D-ASPARTATE IN THE MAMMALIAN BODY

Hiroshi Homma*
Kitasato University
School of Pharmaceutical Sciences
5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, JAPAN*FAX: 03-5791-6381
E-mail address: hommah@pharm.kitasato-u.ac.jp.
(Received 8 August 2002 Accepted 30 August 2002)(Abstract)
After some doubt, it has now been confirmed that D-amino acids occur naturally in mammaliantissues. In this review, I describe our work with D-aspartate. Immunohistochemical staining reveals that
D-aspartate is found in specific cells at distinct periods during the development of rat brain, adrenal,
pineal, and pituitary glands, and testis. D-Aspartate appears to be synthesized by the pituitary glandand testis and then secreted into the vascular system which transports it to tissues such as the adrenal
and pineal glands, which take it up using the L-Glu transporter. The D-aspartate synthesized by theanterior lobe of the pituitary gland may stimulate prolactin-producing cells to secrete more prolactin in
an autocrine and paracrine fashion. In the testis, D-aspartate produced inside the seminiferous tubules
may act in a paracrine fashion on Leydig cells that reside outside the tubules to increase their
testosterone production by stimulating their expression of Steroidogenic Acute Regulatory protein gene.
D-Aspartate in the pineal gland, apparently primarily derived from outside the tissue, suppresses themelatonin secretion by the parenchymal cells (pinealocytes). Studies with cultured mammalian celllines reveal that intracellular D-aspartate concentrations appears to change during the cell cycle andcan be regulated by the cell density of the culture. These studies also showed that mammalian cells
contain all the molecular components needed to regulate D-aspartate homeostasis, as they canbiosynthesize, release, take up, and degrade D-aspartate. D-Aspartate thus appears to function in the
mammalian body as a novel type of a messenger.
(Key

Words)

D-aspartate,enantioseparation,

immunohistochemical

localization,

melatonin,

testosterone, steroidogenic acute regulatory protein, elongate spermatid, prolactin, cell density
observations on where and when during the
Variousimportantfunctionsdevelopment of the mammalian body this isomer

appear to be performed only by the L-forms ofis found. We also describe the evidence thatamino acids, not the D-forms, and D-amino

reveals the mechanisms that influence D-Asp

acids

levelshave

unnaturalamino

consequently
isomers

or

acid

been

regardedlaboratoryasartifacts.

in

cells

or

withintissuesandthebiological activities of this isomer.

However, recent reports have demonstrated thatavarietyofD-amino

acids

do

occur

in

mammalian tissues in their free form or as

1.

Quantification

of

D-Asp

in

different

mammalian tissues during development

protein components. The physiological roles D-

Enantioseparation and detection ofamino acids play in mammals are thus currently

D-Asp

being investigated. We have been studying D-

derived from mammalian tissues is difficult

aspartatebecause of the large amounts of L-Asp and other(D-Asp)andreview

Viva Origino 30 (2002) 204 - 215© 2002 by SSOEL Japan

here

our

204

in

the

complexbiologicalsamplesViva Origino 30 (2002) 204 - 215
biomatrices

presentinthesetissues.

To

shown that high concentrations of D-Asp areovercome these difficulties, we have establishedpresent in the cerebral hemisphere of rat andand now routinely employ a highly sensitive

chick embryos and that these levels decline

method with which we can quantify the D-Asp

rapidly after delivery [1, 6, 7]. This pattern also

in mammalian tissues [1]. This method involves

occurs in humans as at week 14 of humanthe fluorogenic derivatization of amino acids

gestation, as much as 60% of the total Asp in theusing

4-fluoro-7-nitro-2,1,3-benzoxadiazole

prefrontal cortex is in the D-form [8]. The D-(NBD-F) (Fig. 1a), after which the Asp fraction

form levels then rapidly decrease to trace levels

is isolated by reverse-phase HPLC employing

at birth and remain low thereafter. In contrast,

an octadecylsilica column. This is followed by

D-Asp concentrations in the rat pineal gland andthe enantioseparation of D-Asp from L-Asp bytestis increase as development proceeds and inusing the Pirkle-type chiral columns OA2500 or

adult rats the D% (proportion of D-Asp in the

OA3100 (Fig.1 b). To reduce the assay time, we

total Asp) is as high as 30-40%. In the rat

have recently also developed a new HPLC

adrenal gland, D-Asp levels transiently increasesystem in which the two chromatographic steps

at 3 weeks of age to a D% of over 40%, after

are linked by an automated column-switchingwhich they decrease to the levels seen in adults

system [2].

