BRAIN RESEARCH

ELSEVIER Brain Research 703 (1995) 86-92

Research report

Inhibition of potassium-stimulated acetylcholine release from rat brain cortical slices by two high-affinity analogs of vesamicol

Ricardo Maurlcio Le~o a, Marcus Vinicius Gomez b, Brian Collier c, Marco Ant6nio M~ximo Prado b, *

a Departamento de Bioqulmica e Imunologia, 1CB-UFMG, Belo Horizonte, MG 31160-970, Brazil b Departamento de Farmacologia, ICB-UFMG, Belo Horizonte, MG 31160-970, Brazil ¢ Department of Pharmacology and Therapeutics, McGill University, Montrdal, Canada

Accepted 8 August 1995

Abstract

In this work, we investigated the effects of two structural analogs of the drug vesamicol, which inhibits the vesicular acetylcholine (ACh) transport, on the potassium-stimulated release of ACh from rat brain cortical slices. These vesamicol analogs, 4-aminobenzove- samicol (ABV) and (trans)-cyclohexovesamicol (transDec), were almost as potent as vesamicol in inhibiting the evoked release of ACh from cortex slices. Similar to vesamicol, the presence of these analogues inhibited the ability of ACh newly-synthesized from [3H]choline to become releasable. However, vesamicol's action was reversible, while ABV and transDec caused a persistent block of this [3H]ACh release. In addition, vesamicol did not affect the release of pre-stored [3H]ACh, but ABV and transDec partially inhibited the release of [3H]ACh in this condition, suggesting that the two latter drugs may alter some of the steps posterior to the entry of [3H]ACh into synaptic vesicles. The rank order of potency for these drugs to reduce ACh release (vesamicol = transDec > ABV) is close to the rank order for inhibition of ACh vesicular transport (transDec > vesamicol > ABV), but is completely different from the order of affinities of these drugs for the vesamicol receptor (ABV > transDec > > vesamicol). These results suggest that although these two vesamicol analogs are able to block ACh release due to their effects on the vesicular transport system, they may have other unexpected actions not shared by vesamicol.

Keywords: Acetylcholine release; Vesamicol; 4-Aminobenzovesamicol; (trans)-Cyclohexovesamicol; Synaptic vesicle; Vesicular transport

1. Introduction

The drug (trans)-2-(4-phenylpiperidino)cyclohexanol

(AH 5183; vesamicol) inhibits the vesicular uptake of acetylcholine (ACh) by isolated synaptic vesicles from the electric organ of Torpedo via an allosteric interaction with the vesicular transport system [2,5]. The vesicular transport machinery probably corresponds to a multiprotein complex that contains a synaptic vesicle specific proteoglycan [3,6]. More recently, a ves~imicol binding protein has been cloned; it has homology to a family of monoamine vesicu- lar transporters, and it confers upon fibroblasts the ability to accumulate ACh in vesicle-like structures [8,26].

In accordance with the role of synaptic vesicles in the release of neurotransmitters, vesamicol inhibits the output of ACh in almost all of the preparations tested (see Ref.

* Corresponding author. Fax: (55) (31) 441-0835.

0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(95)01069-6

[18] for a comprehensive review). Several characteristics of the vesamicol effect in intact preparations, such as enantioselectivity, effective concentrations, and reduction of vesicular ACh, support the notion that the decrease in ACh release reflects the effect of the drug on the vesicular ACh transport system [18].

The investigation of the relationship between the struc- ture of vesamicol and its pharmacological action, by the synthesis of several vesamicol analogs with varied poten- cies as inhibitors of the vesicular transporter [25], has provided analogs with unusually slow rates of dissociation and high affinity to the vesamicol receptor on synaptic vesicles of the Torpedo electric organ [23,24]. Two of these analogs, the 4-aminobenzovesamicol (ABV) and the (trans)-cyclohexovesamicol (transDec), had the highest affinity to the vesamicol receptor so far described, with affinity constant values in the picomolar range [23], mak- ing them attractive for mapping the cholinergic nervous system and studying cholinergic function [15,16,28].

