Dual effects of botulinum neurotoxin A on the secretory stages of chromaffin cells

European Journal of Neuroscience, Vol. 10, pp. 3369–3378, 1998 © European Neuroscience Association

Dual effects of botulinum neurotoxin A on the secretorystages of chromaffin cells

Anabel Gil, Salvador Viniegra and Luis M. GutierrezDepartamento de Neuroquımica, Instituto de Neurociencias and Facultad de Medicina, Universidad Miguel Hernandez,Campus de San Juan, 03550 Alicante, Spain

Keywords: amperometry, bovine adrenomedullary cells, catecholamines, exocytosis, snare hypothesis

Abstract

Truncation of the C-terminal domain of the synaptosomal associated protein of 25 kDa (SNAP-25) by botulinumneurotoxin A (BoNT A) has been shown to block neuroendocrine cell secretion. It is unclear, however, if toxinmechanism involved the affection of a single or more events of the exocytotic cascade. BoNT A inducedchanges in both the degree of inhibition and the kinetics of catecholamine secretion from populations of culturedbovine chromaffin cells. Ca21-dependent secretion from digitonin-permeabilized cells showed partial inhibitionassociated with the alteration of a slow secretory phase at different toxin concentrations. In contrast, in intactcells stimulated by depolarization, cell treatment with low concentrations (1 nM) of the toxin affected the latephase of secretion, whereas 100 nM BoNT A-poisoned cells showed an alteration even of fast components. Thehigh degree of inhibition associated with fast secretory component alteration was dependent on Ca21 entrythrough the Ca21 channels, as it was absent from cells permeated with the A23187 Ca21 ionophore. Vesiclepools implicated in the effect of BoNT A on the secretory response from single cells were identified usingamperometry. These studies supported the macroscopic view by showing that secretion from BoNT A-treatedpermeabilized cells presented specific inhibition of late vesicle fusions. Intact cells showed alterations in the latevesicle pool (t1

25 39 s) recruited during prolonged or repetitive KCl depolarizations using 1 nM BoNT A-treated

cells as well as in an intermediate kinetic pool (t12

5 18 s) at higher toxin concentrations (100 nM). The fasterresolved component (t1

25 8 s) or the membrane fusion event itself were not affected. Our results demonstrate

that removal of the last nine C-terminal amino acids of SNAP-25 by BoNT A has a specific effect on twodifferent and distal secretory stages in chromaffin cells.

Introduction

Study of the exocytotic process taking place in neurons and endocrinecells is essential to our understanding of how these cells accomplishtheir physiological role. It is now clear through work carried out ona wide variety of cellular systems that secretory pathways sharecommon elements forming the molecular machinery for vesicletrafficking, docking and regulated fusion (Sudhof 1995; Bennet &Scheller, 1993; Bark & Wilson, 1994). The emerging SNAP receptor(SNARE) hypothesis explains the high degree of control and specifi-city of the exocytotic process as a cascade of protein–proteininteractions. Vesicle docking is initiated by the interaction of synapto-brevin, present in the membrane of storage vesicles (v-SNARE),with syntaxin and SNAP-25, target proteins attached to the plasmamembrane (t-SNARES, Sollneret al., 1993; Horikawaet al., 1993).The 7 S complex which is formed, binds NSF (N-ethilmaleimide-sensitive factor) and SNAPs (soluble NSF-attached proteins), forminga 20 S complex which after adenosine triphosphate (ATP) hydrolysiswill energize the vesicle to a primed state. These vesicles constitutethe ‘front line’ producing the exocytotic burst released in responseto transitory Ca21 signals, which also accelerate vesicle transitionsthrough the maturation processes, resulting in membrane fusion and

Correspondence: Luis M. Gutierrez, as above. E-mail: [email protected]

Received 27 January 1998, revised 28 May 1998, accepted 9 June 1998

neurotransmitter release (Von Ruden & Neher, 1993; Heinemannet al., 1994).

The specific cleavage of docking complex proteins by clostridialneurotoxins has been an essential tool in understanding the exocytoticmachinery at a molecular level as well as the events taking placeduring vesicle maturation and membrane fusion (for a review seeMontecucco & Schiavo, 1994). In particular, SNAP-25, is proteolizedat the C-terminal domain by botulinum neurotoxin (BoNT) A and E(Blasi et al., 1993; Schiavoet al., 1993), resulting in the inhibitionof secretion in both neuronal and endocrine cellular systems. Inadrenomedullary chromaffin cells and the related PC12 line, consider-able efforts have been made to clarify the role of SNAP-25 in thesecretory process culminating in two main interpretations of theBoNT A effects as being either a partial inhibition of secretion inpermeabilized cells (Lawrenceet al., 1994), related to the alterationof a late ‘docking’ step (Gutie´rrez et al., 1997) or an almost totalinhibition of intact cell exocytosis (Knight, 1986; Lawrenceet al.,1996) that has been attributed to the action of BoNT A on earlypostdocking events (Banerjeeet al., 1996; Misonouet al., 1996;Lawrenceet al., 1997).

3370 A. Gil et al.

We have taken advantage of amperometry as a high resolutiontechnique for the study of exocytosis in individual cells therebyallowing analysis of the global and fusion kinetics of secretion(Schroederet al., 1996; Oberhauseret al., 1996) from control (non-treated) and BoNT A-poisoned chromaffin cells. Our results obtainedin both individual cells and populations of bovine chromaffin cellsdemonstrate a dose-dependent dual effect of the toxin on the secretionfrom intact cells. BoNT A could affect both a late docking event andan early prefusion step taking place after almost complete primingof the vesicles. The present results imply that the last nine residuesof the C-terminal region of SNAP-25 selectively cleaved by BoNT Aare essential for the participation of the protein in at least two distinctsteps of the exocytotic cascade, and that BoNT A is an excellent toolfor investigating the characteristics of these two distal secretory stages.

Materials and methods

Reagents

[3H]-noradrenaline (14 Ci/mmol) was obtained from NEN, Dupont(Wilmington, DE, USA). All other reagents including purified BoNT Awere obtained from Sigma Chemical Co. (Madrid, Spain). Carbonfibres were from Thornell P-55, Amoco Corp. (Greenville, SC, USA).

