Asp. Lower left panel: the samesample as in the upper panel, but after treatment with D-AspO. The peak corresponding to D-Asp disappears because ofoxidation by D-AspO. Upper right panel: analysis of a rat adenohypophysis extract. The peaks in the chromatogram correspondto 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 modified asfollows. In three Eppendorf tubes (marked sample, sampleplus internal standard, and blank sample) was added 20 lof the purified sample. To the tube marked sample plusinternal standard was added 5 l of NMDA standard at theconcentration of 0.02 mM (100 pmol). To each tube wasadded 20 l of POD-tyramine assay mixture prepared bymixing 20 mg of tyramine hydrochloride (Sigma) in 5 ml of0.2 M Tris-HCl, pH 8.2, plus 20 l of POD (peroxidase 2000U/ml). To the tubes marked sample and sample plusinternal standard was added 2 l of purified D-AspO (2

mg/ml; 20 U/ml), and all tubes were incubated at 37C for30 min. After incubation, 1 ml of 0.1 M Tris-HCl, pH 8.5, wasadded to each tube. The fluorescence of the sample andsample plus internal standard was read against the blanksample using the excitation wavelength of 320 nm andemission wavelength 415 nm. To calculate the concentrationof NMDA in the sample, the following formula was applied:

pmole of NMDA in 30 l of sample As

A(S1) As 100

where AS absorbance of the sample; A(S I) absorbanceof 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 be-tween NMDA and D-AspO as shown by the reaction:

NMDAO2 H2OOD-AspO

-oxaloacetateH2O2 CH3-NH2

The CH3-NH2 produced was determined quantitatively byHPLC after its reaction with OPA-mercaptoethanol. In two 200l Eppendorf tubes (marked sample and blank sample) wasplaced 20 l of sample as purified above and 20 l of 0.1 Mborate buffer, pH 8.2. Then 1 l of purified D-AspO (2 mg/ml;

20 U/ml) was added to the sample and both tubes wereincubated at 37C for 20 min. The blank sample tube was thenkept at 0C. Four microliters of OPA-mercaptoethanol reagent(consisting of 10 mg OPA in 1 ml methanol and 1 ml of 0.2 Mborate buffer pH 9.5 and 20 l of -mercaptoethanol) wasadded to the sample and mixed. After 2 min (needed to obtaincomplete derivatization of the amino acids), 20 l of derivatizedsample was injected onto a C-18 Supelcosil HPLC column(0.4525 cm, Supelco, Inc.) using the Beckman-Gold HPLCsystem. The column was eluted with a gradient consisting ofsolvent A (10% acetonitrile in 30 mM sodium acetate buffer, pH5.5) and solvent B (70% acetonitrile in 30 mM sodium acetatebuffer, pH 5.5) using the following gradient program: 040% Bover 15 min; to 100% B in 4 min, staying at 100% B for 3 minand back 0% B in 1 min, at a flow rate of 1.2 ml/min. CH3-NH2

was detected fluorometrically at an excitation wavelength of 330nm and an emission wavelength of 450 nm. The CH3-NH2eluted as a sharp peak at the retention time of 21.2 min, wellseparated from the other amino acids (Fig. 3). After running thesample, 5 l of OPA-mercaptoethanol was added to the blanksample and chromatographed as with the sample. The differ-ence of the peak areas obtained between the sample and theblank sample gave the net amount of the area due to themethylamine generated by the action of the D-AspO. To quan-tify the concentration of NMDA in the sample, a standard curveof 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 NMDAin amounts as small as 1 pmol.

Method 3: GC-MS

This method was based on the direct measurement of NMDAby GC-MS and used to confirm the occurrence of NMDA inneuroendocrine tissues. The analyses were performed with aHewlett Packard gas chromatograph 5890 Series II Plus. Thegas chromatograph was linked via a direct capillary columnHP5-MS (cross-linked 5% PH Me siloxane) (30 m x 0.25 mm,0.25 M film thickness) to a Hewlett Packard 5989B massspectrometer. Helium flow at 1 ml/min was used as a carriergas. For this purpose, 30 l of the sample (purified as above)

was dried and mixed with 5 l of decanoic acid (as internalstandard) at a concentration of 1 ng/l, 5.8 pmol/l, inchloroform. The mixture was dried under nitrogen flow.Then 20 l of MTBSTFA used as derivatizing agent forcarboxylic groups, was added and heated at 80C for 45 minin a sealed vial. Finally, 6 l of the tert-butyldimethylsilylderivatized sample was injected into the GC-MS system with asplit ratio 1:6. The programmed column temperature was120300C, 3C/min. Injector and detector were at 240C. AGC-EIMS (electron impact mass spectrum) of a standardconsisting of derivatized decanoic acid and NMDA was regis-tered in the 50550 amu (m/z) range in order to determinethe GC retention time (R.T.) and fragmentation of thesederivatized substances. Under these conditions, R.T. for thederivatized decanoic acid and NMDA was 17.89 and 23.86min, respectively. Since previous analyses have revealed thatNMDA levels present in the tissues were undetectable usingthe above scan conditions, the more sensitive single ionmonitoring (SIM) technique was adopted. Direct introduc-tion EIMS spectra of decanoic acid and NMDA standards

were run (data not shown), and fragments for the SIMtechnique were chosen. The SIM fragments used for decanoicacid were m/z 229, 230, 231, obtained from 286, 287, 288(which are MW, MW1, MW2) 57, which is the tert-butylradical fragment, -C(CH3)3, from the derivatizing group (Fig.4, CL) and for derivatized NMDA, m/z 318, 319, 320 obtainedfrom 375, 376, 377 (MW, MW1, MW2) 57 (Fig. 4, DL).These SIM fragments were chosen because the 57 fragmentis characteristic and a well-known radical fragment of eachderivatized compound with NTBSTFA. Naturally, the reten-tion times of the GC peaks for decanoic acid and NMDAobtained in the SIM run are the same as those obtained in theScan run (Fig. 4, AL).

