The effect of sodium on depolarization-induced calcium uptake and acetylcholine release by synaptosomes

The influence of external sodium concentration on potassium (depolarizing agent)-stimulated calcium uptake and Ca⁺-dependent acetylcholine release by rat cerebral cortex synaptosomes has been studied. It was found that increased sodium concentration decreases both the Ca²⁺ uptake and the acetylcholine release, whereas a low external sodium concentration is stimulatory.     

The Synthesis and Release of Acetylcholine by Depolarized Hippocampal Slices Is Increased by Increased Choline Available In Vitro Prior to Stimulation

The objective of these experiments was to determine whether preincubating hippocampal slices with choline provides precursor that can be used during a subsequent incubation to support or enhance the synthesis of acetylcholine (ACh). Slices were preincubated for 60 min with 0, 10, 25, or 50 microM choline, washed, resuspended, and then incubated for 10 min in choline-free buffer containing 4.74 (Krebs-Ringer bicarbonate, KRB) or 25 mM KCl. The tissue contents of ACh and choline were determined prior to and after the preincubation, as well as after the incubation; the amounts of ACh and choline released were measured, and ACh synthesis was calculated. Preincubation in the absence of choline increased the tissue content of ACh to 242% of original levels; preincubation with 10 microM choline did not lead to a further increase, but preincubation with 25 or 50 microM choline increased the ACh content to 272% of original levels, significantly greater than that of slices preincubated with either 0 or 10 microM choline. When tissues were subsequently incubated for 10 min with either KRB or 25 mM KCl, ACh release from slices preincubated with 50 microM choline was greater than from slices preincubated with 0, 10, or 25 microM choline. Incubation of slices with KRB did not alter the tissue content of ACh, but when tissues were incubated with 25 mM KCl, the ACh content of slices preincubated with 0 or 10 microM choline decreased significantly, whereas that of slices preincubated with 25 or 50 microM choline did not.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of streptozotocin-induced diabetes on acetylcholine metabolism in rat brain

The main objective of this study was to determine whether uncontrolled hyperglycemia, as a consequence of diabetes, altered the metabolism of acetylcholine (ACh) in rat brain. To accomplish this, rats received injections of streptozotocin (STZ, 60 mg/kg, i.v.) or vehicle, and were maintained for up to 7 weeks after the injections. Various indices of ACh metabolism were determined in striatum and hippocampus, two brain regions densely innervated by cholinergic neurons. STZ induced diabetes in 96% of the rats injected, as evidenced by glucose spillage into the urine within 48 hours. Serum glucose levels increased to 326% of control values by 1 week and remained at this level for the duration of the study. The steady-state concentrations of ACh and choline, determined in brain tissue from animals killed by head-focused microwave irradiation, did not differ between the control and STZ-injected groups. However, the synthesis and release of neurotransmitter by striatal slices, measured in vitro, decreased in a time-dependent manner. Although the basal release of ACh was unaltered at 1 week, neurotransmitter release decreased significantly by 21% at 5 weeks and by 26% at 7 weeks. The release of ACh evoked by incubation with 35 mM KCl was inhibited significantly by 20% at all time points studied. ACh synthesis by slices incubated under basal conditions decreased by 13% and 27% at 5- and 7-weeks, respectively, the latter significantly less than controls. Synthesis by striatal slices incubated with 35 mM KCl was inhibited by 17% at 7 weeks. Although the synthesis and release of ACh by hippocampal slices from diabetic animals tended to be less than controls, these alterations were not statistically significant. Investigations into the mechanism(s) mediating the deficit in ACh synthesis exhibited by striatal slices indicated that it did not involve alterations in precursor choline availability, nor could it be attributed to alterations in the activities of the synthetic or hydrolytic enzymes choline acetyltransferase or acetylcholinesterase; rather, the decreased turnover of ACh may be secondary to other STZ-induced, hyperglycemia-mediated neurochemical alterations.     

Effects of various experimental manipulations on neostriatal acetylcholine and dopamine release

The release of endogenous acetylcholine and dopamine and the appearance of their metabolites, choline and dihydroxyphenylacetic acid (DOPAC), from neostriatal slices prepared from Fischer 344 rats was examined under various experimental conditions. There was a dose-dependent increase in the amount of neurotransmitter or metabolite as the medium potassium concentration was increased from 5 to 50 mM. Over an eight minute period in Krebs Ringer bicarbonate buffer containing 25 mM potassium, the rate of release of acetylcholine was 6 to 13 times greater than that of dopamine. The dopamine endogenous to the slice preparation appeared to have little effect on the release of endogenous acetylcholine since manipulations that significantly altered dopamine release (depletion with 6-hydroxydopamine or uptake inhibition with nomifensine) had minimal effects on the cholinergic neurons. In contrast, increasing the endogenous acetylcholine in the preparation by inhibiting acetylcholinesterase resulted in a 1.2 to 12 fold increase in dopamine release depending upon the incubation time and the potassium concentration. These studies indicate that within the neostriatal slices there is minimal influence of the endogenous dopamine on the cholinergic neurons, whereas the extracellular acetylcholine can influence dopamine release when its concentration is increased by inhibition of acetylcholinesterase.     

