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* 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;
| 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
1Correspondence: Stazione Zoologica ‘A. Dohrn’, Villa Comunale 1, 80121 Napoli, Italy. E-mail: daniello{at}alpha.snz.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: D-Asp • NMDA • methyltransferase • S-adenosylmethionine • NMDA synthase • testosterone • progesterone • endocrine glands • nervous system
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2)
. Later this amino acid was found in the tissues of
many other invertebrates (3
4
5)
and vertebrates. In
vertebrates, this amino acid occurs in the nervous tissues of chicken
(6)
, rat (7
8
9)
, and human (10
, 11)
. In humans, it is present in brains of embryos
(10)
and adults (11)
, as well as in the
cerebrospinal fluid (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
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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-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.):
D-aspartic 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), ß-mercaptoethanol, tyramine (4-[2-aminoethyl] phenol;
4-hydroxyphenethylamine; tyrosamine 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 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]methyl-NMDA (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, Italy). Decanoic acid was
purchased from Aldrich (Milwaukee, Wis.). Cation exchange resin (AG
50W-X8, H+ form, 100–200 mesh, 60–150 µM
size) was obtained from Bio-Rad Laboratories (Hercules, Calif.).
N-Methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide (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
purified 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 defined as the amount
of the enzyme capable of oxidizing 1 µmol of D-Asp in 1 min at 37°C
in 1 ml of assay mixture containing D-Asp at the concentration of 10
µmol/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
19
20)
.
Animals
Wistar rats were purchased from Charles River Laboratories
(Como, Italy) and housed, three per cage, in a controlled environment
animal facility at 24°C 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 final pH of 7.4. The final volume
was brought to 200 ml with distilled water.
Sample purification
To determine reliably the concentration of D-Asp and NMDA in
tissue extracts, the samples were subjected to four steps of
purification.
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 37°C for 30 min, and centrifuged at 5000
g for 30 min to obtain serum. Two milliliters of serum were
homogenized with 20 ml 0.2 M TCA.
Step 2. Purification on cation exchange resin
The supernatant so obtained was applied to a cation
exchange 1 x 3 cm column, AG 50 W-X8 resin,
H+ form, 100–200 mesh, 63–150 µm (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 finally 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
40–50°C 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 µl) was used to
determine D-Asp; the other was further purified and used to determine
NMDA as described below.
Step 3: OPA treatment and C-18 cartridge purification
950 microliters of the sample obtained from cation
exchange resin purification (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 5–20 µl of 1 M NaOH and
incubated at 37°C for 30 min. The mixture was brought to pH 2.5 with
10–100 µl of 1 M HCl, cooled at 0°C, 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 40–50°C (or using a
rotoevaporator). The residue was dissolved in 100 µl 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 purification
The sample obtained as described was subjected to further
purification by TLC in order to obtain NMDA completely free of traces
of D-Asp or D-Glu. The sample (100 µl) was applied to a TLC cellulose
plate (20x20 cm, 0.5 mm thickness, PSC-Fertigplatten, art. 15275,
Merck) using 20 µl 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 purified 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 purified 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 chromatography-mass
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 µl of
distilled water and used for NMDA determination.
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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)
, modified as follows: 5–20 µl of the sample
obtained after purification on cation exchange resin (Step 2, sample
purification) was mixed with 2–10 µl 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 final
volume of 100 µl. Finally, 5 µl of OPA-NAC reagent (prepared by
mixing 10 mg of OPA and 10 mg of NAC in 4 ml 50% methanol) was added;
after 2 min, 20 µl was injected onto a C-18 Supelcosil HPLC column
(0.45x25 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 flow rate of
1.2 ml/min. Amino acid derivatives were detected fluorometrically using
an excitation wavelength at 330 nm excitation and 450 nm emission. A
standard curve was obtained under the same conditions using 5 µl 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 µl of purified D-AspO for 15 min at 37°C and
chromatographed as above. The total disappearance or the reduction of
area peak corresponding to D-Asp elution peak confirmed 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 µl of the derivatized
sample.
|
NMDA determination
Three methods were used in combination to determine NMDA.
