Volume 270,
Number 38,
Issue of September 22, pp. 22314-22320, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Vesicular L
-Glutamate Transporter in Microvesicles from Bovine Pineal
Glands
DRIVING FORCE, MECHANISM OF CHLORIDE ANION ACTIVATION, AND
SUBSTRATE SPECIFICITY (*)
(Received for publication, May 16, 1995; and in revised form, July 19,
1995)
Yoshinori
Moriyama
(1), (§),
Akitsugu
Yamamoto
(2)From the
(1)Marine Biological Laboratory, Graduate
Department of Gene Sciences, Faculty of Sciences, Hiroshima University,
Mukaishima, Hiroshima 722, Japan and the
(2)Department of Physiology, Kansai Medical
University, Moriguchi, Osaka 570, Japan
ABSTRACT
- INTRODUCTION
- EXPERIMENTAL PROCEDURES
- RESULTS
- DISCUSSION
- FOOTNOTES
- ACKNOWLEDGEMENTS
- REFERENCES
ABSTRACT 
Pinealocytes, endocrine cells that synthesize and secrete
melatonin, possess a large number of synaptic-like microvesicles (MVs)
containing the L-glutamate transporter (Moriyama, Y., and
Yamamoto, A.(1995) FEBS Lett., 367, 233-236). In this
study, the L-glutamate transporter in MVs isolated from bovine
pineal glands was characterized as to its driving force, requirement of
anions, and substrate specificity. Upon the addition of ATP, the MVs
accumulated L-glutamate. The uptake was significantly
dependent on the extravesicular Cl
concentration,
being negligible in the absence of Cl and maximum at
2-5 mM and decreasing gradually at 20-100
mM. The membrane potential (inside positive) was maximum at
0-10 mM Cl and then decreased
gradually depending on the Cl concentration, whereas
a pH gradient was practically absent without Cl and
increased gradually up to 100 mM Cl.
Ammonium acetate or nigericin plus K
, a dissipator of
a pH gradient, had little effect on or was slightly stimulatory toward
the uptake, whereas valinomycin plus K inhibited both
formation of the membrane potential and the glutamate uptake to similar
extents. The ATP- and Cl-dependent glutamate uptake
was inhibited by fluoride, iodide, or thiocyanate, without vacuolar
H-ATPase being affected. An anion channel blocker,
4,4`-diisothiocyanatostilbene-2,2`-disulfonic acid, similarly inhibited
the glutamate uptake in a Cl protectable manner.
Furthermore, ATP- and glutamate-dependent acidification of MVs was
observed when 4 mM Cl was present. Among
more than 50 kinds of glutamate analogues tested, only a few compounds,
including 1-aminocyclohexane-trans-1,3-dicarboxylic acid,
caused similar acidification. A good correlation was observed between
the acidification and the inhibition of glutamate uptake by glutamate
analogues. These results indicated that 1) the major driving force of
the glutamate uptake is the membrane potential, 2) Cl regulates the glutamate uptake, probably via anion-binding
site(s) on the transporter, and 3) the transporter shows strict
substrate specificity. Hence, the overall properties of the vesicular
glutamate transporter in the MVs well matched those of the synaptic
vesicle glutamate transporter. We concluded that the vesicular
glutamate transporter, being similar if not identical to the neuronal
counterpart, operates in endocrine cells.
INTRODUCTION
Pinealocytes are parenchymal endocrine cells of pineal glands
that synthesize and secrete melatonin into the
blood(1, 2, 3) . At least two kinds of
secretory vesicle-like organelles have been identified in pinealocytes:
dense core granules, which are speculated to be involved in the storage
and secretion of melatonin and some neuropeptides such as arginine
vasotocin(3, 4) , and a large number of synaptic-like
microvesicles (MVs) (
)containing synaptophysin(5) .
Although histochemical evidence indicated that MVs are distinct from
neuronal synaptic vesicles in their lack of synapsin I, the possible
participation of MVs in some secretory pathways in pinealocytes was
also speculated(5) .