Chromaffin cell preparation and culture

Chromaffin cells were prepared from bovine adrenal glands bycollagenase digestion and further separated from debris and erythro-cytes by centrifugation on Percoll gradients as described (Almazanet al., 1984; Gomiset al., 1994). Cells were maintained in monolayercultures using Dulbecco’s modified Eagle’s medium (DMEM) supple-mented with 10% foetal calf serum, 10µM cytosine arabinoside,10 µM 5-fluoro-29-deoxyuridine, 50 IU/mL penicillin and 50µg/mLstreptomycin. Cells were harvested at a density of 150 000 cells/cm2

in either 96 well/plates or in 35 mm Petri dishes (Costar Corp.Cambridge, MA, USA) and used between the third and sixth dayafter plating.

Intoxication of adrenomedullary cultures with botulinumneurotoxin A and [3H]noradrenaline release experiences

Twenty-four hours after plating, chromaffin cell cultures were treatedwith BoNT A at the different concentrations specified in figurelegends diluted in a low-ionic-strength buffer of composition (in mM):NaCl, 5; KCl, 4.8; KH2PO4, 1.2; MgCl2, 1.2; CaCl2, 2.2; glucose,11; sucrose, 220; bovine serum albumin, 0.5% (mass/vol.); HEPES,15; pH 7.4; as described by Lawrenceet al., 1996. After 24 h at37 °C, the buffer was removed and cells were equilibrated in DMEMmedium for 24 h before secretory experiments were performed.

For cell population experiences, chromaffin cells (50 000 cells/6.4-mm-diameter wells) were incubated with [3H]noradrenaline (1 mCi/mL) in DMEM during 4 h. Culture medium was replaced by Krebs/HEPES (K/H) basal solution with the following composition (in mM):NaCl, 134; KCl, 4.7; KH2PO4, 1.2; MgCl2, 1.2; CaCl2, 2.5; glucose,11; ascorbic acid, 0.56; HEPES, 15; and pH adjusted to 7.4 using aNaOH solution. Then, cells were washed four times with this mediumprior to the incubation of monolayers with the secretory media.Catecholamine secretion was induced for the different periods speci-fied in figure legends with K/H basal solution, K/H high potassium(replacing isosmotically NaCl by 59 mM KCl), 100µM acetylcholine(ACh) chloride in basal medium or by 10µM A23187 in the samemedium. In other experiences, cell permeabilization was accomplishedwith 10 µM digitonin in 20 mM Pipes, pH 6.8 with 140 mM monoso-dium glutamate, 2 mM MgCl2, 2 mM Mg-ATP, and 5 mM EGTA. In

© 1998 European Neuroscience Association,European Journal of Neuroscience, 10, 3369–3378

this case basal secretion was measured in 5 mM EGTA, whereasstimulated secretion was measured in a medium containing 10µM

buffered Ca21 solution. In both types of experiments with intact orpermeabilized cells, media were collected and the remaining cellswere lysed with 2% sodium dodecyl sulphate. Secreted and totalcatecholamines were quantified by liquid scintillation spectrophotome-try and secretion was expressed as percentage of total catecholaminecontent found into the cells. In the figures, secretory activity wasgiven as net [3H]noradrenaline release (stimulated minus the basalsecretion) in each experimental condition.

Amperometric determination of exocytosis from culturedchromaffin cells

To study secretory activity from individual cells, the isolated cellswere plated in the centre of 30-mm-diameter Costar dishes at thesame density employed in the cell population experiences. Carbon-fibre electrodes insulated with polypropylene and with 14-µm-dia-meter tips were employed to monitor the release of the catecholaminecontent from individual chromaffin granules. Briefly, the electrodeswere positioned in close apposition to the surface of the cells withhigh precision hydraulic micromanipulation after observing cellmembrane deformation using Hoffman optics (Modulation Optics,Greenvale, NY, USA) mounted in an Axiovert 135 inverted-stagemicroscope (Zeiss, Oberkochen, Germany). Electrical connectionwas accomplished with mercury and the amperometric potential of1650 mV vs. an Ag/AgCl bath reference electrode was applied usingan Axopatch 200A amplifier (Axon Instruments Inc., Foster City,CA, USA). Current product of the catecholamine oxidation wasdigitized with an A/D converter (ITC-16, Instrutech Corp., GreatNeck, NY, USA) and recorded at 200µs/point using the programPulseControl (Herrington & Bookman, 1994) running on top of thegraphical software Igor Pro (Wavematrics, Lake Oswego, OR, USA)in a PowerMac 7100 computer. Experiments were performed withthe solutions described in the previous section. Cells were superfusedwith basal medium and the stimuli or permeabilization media wereapplied through a valve-controlled puffer tip commanded by theacquisition software and disposed near the studied cells.

Curve fitting to non-linear models provided or implemented insoftware (Igor Pro and Graphpad Prism) were employed to analysedata. Individual spike characteristics were analysed using Igor-Promacros supplied by Drs Ricardo Borges and Fernando Segura,allowing for the peak detection, integration and kinetic parametercalculations. After detection, only well-defined narrow peaks withamplitude higher than 5 pA were employed to build event histogramsensuring that the vesicle fusions analysed were produced in theelectrode proximity.

The Student’st-test for paired samples or theF-test was used toestablish statistical significance among the presented data. All datawere expressed as mean6 SEM from experiments performed in anumber (n) of plate wells (population experiences) or individual cells.Presented data represent experiments performed with cells from atleast two different cultures.

Results

Cell population experiments suggest dual effects of botulinumneurotoxin A on the secretion from intact chromaffin cells

We decided to use [3H]noradrenaline release from cultured bovinechromaffin cells to establish the concentrations of BoNT A requiredto alter the secretory responses after prolonged exposures (24 h) tothe toxin in a low-ionic-strength that has been shown to result in the

