From the Center for Basic Neuroscience, Department of Molecular
Genetics and Howard Hughes Medical Institute, The University of Texas
Southwestern Medical Center, Dallas, Texas 75235
Newly synthesized phosphatidylinositol phosphates
have been implicated in many membrane-trafficking reactions. They are
essential for exocytosis of norepinephrine in PC12 cells and chromaffin cells, suggesting a function in membrane fusion. We have now studied the role of phosphatidylinositol phosphates in synaptic vesicle exocytosis using synaptosomes. Under conditions where phosphorylation of phosphatidylinositols is blocked, norepinephrine secretion was
nearly abolished whereas glutamate and GABA release was still elicited.
Thus phosphatidylinositides are essential only for some membrane fusion
reactions, and exocytotic release mechanisms differ between
neurotransmitters.
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INTRODUCTION |
At synapses, similar mechanisms of synaptic vesicle exocytosis are
thought to effect the secretion of different neurotransmitters (e.g. glutamate,
GABA,1 and norepinephrine;
reviewed in Refs. 1-4). The same vesicle proteins are present in
presynaptic nerve terminals independent of neurotransmitter type
(e.g. see Refs. 5-8). In norepinephrine-secreting chromaffin cells and in PC12 cells, phosphorylation of
phosphatidylinositols by PI 4-kinase is required for
Ca2+-triggered exocytosis (9-12). In these cells, PI
4-kinase can be potently inhibited by phenylarsine oxide (PAO),
resulting in a block of exocytosis (11, 12). Several proteins involved in synaptic vesicle exocytosis bind to the products of PI 4-kinase activity, PIP and PIP2 (13-17). Furthermore,
phosphoinositides have also been implicated in a number of other
membrane trafficking reactions (reviewed in Refs. 18-20). Together
these findings suggested that phosphorylation of PI is essential for
exocytosis and membrane fusion. To explore the role of PI
phosphorylation in neurotransmitter release, we have now studied
neurotransmitter release from synaptosomes. We demonstrate that
inhibition of PIP and PIP2 synthesis results in
dramatically different effects for different neurotransmitters and that
PIP and PIP2 synthesis is not a requirement for most synaptic exocytosis.
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EXPERIMENTAL PROCEDURES |
Treatments and Phospholipid Analysis of
Synaptosomes--
Synaptosomes were prepared as described (21) and
resuspended in aerated (95% O2, 5% CO2)
ice-cold Krebs-bicarbonate buffer, pH 7.4 (composition in
mM: NaCl 118, KCl 3.5, CaCl2 1.25, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, glucose 11.5, and HEPES-NaOH 5) or phosphate-free Krebs-bicarbonate buffer (for 32P-labeling
experiments). Synaptosomes were 32P-labeled for 1.5 h
at 35 °C in a 95% O2, 5% CO2 atmosphere
with [32P]orthophosphate (1 mCi/ml). After labeling, PAO
(from Aldrich) freshly made in Me2SO or Me2SO
alone (<1% of total volume) was added, and synaptosomes were
incubated for the indicated times in triplicate. Reactions were stopped
on ice. Lipids were extracted with 3.75 volumes of
chloroform:methanol:concentrated HCl (100:200:1). After 10 min on ice,
10 µg of phosphatidylethanolamine (Sigma) was added as a carrier, and
phase partitioning was induced by with 1.25 volumes of chloroform and
of 0.1 N HCl. The chloroform phase with phospholipids was
washed twice with cold methanol:0.1 N HCl (1:1). Equal
amounts of the extracts were loaded on TLC plates with phospholipid
standards (10-20 µg each). TLC plates were developed in
1-propanol:H2O:concentrated NH4OH (65:20:15) and analyzed by autoradiography; phospholipid standards were identified with iodine vapors. 32P incorporation was quantified with a
PhosphorImager (Molecular Dynamics, CA).
Measurements of [3H]Glutamate,
[3H]GABA, and [3H]Norepinephrine
Release--
Synaptosomes were incubated for 5 min with 140 nM [3H]glutamate (specific activity, 15 Ci/mmol), 130 nM [3H]norepinephrine (46.8 Ci/mmol), or 66 nM [3H]GABA (90 Ci/mmol).