Since the GC-MS technique does not distinguish betweenNMDA and NMLA (N-methyl-L-aspartate), each sample wasalso analyzed after treatment with D-AspO. Fifteen microlitersof 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 for20 min at 37C, then filtered on a membrane with a cutoff of30 kDa (microcon filter, Amicon) in order to separate theD-AspO from the smaller molecules. The filtrate was mixed

with 5 l of decanoic acid, dried, derivatized, and analyzed byGC-MS under the same conditions as above. Since the D-

AspO oxidizes NMDA and not NMLA (18, 2122), the

difference in abundances of the peak between before andafter incubation with D-AspO indicated the amount of NMDApresent in the standard (Fig. 4, BL) and the samples (Fig. 4,BR). To verify that the reduction of the NMDA peak wasactually due to the action of D-AspO and not due to sampleinjection errors, the internal standard decanoic acid was usedas a general reference point.

Determination of L-amino acids and other D-amino acids

The determination of each L-amino acid was performedaccording the method of Godel et al. (23), and determinationof other D-amino acids was performed using the method ofOkuma and Abe (24).


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Biosynthesis of NMDA: in vivo and in vitro studies

Since D-Asp has been well documented to be an endogenousmolecule (117) and involved in neuroendocrine activity (13,1617), and NMDA is the methylated form of D-Asp, wehypothesized that there exists a biosynthesis for NMDA. To

validate this hypothesis, in vivoand in vitroexperiments wereperformed. The in vivo experiments consisted of injectingintraperitoneally (i.p.) into a rat a solution of 0.5 M D-Asp ata dose to obtain 2 mol/g body weight of animal. One hourafter injection, the rat was killed and tissues were processedfor purification and determination of NMDA, as describedabove. The in vitro experiments consisted of incubating at37C for 60 min with shaking a homogenate of 200 mg oftissue with 1 ml of phosphate buffer saline (PBS) solutioncontaining 10 mg/ml of BSA, 20 mM sodium D-aspartate, 10mM sodium EDTA (used as metalloprotease inhibitor), 20mM sodium and potassium tartrate (used as inhibitor formammalian D-AspO), and 5 mM of S-adenosyl-L-methionine(AdoMet). After incubation, 1 ml of 1 M TCA was added to

the assay mixture and centrifuged at 30,000 g. The superna-tant was subjected to the purification of NMDA as describedabove and analyzed for NMDA. Parallel experiments werealso performed in presence of S-adenosyl-L-homocysteine(AdoHcy) at the concentration of 10 mM, as an inhibitor forthe methyltransferase.

Effects of D-Asp on LH and GH release: in vivo and in vitrostudies

To study the effects of D-Asp on LH and GH release, in vivoand in vitro experiments were performed. The in vivo exper-iments consisted of i.p. injection of a solution of 0.5 M sodiumD-aspartate, pH 7.4, into 85-day-old male rats using an appro-priate volume to inject 2.0 mol/g body weight. After 1 h and5 h, the animals were killed by decapitation. Blood wascollected, incubated at 37C for 30 min, and then centrifugedfor 30 min at 3000 g. Serum was separated from the red cellsand used for hormonal analyses. Solid tissues were removed,homogenized, and purified as described above, then used to

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 ofNMDA) derivatized with OPA-mercaptoethanol and fluorescence detection. Dashed line represents the HPLC gradientprogram. Note that NMDA is not seen because NMDA does not react with OPA. Middle left panel: same sample of standardamino 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 sampleof standard amino acids as in left top panel, to which 100 pmol of authentic methylamine was added and incubated withD-AspO before HPLC analysis. Upper right panel: analysis of rat hypothalamus extract after purification (cation exchangeresin, OPA treatment and TLC separation). The peaks observed are traces of amino acids still present in the sample afterpurification (each peak represents 0.51% of the each amino acid originally present in the tissue extract). Middle rightpanel: same sample as upper panel, but after treatment with D-AspO, showing the peak of methylamine coming from theD-AspO oxidation of NMDA.


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determine D-Asp and NMDA. Parallel experiments were alsoconducted using other D- and L- amino acids instead ofD-Asp.

In vitroexperiments were conducted: as soon as the animalwas killed, the pituitary gland was separated from the neuro-hypophysis and cut into four portions by making longitudinaland vertical cuts. The four specimens were incubated at 37Cunder gentle shaking in 2 ml of a medium consisting of PBScontaining 5 mg/ml BSA and D-Asp at concentrations from0.2 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 forRIA determination of LH and GH. Other experiments wereconducted under these same conditions in which the assay

mixture also contained 0.1 mM D-AP5, used as an antagonistof NMDA receptors.