Somatostatin stimulates striatal acetylcholine release by glutamatergic receptors: an in vivo microdialysis study

The modulation of striatal cholinergic neurons by somatostatin (SOM) was studied by measuring the release of acetylcholine (ACh) in the striatum of freely moving rats. The samples were collected via a transversal microdialysis probe. ACh level in the dialysate was measured by the high performance liquid chromatography method with an electrochemical detector. Local administration of SOM (0.1, 0.5 and 1 microM) produced a long-lasting and concentration-dependent increase in the basal striatal ACh output. The stimulant effect of SOM was antagonized by the SOM receptor antagonist cyclo(7-aminopentanoyl-Phe-D-Trp-Lys-Thr[BZL]) (1 microM). In a series of experiments, we studied the effect of 6,7-dinitroquinoxaline-2, 3-dione (DNQX), a selective non-NMDA (N-methyl-D-aspartate) glutamatergic antagonist, on the basal and SOM-induced ACh release from the striatum. DNQX, 2 microM, perfused through the striatum had no effect on the basal ACh output but inhibited the SOM (1 microM)-induced ACh release. The non-NMDA glutamatergic receptor antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylendioxy-5H-2,3- benzodiazepine (GYKI-52466), 10 microM, antagonized the SOM (1 microM)-induced release of ACh in the striatum. Local administration of the NMDA glutamatergic receptor antagonist, 2-amino-5-phosphonopentanoic acid (APV), 100 microM, blocked SOM (1 microM)-evoked ACh release. Local infusion of tetrodotoxin (1 microM) decreased the basal release of ACh and abolished the 1 microM SOM-induced increase in ACh output suggesting that the stimulated release of ACh depends on neuronal firing. The present results are the first to demonstrate a neuromodulatory role of SOM in the regulation of cholinergic neuronal activity of the striatum of freely moving rats. The potentiating effect of SOM on ACh release in the striatum is mediated (i) by SOM receptor located on glutamatergic nerve terminals, and (ii) by NMDA and non-NMDA glutamatergic receptors located on dendrites of cholinergic interneurones of the striatum.     

Receptor-mediated presynaptic facilitation of quantal release of acetylcholine induced by pralidoxime inAplysia

1. Possible interactions of contrathion (pralidoxime sulfomethylate), a reactivator of phosphorylated acetylcholinesterase (AChE), with the regulation of cholinergic transmission were investigated on an identified synapse in the buccal ganglion of Aplysia californica. 2. Transmitter release was evoked either by a presynaptic action potential or, under voltage clamp, by a long depolarization of the presynaptic cell. At concentrations higher than 10(-5) M, bath-applied contrathion decreased the amplitude of miniature postsynaptic currents and increased their decay time. At the same time, the quantal release of ACh was transiently facilitated. The facilitatory effect of contrathion was prevented by tubocurarine but not by atropine. Because in this preparation, these drugs block, respectively, the presynaptic nicotinic-like and muscarinic-like receptors involved in positive and negative feedback of ACh release, we proposed that contrathion activates presynaptic nicotinic-like receptors. 3. Differential desensitization of the presynaptic receptors is proposed to explain the transience of the facilitatory action of contrathion on ACh release. 4. The complexity of the synaptic action of contrathion raises the possibility that its therapeutic effects in AChE poisonings are not limited to AChE reactivation.     

A prolonged after-effect of intense synaptic activity on acetylcholine in a sympathetic ganglion.

The findings was confirmed that there is a "rebound" increase of stored acetylcholine (ACh) in cat superior cervical ganglia conditioned by prolonged preganglionic stimulation at a frequency high enough to cause initial depletion of the store. Ganglia removed immediately after 60 min of continuous or interrupted stimulation at 50 Hz, with chloralose as anesthetic, contained about 30% more ACh than their unconditioned controls; the rebound rose to about 60% after 15 min of rest and then subsided with an apparent half-time of about 2 h. Tests with hemicholinium, combined with hexamethonium or tubocurarine, showed that rebound ACh was located presynaptically and could be released by nerve impulses; but conditioned ganglia perfused with an eserine-containing medium did not release more ACh than their unconditioned controls, except in circumstances in which the mobilization of ACh from a reserve store appeared to be the rate-limiting process for release. The appearance of rebound ACh during and after conditioning stimulation was suppressed by hexamethonium and by tubocurarine, neither of which has much effect on ACh turnover in ganglia excited at lower frequencies, but not only by atropine, noradrenaline, or phenoxybenzamine. The formation of rebound ACH is thus contingent on the postsynaptic nicotinic response to released ACh, and may represent an augmentation of the transmitter store in structures remote from the release sites.     