Very recently, we established a
procedure for isolating MVs from bovine pineal glands and found that
these vesicles were actually devoid of synapsin I but possess
synaptotagmin and synaptobrevin 2, proteins necessary for vesicular
transport(6) . Furthermore, the MVs specifically accumulated L-glutamate in an energy-dependent manner(6) . These
results suggested that MVs are the organelles in pinealocytes that
store and secrete L-glutamate. Therefore, it seems likely that
pinealocytes possess novel glutamate-evoked signal transducing systems.
To reveal the entire features of the putative MV-mediated signal
transduction systems in pinealocytes, at first we focused on the
glutamate transporter in MVs. So far, the ATP-dependent vesicular
glutamate transporter has been identified in brain synaptic
vesicles(7, 8, 9, 10, 11, 12, 13) .
Because the glutamate transporter in pineal MVs is the first example of
a vesicular glutamate transporter outside neuronal cells, it is of
interest to determine the mechanistic difference or similarity between
glutamate transporters of the neuronal and endocrine origins. In this
study, we characterized the glutamate transporter in pineal MVs and
obtained evidence that it is quite similar to that in brain synaptic
vesicles.
EXPERIMENTAL PROCEDURES
Isolation of MVs from Pineal Glands
The details
are given in (6) . In brief, bovine pineal glands (about 50
glands for one preparation) obtained from a local slaughterhouse within
a few hours after killing were washed, cut into small pieces with
scissors, suspended in 80 ml of SME buffer (20 mM MOPS-Tris
(pH 7.0) containing 0.3 M sucrose, 5 mM EDTA, 5
µg/ml pepstatin A, and 5 µg/ml leupeptin), and then homogenized
in a Dounce homogenizer. The homogenate was centrifuged at 900 
g (R
) for 10 min, and then the
resultant supernatant was centrifuged at 11,000 g (R) for 20 min. The resultant supernatant
was centrifuged at 130,000 g for 40 min. The resultant
pellet was washed once, suspended in 2.5 ml of SME buffer (crude MV
fraction), and then applied to a discontinuous sucrose density gradient
(0.7 ml of 1.8, 1.2, 1.0, 0.8, 0.6, and 0.4 M sucrose
containing 5 µg/ml pepstatin A and 5 µg/ml leupeptin). After
centrifugation at 207,700 g (R)
for 3 h, four distinct bands were obtained. The top broad white band
was collected, diluted 5-fold with SME buffer, and then centrifuged at
139,000 g for 40 min. The pellet (purified MVs) was
suspended in SME buffer and stored at -85 °C until use. More
than 95% of the vesicles in the final preparation were MVs (relatively
clear small vesicles with an average diameter of 50 nm containing
synaptophysin and V-ATPase), as revealed on conventional and
immunoelectron microscopy using an anti-synaptophysin monoclonal
antibody(6) . The MVs were distinct from synaptic vesicles in
their lack of synapsin I(6) .
Transport Assay
Unless otherwise specified, the
uptake of radioactive L-glutamate by MVs was assayed in 0.5 ml
of 20 mM MOPS-Tris (pH 7.0) containing 4 mM KCl, 0.3 M sucrose, 2 mM magnesium acetate, 2 mM ATP
(Tris salt, pH 7.0), and MVs (about 50 µg of protein) (standard
assay conditions). The assay was started at 30 °C by the addition
of radioactive glutamate (2.5 µCi, 0.1 mM). Aliquots (100
µl) were taken at intervals and filtered through 0.45 mM Millipore filters (type HA). Ater extensive washing, the remaining
radioactivity on the filters was counted with a liquid scintillation
counter.The formation of 
pH (inside acidic) was measured by
means of acridine orange fluorescence quenching as described
previously, the excitation and emission wavelength pair being 492 nm
and 540 nm, respectively(11) . 