at 8 weeks of age [3, 9]. In the rat pituitary
gland, D-Asp is predominantly present in theanterior lobe and its levels increase gradually toa D% of 5% at eight weeks of age [5]. In
addition, there is a gender difference in the DAsp concentrations in the pituitary gland as
females have 1.4-fold higher D-Asp levels thanmales [1].
2. Localization and biological activities of DAsp in rat tissues
As

described

above,

D-Asp

accumulates in particular regions of mammaliantissues and its levels change markedly during
development. To define exactly where D-Asp
occurs in each tissue, we raised a specific antiD-Asp

antibody

analysisoffor

immunohistochemical

the tissues[3-5, 10-12]. Thisrevealed possible biological functions of D-Asp

Fig. 1 (a) Fluorogenic derivatization of an amino

and set the stage for other experiments aimingacid with NBD-F

to determine whether the D-Asp in a particular(b) Structures of Pirkle-type chiral columns,
Sumichiral OA-2500 (S) and OA-2500 (R).

tissue has been synthesized by the tissue or has
been obtained from elsewhere.

We

have

used

this

methodtoquantitate the D-Asp levels and their changes

2.1. Brain

during development in a variety of rat tissues,

The quantitative analysis indicatedincluding the brain, the pineal, pituitary, andthat in several regions of the brain of human or

adrenal glands, and the testis [1, 3-5]. The D-

rat

Asp

during development and decrease rapidly after

levelsineachdevelopment-related

tissuepatterns.

follow

uniqueRegardingembryos, D-Asp

levelsrisetransiently

thedelivery [1, 6-9]. Probing of the rat embryonicbrain, various reports including our own havebrain with the anti-D-Asp antibody at different

205Viva Origino 30 (2002) 204 - 215
stages of development revealed for the first timein this organ (see Section 2.4). With regard tothat D-Asp first emerges in the hindbrain, after

the adrenal medulla of adult rat (eight weeks ofwhich it spreads into the forebrain and thenage), D-Asp IR appears in the cytoplasm ofthroughout the whole brain [11]. Confirming theadrenaline-storing cells and is negligible inresults

noradrenaline-storing cells [3, 13].

of

the

immunoreactivity

quantitative
(IR)

of

analysis,

D-Asp

the

markedlydiminishes after parturition [11].

As will be discussed in more detailbelow, D-Asp appears to be biosynthesized inWith regard to brain cell-specificthe mammalian

body, although

the precise

expression of D-Asp, D-Asp first occurs in thesynthetic route remains to be elucidated (seecell bodies of neuronal cells that had migrated

Section 3.1). Recent reports including our own

into the outer layer of the neuronal epithelium.

suggest that D-Asp is synthesized in certain

Thereafter

specific tissues, released into

itneuroblasts

appears
and

in

the

processes

seems

to

be

of

the vascular

localized

system, and then taken up by particular cells of

exclusively in axons once the distinct axon

various tissues by the L-Glu transporter, which

layer

the

has an affinity for D-Asp as well as for L-Glu

intracellular localization of D-Asp in neuronaland L-Asp [12, 14, 15]. Several observations

cells alters during differentiation, especiallysuggest that the adrenal gland is one of theduring the process of neurite extension. Thistissuessuggests that D-Asp plays a physiologicallysynthesizing it [12]. First, D-Asp administeredsignificant

intraperitoneally

had

been

role

established.

in

the

Thus,

development

andneurogenesis of the embryonic rat brain [11].

that

takes

up

D-Asp

(“exogenous

ratherD-Asp”)

thanisincorporated into the same region of the tissue
where “endogenous D-Asp” is found. Second,

2.2. Adrenal glandThe

the transient increase in D-Asp levels during thequantitative analysis

revealeddevelopment of the tissue coincides with athat in the rat adrenal gland, D-Asp levels at one

transiently increased expression of the Glu

week of age are relatively low but increasetransporter. Third, the Glu transporter in themarkedly at three weeks of age, after which aadrenal gland is found largely on the D-Asp-

rapidcontaining cells.

declineoccurs[3,

9].

