Two modes of exocytosis from synaptosomes are differentially regulated by protein phosphatase types 2A and 2B

Two modes of exocytosis from synaptosomes are differentially

regulated by protein phosphatase types 2A and 2B

Monique L. Baldwin, John A. P. Rostas and Alistair T. R. Sim

School of Biomedical Sciences, University of Newcastle and Clinical Neuroscience Program, Hunter Medical Research Institute,

Callaghan, New South Wales, Australia

Abstract

The inhibitors okadaic acid (OA), fostriecin (FOS) and

cyclosporin A (CsA), were used to investigate the roles of

protein phosphatases in regulating exocytosis in rat brain

synaptosomes by measuring glutamate release and the

release of the styryl dye FM 2-10. Depolarization was induced

by 30 mM KCl, or 0.3 mM or 1 mM 4-aminopyridine (4AP). OA

and FOS produced a similar partial inhibition of KCl- and

0.3 mM 4AP- evoked exocytosis in both assays, but had little

effect upon exocytosis evoked by 1 mM 4AP. In contrast, CsA

had no effect upon KCl- and 0.3 mM 4AP-evoked exocytosis,

but significantly enhanced glutamate release but not FM 2-10

dye release evoked by 1 mM 4AP. None of the phosphatase

inhibitors changed calcium signals from FURA-2-loaded syn-

aptosomes either before or after depolarization. Pretreatment

with 100 nM phorbol 12-myristate 13-acetate abolished the

inhibitory effect of OA on exocytosis induced by 0.3 mM 4AP.

Taken together, these results show that exocytosis from

synaptosomes has a phosphatase-sensitive and phospha-

tase-insensitive component, and that there are two modes of

phosphatase-sensitive exocytosis that can be elicited by dif-

ferent depolarization conditions. Moreover, these two modes

are differentially sensitive to phosphatase 2A and 2B.

Keywords: exocytosis, FM 2-10, glutamate, kiss-and-run,

protein phosphatase, synaptic vesicle.

J. Neurochem. (2003) 85, 1190–1199.

The modulation of synaptic vesicle cycling is one of the

critical cellular mechanisms that controls synaptic transmis-

sion, regulating the supply of releasable transmitter at the

nerve terminal. Once synaptic vesicles have fused with the

plasma membrane to release neurotransmitters (exocytosis),

they must be recycled so that they can be refilled and

undergo the next round of release. Full synaptic vesicle

fusion, whereby vesicles collapse completely into the plasma

membrane, internalize by endocytosis and then recycle

through the endosome (Alvarez et al. 1993; Ales et al.

1999), is the classical model of exocytosis and the principal

mechanism under normal stimulation conditions (De Camilli

and Takei 1996). However, an alternative mechanism has

been suggested whereby vesicles fuse only transiently with

the plasma membrane and are rapidly retrieved (Valtorta

et al. 2001). In this mode of release, referred to as ‘transient

fusion’ or ‘kiss-and-run’, vesicles fuse with the plasma

membrane by forming a transient fusion pore while preser-

ving vesicle integrity. After detachment, intact vesicles can

fuse again with the plasma membrane without any preceding

endosomal fusion. Kiss-and-run has been demonstrated by

analysis of secretion from neuroendocrine and chromaffin

cells by combining whole-cell capacitance methods and

amperometry to measure vesicle fusion and catecholamine

release of vesicle contents (Alvarez et al. 1993; Zhou et al.

1996). The advantage of a kiss-and-run mechanism is a rapid

cycling between a fusion state and a non-fusion state, thus

accelerating the turnover of the limited pool of synaptic

vesicles in neurons (Cousin and Robinson 1999).

Studies using the lipophilic, but membrane impermeable,

fluorescent styryl dyes in combination with the measurement

of soluble neurotransmitter release have made possible the

investigation of kiss-and-run in neurons (Henkel and Betz

1995; Klingauf et al. 1998). When the styryl dye FM 2-10

reversibly partitions into the outer leaflet of exposed plasma

Received December 15, 2002; revised manuscript received January 23,

2003; accepted February 9, 2003.

Address correspondence and reprint requests to Associate Professor A.

T. R. Sim, School of Biomedical Sciences, University of Newcastle and

Clinical Neuroscience Program, Hunter Medical Research Institute,

Callaghan, NSW 2308, Australia.

E-mail: [email protected]

Abbreviations used: 4AP, 4-aminopyridine; CsA, cyclosporin A; FOS,

fostriecin; OA, okadaic acid; PKC, protein kinase C; PMA, phorbol

12-myristate 13-acetate; PP1, protein phosphatase 1; PP2A, protein

phosphatase 2A; PP2B, protein phosphatase 2B.