BoNT A and the secretory stages of chromaffin cells 3371

FIG. 1. BoNT A inhibition of catecholamine release from populations of cultured chromaffin cells. In these experiments cells were poisoned with differentconcentrations of BoNT A in a low-ionic-strength buffer during 24 h. The cells were then incubated in DMEM culture medium for an additional 24 h beforesecretion experiments were performed after 4 h of [3H]noradrenaline uptake. (A) Dose–response of BoNT A effect on intact and digitonin-permeabilized cells.Secretion was stimulated in intact cells by incubation with 59 mM KCl medium during 5 min and in digitonin-permeabilized cells by 10 min incubation with a10 µM free [Ca21] buffer. Net secretion (stimulated minus basal) was normalized to the control release value in the absence of BoNT A treatment. Continuousline represents the best fit to a two-site model of the dose–response curve for intact cells, dashed line the best fit to a single site for the data corresponding topermeabilized cells. Data represented are mean6 SEM from experiments performed in nine wells. (B) Maximal inhibition of BoNT A effect on secretionelicited by different stimuli. Cells were incubated with 100 nM of BoNT A as described above. Intact cells were stimulated by 5 min incubation with a highKCl medium or with 100µM acetylcholine chloride (ACh) in basal K/H medium. Cell permeation was accomplished using digitonin, as above, or by 10 minincubation with a K/H medium containing 10µM of the Ca21-ionophore A23187. Data are expressed as the percentage of inhibition with respect to the secretionevoked in control non-treated cells. Data are mean6 SEM from experiments performed in six wells.

effective uptake of BoNT A (Marxenet al., 1991). Initially, westudied catecholamine secretion from cells incubated with differenttoxin concentrations ranging from 1 pM to 100 nM and stimulated bytreatment during 10 min with a 10µM digitonin permeabilizationmedium in the presence or absence of buffered 10µM [Ca21].In these conditions, a 50% maximal inhibition was reached atconcentrations over 5 nM and the dose–response curve was well fittedby a single site model (Fig. 1A, solid line,R2 5 0.99). This resultcontrast with the inhibition caused by the toxin on intact cellsstimulated by depolarization with 59 mM KCl medium applied during5 min. In the latter case, Fig. 1A shows that the control response wassignificantly inhibited by concentrations as low as 50 pM and reachedmaximal inhibition at concentrations of 10 nM and over. The dose–response curve did not fit well in with a model of a single site oftoxin action (R2 5 0.89), instead the curve, that expanded for 3–4orders in magnitude of concentration, fits significantly better to atwo-site model (Fig. 1A, dashed line,R2 5 0.98), obtainingP , 0.007when the two models were compared using theF-test. Figure 1Bshows that the different action of BoNT A in the secretion frompermeabilized and intact cells could be extended to other secretorystimuli, as the secretion elicited in intact cells by either directdepolarization or through the nicotinic receptor mediated pathways(by acetylcholine stimulation) was highly affected (75–85% inhibition)whereas the release induced by cell permeation using digitonin orthe calcium ionophore A23187 was decreased to 40–50% of the levelobtained for control non-treated cells.

The possible existence of two sites for BoNT A action on thesecretion of intact cells was further investigated by studying thesecretion kinetics of the cells previously incubated with either 1 nM

toxin (thus, saturating the high-affinity site) or 100 nM (that saturatesboth sites). Figure 2A, shows that the secretion of cells incubatedwith 1 nM toxin (open triangles) was not affected during the initial30 s of release, as it was similar to the net [3H]noradrenaline releasefound under control conditions (open circles), after this periodsecretion was completely abolished, reaching 55% inhibition at thelonger period assayed (5 min). Conversely, the time-course of secretionfor cells incubated with 100 nM BoNT A (close triangles) was altered


at the shortest time assayed (30 s) where a 65% decrease was obtainedover the control value, with catecholamine release not significantlyincreasing at later periods, reaching a maximal inhibition of 85% at5 min incubations. Thus, it is clear that by poisoning the cells with1 nM of BoNT A we may alter the late components of the intactchromaffin cell secretory response whereas at higher concentrationthe toxin also affects the initial phase of exocytosis. Instead, thecatecholamine secretion found in cells stimulated by cell permeationusing digitonin in the presence of 10µM free [Ca21] during 1 minwere not different when both toxin concentrations were employed(Fig. 2,B). Thus, taking together these data suggest that in permeatedcells the toxin inhibited slow secretory components recruited after1 min stimulation, whereas in stimulated intact cells in addition tothis slow phase of secretion affected by the toxin using a 1 nM

concentration there are additional fast secretory components alteredat higher concentrations of BoNT A.

Botulinum neurotoxin A specifically affects late vesicle fusionsin digitonin-permeabilized cells

One interesting possibility arising from the experiences performed incell populations, is that intoxication with different concentrations ofBoNT A could be used to distinguish between the kinetic componentsof the secretory response. In order to study these componentsand obtain a view complementary to that provided by populationexperiments, we studied the kinetic properties of the secretoryresponse of individual cells by amperometry. Using carbon-fibreelectrodes of 14µm diameter we obtained responses to cell permeationin the presence of Ca21 composed of the individual vesicle fusionsinduced by the stimulus (Fig. 3A). These traces allowed us to studyboth, the BoNT A effects on the global kinetics of the secretoryprocess through integration of amperometric currents and also thepossible alteration of the exocytotic fusion process by analysing theproperties of individual amperometric spikes. Single cells werepermeabilized by superfusion during 30 s with basal K/H solutioncontaining 10µM digitonin and the secretory responses monitoredusing amperometry. Figure 3A depicts examples of the secretoryresponse of control and BoNT A-treated cells. Clearly, the most

3372 A. Gil et al.

FIG. 2. Temporal course of secretion from BoNT A-treated intact and digitonin-permeabilized cells. BoNT A intoxication was performed as described in Fig. 1.(A) Depolarizing-evoked secretion from intact cells. The figure shows net [3H]noradrenaline release (stimulated minus basal) from control non-poisoned cells(open circles), 1 nM BoNT A treated cells (n), and 100 nM BoNT A treated cells (m). Secretion in basal non-depolarizing media was 3.16 0.2% of totalcatecholamine content and in 59 mM KCl stimulated cells was 9.66 0.6%. (B) Secretion from permeabilized cells. Digitonin permeabilization was performedas described earlier and net release corresponds to control, 1 and 100 nM BoNT A-treated cells incubated during 1 or 10 min in the calcium-containingpermeabilization medium. Data are mean6 SEM from six wells.