Buffers for the norepinephrine experiments and experiments in which
glutamate and norepinephrine release were measured in the same
synaptosomes also contained 0.4 mM ascorbic acid, 30 µM EDTA, and 10 µM pargyline.
3H-Loaded synaptosomes (0.1 ml) were trapped on glass fiber
filters (GF/B, Whatman), overlaid with 50 µl of a 50% Sephadex G-25
slurry, and superfused at 33 °C with Krebs-bicarbonate buffer (flow
rate, 0.8 ml/min) under continuous aeration with 95% O2,
5% CO2. For sucrose-triggered neurotransmitter release
experiments, synaptosomes were superfused with Ca2+-free
Krebs-bicarbonate buffer containing 0.1 mM EGTA. After 12 min of washing, two 1-min fractions were collected to determine base-line release. We then evoked release from synaptosomes by the
following agents: 1) 25 mM KCl for 30 s; 2) 5 µM ionomycin for 30 s; 3) 0.5-3 nM
-latrotoxin for 1 min; 4) 0.5 M sucrose for 30 s in
Ca2+-free Krebs-bicarbonate buffer. All stimuli were
applied by rapid switching of the superfusion lines between regular and
stimulation buffers. The amounts of [3H]glutamate,
[3H]norepinephrine, or [3H]GABA released
into the superfusate and remaining in the synaptosomes at the end of
the experiment were quantified by liquid scintillation counting.
Fractional neurotransmitter release was calculated by dividing the
amount of neurotransmitter released during a time interval by the
amount of transmitter remaining in the synaptosomes at that time. To
obtain the total GABA, glutamate, and norepinephrine release induced by
a given stimulus, the evoked release above baseline was integrated over
the time of the experiment. To test the effect of PAO on release,
synaptosomes were treated with PAO in Me2SO,
Me2SO alone (control; <1% of total volume), or vanadyl hydroperoxide (VOOH; prepared as a complex with 1,10-phenanthroline as
in Ref. 22) for 20 min at 35 °C in Ca2+-containing
aerated (95% O2, 5% CO2) Krebs-bicarbonate
buffer. During the last 5 min, 3H-labeled neurotransmitters
were added for loading the synaptosomes, which were then used for
release measurements as described above. PAO treatment partly inhibited
[3H]norepinephrine and [3H]GABA but not
[3H]glutamate uptake. To control for this and other
possible indirect effects, we performed experiments in which
synaptosomes were first loaded with 3H-labeled
neurotransmitters and then treated with PAO, with identical results to
those shown here. For each experiment, results are expressed in percent
release compared with control conditions.
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RESULTS |
PAO Blocks PIP and PIP2 Synthesis in
Synaptosomes--
We labeled synaptosomes with
32Pi and treated them with PAO or control
buffer. The synaptosomes were then incubated under various control and
stimulation conditions, and their phospholipids were analyzed by TLC.
Three major 32P-labeled phospholipids were observed and
identified with unlabeled phospholipid standards as PIP,
PIP2, and closely co-migrating PI and phosphatidic acid
(data not shown). The preferential labeling of PI, PIP, and
PIP2 in synaptosomes agrees well with the high turnover
rate of PIPs (23-25). We then tested the effect of PAO on PIP and
PIP2 synthesis in synaptosomes. PAO caused a major inhibition of 32P-labeling of PIP and PIP2 but
induced only marginal changes in PI and phosphatidic acid (Fig.
1 and data not shown). Stimulation of
synaptosomes by
-latrotoxin or KCl depolarization elicited moderate
decreases in the levels of 32P-labeled PIP and
PIP2 in the absence of PAO but had no effect on the
inhibition of PI and PIP phosphorylation by PAO (data not shown). We
did not determine at which positions the phosphatidylinositol rings are
phosphorylated in these experiments. However, similar studies in
chromaffin cells showed that the majority of PIP and PIP2 is phosphorylated at the 4- and 5-positions (11,
12). These results argue that, as in chromaffin cells, PAO
inhibits phosphorylation of PIP and PIP2 at the 4- and
5-positions of the inositol ring in synaptosomes (11, 12).