Journal of Neurochemistry, 2003, 85, 1190–1199 doi:10.1046/j.1471-4159.2003.01779.x

1190 � 2003 International Society for Neurochemistry, J. Neurochem. (2003) 85, 1190–1199

membrane its fluorescence increases. By endocytosis, the dye

is able to label the luminal surface of the synaptic vesicle

and, during subsequent exocytosis, is lost to the extracellular

medium, accompanied by a decrease in fluorescence (Cousin

and Robinson 1999). Release of neurotransmitter is detect-

able whether the nerve terminal uses full fusion or kiss-and-

run, as neurotransmitter is soluble within the synaptic vesicle

and can diffuse rapidly through an open pore. However, only

the relatively slow process of full fusion would allow

complete dye loss from the membrane, whereas in the

relatively rapid kiss-and-run mode the synaptic vesicles are

internalized before the dye can fully departition and escape

from the membrane (Klingauf et al. 1998).

By comparing the relative ability of the glutamate release

and styryl dye release assays to measure exocytosis, Cousin

and Robinson (2000) were able to distinguish between two

different modes of glutamate release from isolated nerve

terminals (synaptosomes); 30 mM KCl and 0.3 mM 4-ami-

nopyridine (4AP) evoked exocytosis by a mechanism that

was readily detectable with both the glutamate and FM 2-10

release assays, and was therefore assumed to be occurring

with the full fusion mode of release. However, 1 mM 4AP

recruited a second mode of release that was detectable using

the glutamate assay but not the FM 2-10 assay; this was

proposed to be kiss-and-run. This second mode was

proposed to be largely mediated by protein kinase C (PKC)

(Cousin and Robinson 2000).

Phosphorylation and dephosphorylation of nerve terminal

proteins is known to regulate neurotransmitter release, and

experiments using membrane-permeable inhibitors of protein

phosphatases suggest that serine/threonine protein phospha-

tases are positive regulators of this release (Sim et al. 1991;

Verhage et al. 1995; Vickroy et al. 1995; Issa et al. 1999;

Storchak et al. 2001). However, the relative importance of

the different serine/threonine protein phosphatases in the full

fusion and kiss-and-run modes of release is not known. The

three major serine/threonine phosphatases are protein phos-

phatase 1 (PP1), 2A (PP2A) and 2B (PP2B), all three of

which are found in rat brain synaptosomes (Sim et al. 1993).

Okadaic acid (OA), an inhibitor of protein phosphatases PP1,

PP2A and to a lesser extent PP2B (Haystead et al. 1989;

Ishihara et al. 1989), inhibits depolarization-induced glu-

tamate release from isolated nerve terminals (synaptosomes)

(Verhage et al. 1995; Vickroy et al. 1995; Issa et al. 1999).

However, it is impossible to ascertain which class of protein

phosphatase is being affected in this attenuation of transmit-

ter release, as OA causes partial or complete inhibition of

several protein phosphatases. The use of selective inhibitors

is necessary to resolve this issue.

We have used the highly selective, membrane-permeable

protein phosphatase inhibitors fostriecin (FOS) and cyclosp-

orin A (CsA) to delineate the class of protein phosphatases

likely to be involved in the release of neurotransmitter from

synaptosomes. Synaptosomes have been used extensively to

study the mechanisms of synaptic neurotransmitter release

because they remain metabolically active and preserve the

ability to synthesize and release neurotransmitters. Our

results support the argument for the presence of two modes

of exocytosis in synaptosomes and, more importantly, show

that PP2A and PP2B have distinct roles in regulating

exocytosis from synaptosomes by these two modes.

Experimental procedures

Materials

OAwas obtained from Alexis Biochemicals (San Diego, CA, USA);

FOS and CsA were obtained from Calbiochem (San Diego, CA,

USA). FM 2-10 and FURA-2 AM were from Molecular Probes

(Eugene, OR, USA). All other reagents were obtained from the

Sigma Chemical Co. (St Louis, MO, USA).

Glutamate release assay

Synaptosomes were prepared from rat brain cerebral cortex by

centrifugation on discontinuous Percoll gradients (fractions 3 and 4

were combined) as described previously (Dunkley et al. 1986).

Under the conditions used, synaptosomes remain viable for several

hours and respond to multiple depolarization and repolarization

signals (Dunkley et al. 1986). The glutamate release assay was

performed using enzyme-linked fluorescent detection of released

glutamate (Nicholls and Sihra 1986). In brief, synaptosomes were

stored on ice and were diluted to 500 lg protein in 2 mL Krebs-like

solution (118 mM NaCl, 5 mM KCl, 25 mM NaHCO3, 1 mM MgCl2,

10 mM Glucose, pH 7.4) at 37�C. Experiments were started after

addition of 1 mM NADP+. Fifty units of glutamate dehydrogenase

were added after 1 min, and the synaptosome suspension was treated

after 4 min with either KCl (30 mM) or 4AP (0.3 or 1 mM).

