Botulinum neurotoxin inhibits the release of newly synthesized acetylcholine from torpedo electric organ synaptosomes

Neurochem. lnt. Vol. 12, No. 4, pp. 439-445, 1988

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BOTULINUM N E U R O T O X I N INHIBITS THE RELEASE OF NEWLY SYNTHESIZED ACETYLCHOLINE FROM TORPEDO ELECTRIC O R G A N SYNAPTOSOMES J. MAR$AL*, C. SOLSONA,X. RABA~EDAand J. BLASI 13g'partamentde Biologia C¢l.lulari Anatomla Patolbgica,Facultat de Medicina, Hospital de Bellvitge, Universitat de Barcelona, Barcelona, Catalonia, Spain

(Received 23 July 1987; accepted 27 October 1987) Almraet--In previous reports, we have shown that botulinum neurotoxin inhibits acetyicholine release from Torpedo marmorata electric organ and from its synaptosomal fraction. Here, we have focussed our attention on the study of the effect of botulinum neurotoxin on the metabofism of acetylcholine, namely, the precursors supply, the synthesis activity and the storage of the neurotransmitter into nerve endings isolated from Torpedo electric organ. Radiolabelled acetylcholine precursors (acetate and choline) uptake, choline O-acetyltransferase activity, and the compartmentalization of the transmitter into the synaptosomes were not modified by botulinum neurotoxin. When labelled nerve endings were depolarized by K +, the specific radioactivity of acetyicholine in the free pool fell markedly, but the specific radioactivity in the bound pool remained constant. Botulinum neurotoxin prevented this K+-induced decrease of specific radioactivity in the free pool.

The electric organ of Torpedo marmorata is a pure cholinergic innervated tissue, sharing embryological and most physiological properties with the neuromuscular system. From this tissue a subcellular fraction of pure cholinergic synaptosomes can be obtained (Morel et al., 1977), which is a very suitable preparation to study the mechanisms of acetylcholine (ACh) release and metabolism. ACh metabolism is a carefully regulated process (see Tui~ek, 1985 for a review), controlled mainly by the enzyme choline O-acetyltransferase (EC 2.3.1.6) (CHAT). This enzyme converts acetyl-CoA and choline (Ch) to ACh. Ch is supplied by a high affinity Ch uptake carrier at the presynaptic membrane of the cholinergic nerve terminal. ACh is released to the synaptic cleft, and liydrolysed by acetylcholinesterase to Ch and acetate. Acetate, like Ch, is taken up by the motor nerve ending, and used to acetylate CoA, for the formation of new ACh. This process of synthesis of the neurotransmitter takes place in the cytoplasm of the terminal, but part of the formed ACh is stored in *Address correspondence to: Professor J. Marsal, Departament de Biologia Cellular i Anatomia Patoitgica, Facultat de Medicina, Universitat de Barcelona, Casanova 143, 08036-Barcelona, Catalonia, Spain. 439

the synaptic vesicles until its utilization as a neurotransmitter. Consequently, there are two pools of ACh, a bound (vesicular) pool and a free (nonvesicular) pool of neurotransmitter. Botulinum neurotoxin type A (BoNTx) is a very specific toxin which inhibits the quantal release of ACh from the neuromuscular junction (see Simpson, 1986, for a review), and from the electric organ of Torpedo marmorata (Dunant et ai., 1987). We have recently reported that this toxin, as well as tetanus toxin, impair the release of ACh from the isolated nerve terminals of the electric organ of Torpedo (Marsal et ai., 1987; Rabasseda et al., 1987). This toxin acts on the neurotransmitter release, but does not modify the excitation component of the excitation-secretion coupling process. This has been shown by studying the changes in membrane potentials (Marsal et ai., 1987, in Torpedo synaptosomes) and currents (Dreyer et al., 1983, in poisoned neuromuscular junction) during depolarization, and the depolarization-induced calcium entry into the poisoned synaptosomes (Marsal et al., 1987), triggering process of neurotransmission. Thus, BoNTx could act on the metabolism of ACh. We have used a preparation of pure cholinergic synaptosomes from Torpedo electic organ to study the action of BoNTx

