INTRODUCTION

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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.

	EXPERIMENTAL PROCEDURES

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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.

	RESULTS

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PAO Blocks PIP and PIP₂ Synthesis in Synaptosomes-- We labeled synaptosomes with ³²P_i 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).

View larger version (17K): [in this window] [in a new window]   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.

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.

View larger version (30K): [in this window] [in a new window]   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.

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.

View larger version (46K): [in this window] [in a new window]   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.

View larger version (55K): [in this window] [in a new window]   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.

View larger version (53K): [in this window] [in a new window]   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.

	DISCUSSION

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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.