Báo cáo khóa học: Glutamate signaling in peripheral tissues ppt

REVIEW ARTICLE
Glutamate signaling in peripheral tissues
Eiichi Hinoi, Takeshi Takarada, Taichi Ueshima, Yuriko Tsuchihashi and Yukio Yoneda
Laboratory of Molecular Pharmacology, Kanazawa University Graduate School of Natural Science and Technology, Kanazawa,
Ishikawa, Japan
The hypothesis that
L
-glutamate (Glu) is an excitatory
amino acid neurotransmitter in the mammalian central
nervous system is now gaining more support after the suc-
cessful cloning of a number of genes coding for the signaling
machinery required for this neurocrine at synapses in the
brain. These include Glu receptors (signal detection), Glu
transporters (signal termination) and vesicular Glu trans-
porters (signal output through exocytotic release). Relatively
little attention has been paid to the functional expression
of these molecules required for Glu signaling in peripheral
neuronal and non-neuronal tissues; however, recent
molecular biological analyses show a novel function for Glu
as an extracellular signal mediator in the autocrine and/or
paracrine system. Emerging evidence suggests that Glu
could play a dual role in mechanisms underlying the main-
tenance of cellular homeostasis – as an excitatory neuro-
transmitter in the central neurocrine system and an
extracellular signal mediator in peripheral autocrine and/or
paracrine tissues. In this review, the possible Glu signaling
methods are outlined in specific peripheral tissues including
bone, testis, pancreas, and the adrenal, pituitary and pineal
glands.
Keywords: autocrine; glutamate; glutamate receptor;
glutamate transporter; neurotransmitter; paracrine; vesicular

glutamate transporter; peripheral tissues.
Glutamate signaling molecules
Glutamate receptors
L
-Glutamate (Glu) is accepted as an excitatory amino acid
neurotransmitter in the mammalian central nervous sys-
tem (CNS). Receptors for Glu (GluRs) are categorized
into two major classes, metabotropic (mGluRs) and
ionotropic (iGluRs) receptors, according to their differen-
tial intracellular signal transduction mechanisms and
molecular homologies (Fig. 1) [1–3]. mGluRs are further
divided into three distinct subtypes containing seven
transmembrane domains, including group I (mGluR1
and mGluR5), group II (mGluR2 and mGluR3) and
group III (mGluR4, mGluR6, mGluR7 and mGluR8), in
line with each receptor’s exogenous agonists and intracel-
lular second messengers [4,5]. The group I subtype
stimulates formation of inositol 1,4,5-triphosphate and
diacylglycerol, while both group II and III subtypes
induce reduction of intracellular cyclic AMP (cAMP). On
the basis of sequence homology and agonist preference,
the latter iGluRs are classified into N-methyl-
D
-aspartate
(NMDA),
DL
-a-amino-3-hydroxy-5-methylisoxasole-4-
propionate (AMPA), and kainate (KA) receptors, which
are associated with ion channels permeable to particular
cations [6,7].

NMDA receptor channels. These channels are highly
permeable to Ca
2+
, with sensitivity to blockade by Mg
2+
in a voltage-dependent manner [8,9]. Functional NMDA
receptor channels are comprised of heteromeric assemblies
between the essential NR1 subunit and one of four different
NR2 (A–D) subunits, in addition to one of two different
NR3 (A–B) subunits. Expression of the NR2 subunit alone
does not lead to composition of functional ion channels in
any expression system, while coexpression of each NR2
subunit with an NR1 subunit results in expression of
functional channels permeable to Ca
2+
in mammalian cells
[10]. In contrast, NR3 subunits are a dominant-negative
family that suppresses opening of functional NMDA
receptor channels [11–13].
AMPA/KA receptor channels. AMPA receptor channels
consist of different subunits (GluR1–4), whereas KA
receptor channels are constructed using combinations of
GluR5–7 subunits. Both KA1 and KA2 subunits are
thought to participate in the expression of functional, high
affinity KA receptor channels along with other KA receptor
subunits [7]. GluR d subunits are cloned by homology
screening and classified as iGluRs on the basis of sequence
Correspondence to Yukio Yoneda, Laboratory of Molecular
Pharmacology, Kanazawa University Graduate School of Natural
Science and Technology, 13–1 Takara-machi, Kanazawa,

Ishikawa 920–0934, Japan. Tel.:/Fax: + 81 76 234 4471;
E-mail:
Abbreviations: AP1, activator protein-1; AMPA,
DL
-a-amino-3-hyd-
roxy-5-methylisoxasole-4-propionate; Asp, aspartate; CBFA1, core
binding factor a)1 ; CNS, central nervous system; EAAC1, excitatory
amino acid carrier 1; EAAT, excitatory amino acid transporter;
GLAST, glutamate aspartate transporter; GLT-1, glutamate trans-
porter-1; Glu, glutamate; GluR, glutamate receptor; iGluR, iono-
tropic glutamate receptor; KA, kainate; mGluR, metabotropic
glutamate receptor; MK-801, dizocilpine; NMDA, N-methyl-
D
-aspartate; t-ACPD, (+/–)-1-aminocyclopentane-trans-
1,3-dicarboxylic acid; VGLUT, vesicular glutamate transporter.
(Received 28 August 2003, accepted 4 November 2003)
Eur. J. Biochem. 271, 1–13 (2004) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03907.x
similarity. Using different pharmacological analyses and
expression in transfected cells [14,15] researchers believe that
these d subunits are thought to be orphan receptors. In the
CNS, these iGluR and mGluRs are thought to mediate
excitatory neurotransmission and to play key roles in
synaptogenesis, formation of neuronal circuitry and to be
involved in mechanisms for synaptic plasticity such as
learning and memory [16,17]. Excessive activation of GluRs
is thought to participate in the neurodegeneration following
a wide range of neurological insults including ischemia,
trauma, hypoglycemia and epileptic seizures. Chronic
neurodegenerative disorders such as Alzheimer’s disease,
Huntington’s chorea, AIDS encephalopathy and

amyotrophic lateral sclerosis may also involve neuronal
cell death induced by Glu [18–20].
Glutamate transporters in the plasma membrane
In the CNS, Glu transporters are essential for the termin-
ation of signal transduction mediated by Glu as well as for
the prevention of neurotoxicity mediated by this endo-
genous excitotoxin. These transporters maintain a 10 000-
fold gradient of intracellular Glu (3–10 m
M
) to extracellular
Glu (0.3–1 l
M
), which is driven by the ionic gradients
generated by ion-exchanging pumps such as Na
+
/K
+
-
ATPase [21,22]. These Glu transporters are classified into
five different subtypes including glutamate aspartate trans-
porter (GLAST) (EAAT1; excitatory amino acid transpor-
ter 1) [23,24], glutamate transporter-1 (GLT-1) (EAAT2)
[25], excitatory amino acid carrier (EAAC1) (EAAT3) [26],
EAAT4 [27], and EAAT5 [28] to date (Table 1). These Glu
transporters display heterologous regional and cellular
expression profiles. Both GLAST and GLT-1 are localized
to astrocytes, with GLAST predominating in the cerebellum
and GLT-1 in the cortex and forebrain, respectively [29].
These transporters are believed to sequester the majority of
extracellular Glu released from neurons. In astrocytes,

extracellular Glu is incorporated and converted into gluta-
mine by glutamine synthetase, and glutamine is shuttled
back to neurons for the resynthesis of Glu in neurons [30].
On this astrocyte/neuron, glutamate/glutamine hypothesis
for the neurotransmitter compartment, astrocytes play an
important role in mechanisms underlying the disposition of
Glu at synaptic vesicles through vesicular transporters and
the recycling of Glu for exocytotic release. EAAC1 is
localized to neurons throughout the CNS [31], whereas
EAAT4 localization is largely restricted to cerebellar
Purkinje cells [27]. EAAT5 has been shown to exclusively
reside in the retina [28] and more specifically on photo-
receptor and bipolar rod and cone cells [32].
Vesicular glutamate transporters
The proteins responsible for vesicular glutamate transport
have only recently been identified when it was recognized
that putative inorganic phosphate transporters mediate
Glu transport for condensation reactions in synaptic
vesicles (Table 1). Within the CNS, vesicular glutamate
transporter (VGLUT) isoforms-1 [33,34] and -2 [35,36]
appear restricted to known glutamatergic neurons and
exhibit a striking complementary pattern of expression at
excitatory synapses [37,38], suggesting that they might
define the excitatory neuronal phenotype. Indeed, heterol-
ogous expression of VGLUT1 or VGLUT2 suffices to
convert inhibitory neurons to excitatory ones [34,39,40].
The expression of VGLUT1 and VGLUT2 would
account for the exocytotic release of Glu in all known
glutamatergic neurons, while VGLUT3 is expressed in a
number of cell types suggested previously to release Glu

through exocytosis, among them, dopaminergic, GAB-
Aergic and serotonergic neurons and astrocytes [41,42].
These previous findings give support to the hypothesis
Table 1. Classification of glutamate transporters and vesicular glutam-
ate transporters. Glu transporters are classified into five different sub-
types including GLAST (EAAT1), GLT-1 (EAAT2), EAAC1
(EAAT3), EAAT4 and EAAT5, which are responsible for the ter-
mination of signal transduction mediated by Glu and for the preven-
tion of neurotoxicity mediated by this endogenous excitotoxin.
Vesicular Glu transports are divided into three groups, VGLUT1,
VGLUT2 and VGLUT3. Expression of VGLUT suffices for definition
of a glutamatergic phenotype in neurons.
Transporter
Amino acid
length (aa)
Tissue
distribution
Glutamate transporters subtype
GLAST 543 Brain, retina, testis, bone
GLT-1 576 Brain, retina, liver
EAAC1 524 Brain, intestine, kidney,
retina, liver, heart
EAAT4 564 Brain, placenta
EAAT5 560 Retina, liver
Vesicular glutamate transporter subtypes
VGLUT1 560 Brain, bone
VGLUT2 558 Brain, pancreas,
pineal gland
VGLUT3 589 Brain, retina, liver
Fig. 1. Classification of glutamate receptors. GluRs are divided into

two major classes, metabotropic (mGluRs) and ionotropic (iGluRs)
receptors, according to their differential intracellular signal transduc-
tion mechanisms as well as sequence homologies. There are three
distinct subtypes of mGluRs, including groups I, II and III, classified
based on sensitivity to exogenous agonists and intracellular second
messengers employed. iGluRs are subclassified, on the basis of
sequence homologies and agonist preference, into NMDA, AMPA
and KA receptors, which are all associated with ion channels per-
meable to particular cations.
2 E. Hinoi et al. (Eur. J. Biochem. 271) Ó FEBS 2003
that expression of either VGLUT would suffice for the
definition of a glutamatergic phenotype in endocrine cells
[43,44] as also shown in neurons [33,34].
Glutamate signaling in peripheral tissues
Relatively little attention has been paid to functional
expression of Glu signaling molecules in peripheral tissues.
Evidence is emerging for a role of Glu as an extracellular
signal mediator in the autocrine and/or paracrine system, in
addition to an excitatory amino acid neurotransmitter role
in the CNS [45]. We have shown previously that specific
binding sites of [
3
H]Glu are localized with high affinity,
selectivity and quisqualate sensitivity in rat adrenal [46] and
pituitary [47] glands, in addition to the brain. Quisqualate-
sensitive, Cl
–
/Ca
2+
-dependent [