Effects of membrane peroxidation on [3H]acetylcholine release in rat cerebral cortical synaptosomes

[³H]Acetylcholine (ACh) release, malonaldehyde formation and⁴⁵calcium-uptake were measured in rat cerebral cortical nerve terminal that were exposed to various concentrations of ferrous and ascorbate ions. At a constant molar ratio of 25:1, ferrous:ascorbate, these ions increased malonaldehyde (MA) synthesis in a concentration-dependent manner. Treatment with these ions in the same ratio also induced a dose-related inhibition of the K⁺-depolarization-induced release of newly synthesized [³H]ACh. Combined exposure to Fe²⁺/ascorbate also reduced calcium ionophore A23187-induced [³H]ACh release. Neither ferrous nor ascorbate ions alone altered depolarization-or ionophore-induced [³H]ACh release over this concentration range. Depolarization- and A23187-induced⁴⁵calcium uptake were not affected by peroxidation, suggesting that membrane peroxidation influenced some process in the release-process subsequent to calcium influx in a manner similar to what is observed during aging.     

Halothane enhances acetylcholine release by decreasing dopaminergic activity in rat striatal slices

The present study investigated the effect of halothane on acetylcholine (ACh) and dopamine (DA) release from the rat striatum. Halothane decreased DA release in a concentration-dependent manner, while increased ACh release. In our previous investigation, a volatile anesthetic, halothane, inhibited DA release from the rat striatal slices in a concentration-dependent manner. Although the release of ACh from cholinergic interneurons is tonically modulated by DA in the striatum, the effect of halothane on the relationship between the release of ACh and DA has not been discussed. Using double-labeled techniques, we investigated the effect of halothane on ACh and DA release simultaneously. The slices were incubated with [14C]-choline and [3H]-DA and superfused with modified Krebs solution containing 1 microM of hemicholinium-3. We applied electrical field stimulation (2 Hz, 240 shocks), and the amount of the release of radioactivity evoked by stimulation was calculated by subtraction of the basal radioactive outflow from the total outflow at the beginning of the respective stimulation periods. The effects of drugs on the release were expressed as the ratio of stimulation-evoked fractional releases (FR), measured in the presence and absence (FRS2/FRS1) of the drug. Halothane decreased DA release in a concentration-dependent manner (FRS2/FRS1=0.767+/-0.021, 0.715+/-0.026, 0.671+/-0.014 and 0.639+/-0.033 at the concentration of 0, 0.5, 2 and 4%, respectively), while ACh release showed a biphasic change in the presence of different concentrations of halothane. The release of ACh was significantly increased at the concentration of 2%, but not at 0.5 or 4%. Halothane failed to increase the release of ACh in striatal slices after lesion by 6-OH-dopamine. The application of amphetamine reduced the release of ACh and abolished the effect of halothane. These results indicate that the effect of halothane on ACh release is indirect: it increases the release by attenuating the inhibitory effect of DA released from the nigro-striatal pathway. The nonsynaptic interaction between DA and ACh release is involved in the effect of halothane on ACh release.     

Differential inhibition of the release of endogenous and newly synthesized acetylcholine from Torpedo synaptosomes by presynaptic muscarinic receptors

Activation of Torpedo presynaptic muscarinic acetylcholine (ACh) receptors with the agonist oxotremorine (20 μM) results in the inhibition of Ca²⁺-dependent release of endogenous ACh from Torpedo synaptosomes. This effect is reversed by the muscarinic antagonist atropine (1 μM) which, by itself, has no effect. In contrast, under the same conditions the amount of newly synthesized radiolabeled [³H]ACh released is not affected by muscarinic ligands. These findings suggest that presynaptic muscarinic inhibition in the Torpedo is due to interference with the mobilization of ACh from a storage pool.     