Fluorescence at 460 nm emission was measured continuously in a

Perkin-Elmer (Shelton, CT, USA) LS-50B spectrofluorimeter using a

four-cuvette holder that enabled four samples to be run simulta-

neously. A typical experiment consisted of two control conditions,

and two treated conditions, each in the presence and absence of Ca2+

(1.2 mM), thus enabling each sample to be measured against its own

control simultaneously. Glutamate release was quantitated by the

addition of 10 nmol glutamate at the end of each run, which acted as

an internal standard that allowed comparison to be made between

experiments. Data are presented as the Ca2+-dependent glutamate

release (expressed in nmoles per milligram protein), measured at

different time points after stimulation (100, 200, 300 and 400 s), and

calculated as the difference between release measured in the presence

and absence of added Ca2+. In experiments in which inhibitors were

used, the synaptosomes were preincubated with either OA (0.01–

10 lM), FOS (0.1 or 1 lM), CsA (10 lM) or phorbol 12-myristate13-acetate (PMA) (100 nM), for 5, 5, 15 or 7 min respectively, before

depolarization with either KCl or 4AP.

None of the drugs used altered basal release (in the presence or

absence of added Ca2+), and none had any effect on either KCl- or

4AP-evoked Ca2+-independent release (data not shown).

Styryl dye release assay

Synaptic vesicle fusion with the plasma membrane was measured

using release of the fluorescent dye FM 2-10, according to Cousin

Protein phosphatase 2A and 2B in exocytosis 1191

� 2003 International Society for Neurochemistry, J. Neurochem. (2003) 85, 1190–1199

and Robinson (2000). In brief, synaptosomes were stored on ice and

were diluted to 700 lg protein in 2 mL Ca2+-containing Krebs-like

solution (as used in the glutamate release assay) at 37�C. FM 2-10

(100 lM) was added after 3 min, and endocytotic uptake of

membrane-bound FM 2-10 was stimulated 1 min later with

30 mM KCl. After 2 min to allow internalization of the dye,

synaptosomes were pelleted in a microfuge for 1 min and washed

twice in Krebs-like solution containing 1 mg/mL bovine serum

albumin. Synaptosomes were then resuspended in Krebs-like

solution (either plus or minus 1.2 mM Ca2+) at 37�C, and release

induced by the addition of either KCl (30 mM) or 4AP (0.3 or

1 mM). Release of accumulated FM 2-10 was measured as the

decrease in fluorescence upon release of the dye into solution

(excitation 488 nm, emission 540 nm). Data are presented as the

Ca2+-dependent decrease in FM 2-10 fluorescence and calculated as

the difference between release measured in the presence of 1.2 mM

Ca2+ and that in the presence of 1.2 mM EGTA. Any drugs were

added after the dye-loading procedure, and the synaptosomes were

preincubated with OA (0.01–10 lM), FOS (0.1 lM), CsA (10 lM) orPMA (100 nM) for 5 min before depolarization with either KCl or

4AP. For experiments with PMA and OA, PMAwas added first, and

OA was added 30 s later.

Calcium measurements

Intrasynaptosomal calcium levels were measured using FURA-2

fluorescence according to Brent et al. (1997). FURA-2 was prepared

as a 1-mM stock solution in dimethyl sulfoxide and 0.5% pluronic

acid. In brief, synaptosomes (2 mg protein) were incubated with

gentle shaking for 40 min at 37�C in 2 mL control buffer containing

5 lM FURA-2. The suspensions were then washed in ice-cold

Krebs-like buffer and centrifuged for 2 min to remove extrasyna-

ptosomal FURA-2. Synaptosomes were then stored on ice and

diluted to 600 lg protein in 2 mL Ca2+-containing Krebs-like

solution at 37�C, adjusted to give a final concentration of 1.2 mM

Ca2+ in the cuvette. Measurements were made by alternating the

excitation wavelengths of 340 and 380 nm (the 340 : 380 nm

fluorescence ratio); fluorescent emission was monitored at 510 nm.

Calibration of the fluorescence signals was performed at the end of

each experiment by adding digitonin (200 lM) to obtain Fmaxfollowed by EGTA (2 mM) to obtain Fmin (Grynkiewicz et al.

1985). Extrasynaptosomal FURA-2 was determined for each

synaptosomal preparation by adding MnCl2 (10 mM) at the end of

each experiment to quench the extracellular fluorescence and was

stable for the duration of the experiments. Intracellular [Ca2+] was

calculated by the equation of Grynkiewicz et al. (1985), using a KD

of 81 nM for the Ca2+–FURA-2 complex derived under our

laboratory conditions by scanning the fluorescent response of

FURA-2 to different concentrations of Ca2+ (0–39.8 lM). In

experiments in which drugs were used, the synaptosomes were

preincubated with OA (0.1 lM), FOS (0.1 lM) or CsA (10 lM), for5, 5 or 15 min respectively, before depolarization with either KCl or

4AP. As basal calcium levels in synaptosomes gradually increased

during the 3–4 h involved in a series of experiments (86–117 nM

Ca2+), the sequence of application of drugs was varied between

experiments, and drug-treated samples were compared with control

samples measured immediately before or after the drug-treated

samples. There was no difference in drug effects associated with the

age of the synaptosomes.