FIG. 3. BoNT A selectively inhibits late fusion events in individual digitonin-permeabilized cells. In these experiments secretion was monitored by amperometryusing 14-µm carbon-fibre electrodes in close apposition with the cell surface. (A) Amperometric current traces representative of experiments performed incontrol and BoNT A-treated cells. Traces obtained from control non-treated cells, 1 nM and 100 nM BoNT A treated cells stimulated by rapid superfusion during30 s with 10µM digitonin in K/H basal media are shown. (B) Cumulative integrals from control and BoNT A-treated cells. Conditions were as described in(A), after individual response integration the average curve was obtained from control (n 5 13), 1 nM BoNT A (n 5 12) and 100 nM BoNT A (n 5 20) treatedcells. Normalized curves for kinetic comparison are shown in the inset.

evident effect of the cell intoxication is the drastic decrease in thenumber of late vesicles fusions occurring 40 s after cell permeation,being similar the responses of the cells treated with 1 and 100 nM

BoNT A concentrations. In order to gain further information thecurves were fitted to equations composed of one or more Boltzmancomponents representing the different granule ‘pools’ mobilized


during cell stimulation. The control curve was best-fitted to threeBoltzman components giving a significance ofP , 0.0001 whencompared by using theF-test to the best fit to two components. Thefit was further checked by comparison with the best fit to fourBoltzman components, in this case no statistical improvement wasobtained (P . 0.05) over the simpler equation. We obtained half-


FIG. 4. Effect of BoNT A on the secretion of individual chromaffin cells. Amperometry was performed as described in the previous figures. (A) Amperometriccurrent traces representative of experiments performed in control and BoNT A-treated cells. Traces obtained from control non-treated cells, 1 nM and 100 nMBoNT A treated cells stimulated by rapid superfusion during 1 min with a 59 mM KCl solution are shown. (B) Cumulative integrals. Conditions were asdescribed in (A). The average curve was obtained for control (n 5 30, continuous line), 1 nM (n 5 9, pointed line) and 100 nM BoNT A (n 5 10, dashed line)treated cells. Current integral values (mean6 SEM pC) at 1 min were 4696 82 (control), 2596 56 (1 nM BoNT A) and 1506 25 (100 nM BoNT A. The insetshows normalized data for kinetic comparison.

time constants of 15, 28 and 75 s, and the average cumulativeintegral obtained from cells intoxicated with 100 nM BoNT A wascharacterized by a 90% decrease in the proportion of the slowercomponent compared with that found in control cells while it did notaffect the faster components. The great specificity of the toxin inaffecting the slower component of the secretory response fromdigitonin-permeabilized chromaffin cells was evident when the curveswere normalized (see Fig. 3B, insert), the control average cumulativeintegral is characterized by the presence of a slow phase of secretionthat was absent from the secretion obtained in cells treated with 1 or100 nM BoNT A. Cumulative curves obtained from cells treated withboth concentrations of BoNT A almost superimpose indicating thatthey were kinetically similar.

Botulinum neurotoxin A modifies the size of granule ‘pools’recruited by cell depolarization of intact cells

We next addressed if different concentrations of BoNT A readilyaffect the size of distinct vesicle ‘pools’ recruited by stimulation ofintact chromaffin cells as was suggested by the data obtained withcell populations in contrast with the specific alteration of a singlelate component demonstrated in permeabilized cells. Representativeamperometric current traces of control and BoNT A treated cellsstimulated by superfusion with 59 mM KCl are depicted in Fig. 4A;note that the cells incubated with 1 nM lacked late fusion spikesduring prolonged exposure to a depolarizing stimulus, in contrast tothe relative abundance of these spikes in control non-treated cells.Incubation of the cells with higher concentrations of toxin (100 nM)led to an enhancement of this effect resulting not only in the absenceof late granule fusions but also a decrease in the number of eventsrecruited at earlier times (between 10 and 20 s of stimulation).Figure 4B shows the average cumulative integral from experimentsperformed in control cells (n 5 30) and cells incubated with 1 nM

(n 5 9) and 100 nM (n 5 10) concentrations of BoNT A. Controlcurve was best-fitted again to three Boltzman components (Table 1)giving a significance ofP , 0.0001 when compared by using the


F-test to the best fit to two components. Half-times for thesecomponents were 8.7, 18.6 and 38.8 s (two times faster than secretionfrom permeabilized cells) and account for 19, 38 and 43%, respect-ively, of the total vesicle release obtained during 1 min depolarizations.Interestingly, the preincubation of the cells with 1 or 100 nM BoNT Aaltered the average cumulative integral (Fig. 3B, dashed lines) thatsaturates after 30 s of stimulation (see the normalized graph depictedin Fig. 3B inset). Both curves were best-fitted to two Boltzmancomponents with half-times close to 9 and 19 s (see parameters inTable 1), and differed only in the proportion of the components,decreasing the relative importance of the later ‘pool’ at 100 nM toxinconcentration. Thus, it is clear that the main effect of the toxin evenat low concentrations is to prevent late vesicle fusions detected undercontrol conditions (half-time of 39 s) and that higher concentrationof the toxin (100 nM) affected even the population of fusions occurringat an intermediate time (half-time of 19 s), all these data beingconsistent with the cell population experiments.

Early and late stages of the secretory process affected by thebotulinum neurotoxin A in intact cells

In order to study if the initial secretory ‘pools’ were affected by thetoxin, shorter 10 s depolarizations were applied and the amperometricresponses analysed in control and 100 nM BoNT A-incubated cells.Current integration and average, produced curves that fit to a singleBoltzman model, with maximum values of 1446 21 and 1116 18 pC,respectively (data not shown). In addition the curves overlap whennormalized; therefore, we can say that BoNT A have no, or at mosta minor effect on the initial vesicle ‘pool’ mobilized by transientstimulation.

To assess further the inhibition of the late components of thesecretory response induced by BoNT A, the control and toxin-treatedcells where stimulated by repetitive transient 10 s depolarizations.The rationales considered that the first pulse would deplete initial‘pools’ and subsequent pulses would recruit intermediary and latevesicle fusions. Figure 4A shows examples of the secretory responses

3374 A. Gil et al.

TABLE 1. Secretory components identified from curve fitting parameters for amperometric cumulative integrals in the presence and absence of BoNT A

Faster ‘pool’ Intermediate Slow ‘pool’

Experimental condition (n) t12

(s) B (pC) K(s) t12

(s) B (pC) K(s) t12

(s) B (pC) K(s)

Control (30) 8.7 90.4 1.9 18.6 175.4 4.3 38.8 201.3 8.5Vesicles secreted/cell 235 456 523BoNT A 1 nM (9) 8.7 81.7 2.3 19.6 174.6 4.1 no detectedVesicles secreted/cell 212 454BoNT A 100 nM (10) 9.6 71.6 2.2 20.2 74.6 3.8 no detectedVesicles secreted/cell 186 194

Averaged cumulative integrals were fitted by non-linear regression to equations composed of Boltzman distributions: C (t)5 B/[1 1 exp(t12– t)/K)]. The vesicles

secreted in each ‘pool’ were calculated taking the maximum charge value corresponding to this component (B), and assuming that 14µm diameter electrodescover 22% of the surface of a typical 15µm diameter spherical chromaffin cell and that the average vesicle charge is 1.756 0.08 pC (n 5 450) under ourrecording conditions.