(inside positive)
was measured by means of oxonol-V fluorescence quenching (excitation,
580 nm; emission, 620 nm) (11) .
Chemicals
L-[2,3-
H]Glutamate
(638.3 GBq/mmol) was purchased from Amersham Corp. The glutamate
analogues listed in Table 3and Table 4were from Tocris
Neuramin, except for methyleneglutamate, 
-ethylglutamate,
methylglutamate, aspartate, and -aminobutyrate, which were from
Sigma. ATP (Tris salt) was from Sigma. DIDS was obtained from Dojindo
Laboratories (Japan). Other chemicals were of the highest grade
commercially available.
RESULTS
Requirement of ClforL-Glutamate Uptake by Pineal MVs
MVs
purified from bovine pineal glands took up L-glutamate, which
was coupled with an electrochemical proton gradient established by
V-ATPase(6) . During the assaying glutamate uptake by pineal
MVs, we noticed that a low concentration of Cl significantly enhanced the L-glutamate uptake, as shown
in Fig. 1A. Cation species were not responsible for the
activation (data not shown). The Cl concentration
required for the maximum glutamate uptake was 2-5 mM,
with the uptake decreasing gradually at higher concentrations (Fig. 1B). Only 6% of the maximum activity was observed
in the absence of Cl (Fig. 1B).
Chloride and bromide anions were equally effective for the activation (Fig. 1C). The nitrate, iodide, and gluconate ions were
slightly effective, whereas the acetate and bicarbonate ions were not
effective, and the fluoride and thiocyanate ions were inhibitory (Fig. 1C). These results indicated that the
ATP-dependent glutamate uptake by pineal MVs is anion-sensitive and
requires Cl.
Figure 1:
Chloride anion-stimulated L-glutamate uptake into MVs. A, the time course of
ATP-dependent L-glutamate uptake was measured as described
under ``Experimental Procedures'' in the presence (open
circles) or absence (closed circles) of 4 mM KCl. B, the Cl dose dependence of the
activation of glutamate uptake at 5 min was measured according to the
standard procedure in the presence of the indicated concentrations of
KCl. C, the effects of various anions were assayed as
described above except that the indicated potassium salts (4 mM each) were used instead of KCl. The results were expressed as
activation fold, taking the glutamate uptake in the absence of salts
(sucrose) as 1.0, and are presented as the means ± S.E. (average
of 4 different experiments).
The requirement of a low
concentration of Cl may be one of the significant
properties of the glutamate uptake. The following alternative
mechanisms may possibly explain the activation: one is direct
interaction of Cl with the glutamate transporter and
another is the necessity of Cl for the formation of
an electrochemical proton gradient, because V-ATPase is an
anion-sensitive proton pump(14, 15, 16) . To
clarify the role of Cl in the glutamate uptake, we
next analyzed the effects of anions on ATP-dependent membrane
energization.
Characteristics of ATP-dependent Membrane Energization of
Pineal MVs
Fig. 2shows the
Cl-dependence of ATP-dependent formation of pH
(inside acidic) and (inside positive) in MVs, as measured
with fluorescent indicators. A proton conductor,
3,5-di-tert-butyl-4-hydroxybenzylidene malononitrile (SF6847)
(0.5 µM), or a V-ATPase inhibitor, bafilomycin A1 (50
nM), inhibited the formation of both pH and ,
confirming that V-ATPase formed an electrochemical proton gradient
across the MV membrane (Fig. 2, A and B). The
formation of pH was dependent on the exogenous Cl concentration; a little pH was generated in the absence of
Cl, but it increased linearly up to 20 mM and then gradually to 100 mM Cl (Fig. 2C). On the other hand, was
highest at 0-10 mM Cl and then
decreased gradually depending on the Cl concentration (Fig. 2C). These results indicated that the formation
of prevented the formation of pH electrically in the
absence of Cl, and that the collapse of
due to Cl uptake, possibly through an anion channel
or anion transporter, induced further acidification, as in the case of
other endomembrane systems(16, 17, 18) . This
was confirmed by the observation that Br, another
substrate of the endomembrane anion channel (transporter)(19) ,
also induced the ATP-dependent acidification, whereas other anions,
such as sulfate or acetate, which are not substrates for the anion
channel, did not (Fig. 2A).