Results

Experimental approach

Exocytosis from isolated synaptosomes was measured using

two complementary approaches. Results from the measure-

ment of glutamate and FM 2-10 release in response to three

different stimuli are shown in Fig. 1. Although the time

course of exocytosis is different for the two assays, this is a

reflection of the nature of the assays used rather than an

inherent difference in the mechanisms of exocytosis of

glutamate and FM 2-10. Specifically, the exocytosis of

glutamate is slower than that of FM 2-10 as a consequence

of the dilution of released glutamate into a large volume of

buffer. This results in submaximal conditions for the

glutamate dehydrogenase used in the assay. The signal

generated over minutes by the assay therefore represents

enzymatic amplification of the glutamate released within

seconds. FM dye is released more slowly than glutamate

owing to the requirement for it to departition from the

membrane before a signal is generated. However, it is

measured directly, producing a signal in seconds, and

therefore has a shorter time course than measurement of

glutamate.

Measurement of glutamate release clearly restricts the

measurement of exocytosis to glutamatergic synaptosomes,

although these do represent a large proportion of the

synaptosomes in the preparation. Measurement of FM 2-10

dye release, on the other hand, is independent of the

neurotransmitter, and represents exocytosis from all syna-

ptosomes. In order to restrict analysis to exocytosis rather

than signals generated by non-specific leaching or bleaching

of the dye, results are reported as only the Ca2+-dependent

release.

Two alternate modes of depolarization were studied (KCl

and 4AP) to investigate whether any effects of the phospha-

tase inhibitors on glutamate release were due to actions on

events associated with exocytosis, as opposed to the

depolarization-activated steps that trigger exocytosis. KCl

produces a clamped depolarization, whereas 4AP, at the

Fig. 1 Ca2+-dependent glutamate (a) and FM 2-10 (b) release evoked

by 30 mM KCl, 0.3 mM 4AP and 1 mM 4AP. Arrowhead shows when

depolarizing agents were added. Each point is the mean ± SEM of

3–4 independent experiments.

1192 M. L. Baldwin et al.


concentrations used, leads to propagation of repetitive action

potentials by blocking both A type and delayed rectifier

potassium channels, thereby preventing correction of spon-

taneous depolarizations that occur from ion diffusion (Nich-

olls 1993). Levels of Ca2+-dependent glutamate release

evoked by 30 mM KCl and 0.3 mM 4AP were similar,

whereas depolarization with 1 mM 4AP evoked more release

(Fig. 1a). However, the depolarization-stimulated FM 2-10

release was similar for all depolarizing stimuli (Fig. 1b).

These results are consistent with the findings of Cousin and

Robinson (2000) that the slow rate of diffusion of FM 2-10

dye limits its capacity to detect short-duration modes of

exocytosis, suggesting that the increased exocytosis promo-

ted by higher 4AP concentrations represents a ‘kiss-and-run’-

like mode.

Regression analysis of FM 2-10 release indicated that the

data were consistent with two phases of release. The biphasic

nature of exocytosis from synaptosomes has been well

documented although the interpretation of this remains

unclear. Analysis showed that the initial rate constant for

each of the three depolarization conditions was similar

(s ¼ 4–6 s). Importantly, these kinetics of FM dye release

derived from a population of synaptosomes are within the

same order of magnitude as that observed in a single

presynaptic bouton from an intact neuron (Ryan et al. 1993).

Effects of OA and FOS: role of PP1 and PP2A

in exocytosis

The protein phosphatase inhibitor OA has previously been

shown to inhibit KCl-evoked glutamate release in synapto-

somes (Verhage et al. 1995). In this study OA inhibited this

release by a mean ± SEM of 58 ± 5% at 100 s (Fig. 2a).

Investigation of the concentration dependence of this inhi-

bition of glutamate release showed that inhibition was

maximal at 0.1 lM; increasing the OA concentration 100-

fold to 10 lM did not significantly change this inhibition

(data not shown). This also suggests that PP2B, which is

inhibited by OA at 10 lM but not at 0.1 lM (Cohen 1989),

has little role in regulating release under these conditions.

However, it cannot be determined whether the major

inhibitory effect on release seen at lower OA concentrations

results from inhibition of PP2A and/or PP1. FOS (0.1 lM), apotent and selective inhibitor of PP2A (Evans and Simon

2001), inhibited glutamate release to a similar extent as OA

(Fig. 2b). Increasing the concentration of FOS to 1 lM did

not change the extent of inhibition (data not shown). There

was no significant difference (p > 0.24, Student t-test)

between the level of inhibition of Ca2+-dependent release

by OA and FOS at any time after depolarization. The effect

of OA and FOS on glutamate release evoked by 0.3 mM 4AP

(Figs 2c and d) followed the same pattern as that observed

following KCl-evoked release; OA (0.1 lM) and FOS

(0.1 lM) inhibited Ca2+-dependent release to a similar extent.These results suggest a major role for PP2A in the regulation

of KCl- and 0.3 mM 4AP-evoked glutamate release. In

contrast, the effect of phosphatase inhibition on release

evoked by 1 mM 4AP was markedly different: OA and FOS

(Figs 2e and f respectively) had little or no effect on Ca2+-

dependent glutamate release.