R.M. Le~o et al. / Brain Research 703 (1995) 86-92 87

Although the vesamicol analogs are relatively well characterized for their binding to Torpedo cholinergic synaptic vesicles and for inhibition of vesicular ACh trans- port, there has been less information about their effects on the release of ACh, particularly for mammalian prepara- tions. In the present work, we investigated the effect of the two analogs with high affinity for the vesamicol receptor, ABV and transDec, on the potassium-induced release of ACh from rat brain cortical slices. We report here that ABV and transDec inhibit the potassium-evoked ACh release with some characteristics distinct from those ob- served with vesamicol. "[lais work has been previously presented in abstract form [19].

2. Materials and method,~

2.1. Materials

Materials were as follows: ACh chloride; ATP; choline chloride; dithiothreithol (DTT); 3-heptanone; 5*amino- 2,3-dihydro-l,4-phthalazinedione (luminol); diethyl-p- nitrophenyl phosphate (paraoxon); physostigmine free base; 1,4-bis[5-phenyl-2-oxazolyl]-benzene-2,2'-p-phenylene- bis[5-phenyl oxazole] (POPOP); 2,5-diphenyloxazol (PPO); sodium tetraphenylborate (TPB); trichloroacetic acid (TCA); acetylcholinesterase (EC 3.1.1.7); choline kinase (EC 2.7.1.32); choline oxidase (EC 1.1.3.17) and mi- croperoxidase MP-11 were from Sigma Chemical Co. (St. Louis, MO). [Methyl-3H]choline chloride (1 mCi/ml, 75 Ci/mmol) was purchased from Amersham International (UK). The racemates of vesamicol, ABV and transDec, were a gift from Dr. Stanley M. Parsons and Dr. Gary A. Rogers (Department of Chemistry and the Neuroscience Research Institute, University of California, Santa Barbara, CA). All other chemicals and reagents were of analytical grade.

2.2. Release of ACh

Wistar rats of either sex weighing 200 to 300 g were killed by decapitation. The brains were quickly removed and the cerebral cortices dissected. The slices (0.5 × 0.5 mm) were prepared with ~t Mcllwain tissue chopper. Forty mg of slices were incubated in 0.6 ml Krebs-bicarbonate medium containing NaC1 120 mM; KC1 4.6 mM; CaC12 2.5 mM; MgSO 4 1.2 mM; KH2PO 4 1.2 mM; NaHCO 3 25 mM; choline chloride 10 ,t~M; glucose 10 mM; physostig- mine 10 /~M, equilibrated with a mixture of 95% 0 2 and 5% CO 2 to give a pH of ,7.4 at 37°C. The slices were first pre-incubated during 30 min, and after changing the medium they were, when required, exposed to drugs at several concentrations. After 20 min, the slices were changed to fresh medium containing 33 mM KC1 (iso- osmotically replacing NaC1) in the absence or the presence of different concentrations of the drugs for a further 60

min; after that the medium was separated from the slices by centrifugation. TCA was added to the supernatant to a final concentration of 5% and samples kept at - 20°C until processing.

2.3. Assay of ACh

After extracting the TCA with several washes of H20- saturated ether, the ACh was assayed using the chemilumi- nescent assay [13] with the modifications for the ACh determination in physostigmine-containing samples [21]. Briefly, physostigmine was extracted by successive washes of the sample with benzene, after which 30 /zl of the sample were added to 1 ml of reaction mixture containing choline oxidase 1.5 U, microperoxidase 20/xg, luminol 10 /zM and glycine buffer 67 mM, pH 8.6. The light pro- duced by the reaction was analyzed in a BioOrbit 1750 luminometer. After an initial peak of light, corresponding to the oxidation of choline, 1.6 U of acetylcholinesterase were added to the reaction and the light emitted recorded. ACh standards were used to quantify the ACh of the samples.