440

.! MarsAt ~/ at

on the metabolism of ACh from presynaptic nerve endings, that is, the supply o f precursors for ACh synthesis, the synthesis activity and the storage o f thc neurotransmitter.

added. This produced two phases, one hydrophobiL ~phase, containing ACh, and one hydrophilic phase containing acetate and other acetate compounds. Scintillation ~)lution (toluene with 5 g/t PPO and 0.2 g/I POPOP) was added and radioactivity of the hydrophobic phase was counted m a Beckman Scintillation Counter.

EXPERIMENTAL PROCEDURES

Isolation o/" synaptosomes Pure chotinergic synaptosomes were purified from homogenates of fresh Torpedo marmorata electric organs as described by Morel et al. (1977). Synaptosomes were recovered in a physiological saline solution containing (mmol/l): NaCI, 280; KC1, 3; CaC12, 3.4; MgCI 2, 1.8; HEPES/NaOH, pH 6.8, 3.6; sucrose, 400 and glucose, 5.5, adjusted to pH 7.0 by adding NaHCO 3 (about 5 mmol/l, final concentration). All steps were made at 4~C. The synaptosomal band represents 0.4 _+ 0.01 g of initial tissue/ml (n = 17)~ Lactate dehydrogenase activity To study the integrity of synaptosomes, occluded lactate dehydrogenase (EC 1.1.1.27) (LDH) activity was measured according to Johnson and Whittaker (1963). Choline uptake 200 #1 of synaptosomal preparation were incubated with 300/~I of physiological saline solution, in which the final concentration of non-labelled Ch was adjusted to 100/~mol/1. The preexisting external Ch concentration (about 100/~mol/l) in the synaptosomal suspension was estimated by the chemiluminescent method (Israel and Lesbats, 1981). [3H]Ch was added (7.SGBq//~mol, final concentration) and samples were incubated for 10min at room temperature, with or without BoNTx (125 pmol/l, in 2.5 g/l glelatin solution). In non-treated synaptosomes, the volume of BoNTx was replaced by physiological medium containing gelatin (2.5g/1) and bovine serum albumin (20/~g/1). Then, synaptosomes were filtered through Mitlipore filters (1.2 ,urn pore size) in a perfusion chamber. Filters were washed with 20 ml of physiological saline solution, air dried and put into a vial with 20 ml of scintillation solution (Toluene/Triton X-100 (3/1, vol/vol) containing 5 g/l PPO, 0.2 g/l POPOP) for counting. Non-specific Ch binding to filters was calculated and substracted in quantitative Ch uptake measurements.

4cetylcholine determination We used a chemiluminescent method described by Israel and Lesbats (1981) to measure ACh levels. This method is based on enzymatic conversion of ACh to Ch by acetylcholinesterase. Ch is then hydrolysed to Betaine plus H202 by the choline oxidase enzyme, and the hydrogen peroxide is detected by the luminol-peroxidase (EC I.I 1.17) luminescent reaction. Acetycholine compartmentation To measure total ACh content, samples of synaptosomal fraction, either, BoNTx poisoned (125 pmol/l, 10min) or non-poisoned (with bovine serum albumin (20/~g/1, final concentration) replacing BoNTx in the same gelatin medium) were treated with trichoroacetic acid (TCA) 5 g/I, final concentration). TCA was washed with successive ether (H20 saturated) extractions to reach a pH of 4, To measure the occluded (vesicular) content of ACh, samples were frozen, by immersion in liquid N,, and thawed during 30 min at room temperature. Plasma membranes were broken, and non-vesicular ACh was hydrolysed by the synaptosomal acetylcholinesterase. Finally, samples were treated with TCA and processed as before. ACh contents were measured with the choline oxidase (EC 1.1.99.1) luminescent method. The difference between total and bound ACh was free ACh. For the measurement of ACh compartments in depolarized by K ~, KCt (100 mmol/l, final concentration) was added during 30sec before freezethawing or TCA addition.