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Fig. 1.
PAO inhibits PI and PIP phosphorylation in
synaptosomes. A, time course of PAO action.
32P-Labeled synaptosomes were treated with 30 µM PAO for the indicated times. 32P-Labeled
PIP and PIP2 were analyzed by TLC and quantified by
PhosphorImager measurements. B, PAO concentration
dependence. Synaptosomes were incubated with various concentrations of
PAO for 20 min, and the levels of PIP and PIP2 were
quantified. Data are means ± S.E. from a single experiment
repeated multiple times with similar results.
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To optimize the PAO treatment conditions, we measured the time and dose
dependence of the effect of PAO (Fig. 1). Synaptosomes were incubated
with different concentrations of PAO for various time periods.
32P-Labeled PIP and PIP2 were quantified using
TLC and PhosphorImager detection. Inhibition of PI and PIP
phosphorylation by PAO reached a maximum after 20 min and was almost
complete at PAO concentrations of more than 3 µM PAO
(Fig. 1).
Ca2+-dependent and
Ca2+-independent Release of Norepinephrine, Glutamate, and
GABA from Synaptosomes--
To study release, we loaded synaptosomes
with 3H-labeled glutamate, GABA, and norepinephrine, placed
them in superfusion chambers, and monitored neurotransmitter secretion
under continuous superfusion. Release was stimulated by four procedures
that act on different components of the release machinery: moderate KCl
depolarization (K) to open voltage-gated Ca2+ channels
(26), ionomycin (I), a Ca2+ ionophore, hypertonic sucrose
that induces exocytosis of docked vesicles from the readily releasable
pool (27), and
-latrotoxin (L) that triggers release by an unknown
mechanism. We found that KCl and ionomycin induced secretion of
glutamate, GABA, and norepinephrine from synaptosomes in a strictly
Ca2+-dependent manner (Fig.
2). KCl-evoked release was transient
whereas ionomycin caused a prolonged effect, probably because as a
Ca2+ ionophore, ionomycin induces a long lasting increase
in intracellular Ca2+ that results in the continuous
recruitment of vesicles for exocytosis.
-Latrotoxin triggered
neurotransmitter release by a Ca2+-independent high
affinity interaction (EC50
3 nM). Its action was inhibited by low concentrations of La3+,
indicating a specific mechanism (Fig. 2). Sucrose caused massive transient neurotransmitter release (data not shown). Preliminary studies showed that sucrose is more potent in releasing
neurotransmitters from synaptosomes than KCl depolarization but
that both secretagogues act on the same neurotransmitter
pools.2 Sucrose-triggered
release similar to electrophysiological studies is partially inhibited
by tetanus toxin.2 Together these experiments suggest that
the synaptosomes can be used to probe secretion of different types of
neurotransmitters elicited by stimuli acting on distinct parts of the
release machinery.

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Fig. 2.
Characterization of norepinephrine,
glutamate, and GABA release from synaptosomes. Synaptosomes
preloaded with [3H]norepinephrine (top),
[3H]glutamate (middle), or
[3H]GABA (bottom) were superfused with aerated
Krebs-bicarbonate buffer. Released neurotransmitters were measured in
the efflux and are plotted as fractional release (see "Experimental
Procedures"). In the experiments on the left, release was
stimulated by brief pulses of 25 mM KCl (K) and
5 µM ionomycin (I) in regular or
Ca2+-free buffer. In the experiments on the right,
secretion was evoked by application of 25 mM KCl
(K) and 3 nM -latrotoxin (L) in
Krebs-bicarbonate buffer containing or lacking 50 µM
La3+. Note the scale differences between graphs.