2.4. Release of [3H]ACh

The slices were prepared as described above. The la- belling of the tissue was done as described elsewhere [12]. Forty mg of slices were first depleted of endogenous ACh by 30 min of incubation in Tyrode's medium containing 50 mM of KC1 (composition of normal Tyrode's medium: NaCI 136 raM; KCI 2.7 mM; CaC12 1.35 mM; Tris-HC1 10 mM; glucose 5.5 raM; pH 7.4, for the high potassium medium the NaC1 was replaced isoosmotically with KC1) containing paraoxan 15 ~M. In order to label the internal stores of ACh, the medium was then replaced by 3 ml of normal Tyrode's medium containing 0.06 /xCi of [3H]choline chloride (free of choline carrier), and the slices were incubated for 35 min with or without 1 /zM of the vesamicol or analogue. After washing the excess of ra- dioactivity with 2 ml of Tyrode's medium containing 1 /xM of choline, the slices were transferred to fresh medium that contained KC1 (33 mM or 2.7 mM) in the presence or absence of 1 /zM of the different drugs. The tissue was then incubated for 30 min and, at the end of the incuba- tion, the slices were separated from the supernatant by centrifugation. A 500 /xl aliquot of the supernatant was used to determine the total 3H-labelled material released, and the radioactivity present in the slices was extracted with 5% TCA.

2.5. Determination of [ 3H]ACh

The quaternary amines were extracted from the medium following the method described by Fonnum [10] using 10 mg/ml of tetraphenylboron (TPB) in 3-heptanone. The amines were recovered to an aqueous phase using 20

88 R.M. Legto et al. / Brain Research 703 (1995) 86-92

m g / m l of AgNO 3. The silver was precipitated with 1 M MgCI 2 and the samples dried in a vacuum concentrator. The [3H]ACh was separated from [3H]choline using the choline kinase assay of Goldberg and McCaman [11], as described in [20]. The samples were reconstituted in 32/x l of a mixture containing ATP 0.8 mM; MgC12 11.5 mM; DqT 5 mM; choline chloride 10 ~M; Tris-HCl 50 mM (pH 8.0) and 0.0025 U of choline kinase, and incubated at 30°C for 30-40 min. The reaction was stopped with 200 /xl of ice-cold water and the [3H]ACh was separated from the [3H]phosphorylcholine by shaking the mixture with 230 /xl of TPB/3-heptanone 10 mg/ml . Acetylcholine was recovered in the organic phase while the phosphoryl- choline remained in the aqueous phase. Aliquots of each phase were taken and the radioactivity measured. The correction for recovery of each amine was done by extract- ing and assaying radiolabelled standards of choline and ACh.

Radioactivity was determined by liquid scintillation spectrophotometry in a Packard TR-600 liquid scintillation spectrophotometer. Aliquots of the samples were added to minivials containing 3 ml of modified Bray's cocktail (composition per liter: absolute ethanol 300 ml; dioxan 300 ml; toluene 300 ml; naphthalene 70 g; POPOP 200 mg; PPO 5 g, Triton X-100 100 ml). The counting effi- ciency was corrected by a ratio method and readings transformed to dpm.

2.6. Statistics and curve fitting

Each experiment was repeated at least three times in duplicate. Analysis of variance was performed to access the significance of the differences observed between differ- ent conditions in the same experiment. A P value of less than 0.05 was considered to be significant.

The IC50 values were obtained by fitting dose-effect curves with the logistic equation using the Sigma Plot software (Jandel Scientific).

3. Results

3.1. Dose-response inhibition of ACh release

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Fig. 1. D o s e - r e s p o n s e curves for the inhibition o f potass ium-st imulated

release of ACh by vesamicol (a), ABV (b) and transDec (c). The slices were incubated with Krebs-bicarbonate medium containing distinct con- centrations of each drug 20 min before and during the stimulation with 33 mM K ÷ for 1 h. The curve fitting was obtained using a logistic equation. The graph shows the logarithm of the molar concentration of the drugs versus the normalized stimulated ACh released.

in a concentration-dependent fashion (Fig. la, with an estimated IC50 of 11 nM).

The two vesamicol analogs, ABV and transDec, also inhibited the K+-stimulated release of ACh from brain cortical slices (Fig. lb and c). The calculated IC50 were 65 and 14 nM, for ABV and transDec, respectively.

3.2. Effect of vesamicol analogs on the release of newly- synthesized [ 3 H]A Ch

The vesamicol analogs are known to block the transport of ACh into synaptic vesicles, and consequently, they should reduce the amount of neurotransmitter released during prolonged stimulation. Therefore, we have com- pared the release of ACh by depolarized brain slices exposed to different concentrations of vesamicol, ABV, or transDec with the ACh released by control slices, which were stimulated in the absence of any drugs.