Immunohistochemical analysis revealed that DAsp emerges in different cell types at distinct

2.3. Pineal glandperiods during development [3]. At one and

The adult rat pineal gland has very

three weeks of age, D-Asp IR is intense in thehigh D-Asp levels and IR analysis revealed acytoplasm of cells in the zona fasciculate andregional difference in that intense IR is evident

zona reticularis, the two innermost layers of thein the distal (caudal) portion of the gland but

adrenal cortex, but is negligible in the zona

little staining is found in the proximal (rostral)glomerulosa, the outermost layer of the cortex.

region [10]. In addition, D-Asp is concentrated

However, at eight weeks of age, these corticalin the cytoplasm of pinealocytes, the cell typeIR patterns are reversed as the cytoplasmic IR inthat constitutes approximately 80% of the cells

the zona fasciculate and zona reticularis cells

in this gland and that synthesizes and secretes

has disappeared and the zona glomerulosa now

melatonin. As the pinealocytes in the distal

shows intense IR. The cortical zones differ in

portion of the gland are believed to be closely

functionglomerulosa secretes

involved in the synthesis and secretion ofmineralocorticoids while the inner zones secrete

melatonin, the localization of D-Asp in thisglucocorticoids. The distinct temporal and site-

portion suggests that this amino acid may be

specific patterns of D-Asp in the adrenal gland

involved in this process. Supporting this notion

suggesttheis that cultures of primary rat pinealocytes

development and maturation of steroidogenesis

exposed to D-Asp reduce their norepinephrine-

as

that

the

zona

D-Asp

is

involvedin206

Viva Origino 30 (2002) 204 - 215
induced secretion of melatonin in a dosedependent manner[16]. D-Asp

2.4. Testis

may affectA cross-section of the seminiferous

melatonin secretion through the activation of atubules

subtype(s) of L-Glu receptor, because it has an

spermatogonia along the basement membrane,affinity for the receptor [17-19].

one or several layers of spermatocytes further

of

the

testiswill

revealafew

Like the adrenal gland, the pineal

towards the lumen, and a group of spermatids

gland appears to take up D-Asp synthesizednext to the tubule lumen. Thus, as the germ cells

elsewhere rather than synthesize it itself. This isprogress through the various stages of theirsupported

D-Asp

development, they move from the basal side ofsynthesis is minimal to non-existent in cultured

the tubule towards the lumen. There are also

primary pinealocytes and that these cells areinterstitialhighly efficient in taking up exogenous D-Asp

tubules, such as Leydig cells that synthesize and

from

the

secrete testosterone (Fig. 2). In the adult rat

pinealocytes express the L-Glu transporter [20].

testis, D-Asp IR is noted in the cytoplasm of

by

the
It

the

observation

medium
has

[16].

been

shown

that

Moreover,that

cultured

germ

cells

cells,

outsideparticularly

the

in

seminiferoustheelongate

pinealocytes loaded with D-Asp will releasespermatids, the most mature of the germ cells

their[4]. The IR intensity in each seminiferous

D-Asp

norepinephrine

when

they

[16].

are stimulated

Norepinephrine

by
also

tubulediffers

becauseeach

tubularcross-

induces pinealocytes to secrete melatonin. Thus,

section contains germ cells at a different stage

the norepinephrine-induced release of D-Asp byofpinealocytes

feedbackrecognized in cells other than germ cells,

mechanism by which norepinephrine regulates

including Sertoli cells, Leydig cells, and otherits ability to induce melatonin secretion.

testicular cells (Fig 2).