3
H]Glu binding is also seen
in bovine pineal membranes [48]. Both NMDA and
L
-Glu
induce a rapid contraction of the guinea pig ileum in a
manner sensitive to NMDA antagonists [49–51]. Contrac-
tion is also induced by Glu in rat bronchial smooth muscle
with pharmacological profiles different from those for any
known GluRs in the brain [52]. In addition to these
pioneering previous studies, recent molecular biological
analyses give support to the expression of particular Glu
signaling molecules in a variety of different neuronal and
non-neuronal peripheral locations. These include bone
(osteocyte, osteoblast, and osteoclast) [53–55], testis
[56,57], pancreas [44,58,59], adrenal gland [60,61], pituitary
gland [62,63], pineal gland [64,65], taste buds [66], lung [67],
hepatocyte [68], thymus [69], cerebral endothelium [70],
megakaryocytes [71], keratinocytes [72], lymphocytes [73],
platelets [74], and heart [75]. In this article, we focus on Glu
signaling machinery expressed in particular tissues.
Bone
Two distinct cell types are known to coordinate to regulate
bone formation and maintenance, these are bone-forming
osteoblast and bone-resorbing osteoclasts. An imbalance
between these cells leads to pathogenesis and to etiologies of
certain metabolic bone diseases including osteoporosis,
Paget’s disease, and osteopetrosis [76,77]. The balancing
mechanism, at least in part, involves endocrine control (via
estrogen and parathyroid hormone) and paracrine (auto-

crine) control (via interleukin, insulin-like growth factor and
fibroblast growth factor) [76,77]. Recent studies have raised
the possibility that Glu may be one of the endogenous
paracrine (autocrine) factors used for intercellular commu-
nications in bone cells [53,54] (Fig. 2).
In mammalian bone, NMDA receptors are also expressed
in osteoblasts and osteoclasts as revealed by RT-PCR, in situ
hybridization, immunohistochemistry, and electrophysio-
logy [54,55,78–81]. Further studies reveal the expression of
functional NMDA receptor channels in osteoblasts and
osteoclasts. The addition of an NMDA receptor antagonist
inhibits cell differentiation in cultured osteoclasts [55,82],
while Glu induces elevation of intracellular free Ca
2+
in a
manner sensitive to antagonism by the NMDA receptor
antagonist, dizocilpine (MK-801) in the human osteoblastic
cell lines MG63 and SaOS-2 [83]. In our hands, however,
expression of mRNA was seen for GLT-1 and EAAT4, but
not for either other Glu transporters or any GluR subtypes
including NMDA receptors, in mouse primary cultured
osteoclasts differentiated from monocyte/macrophage pro-
genitor cells purified by Ficoll density gradient centrifugation
(E. Hinoi, T. Takarada, M. Inoue and Y. Yoneda, unpub-
lished data).
In addition to NMDA receptors, osteoblasts constitu-
tively express mRNA for non-NMDA receptors such as the
GluR3 subunit of AMPA receptors and KA1 and KA2
subunits of KA receptors [84]. AMPA receptors modulate
the exocytotic release of Glu from cultured osteoblasts

[85,86]. An antagonist for AMPA receptors is shown to
significantly inhibit the release of endogenous Glu in a
concentration-dependent manner in MG-63 osteosarcoma
cells [85], whereas AMPA facilitates the release of endo-
genous Glu from cultured osteoblasts in the presence of the
inhibitor of AMPA receptor desensitization cyclothiazide
[86]. Moreover, constitutive expression is also shown for
particular mGluR (mGluR4 and mGluR8) that are coupled
negatively to adenylyl cyclase thus inhibiting the formation
of cAMP stimulated by forskolin in cultured rat calvarial
osteoblasts during different developmental states [79], and
prior stimulation of mGluR by (+/–)-1-aminocyclopen-
tane-trans-1,3-dicarboxylic acid (t-ACPD) leads to reduc-
tion of NMDA-induced whole cell current in cultured rat
femoral osteoblasts [87]. Of the EAAT subtypes cloned to
date, both GLAST and GLT-1 are shown to reside in bone
with respect to mRNA and corresponding proteins, without
expression of immunoreactive EAAC1 [53,88]. Mechanical
Fig. 2. Glutamate signaling in bone. Two dis-
tinct cell types are known to coordinate to
regulate bone formation and maintenance of
bone; bone-forming osteoblast and bone-
resorbing osteoclasts. Particular functional
GluRs are identified in osteoblasts (NMDA
receptors, AMPA receptors and mGluRs) as
well as in osteoclasts (NMDA receptors).
GLAST sensitive to mechanical stress is
expressedinbothosteoblastsandosteocytes,
while VGLUTs are responsible for Glu release
from intracellular vesicular constituents

through activation of particular iGluR sub-
types present in osteoblasts.
Ó FEBS 2003 Glutamate signaling in peripheral tissues (Eur. J. Biochem. 271)3
loading is shown to down-regulate GLAST expressed in
osteocytes when determined by immunohistochemistry [53].
In GLAST knockout mice, however, no marked differences
are seen in a variety of phenotypes including mandible
and long bone size, morphology, trabeculation, regions of
muscle attachment, resorption lacunae, and areas of
formation vs. resorption of bone, compared with wild-type
siblings [89]. In addition to Glu transporters expressed at the
cell surface, both RT-PCR and immunohistochemical
analyses reveal constitutive expression of membrane and
intracellular molecular machinery generally required for
vesicular release of a central neurotransmitter in osteoblastic
cell lines as well as primary cultured osteoblasts [90]. We
have also demonstrated constitutive expression of particular
VGLUT in cultured osteoblasts irrespective of maturity of
cultured osteoblasts [86].
Core binding factor a)1 (CBFA1) is a transcription factor
essential for the growth of osteoblasts and is a master
regulator at the differentiation stage [91,92]. Disruption of
CBFA1 prevents skeltogenesis and heterozygous mutations
lead to cleidocranial dysplasia, an autosomal dominant
disorder [93,94]. CBFA1 is also shown to regulate expression
of several genes such as collagenase-3, type (I) collagen,
osteoprotegerin, bone sialoprotein, osteopontin,andalkaline
phosphatase in addition to osteocalcin [95–97]. We have also
recently demonstrated the exacerbation of osteoblastic
differentiation by different NMDA receptor antagonists

[55]. Sustained exposure to MK-801 invariably leads to
marked inhibition of expression of both mRNA and
corresponding protein for the master regulator of bone
differentiation CBFA1 (in addition to its DNA binding
activity) in primary cultured rat calvarial osteoblasts when
determined after the stage of cellular differentiation [55]. The
antagonist not only inhibits temporal marked expression of
CBFA1 seen at the stage of cell differentiation but also
prevents Ca
2+
accumulation only when added before the
period of cell differentiation. The lack of effects of
MK-801 on Ca
2+
accumulation and cell numbers through-
out cell growth, when added after the stage of cell differen-
tiation, gives support to the proposal that NMDA receptors
predominantly modulate cell differentiation rather than
proliferation through a mechanism associated with expres-
sion of CBFA1 during cellular maturation in osteoblasts.
As mentioned above, bone cells could express all mole-
cular machinery required for Glu signaling in the CNS. Glu
signals in bone, as seen at glutamatergic synapses in the brain,
could be dispatched through exocytotic release from vesicles
expressing VGLUT for condensation in vesicular particles,
transduced specifically via both iGluRs and mGluRs and
terminated by Glu transporters. Moreover, bone is supposed
to have sympathetic and glutamatergic innervations
[98–100]. Accordingly, Glu could have a pivotal dual role
in mechanisms associated with maintenance of homeostasis

as an excitatory neurotransmitter (toward neurocrine pro-
cesses in the CNS) and as a trophic factor (toward autocrine
and/or paracrine processes in bone).
Testis
A testis consists of seminiferous tubules and interstitial cells,
specifically Leydig cells, having unique functions in the male
body – the generation of gametes and the production and
controlled release of sex steroids [101–103]. The seminifer-
ous tubules include germ cells as well as somatic cells such as
Sertoli cells. Spermatogenesis in mammals is characterized
by intense and continuous proliferative activity with a
complex pattern of mitotic divisions in different types of
spermatogonia, culminating in meiotic cleavage of primary
spermatocytes to produce noncycling haploid cells (sperm-
atids). Development of male germ cells in the seminiferous
tubules is dependent on the action of pituitary gonando-
tropins and androgens secreted from Leydig cells. The entire
process of germ cell development, moreover, also relies on
Sertoli cells for structural and nutritional support. For
instance, germ cells are segregated from the systemic
circulation because of the blood–testis barrier created by
tight junctions between Sertoli cells near the basal lamina
[104,105] as seen with astroglial cells for the blood–brain
barrier.
In rat testis tissue, mRNA and corresponding proteins
are constitutively and functionally expressed for both
mGluR1 and mGluR5, but not for mGluR2 and mGluR3
[56]. Immunohistochemical and molecular biological ana-
lyses reveal the expression of NR1, GluR2/3, and mGlu
R2/3 proteins in kidney, liver, lung, spleen, and testis