The control slices released 22 _ 1 nmol of ACh per gram of tissue during 60 min depolarization with 33 mM KC1. As expected, vesamicol inhibited the release of ACh

The above experiments suggested that ABV and trans- Dec might inhibit the release of ACh due to their effects on the vesicular transport of transmitter. In order to test this point, we measured the release of [3H]ACh from tissue that had synthesized the ester from [3H]choline in the presence or absence of the drugs, with the rationale that any [3H]ACh recently synthesized in the cytosol [9] had to enter synaptic vesicles before being released.

The slices were labelled in the presence of different drugs, as indicated in methods, and the release of [3H]ACh induced by 33 mM KCI was compared to that obtained

R.M. Le~o et al. / Brain Research 703 (1995) 86-92 89

from control slices that were labelled in the absence of any drugs. Cortical slices depolarized in control conditions released on average 200 d p m / m g of tissue, a twofold increase over the resting release of [3H]ACh. Vesamicol (1 /xM) decreased this release of [3H]ACh by about 50% (Fig. 2a; P < 0.01). The two vesamicol analogs, ABV and transDec in concentrations that are maximally effective to inhibit the release of endogenous ACh (1 /xM) also dimin- ished the release of newly-synthesized transmitter (Fig. 2b and c). These drugs, however, had a slightly larger effect than had vesamicol; ABV blocked by 75% and transDec by 80% the K÷-stimulated release. This effect could not be attributed to a decrease on the uptake of [3H]choline at the nerve terminals, since the amount of radiolabelled material incorporated into slices was not significantly different for the different treatments when compared to control condi- tions (Fig. 2a, b and c).

In order to test if the inhibition of the release of [3H]ACh had characteristics similar to those known for the inhibition of vesicular ACh transport, we evaluated the reversibility of action of vesamicol, ABV, and transDec. The vesamicol analogs interact with the vesamicol receptor in a quasi-irreversible manner [23], while vesamicol seems to bind to its receptor reversibly [4,23]. Therefore, we

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Fig. 2. Effect of 1 /.~M of vesamicol (a), ABV (b) and transDec (c) in the total release of [3H]ACh and in the uptake of [3H]choline. Forty mg of slices were labelled with 0.06 p, Ci of [3H]choline in the presence of 1 /.tM of each drug during 35 rain. The radioactive choline was washed out and the slices incubated with Tyrode's medium with 33 mM K + during 30 min in the presence of 1 p,M of each drug. The bars represent the mean+S.E.M, of at least 3 experiments performed in duplicate. The [3H]ACh released by potassium in the presence of each drug was significantly different from the [3H]ACh released in their absence (P < 0.01). The uptake of [3H]choline in the controls without drugs were not statistically different in the presence of the drugs.

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Fig. 3. Reversibility of the effect of vesamicol (a), ABV (b) and transDec (c). Forty mg of slices were labelled with 0.06 gCi of [3H]choline in the presence of 1 /zM of each drug during 35 min. The radioactive choline and the drugs were washed out and the slices incubated with Tyrode's medium with 33 mM K + during 1 h. The bars represent mean + S.E.M. of at least 3 experiments performed in duplicate. * Significantly different from K + (P < 0.01).

performed an experiment similar to the one described above, where the slices were initially exposed to the drugs (1 /zM) and labelled with [3H]choline, but then, they were washed and stimulated in the absence of any drugs. In this condition, there was no significant difference between the release of [3H]ACh from control tissue that had been exposed to vesamicol (Fig. 3a).

The drugs ABV and transDec showed a different pat- tern, and decreased the output of radiolabelled ACh in this condition by an amount that was similar to that evident when they were maintained during the entire experiment (Compare Fig. 3b with Fig. 2b and Fig. 3c with Fig. 2c; P > 0.05).