Effects of D-Asp on GnRH synthesis and release

This experiment was undertaken to ascertain whether D-Aspinduced the release and the synthesis of GnRH from isolatedhypothalamus. Hypothalami collected from four adult malerats were each cut into four portions and incubated at 37Cfor 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 mMsodium and potassium tartrate, and 2 mM D-aspartic acid.After incubation, the medium was centrifuged at 1000 g, andthe supernatant was homogenized with 8 ml of 100% meth-anol and centrifuged at 30,000 g. The supernatant was driedby using a rotoevaporator at 2530C. The residue wasdissolved in 1 ml of distilled water and passed through a C-18Sep-Pak cartridge (C-18 of 1 g size, Waters). After the samplehad been absorbed, the cartridge was washed with 5 ml of15% methanol. This eluent was discarded. The cartridge wasthen 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 bufferand used to determine GnRH by HPLC analyses. For HPLCanalyses, 50 l of the sample was injected onto a C-18

supelcosil HPLC column (0.4525 cm, Supelco, Inc.) using aBeckman-Gold HPLC system. The column was eluted with agradient of solvent A consisting of 5% acetonitrile in 30 mMsodium acetate buffer, pH 5.5, and solvent B consisting of70% acetonitrile in 30 mM sodium acetate buffer, pH 5.5.The gradient program consisted of 050% B over 22 min;then to 100% B over 3 min, staying at 100% B for 2 min, andfinally back 0% B over 1 min, at a flow rate of 1.2 ml/min. TheGnRH was detected using a wavelength of 215 nm. A standardcurve was performed under the same conditions by injecting50 l of synthetic GnRH (Sigma) at concentrations between10 and 100 pg/ml. The peak of GnRH eluted at the retentiontime 16.8 min (Fig. 6). To verify that the peak attributed by usas GnRH in the sample was really this compound, theremaining 50 l of the sample were treated with 20 l of a

solution of the antibody against mammalian GnRH andincubated overnight at 4C. The GnRHantibody complex

was then centrifuged on a molecular weight filter with a cutoffof 30,000 (microcon filter, Waters) and the filtrate wasinjected on the HPLC. In this last case the GnRH-antibodycomplex does not pass through the filter because of its highmolecular weight. Consequently, the peak previously ob-served at the elution time corresponding to the GnRHdisappears (Fig. 6).

Radioimmunoassays

Concentrations of LH, GH, and TSH in serum and in themedium from the in vitro experiments, as well as in the

pituitary homogenate, were determined by double-antibodyautoimmunoassay methods using the rat reagent kits pur-chased from Amersham Life Science (Buckinghamshire,U.K.) with magnetic separation, according to the suggestedassay procedures. The assay system uses a high specific activity[125I] tracer, together with a highly specific and sensitiveantiserum. The sensitivity of the assay was in the range 0.08 to5.0 ng per tube. The serum samples, derived from the in vivoexperiments, were examined undiluted and after 1:2, 1:4, 1:8,and 1:16 dilution with PBS. Tissue incubation media from invitro experiments of the indicated periods were diluted withPBS 1:1000, 1:10,000, and 1:100,000 and then used for thehormonal analyses. Serum testosterone, progesterone, 17-estradiol, androstenedione, 17-hydroxyprogesterone, corti-

Figure 4. GC-MS analyses of NMDA. Left panels (standards):AL shows the gas chromatographic profile of the derivatizedinternal standard decanoic acid (1.16 pmol; R.T.17.89 min)and NMDA (27.7 pmol; R.T.23.86 min). BL is the samestandard mixture as AL, but after treatment with D-AspO(note the disappearance of the peak at R.T. 23.86 corre-sponding to NMDA). CL and DL show the SIM mass spectrumof 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/z318, 319, 320). Right panels (tissue sample): analyses of apurified sample from rat hypothalamus plus decanoic acidunder the conditions depicted in the left panel. AR shows thegas chromatographic profile of decanoic acid and otherpeaks from the tissue extract (the peak corresponding toNMDA is well shown). BR is the same sample after thetreatment with D-AspO, showing the disappearance of theNMDA peak. CR and DR show the SIM mass spectrum of theGC peak of the internal standard (decanoic acid) and of thepeak corresponding to NMDA, respectively.


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sol, 3,5,3-triiodothyronine (T3), and 3,5,3,5-tetraiodothyro-nine (T4) were also assayed by radioimmunoassay using thereagent kits for human blood purchased from BiochemicalImmunosystem Company (Milan, Italy).

Statistical analysis

The results described in the text are expressed as the mean sd. Statistical analyses were performed using the SPSS soft-

ware package (v 8.0), running on a Pentium II computerequipped with Windows 95 operating system.

RESULTS

Endogenous occurrence of D-Asp in rat tissues andits accumulation in response to acute treatment

Using a specific and sensitive HPLC method todetermine D-Asp based on the diastereomeric sepa-ration of D-Asp from the other amino acids (Fig. 2),associated with the use of D-aspartate oxidase, wehave determined the endogenous occurrence ofD-Asp in the neuroendocrine and other tissues ofrat. The results obtained confirmed that rat tissuespossess D-Asp, as we and others had previouslyreported (614), and that among various tissues thisamino acid is concentrated mostly in the endocrineglands: adenohypophysis, testes, and adrenal, whereits concentration corresponds to a mean value of114 18, 90 14, and 78 8 nmol/g tissue,respectively (Table 1). The nervous tissues possess anamount of D-Asp four- to fivefold lower than endo-crine glands (between 1526 nmol/g) except for thehypothalamus, where this amino acid reaches a

mean value of 53 9 nmol/g tissue. The othertissues (liver, kidney, muscle, and blood) show verylow amounts of D-Asp (28 nmol/g tissue).