Method 1: Enzymatic fluorometric
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:
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and modified as
follows. In three Eppendorf tubes (marked ‘sample’, ‘sample plus
internal standard’, and ‘blank sample’) was added 20 µl of the
purified sample. To the tube marked ‘sample plus internal standard’
was added 5 µl of NMDA standard at the concentration of 0.02 mM (100
pmol). To each tube was added 20 µl 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 µl of POD (peroxidase 2000 U/ml). To
the tubes marked ‘sample’ and ‘sample plus internal standard’ was
added 2 µl of purified D-AspO (2 mg/ml; 20 U/ml), and all tubes were
incubated at 37°C for 30 min. After incubation, 1 ml of 0.1 M
Tris-HCl, pH 8.5, was added to each tube. The fluorescence 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:
 |
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:
 |
The CH3-NH2 produced
was determined quantitatively by HPLC after its reaction with
OPA-mercaptoethanol. In two 200 µl Eppendorf tubes (marked ‘sample’
and ‘blank sample’) was placed 20 µl of sample as purified above
and 20 µl of 0.1 M borate buffer, pH 8.2. Then 1 µl of purified
D-AspO (2 mg/ml; 20 U/ml) was added to the sample and both tubes were
incubated at 37°C for 20 min. The blank sample tube was then kept at
0°C. 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 µl of ß-mercaptoethanol) was added to the sample and mixed.
After 2 min (needed to obtain complete derivatization of the amino
acids), 20 µl of derivatized sample was injected onto a C-18
Supelcosil HPLC column (0.45x25 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: 0–40% 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 flow rate of 1.2 ml/min.
CH3-NH2 was detected
fluorometrically 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 µl 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
0.1–1.0 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.
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Method 3: GC-MS
This method was based on the direct measurement of NMDA by GC-MS
and used to confirm 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 µM film thickness) to a Hewlett Packard 5989B mass
spectrometer. Helium flow at 1 ml/min was used as a carrier gas. For
this purpose, 30 µl of the sample (purified as above) was dried and
mixed with 5 µl of decanoic acid (as internal standard) at a
concentration of 1 ng/µl, 5.8 pmol/µl, in chloroform. The mixture
was dried under nitrogen flow. Then 20 µl of MTBSTFA used as
derivatizing agent for carboxylic groups, was added and heated at
80°C for 45 min in a sealed vial. Finally, 6 µl of the
tert-butyldimethylsilyl derivatized sample was injected into the GC-MS
system with a split ratio 1:6. The programmed column temperature was
120–300°C, 3°C/min. Injector and detector were at 240°C. 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, MW+1,
MW+2) –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, MW+1, MW+2) –57 (Fig. 4DL
). 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. 4AL
).
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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 µl
of 0.02 M borate buffer (pH 8.2) and 1 µl of D-AspO. The mixture was
incubated for 20 min at 37°C, then filtered on a membrane with a
cutoff of 30 kDa (microcon filter, Amicon) in order to separate the
D-AspO from the smaller molecules. The filtrate was mixed with 5 µl
of decanoic acid, dried, derivatized, and analyzed by GC-MS under the
same conditions as above. Since the D-AspO oxidizes NMDA and not NMLA
(18
, 21
22)
, 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. 4BL
) and the
samples (Fig. 4BR
). 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)
.
Biosynthesis of NMDA: in vivo and in
vitro studies
Since D-Asp has been well documented to be an endogenous
molecule (1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17)
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 µmol/g body weight of animal. One hour after
injection, the rat was killed and tissues were processed for
purification and determination of NMDA, as described above. The
in vitro experiments consisted of incubating at 37°C 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 the assay mixture and
centrifuged at 30,000 g. The supernatant was subjected to
the purification 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 µmol/g body weight. After
1 h and 5 h, the animals were killed by decapitation. Blood
was collected, incubated at 37°C 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 purified as described above, then used to 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 37°C 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 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 37°C 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 25–30°C. 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 µl of 0.1 M
phosphate buffer and used to determine GnRH by HPLC analyses. For HPLC
analyses, 50 µl of the sample was injected onto a C-18 supelcosil
HPLC column (0.45x25 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 finally back 0%
B over 1 min, at a flow 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 µl 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
µl of the sample were treated with 20 µl of a solution of the
antibody against mammalian GnRH and incubated overnight at 4°C. The
GnRH–antibody complex was then centrifuged on a molecular weight
filter with a cutoff of 30,000 (microcon filter, Waters) and the
filtrate was injected on the HPLC. In this last case the GnRH-antibody
complex does not pass through the filter because of its high molecular
weight. Consequently, the peak previously observed at the elution time
corresponding to the GnRH disappears (Fig. 6)
.
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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 specific
activity [125I] tracer, together with a highly
specific 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, 17ß-estradiol,
androstenedione, 17-hydroxyprogesterone, cortisol,
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 |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, 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 confirmed that rat tissues
possess D-Asp, as we and others had previously reported
(6
7
8
9
10
11
12
13
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 fivefold
lower than endocrine glands (between 15–26 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
µmol/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
(134±24 nmol/g tissue 1 h after D-Asp injection and 195±39
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 (1500±250 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 D-Met) were not significantly taken up
by the adenohypophysis or other neuroendocrine tissues (data not
shown), indicating that D-Asp is the only D-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
µmol/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 significantly 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 (P<0.01), and after
5 h reached 2.6-fold the basal level (P<0.01) (Table 2)
. Similarly, LH increased significantly (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 significant.