[106,107]. In addition to functional expression of GluRs,
Glu transporters are also expressed in testis tissue. A
Northern blot study showed expression of mRNA for a
novel Glu transporter later found to be identical to
GLAST [22,23] in rat forebrain, cerebellum, and testis
[108]. Expression of mRNA is also shown for a neuron-
specific Glu transporter (later identified as EAAC1)
isolated from human brain cDNA libraries in human
brain, liver, muscle, ovary and testis using Northern blot
analysis [109]. Figure 4 shows our recent findings on the
distribution profiles of different Glu transporters expressed
in rat testis. In Fig. 3A, a schematic representation is
shown for histological characteristics of testis tissue. In
these experiments, rat testes were removed and frozen
sections dissected with subsequent fixation with parafor-
maldehyde for detection of localization of each mRNA
using DIG-labeled cRNA probe. Insituhybridization
evaluation demonstrated clearly the localized expression of
mRNA for GLAST in the interstitial space of rat testis
(Fig. 3b). Constitutive expression is seen with mRNA for
GLT-1 in elongated spermatids of rat testis, while expres-
sion of EAAT5 mRNA is found in the basal compartment
of seminiferous tubule in rat testis. However, mRNA
expression is not seen for EAAC1 and EAAT4 in any
particular structures of rat testis.
D
-Aspartate (
D
-Asp) endogenously occurs in rat adeno-
hypophysis, testis, adrenal gland, and brain in a rank order

of decreasing levels, while an intraperitoneal injection of
exogenous
D
-Asp induced a marked increase in its accu-
mulation in these neuroendocrine tissues [110]. Exposure of
cultured rat Leydig cells to
D
-Asp selectively stimulates
testosterone synthesis after its incorporation into cells
through GLAST but exposure to
L
-and
D
-Glu,
L
-Asp
and
L
-and
D
-aspargine is ineffective [57]. The prevailing
view, that all Glu transporters cloned to date are unable to
differentiate between
L
-and
D
-Asp as a substrate, argues in
favor of a role for endogenous
D
-Asp in testosterone

synthesis following incorporation through particular Glu
transporters expressed at surfaces of Leydig cells.
4 E. Hinoi et al. (Eur. J. Biochem. 271) Ó FEBS 2003
Pancreas
The islet of Langerhans, a pancreatic miniature organ for
several hormones regulating the blood glucose level, is
composed of four major types of endocrine cells, including
glucagon-secreting alpha (a) cells, insulin-secreting beta (b)
cells, somatostatin-secreting d cells, and pancreatic polypep-
tide-secreting F cells. The B cells are the most numerous
and are concentrated in the center of the islet, while the
a cells are larger and less numerous and located at the
periphery of the islet [111,112].
By molecular biological, electrophysiological, and
immunohistochemical studies, it has been reported that
these islet cells should express functional Glu receptors
[58,59,113–119], Glu transporters [120,121] and VGLUT
[43,44,122], suggesting that Glu could function as an
intercellular signal mediator in the islet (Fig. 4). Several
independent lines of evidence indicate that iGluR stimu-
lation positively modulates secretion of both glucagon and
insulin in pancreatic islets. Insulin secretion is stimulated
by AMPA, KA and NMDA in MIN6 cells derived from
insulin-secreting pancreatic b cells [58], and also induced
by both AMPA and KA, but not by NMDA, in isolated
pancreatic islets [113,123]. Similarly, Glu stimulates glu-
cagon secretion via AMPA receptors in rat isolated
pancreatic islets [124]. It has been demonstrated that
mGluRs also participate in hormone secretion from
pancreatic islets. Agonists for group I and II mGluR

subtypes increase the release of insulin in the presence of
glucose at low concentrations, whereas a group III
mGluR agonist inhibits insulin release determined at high
glucose concentrations [119]. The activation of group III
mGluR subtype expressed in pancreatic a cells leads to
inhibition of glucagon release [59]. A high-affinity Glu
transporter expressed in pancreatic islets of Langerhans
modulates insulin secretion stimulated by glucose [121].
Besides expression in neuronal cells, moreover, both
VGLUT1 and VGLUT2 are expressed preferentially in
aTC6 cells, clonal pancreatic a cells,aswellasa cells
isolated from Langerhans islets [43,44]. Low glucose
conditions result in cosecretion of stoichiometric amounts
of Glu and glucagon from aTC6 cells and isolated islets,
Fig. 3. Analyses on localization of Glu trans-
porters in rat testis. (A) Schematic represen-
tation of the histological characteristics of
testis. (B) Rat testicular frozen sections were
fixed with paraformaldehyde and then incu-
bated with DIG-labeled cRNA probe for
GLAST, GLT-1, EAAC1, EAAT4 and
EAAT5 for in situ hybridization. Typical
micrographs are shown in the figures with
similar results in three independent experi-
ments. Scale bars, 50 lm.
Ó FEBS 2003 Glutamate signaling in peripheral tissues (Eur. J. Biochem. 271)5
which triggers GABA secretion from B cells through
GluRs in the isolated islets [44].
Adrenal gland
Adrenal gland consists of two concentric layers; adrenal

cortex which is a yellow peripheral layer, and adrenal
medulla which is a reddish-brown central layer. Adrenal
cortex has the characteristics of steroid-synthesizing tissues,
with subdivision into the three concentric layers – the zona
glomerulosa, the zona fasciculate and the zona reticularis.
The zona glomerulosa secrets mineralocorticoids such as
aldosterone (involved in the maintenance of balance
between electrolytes and water), while the zona fasciculata
and probably the zona reticularis secret the glucocorticoids
corticosterone and cortisol (both relevant to the regulation
of metabolism of carbohydrates, proteins, and fats).
Adrenal medulla is composed of polyhedral parenchymal
cells and supported by a reticular fiber network, and secretes
both adrenaline and noradrenaline [125,126].
The role of Glu in adrenal glands has been investigated in
independent research laboratories including ours. In addi-
tion to Glu [127], iGluR agonists (NMDA, AMPA and
KA) and an mGluR agonist (t-ACPD) [128] all stimulate
catecholamine release from adrenal glands. We have shown
previously the localization of [
3
H]Glu binding sites with
stereoselectivity, high affinity and saturability in rat adrenal
medulla [46,129]. Adrenal [
3
H]Glu binding sites are solubi-
lized by detergents with pharmacological profiles similar to
those found in membrane preparations [130,131]. On
sections of rat [132] and bovine [133] adrenal glands,
[

3
H]Glu binding sites are of high affinity, rapid, saturable,
reversible, stereospecific, and specific to a single population.
In addition to these early studies using conventional
techniques, recent molecular biological analyses support
the presence of particular iGluR subunits in adrenal glands.
Rat adrenal cortex expresses AMPA receptors [134], but
adrenal medulla expresses NMDA receptors [135] as
revealed by in situ hybridization. We could also detect both
mRNA and corresponding proteins for NR1, NR2C and
NR2D in rat adrenal medulla [61]. Furthermore, an
intraperitoneal injection of a blocker of NMDA receptor
channels, but not NMDA itself, leads to a marked increase
in DNA binding activity of the nuclear transcription factor
AP-1 in rat adrenal glands [61]. Single episodes of immo-
bilization stress induces a significant increase in NR1
mRNA expression in adrenal glands [136], while a single
dose of morphine decreases NR1 mRNA in adrenal glands
but not in hippocampus [137]. The prior systemic admin-
istration of NMDA prevents the marked increase in adrenal
AP1 DNA binding induced by subsequent stress [138]. In
rat adrenal glands, the expression of GLAST, which has an
affinity for
D
-Asp, is transiently increased at 3 weeks of age
[139] and localization patterns are almost coincident with
those of endogenous
D
-Asp within the glands. Adrenal
glands receive sympathetic efferent and afferent innerva-

tions in addition to the typical cholinergic innervation
[140,141]. A mechanism for Glu release is detected in bovine
adrenal chromaffin cells [142] and glutamatergic innervation
is also found in sympathoadrenal neurons [143,144].
Pituitary gland
The pituitary gland is connected to the hypothalamus at the
base of the brain and consequentially important anatomical
and functional relationships exist. During embryogenesis,
the pituitary gland develops from the oral ectoderm and
also nerve tissue. The pituitary tissue from nerve tissue is
referred to as the neurohypophysis and consists of the pars
nervosa and infundibulum, while the pituitary gland derived
from oral ectoderm is known as the adenohypophysis and
has three different areas – the pars distalis or anterior lobe,
the pars tuberalis and the pars intermedia [145,146]. A
portion of the pars intermedia is sometimes included in the
terminology Ôposterior lobeÕ.
Molecular biological and immunohistochemical analyses
demonstrated the presence of both non-NMDA [62,147,
148] and NMDA receptors [149,150], while studies using
autoradiographic [151] and receptor binding [47,152] tech-
niques showed the localization of [
3
H]Glu binding in the
pituitary gland. Several independent lines of evidence for
direct regulation by Glu of hormone secretion in isolated
anterior pituitary cells are available in the literature. The
secretion of prolactin is stimulated by Glu in a manner
sensitive to prevention by MK-801 in primary cultures of
anterior pituitary cells [153,154]. Both NMDA and KA

facilitate the secretion of growth hormone in dispersed
anterior pituitary cells [155], while KA is effective in
stimulating gonandotropin secretion from the anterior
pituitary [156]. In single primary cultured rat anterior
pituitary cells, Glu induces a marked increase in cytosolic
free Ca
2+
concentration by a nonsynaptic mechanism [157].
These previous findings show functional expression of
particular subtypes of GluRs in the pituitary gland. We
have also demonstrated that the rat pituitary gland contains
both mRNA and corresponding proteins for GluR6/7
subunits of KA receptors in addition to specific binding sites
of [
3
H]KA [63]. Moreover, an intraperitoneal injection of
KA results in increased AP1 DNA binding in rat pituitary
gland as well as hippocampus [63].
Fig. 4. Glutamate signaling in pancreas. The islet of Langerhans, a
pancreatic miniature organ, is composed of four major types of
endocrine cells, including a, b, d and F cells. Particular functional
GluRs, which could modulate secretion of hormones, are identified in
a cells (AMPA receptors, KA receptors and mGluRs), in b cells
(AMPA receptors), and in d cells (KA receptors), respectively. High-
affinity Glu transporters are expressed in both a and d cells, while
VGLUTs are preferentially expressed in a cells.
6 E. Hinoi et al. (Eur. J. Biochem. 271) Ó FEBS 2003
Pineal gland
Mammalian pineal gland consists of several types of cells,
principally pinealocytes and astroglial cells. Pinealocytes

are endocrine cells with the ability to synthesize and secret
melatonin, a hydrophobic hormone that mediates many
physiological functions such as circadian rhythms and
seasonal reproduction [158,159].
[
3
H]Glu binding is also seen in bovine [48] and rat
[160] pineal membranes. Exogenous Glu leads to inhibition
of the activities of serotonin N-acetyltransferase [161] and
hydroxyindole O-methyltransferase [162], with concomit-
ant suppression of both synthesis and secretion of mela-
tonin [163,164]. Furthermore, expression of particular
GluRs has been confirmed in the mammalian pineal gland
in molecular biological and immunohistochemical studies
to date. These include GluR delta [165], AMPA [166], KA
[167], NMDA [167], group I mGluR [168,169], and group
II mGluR [64,169]. Activation of mGluR3 negatively
regulates noradrenaline-dependent serotonin N-acetyl-
transferase activity and melatonin synthesis in rat pinealo-
cytes [64], while GluR1 triggers exocytosis mediated by
microvesicles (counterparts of the neuronal synaptic ves-
icles) of Glu through activation of
L
-type Ca
2+
channels in
rat pinealocytes [65]. Immunoreactive Glu is enriched in
pinealocytes of gerbil pineal gland [170], and pinealocytes
accumulate Glu in microvesicles [171] for secretion through
Ca