When exogenous D-Asp was administered to ratsvia i.p. injection (2.0 mol/g body weight), it accu-mulated predominantly in the endocrine glands andespecially in the adenohypophysis. One hour afterinjection of D-Asp, the adenohypophysis accumu-lated 3200 nmol/g tissue (28-fold higher than the

basal value); 5 h after treatment, this gland hadaccumulated 5250 nmol (46-fold higher than thebasal value) (Table 1). The testes and adrenal alsopresented this phenomenon, but to a much lowerextent than the adenohypophysis. D-Asp accumula-tion in the brain was low compared to the adenohy-pophysis, because the brain barrier actively preventsa large amount of D-Asp from entering (25). How-ever, it is interesting to observe that the hypothala-mus is a brain region capable of binding exogenousD-Asp (13424 nmol/g tissue 1 h after D-Asp injec-tion and 19539 nmol/g after 5 h) (Table 1). These

last data are of particular interest in that the hypo-thalamus and the adenohypophysis are directly con-nected in the control of the pituitary hormonesecretion. The results also showed that 24 h after theinjection of D-Asp, the adenohypophysis still stronglybound D-aspartate at an elevated concentration(1500250 nmol/g tissue), whereas in other tissuesthe D-Asp level decreased to near basal values. OtherD-amino acids investigated (D-Ala, D-Glu, and D-Met) were not significantly taken up by the adeno-hypophysis or other neuroendocrine tissues (datanot 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

Endogenous occurrence ofD-Asp in rat tissues

(nmol/g tissue)

D-Asp accumulation after i.p. injectiona (nmol/g tissue)Time post treatment

1 h 5 h 24 h

Endocrine glandsAdenohypophysis 114 18 3200 460 5250 690 1500 250Testes 90 14 210 65 370 85 120 24

Adrenal 78 8 220 58 345 67 91 25Brain

Hypothalamus 53 9 134 24 195 39 60 12

Hippocampus 26 5 60 10 76 18 35 8Cerebellum 18 4 62 9 70 15 28 6Frontal lobe 15 3 54 8 64 12 18 4Parietal lobe 18 4 45 6 58 11 17 4Occipital lobe 21 5 44 7 63 14 20 3

Other tissuesBlood 2 1 252 48 80 15 7 3Liver 7 4 224 44 90 21 9 4Kidney 8 6 212 38 120 32 12 3Hind leg muscle 3 2 15 5 14 4 4 2

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/gbody weight. The rats were injected at about 10 a.m. and killed after the times indicated. Data were obtained using the specific HPLC methodfor 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 adenohy-pophysis.

Effects of D-Asp on hormone release: in vivo andin vitro studies

Since D-Asp occurs at high concentrations in theadenohypophysis as an endogenous natural com-pound; since this gland responds positively by accu-mulating this amino acid, we hypothesized it couldbe 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/gbody weight. After 1 and 5 h, the levels of someadenohypophysial and gonadal hormones were mea-sured in the blood. The results indicated that GH,LH, and the gonadal hormones testosterone andprogesterone were significantly increased in theblood in response to D-Asp injection (Table 2). Onehour after the injection of D-Asp, GH concentrationin plasma increased 1.96-fold over basal levels

(P0.01), and after 5 h reached 2.6-fold the basallevel (P0.01) (Table 2). Similarly, LH increasedsignificantly (2.1- and 2.5-fold over basal level 1 and5 h after injection, respectively) (Table 2). Testoster-one and progesterone also increased in the blood,but only 5 h after D-Asp treatment was this increasesignificant. Testosterone rose 3.36-fold higher thanthe basal serum levels (P0.01), whereas progester-one increased 2.72-fold (P0.01). Other hor-mones17-estradiol, androstenedione, 17-hydroxyprogesterone, cortisol, thyroid-stimulatinghormone (TSH), 3,5,3-Triiodothyronine (T3), and

3,5,3,5-tetraiodothyronine (T4,)were not af-fected by D-Asp injection (Table 2). The free aminoacids L-Asp, D- and L-Glu, D-, and L-Ala, and D- and

L-Met were tested under the same conditions asD-Asp; they did not induce any significant increase ofthe above mentioned hormones (results not shown).

To verify whether D-Asp has a direct effect on theadenohypophysis, experiments in vitro were per-formed on this isolated gland (see Materials andMethods). These experiments indicate that D-Asp isable to increase the in vitrorelease of GH, but not ofLH (data not shown); the results show that D-Asp is

not the direct effector of LH release in vivo, butD-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 therelease of hypothalamus and adenohypophysis hor-mones (2650). Since NMDA is a molecule biochem-ically very similar to D-Asp, which is the methylatedform of D-Asp, we hypothesized that NMDA could bepresent in neuroendocrine tissues as an endogenous

compound and D-Asp as its natural precursor. Pre-vious attempts to determine this amino acid in rattissues indicated that NMDA was present in very lowamounts, and it was necessary to accurately purify thesample by cation exchange resin, OPA treatment,purification on C-18 cartridge, and TLC (Fig. 1) inorder to detect the small amount of NMDA withoutinterference by other compounds present in thesample. To determine the NMDA concentration, weset up an enzymatic fluorometric method based onthe determination of the hydrogen peroxide gener-ated from the reaction between NMDA and D-AspO,

and an enzymatic HPLC method based on determi-nation of methylamine generated by the oxidation ofNMDA 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 injection2 mM NaCl)