FIG. 5. BoNT A affects granule recruitment by repetitive depolarizations. The capability of cells to recruit late ‘pools’ was studied by using six successivestimulations of 10 s depolarization. (A) Typical traces obtained from control, 1 nM and 100 nM BoNT A poisoned cells. (B) Rate of vesicle recruitment calculatedfrom cells under the conditions described in (A). The cumulative integral of the first depolarization was normalized to 100% (control response) and the secretionobtained in successive pulses expressed as a percentage of the initial response. Data are mean6 SEM from experiments performed on nine (Control), nine(1 nM BoNT A) and eight (100 nM BoNT A) cells.

from individual cells under each experimental condition, the gradualdepletion of releasable vesicle ‘pools’ was evident in control cellsafter completion of six depolarizations. Pretreatment of the cells witheither 1 or 100 nM BoNT A increased the rate of vesicle depletion,very few fusions could be observed after three depolarization pulses(Fig. 5A). A quantification of this effect by current integration ineach pulse is given in Fig. 4B, the first pulse after the test depolariza-tion (100%) is greatly inhibited in 1 nM (60% of control,n 5 9)and 100 nM BoNT A-treated cells (79%,n 5 6). Almost completeinhibition is obtained in subsequent pulses with both toxin concentra-tions. These results suggest that the different degree of inhibitionobserved between the two concentrations of the toxin during the firstpulse after the test was due to the fact that intermediate releasable‘pools’ were differently affected by both concentrations whereas thesubsequent total inhibition reflected the high level of alteration of thelate ‘pool’ recruitment.

Stimuli recruiting the lower proportion of the late vesicle ‘pool’are less affected by low concentrations of botulinumneurotoxin AAn interesting prediction of the concentration-dependent dual effectsof BoNT A on the secretory stages of intact neuroendocrine cells is


that stimuli recruiting different proportions of late components wouldbe differently affected by toxin treatment. Continuous stimulationwith the physiological agent acetylcholine at 100µM concentrationresulted in a secretory pattern (Fig. 6A, lower trace) that representeda smaller proportion of late vesicle fusions when compared withdirect depolarization (Fig. 6A, upper trace). The average currentintegral from experiments performed in 25 cells showed a moresteady slope than that obtained for 59 mM KCl stimulation (Fig. 6A).This cumulative integral was best-fitted to a 3 Boltzman componentequation with half-time constants of 8.5, 18.6 and 35.8 s, veryconsistent with those reported above for KCl stimulation, but withthe proportion of the late component accounting for only 21% of thetotal release at 1 min, half of that found for the depolarizing stimulus.The differences between both stimuli were evident when sensitivityto low concentrations of BoNT A was analysed using multiple-pulseprotocols (Fig. 6B). The rate of vesicle ‘pool’ recruitment was lessaffected when using ACh in transient 10 s stimulations. Moreover,cell treatment with 1 nM BoNT A had only minor affects on the rateof vesicle fusion recruitment as can be seen in Fig. 6B. However,incubation with 100 nM of toxin greatly affected the ability to recruitvesicle ‘pools’. Taking together these data, it seems reasonable that byusing stimuli recruiting smaller proportions of late vesicle releasable


FIG. 6. Cell stimulation with acetylcholine (ACh) is less affected by low concentrations of BoNT A. (A) Responses to direct depolarization or ACh recruitdifferent proportions of late components. Cells were stimulated by 1 min exposure to secretagogues. Representative traces and average cumulative integralsfrom experiments performed on cells treated with 59 mM KCl (n 5 30, continuous line) and 100µM ACh (n 5 25, dashed line). (B) Repetitive 10 s pulses withACh demonstrating the minor effects of BoNT A at a 1 nM concentration. Experiments were performed and averaged as described in the previous figure butusing 100µM ACh as the stimulus. Representative traces and the rate of recruitment from experiments performed on eight cells under each experimentalcondition are shown.

‘pools’ we may find lower sensitivity to BoNT A, reflecting theability of the toxin to block preferentially late fusion events whenlow concentrations are employed (1 nM). Alternatively, the highdegree of inhibition found at concentrations in the order of 100 nM

may be the result of a dual effect of the toxin that at theseconcentrations affects not only late stages of secretion but also earlierprocesses.

Membrane fusion stages and catecholamine release are notaffected by the toxin

Once analysed the effect of BoNT A treatment on the kineticcomponents of the secretory response in chromaffin cells it isinteresting to study if the truncation of the C-terminal domain ofSNAP-25 caused by the toxin (Blasiet al., 1993; Schiavoet al.,1993) could be also affecting the membrane fusion event itself. Anapproach to study this problem is to analyse the amperometric singleevent properties of control and toxin-treated intact cells, as everystep of the fusion process is reflected in the ‘spike’ characteristics(Schroederet al., 1996). More than 200 well-defined narrow individualfusion events from KCl responses were analysed from control and100 nM BoNT A treated cells, half-width and ‘spike’ charge weremeasured and binned to generate the distribution histograms depictedin Fig. 7. Half-width histograms fit to Lorentzian distributions centredat t1

2of 18.66 0.7 ms for control and 20.06 0.7 ms for BoNT A-

treated cells, this difference not being statistically significant. In asimilar way event charge (Q) was studied after integration, bothdistributions being centred at the same value of 0.66 0.1 pC. In


addition, we analysed the number of events that presented a well-defined ‘foot’ feature under each experimental condition. Six to 7%of the events presented this characteristic in both control and 100 nM

BoNT A-poisoned cells. Taking into account all these data it appearsthat the ‘spike’ shape and size characteristics remain relativelyunchanged after BoNT A-treatment. It is therefore unlikely that thealteration of SNAP-25 caused by the toxin is affecting the very lastmembrane fusion steps leading to catecholamine release.

