(Received for publication, May 16, 1995; and in revised form, July 19, 1995)
From the
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.
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.
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) .
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.
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.
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.
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.
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.
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.