2+
-dependent exocytosis [172–174]. In mammalian
pinealocytes, expression of VGLUT2 is confirmed in
addition to expression in Langerhans islets [43]. Extracel-
lular Glu is sequestered by pinealocytes through a GLT-1
type, Na
+
-dependent Glu transporter expressed in the
plasma membrane [175], whilst immunoreactivity to
GLAST is detected in interstitial glial cells but not in
pinealocytes [176].
Other tissues
In rat hepatocytes, the mGluR agonists, t-ACPD and
quisqualate, not only stimulate polyphosphoinositide
hydrolysis, with an antagonist for group I mGluR subtype
protecting against hypoxic damage [68]. Constitutive
expression of particular iGluRs, including NR1, NR2D,
KA2, and GluR delta-1 subunits [177], is found in lingual
epithelium obtained from rat foliate and vallate papillae, in
addition to truncated a variant of mGluR4a [66,178]. These
GluRs may be responsible for sensory transduction of
ÔumamiÕ taste in addition to the taste-specific receptor,
T1R1/T1R3 dimer, which is coupled to G-protein for
activation by dietary Glu [179]. The expression of both
group I and group II mGluRs is demonstrated in mouse
thymus, isolated thymocytes and thymic stromal cell line
[69]. The quisqualate-sensitive binding of [
3
H]Glu is detected
in T lymphocytes [180], whereas in human lymphocytes,

Glu and other iGluR agonists potentiate the rise of
intracellular free Ca
2+
concentrations induced by the
mAb anti-CD(3) or phytohaemagglutinin [73]. In human
platelets, Glu or NMDA is antagonistic for the aggregating
activity of arachidonic acid, with detection of both
NMDA-displaceable [
3
H]Glu binding and [
3
H]MK-801
binding [74,181]. In megakaryocytes, both mRNA and
proteins for NMDA receptors are expressed in addition to
autoradiographic binding of [
3
H]MK-801. Blockade of
NMDA receptors, by specific antagonists, inhibits the
phorbol myristate acetate-induced increases in cellular size,
CD41 expression and mutual adhesion in MEG-01 clonal
megakaryoblastic cells [71]. Both NMDA and non-NMDA
receptors are expressed in rat heart [75], while the stimula-
tion of iGluRs by Glu, NMDA, AMPA and KA invariably
leads to an increase in intracellular Ca
2+
oscillation
frequency in cultured rat myocardial cells [182]. Moreover,
mGluRs (mGluR1, mGluR2/3, and mGluR5) are also
expressed in rat heart [183]. Expression of NMDA receptors
has been demonstrated in lung [184,185] and NMDA elicits

acute high-permeability edema in perfused rat lungs [67].
Nitric oxide synthase inhibitors and the neuropeptide
vasoactive intestinal peptide protect against injuries medi-
ated by NMDA receptors in lung [186]. In whole guinea-pig
lungs perfused via the trachea, NMDA increases airway
perfusion pressure in a manner that is totally abolished by
MK-801 [187]. Expression of particular GluRs is shown in
skin including keratinocytes for NMDA and AMPA
receptors [72,188] and melanocytes for mGluR5 [189].
Blockade of NMDA receptors expressed in keratinocytes,
induces stratification and other characteristics of differen-
tiation [72]. Another excitatory amino acid,
L
-Asp, inhibits
acid secretion stimulated by histamine, without affecting
that by oxotremorine and pentagastrin in an everted
preparation of isolated rat stomach [190], while an NMDA
receptor antagonist blocks the inhibitory effect of
L
-Asp on
histamine-stimulated acid secretion [190].
Conclusions
It appears that the molecular machinery required for Glu
signaling, which includes iGluRs, mGluRs, Glu transport-
ers and VGLUTs, are constitutively expressed to play a role
crucial for maintenance of the functionality and integrity
in several neuronal and non-neuronal peripheral tissues
(Table 2). Circulating Glu seems to be sufficient for
saturation of any of the subtype of iGluRs and mGluRs
expressed at peripheral locations with intimate contacts to

the blood supply. One possibility is that peripheral GluRs
could be under the influence of tonic stimulation by Glu in
plasma, whereas an alternative explanation is that circula-
ting Glu may not easily gain access to peripheral GluRs due
to particular protection mechanisms against the direct
exposure to blood supply. From this point of view, the exact
origin of an endogenous agonist is still unclear in contrast to
the conclusive evidence for expression of mRNA for
particular iGluR and mGluR subtypes as well as for
VGLUTs in peripheral tissues.
In any case, Glu seems to play a dual role in mechanisms
underlying maintenance of homeostasis – an excitatory
neurotransmitter in the central neurocrine system and an
extracellular signal mediator in the peripheral autocrine
and/or paracrine process. Searches for ligands at GluRs and
Glu transporters may need to be rerun with regard to
permeability to the brain across blood–brain barrier.
Nonpermeable compounds could be of a great benefit as
therapeutic drugs without serious central side-effects for a
variety of disorders associated with malfunction of Glu
signaling in the periphery. In contrast to the prevailing view,
a new concept could be required for classification of
Ó FEBS 2003 Glutamate signaling in peripheral tissues (Eur. J. Biochem. 271)7
extracellular signal mediators between different cells for
neurocrine, endocrine, autocrine, and paracrine communi-
cations.
Acknowledgements
This work was supported in part by Grants-in-Aids for Scientific
Research to E.H. and Y.Y. from the Ministry of Education, Culture,
Sports, Science and Technology, and to Y.Y. from the Ministry of

Health, Labour and Welfare, Japan, respectively.
References
1. Hollmann, M., O’Shea-Greenfield, A., Rogers, S.W. & Heine-
mann, S. (1989) Cloning by functional expression of a member of
the glutamate receptor family. Nature 342, 643–648.
2. Nakanishi, N., Shneider, N.A. & Axel, R. (1990) A family of
glutamate receptor genes: evidence for the formation of hetero-
multimeric receptors with distinct channel properties. Neuron 5,
569–581.
3. Yoneda, Y., Kuramoto, N., Kitayama, T. & Hinoi, E. (2001)
Consolidation of transient ionotropic glutamate signals through
nuclear transcription factors in the brain. Prog. Neurobiol. 63,
697–719.
4. Masu,M.,Tanabe,Y.,Tsutida,K.,Shigemoto,R.&Nakanishi,
S. (1991) Sequence and expression of a metabotropic glutamate
receptor. Nature 349, 760–765.
5. Tanabe,Y.,Masu,M.,Ishii,T.,Shigemoto,R.&Nakanishi,S.
(1992) A family of metabotropic glutamate receptors. Neuron 8,
169–179.
6. Wisden, W. & Seeburg, P.H. (1993) Mammalian ionotropic
glutamate receptors. Curr. Opin. Neurobiol. 3, 291–298.
7. Hollmann, M. & Heinemann, S. (1994) Cloned glutamate
receptors. Ann. Rev. Neurosci. 17, 31–108.
8. Reynolds, I.J. & Miller, R.J. (1988) Multiple sites for the reg-
ulation of the N-methyl-
D
-aspartate receptor. Mol. Pharmacol.
33, 581–584.
9. MacDonald, J.F. & Nowak, L.M. (1990) Mechanisms of
blockade of excitatory amino acid receptor channels. Trends.

Pharmacol. Sci. 11, 167–172.
10. Lynch, D.R. & Guttmann, R.P. (2001) NMDA receptor phar-
macology: perspectives from molecular biology. Curr. Drug
Targets 2, 215–231.
11. Das, S., Sakaki, Y.F., Rothe, T., Premkumar, L.S., Takasu, M.,
Crandall, J.E., Dikkes, P., Conner, D.A., Rayudu, P.V., Cheung,
W., Chen, H.S., Lipton, S.A. & Nakanishi, N. (1998) Increased
NMDA current and spine density in mice lacking the NMDA
receptor subunit NR-3A. Nature 393, 377–381.
12. Nishi, M., Hinds, H., Lu, H.P., Kawata, M. & Hayashi, Y.
(2001) Motoneuron-specific expression of NR-3B, a novel
NMDA-type glutamate receptor subunit that works in a domi-
nant-negative manner. J. Neurosci. 21, 1–6.
13. Matsuda, K., Kamiya, Y., Matsuda, S. & Yuzaki, M. (2002)
Cloning and characterization of a novel NMDA receptor subunit
NR3B: a dominant subunit that reduces calcium permeability.
Mol. Brain Res. 100, 43–52.
14. Araki, K., Meguro, H., Kushiya, E., Takayama, C., Inoue, Y. &
Mishina, M. (1993) Selective expression of the glutamate receptor
channel delta 2 subunit in cerebellar Purkinje cells. Biochem.
Biophys. Res. Commun. 197, 1267–1276.
15. Lomeli, H., Sprengel, R., Laurie, D.J., Kohr, G., Herb, A. &
Seeburg, P. (1993) The rat delta-1 and delta-2 subunits extend
the excitatory amino acid receptor family. FEBS Lett. 315, 318–
322.
16. Scheetz, A.J. & Constantine-Paton, M. (1994) Modulation of
NMDA receptor function: implications for vertebrate neural
development. FASEB J. 8, 745–752.
17. Riedel, G., Platt, B. & Micheau, J. (2003) Glutamate receptor
function in learning and memory. Behav. Brain Res. 140, 1–47.

18. Choi, D.W. (1988) Glutamate neurotoxicity and diseases of the
nervous system. Neuron 1, 623–634.
19. Goff, D.C. & Wine, L. (1997) Glutamate in schizophrenia: clin-
ical and research implications. Schizophr. Res. 27, 157–168.
20. Cull-Candy, S., Brickley, S. & Farrant, M. (2001) NMDA
receptor subunits: diversity, development and disease. Curr. Opin.
Neurobiol. 11, 327–335.
21. Schousboe, A. & Divac, I. (1979) Difference in glutamate uptake
in astrocytes cultured from different brain regions. Brain Res.
177, 407–409.
22. Danbolt, N.C. (2001) Glutamate uptake. Prog. Neurobiol. 65,
1–105.
23. Storck, T., Schulte, S., Hofmann, K. & Stoffel, W. (1992)
Structure, expression, and functional analysis of a Na
+
-
dependent glutamate/aspartate transporter from rat brain. Proc.
NatlAcad.Sci.USA89, 10955–10959.
24. Tanaka, K. (1993) Cloning and expression of a glutamate
transporter from mouse brain. Neurosci. Lett. 159, 183–186.
25. Pines, G., Danbolt, N.C., Bjoras, M., Zhang, Y., Bendahan, A.,
Eide, L., Koepsell, H., Storm-Mathisen, J., Seeberg, E. & Kan-
ner, B.I. (1992) Cloning and expression of a rat brain
L
-glutamate
transporter. Nature 360, 464–467.
26. Kanai, Y. & Hediger, M.A. (1992) Primary structure and func-
tional characterization of a high-affinity glutamate transporter.
Nature 360, 467–471.
27. Fairman, W.A., Vandenberg, R.J., Arriza, J.L., Kavanaugh,