Time post treatment

1 h 5 h

Growth hormone (GH) 31.6 6.5 61.6 9.6* 82.5 17.8*Luteinizing hormone (LH) 3.7 0.6 7.8 1.1* 9.1 1.3*Testosterone 5.5 1.3 7.5 2.6 18.5 5.4*Progesterone 10.8 3.3 14.5 6.2 29.4 6.7*17-Estradiol 2.5 0.5 2.7 0.6 2.8 0.717-Hydroxyprogesterone 20.1 5.2 20.9 5.2 22.3 4.8

Androstenedione 1.0 0.2 1.2 0.4 1.4 0.3Cortisol 7.0 2.3 7.3 1.1 7.8 1.2Thyroid-stimulating hormone (TSH) 6.6 1.9 6.4 2.9 8.5 3.53,5,3-Triiodothyronine (T3) 1.0 0.2 1.1 0.3 1.4 0.53,5,3,5-Tetraiodothyronine (T4) 50.5 9.4 61.3 10.5 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 administer2.0 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 ttest; *P0.01 compared with control (NaCl 2 mol/g body weight animal).


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methods, the highest concentration of endogenousNMDA occurred in the adenohypophysis (6.81.1and 6.90.8 nmol/g tissue by the enzymatic fluoro-metric and HPLC methods, respectively), followedby the hypothalamus (4.40.6 and 4.30.4 nmol/gtissue), testis (3.50.5 and 3.40.4 nmol/g tissue)and frontal cortex (1.50.3 and 1.40.3 nmol/gtissue) (Table 3). In the kidney, muscle, and serum,NMDA was either very low or almost undetectable.

To further validate the results obtained by thesetwo methods, analysis was conducted on the samesamples by GC-MS. Results obtained by this tech-nique confirmed that NMDA is present in the sametissues tested above (Table 3). In this case, since theGC-MS technique does not provide the opportunityto quantify the exact amount of NMDA, we have onlyreported the presence or the absence of this aminoacid as indicated by the symbol or (Table 3).Figure 4 shows typical examples of the GC-EIMSanalyses conducted on a standard of NMDA plus astandard of decanoic acid (Fig. 4, AL, CL, DL) and on

a sample of rat hypothalamus plus standard decanoicacid (Fig. 4, AR, CR, DR). A GC peak at R.T. of 23.86min, corresponding to NMDA, was observed in boththe standard and the tissue samples. In each case,this GC peak shows the same SIM abundance at m/z318, 319, 320, characteristic of NMDA. In addition,when the standard and/or sample was treated withD-AspO and subjected to GC-MS analysis, the GCpeak at 23.86 min (corresponding to NMDA) disap-peared or was significantly reduced (Fig. 4, BL, BR).

Biosynthesis of NMDA: in vivo and in vitro studies

When D-Asp was acutely administrated to adult malerats (i.p. 2 mol/g body weight), a significant in-crease of NMDA in the endocrine glands and brainwas found 60 min after D-Asp injection (Fig. 5).

Compared to other tissues, the hypothalamus regis-tered the largest increase in NMDA, correspondingto 11.2-fold over the basal value (48.25.8 nmol/gvs. 4.30.8 nmol/g basal level; P0.01). In theadenohypophysis, the increase was 2.6-fold(15.42.2 nmol/g vs. the 5.91.5 nmol/g basalvalue; P0.01), in the brain 2.6-fold (3.40.8nmol/g vs. the 1.30.4 nmol/g basal value; P0.05),and in the testis the increase was 2.5-fold (8.51.9nmol/g vs. the 3.40.8 nmol/g the basal value;P0.05) (Fig. 5).

These data as a whole led us to hypothesize thatthe NMDA increase in these tissues was due to thetransformation of D-Asp into NMDA, supporting thehypothesis that an enzyme capable of synthesizingNMDA from D-Asp could exist. Therefore, we con-ducted in vitro experiments in which rat tissue ho-mogenates were incubated with D-Asp and S-adeno-syl-L-methionine (AdoMet, the universal methyldonor in transmethylation reactions), and NMDA inthe medium was determined. The results show that asynthesis of NMDA occurs, with the highest NMDAproduction 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 toobtain NMDA synthesis. In fact, if the assay mixturecontained only D-Asp, the amount of NMDA synthe-sized was very low (30- to 40-fold lower) comparedwith previous conditions. In addition, incubationwith only AdoMet or without D-Asp showed noNMDA biosynthesis. The specificity of this reactionwas confirmed by the addition of the transmethyla-tion inhibitor S-AdoHcy to the incubation medium.In this case, addition of AdoHcy to the assay mixture

Figure 5. In vivo biosynthesis of NMDA. The experimentconsisted of an i.p. injection of sodium D-aspartate solution(0.5 M, pH 7.4) to adult male rat (85 days old) using anappropriate volume to administer 2.0 mol/g body weight.

After 1 h tissues were taken from the animals, processed asdetailed under Materials and Methods, and subjected toHPLC analysis for NMDA through the determination ofmethylamine. The values represent the mean sd obtainedfrom 6 determinations. *P0.01 **P0.05.