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Effect of Blocking PIP and PIP2 Synthesis on
Neurotransmitter Release--
We treated synaptosomes with 3 µM PAO or control buffer and measured norepinephrine and
glutamate release stimulated by KCl, ionomycin, and
-latrotoxin
(Fig. 3A). After PAO
treatment, norepinephrine secretion was blocked whereas glutamate
release was unchanged. This was a surprising result because it suggests
that glutamate and norepinephrine release may be mechanistically
different, although both are Ca2+-dependent
(Fig. 2). To exclude the possibility that the distinct effects of PAO
were caused by differences between experimental conditions, we measured
norepinephrine and glutamate release in the same preparation of
PAO-treated and control synaptosomes (Fig. 3B). The PAO
concentrations used were high enough to assure a virtually total
inhibition of PI and PIP phosphorylation (Fig. 1). Again,
norepinephrine release was inhibited whereas glutamate release was not.
Thus PAO has opposite effects on the exocytosis of norepinephrine- and
glutamate-containing vesicles.

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Fig. 3.
PAO inhibits synaptosomal norepinephrine
release but not glutamate release. A, representative
experiments of [3H]norepinephrine (left
panels) and [3H]glutamate release
(right panels) from control and PAO-treated
synaptosomes triggered by short pulses of 25 mM KCl
(K), 5 µM ionomycin (I), or 3 nM -latrotoxin (L). B, direct
comparison of [3H]norepinephrine and
[3H]glutamate release from the same preparation of
synaptosomes. Synaptosomes were incubated with 10 µM PAO
or control buffer, divided into two aliquots, and loaded with
[3H]norepinephrine or [3H]glutamate prior
to stimulation with 25 mM KCl or 3 nM
-latrotoxin. Release measurements were performed side by side in the
same experiment. Data shown are duplicate determinations in a
representative experiment.
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The possibility that norepinephrine release but not glutamate release
is inhibited under conditions that block PI and PIP phosphorylation is
interesting because it implies that PI and PIP phosphorylation is not
universally required for exocytosis. In addition, if the components of
the release machinery differ between types of neurotransmitters, their
exocytotic mechanisms must be distinct. To affirm these conclusions, we
performed a large-scale study of the effects of PAO on synaptic vesicle
exocytosis. Three transmitters, norepinephrine, glutamate, and GABA,
were analyzed in parallel under PAO treatment conditions that block PI
and PIP phosphorylation. Because PAO is also a tyrosine phosphatase inhibitor (11, 12), we treated the synaptosomes with a strong tyrosine
phosphatase inhibitor, vanadyl hydroperoxide (VOOH), as a further
control (18). We conducted multiple independent experiments,
normalized their results, and expressed the integrated release as
percent of control (Figs. 4 and
5). Overall, the data confirm that PAO
selectively inhibits exocytosis of norepinephrine containing synaptic
vesicles independent of which secretory agent is used, without a
consistent inhibitory effect on GABA or glutamate release.

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Fig. 4.
Differential effects of PAO on
Ca2+-dependent norepinephrine, glutamate, and
GABA secretion. Transmitter release from synaptosomes preloaded
with [3H]norepinephrine, [3H]glutamate, or
[3H]GABA was stimulated by KCl (top) or
ionomycin (bottom). Synaptosomes were analyzed after
treatment with control buffer, two concentrations of PAO (3 and 10 µM PAO for norepinephrine and GABA, and 10 and 30 µM PAO for glutamate), or 100 µM VOOH
(additional control). The total release evoked by each stimulation was
calculated by integration of the release peak (Fig. 2) and normalized
by comparison with the total release observed in control synaptosomes
from the same experiment. Data shown are means ± S.E. from
multiple experiments.
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Fig. 5.
PAO inhibits synaptosomal secretion of
norepinephrine but not glutamate or GABA induced by hypertonic sucrose
or -latrotoxin. Exocytosis was induced by 3 nM
-latrotoxin (top) or 0.5 M sucrose
(bottom) in experiments similar to those described in Figs.
2 and 4, except that sucrose was applied in Ca2+-free
Krebs-bicarbonate buffer. Treatments of synaptosomes and calculations
of release were as shown in Fig. 4. Note that the only norepinephrine
release is significantly inhibited by PAO under any stimulation
condition. Data shown are means ± S.E. from multiple
experiments.