Figure 2:
Effects of Cl and other
anions on the ATP-dependent formation of pH and in
pineal MVs. A, ATP-driven fluorescence quenching of acridine
orange was measured in 20 mM MOPS-Tris buffer (pH 7.0)
containing 0.3 M sucrose, 2 mM magnesium acetate, 2
µM acridine orange, and 20 µg of protein of purified
MVs in the presence of the indicated salts (0.1 M), if
otherwise stated. In an experiment shown in K-acetate + KCl (4
mM) and KCl + K-acetate, potassium acetate
(0.1 M) plus KCl (4 mM) and KCl plus potassium
acetate (0.1 M each) were included in the assay mixture,
respectively. The increase in the acridine orange fluorescence on the
addition of ATP was omitted from the figure because it is an artifact
due to the interaction of the dye and ATP(46) . B,
oxonol-V fluorescence quenching was measured in the same buffer as in A except that 5 µM oxonol-V was used instead of
acridine orange. C, acridine orange (closed circles)
and oxonol-V (open circles) fluorescence quenching were
measured in the presence of the indicated concentrations of KCl and
expressed as relative values, taking maximum quenching as 100%. Other
additions: 1.0 mM ATP, 50 nM bafilomycin A1, and 5
mM ammonium acetate.
Energetics of ATP-dependent and
Cl-activated Glutamate
Uptake
The dose-dependent effects of Cl on the formation of pH and differed from those
of the glutamate uptake (Fig. 1C), indicating clearly
that pH and are not simply linked to the
Cl-activated glutamate uptake. Two alternative
possibilities as to the role of Cl can be considered.
One is that is the driving force and Cl acts as a co-factor or a coupling anion for the uptake. The other
possibility is that is a major driving force but that the
small pH present at a low concentration of Cl is
also necessary for the uptake.Then, we compared the effects of
ionophores and the ammonium ion on the glutamate uptake, pH and
(Table 1). To control the magnitude of pH and
due to ionophores, 0.1 M potassium acetate was
included in the assay medium as a K source, because
this salt did not affect the Cl-stimulated glutamate
uptake. Nigericin or ammonium acetate dissipated pH without
affecting or slightly increasing . Under these conditions,
glutamate uptake was not affected or slightly stimulated. Valinomycin,
on the other hand, partially abolished and inhibited
glutamate uptake to similar extents. The combination of valinomycin and
nigericin or ammonium acetate dissipated both pH and ,
resulting in almost complete inhibition of the glutamate uptake. These
results indicated that the magnitude of glutamate uptake into MVs is
correlated with the magnitude of but not that of pH.
Thus, seems to be the driving force for glutamate uptake in
pineal MVs, although we could not completely rule out the participation
of a small pH in the glutamate transport.
Characteristics of a Putative Anion-binding Site in the
Glutamate Transporter
The stimulatory effect of
Cl, therefore, suggests the presence of a
Cl-binding site in the glutamate transporter. To
reveal the properties of this putative Cl-binding
site, we investigated the effects of various anions on the
Cl-stimulated glutamate uptake. As shown in Fig. 3A, the fluoride, iodide, thiocyanate, and nitrate
ions were all inhibitory, whereas other anions had little effect. The
inhibitory effect of the nitrate ion was due to a decrease in the
magnitude of the driving force (loss of ) due to inhibitin
of V-ATPase (Fig. 3B). On the other hand, the fluoride
and thiocyanate ions under the concentrations used in this experiment
had little effect on (Fig. 3B), suggesting
that the inhibition was due to direct interaction with the glutamate
transporter. The iodide ion had a similar effect (not shown). The
inhibition was reversible, and full activity was recovered after
washing of the membranes (not shown).