To investigate whether the effects of OA and FOS were

specific for glutamate release or more generally applicable to

the mechanisms of synaptic exocytosis, we next investigated

FM 2-10 release and compared the results with the profile of

glutamate release under the same conditions. The effects of

the phosphatase inhibitors OA and FOS on depolarization-

stimulated FM 2-10 release are shown in Fig. 3. OA and

FOS both inhibited FM 2-10 release in the same manner as

that observed for glutamate release. Inhibition was greatest

when release was evoked by KCl or 0.3 mM 4AP (Figs 3a–d):

31 ± 4 and 28 ± 2% inhibition of KCl-evoked release, and

30 ± 3 and 31 ± 3% inhibition of 0.3 mM 4AP-evoked

release respectively (3 min after depolarization). Kinetic

analysis showed that the rate constants (s) of release were not

Fig. 2 Effect of OA and FOS on Ca2+-dependent release of glutamate

in synaptosomes depolarized by 30 mM KCl (a and b), 0.3 mM 4AP (c

and d), and 1 mM 4AP (e and f). Synaptosomes were preincubated

with either 0.1 lM OA (a, c and e; dotted line) or 0.1 lM FOS (b, d and

f; dashed line) before depolarization. Each point is the mean ± SEM of

3–4 independent experiments.



affected by the inhibitors (not shown); rather, as indicated by

the figures, the effect was on the extent of the first phase. In

contrast to release measured in response to 30 mM KCl or

0.3 mM 4AP, neither OA nor FOS significantly affected

release evoked by 1 mM 4AP (Figs 3e and f).

It has been shown that PMA greatly increases glutamate

release stimulated by 0.3 mM 4AP, but not FM 2-10 release;

this has been interpreted as resulting from a switch from full

fusion to kiss-and-run release (Cousin and Robinson 2000).

Although PMA has a number of targets, this effect of PMA is

assumed to be related to activation of PKC as it is blocked by

inhibition of PKC (Cousin and Robinson 2000). Consistent

with these results, we found that PMA increased 0.3 mM

4AP-evoked Ca2+-dependent glutamate release by 103 ± 8%

but had no effect on FM 2-10 release (Fig. 4). In addition,

OA (0.1 lM) produced no change in 0.3 mM 4AP-evoked

release in the presence of PMA for both assays, whereas it

reduced 0.3 mM 4AP-evoked release of glutamate and

FM 2-10 by 64 ± 7% and 30 ± 3% respectively in the

absence of PMA (Fig. 2c and Fig. 3c respectively). Taken

together, these results are consistent with the hypothesis

that PMA induces a switch from full fusion to kiss-and-run as

the predominant mode of release (Cousin and Robinson

2000).

Effects of CsA: role of PP2B in exocytosis

To further investigate the role of different phosphatases in

exocytosis we investigated the effect of the PP2B-selective

inhibitor CsA (Liu et al. 1991) on both glutamate and FM2-

10 release. Using this inhibitor, we observed no significant

change in Ca2+-dependent glutamate release following

KCl- or 0.3 mM 4AP-induced depolarization (Figs 5a and b

respectively). This is consistent with our findings that 10 lMOA (which also inhibits PP2B) had no greater effect on

glutamate release than 0.1 lM OA, suggesting that PP2B

plays little role in regulating the release of glutamate evoked

by KCl or 0.3 mM 4AP. In contrast, CsA significantly

increased glutamate release evoked by 1 mM 4AP, and the

degree of stimulation increased with time reaching 59 ± 6%

above control values by 400 s (Fig. 5c).

CsA did not affect FM 2-10 release under any of the

depolarization conditions used (Fig. 6). The lack of effect on

KCl- and 0.3 mM 4AP-stimulated release correlates well with

results for glutamate release. However, CsA greatly increased

Ca2+-dependent glutamate release stimulated with 1 mM

4AP, whereas FM 2-10 release was virtually unaffected.

This is consistent with CsA affecting a second mode of

exocytosis that is less apparent in the FM 2-10 assay.

Fig. 3 Effect of OA and FOS on Ca2+-dependent release of FM 2-10 in

synaptosomes depolarized by 30 mM KCl (a and b), 0.3 mM 4AP (c and

d), and 1 mM 4AP (e and f). Arrowhead shows when depolarizing agent

was added. Synaptosomes were preincubated with either 0.1 lM OA

(a, c and e) or 0.1 lM FOS (b, d and f) before depolarization. C, control.

Each point is the mean ± SEM of 3–4 independent experiments.

Fig. 4 Effect of OA on 0.3 mM 4AP-evoked glutamate release (a) and

FM 2-10 release (b) in the presence of PMA. Arrowhead shows when

depolarizing agent was added. Each point is the mean ± SEM of 3–4

independent experiments.