Fig.2 D-Asp in the rat testis(a) Representative illustration of the adult rat testis.
Seminiferous tubules include Sertoli cells and germ
cells that are in various stages of their development.
D-Asp is primarily localized in the cytoplasm ofgerm cells, particularly in the region rich in elongate
spermatids (shown in red). D-Asp is apparently

synthesized inside the seminiferous
tubules and secreted out of the tubules into
the interstitial space where it stimulates
Leydig cells to increase testosterone
production by stimulating StAR gene
expression. However, exogenously
administered D-Asp accumulates in theinterstitial spaces of the testis rather thanbeing incorporated into the inside of thetubules (blue arrow). (b)Immunohistochemical staining of D-Asp
in the adult rat testis (eight weeks of age)with anti-D-Asp antibody.
Immunoreactivity was visualized with theperoxidase-antiperoxidase complex anddiaminobenzidine and is therefore shownas brown signals. Counter-staining withhematoxylin and eosin was also carried out.
D-Asp is localized in the central areas ofthe seminiferous tubules, mainly aroundthe region rich in elongate spermatids.
Details are described in [4] Bar: 100m,
anti-D-Asp antibody: 1:100. (c) Adult
male rats (eight weeks of age) were
injected intraperitoneally with D-Asp (1.0 mol/g
antibody. Exogneous D-Asp (brown) accumulated in
the testis by being taken up from the vascular system
is primarily localized in the interstitial space and is
not incorporated into the seminiferous tubeles. Bar:
100m, anti-D-Asp antibody: 1:1000. Details aredescribed in [4].

may

representa207development.

AlmostnostainingisViva Origino 30 (2002) 204 - 215

To confirm these patterns of D-Asp

tubules(Lee

andHomma,

unpublishedlocalization in the testis, toxic chemicals wereobservation,

applied to rats to eliminate specific testicularconcentrations are found in rat testicular venous

cell

example,blood plasma [21] support the notion that D-Asp

administration of methoxyacetic acid causes theis produced within the seminiferous tubules of

specific loss of pachytene spermatocytes andthe testis and that it is then secreted out of theelongate spermatids three and 20 days later,

tubules.

populations

[4].

For

Fig.

2).

That

high

D-Asp

respectively. Three days after methoxyacetic

It appears that the secreted testicularacid treatment, when pachytene spermatocytes

D-Asp acts to modulate testosterone synthesisareby Leydig cells. Treatment of purified rat

specificallydeletedbut

theelongate

spermatid numbers show no significant change,

LeydigL-Asparehuman chorionic gonadotropin (hCG)-induced

reduced but D-Asp levels are unchanged. This is

testosterone synthesis [22]. The increase isconsistentwithdependent upon the culture period with D-Asp,

observationthatlevels

and

total
the

proteinlevelsimmunohistochemical

pachytene

spermatocytes

cells

with

D-Asp

directly

increasewith considerable stimulation being observed

contain a relatively low amount of D-Asp. In

after

contrast, twenty days after the treatment, when

Testosterone

the elongate spermatid numbers are severely

stimulated by D-Asp even in the absence of hCG

depleted, a significant decrease in D-Asp levels

stimulation, and D-Asp and hCG appear to haveis observed together with reduced L-Asp andsynergistic

protein levels [4]. These observations confirm

testosterone production. This stimulation isthat elongate spermatids contain higher levels

independent of the L-Glu receptor on rat Leydig

of D-Asp than the germ cells at earlier stages ofcells, as L-Glu, L-Asp and various synthetic

development.

agonists of the receptor do not have the same

three

hr

of

culturingsecretionrather

by

than

with

Leydigadditive

D-Asp.
cells

effectsison

It appears that when the elongate

effect as D-Asp. In addition, Glu receptor

spermatids are released as free spermatozoa into

antagonists do not suppress the stimulatory

the epididymides to continue their development,

effect of D-Asp. However, it appears that rat

they

the

Leydigseminiferous tubule, because D-Asp levels insubtypethe rat epididymides and epididymal fluid are

specifically mediate their D-Asp uptake. L-very low compared with those of the testis [4].