Acetate uptake Acetate uptake was performed with the same procedure as for Ch uptake measurement, but with [~4C]acetate (50/~mol/1; 1.85 GBq/mmol) substituting for [3H]Ch. Preexisting external acetate concentration was not determined.

Specific radioactivity o f acetylcholine compartmentatmn Poisoned (BoNTx, 125pmol/l) or non-poisoned synaptosomes (with bovine serum albumin (20 #g/l) in gelatin (2.5 g/l) containing physiological solution) were incubated at room temperature during 1 h with [~4C]acetate (50/~ mol/l). In these conditions, 95% of incorporated radioactivity was found as [14C]ACh (Morel et al., 1977; Suszkiw et al., 1978). Samples were then processed as for ACh compartment measurements. Finally, [~4C]ACh was extracted and measured with the chemiluminescent method, and counted for radioactivity. For the latter, the sodium tetraphenylborate method of Fonnum (1975) was used, as for ChAT activity determination.

Choline acetyltransferase determination The radiochemical method of Fonnum (1975) was used. Synaptosomes were treated with BoNTx (125 pmol[1) in a medium containing 2.5g/1 gelatin, during 10min. Non treated cholinergic terminals were exposed, during the same time, to bovine serum albumin (20/~g/1) in the same medium as BoNTx. After this treatment, synaptosomes were lysed in a Na2HPO4/NaH2PO4 buffer, pH 7.4, 100 mmol/l containing Triton X-100 (300 ml/l), acetyl-CoA (0.96 mmol/l), Ch (10 retool/l) and eserine (0.36 retool/l). ['4C]Acetyl-CoA was added to start [~4C]ACh synthesis. After 15 rain of incubation, at room temperature, Na2HPOJNaH2PO4 10mmol/1 buffer pH7.4 (Sml) and sodium tetraphenylborate (10g/l) in acetonitrile solution (2ml) were

Specific radioactivity o f the ACh released We used a perfusion chamber described by Morel et al. (1979). Labelled synaptosomes were placed in the perfusion chamber (Swinnex), occluded by a Millipore filter (1.2/~m pore size). Synaptosomes were perfused at a ratio of 2 ml/min with the physiological saline solution. After 30 rain washing, a modified saline solution containing 100 mmol/1 KCI (where KC1 replaces an equivalent amount of NaCI) was perfused, and drops were collected (two drops per tube). Ch was measured by the chemiluminescent reaction in each tube, because as reported by Dunant et al. (I 980), there is only release of ACh, and not of Ch, in this tissue on stimulation. [~4C]Acetate was also counted in each tube. Specific radioactivity (SRA) was calculated by dividing

Botulinum toxin and acetylcholine storage [~4C]acetate by Ch contained in the drops collected at the peak of release. Thus, results reflect SRA at the peak of release.

Statistics All results are given as means + SEM and treated for statistical significance by the Student's unpaired t-test. Materials Toxin crystal preparations were obtained from a culture of Ciostridium botu//num type A (NCTC 2916) by methods previously described (Duff et al., 1957; Sugiyama et al., 1977). Briefly, after acid precipitation of the culture (kindly provided by Dr Aurora Casanova, Department of Bacteriology, Hp. llellvitge, Barcelona, Spain) the toxin extract was purified by ion exchange methods in DEAE-cellulose chromatography. Haemagglutinin was removed from the toxin complex by affinity chromatography and DEAEcellulose chromatography as described by Moberg and Sugiyama (1978) and Tse et al. (1982). A mouse LDs0 of the neurotoxin represents 0.66ng/kg. BoNTx was stored in gelatin (2.5g/1) solution. Torpedo marmorata specimens were caught from the Catalan coast and maintained alive in sea water. [methyl-3H]Choline (3 TBq/mmol), [1-14C]acetate (I.85 GBq/mmol) and [14C]acetyl-CoA (2 GBq/mmol) were supplied by the Radiochemical Centre, Amersham, U.K. HEPES (N-2-hydroxy-ethyl-piperazin-N'-2-ethane-sulfonic acid), choline oxidase, horseradish peroxidase, acetylcholinesterase (type VI) (EC 3.1.1.7) and luminol (5-amino-2,3-dihydro-l,4-phtalazinedione) were purchased from Sigma Biochemical Company, U.S.A. Choline chloride, sodium tetraphenylborate and ether were from Merck (West Germany); Triton X-100, PPO (2,5-di-phenyloxazole) and POPOP (1,4-di-[2-(5-phenyl-oxazolyl)]benzene) from Koch Light, England. RESULTS