Discussion

Size of the vesicle ‘pools’ mobilized during stimulation ofintact chromaffin cells

Several studies using capacitance measurements (Neher & Marty,1982) have attempted to identify the vesicle ‘pools’ governingsecretion from neuroendocrine chromaffin cells (Neher & Zucker,1993; Heinemannet al., 1994; Horrigan & Bookman, 1994; Parsonset al., 1995).These studies established the basis for our knowledgeon granule recruitment during cell stimulation; however, due to thefact that ‘patch-clamped’ cells ‘leak’ molecular components that maybe essential for exocytosis (Morgan & Burgoyne, 1992) and thatincreases in membrane capacitance are often not due to granule fusion(Oberhauseret al., 1996), it is desirable to study the size of releasablevesicle ‘pools’ using alternative techniques. Amperometry in intactcells provides one such alternative even though kinetics are affectedby the imposition of limiting diffusion step due to the application ofsecretagogues through perfusion systems. Overall, our kinetics are in

3376 A. Gil et al.

FIG. 7. Effect of BoNT A-poisoning on the characteristics of single fusionevents. Individual ‘spikes’ were analysed from the depolarization responsesof control (non-treated) and 100 nM BoNT A-poisoned cells. (A) BoNT Aaction on the ‘spike’ half-width time. Half-width was measured as thedifference between rising and falling half height times for each spike. Datawere binned at 4 ms intervals. (B) Vesicle charge distribution. Even chargewas calculated by trapezoidal integration and histograms were built using0.2 pC bin size. Results shown are from control (263 events) and BoNTA-treated cells (207 events).

the seconds range described by other authors using electrochemicaldetection (Kim & Westhead, 1989) or luciferin–luciferase ATPmonitoring (Cen˜a & Rojas, 1990) and far from the tens of ms scaleprovided in capacitance measurements. Calculation of secretory poolsize can easily be accomplished by using the averaged currentintegrals obtained from amperometric measurements in control cells(4666 82 pC, 1 min depolarization), and as we have used 14-µm-diameter carbon-fibre electrodes in close apposition with the cells,we can estimate that secretion was recorded from 22% of the totalcell area of a typical 15µm in diameter round chromaffin cells (thusthe averaged total secretion is about 21356 370 pC/cell). Finally, wecan express the integral current in terms of fused vesicles knowingthat the averaged ‘spike’ integral has a value of 1.756 0.08 pC/vesicle under our recording conditions (n 5 450), giving about 1200vesicles released per cell during 1 min of prolonged depolarization.Given that value we can calculate the respective sizes of the threevesicle components mobilized during prolonged depolarization, whichare characterized by their best fit to Boltzman components as describedin results (Fig. 4 and Table 1). As such, 230 vesicles are released inthe early detected component (t1

25 8.7 s), 460 granules in the inter-

mediate ‘pool’ (t125 18.6 s) and 510 vesicles in the slower component

(t125 38.8 s), these data have been summarized in scheme 1. There

is a good correlation between the size of the fast component detectedin the present work (release of granule populations D, D9... in scheme


1) and the reported value of about 200 vesicles governing the fusionstep studied by repetitive depolarizations or photolysis of caged Ca21

(Neher & Zucker, 1993; Heinemannet al., 1994). Based on theamount of released vesicles, this component may be formed by thedifferent fast and slow ‘subpools’ described in isolated cells or tissueslices (Horrigan & Bookman, 1994; Gilliset al., 1996; Moser &Neher, 1997) and constitute the initial set of about 200 vesiclesreleased in chromaffin cells stimulated by rapid perfusion (Cen˜a &Rojas, 1990). The heterogeneous composition of this ‘pool’ wasconfirmed when an estimated 40–50 vesicles were released in responseto a 500 mM sucrose hypertonic solution (20.26 3 pC, n 5 9, Giland Gutierrez, unpublished data), procedure that defines readilyreleasable pools in neurons (Stevens & Tsujimoto, 1995). Aftercompletion of the first burst, the subsequent may represent the fusionof ‘docked’ granules undergoing ‘priming’ processes (scheme 1 poolsB, C, C9...), and as such the 690 vesicles forming both the first andsecond components released during 30–40 s after cell depolarizationmight be the result of ‘docked’ granule depletion. This result is ingood agreement with the 450 (Burgoyne 1991) to as much as 800‘docked’ granules (Parsonset al., 1995) reported elsewhere andsuggests that only a fraction of these membrane-attached vesiclesconstitute the ‘ready to go’ reservoir. In addition, the depletion ofthis two initial components taking about 35–40 s in permeabilized orintact cells (see Figs 3 and 4) is in close agreement with the timerequired for the complete release of morphologically ‘docked’ vesiclesin melanotrophs and chromaffin cells (Parsonset al., 1995). Therefore,the slower component detected in our recordings (t1

25 38.8 s) is likely

to represent the vesicles that may undergo ‘docking’ (component Ain scheme 1) and complete the reserve ‘pool’ thereby allowing thecells to release more than 1000 vesicles during repetitive or prolongedstimulations (Neher & Zucker, 1993).

(Scheme 1)

These components could be also identified in the slower secretoryresponse of permeabilized cells in proportions very similar to thosefound in intact cells, indicating that at the temporal resolution of ouramperometric responses we could readily identify three separatecomponents in the secretory response of chromaffin cells.

A model of action of botulinum toxin A on the different stagesof secretion

In addition to determining the timing and size of the vesicularcomponents mobilized by stimulation of ‘intact’ and permeabilizedcells, our studies in populations and individual cells have allowed usto characterize the dual effect of BoNT A on the exocytotic stagesin neuroendocrine chromaffin cells. Thus, by using 1 nM concentrationof BoNT A the secretory response of treated cells resembled that ofthe control non-poisoned cells during the first 30 s of stimulation, butwas completely abolished after this period. Single cell amperometricresponses have shown in a very graphic way that the toxin preventsgranule fusions associated with the late component of secretion (t1

25

38.8 s, component A). Curve fitting of the average cumulative integralfrom 1 nM BoNT A-poisoned cells showed a high specificity foraffecting this component, obtaining an average maximum chargevalue of 2586 56 pC, that represents the fusion of 670 vesicles. Thenon-affected release which corresponded very well in timing and size


to the secretion of the total population (97%) estimated for ‘docked’granules, indicates that low concentrations of the toxin inhibittransition A→B in scheme 2. This observation is consistent with theeffects reported when peptides mimicking the C-terminal region ofSNAP-25 were assayed. These peptides which have a similar actionto BoNT A in preventing binding of the endogenous protein to otherpolypeptides that form the ‘docking’ complex, cause inhibition of thelate components of catecholamine release and the accumulation ofvesicles in ‘predocked’ states (Gutie´rrezet al., 1995; Gutie´rrezet al.,1997), also observed in BoNT A-treated cells (Gutie´rrezet al., 1997).The specific alteration of the ‘docking’ process caused by lowconcentrations of BoNT A may reflect the higher sensitivity of freeSNAP-25 molecules to the toxin or alternatively, the participation oflow-affinity 7 S complexes during these initial exocytotic events.Higher concentrations of the toxin would be required to affect theC-terminal region of SNAP-25 when the protein is ‘tightly’ associated,forming the ‘matured’ 20 S complex which participates in late stagesof the exocytotic process (Hayashiet al., 1994; Pellegriniet al., 1995).