TABLE 3. Endogenous occurrence of NMDA in rat tissuesa

Enzymaticfluorometric

methodEnzymatic

HPLC methodGC-MSmethod

nmol/g tissue

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

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


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caused a strong inhibition of NMDA synthesis (Table4). To verify that the NMDA found really came fromthe biosynthesis and was not a contaminant in theD-Asp used, the D-Asp was analyzed for the presenceof 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-Aspalone elicits GH release but not LH. On the otherhand, since in the in vivoexperiments we observed thatLH was released in response to D-Asp injection, wededuced that D-Asp could play a role in promoting thesynthesis of another compound (i.e., NMDA), which isthe molecule responsible for LH release throughGnRH. Therefore, we tested the action of D-Asp andNMDA, individually or together, on the isolated ade-nohypophysis or adenohypophysis plus hypothalamus

in hormone release. Results indicated that using 1 mMD-Asp (the concentration at which D-Asp exhibits themaximum activity in inducing hormone release), GHrose 2.61-fold compared to the control (82.59.8ng/ml medium vs. 31.65.5 ng/ml, P0.01) (Table5). LH release also was found to increase, but not

significantly. However, using NMDA alone (0.1 mM),LH release becomes significant over basal levels(6.81.2 ng/ml medium vs. 3.70.6 ng/ml of thecontrol). In addition, when D-Asp and NMDA wereused together, a more significant increase in the re-lease of LH was observed compared to the action ofNMDA alone (8.81.9 ng/ml medium vs. 3.70.6

ng/ml of its control). However, when the adenohy-pophysis was coincubated together with the hypothal-amus and D-Asp, a more significant increase of LH wasregistered (14.53.5 ng/ml medium vs. 8.42.3ng/ml of its control) (Table 5). In addition, whenD-Asp is incubated with the adenohypophysis and thehypothalamus together with NMDA, then LH releasein the medium was further increased (25.44.2 ng/mlmedium). Our interpretation of these results is thatD-Asp is converted to NMDA (see Table 4), which inturn induces release of the GnRH from the hypothal-amus, as previously inferred (see refs 34, 37, 47, 50).

GnRH then induces the release of LH from the pitu-itary gland. This interpretation is further confirmed bythe fact that when the hypothalamus is incubated withD-Asp, both NMDA and GnRH are increased (Table 4;Fig. 6). To know whether the action of these two aminoacids is mediated by the NMDA receptors, additional

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 D-Asp

OnlyAdoMet

Without D-Aspand AdoMet

D-Asp AdoMet AdoHcy

Hypothalamus 31.4 5.9 0.9 0.3 0.1 0.1 5.8 1.5Brain (frontal cortex) 17.3 3.2 0.8 0.3 0.1 0.1 3.5 1.1

Adenohypophysis 15.3 3.5 0.7 0.3 0.1 0.1 3.2 0.9Liver 13.8 3.4 0.5 0.2 0.1 0.1 2.3 0.5

Kidney 0.1 0.1 0.1 0.1 0.1

aAfter the experiment, the sample was purified 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-L-homocysteine.

TABLE 5. In vitro effects of D-Asp and NMDA on GH and LH release from isolated adenohypophysis and adenohypophysis plus thehypothalamus of rata

Incubation medium

Adenohypophysis Adenohypophysis hypothalamus

GH LH GH LH

(ng/ml medium) (ng/ml medium)

Control (1 mM NaCl) 31.6 5.5 3.7 0.6 80.2 11.4* 8.4 2.1*D-Asp 1 mM 82.5 9.8* 4.3 1.0 120.2 20.3* 14.5 3.5*NMDA 0.1 mM 90.3 10.2* 6.8 1.2* 150.4 20.4* 25.4 4.2*D-Asp 1 mM NMDA 0.1 mM 110.3 14.6* 8.8 1.9* 189.6 22.5* 25.3 4.7*D-Asp 1 mM D-AP5 0.1 mM 42.3 6.1 4.0 0.9 88.2 13.7 9.2 2.2

NMDA 0.1 mM D-AP5 0.1 mM 42.4 9.5 4.0 0.7 110.5 15.7 11.5 2.2

D-Asp 1 mM NMDA 0.1 mM D-AP5 0.1 mM 50.3 7.5 4.1 0.6 90.3 10.5 12.3 2.4

a The experiment consisted of incubating the adenohypophysis alone or together with the hypothalamus in 2 ml of the medium indicatedat 37C for 60 min under moderate agitation. Hormone concentrations in the medium were measured using the RIA method. Values representthe mean sd from 5 experiments. Statistical analyses were performed using the Students unpaired ttest. *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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experiments were conducted using D-AP5, a specificNMDA receptor antagonist. The results indicate thatD-AP5 at a minimal concentration of 0.1 mM produceda significant inhibition in the release of the above-mentioned hormones (Table 5), indicating that theactions of D-Asp and NMDA are mediated by theNMDA receptors.

Effects of D-Asp on the release of gonadotropin-releasing hormone

The experiments so far described showed that D-Asprepresents the precursor for NMDA synthesis. Inaddition, NMDA elicits release of LH (2635, 3739,4143, 4550) and GH (36, 39, 41, 4449) in malerats, as previously inferred by other authors. It ap-pears that D-Asp and/or NMDA are involved inpituitary LH release through hypothalamic GnRH.To verify this hypothesis, we tested the effects of

D-Asp on the hypothalamus in the in vitrorelease ofGnRH. Results showed a significant increase ofGnRH (Fig. 6). In fact, a significant amount ofGnRH was detected in response to D-Asp treatment(Fig. 6C, peak at retention time 16.8 min). In thehypothalamus incubated with NaCl instead of D-Asp,this peak was almost undetectable (data not shown).To unequivocally identify GnRH, the sample wastreated with the antibody anti mGnRH. With thesample (Fig. 6D) and in the case of standard GnRH(Fig. 6B), the peak corresponding to elution time ofGnRH disappeared.