Choline acetyltransferase activity measurement The activity of C h A T was measured in non-treated and BoNTx-treated synaptosomes (Table 1). BoNTx (125 pmol/I, 10 min) did not alter the activity of the enzyme.

L D H occluded activity To assess integrity of the synaptosomal fraction after BoNTx poisoning, we have studied the L D H occluded activity in both, control and BoNTx treated synaptosomes. As seen in Table 1, BoNTx (125 pmol/l, 10rain) poisoning did not modify occluded L D H activity, either, after 10rain of incubation with the neurotoxin, or after 60 min.

Choline and acetate uptake The uptake of the precursors for ACh synthesis was studied under the action of BoNTx. Table 1 also shows the action of the toxin on the uptake of Ch and [x'C]acetate. BoNTx (125pmol/l, 10rain) did not affect, either, Ch or acetate uptake.

441

Table 1. Effect of BoNTx on the uptake of ACh precursors and on ACh synthesis Non-poisoned Poisoned LDH occluded activity (per cent of total) (n = 3) 10rain of poisoning 76.7 + 5.1 74.2 _+5.3 60 rain of poisoning 73.8 + 5.9 70.5 _+4.2 [14C]Acetate uptake (Bq/min/g initial tissue) (n =3) 12.0+ 0.7 11.9_+ I.I Choline uptake (pmol Ch/min/gi tiss) (n ~ 3) 117.0 _+17 116.6 + 24 ChAT activity (nmol/g/h) (n = 3) 442 + 41 486 :t: 63 BoNTx does not affect LDH occluded activity in the synaptosomal fraction. The effect of BoNTx on the uptake of ACh precursors was investigated using radiolahelled substrates ([3H]Ch (0.25/~mol/1)and [~L']acetate(50/Jmol/I, final concentrations). BoNTx does not alter the ACh precursors uptake. Finally, the activity of the ACh synthesizingenzyme(CHAT)was measured using [14C]acetyI-CoAand Ch as substrates. BoNTx does not modifythis activity.In non-poisonedsynuptosomes,the volume of BoNTx was replaced by bovine serum albumin (20/~g/l), in physiologicalsaline solution containing 2.5 g/I of gelatin.

ACh compartments measurement The action of BoNTx on the two pools of ACh, the b o u n d pool and the free pool, was studied at rest and after 30s of K+-depolarization. BoNTx did not modify the ACh content of any pool under resting conditions (Table 2). After stimulation of synaptosomes during 30 s by high external potassium concentration, the A C h content and compartmentation were not significantly altered. Even, after poisoning and potassium stimulation, we could not detect any difference with respect to the controls (Table 2).

Specific radioactivity o f ACh compartments When synaptosomes were incubated with [~4C]acetate, as much as 95% of incorporated radioactivity was found as ACh (Morel et al., 1977; Suszkiw et al., 1978; Michaelson et al., 1986). Table 2 also shows SRA in A C h compartments. SRA of A C h in both pools was different, SRA in the free pool being 2.6 fold higher than that found in the b o u n d pool. In labelled synaptosomes depolarized by high external potassium concentration during 30 s, SRA of total and b o u n d A C h were unchanged whereas SRA of the free pool of neurotransmitter fell 3 fold. When labelled synaptosomes were treated with BoNTx (125 pmol/l, 10 rain), no modification on the pattern of SRA of ACh pools was observed, but BoNTx abolished the K + -induced decrease of SRA in the u n b o u n d pool (Table 2).