M.P. & Amara, S.G. (1995) An excitatory amino-acid
transporter with properties of a ligand-gated chloride channel.
Nature 375, 599–603.
28. Arriza, J.L., Eliasof, S., Kavanaugh, M.P. & Amara, S.G. (1997)
Excitatory amino acid transporter 5, a retinal glutamate trans-
Table 2. Expression of Glu signaling molecules in several neuronal and
non-neuronal peripheral tissues. iGluR (A, AMPA receptors; K, KA
receptors; N, NMDA receptors); mGluR (I, group I mGluR; II, group
II mGluR; III, group III mGluR); GluT (1, GLAST; 2, GLT-1; 3,
EAAC1; 4, EAAT4; 5, EAAT5); VGLUT (1, VGLUT1; 2, VGLUT2;
3, VGLUT3). ND, not determined; ?, unidentified.
Tissue/cell iGluR mGluR GluT VGLUT
Osteoblast + (A,K,N) + (I, II) + (1) + (1)
Osteoclast + (N) ND ND ND
Osteocyte ND ND + (1) ND
Testis + (A,K,N) + (I, II) + (1,2,5) ND
Pancreas + (A,K,N) + (I, II, III) + (2) + (1,2)
Adrenal gland + (A,K,N) ? + (1) ND
Pituitary gland + (A,K,N) ND ND ND
Pineal gland + (A,K,N) + (I, II) + (1,2) + (2)
Hepatocyte ND +(I) ND ND
Lingual
epithelium
+ (K,N) + (III) ND ND
Thymus ND +(I, II) ND ND
T lymphocyte ? ND ND ND
Platelet ? ND ND ND
Megakaryocyte + (N) ND ND ND
Heart + (A,K,N) + (I, II) + (1,3) ND
Lung + (N) ND ND ND

Keratinocyte + (A,N) ND + (2,3) ND
Melanocyte ND +(I) ND ND
8 E. Hinoi et al. (Eur. J. Biochem. 271) Ó FEBS 2003
porter coupled to a chloride conductance. Proc. Natl Acad. Sci.
USA 94, 4155–4160.
29. Lehre, K.P., Levy, L.M., Ottersen, O.P., Storm-Mathisen, J. &
Danbolt, N.C. (1995) Differential expression of two glial gluta-
mate transporters in the rat brain: quantitative and
immunocytochemical observations. J. Neurosci. 15, 1835–1853.
30. Hamberger, A., Chiang, G.H., Sandoval, E. & Cotman, C.W.
(1979) Glutamate as a CNS transmitter. II. Regulation of
synthesis in the releasable pool. Brain Res. 168, 531–541.
31. Kanai, Y., Bhide, P.G., DiFiglia, M. & Hediger, M.A. (1995)
Neuronal high-affinity glutamate transport in the rat central
nervous system. Neuroreport 6, 2357–2362.
32. Pow, D.V. & Barnett, N.L. (2000) Developmental expression
of excitatory amino acid transporter 5: a photoreceptor and
bipolar cell glutamate transporter in rat retina. Neurosci. Lett.
280, 21–24.
33. Bellocchio, E.E., Reimer, R.J., Fremeau, R.T. Jr & Edwards,
R.H. (2000) Uptake of glutamate into synaptic vesicles by an
inorganic phosphate transporter. Science 289, 957–960.
34. Takamori, S., Rhee, J.S., Rosenmund, C. & Jahn, R. (2000)
Identification of a vesicular glutamate transporter that defines a
glutamatergic phenotype in neurons. Nature 407, 189–194.
35. Bai, L., Xu, H., Collins, J.F. & Ghishan, F.K. (2001) Molecular
and functional analysis of a novel neuronal vesicular glutamate
transporter. J. Biol. Chem. 276, 36764–33679.
36. Varoqui, H., Schafer, M.K., Zhu, H., Weihe, E. & Erickson, J.D.
(2002) Identification of the differentiation-associated Na+/PI

transporter as a novel vesicular glutamate transporter expressed
in a distinct set of glutamatergic synapses. J. Neurosci. 22,
142–155.
37. Fremeau, R.T. Jr, Troyer, M.D., Pahner, I., Nygaard, G.O.,
Tran, C.H., Reimer, R.J., Bellocchio, E.E., Fortin, D., Storm-
Mathisen, J. & Edwards, R.H. (2001) The expression of vesicular
glutamate transporters defines two classes of excitatory synapse.
Neuron 31, 247–260.
38. Herzog,E.,Bellenchi,G.C.,Gras,C.,Bernard,V.,Ravassard,P.,
Bedet, C., Gasnier, B., Giros, B. & El Mestikaway, S. (2001) The
existence of a second vesicular glutamate transporter specifies
subpopulations of glutamatergic neurons. J. Neurosci. 21,
RC181.
39. Takamori, S., Rhee, J.S., Rosenmund, C. & Jahn, R. (2001)
Identification of differentiation-associated brain-specific phos-
phate transporter as a second vesicular glutamate transporter
(VGLUT2). J. Neurosci. 21, RC182.
40. Kaneko, T. & Fujiyama, F. (2002) Complementary distribution
of vesicular glutamate transporters in the central nervous system.
Neurosci. Res. 42, 243–250.
41. Schafer,M.K.,Varoqui,H.,Defamie,N.,Weihe,E.&Erickson,
J.D. (2002) Molecular cloning and functional identification of
mouse vesicular glutamate transporter 3 and its expression in
subsets of novel excitatory neurons. J. Biol. Chem. 277, 50734–
50748.
42. Fremeau, R.T. Jr, Burman, J., Qureshi, T., Tran, C.H., Proctor,
J., Johnson, J., Zhang, H., Sulzer, D., Copenhagen, D.R., Storm-
Mathisen, J., Reimer, R.J., Chaudhry, F.A. & Edwards, R.H.
(2002) The identification of vesicular glutamate transporter 3
suggests novel modes of signaling by glutamate. Proc. Natl Acad.

Sci. USA 99, 14488–14493.
43. Hayashi, M., Otsuka, M., Morimoto, R., Hirota, S., Yatsushiro,
S., Takeda, J., Yamamoto, A. & Moriyama, Y. (2001) Differ-
entiation-associated Na
+
-dependent inorganic phosphate
cotransporter (DNPI) is a vesicular glutamate transporter in
endocrine glutamatergic systems. J. Biol. Chem. 276, 43400–
43406.
44. Hayashi, M., Yamada, H., Uehara, S., Morimoto, R.,
Muroyama, A., Yatsushiro, S., Takeda, J., Yamamoto, A. &
Moriyama, Y. (2003) Secretory granule-mediated co-secretion
of
L
-glutamate and glucagon triggers glutamatergic signal
transmission in islets of Langerhans. J. Biol. Chem. 278, 1966–
1974.
45. Skerry, T.M. & Genever, P.G. (2001) Glutamate signalling in
non-neuronal tissues. Trend Pharmacol. Sci. 22, 174–181.
46. Yoneda, Y. & Ogita, K. (1986) Localization of [
3
H]glutamate
binding sites in rat adrenal medulla. Brain Res. 383, 387–391.
47. Yoneda, Y. & Ogita, K. (1986) [
3
H]Glutamate binding sites in the
rat pituitary. Neurosci. Res. 3, 430–435.
48. Govitrapong, P., Ebadi, M. & Murrin, L.C. (1986) Identification
of a Cl
–

/Ca
2+
-dependent glutamate (quisqualate) binding site in
bovine pineal organ. J. Pineal Res. 3, 223–234.
49. Moroni, F., Luzzi, S., Franchi-Micheli, S. & Zilletti, L. (1986)
ThepresenceofN-methyl-
D
-aspartate-type receptors for glu-
tamic acid in the guinea-pig myenteric plexus. Neurosci. Lett. 68,
57–62.
50. Luzzi, S., Zilletti, L., Franchi-Micheli. A.M. & Moroni, F. (1988)
Agonists, antagonists, and modulators of excitatory amino acid
receptors in the guinea-pig myenteric plexus. Br.J.Pharmacol.
95, 1271–1277.
51. Shannon, H.E. & Sawyer, B.D. (1989) Glutamate receptors of
N-methyl-
D
-aspartate subtype in the myenteric plexus of guinea-
pig ileum. J. Pharmacol. Exp. Ther. 251, 518–523.
52. Aas, P., Tanso, R. & Fonnum, F. (1989) Stimulation of peri-
pheral cholinergic nerves by glutamate indicates a new peripheral
glutamate receptor. Eur. J. Pharmacol. 164, 93–102.
53. Mason, D.J., Suva, L.J., Genever, P.G., Patton, A.J., Steuckle,
S., Hillam, R.A. & Skerry, T.M. (1997) Mechanically
regulated expression of a neural glutamate transporter in bone: a
role for excitatory amino acids as osteotropic agents? Bone 20,
199–205.
54. Chenu, C., Serre, C.M., Raynal, C., Burt-Pichat, B. & Delmas,
P.D. (1998) Glutamate receptors are expressed by bone cells and
are involved in bone resorption. Bone 22, 295–299.

55. Hinoi, E., Fujimori, S. & Yoneda, Y. (2003) Modulation of cel-
lular differentiation by N-methyl-
D
-aspartate receptors in osteo-
blasts. FASEB J. 17, 1532–1534.
56. Storto, M., Sallese, M., Salvatore, L., Poulet, R., Condorelli,
D.F., Dell’Albani, P., Marcello, M.F., Romeo, R., Piomboni, P.,
Barone,N.,Nicoletti,F.,DeNicoletti,F.&Blasi,A.(2001)
Expression of metabotropic glutamate receptors in the rat and
human testis. J. Endocrinol. 170, 71–78.
57. Nagata, Y., Homma, H., Lee, J.A. & Imai, K. (1999)
D
-Aspartate
stimulation of testosterone synthesis in rat Leydig cells. FEBS
Lett. 444, 160–164.
58. Gonoi, T., Mizuno, N., Inagaki, N., Kuromi, H., Seino, Y.,
Miyazaki, J. & Seino, S. (1994) Functional neuronal ionotropic
glutamate receptors are expressed in the non-neuronal cell line
MIN6. J. Biol. Chem. 269, 16989–16992.
59. Tong, Q., Ouedraogo, R. & Kirchgessner, A.L. (2002) Locali-
zation and function of group III metabotropic glutamate
receptors in rat pancreatic islets. Am. J. Physiol. Endocrinol.
Metab. 282, 1324–1333.
60. Kristensen, P. (1993) Differential expression of AMPA glutamate
receptor mRNA in the rat adrenal glands. FEBS Lett. 332, 14–18.
61. Hinoi,E.,Fujimori,S.,Nakamura,Y.,Balcar,V.J.,Kubo,K.,
Ogita, K. & Yoneda, Y. (2002) Constitutive expression of het-
erologous N-methyl-
D
-aspartate receptor subunits in rat adrenal