Cysteine sulfinic acid inhibits D-Asp uptake byThis suggests that most of the D-Asp in thethis transporter and when cultured Leydig cells

elongate

leave

their

D-Asp

spermatids

behindcells

express

denoted

a

as

L-Glu

GLAST

transporter
that

may

the

are treated with this inhibitor, both the uptake

in

the

of D-Asp and the D-Asp-mediated stimulation

seminiferous tubules when the cells expel mostof testosterone production is decreased in aof their cytoplasm in the tubules and are

dose-dependent

released into the epididymides. Thus, D-Asp

testosterone production also correlates wellappears to play a significant role(s) in the testis

with the amount of D-Asp taken up. Thus, itrather than in the spermatozoa.

appears that Leydig cells take up D-Asp and thatfragment

left

in

in

cytoplasmic

isbehindremaining

The germ cells in the seminiferous

manner.

The

increaseinthis stimulates testosterone production.

tubules of the testis, unlike the adrenal andThemechanism

by

which

D-Asp

pineal gland cells, appears to synthesize theirincreases Leydig cell testosterone productionown

was assessed by examining the effect of D-Asp

D-Asp

administeredbecauseD-Asp

intraperitoneallyaccumulates

in

the

on various stages of the testosterone synthetic

interstitial spaces of the testis (i.e. outside thepathway [22]. After hCG challenge of Leydig

tubules) rather than being incorporated into thecells, D-Asp has no effect on the levels of208Viva Origino 30 (2002) 204 - 215
cAMP, suggesting that D-Asp exerts its effectmembrane, a key and rate-limiting process indownstream of the point cAMP is formed

the(Fig.3). For testosterone synthesis, the process

Acuteof cholesterol translocation from the outer todemonstrated to be a key regulatory factor in

the inner mitochondrial membrane is a rate-

this process and D-Asp is shown to actuallylimiting step and overall rate of the synthesis isenhance the steady state mRNA and protein

limited by the availability of cholesterol tolevels of this factor [23]. Thus, D-Asp appears

intramitochondrial P450scc (Fig. 3).

to increase testosterone production in rat Leydig

steroidogenesis.
Regulatory

Recently

Steroidogenic

protein(StAR)

wascells by stimulating StAR gene expression.
It is likely that the D-Asp secreted bygerm cells also acts to regulate physiological
functions in other tissues (e.g. the adrenal
gland) in an endocrine fashion by entering thevenous blood and traveling via the vascularsystem to tissues that can take it up.
2.5. Pituitary gland
The pituitary gland consists of threedifferent lobes, namely, an anterior lobe madeup of endocrine cells, an intermediate lobe, anda posterior lobe made up of neuronal axons andFig.3 D-Asp action on Leydig cells in the testis.
In Leydig cells, cholesterol is transported across theouter mitochondrial membrane to the innermitochondrial membrane where it is converted topregnenolone by P-450scc. Pregnenolone is thenconverted to testosterone via the steroidogenic
pathway in the endoplasmic reticulum (ER). The
process of translocation of cholesterol to the innermitochondrial membrane is the rate-limiting step for
testosterone biosynthesis. This step is activated in
Leydig cells when human chorionic gonadotropin(hCG) binds to the luteinizing hormone (LH)receptor. Stimulation of this receptor results inincreased levels of cAMP, which then stimulates
Steroidogenic Acute Regulatory (StAR) protein gene
expression.
The
StAR
proteinsubsequently
facilitates the translocation of cholesterol to theinner mitochondrial membrane and increases
testosterone production. D-Asp appears to be taken
up by the Leydig cells via their L-Glu transporters
after which it increases StAR gene expression by anas yet undefined mechanism.

glial cells. In the posterior lobe of adult rats,
D-Asp IR is spread homogeneously over an area
apparently corresponding to that occupied byneuronal axons, while in the anterior lobe
intense IR is scattered throughout the cytoplasmof the endocrine cells [5, 13]. HPLC analysis
indicates that the D-Asp levels in the anterior
lobe are approximately seven-fold higher thanthose in the posterior lobe [5]. The anterior lobeof the rat pituitary gland is constituted by atleast six different cells types. One is thefolliculo-stellate cell type and the remaining
five types produce and secrete their distinct
hormones, namely, growth hormone, prolactin,
ACTH,thyroid

stimulating

gonadotropic hormone.