Figure 3:
Effects of anion species on
Cl-stimulated glutamate uptake. A, the
ATP-dependent glutamate uptake at 5 min in the presence of 4 mM KCl plus the indicated potassium salts (4 mM) is shown as
the means ± S.E. (average of 4 experiments). The degree of
, as measured by means of oxonol-V fluorescence quenching
under similar conditions, is also shown on the right as
relative values. B, the F,
I, SCN, and NO3 dose dependences of the inhibition of
Cl-stimulated glutamate uptake (closed
circles) and (open circles) in the presence
of 4 mM KCl are shown.
To find compounds that
interact with the anion-binding site(s) irreversibly or that are more
potent than thiocyanate, we tested several kinds of anion channel
blockers. DIDS was found to be a strong inhibitor of the glutamate
uptake (Table 2). DIDS also inhibited
V-ATPase(19, 20) , the ID
value being 3.8
µM. However, the ID value on inhibition of
the glutamate uptake was only 0.3 µM. Furthermore,
inhibition of the glutamate uptake by DIDS was prevented by
Cl but not by acetate (Table 2). Taken
together, these results suggested that the Cl-binding
site(s) in the glutamate transporter can be occupied by
F, thiocyanate, or DIDS. The chloride-binding site(s)
may be important for regulation of the uptake activity.
Substrate Specificity and Inhibitors of the
Cl-activated Glutamate
Transporter
It is difficult to elucidate the substrate
specificity of the glutamate transporter, because only a limited number
of radioactive glutamate analogues is available commercially. Even in
the case of the synaptic vesicle glutamate transporter, the substrate
specificity was evaluated indirectly by measuring the spectrum of
inhibition of glutamate uptake in the presence of glutamate
analogues(7, 22) . As in the case of the brain
synaptic vesicle glutamate
transporter(9, 10, 11) , we noticed that the
combined addition of glutamate and ATP caused acidification in MVs when
4 mM Cl was present in the assay medium (Fig. 4). The acidification was sensitive to bafilomycin A1 (50
nM). Furthermore, the rate of acidification became saturated
depending on the glutamate concentration, the apparent K
value being 3 mM, which is comparable with the K value (1.3 mM) obtained using the
radioisotopes(6) . Some glutamate analogues, such as
1-aminocyclohexane-trans-1,3-dicarboxylic acid, showed similar
ATP and Cl-dependent acidification (Fig. 4d). These results indicated that the
Cl and glutamate-dependent acidification reflected
glutamate uptake by MVs; the uptake of the glutamate anion through the
glutamate transporter dissipated , which in turn accelerated
the formation of pH. Therefore, we can estimate the kinetic
parameters for the uptake of glutamate analogues by measuring the rate
of analogue-evoked acidification.
Figure 4:
Glutamate-dependent acidification of MVs.
Acridine orange fluorescence was measured in 2 ml of 20 mM MOPS-Tris (pH 7.0) buffer containing 0.3 M sucrose, 2
mM magnesium acetate, 20 µg of MVs, and 2 µM acridine orange in the absence (b) or presence (a, c, and d) of 4 mM KCl. The
additions were as follows: 1 mM ATP, 5 mML-glutamate, 5
mM1-aminocyclohexane-trans-1,3-dicarboxylic acid (ACHD), and 5 mM ammonium
chloride.
Table 3shows the degree of
acidification and the K values obtained on
glutamate analogue-induced acidification as described above. Aspartate,
a substrate for the Na-dependent glutamate transporter
in plasma membranes(23) , was not effective. Again, cyclic
glutamate analogues were relatively good substrates, with higher
affinity than glutamate. The cis forms of these cyclic
compounds were all ineffective. Four other compounds (D-glutamate, 
-methyl-D,L-glutamate,
-methylene-D,L-glutamate, and D,L-2-amino-4-phosphonobutyric acid) caused low
acidification, suggesting that these compounds act as less efficient
substrates.