Effects of phosphatase inhibition on calcium levels

To test the possibility that the effects of protein phosphatase

inhibition on exocytosis were via modulation of calcium

influx, mean calcium levels were measured using the

fluorescent dye, FURA-2. Figure 7 shows that, under the

conditions used, none of the inhibitors changed synapto-

somal calcium levels under basal conditions or after depo-

larization with either KCl or 4AP (0.3 mM and 1 mM). The

increase in levels of calcium induced by depolarization with

1 mM 4AP, the stimulus that induces the kiss-and-run-like

mode of exocytosis, was significantly greater than that

induced by depolarization with 0.3 mM 4AP, the stimulus

that primarily induces the full fusion mode of exocytosis

(p < 0.0001, Student’s t-test). There was no significant

difference between the calcium levels induced by depolar-

ization with 0.3 mM 4AP or KCl, both of which induce

exocytosis by full fusion.

Discussion

Isolated synaptosomes provide a useful model with which to

study synaptic exocytosis. Although the measurement of

exocytosis and endocytosis in all in vitro preparations does

not fully match the millisecond timescale that occurs in vivo,

the molecular machinery underlying exocytosis is the same

and all in vitro approaches, including electrophysiological

ones, take advantage of artificially reduced rates to probe

these molecular mechanisms. Interestingly, the rate constant

of diffusion of FM dye in the present study, which measures

a population of nerve terminals, is similar to that seen by

direct imaging of individual hippocampal neurons (Ryan

et al. 1993).

The inhibition of KCl-evoked release by OA has been

observed in several cell types (Verhage et al. 1995; Vickroy

et al. 1995; Issa et al. 1999), but no previous release studies

Fig. 5 Effect of selective inhibition of PP2B on Ca2+-dependent

release of glutamate in synaptosomes depolarized by 30 mM KCl (a),

0.3 mM 4AP (b) and 1 mM 4AP (c). Synaptosomes were preincubated

with 10 lM CsA (dotted line) before depolarization. Each point is the

mean ± SEM of 3–4 independent experiments.

Fig. 6 Effect of selective inhibition of PP2B on Ca2+-dependent

release of FM 2-10 in synaptosomes depolarized by 30 mM KCl (a),

0.3 mM 4AP (b) and 1 mM 4AP (c). Arrowhead shows when depolar-

izing agent was added. Synaptosomes were preincubated with 10 lM

CsA before depolarization. C, control. Each point is the mean ± SEM

of 3–4 independent experiments.



have examined the effects of specifically inhibiting PP2A.

We have now been able to do this using FOS, a PP2A

inhibitor that is 10 000-fold more selective for PP2A than for

PP1 (Walsh et al. 1997). Our finding that FOS inhibited KCl-

and 0.3 mM 4AP-evoked glutamate release to a similar extent

as OA shows that the principal phosphatase regulating

release under these conditions is PP2A, although a role for

PP1 cannot be ruled out. The results with FM 2-10

strengthen this interpretation, depicting a similar pattern of

effect of OA and FOS on release evoked by KCl and 0.3 mM

4AP. The lack of effect of PP2B inhibition using the specific

inhibitor CsA (Liu et al. 1991) confirmed that PP2B plays

little role in release under these conditions. In view of the

well recognized role of PP2B in endocytosis (Liu et al.

1994), one might have expected to see some effect of PP2B

inhibition on the release of glutamate. The fact that PP2B

inhibition had no observable effect on release indicates that

glutamate release measurements under these experimental

conditions primarily reflect steps in the synaptic vesicle

fusion cycle associated with recruitment of release-ready

vesicles and fusion with the membrane, rather than the whole

synaptic vesicle cycle. It is important to note that maximal

inhibition of PP2A evoked by KCl- and 0.3 mM 4AP evoked

release does not completely inhibit release. Therefore,

exocytosis from synaptosomes has both phosphatase-sensi-

tive and phosphatase-insensitive components.

Cousin and Robinson (2000) reported no increase in

FM 2-10 release as the concentration of 4AP was increased

from 0.3 to 1 mM, despite an increase in glutamate release

under the same conditions. Our results follow the same

pattern and are therefore consistent with the interpretation

that a different mechanism of release is recruited at higher

concentrations of 4AP. Our results with phosphatase inhib-

itors reveal that under these different depolarization condi-

tions exocytosis also shows a differential pharmacological

sensitivity. This further strengthens the hypothesis that

different mechanisms of release occur under different

depolarization conditions. Specifically, OA and FOS inhib-

ited exocytosis evoked by KCl and 0.3 mM 4AP, but not that

evoked by 1 mM 4AP, whereas CsA increased glutamate

release evoked by 1 mM 4AP but had no effect on release

evoked by 0.3 mM 4AP and KCl.