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When release was induced by KCl depolarization, PAO at low
concentrations (3 µM) severely depressed norepinephrine
secretion but had no effect on glutamate release even at 10-fold higher levels (30 µM PAO; Fig. 4). PAO also partially inhibited
GABA release stimulated by KCl. Norepinephrine release triggered by ionomycin, the second Ca2+-dependent and
probably most powerful secretagogue used here (Fig. 2), was also
inhibited by PAO. Again, glutamate secretion was unaffected. With
ionomycin, GABA release was also unchanged by PAO (Fig. 4). In
addition, norepinephrine secretion triggered by
-latrotoxin or by
sucrose was severely inhibited by 3 µM PAO (Fig. 5). With
both stimulation agents, we again observed no effect of PAO at
concentrations of up to 30 µM on glutamate secretion. Furthermore, PAO exerted no significant inhibition of GABA release stimulated by either hypertonic sucrose or
-latrotoxin.
In the design of our experiments, PAO treatments preceded the uptake of
labeled neurotransmitters for the release measurements. To exclude the
possibility that PAO changes the disposition of neurotransmitters after
uptake into the synaptosomes, we performed release measurements in
which the order of PAO treatment and neurotransmitter uptake was
reversed. With this protocol, PAO still did not inhibit glutamate
release but strongly suppressed norepinephrine secretion (data not
shown). It is also unlikely that "glutamatergic" and "GABAergic" nerve terminals are selectively protected from the inhibitory effects of PAO. These two transmitters account for more than 90% of all synapses in brain cortex. Thus the changes in PIP
and PIP2 observed by TLC with PAO treatment must
affect glutamatergic and GABAergic synaptosomes.
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DISCUSSION |
In the current experiments, we use synaptosomes to study the
release of three neurotransmitters, norepinephrine, glutamate, and
GABA. We applied PAO to inhibit PIP and PIP2 synthesis and studied the effect of this inhibition on release. Our data show that
norepinephrine secretion elicited with four stimulation protocols is
severely inhibited by PAO. In contrast, PAO had no effect on glutamate
secretion stimulated by all four stimulation protocols. PAO did not
inhibit GABA release stimulated by ionomycin, sucrose, and
-latrotoxin, and moderately inhibited GABA release evoked by KCl. We
used PAO concentrations that cause a nearly complete inhibition of PI
and PIP phosphorylation. Thus glutamate and GABA secretion do not
require newly synthesized PIP and PIP2. In contrast, similar to PC12 and chromaffin cells (9-12), PI and PIP
phosphorylation is essential for norepinephrine secretion. The fact
that we employed different means of stimulating release ensures that
the effects observed are not artifacts of a particular stimulation
method.
These results have implications for our understanding of
neurotransmitter release and membrane fusion. First, the data show that
there are transmitter-specific differences in the mechanism of
exocytosis. No such differences have been observed in the protein composition of synapses (e.g. see Refs. 5-8). The selective
requirement for phosphoinositides in norepinephrine but not glutamate
and GABA release may reflect fundamental differences between exocytosis of dense-core synaptic vesicles containing catecholamines and clear
synaptic vesicles containing glutamate and GABA.
Second, exocytosis of norepinephrine-containing vesicles requires PIP
phosphorylation at a step that precedes Ca2+ action. We
found that PAO equally inhibits Ca2+-dependent
release induced by KCl and ionomycin and Ca2+-independent
release evoked by hypertonic sucrose, which triggers exocytosis of all
docked synaptic vesicles by an unknown mechanism prior to
Ca2+ entry (27).
Third, phosphorylation of PI and PIP is not universally involved in
exocytosis. Although PAO could have multiple actions, our data document
that it acts as an effective inhibitor of PI and PIP phosphorylation in
synaptosomes. The undiminished capacity of synaptosomes to secrete
glutamate and GABA in the absence of significant PI and PIP
phosphorylation is striking. It thus is unlikely that PIP and
PIP2 function as general signals or lipidic mediators in
membrane fusion.
We thank Dr. G. Lonart for setting up the
synaptosomal release assays, Dr. K. Ichtchenko for
-latrotoxin, and
Drs. J. L. Goldstein and M. S. Brown for advice.