Inhibition of glutamate uptake by the same compounds was
also examined (Table 4). Consistent with the acidification,
cyclic glutamate analogues (trans form) strongly inhibited the
glutamate uptake. Compounds causing acidification always inhibited
glutamate uptake with a similar order of effectiveness. Other
compounds, including sulfur-containing amino acids, which inhibited
glutamate uptake in synaptic vesicles(22, 24) ,
neither affected the uptake nor caused acidification (Table 3).
At more than 5 mM, these sulfur-containing amino acids
partially inhibited the glutamate uptake, but the inhibition was due to
the decreased driving force upon inhibition of V-ATPase (data not
shown). The following glutamate analogues also neither affected
glutamate uptake nor caused acidification: L-glutamate amide, D,L-hydroxyglutaric acid,
2,4-dinitrophenyl-L-glutamate, N-phthaloyl-D,Lglutamic acid, N-carboxymethylglutamic acid, N-acetyl-L-glutamic acid, N-phenacetyl-D,Lglutamic acid, N-methylpyro-D,Lglutamic acid, N-isopropylpyro-D,Lglutamic acid,
ethyl-D,scap]l2-pyrrolidone-5-carboxylate, N-trimethyl-D,L-glutamate,
butyl-D,Lpyroglutamate, N-dimethyl-D,Lhydroxyglutamate, N-caproyl glutamate, 
-oxyglutamate,
-hydroxyglutamate, N-benzyloxycarbamorylglutamate, N-toluenesulfonyl-L-glutamate, and S-carboxymethyl-L-cysteine.
1-Amino-trans-3-phosphocyclopropane carboxylic acid inhibited
about 40% of the glutamate uptake without acidification (Table 3), suggesting that this compound may bind to the
transporter but not be transported. Taken together, these results
suggested that the glutamate transporter in pineal MVs shows strict
substrate recognition; a compound on the replacement of an amino group
and a change in the carbon chain length of the L-glutamate
moiety ceased to be a substrate. The transporter prefers a partially
folded conformation of a substrate rather than an extended one. It
seems likely that the transporter recognizes the configuration of the
1,3-carboxylates and 1-amino groups of cyclic glutamate analogues but
is indifferent as to the length of the carbon chain on the opposite
side of the cyclic molecules.
DISCUSSION
Glutamate, an excitatory neurotransmitter, is stored in
synaptic vesicles, is extruded into the synaptic cleft upon
stimulation, and binds to its receptors present in postsynaptic
membranes so as to transmit signals
intercellularly(25, 26) . The vesicular glutamate
transporter in synaptic vesicles is responsible for the storage of
glutamate in
neurons(27, 28, 29, 30) . Like other
neurotransmitter transporters, the glutamate transporter is
energetically coupled with V-ATPase (27, 28, 29, 30) but uses
as the major driving
force(9, 10, 11, 31, 32) .
The chloride anion is suggested to regulate the transport activity via
anion-binding site(s)(21) . However, the properties of this
transporter at the molecular level remain obscure because neither
purification of the transporter nor cloning of its cDNA have been
successful. One of the aims of this study was to find a new approach
for this fascinating transporter, because a comparative study will be
possible if the vesicular glutamate transporter in pineal MVs resembles
the synaptic vesicle counterpart.
As summarized, the glutamate
transporter in pineal MVs is driven by (positive inside) ( Fig. 2and Table 1). The spectrum of the requirement of
anions for the uptake activity was essentially the same as that of the
synaptic vesicle counterpart(7) . The activation by
Cl or Br could be attributed to
their direct interaction with the transporter, presumably via an
anion-binding site ( Fig. 3and Table 2). At present, it is
difficult to prove that Cl acts as a counter ion for
chloride and glutamate co-transport, because we can not demonstrate
Cl movement through either the glutamate transporter
or the Cl channel in MVs. Furthermore, we showed that
the substrate specificity of the vesicular glutamate transporter was
quite similar to that of the synaptic vesicle counterpart, as judged
from the inhibition of glutamate uptake by glutamate
analogues(7, 22) . The overall properties of the
glutamate transporters in synaptic vesicles and pineal MVs are similar
to each other. We concluded that a glutamate transporter that is
similar, if not identical, to the synaptic vesicle counterpart operates
in MVs. Thus, pineal MVs constitute another experimental system for
studies on the vesicular glutamate transporter.