442

J N,IARSAL et al. Table 2. Effect of BoNTx on the storage of ACh on isolated synaptosomes at rest and after potassium stimulation Total

Bound

Free

Control synaptosomes Resting conditions (n = 5)

corn SRA

39.7 ± 0.7 t 2.9 + 2.7

25.2 : 2.6 8.4 ± 1.8

145 = 1.0 22. I :~ 4. I

KCI (100 mmol/l, 3(1 s) (n = 5)

cont SRA

37.8 ± 0.7 9.7 ± 2.6

26.5 =2 2.7 11.8 ± 1.7

t 1.3 ~ 1.8 7.0 ~: 3.3*

Resting conditions (n = 5)

cont SRA

39.3 + 1.7 t2.7 + 2.5

25.6 -Z-_5.1 9.2_+ 1.7

13.7 _+ 1. I 22.5 + 4.0

KCI (100 mmol/k 30s) (n = 5)

cont SRA

37.0 _+ 1.3 11.6 + 3.0

24.5 _+ 2.5 8.2 _+ 1.4

12.5 _+_1.0 17.5 + 4.4

BoNTx poisoned synaptosomes

cont, content (nmol/g initial tissue); SRA, specific radioactivity (Bq/nmol). ACh contents and SRA of ACh compartments were measured. Synaptosomes were prelabelled with [~4C]acetate (50 pmol/1) during 60 min. In this condition, as much as 95% of incorporated acetate is recovered as ACh. After this prelabelling period, a portion of the labelled synaptosomal fraction was treated with BoNTx (125 pmol/l, 10min, in a medium containing 2:5 g/l of gelatin), and the rest (control synaptosomes) with the same gelatin containing medium, supplemented with bovine serum albumin (20#g/I). Compartments were measured after submitting aliquots of the synaptosomal fraction to a freczing-thawing cycle and TCA (for measuring bound ACh), or TCA treatment alone (for measuring total ACh). Free ACh was the difference between the two mentioned pools. SRA was calculated dividing Bq of the [~4C]acetate incorporated to ACh by nmol of ACh/g initial tissue measured by the chemiluminescent method. Experiments had been done in synaptosomes at rest, as well as, K +-depolarized (100 mmol/1, final concentration). Statistical significance (respect to the control at rest): *P < 0.01.

Specific radioactivity of the ACh released In order to explore the source o f the ACh released during the nerve terminal depolarization, SRA of the ACh secreted by K + -stimulation of the synaptosomes, either, in control or during BoNTx poisoning, was measured. Previously, Michaelson et al. (1986) have demonstrated that most of the radioactivity ( > 95%) is recovered as labelled ACh. In the analysis of eluted drops from the perfusion device, the SRA has been calculated by dividing radioactivity from [~4C]acetate by the molar amount of choline present in the drops collected at the peak of release triggered by KCI. SRA of collected drops was higher than that found in the intrasynaptosomal ACh compartments, but no difference was detected between the levels of SRA of the released ACh from control synaptosomes and poisoned nerve endings whereas choline released after BoNTx poisoning represented about 30% of choline released from the controls (Table 3).

on the precursors uptake, on the synthesis (CHAT activity), and on the transmitter storage. Substrates for ChAT activity are acetyl-CoA and Ch, and the precursor for acetyl-CoA synthesis, at the electric organ of Torpedo, is acetate (Israel and Turek, 1974). To explore whether BoNTx impairs the supply of acetate and Ch to the cholinergic terminals, we have studied the uptake of acetate and Ch by incubating synaptosomes with [~4C]acetate or [3H]Ch (Morel et al., 1977). Previously, two groups have demonstrated that botulinum toxin (BoTx) does not directly inhibit the Ch uptake in brain synaptosomes (Wonnacott and Marchbanks, 1976; Gundersen and