Our results also reveal the ‘postdocked’ nature of the secretorystage affected by incubation of the cells with higher concentrationsof BoNT A (over 10 nM). The best-fit for the average cumulativeintegral obtained when stimulating the cells treated with 100 nM

of toxin gave a value of 370 vesicles released during prolongeddepolarizations, leaving the faster component relatively unaffected(less than 20% inhibition) and strongly limiting the vesicles releasedas an intermediary ‘pool’ (t1

25 18 s). These data imply that the altered

stage is a late process taking place after the first secretory burst andthat the granules released are ‘docked’ and have undergone part ofthe ‘priming’ processes (inhibition of transition C9→D in scheme 2).In addition, the absence of this alteration in the secretion of digitoninand A23187 permeated cells revealed the Ca21 sensitivity of this latesecretory event that senses the high and focal Ca21 rise occurringafter the opening of voltage-dependent Ca21 channels in stimulatedintact cells.

(Scheme 2)

The data presented here give further support to the biochemicalevidence for a postdocking role of SNAP-25 presented by severalgroups by using the model of permeabilized chromaffin and PC12cells treated by BoNT E (Banerjeeet al., 1996; Misonouet al., 1996;Lawrenceet al., 1997). While these works established the importanceof C-terminal residues (181–197) and their dissociation from synaptob-revin in late postpriming events involving SNAP-25, our data demon-strate the importance of the nine C-terminal residues cleaved byBoNT A in the participation of this protein in two different stages ofCa21-dependent exocytosis. As factors such as temperature, pH and[Ca21] influence some of the molecular transitions that constitutemembrane-attached vesicle maturations, and there is no preciseunderstanding of the nature and sequence of these events, furthercharacterization of the late secretory events and mechanisms under-lying the regulation of vesicle ‘pools’ will be essential to understandingof exocytotic process taking place in neuroendocrine cells anddetermining what differences it has with synaptic transmission, despitethe molecular components they have in common.


Acknowledgements

We thank M.A. Company for his technical assistance and S. Ingham for art-work and style corrections. The kind supply of Igor-based programs to performsingle event analysis by Drs Ricardo Borges and Fernando Segura from theUniversidad de la Laguna (Tenerife, Spain) is greatly acknowledged. Thiswork was supported by a Grant from the Spanish Direccio´n General deInvestigacio´n Cientıfica y Tecnica (DGICYT, PM 95–0110). A.G. was arecipient of a research aid from Instituto Juan Gil-Albert.

Abbreviations

BoNT A botulinum neurotoxin ADβH dopamine-β-hydroxylaseNSF N-ethylmaleimide-sensitive fusion proteinSNAP soluble NSF attachment proteinSNAP-25 synaptosomal associated protein of 25 kDaSNARE SNAP receptort-SNARE target-SNAREVAMP vesicle-associated membrane proteinv-SNARE vesicle SNARE

References

Almazan, G., Aunis, D., Garcia, A.G., Montiel, C., Nicolas, G.P. & Sanchez-Garcia, P. (1984) Effect of collagenase on the release of [3H]-noradrenalinefrom bovine cultured adrenal chromaffin cells.Br. J. Pharmacol., 81,599–610.

Banerjee, A., Kowalchy, J.A., DasGupta, B.R. & Martin, T.F.J. (1996) SNAP-25 is required for a late post-docking step in Ca21-dependent exocytosis.J. Biol. Chem., 271, 20227–20230.

Bark, I.C. & Wilson, M.C. (1994) Regulated vesicular fusion in neurons:snapping together the details. Proc. Natl Acad. Sci. USA, 91, 4621–4624.

Bennett, M. & Scheller, R. (1993) The molecular mechanism for secretion isconserved from yeast to neurons.Proc. Natl Acad. Sci. USA., 90, 2559–2563.

Blasi, J., Chapman, E.R., Link, E., Binz, T., Yamasaki, S., De Camilli, P.,Sudhof, T.C., Niemann, H. & Jahn, R. (1993) Botulinum neurotoxin Aselectively cleaves the synaptic protein SNAP-25.Nature, 365, 160–163.

Burgoyne, R.D. (1991). Control of exocytosis in adrenal chromaffin cells.Biochem. Biophys. Acta, 1071, 174–202.

Cena, V. & Rojas, E. (1990) Kinetic characteristics of calcium-dependent,cholinergic receptor controlled ATP secretion from adrenal medullary cells.Biochem. Biophys. Acta, 1023, 213–222.

Gillis, K.D., Moβner, R. & Neher, E. (1996) Protein kinase C enhancesexocytosis from chromaffin cells by increasing the size of the readillyreleasible pool of secretory granules.Neuron, 16, 1209–1220.

Gomis, A., Gutierrez, L.M., Sala, F., Viniegra, S. & Reig, J.A. (1994)Ruthenium red inhibits selectively chromaffin cell calcium channels.Biochem. Pharmacol., 47, 225–231.

Gutierrez, L.M., Canaves, J., Ferrer-Montiel, A.-V., Reig, J.A., Montal, M. &Viniegra, S. (1995) A peptide that mimics the carboxy-terminal domain ofSNAP-25 blocks Ca21 -dependent exocytosis in chromaffin cells.FEBSLett., 372, 39–43.

Gutierrez, L.M., Viniegra, S., Rueda, J., Ferrer-Montiel, A.-V., Ca`naves, J. &Montal, M. (1997) A peptide that mimics the C-terminal sequence ofSNAP-25 inhibits secretory vesicle docking in chromaffin cells.J. Biol.Chem., 272, 2634–2639.