The release of glutamate is detectable whether the nerve

terminal uses the full fusion or kiss-and-run-like mode of

exocytosis because glutamate is soluble within the synaptic

vesicle and able to diffuse rapidly into the extracellular

space, even if the vesicle remains fused for only a short

period (as happens during kiss-and-run). However, the loss

of FM 2-10 styryl dye from the luminal surface of the vesicle

membrane is slow, so that only the relatively long duration of

full fusion would allow substantial dye loss from the

membrane. In the relatively short duration of the kiss-and-

run mode of exocytosis the synaptic vesicles are internalized

before most of the dye can escape from the membrane.

It is therefore proposed that the mode of release seen at

high concentrations of 4AP represents a kiss-and-run-type

Fig. 7 Effect of protein phosphatase inhib-

itors on calcium levels in synaptosomes

depolarized with 30 mM KCl (a, b and c),

0.3 mM 4AP (d, e and f) or 1 mM 4AP (g, h

and i). Each panel contains average traces

for 3–5 experiments normalized to a com-

mon starting calcium level. Control (filled

circle) and drug treatment (open square)

traces are both plotted but are virtually co-

incident. Addition of drug and depolarizing

agent are indicated by the downward and

upward arrowheads respectively.



mechanism because it is observed with the glutamate release

assay but not with the FM 2-10 release assay. Furthermore,

we propose that PP2A is a positive regulator of the full

fusion mode of exocytosis, shown by the ability of PP2A

inhibitors (OA and FOS) to inhibit both glutamate and

FM 2-10 release stimulated by KCl or 0.3 mM 4AP. However,

PP2A appears to have little role to play in the kiss-and-run-

like mode of exocytosis, as indicated by the lack of effect of

OA and FOS on glutamate and FM 2-10 release evoked by

1 mM 4AP. The differential ability of the two measures of

exocytosis to detect the effects of PP2B inhibition on 1 mM

4AP-evoked release suggests that PP2B is involved in

regulating the kiss-and-run-like mode of exocytosis, in

addition to its known role in endocytosis (Liu et al. 1994).

The differential effect of PP2B inhibition on glutamate

release induced by 30 mM KCl and 1 mM 4AP was first

noted by Nichols et al. (1994) but, in that study, the

difference was assumed to be due to differences in the mode

of depolarization. Our results show that this is not the case:

changing the mode of depolarization does not change the

sensitivity to the phosphatase inhibitors, as long as the same

mechanism of release is activated. Using two different

depolarization stimuli, KCl and 0.3 mM 4AP, each of which

induces full fusion release, no sensitivity to the PP2B

inhibitor CsA was observed. However, sensitivity to the

PP2B inhibitor was seen when the same depolarizing agent

(4AP) was used at a higher concentration (1 mM) because a

different mode of exocytosis that is sensitive to PP2B action

was induced.

Valtorta et al. (2001) discussed the possibility of both

modes of release operating in synapses, with a switch to kiss-

and-run occurring as vesicles acquire the ‘competence’ to

release neurotransmitter in this way. By using amperometry,

Ales et al. (1999) showed that, in response to an increase in

extracellular Ca2+, chromaffin cells shifted from the full

fusion mode to the kiss-and-run mechanism. During kiss-

and-run events the fusion pore appeared to expand briefly to

a large size, allowing for rapid and complete transmitter

release. There was no size difference between vesicles that

underwent full fusion and those that released transmitter by

the kiss-and-run mechanism. This shows that vesicle cycling

can be consistent with both full fusion and transient fusion.

The rise in intracellular Ca2+ can be induced by increasing

the concentration of Ca2+ in the incubation medium or by

applying robust electrical stimulation paradigms (von Gers-

dorff and Matthews 1994; Ales et al. 1999; Pyle et al. 2000;

Beutner et al. 2001), but also after the application of phorbol

esters (Cousin and Robinson 2000). Phorbol esters, which

activate PKC, increase the release of neurotransmitter in

many neuronal systems (Malenka et al. 1986; Gerber et al.

1989; Barrie et al. 1991; Coffey et al. 1993; Capogna et al.

1995, 1999; Stevens and Sullivan 1998; Chen et al. 1999;

Cousin and Robinson 1999; Yawo 1999), including syna-

ptosomes. Although PMA has a number of targets, the

enhancement of exocytosis is blocked by inhibitors of PKC

(Cousin and Robinson 2000). Activation of PKC in chrom-

affin cells accelerated fusion pore expansion and subsequent

pore closure and vesicle retrieval (Graham et al. 2000).

Is the additional mode of exocytosis induced in synapto-

somes at high 4AP in addition to full fusion, or does the

predominant mode of release switch from one mode to

another? Our studies with phosphatase inhibitors add support

to the latter conclusion. As FOS or OA inhibited release

evoked by 0.3 mM but had no effect on release evoked by

1 mM 4AP, the simplest interpretation is that, by increasing

the concentration of 4AP, a switch has occurred in the

phosphatase-sensitive component of release from full fusion

to the kiss-and-run-like mode. Cousin and Robinson (2000)

reported that PMA induced a switch in the predominant

mode of release from full fusion to kiss-and-run when

synaptosomes were stimulated by 0.3 mM 4AP. We have

confirmed these results and now show that, in the presence of

PMA, OA produced no change in 0.3 mM 4AP-evoked

release; however, in the absence of PMA, OA inhibited

0.3 mM 4AP-evoked release. These results are further

evidence for the interpretation that a switch occurs in the

predominant mode of release under these conditions, at least

for the phosphatase-sensitive component of release.