medulla. J. Neurosci. Res. 68, 36–45.
62. Kiyama, H., Sato, K. & Tohyama, M. (1993) Characteristic
localization of non-NMDA type glutamate receptor subunits in
the rat pituitary gland. BrainRes.Mol.BrainRes.19, 262–268.
63. Hinoi, E. & Yoneda, Y. (2001) Expression of GluR6/7 subunits
of kainate receptors in rat adenohypophysis. Neurochem. Int.
38, 539–547.
Ó FEBS 2003 Glutamate signaling in peripheral tissues (Eur. J. Biochem. 271)9
64. Yamada, H., Yatsushiro, S., Ishio, S., Hayashi, M., Nishi, T.,
Yamamoto, A., Futai, M., Yamaguchi, A. & Moriyama, Y.
(1998) Metabotropic glutamate receptors negatively regulate
melatonin synthesis in rat pinealocytes. J. Neurosci. 18, 2056–
2062.
65. Yatsushiro, S., Yamada, H., Hayashi, M., Yamamoto, A. &
Moriyama, Y. (2000) Ionotropic glutamate receptors trigger
microvesicle-mediated exocytosis of
L
-glutamate in rat pine-
alocytes. J. Neurochem. 75, 288–297.
66. Chaudhari, N., Landin, A.M. & Roper, S.D. (2000) A metabo-
tropic glutamate receptor variant functions as a taste receptor.
Nat. Neurosci. 3, 113–119.
67. Said, S.I., Berisha, H.I. & Pakbaz, H. (1996) Excitotoxicity in the
lung: N-methyl-
D
-aspartate-induced, nitric oxide-dependent,
pulmonary edema is attenuated by vasoactive intestinal peptide
and by inhibitors of poly (ADP-ribose) polymerase. Proc. Natl.
Acad. Sci. USA 93, 4688–4692.
68. Storto, M., de Grazia, U., Knopfel, T., Canonico, P.L., Copani,

A., Richelmi, P., Nicoletti, F. & Vairetti, M. (2000) Selective
blockade of mGlu5 metabotropic glutamate receptors protects
rat hepatocytes against hypoxic damage. Hepatology 31, 649–
655.
69. Storto, M., de Grazia, U., Battaglia, G., Felli, M.P., Maroder,
M., Gulino, A., Ragona, G., Nicoletti, F., Screpanti, I., Frati, L.
& Calogero, A. (2000) Expression of metabotropic glutamate
receptors in murine thymocytes and thymic stromal cells.
J. Neuroimmunol. 109, 112–120.
70. Krizbai, I.A., Deli, M.A., Pestenacz, A., Siklos, L., Szabo, C.A.,
Andras, I. & Joo, F. (1998) Expression of glutamate receptors on
cultured cerebral endothelial cells. J. Neurosci. Res. 54, 814–819.
71. Genever, P.G., Wilkinson, D.J., Patton, A.J., Peet, N.M., Hong,
Y., Mathur, A., Erusalimsky, J.D. & Skerry, T.M. (1999)
Expression of a functional N-methyl-
D
-aspartate-type glutamate
receptor by bone marrow megakaryocytes. Blood 93, 2876–2883.
72. Genever, P.G., Maxfield, S.J., Kennovin, G.D., Maltman, J.,
Bowgen, C.J., Raxworthy, M.J. & Skerry, T.M. (1999) Evidence
for a novel glutamate-mediated signaling pathway in keratino-
cytes. J. Invest. Dermatol. 112, 337–342.
73. Lombardi, G., Dianzani, C., Miglio, G., Canonico, P.L. &
Fantozzi, R. (2001) Characterization of ionotropic glutamate
receptors in human lymphocytes. Br. J. Pharmacol. 133, 936–944.
74. Franconi, F., Miceli, M., De Montis, M.G., Crisafi, E.L., Ben-
nardini, F. & Tagliamonte, A. (1996) NMDA receptors play an
anti-aggregating role in human platelets. Thromb. Haemost. 76,
84–87.
75. Gill, S.S., Pulido, O.M., Mueller, R.W. & McGuire, P.F. (1998)

Molecular and immunochemical characterization of the iono-
tropic glutamate receptors in the rat heart. Brain Res. Bull. 46,
429–434.
76. Ducy, P., Schinke, T. & Karsenty, G. (2000) The osteoblast: a
sophisticated fibroblast under central surveillance. Science 289,
1501–1504.
77. Teitelbaum, S.L. (2000) Bone resorption by osteoclasts. Science
289, 1504–1508.
78. Patton, A.J., Genever, P.G., Birch, M.A., Suva, L.J. & Skerry,
T.M. (1998) Expression of an N-methyl-
D
-aspartate-type
receptor by human and rat osteoblasts and osteoclasts suggests
a novel glutamate signaling pathway in bone. Bone 22,
645–649.
79. Hinoi, E., Fujimori, S., Nakamura, Y. & Yoneda, Y. (2001)
Group III metabotropic glutamate receptors in cultured rat
calvarial osteoblasts. Biochem. Biophys. Res. Commun. 281,
341–346.
80. Espinosa,L.,Itzstein,C.,Cheynel,H.,Delmas,P.D.&Chenu,C.
(1999) Active NMDA glutamate receptors are expressed by
mammalian osteoclasts. J. Physiol. 518, 47–53.
81. Gu, Y., Genever, P.G., Skerry, T.M. & Publicover, S.J. (2002)
The NMDA type glutamate receptors expressed by primary rat
osteoblasts have the same electrophysiological characteristics as
neuronal receptors. Calcif. Tissue Int. 70, 194–203.
82. Peet, N.M., Grabowski, P.S., Laketic-Ljubojevic, I. & Skerry,
T.M. (1999) The glutamate receptor antagonist MK801 mod-
ulates bone resorption in vitro by a mechanism predominantly
involving osteoclast differentiation. FASEB J. 13, 2179–2185.

83. Laketic-Ljubojevic, I., Suva, L.J., Maathuis, F.J., Sanders, D. &
Skerry, T.M. (1999) Functional characterization of N-methyl-
D
-aspartic acid-gated channels in bone cells. Bone 25, 631–637.
84. Hinoi, E., Fujimori, S., Takemori, A., Kurabayashi, H.,
Nakamura, Y. & Yoneda, Y. (2002) Demonstration of expres-
sion of mRNA for particular AMPA and kainate receptor sub-
units in immature and mature cultured rat calvarial osteoblasts.
Brain Res. 943, 112–116.
85. Genever, P.G. & Skerry, T.M. (2001) Regulation of spontaneous
glutamate release activity in osteoblastic cells and its role in dif-
ferentiation and survival: evidence for intrinsic glutamatergic
signaling in bone. FASEB J. 15, 1586–1588.
86. Hinoi,E.,Fujimori,S.,Takarada,T.,Taniura,H.&Yoneda,Y.
(2002) Facilitation of glutamate release by ionotropic glutamate
receptors in osteoblasts. Biochem. Biophys. Res. Commun. 297,
452–458.
87. Gu, Y. & Publicover, S.J. (2000) Expression of functional
metabotropic glutamate receptors in primary cultured rat os-
teoblasts. Cross-talk with N-methyl-
D
-aspartate receptors.
J. Biol. Chem. 275, 34252–34259.
88. Huggett, J., Vaughan-Thomas, A. & Mason, D. (2000) The
open reading frame of the Na
+
-dependent glutamate transporter
GLAST-1 is expressed in bone and a splice variant of
this molecule is expressed in bone and brain. FEBS Lett. 485,
13–18.

89. Gray, C., Marie, H., Arora, M., Tanaka, K., Boyde, A., Jones, S.
& Attwell, D. (2001) Glutamate does not play a major role in
controlling bone growth. J. Bone Miner. Res. 16, 742–749.
90. Bhangu, P.S., Genever, P.G., Spencer, G.J., Grewal, T.S. &
Skerry, T.M. (2001) Evidence for targeted vesicular glutamate
exocytosis in osteoblasts. Bone 29, 16–23.
91. Ducy,P.,Zhang,R.,Geoffroy,V.,Ridall,A.L.&Karsenty,G.
(1997) Osf2/Cbfa1: a transcriptional activator of osteoblast dif-
ferentiation. Cell 89, 747–754.
92. Komori, T., Yagi, H., Nomura, S., Yamaguchi, A., Sasaki, K.,
Deguchi, K., Shimizu, Y., Bronson, R.T., Gao, Y.H., Inada, M.,
Sato, M., Okamoto, R., Kitamura, Y., Yoshiki, S. & Kishimoto,
T. (1997) Targeted disruption of Cbfa1 results in a complete lack
of bone formation owing to maturational arrest of osteoblasts.
Cell 89, 755–764.
93. Otto, F., Thornell, A.P., Crompton, T., Denzel, A., Gilmour,
K.C., Rosewell, I.R., Stamp, G.W., Beddington, R.S., Mundlos,
S., Olsen, B.R., Selby, P.B. & Owen, M.J. (1997) Cbfa1, a can-
didate gene for cleidocranial dysplasia syndrome, is essential for
osteoblast differentiation and bone development. Cell 89,
765–771.
94. Mundlos, S., Otto, F., Mundlos, C., Mulliken, J.B., Aylsworth,
A.S., Albright, S., Lindhout, D., Cole, W.G., Henn, W., Knoll,
J.H., Owen, M.J., Mertelsmann, R., Zabel, B.U. & Olsen, B.R.
(1997) Mutations involving the transcription factor CBFA1
cause cleidocranial dysplasia. Cell 89, 773–779.
95. Komori, T. (2000) A fundamental transcription factor for bone
and cartilage. Biochem. Biophys. Res. Commun. 276, 813–816.
96. Karsenty, G. (2000) Role of Cbfa1 in osteoblast differentiation
and function. Semin. Cell Dev. Biol. 11, 343–346.