3.3. Effect of vesamicol analogs on the release of pre-stored [3H]ACh

In all of the above experiments, the results are compati- ble with the hypothesis that vesamicol and its analogs decrease the release of ACh due to their interaction with the vesamicol receptor and inhibition of the vesicular ACh transport. It is known that vesamicol does not inhibit the release of [3H]ACh when the tissue is exposed to the drug after labelling the internal stores [1,17,27]. This is an important evidence that vesamicol does not interfere with the transmitter release machinery, and that the ACh re- leased has a vesicular origin. In the next set of experi- ments, we tested whether ABV and transDec had any effect on the release of pre-stored [3H]ACh. Thus, the

90 R.M. Le~o et al. / B r a i n Research 703 (1995) 8 6 - 9 2

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Fig. 4. The effect of 1 /xM of vesamicol (a), ABV (b) and transDec (c) on the release of prelabelled [3H]ACh. Forty mg of slices were labelled with 0.06/zCi of [3H]choline in the absence of drugs during 35 rain. The radioactive choline was washed out and the slices incubated with Tyrode's medium with 33 mM K ÷ during 1 h in the presence of 1 ~M of each drug. The bars represent the mean + S.E.M. of at least 3 experiments performed in duplicate. * * Significantly different from K +, P < 0.05.; • significantly different from K +, P < 0.01.

slices were labelled in the absence of any drug, washed and then stimulated in the presence of 1 /zM of vesamicol or its analogs.

In agreement with previous work, vesamicol did not alter the secretion of [3H]ACh in this condition (Fig. 4a). Brain slices stimulated in the presence of ABV showed a small, but significant, decrease in the amount of [3H]ACh released when compared to slices that were not exposed to the drug (Fig. 4b; P < 0,05). TransDec had a more robust effect and the [3H]ACh released induced by 33 mM KC1 was 50% of that from control slices (Fig. 4c, P < 0.01). The reduction of the release of [3H]ACh in this last condition was significantly different from that observed when the drugs were introduced before the exposure to radiolabelled choline ( P < 0.05).

4. Discussion

The hypothesis that ACh needs to penetrate synaptic vesicles before being released has been strongly supported since the discovery that vesamicol inhibits the transport of ACh into the synaptic vesicles. The subsequent biochemi- cal, pharmacological and molecular characterization of the vesicular ACh transport system has added more evidence for the vesicular origin of released ACh [6,18,26]

Several analogs of vesamicol have been synthesized with the objective of studying the pharmacology of the

vesicular ACh transport [25]. These studies revealed inter- esting relationships between the structure of the molecule of vesamicol and its action on Torpedo synaptic vesicles [14,23,25]. The addition of groups such as the benzyl or the cyclohexyl rings to the cyclohexanol stabilizes the conformation of the molecule in a diaxial position of the amino and alcohol groups. This change increases consider- ably the affinity for the vesamicol receptor, lowering the rates of dissociation for the analogs ABV and transDec [231.

The present experiments had two major objectives: (1) to estimate the potencies of ABV and transDec to inhibit the release of ACh from mammalian nervous tissue, and (2) to investigate whether the characteristics of inhibition of ACh release by these two analogs were similar to those presented by vesamicol.

The vesamicol analogs ABV and transDec inhibited the potassium-stimulated release of ACh from rat brain corti- cal slices in a dose-dependent manner, with potencies (IC50) close to the value obtained for vesamicol (Fig. 1). These values (65 nM for ABV and 14 nM for transDec) were similar to those obtained for inhibition of the trans- port of ACh in isolated synaptic vesicles of Torpedo (100 nM for ABV and 10 nM for transDec from Ref. [25]). However, they are much greater than the affinity constant values for the binding of ABV and transDec in synaptic vesicles (6.5 and 9.0 pM for ABV and transDec, respec- tively, from Ref. [23]). The relationship between affinity constants and potencies for these two analogs is in sharp contrast with the relation between the affinity constant for vesamicol and its potency to interfere with cholinergic function ( K i 1 nM Ref. [23], IC50 40 nM Ref. [25] for vesicular transport, and IC50 11 nM present study for ACh release).