hormone,andDouble staining with

antibodies against D-Asp and prolactin revealedTosurmount

thishydroxycholesterol,

limitation,afreely22(R)accessible

substrate to P450scc can be supplied to measure
the ultimate capacity of testosterone productionin the cells. In the presence of this cholesterol
derivative, D-Asp does not increase testosterone
production any more, suggesting that D-Asp
accelerates

theprocesstranslocation

to

the

inner

of

cholesterol
mitochondrialthat the D-Asp-positive cells in the anterior lobe
of

the

rat

pituitary

gland

are

prolactin-

producing cells or another closely related cell
type

[5].

This

is

supportedbyseveralobservations. First, the morphological featuresof the D-Asp-positive cells as revealed byelectron microscopy are similar to those of
prolactin-producing cells. Second, D-Asp levelsin the pituitary gland are increased by estrogen209Viva Origino 30 (2002) 204 - 215
implantation, which is also known to increasethat PC12 cells contain D-Asp and that the

prolactin-producing cell numbers and serum

cellular levels of D-Asp increase with theprolactinlevels.

Third,

D-Asp

levelsandduration of culture. As this amino acid was notcellnumbersarebothadded to the culture medium, our observations

prolactin-producing

higher in females than in males [5].

demonstrate for the first time that D-Asp is

It appears that the anterior lobe of theindeedsynthesizedbymammaliancells.

pituitary gland synthesizes most of its own D-

Exogenous D-Asp is not taken up into the cells

Asp because intraperitoneally administered D-

becauseAsp entering the pituitary gland is incorporated

transporter, confirming that the biosynthesis

mainlyinto

takes place inside the cells. The D-Asp in PC

Furthermore,cells constitutes 12-14% of the total Asp andchanges in Glu transporter expression that occur

other D-amino acids have not been detected. Rat

during pituitary gland development do not

pituitary tumor cells (GH 3 ) have also beencorrelate with development-related changes infound to synthesize D-Asp [24] (see also

D-Asp levels. Moreover, prolactin-producing

Section 2.5), unlike mouse 3T3 fibroblasts andcells do not express any of the Glu transporters

human neuroblastoma NB-1 cells [28].

intoendothelial

prolactin-producing

cells

cells,
[12].

not

the

cells

do

Althoughcharacterized to date. Consistent with these

not

the

express

L-Glu

mammalianD-Asp

observations is that we found by HPLC analysis

synthetic pathway is yet to be defined, theand immunocytochemical staining that D-Asp is

enzyme that is most likely to be responsible for

produced by a prolactin-producing clonal strain

its biosynthesis is Asp racemase. Asp racemaseof the rat pituitary tumor cell line GH 3 [24].

from shellfish [29] and archaea [30, 31] has

Furthermore, D-Asp dose-dependently enhances

been characterized and genes encoding Asp

thyrotropin-releasing

racemasehormone-induced

have

been

prolactin secretion from the cells [24]. Recent

hyperthermophilicarchaeareports also show that isolated pituitary glands

concentrations of D-Asp are observed in theseor dispersed anterior pituitary cells incubatedlivingwith D-Asp increase their prolactin secretion

racemases are responsible for this. The enzymes[25, 26]. Thus, it appears that the anterior

from the hyperthermophilic archaea are Asp-

pituitary gland synthesizes D-Asp, which thenspecific

acts in an autocrine or paracrine fashion tophosphate but we recently found a different Aspenhance the secretion of prolactin and otherracemase in an acidothermophilic archaeon that

hormones.

is dependent on pyridoxal 5’-phosphate andorganisms

and

and

it

independent

cloned
[30,
is

of

from

31].
believed

pyridoxal

High
the

5’-

relatively insensitive to a SH-modifying reagent,
3. D-Asp in mammalian cells

unlike the enzymes in the hyperthermophilicarchaea [32].