97. Yang, X. & Karsenty, G. (2002) Transcription factors in bone:
developmental and pathological aspects. Trends Mol. Med. 8,
340–345.
10 E. Hinoi et al. (Eur. J. Biochem. 271) Ó FEBS 2003
98. Hohmann, E.L., Elde, R.P., Rysavy, J.A., Einzig, S. & Gebhard,
R.L. (1986) Innervation of periosteum and bone by sympathetic
vasoactive intestinal peptide-containing nerve fibers. Science 232,
868–871.
99. Serre, C.M., Farlay, D., Delmas, P.D. & Chenu, C. (1999) Evi-
dence for a dense and intimate innervation of the bone tissue,
including glutamate-containing fibers. Bone 25, 623–629.
100. Takeda, S., Elefteriou, F., Levasseur, R., Liu, X., Zhao, L.,
Parker, K.L., Armstrong, D., Ducy, P. & Karsenty, G. (2002)
Leptin regulates bone formation via the sympathetic nervous
system. Cell 111, 305–317.
101. Fujisawa, M. (2001) Cell-to-cell cross talk in the testis. Urol. Res.
29, 144–151.
102. Cheng, C.Y. & Mruk, D.D. (2002) Cell junction dynamics in the
testis: Sertoli–germ cell interactions and male contraceptive
development. Physiol. Rev. 82, 825–874.
103. Silva, F.R., Leite, L.D. & Wassermann, G.F. (2002) Rapid signal
transduction in Sertoli cells. Eur. J. Endocrinol. 147, 425–433.
104. Dym, M. & Fawcett, D.W. (1970) The blood–testis barrier in the
rat and the physiological compartmentation of the seminiferous
epithelium. Biol. Reprod. 3, 308–326.
105. Holash, J.A., Harik, S.I., Perry, G. & Stewart, P.A. (1993) Barrier
properties of testis microvessels. Proc.NatlAcad.Sci.USA90,
11069–11073.
106. Gill, S.S. & Pulido, O.M. (2001) Glutamate receptors in periph-
eral tissues: current knowledge, future research, and implication

for toxicology. Toxicol. Pathol. 29, 2089–2223.
107. Gill, S.S., Mueller, R.W., McGuire, P.F. & Pulido, O.M. (2000)
Potential target sites in peripheral tissues for excitatory neuro-
transmission and excitotoxicity. Toxicol. Pathol. 28, 277–284.
108. Tanaka, K. (1993) Expression cloning of a rat glutamate trans-
porter. Neurosci. Res. 16, 149–153.
109. Shashidharan, P., Huntley, G.W., Meyer, T., Morrison, J.H. &
Plaitakis, A. (1994) Neuron-specific human glutamate transpor-
ter; molecular cloning, characterization and expression in human
brain. Brain Res. 662, 245–250.
110. D’Aniello, A., Di Flore, M.M., Fisher, G.H., Milone, A., Seleni,
A., D’Aniello, S., Perna, A.F. & Ingrosso, D. (2000) Occurrence
of
D
-aspartic acid and N-methyl-
D
-aspartic acid in rat neuro-
endocrine tissues and their role in the modulation of lutenizing
hormone and growth hormone release. FASEB J. 14, 699–714.
111. Bramblett, D.E., Huang, H.P. & Tsai, M.J. (2000) Pancreatic islet
development. Adv. Pharmacol. 47, 255–315.
112. Moldovan, S. & Brunicardi, F.C. (2001) Endocrine pancreas:
summary of observations generated by surgical fellows. World J.
Surg. 25, 468–473.
113. Inagaki, N., Kuromi, H., Gonoi, T., Okamoto, Y., Ishida, H.,
Seino, Y., Kaneko, T., Iwanaga, T. & Seino, S. (1995) Expression
and role of ionotropic glutamate receptors in pancreatic islet cells.
FASEB J. 9, 686–691.
114. Molnar, E., Varadi, A., McIlhinney, R.A. & Ashcroft, S.J. (1995)
Identification of functional ionotropic glutamate receptor pro-

teins in pancreatic beta-cells and in islets of Langerhans. FEBS
Lett. 371, 253–257.
115. Weaver, C.D., Yao, T.L., Powers, A.C. & Verdoorn, T.A. (1996)
Differential expression of glutamate receptor subtypes in rat
pancreatic islets. J. Biol. Chem. 271, 12977–12984.
116. Liu, H.P., Tay, S.S. & Leong, S.K. (1997) Localization of glu-
tamate receptor subunits of the alpha-amino-3-hydroxy-5-
methyl-4-isoxazolepropionate (AMPA) type in the pancreas of
newborn guinea pigs. Pancreas 14, 360–368.
117. Weaver, C.D., Partridge, J.G., Yao, T.L., Moates, J.M.,
Magnuson, M.A. & Verdoorn, T.A. (1998) Activation of
glycine and glutamate receptors increases intracellular calcium in
cells derived from the endocrine pancreas. Mol. Pharmacol. 54,
639–646.
118. Morley, P., MacLean, S., Gendron, T.F., Small, D.L., Tremblay,
R.,Durkin,J.P.&Mealing,G.(2000)Pharmacologicaland
molecular characterization of glutamate receptors in the MIN6
pancreatic beta-cell line. Neurol. Res. 22, 379–385.
119. Brice, N.L., Varadi, A., Ashcroft, S.J. & Molnar, E. (2002)
Metabotropic glutamate and GABA (B) receptors contribute to
the modulation of glucose-stimulated insulin secretion in pan-
creatic beta cells. Diabetologia 45, 242–252.
120. Manfras, B.J., Rudert, W.A., Trucco, M. & Boehm, B.O. (1994)
Cloning and characterization of a glutamate transporter cDNA
from human brain and pancreas. Biochim. Biophys. Acta. 1195,
185–188.
121. Weaver, C.D., Gundersen, V. & Verdoorn, T.A. (1998) A high
affinity glutamate/aspartate transport system in pancreatic islets
of Langerhans modulates glucose-stimulated insulin secretion.
J. Biol. Chem. 273, 1647–1653.

122. Bai, L., Zhang, X. & Ghishan, F.K. (2003) Characterization of
vesicular glutamate transporter in pancreatic alpha – and beta-
cells and its regulation by glucose. Am.J.Physiol.Gastrointest.
Liver Physiol. 284, G808–G814.
123. Bertrand, G., Gross, R., Puech, R., Loubatieres-Mariani, M.M.
& Bockaert, J. (1993) Evidence for a glutamate receptor of the
AMPA subtype which mediates insulin release from rat perfused
pancreas. Br. J. Pharmacol. 106, 354–359.
124. Bertrand, G., Gross, R., Puech, R., Loubatieres-Mariani, M.M.
& Bockaert, J. (1993) Glutamate stimulates glucagon secretion
via an excitatory amino acid receptor of the AMPA subtype in
rat pancreas. Eur. J. Pharmacol. 237, 45–50.
125. Rosol, T.J., Yarrington, J.T., Latendresse, J. & Capen, C.C.
(2001) Adrenal gland: structure, function, and mechanisms of
toxicity. Toxicol. Pathol. 29, 41–48.
126. Kemppainen, R.J. & Behrend, E.N. (1997) Adrenal physiology.
Vet. Clin. North Am. Small Anim. Pract. 27, 173–186.
127. Nishikawa, T., Morita, K., Kinjo, K. & Tsujimoto, A. (1982)
Stimulation of catecholamine release from isolated adrenal
glands by some amino acids. Jpn. J. Pharmacol. 32, 291–297.
128. Gonzalez, M.P., Herrero, M.T., Vicente, S. & Oset-Gasque, M.J.
(1998) Effect of glutamate receptor agonists on catecholamine
secretion in bovine chromaffin cells. Neuroendocrinology 67,
181–189.
129. Yoneda, Y. & Ogita, K. (1987) Enhancement of [
3
H]glutamate
binding by N-methyl-
D
-aspartic acid in rat adrenal. Brain Res.

406, 24–31.
130. Yoneda, Y. & Ogita, K. (1987) Solubilization of novel binding
sites for [
3
H]glutamate in rat adrenal. Biochem. Biophys. Res.
Commun. 142, 609–616.
131. Yoneda, Y. & Ogita, K. (1989) Characterization of quisqualate-
sensitive [
3
H]glutamate binding activity solubilized from rat
adrenal. Neurochem. Int. 15, 137–147.
132. Nakamuta, H., Ogita, K., Fukuda, Y., Koida, M. & Yoneda, Y.
(1987) Characterization of [
3
H]glutamate binding sites on frozen
sections from rat adrenal. Neurochem. Int. 10, 565–570.
133. O’Shea, R.D., Marley, P.D., Mercer, L.D. & Beart, P.M. (1992)
Biochemical, autoradiographic and functional studies on a
unique glutamate binding site in adrenal gland. J. Auton. Nerv.
Syst. 40, 71–85.
134. Kristensen, P. (1993) Differential expression of AMPA gluta-
mate receptor mRNAs in the rat adrenal gland. FEBS Lett. 332,
14–18.
135. Watanabe, M., Mishina, M. & Inoue, Y. (1994) Distinct gene
expression of the N-methyl-
D
-aspartate receptor channel subunit
in peripheral neurons of the mouse sensory ganglia and adrenal
gland. Neurosci. Lett. 165, 183–186.
136. Schwendt, M. & Jezova, D. (2001) Gene expression of NMDA

receptor subunits in rat adrenals under basal and stress condi-
tions. J. Physiol. Pharmacol. 52, 719–727.
Ó FEBS 2003 Glutamate signaling in peripheral tissues (Eur. J. Biochem. 271)11
137. Pirnik, Z., Schwendt, M. & Jezova, D. (2001) Single dose of
morphine influences plasma corticosterone and gene expression
of main NMDA receptor subunit in the adrenal gland but not in
the hippocampus. Endocr. Regul. 35, 187–193.
138. Hinoi, E., Fujimori, S., Yoneyama, M. & Yoneda, Y. (2002)
Blockade by N-methyl-
D
-aspartate of elevation of activator
protin-1 binding after stress in rat adrenal gland. J. Neurosci. Res.
70, 161–171.
139. Lee, J.A., Long, Z., Nimura, N., Iwatsubo, T., Imai, K. &
Homma, H. (2001) Localization, transport, and uptake of
D
-aspartate in the rat adrenal and pituitary glands. Arch. Bio-
chem. Biophys. 385, 242–249.
140. Parker, T.L., Kesse, W.K., Mohamed, A.A. & Afework, M.
(1993) The innervation of the mammalian adrenal gland. J. Anat.
183, 265–276.
141. Pyner, S. & Coote, J.H. (1995) Arrangement of dendrites
and morphological characteristics of sympathetic preganglio-
nic neurones projecting to the superior cervical ganglion
and adrenal medulla in adult cat. J. Auton. Nerv. Syst. 52,35–
41.
142. Romero, O., Figueroa, S., Vicente, S., Gonzalez, M.P. & Oset-
Gasque, M.J. (2003) Molecular mechanisms of glutamate release
by bovine chromaffin cells in primary culture. Neuroscience 116,
817–829.