It has been suggested that the low sensitivity of the ACh transport assay could explain the large differences among the measured affinities and potencies for vesamicol analogs [23]. Hence, in analogy to the vesicular transport assay, the high concentrations of vesamicol receptor in our preparation of cortex slices might preclude the measure- ment of potencies for drugs with high affinity for the vesamicol receptor. However, the relationship between receptor occupancy and effect on transport is not alto- gether clear, whether 'spare receptor concepts' apply is not known, and just what fraction of the total receptor popula- tion needs to be occupied to effectively block ACh trans- port in situ has yet to be established [14]. The phenomenon could also reflect the possibility that some of the drugs alter the conformation of the vesamicol receptor in more intact preparations, such as brain slices, in a non-effective way; vesamicol could be then more efficacious than its two high affinity analogs.

It was clear that the three drugs were able to impair the release of newly synthesized [3H]ACh, as shown in Fig. 2, when they were maintained in contact with the slices during the entire experiment. It is somewhat curious that

R.M. Le~o et al. /Brain Research 703 (1995) 86-92 91

the vesamicol effect in this experiment is incomplete; the drug concentration used might be expected to about fully inhibit ACh uptake by vesicles, yet some 50% of control release, and presumably [3H]ACh uptake to vesicles was evident. It is possible that this reflects the vesamicol-insen- sitive synaptic vesicles postulated to exist on the basis of other evidences [7,20], which might continue to turnover their ACh despite the drug's presence. The effect of the ABV and transDec on [3HILACh release was greater than that of vesamicol in this test, reflecting either a difference with respect to these vesamicol-insensitive stores or, more likely, an additional effect of the analogs on ACh release as discussed below.

The action of vesamicol was readily reversible after washout, as observed previously [22], and in accordance with the kinetics of vesamicol binding to its receptor and inhibition of ACh transport [4,23]. In contrast, the effects of ABV and transDec were not altered by washing the tissue in the same way as vesamicol. This result certainly reflects the strong binding of ABV and transDec to the vesamicol receptor in synaptic vesicles [23], producing a long-lasting blockade of the entrance of ACh.

The hypothesis that A B V and transDec block the re- lease of ACh by interfering,, with vesicular ACh transport has been supported by the two distinct lines of evidence discussed above. The drugs blocked ACh release in the same range of concentration as they interfered with the vesicular transport; and they inhibited persistently the re- lease of the transmitter synthesized in their presence. How- ever, the investigation of 1:he action of these vesamicol analogs after labelling the internal stores of ACh reveals another feature of the pharmacology of ABV and transDec. Vesamicol does not interfere with the release of ACh in this protocol (Refs. [1,17,2";'] and Fig. 4a), suggesting that [3H]ACh released by cortical slices upon potassium depo- larization was prepackaged into the synaptic vesicles, oth- erwise vesamicol would inlhibit this release. It was clear that, in contrast to the action of vesamicol, ABV and transDec were able to interfere significantly with the re- lease of pre-packed [3H]ACh (Fig. 4b and c), suggesting that these analogs have additional actions in cholinergic nerve terminals, presumably on transmitter release mecha- nisms 'down-stream' from vesicle loading.

We conclude that ABV and transDec cause a prolonged inhibition of ACh release, probably due to their quasi-irre- versible binding for the vesamicol receptor in rat brain [23]. The rank order of potencies for the three drugs to inhibit the release of ACh (vesamicol = transDec > ABV) was similar to that for inhibiting vesicular transport (trans- Dec > vesamicol > ABV, Ref. [25]), but it did not reflect the rank of affinities of these analogs to the vesamicol receptor (ABV > transDec > > vesamicol, Ref. [23]). In addition, the unexpected effect of these two analogs on the release of pre-packed [3H]ACh may suggest that there is more than one relevant action of ABV and transDec in cholinergic nerve terminals.. At present, it is not known

whether or not the apparent effect of these analogs on the release mechanism is cholinergic-specific, or whether it could be manifested at nerve terminals with other pheno- type. This possibility might have to be considered in the use of these analogs as in vivo imaging ligands [15].

Acknowledgements

We thank Ms. Adriane Pereira, Ms. Anna McNicoll and Mr. Antonio C. Gomes for expert technical assistance, Dr. Luiz Armando De Marco and Dr. M.M. Santoro for help- ful discussions. We are in debt to Drs. S.M. Parsons and G. Rogers for the gift of vesamicol, ABV and transDec. This work was supported by CNPq, FAPEMIG, FINEP (Brazil), and the MRC (Canada).

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