Relationship between the endogenous occurrenceof D-Asp in rat adenohypophysis and GH and LHrelease and synthesis during the day-night circadiancycle

It is widely recognized that rats and many otheranimals show a predominantly nocturnal activity

(51). We, therefore, investigated the possibility thatD-Asp in the rat pituitary gland varied during theday-night circadian cycle and examined whether arelationship exists between the endogenous occur-rence of D-Asp and GH release. Six adult male rats(85 days old) were killed at 10 a.m. and six adultmale rats of the same age were killed at midnight.Blood and pituitaries were collected and treated asabove for D-Asp, GH, and LH determination. Hor-mone release was measured directly in the blood.The results are shown in Fig. 7. A significant differ-ence in D-Asp content in the blood between morn-ing and nocturnal levels is present. During the night,D-Asp concentration in the adenohypophysis wasfound to be increased nearly twofold compared tothat at 10 a.m. (22030 nmol/g adenohypophysis inthe night vs. 11415 nmol/g during the morning;P0.01). These events were paralleled by a signifi-cant increase in blood of GH and LH concentra-tions. In fact, as shown in Fig. 7, blood GH increased1.76-fold (6012 ng/ml serum at night vs. 345ng/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/mlat 10 a.m., P0.01) (Fig. 7).

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


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DISCUSSION

Here we report the occurrence of endogenous D-Aspand NMDA in the nervous system and endocrineglands of the rat and provide the first evidence fortheir neuroendocrine role. D-Asp occurs at levels of1526 nmol/g wet tissue in the brain with theexception of the hypothalamus, where D-Asp con-centration is higher with respect to other brainregions (539 nmol/g wet tissue). In the endocrineglands, D-Asp occurs at a much higher concentrationthan in the brain. In other tissues, D-Asp is found atvery low levels compared to the above tissues. Theadenohypophysis is the gland in which this aminoacid is present at the highest concentration (11418nmol/g wet tissue). In addition, this gland possesses

the capacity to take up and accumulate exogenouslyadministered D-Asp (Table 1). The occurrence ofD-Asp in the endocrine tissue led us to propose thatthis amino acid could be endowed with a neuroen-docrine role. We investigated the in vivo effects ofD-Asp on the release of some hypophysial hormonesin the rat. The results show a significant rise in theplasma levels of GH and LH between 1 and 5 h afteri.p. injection of D-Asp, and of testosterone andprogesterone after 5 h (Table 2). Since the release oftestosterone 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-Aspelicits hormone release, we were intrigued by thestructural similarities between D-Asp and its methyl-ated derivative NMDA, hypothesizing that the latterexcitatory amino acid could be present in neuroen-docrine animal tissues arising from the endogenousD-Asp. This hypothesis was suggested by the widelydocumented ability of NMDA to elicit the release ofGnRH from the hypothalamus (34, 37, 43, 50) as wellas the secretion of adenohypophysial hormones inrat (2628, 3133, 35, 38, 4243, 49), rhesus monkey

(2930), sheep (36), pig (40), rainbow trout (47),barrow (44), ewe (45), mares (46), gilts (48), ovinefetus (50), and pig cultured cells (41). Using twosensitive and specific fluorometric methods devisedin this work, we were able to demonstrate thatNMDA is actually present in the rat neuroendocrinesystem at levels comparable to those of many knownhormones of the hypothalamus-hypophysis axis. Thehighest concentration of this excitatory amino acid

occurs in the adenohypophysis (6.86.9 nmol/g wettissue), followed by hypothalamus, testis, and brain(Table 3). To further confirm the above results andascertain whether NMLA (N-methyl-L-aspartic acid)was also present (in addition to NMDA) in the rattissues investigated, the samples were analyzed byGC-MS in combination with D-AspO treatment. Re-sults show that in all samples analyzed, the peakcorresponding to elution time of NMDA (which alsohas the same elution time as NMLA) almost com-pletely disappeared after D-AspO treatment (Fig. 4).Since D-AspO is able to oxidize NMDA and not

NMLA, these results not only confirmed the pres-ence of NMDA in rat neuroendocrine tissues, butalso indicated that probably no NMLA is present oris at very low concentrations compared to NMDA.

This is the first evidence for the occurrence ofendogenous NMDA in neuroendocrine tissue. Onlyone example of the occurrence of NMDA in livingorganisms has previously been reported (52). Itdescribes the finding of NMDA in muscle extract ofthe blood shell, Scapharca broughtonii. In this mollusk,however, NMDA maximally occurs in muscle tissuesand not in the neuroendocrine tissues. Conse-

quently, it appears that in this animal NMDA couldhave an osmotic function rather than a neuroendo-crine activity.