3.1. Biosynthesis
Until recently, it was not clear whether
mammals can synthesize D-Asp since dietary

3.2. Mechanisms influencing intracellular D-Asp
levels

D-Asp is readily absorbed by the intestine andElucidatingthemechanisms

that

transported via the vascular system into various

affect D-Asp levels in mammalian cells willtissues, where it is taken up by cells carrying

greatly

the L-Glu transporter [14, 15, 27]. We addressed

physiological significance of D-Asp in thethis issue by employing pheochromocytoma

mammalian body. To investigate this issue, we(PC) 12 cells, a cultured mammalian cell line

assessed the intracellular localization of D-Asp

[28].

HPLC

assisttheunderstanding

of

the

analysis,

immunocytochemicalin 2068 and MPT1 cells, which are variant

use

an

enantioselective

strains of PC12 cells and whose D-Asp levels

degradative enzyme (D-Asp oxidase) revealed

are higher than those in PC12 cells [2, 33, 34].

staining,

and

of

210

Viva Origino 30 (2002) 204 - 215
Like PC cells, these strains both synthesize D-

different mechanisms regulate D- and L-Asp

Asp. Immunofluorescent staining of 2068 cells

levels in mammalian cells.

with the anti-D-Asp antibody and confocal laser

Notably,

individual

PC12

cells

scanning microscopy revealed that D-Asp IR

incubated with the anti-D-Asp antibody differ inoccurs in the cytoplasm, particularly around thetheir staining intensity [28]. This individual

nuclei. In addition, there are granular structures

variation is even more pronounced in 2068 cells

bearing fluorescent grains. These observations

and is particularly prominent in round-shapedsuggest that D-Asp is associated with granule-cells that have condensed chromosomes andlike structure(s) in the cytoplasm and that it isappear to be undergoing mitosis [33]. Thus, itreleased by exocytosis. Consistent with thisappears that cellular D-Asp levels alter during

notion is that D-Asp is released from 2068 cells

thewhen the cells are depolarized with a highrequires confirmation and clarification withconcentration of KCl or when the cells are

additional experiments.

incubated with the Ca2+ionophore A23187.
Ca 2 +

cycle,

althoughthisobservation

Thus, mammalian cells are able toalsobiosynthesize, release, and take up D-Asp. It

significantly suppresses D-Asp release [33].

has also been demonstrated that mammals canSeveral reports including our own have alsodegrade D-Asp by the use of D-Asp oxidase, ademonstrated that D-Asp is released by cells [16,peroxisomal enzyme that catalyzes the oxidative35, 36].

deamination of D-Asp to generate oxaloacetate

Depletionofextracellular

ions

cell

It is still unclear at present how

together with hydrogen peroxide and ammonium

cellular D-Asp concentrations are regulated. We

ions. The oxidase prefers acidic D-amino acids

have noted that the cellular levels of D- and L-

such as D-Asp and D-Glu and does not act on

Asp in the variant PC12 cell line MPT1 are

L-Asp [37]. A different type of oxidase, D-regulated by the cell density in the cultureamino acid oxidase, is also present in the

because when cell density increases, D-Asp

peroxisome, which acts on neutral and basic D-

levels increase while L-Asp levels decrease [34].

amino acids but not on acidic D-amino acids

The D- and L-Asp levels in the cells are[37]. These lines of evidence indicate that

dependent on the number of cells inoculated,mammalian cells

thus the cellular levels of D-Asp increase, while

components essential for the regulation of D-

L-Asp levels decrease in the culture plates, in

Asp levels.

contain all the molecularwhich higher numbers of cells are inoculated.
The effect of cell density on D- and L-Asp
levelsisalsoequivalent

In summary, it appears that D-Asp isnumbers of MPT1 cells are inoculated in culture

a novel type of messenger in the mammalianplatesbody (Fig. 4) that is synthesized by specificwithcontrast,

observedwhen

4. D-Asp in the mammalian bodydifferent diameters. In

aretissues and cells (Section 3.1), including thedensityanterior lobe of the pituitary gland (Section 2.5)

decreases L-Asp levels but has almost no effectand the seminiferous tubules of the testis

examined,

however,whenincreasing

PC12

marked

PC12

cell

cells

on D-Asp levels. As MPT1 cells express the L-(Section 2.4). D-Asp is released from the cell