Table 3. Effect of BoNTx on the release of ACh from isolated cholinergic synaptosomcs

KCI (100 mmol/I) (n = 3)

DISCUSSION

As we have previously shown (Marsal et at., 1987), BoNTx inhibits ACh release at pure cholinergic synaptosomes isolated from the electric organ of Torpedo marmorata, without modifying either, membrane potential, or calcium uptake into the nerve terminals. Thus, it was interesting to investigate the action of the toxin on the ACh metabolism, that is,

Ch SRA

Control

Poisoned

1.29 + 0.17 152.1 _+ 12.8

0.35 _+0. t2* 139.7 + 21.2

Ch (nmol/g initial tissue); SRA, specific radioactivity (Bq/nmol). Synaptosomcs prclahened with [ ' ~ C ' ~ ' ~ t e as dcsc-fibcd in E x I ~ i mental Procedures, were placed in a filter chamber occluded by a 0.22/zm pore filter, and washed with physiological solution until a stable base line of Ch and radioactivity in the superfusate was obtained. At this momem, a modified physiological soPation containing 100 mM KC1 was perfused and two drol~ per tube were collected from the peffusion device. Choline a n d radioactivity were evaluated in each tube, a n d the p r e - s t ~ t i o n amounts detected were s u b t r a c t S , Results represent the a~lount of Ch recovered at the peak of reL-'lu~, and its SRA (calculated dividing Bq by Ch in this peak). Statistical sig~nif~ance: *P < 0.01.

Botulinum toxin and acetylcholine storage

Howard, 1978). Bigalke et al. (1978) reported that BoTx reduced the production of [3H]ACh from [3H]Ch in primary cultures derived from rat brain. This finding is reminiscent of the indirect inhibition by BoTx of synaptosomal [3H]Ch transport reported by Gundersen and Howard 0978). Our results better agree with those of Wonnacott and Marchbanks (1976). The synthesis of ACh is the second metabolic step we have studied. Torda and Wolff (1947) reported as much as an 80% inhibition of ACh production in BoTx-treated homogenates of frog brain. Burgen et al. (1949) failed to confirm this observation in minces or in acetone extracts of rat brain. Finally, Tonge et al. (1975) showed that ChAT activity in mouse skeletal muscle was unaffected by BoTx. In pure cholinergic synaptosomes, this report shows that BoNTx does not modify ChAT activity. Our results on the action of BoNTx on the metabolism of ACh indicate that the toxin inhibition of ACh release is not produced by an interference on these synthetic pathways. In one widely accepted view, ACh is synthesized in the nerve terminal cytoplasm, and a portion of the transmitter is loaded into the synaptic vesicles. In Torpedo electric organ, two pools of ACh have been described (Dunant et aL, 1972; Suszkiw, 1980), a bound pool of ACh, related to synaptic vesicles, and a free pool of neurotransmitter. The definition of free ACh is operational, it represents the difference between the total and the bound ACh. Free ACh almost certainly represents the cytoplasmic pool OVeiler et al., 1982), where ACh synthesis occurs. Alternatively, a portion of this pool may be associated to a specific class of fragile synaptic vesicles (VP2), more directly involved in the release of the neurotransmitter, and constantly refilled with transmitter from the cytoplasmic pool (Zimmermann and Whittaker, 1977). In any case, it is the free pool of neurotransmitter which contains the most recently synthesized ACh when the transmitter is labelled with a radioactive precursor, either Ch (Marchbanks and Israel, 1971; Dunant et al., 1972) or acetate (Israel and Tu~ek, 1974). The way to study ACh compartments in the synaptosomal fraction was to open only the plasma membrane, for measuring the bound ACh, or to disrupt all membranes, for measuring total ACh content. The disruption of the synaptosomal plasma membrane has been done by submitting the synaptosomes to a freezing and thawing cycle (Morel et al., 1977; Israel and Lesbats, 1981). There is slightly less ACh in the free (40% of total) than in the bound NCI. 12/4--D