Testosterone rose 3.36-fold higher than the basal serum levels
(P<0.01), whereas progesterone increased 2.72-fold
(P<0.01). Other hormones—17ß-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 significant 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
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
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, purification 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 fluorometric 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 methods, the highest concentration of endogenous
NMDA occurred in the adenohypophysis (6.8±1.1 and 6.9±0.8 nmol/g
tissue by the enzymatic fluorometric and HPLC methods, respectively),
followed by the hypothalamus (4.4±0.6 and 4.3±0.4 nmol/g tissue),
testis (3.5±0.5 and 3.4±0.4 nmol/g tissue) and frontal cortex
(1.5±0.3 and 1.4±0.3 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 confirmed 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. 4AL,
CL, DL
) and on a sample of
rat hypothalamus plus standard decanoic acid (Fig. , 4AR, 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
significantly reduced (Fig. , 4BL,
BR
).
Biosynthesis of NMDA: in vivo and in
vitro studies
When D-Asp was acutely administrated to adult male rats (i.p. 2
µmol/g body weight), a significant increase of NMDA in the endocrine
glands and brain was found 60 min after D-Asp injection (Fig. 5
). Compared to other tissues, the hypothalamus registered the largest
increase in NMDA, corresponding to 11.2-fold over the basal value
(48.2±5.8 nmol/g vs. 4.3±0.8 nmol/g basal level; P<0.01).
In the adenohypophysis, the increase was 2.6-fold (15.4±2.2 nmol/g vs.
the 5.9±1.5 nmol/g basal value; P<0.01), in the brain
2.6-fold (3.4±0.8 nmol/g vs. the 1.3±0.4 nmol/g basal value;
P<0.05), and in the testis the increase was 2.5-fold
(8.5±1.9 nmol/g vs. the 3.4±0.8 nmol/g the basal value;
P<0.05) (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 (31.4±5.9 nmol/ml assay
mixture) followed by brain (17.3±3.3), adenohypophysis
(15.3±3.5), and liver (13.8±3.4) (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 specificity of this reaction was
confirmed by the addition of the transmethylation inhibitor S-AdoHcy to
the incubation medium. In this case, addition of AdoHcy to the assay
mixture 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 (82.5±9.8 ng/ml medium vs.
31.6±5.5 ng/ml, P<0.01) (Table 5
). LH release also was found to increase, but not significantly.
However, using NMDA alone (0.1 mM), LH release becomes significant over
basal levels (6.8±1.2 ng/ml medium vs. 3.7±0.6 ng/ml of the control).
In addition, when D-Asp and NMDA were used together, a more significant
increase in the release of LH was observed compared to the action of
NMDA alone (8.8±1.9 ng/ml medium vs. 3.7±0.6 ng/ml of its control).
However, when the adenohypophysis was coincubated together with the
hypothalamus and D-Asp, a more significant increase of LH was
registered (14.5±3.5 ng/ml medium vs. 8.4±2.3 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 (25.4±4.2 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 confirmed 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 experiments were conducted using D-AP5,
a specific NMDA receptor antagonist. The results indicate that D-AP5 at
a minimal concentration of 0.1 mM produced a significant inhibition in
the release of the above-mentioned 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 gonadotropin-releasing hormone
The experiments so far described showed that D-Asp represents the
precursor for NMDA synthesis. In addition, NMDA elicits release of LH
(26
27
28
29
30
31
32
33
34
35
, 37
38
39
, 41
42
43
, 45
46
47
48
49
50)
and GH (36
, 39
, 41
, 44
45
46
47
48
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 significant increase of GnRH (Fig. 6)
. In
fact, a significant 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.
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 significant 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. (220±30 nmol/g adenohypophysis in the
night vs. 114±15 nmol/g during the morning; P<0.01). These
events were paralleled by a significant increase in blood of GH and LH
concentrations. In fact, as shown in Fig. 7
, blood GH increased
1.76-fold (60±12 ng/ml serum at night vs. 34±5 ng/ml at 10 a.m.,
P<0.01); LH increased 1.75-fold (5.6±0.8 ng/ml of serum at
night vs. 3.2±0.4 ng/ml at 10 a.m., P<0.01) (Fig. 7)
.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. 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 significant 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
27
28
, 31
32
33
, 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 specific fluorometric 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 confirm 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 confirmed 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 first 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 finding 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 confirm 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 synthesizing 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 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 significantly 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 µmol/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.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