Hayashi, T., McMahon, H., Yamasaki, S., Binz, T., Hata, Y., Sudhoff, T.C. &Niemann, H. (1994) Synaptic vesicle membrane fusion complex: action ofclostridial neurotoxins on assembly.EMBO J., 13, 5051–5061.

Heinemann, C., Chow, R.H., Neher, E. & Zucker, R.S. (1994) Kinetics of thesecretory response in bovine chromaffin cells following flash photolysis ofcaged Ca21. Biophys. J., 67, 2546–2557.

Herrington, J. & Bookman, R.J. (1994)Pulse Control V4.3: IGOR XOPS forPatch Clamp Data Acquisition and Capacitance Measurements. Universityof Miami Press. Miami, FL.

Horikawa, H., Saisu, H., Ishizuka, T., Sekine, Y., Tsugita, A., Odani, S. &Abe, T. (1993) A complex of rab3A, SNAP-25, VAMP/synaptobrevin-2and syntaxins in brain presynaptic terminals.FEBS Lett., 330, 236–240.

Horrigan, F.T. & Bookman, R.J. (1994) Releasible pools and the kinetics ofexocytosis in adrenal chromaffin cells.Neuron, 13, 1119–1129.

Kim, K.-T. & Westhead, E.W. (1989) Cellular responses to Ca21 fromextracellular and intracellular sources are different as shown by simultaneous

3378 A. Gil et al.

measurements of cytosolic Ca21 and secretion from bovine chromaffincells.Proc. Natl Acad. Sci. USA, 86, 9881–9885.

Knight, D.E. (1986) Botulinum toxin types A, B and D inhibit catecholaminesecretion from bovine adrenal medullary cells.Febs Lett., 207, 222–226.

Lawrence, G.W., Weller, U. & Dolly, J.O. (1994) Botulinum A and thelight chain of tetanus toxins inhibit distinct stages of Mg.ATP-dependentcatecholamine exocytosis from permeabilised chromaffin cells.Eur. J.Biochem., 222, 325–333.

Lawrence, G.W., Foran, P. & Dolly, J.O. (1996) Distinct exocytotic responsesof intact and permeabilised chromaffin cells after cleavage of the 25-kDasynaptosomal-associated protein (SNAP-25) or synaptobrevin by botulinumtoxin A or B. Eur. J. Biochem., 236, 877–886.

Lawrence, G.W., Foran, P., Mohammed, N., DasGupta, B.R. & Dolly, J.O.(1997) Importance of two adjacent C-terminal sequences of SNAP-25 inexocytosis from intact and permeabilized chromaffin cells revealed byinhibition with botulinum neurotoxins A and E.Biochemistry, 36, 3061–3067.

Marxen, P., Bartels, F., Ahnert-Hilger, G. & Bigalke, H. (1991) Distinc targetsfor tetanus and botulinum A neurotoxins in the signal transducing pathway inchromaffin cells.Naunyn-Schmiedeberg’s Arch. Pharmacol., 344, 387–395.

Misonou, H., Nishiki, T.-I., Sekiguchi, M., Takahashi, M., Kamata, Y., Kozaki,S., Ohara-Imaizumi, M. & Kumakura, K. (1996) Dissociation of SNAP-25and VAMP-2 by MgATP in permeabilized adrenal chromaffin cells.BrainRes., 737, 351–355.

Montecucco, C. & Schiavo, G. (1994) Mechanism of action of tetanus andbotulinum neurotoxins.Mol. Microbiol., 13, 1–8.

Morgan, A. & Burgoyne, R.D. (1992) Exo1 and Exo2 proteins stimulatecalcium-dependent exocytosis in permeabilized adrenal chromaffin cells.Nature, 355, 833–835.

Moser, T. & Neher, E. (1997) Rapid exocytosis in single chromaffin cellsrecorded from mouse adrenal slices.J. Neurosci., 17, 2314–2323.


Neher, E. & Marty, A. (1982) Discrete changes of cell membrane capacitanceobserved under conditions of enhanced secretion in bovineadrenalchromaffin cells. Proc. Natl Acad. Sci. USA, 79, 6712–6716.

Neher, E. & Zucker, R.S. (1993) Ca21-dependent steps in chromaffin cellsecretion.Neuron, 10, 21–30.

Oberhauser, A.F., Robinson, I.M. & Fernandez, J.M. (1996). Simultaneouscapacitance and amperometric measurements of exocytosis: a comparison.Biophys. J., 71, 1131–1139.

Parsons, T.D., Coorssen, J.R., Horstmann, H. & Almers, W. (1995). Dockedgranules, the exocytic burst, and the need for ATP hydrolysis in endocrinecells.Neuron, 15, 1085–1096.

Pellegrini, L., O’Connor, V., Lottspeich, F. & Betz, H. (1995) Clostridialneurotoxins compromise the stability of a low energy SNARE complexmediating NSF activation of synaptic vesicle fusion.EMBO J., 14, 4705–4713.

Schiavo, G., Santucci, A., DasGupta, B.R., Mehta, P.P., Jontes, J., Benfenati,F., Wilson, M.C. & Montecucco, C. (1993). Botulinum neurotoxins serotypesA and E cleave SNAP-25 at distinct COOH-terminal peptide bonds.FEBSLett., 335, 99–103.

Schroeder, T.J., Borges, R., Finnegan, J.M., Pihel, K., Amatore, C. &Wightman, R.M. (1996) Temporally resolves, independent stages ofindividual exocytotic secretion events.Biophys. J., 70, 1061–1068.

Sollner, T., Whiteheart, S.W., Brunner, M., Erdjument-Bromage, H.,Geromanos, S., Tempst, P. & Rothman, J. (1993) SNAP receptors implicatedin vesicle targeting and fusion.Nature, 362, 318–324.

Stevens, C.F. & Tsujimoto, T. (1995) Estimates for the pool size of releasiblequanta at a single synapse and for the time required to refill a pool.Proc.Natl Acad. Sci. USA, 92, 846–849.

Sudhof, T.C. (1995) The synaptic vesicle cycle: a cascade of protein–proteininteractions.Nature, 375, 645–653.

Von Ruden, L. & Neher, E. (1993) A Ca-dependent early step in the releaseof catecholamines from adrenal chromaffin cells.Science, 262, 1061–1065.

Dual effects of botulinum neurotoxin A on the secretory stages of chromaffin cells

Documents

Transcript of Dual effects of botulinum neurotoxin A on the secretory stages of chromaffin cells