Glu transporter but PC12 cells do not, it appears

after its synthesis (Section 3.2) and may act inthat the L-Glu transporter is involved in the cell

an autocrine and paracrine fashion on thedensity-dependent control of the D-Asp content

synthesizing cell itself and/or neighboring cells.

in MPT1 cells. It has been reported earlier thatAn example of such autocrine activity is that inseveral transporter systems of amino acids,theincluding the L-Glu transporter, alter in activity

prolactin-producing cells synthesize D-Asp thatwhen the cell density changes (see references inthen stimulates these cells to secrete more[34]). These lines of evidence also show that211

anterior

lobe

of

the

pituitary

gland,

Viva Origino 30 (2002) 204 - 215

Fig. 4 D-Asp in the mammalian body.
D-Asp is a novel type of messenger in the mammalian body. It appears to be synthesized in the anterior pituitary
gland and the seminiferous tubules of the testis where it stimulates the prolactin-producing cells in the anterior
pituitary gland to secrete more prolactin and the Leydig cells in the testis to increase testosterone production.
D-Asp is probably also released by these tissues and travels via the vascular system to other tissues that take it up
via their L-Glu transporters. These tissues include the adrenal and pineal glands and possibly the brain. In thepineal gland, D-Asp suppresses the ability of pinealocytes, the parenchymal cells of the gland, to secrete
melatonin. In the adrenal gland, it appears to modulate steroidogenesis. The functional significance of D-Asp in
the brain remains to be clarified.

prolactin (Section 2.5). An example of paracrine

functional

activity

However, D-Asp is an agonist for the L-Glu

is

that

D-Asp

secretedbythesignificance

of

this

is

unclear.

seminiferous tubules may stimulate Leydig cells

receptor,

in

their

development and neurogenesis of the brain.

testosterone production by stimulating StAR

Furthermore, since the brain is a steroidogenic

gene expression (Section 2.4). D-Asp secreted

tissue like the testis and adrenal gland [38], D-

by tissues that synthesize it may also act in an

Aspendocrine fashion on other tissues by entering

neurosteroids in the brain similar to the way thisthe vascular system and being taken up by cells

occurs in the latter tissues.

theinterstitial

space

to

increase

which

may

may

modulatebeinvolved

theinproduction

the

of

bearing the L-Glu transporter (Section 3.2). One
example of this is testicular D-Asp, which is

Acknowledgements

known to be secreted into the testicular venous

Our works described in this review

blood plasma (Section 2.4). The tissues thatare supported, in part, by Grants-in-Aid fortake up D-Asp synthesized elsewhere include

Scientific

the adrenal and pineal glands. D-Asp taken up

Education, Science, Sports and Culture of Japan,

by the adrenal gland appears to modulate thethe Science Research Promotion Fund from thesteroidogenesis

organ,

Promotion and Mutual Aid Corporation forsimilar to the way testosterone production by

Private School of Japan, the Japan healththe testis is regulated by D-Asp (Section 2.2).

Sciences Foundation for Research on Health

Similarly, D-Asp taken up from the vascular

Sciences focusing on Drug Innovation, thesystem may suppress melatonin secretion by

Tokyo Biochemical Research Foundation, thepinealocytes, the parenchymal cells

Fugakuthat

occurs

in

that

of

the

pineal gland (Section 2.3).
development

Trust

for

from

the

Medicinal

Ministry

Research,oftheFujisawa Foundation, the Terumo Life Science

The brain contains D-Asp during its
embryonic

Research

but

as

yet

the

Foundation,

theMishimaFoundation

and

the

212

Kaiun

Memorial

Sasakawa

ScientificViva Origino 30 (2002) 204 - 215
Research Grant from the Japan Science Society.

Nishikawa, T., Oka, T., Takahashi, K.,
Mito,

T.,

Takashima,

S.,

Doi,

N.,

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