One potential implication of a switch in the predominant

mode of exocytosis is that release evoked by 0.3 mM 4AP

plus PMA might be increased by PP2B inhibition with CsA

(as seen for 1 mM 4AP). However, for this to be true, the

CsA-sensitive step must be rate limiting even in the presence

of PMA. We found that CsA had no effect on synaptosomes

pretreated with 100 nM PMA (maximal stimulation of

glutamate release) and depolarized with 0.3 mM 4AP (results

not shown). Attempts to repeat the experiment with

submaximal concentrations of PMA were unsuccessful

because of the extremely steep dose–response curve of

PMA (50 nM had no discernible effect on glutamate release).

At which point in the synaptic vesicle cycle are the serine/

threonine phosphatases acting to differentially regulate the

two modes of release? These enzymes are likely to have

many substrate proteins involved in many aspects of the

synaptic vesicle cycle such that no single protein is likely to

be the target. One possibility is that the protein phosphatase

inhibitors modulate exocytosis through modulation of cal-

cium levels. However, experiments using FURA-2 did not

show an effect of any phosphatase inhibitor on calcium levels

under basal or stimulated conditions (Fig. 7). This suggests

that the major role for PP2A and PP2B in regulating

exocytosis is downstream of calcium influx, but their targets

remain unknown. Interestingly, CsA also increased exocyto-

sis from synaptosomes induced by ionomycin (Jovanovic

et al. 2001), which is consistent with a role for PP2B

downstream of calcium entry. Our studies also suggest that

the mode of exocytosis induced by ionomycin is also of the

kiss-and-run type.



Although the molecular mechanisms underlying the two

modes of exocytosis have not been delineated, the emerging

concept is that the two modes involve the same molecular

components (Valtorta et al. 2001). Thus after a vesicle has

fused with the membrane, it reaches a point in the common

pathway when it must decide whether to proceed via the full

fusion or the kiss-and-run mode; this decision is made by

regulatory molecular mechanisms that alter the rates of the

two pathways in response to different stimulatory conditions.

This is interesting in view of recent mathematical modeling

which suggests that the predominant role of protein

phosphatases in the control of intracellular processes is to

regulate the rate and duration of signaling (Heinrich et al.

2002). The simplest interpretation of our results is that PP2A

promotes the step(s) that commit the vesicle to full fusion

and, under normal depolarization conditions, the mechanism

for retrieval of the vesicle by the kiss-and-run-like mode is

not activated. Upon stimulation with a high 4AP concentra-

tion, the alternative pathway for the retrieval of the vesicle by

the kiss-and-run-like mode is activated and the retrieval of

vesicles is held in check by PP2B activity. Alternatively,

PP2A and/or PP2B might be influencing the delivery of

synaptic vesicles to the readily releasable pool or the plasma

membrane. This is consistent with the recent finding that in

response to 1 mM 4AP there is a rapid dephosphorylation of

PP2B-specific sites on synapsin I, a protein involved in

synaptic vesicle trafficking from reserve pools (Jovanovic

et al. 2001). Given that PP2A influences only full fusion and

PP2B influences only the kiss-and-run-like mode of exocy-

tosis, this interpretation of our data would, however, imply

that each mode uses different molecular machinery for the

delivery of synaptic vesicles to the plasma membrane.

We have focused on the phosphatase-sensitive component

of exocytosis and argued that increasing stimulation induces

a switch in the predominant mode of exocytosis. This leaves

open the question as to whether a similar switch occurs in the

phosphatase-insensitive component. Chromaffin cells can

show an increase in kiss-and-run from 20 to 80% of the

release mechanism under conditions of high stimulation

(Ales et al. 1999). As we do not know how many kiss-and-

run cycles occur during a typical assay or what proportion of

the vesicular styryl dye is released during a single fusion

event, we cannot determine the relative proportions of full

fusion and kiss-and-run involved in the phosphatase-insen-

sitive modes of release from synaptosomes using these

techniques. The mechanisms responsible for the switch

between the modes of release, and the identity of the protein

phosphatase-sensitive control points, remain to be deter-

mined.

Acknowledgements

This research was supported by grants from the National Health

Medical Research Council of Australia, a University of Newcastle

postgraduate scholarship to MLB, and infrastructure funding from

NSW Department of Health through the Hunter Medical Research

Institute. We are grateful to Dr Derek Laver for assistance with

linear regression analysis of FM 2-10 release and we also thank

Professor Peter Dunkley and Dr Martı́n Cammarota for helpful

discussions.

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Two modes of exocytosis from synaptosomes are differentially regulated by protein phosphatase types 2A and 2B

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