The presence of NMDA in rat nervous tissues andendocrine glands and its increase in response to D-Aspadministration led us to hypothesize that NMDA isbiosynthesized from its parent compound, D-Asp. Toconfirm our hypothesis, rats were injected with D-Asp,and after 60 min tissues were analyzed for NMDAconcentration. Results indicated that in vivobiosynthe-sis of NMDA occurs and that the hypothalamus is thesite 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 beincreased 11.2-fold compared to basal levels, whereasin the adenohypophysis, brain, and testis NMDA wasincreased by 2.6-, 2.6-, and 2.5-fold, respectively. In vitroexperiments conducted by incubating a tissue homog-enate with D-Asp and AdoMet (the putative methylgroup donor) demonstrated that the conversion ofD-Asp into NMDA effectively occurs, and that in thiscase the hypothalamus is the tissue in which themaximum synthesis of NMDA occurs (Table 4). Thus,these results indicate that an enzyme capable of synthe-

Figure 7. Relationship between adenohypophysis D-Asp oc-currence and GH and LH release during day-night circadiancycle. The results represent mean sd obtained from 6 adultmale rats (85 days old). The determination of the hormonerelease was performed on serum. *P 0.01 compared withrats killed at 10 a.m.


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sizing NMDA is present in animals, and it is a methyl-transferase. That this enzymatic reaction involves amethyltransferase is evidenced by the fact that the invitro synthesis for NMDA is inhibited by AdoHcy, acompound widely known to be an inhibitor for meth-yltransferases (53). Therefore, we propose to call thisenzyme NMDA synthase or, alternatively, S-adenosyl-methionine: D-Asp-N-methyltransferase.

There appears to be some discrepancy concerning

the endogenous presence and the biosynthesis ofNMDA. In fact, we found the maximum concentra-tion of endogenous NMDA in the adenohypophysis(Table 3), whereas the maximum biosynthesis occursin the hypothalamus (Table 4, Fig. 5). Our explana-tion is simply that more NMDA was found endog-enously in the adenohypophysis, because the highestconcentration of D-Asp also occurs in this gland(Table 1), from which NMDA is biosynthesized; onthe other hand, the hypothalamus is the tissue wherethe enzyme NMDA synthase is more concentrated, sothat the most biosynthesis of NMDA occurs in the

hypothalamus.To understand how D-Asp is implicated in the

hormonal action, in vitro experiments were per-formed by incubating isolated adenohypophysis oradenohypophysis plus hypothalamus with D-Asp orNMDA. The results obtained indicated that D-Asphas a direct action in inducing GH release, but notLH release. However, whether adenohypophysis isincubated together with hypothalamus and D-Asp,the release of LH in the medium becomes higherthan that which occurs when only the hypothalamusand adenohypophysis are incubated together, but

without D-Asp. This occurs because D-Asp exerts adouble action: it has a direct action on the hypothal-amus in inducing the synthesis and release of GnRHfrom the hypothalamus, and at the same time is asubstrate for the biosynthesis of NMDA, which moreactively induces the GnRH release, as previouslydemonstrated by others (34, 37, 43, 47, 50).

Previous reports indicated that NMDA involve-ment in hypothalamus and adenohypophysial hor-mones is mediated by NMDA receptors (30, 33, 39).We also performed in vitro experiments in whichD-Asp and NMDA were tested in the presence of

D-AP5, an NMDA receptor antagonist. The observa-tion was that D-AP5 blocked D-Asp-induced releaseof GH from the isolated adenohypophysis, as well asthe action of D-Asp and/or NMDA in the release ofGH and LH from isolated adenohypophysis alone orincubated with the hypothalamus. These data thussuggest that an NMDA receptor-mediated event oc-curs, presumably at the level of the adenohypophysisand the hypothalamus.

It is common knowledge that rodents display anincrease in activity during the night, including sexualactivity (51). Based on this concept, we conducted a

series of experiments designed to clarify the role ofD-Asp in hormone biorhythms, both in secretion andsynthesis. We found that in the basal, unstimulatedstate, D-Asp in the adenohypophysis occurs at asignificantly higher concentration during nighttimethan at daytime (Fig. 7), and a direct relationshipbetween the natural D-Asp occurrence in the adeno-hypophysis and GH and LH secretion was observed.These results strengthen the notion that D-Asp is

implicated in hormone activity and agrees well withthose obtained by Snyder et al. (14), who foundD-Asp to be present at very high concentrations inthe pineal gland (1.2 mol/g tissue), a gland thatregulates the day-night circadian biorhythm.

In conclusion, we provide evidence that D-Asp andNMDA are present as endogenous compounds in themammalian neuroendocrine system, where NMDA issynthesized from D-Asp by a methyltransferase. Bothof these amino acids are involved in the regulation ofGH and LH release from the adenohypophysis andof GnRH from the hypothalamus. Compared with

D-Asp, NMDA elicits the same action, but at aconcentration 100-fold lower than D-Asp. There-fore, we believe that D-Asp has a minor role as aneffector molecule for hormone release, but that itmainly acts as the precursor for NMDA synthesis,which in turn is directly involved in the regulation ofGnRH secretion from the hypothalamus and of GHfrom the adenohypophysis.

We gratefully acknowledge Drs. Margherita Branno,Francesco Aniello, and Francesco Errico, Department ofBiochemistry and Molecular Biology of the Zoological Sta-tion, Naples, Italy, for their help in the preparation ofD-aspartate oxidase obtained by molecular biology usingmRNA from beef kidney. This work was principally supportedby and carried out at the Zoological Station, Naples, Italy.G.H.F. gratefully acknowledges support from NIH grantsNIGMS-MBRS/SCORE SO6GM45455 and FIC-MIRT GM-TW00033.

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Received for publication June 10, 1999.Revised for publication October 22, 1999.

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