443

pool (60% of total). These compartments of ACh are not modified after poisoning by BoNTx. Nevertheless, since we could not exclude that the toxin could alter the loading of synaptic vesicles after the cytoplasmic synthesis (Boroff et al., 1974), it was necessary to study the turnover of ACh in both compartments by exploring the incorporation of labelled precursors to both pools of neurotransmitter. Even in these experiments, we have not found any difference between poisoned and non-poisoned synaptosomes. Labelled ACh is preferentially found in the cytoplasmic pool, SRA of the free ACh being, in our experiments, 2.6 fold higher than that of the bound pool. The SRA of the bound pool is the same either in BoNTx treated or non-treated synaptosomes, thus, BoNTx does not inhibit the loading of ACh into synaptic vesicles. Moreover, we have also investigated the effect of the toxin on the preferentially releasable pool of ACh that, in the Torpedo electric organ (Dunant et al., 1972), is the free compartment. This latter experiment using cholinergic nerve endings isolated from Torpedo electric organ show us that after K + -stimulation, SRA of the free pool of neurotransmitter falls markedly and, therefore, the SRA of the released ACh is greater (7 fold) than SRA of the free pool of neurotransmitter. Similar results (SRA of released ACh 5 fold greater than SRA of free ACh) were obtained by Dunant et al. (1972) when measuring the SRA of the ACh released from electrically stimulated electric organ fragments. The explanation for these facts is rather difficult. The SRA of ACh compartments and collected drops from K+-stimulated synaptosomes has been calculated from results coming from different experimental procedures. Moreover, we cannot exclude that some synaptosomes are metabolically more active than others. Nevertheless, the possibility is open that newly synthesized ACh is not homogeneously distributed among the free pool, where a turning-over fraction of ACh is preferentially released. This hypothesis agrees with the finding reported by Collier (1969) and Potter (1970) that the newly synthesized ACh is the first released. Furthermore, it has been proposed by Perry (1953) and Birks and Macintosh (1961) that a small compartment of "immediately available transmitter" is situated in series between the process of release and the main depot of transmitter. The depletion of newly synthesized ACh at the free pool after stimulation and the greater SRA of neurotransmitter at the collected drop support this non-homogeneous distribution of labelled ACh at the free pool, immediately available for neurotransmitter secretion.

444

J. MARSALC/ a/.

In any case, the K ' -induced fall of S R A at the free A C h is blocked by BoNTx. The action of B o N T x suggests that the preferentially releasable pool of t r a n s m i t t e r is the newly synthesized A C h located at the free pool. F u r t h e r m o r e , BoNTx, even if it reduces the t r a n s m i t t e r release up to 70% (Marsal et al., 1987), does not modify the S R A o f the residual release (30%) after K ~ - s t i m u l a t i o n . Consequently, the A C h released by a K ~-stimulation, either in poisoned or n o n - p o i s o n e d synaptosomes, comes from the same pool. As B o N T x inhibits q u a n t a l release o f A C h at the cholinergic synapses of the electric o r g a n of Torpedo ( D u n a n t et al., 1987), as well as at the n e u r o m u s c u l a r j u n c t i o n (Harris a n d Miledi, 1971), we may speculate that the A C h release which is inhibited by B o N T x at the Torpedo syna p t o s o m e s has, also, a q u a n t a l nature. F r o m this study, we can conclude t h a t B o N T x blocks the release of newly synthesized A C h stored at the free pool. Acknowledgements--We are indebted to Dr P. Art6 and the staff of the "Institut de Cidncies del Mar de Barcelona" (CSIC) for maintaining alive Torpedo marmorata specimens. We are, also, grateful to Dr A. Casanova, for providing us with the culture of Clostridium botulinum. This work was supported by grants from CAICYT (84/2154), CIRIT (AR82/2-73) and FIS (86/186). X.R. is a fellow from FIS.

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