143. Llewellyn-Smith, I.J., Phend, K.D., Minson, J.B., Pilowsky, P.M.
& Chalmers, J.P. (1992) Glutamate-immunoreactive synapses on
retrogradely-labelled sympathetic preganglionic neurons in rat
thoracic spinal cord. Brain Res. 581, 67–80.
144. Llewellyn-Smith, I.J., Minson, J.B., Pilowsky, P.M., Arnolda,
L.F. & Chalmers, J.P. (1995) The one hundred percent hypo-
thesis: glutamate or GABA in synapses on sympathetic pregan-
glionic neurons. Clin.Exp.Hypertens.17, 323–333.
145. Amar, A.P. & Weiss, M.H. (2003) Pituitary anatomy and phy-
siology. Neurosurg. Clin. N. Am. 14, 11–23.
146. Dorton, A.M. (2000) The pituitary gland: embryology, physiol-
ogy, and pathophysiology. Neonatal Netw. 19, 9–17.
147. Petrusz, P. (1994) The glutamate receptor subunit GLuR1 is
expressed in gonadotrophs of the anterior pituitary and is regu-
lated by gonadal feedback. Acta. Biol. Hung. 45, 387–397.
148. Mahesh,V.B.,Zamorano,P.,DeSevilla,L.,Lewis,D.&Brann,
D.W. (1999) Characterization of ionotropic glutamate receptors
in rat hypothalamus, pituitary and immortalized gonadotropin-
releasing hormone (GnRH) neurons (GT1-7 cells). Neuroendo-
crinology 69, 397–407.
149. Petralia, R.S., Yokotani, N. & Wenthold, R.J. (1994) Light and
electron microscope distribution of the NMDA receptor subunit
NMDAR1 in the rat nervous system using a selective anti-pep-
tide antibody. J. Neurosci. 14, 667–696.
150. Bhat, G.K., Mahesh, V.B., Chu, Z.W., Chorich, L.P., Zamor-
ano, P.L. & Brann, D.W. (1995) Localization of the N-methyl-
D
-aspartate R1 receptor subunit in specific anterior pituitary
hormone cell types of the female rat. Neuroendocrinology 62,
178–186.

151. Meeker, R.B., Greenwood, R.S. & Hayward, J.N. (1994) Glu-
tamate receptors in the rat hypothalamus and pituitary.
Endocrinology 134, 621–629.
152. Yoneda, Y. & Ogita, K. (1989) Solubilization of stereospecific
and quisqualate-sensitive activity of [
3
H]glutamate binding in the
pituitary of the rat. Neuropharmacology 28, 611–616.
153. Login, I.S. (1990) Direct stimulation of pituitary prolactin release
by glutamate. Life Sci. 47, 2269–2275.
154. Pampillo, M., Theas, S., Duvilanski, B., Seilicovich, A. &
Lasaga, M. (2002) Effect of ionotropic and metabotropic
glutamate agonists and
D
-aspartate on prolactin release from
anterior pituitary cells. Exp. Clin. Endocrinol. Diabetes 110,
138–144.
155. Niimi,M.,Sato,M.,Murao,K.,Takahara,J.&Kawanishi,K.
(1994) Effect of excitatory amino acid receptor agonists on
secretion of growth hormone as assessed by the reverse hemolytic
plaque assay. Neuroendocrinology 60, 173–178.
156. Zanisi, M., Galbiati, M., Messi, E. & Martini, L. (1994) The
anterior pituitary gland as a possible site of action of kainic acid.
Proc.Soc.Exp.Biol.Medical206, 431–437.
157. Giovannucci, D.R. & Stuenkel, E.L. (1995) Glutamate receptor
agonists modulate [Ca
2+
]i in isolated rat melanotropes. Neuro-
endocrinology 62, 111–122.
158. Paquette, H. (2000) The pineal gland. Neonatal Netw. 19, 9–11.

159. Fukada, Y. & Okano, T. (2002) Circadian clock system in the
pineal gland. Mol. Neurobiol. 25, 19–30.
160. Kus, L., Handa, R.J. & McNulty, J.A. (1993) Characterization of
a[
3
H]glutamate binding site in rat pineal gland: enhanced affinity
following superior cervical ganglionectomy. J. Pineal Res. 14,
39–44.
161. Govitrapong, P. & Ebadi, M. (1988) The inhibition of pineal
arylalkylamine N-acetyltransferase by glutamic acid and its
analogues. Neurochem. Int. 13, 223–230.
162. Ishio, S., Yamada, H., Craft, C.M. & Moriyama, Y. (1999)
Hydroxyindole-O-methyltransferase is another target for
L
-glu-
tamate-evoked inhibition of melatonin synthesis in rat pine-
alocytes. Brain Res. 850, 73–78.
163. Kus, L., Handa, R.J. & McNulty, J.A. (1994) Glutamate
inhibition of the adrenergic-stimulated production of melatonin
in rat pineal gland in vitro. J. Neurochem. 62, 2241–2245.
164. van Wyk, E. & Daya, S. (1994) Glutamate inhibits the iso-
prenaline-induced raise in melatonin synthesis by organ cultures
of rat pineal glands. Med. Sci. Res. 22, 635–636.
165. Yatsushiro, S., Hayashi, M., Morita, M., Yamamoto, A. &
Moriyama, Y. (2000) Glutamate receptor subunit delta2 is highly
expressed in a novel population of glial-like cells in rat pineal
glands in culture. J. Neurochem. 75, 1115–1122.
166. Mick, G. (1995) Non-N-methyl-
D
-aspartate glutamate receptors

in glial cells and neurons of the pineal gland in a higher primate.
Neuroendocrinology 61, 256–264.
167. Sato, K., Kiyama, H., Shimada, S. & Tohyama, M. (1993) Gene
expression of KA type and NMDA receptors and of a glycine
transporter in the rat pineal gland. Neuroendocrinology 58, 77–79.
168. Yatsushiro, S., Yamada, H., Hayashi, M., Tsuboi, S. & Mor-
iyama, Y. (1999) Functional expression of metabotropic
glutamate receptor type 5 in rat pinealocytes. Neuroreport 10,
1599–1603.
169. Pabst, H. & Redecker, P. (1999) Interstitial glial cells of the gerbil
pineal gland display immunoreactivity for the metabotropic
glutamate receptors mGluR2/3 and mGluR5. Brain Res. 838,
60–68.
170. Redecker, P. & Veh, R.W. (1994) Glutamate immunoreactivity is
enriched over pinealocytes of the gerbil pineal gland. Cell Tissue
Res. 278, 579–588.
171. Moriyama, Y. & Yamamoto, A. (1995) Microvesicles isolated
from bovine pineal gland specifically accumulate
L
-glutamate.
FEBS Lett. 367, 233–236.
172. Moriyama, Y. & Yamamoto, A. (1995) Vesicular
L
-glutamate
transporter in microvesicles from bovine pineal glands. Driving
force, mechanism of chloride anion activation, and substrate
specificity. J. Biol. Chem. 270, 22314–22320.
173. Yamada, H., Yamamoto, A., Takahashi, M., Michibata, H.,
Kumon, H. & Moriyama, Y. (1996) The
L

-type Ca
2+
channel is
involved in microvesicle-mediated glutamate exocytosis from rat
pinealocytes. J. Pineal Res. 21, 165–174.
174. Yamada, H., Yamamoto, A., Yodozawa, S., Kozaki, S.,
Takahashi, M., Morita, M., Michibata, H., Furuichi, T.,
Mikoshiba, K. & Moriyama, Y. (1996) Microvesicle-mediated
exocytosis of glutamate is a novel paracrine-like chemical trans-
12 E. Hinoi et al. (Eur. J. Biochem. 271) Ó FEBS 2003
duction mechanism and inhibits melatonin secretion in rat pine-
alocytes. J. Pineal Res. 21, 175–191.
175. Yamada, H., Yatsushiro, S., Yamamoto, A., Hayashi, M., Nishi,
T., Futai, M., Yamaguchi, A. & Moriyama, Y. (1997) Functional
expression of a GLT-1 type Na
+
-dependent glutamate trans-
porter in rat pinealocytes. J. Neurochem. 69, 1491–1498.
176. Redecker, P. & Pabst, H. (2000) Immunohistochemical study of
the glutamate transporter proteins GLT-1 and GLAST in rat and
gerbil pineal gland. J. Pineal Res. 28, 179–184.
177. Chaudhari, N., Yang, H., Lamp, C., Delay, E., Cartford, C.,
Than, T. & Roper, S. (1996) The taste of monosodium glutamate:
membrane receptors in taste buds. J. Neurosci. 16, 3817–3826.
178. Toyono, T., Seta, Y., Kataoka, S., Harada, H., Morotomi, T.,
Kawano, S., Shigemoto, R. & Toyoshima, K. (2002) Expression
of the metabotropic glutamate receptor, mGluR4a, in the taste
hairs of taste buds in rat gustatory papillae. Arch. Histol. Cytol.
65, 91–96.
179. Li,X.,Staszewski,L.,Xu,H.,Durick,K.,Zoller,M.&Adler,E.

(2002) Human receptors for sweet and umami taste. Proc. Natl
Acad. Sci. USA 99, 4692–4696.
180. Kostanyan, I.A., Merkulova, M.I., Navolotskaya, E.V. & Nur-
ieva, R.I. (1997) Study of interaction between 1-glutamate and
human blood lymphocytes. Immunol. Lett. 58, 177–180.
181. Franconi, F., Miceli, M., Alberti, L., Seghieri, G., De Montis,
M.G. & Tagliamonte, A. (1998) Further insights into the anti-
aggregating activity of NMDA in human platelets. Br.J.Phar-
macol. 124, 35–40.
182. Winter, C.R. & Baker, R.C. (1995)
L
-glutamate-induced changes
in intracellular calcium oscillation frequency through
non-classical glutamate receptor binding in cultured rat myo-
cardial cells. Life Sci. 57, 1925–1934.
183. Gill, S.S., Pulido, O.M., Mueller, R.W. & McGuire, P.F. (1999)
Immunochemical localization of the metabotropic glutamate
receptors in the rat heart. Brain Res. Bull. 48, 143–146.
184. Dickman, K.G., Youssef, J.G., Mathew, S.M. & Said, S.I. (2003)
Ionotropic glutamate receptors in lungs and airways: molecular
basis for glutamate toxicity. Am.J.Respir.CellMol.Biol.in
press.
185. Said, S.I., Dey, R.D. & Dickman, K. (2001) Glutamate signalling
in the lung. Trends Pharmacol. Sci. 22, 344–345.
186. Said, S.I., Berisha, H.I. & Pakbaz, H. (1995) N-methyl-
D
-aspar-
tate receptors outside the central nervous system: activation
causes acute lung injury that is mediated by nitric oxide synthesis
and prevented by vasoactive intestinal peptide. Neuroscience 65,

943–946.
187. Said, S.I. (1999) Glutamate receptors and asthmatic airway dis-
ease. Trends Pharmacol. Sci. 20, 132–134.
188. Morhenn, V.B., Waleh, N.S., Mansbridge, J.N., Unson, D.,
Zolotorev, A., Cline, P. & Toll, L. (1994) Evidence for an NMDA
receptor subunit in human keratinocytes and rat cardiocytes.
Eur. J. Pharmacol. 268, 409–414.
189. Frati, C., Marchese, C., Fisichella, G., Copani, A., Nasca, M.R.,
Storto, M. & Nicoletti, F. (2000) Expression of functional mGlu5
metabotropic glutamate receptors in human melanocytes. J. Cell
Physiol. 183, 364–372.
190. Tsai, L.H., Lee, Y.J. & Wu, J. (1999) Effect of excitatory amino
acid neurotransmitters on acid secretion in the rat stomach.
J. Biomed. Sci. 6, 36–44.
Ó FEBS 2003 Glutamate signaling in peripheral tissues (Eur. J. Biochem. 271)13