Pineal MVs contain
synaptobrevin and synaptotagmin, proteins important for vesicular
transport(6) . Recently, we found that pinealocytes express
relatively high concentrations of N-ethylmaleimide-sensitive
fusion protein, Ca
-channel protein, and smg25A (a
small GTP-binding protein). Some of N-ethylmaleimide-sensitive
fusion protein and smg25A was found to be associated with MVs. (
)These results further support that MVs are the organelles
that store L-glutamate and are involved in the
glutamate-evoked signal transduction system. Released glutamate may
bind to the glutamate receptor identified in pinealocytes (33, 34) and may inhibit noradrenaline-stimulated N-acetyltransferase and
hydroxyindole-O-methyltransferase activities, resulting in
inhibition of melatonin synthesis (35, 36, 37) .
Finally, we should point
out the similarity between pinealocytes and retinal photoreceptor
cells. Pinealocytes accumulate glutamate inside MVs and may extrude it
through exocytosis, possibly at process terminals and/or the synaptic
ribbon region ( (6) and this paper). Similarly, retinal
photoreceptor cells use glutamate as a transmitter possibly via
synaptic vesicle-mediated exocytosis at the ribbon synapse (38, 39) . Both types of cells lack of synapsins,
suggesting that pineal MVs and retinal synsaptic vesicles move to their
release sites though a novel mechanism(40) . Furthermore,
pinealocytes, especially from lower vertebrates, can receive
photosignals via photo-receptive molecules such as
pinopsin(41) . These profound mechanistic similarities will be
useful for understanding the mechanism underlying the signal
transduction in pineal glands and retinal cells.
MVs from pancreatic
cells(42) , adrenal chromaffin cells(43) , PC12
cells(44) , and posterior pituitaries (45) have been
shown to accumulate -aminobutyrate, noradrenaline, acetylcholine,
and noradrenaline, respectively. Upon stimulation, these transmitters
may be extruded from the cells and then transmit signals
intercellularly. Therefore, the presence of V-ATPase and the
neurotransmitter transporter in MVs may be one of the usual features of
endocrine cells.
FOOTNOTES
- *
- This study was supported in part
by grants from the Japanese Ministry of Education, Science, and
Culture. The costs of publication of this article were defrayed in part
by the payment of page charges. This article must therefore by hereby
marked ``advertisement'' in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
- §
- To whom correspondence should be addressed.
Tel.: 81-848-44-1143; Fax: 81-848-44-5914.
- ()
- The
abbreviations used are: MV, synaptic-like microvesicle; DIDS,
4,4`-diisothiocyanatostilbene-2,2`-disulfonic acid; pH,
transmembrane pH gradient; , transmembrane potential
difference; V-ATPase, vacuolar H-ATPase; MOPS,
4-morpholinepropanesulfonic acid; oxonol-V,
bis(3-phenyl-5-oxoisoxasol-4-yl)peptamethine oxonol.
- ()
- Moriyama, Y., Yamamoto, A., Tagaya, M., Tashiro,
Y., and Michibata, H.(1995) Neuroreport6, in press.
ACKNOWLEDGEMENTS
We thank Drs. Masamitsu Futai (Osaka University),
Hitoshi Michibata (Hiroshima University), Yutaka Tashiro (Kansai
Medical University), and Tomofusa Tsuchiya (Okayama University) for
critically reading the manuscript and encouragement. We also thank Dr.
Shinji Ohmori (Okayama University) for providing us with some of the
glutamate analogues.
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