Acetylcholine Turnover and Compartmentation in Rat Brain Synaptosomes

Abstract: The turnover of acetylcholine (ACh) in rat brain synaptosomes and its compartmentation in the labile bound and stable bound pools were investigated. The P₂ fraction from rat brain was subjected to three sequential incubations, each terminated by centrifugation followed by determination of ACh concentrations by gas chromatography-mass spectrometry (GCMS): (1) Depletion phase: Incubation of synaptosomes at 37°C for 10 min in Na⁺-free buffer containing 35 mM-KCl reduced the content of both labile bound and stable bound ACh by 40%. (2) Synthesis phase: Incubation at 37°C with 2 μ M -[²H₄]choline resulted in accumulation of labeled and unlabeled ACh in both compartments. Addition of an anticholinesterase had little effect on stable bound ACh but greatly increased the content of labile bound ACh. This excess accumulated ACh was probably due to inhibition of intracellular acetylcholinesterase (AChE), because negligible uptake of ACh from the medium was observed. The effects on ACh synthesis of altered cation concentrations and metabolic inhibitors were examined. (3) Release phase: The tissue was incubated in the presence of 35 mM-KCl, 40 μM-paraoxon, and 20 μM-hemicholinium-3 (HC-3) (to inhibit further synthesis of ACh). Measurements of the compartmental localization of ACh at several time points indicated that ACh was being released from the labile bound fraction. In support of this conclusion, 20 mM-Mg²⁺ reduced ACh release and increased the labile bound ACh concentration.     

Recovery of acetylcholine release and choline acetyltransferase activity after freezing-induced degeneration of rat soleus muscle

In order to study the effect of synaptic contact on the amounts of choline acetyltransferase (ChAT) and acetylcholine (ACh) in the nerve terminals and on their ability to release ACh, a freeze—thaw procedure was developed as a means to induce long lasting degeneration of rat soleus muscle. It was found that 4 days after the freeze—thaw procedure the preparation did not contract upon direct electric stimulation and the level of creatine kinase (CK) was below detection. The preparation contained about 15% of the ChAT activity and 15% of the ACh content of the controls. The ACh release evoked by 50 mM KCl was 25% of controls, but it was, when expressed as a fraction of the ACh content, about twice as high as that in control muscles. At day 12, the preparation still did not contract and the level of CK was less than 5% of controls. The ChAT activity and the ACh content were 40%) and 20% of controls, respectively. However, no release of ACh could be evoked by 50 mM KCl. At days 28 and 58 the preparation contracted upon stimulation of the nerve; the CK activity had recovered to about 20% and the ACh content to 40%, while the ChAT activity did not increase above 40%. The KCl–evoked ACh release had recovered to 20—30% of controls. The results indicate that freezing destroyed muscle cells and most intramuscular nerve branches. Subsequent regeneration of muscle fibres was slow, probably because freezing had killed many satellite cells in the muscle. Because the ChAT activity at day 12 had recovered when CK was almost absent and the preparation failed to contract, we conclude that there was expression of ChAT activity in ‘nerve terminals’ which do not make contact with regenerated muscle cells, although little if any ACh was released from these sites. ©1998 Elsevier Science Ltd. All rights reserved.     

Colchicine-binding activity and neurotubule integrity in a rat sympathetic ganglion

The colchicine-binding activity of rat superior cervical ganglia was examined. Ganglia were cooled and re-warmed in the presence of either Cu²⁺ or of metabolic inhibitors. Electronmicroscopy showed that these treatments depolymerized the neurotubules. This depolymerization of neurotubules did not affect the colchicine-binding activity of ganglion homogenates but caused a two-fold increase in colchicine-binding by whole ganglia. This suggests that colchicine binds only to depolymerized neurotubule subunits and that colchicine-binding by whole ganglia can be used as a measure of polymerization of the neurotubule protein.The major part of the colchicine-binding activity of ganglion homogenates was found in the soluble fraction and was unstable. In the absence of divalent cations, 10⁻⁴ M vinblastine stabilised the soluble colchicine-binding activity.     

The Role of Chloride in Acetylcholine Metabolism

Abstract: The chloride dependence of acetylcholine (ACh) synthesis and release and of choline uptake was studied in synaptosomal preparations from rat brain. The substitution of propionate for chloride, in the presence of 35 m m -potassium, lowered the ACh content of the synaptosomes. However, in the presence of 5 m m -potassium, the ACh level in synaptosomes was reduced, but significantly less so. Propionate had no effect on choline acetyltransferase (EC 2.3.1.6) activity when measured in a standard chloride-containing medium. In the presence of propionate, the spontaneous release of ACh was unchanged, but potassium-stimulated release of ACh was markedly reduced as compared with a chloride-containing medium. The synthesis of ACh, as measured by the net increase in the amount of ACh in the synaptosomes and that released to the medium, was reduced with propionate at 5 m m -potassium and was totally inhibited when the potassium concentration was increased to 35 m m . Choline uptake studies revealed that with propionate only a low-affinity component of the choline transport system existed. Further, the V _max was markedly reduced when the potassium concentration was increased to 35 m m . The results suggest that under certain conditions choline transported by a low-affinity system might provide a substantial source of choline for ACh synthesis.     

The effect of hemicholinium-3 on choline and acetylcholine levels in a sympathetic ganglion.

Pregangliaaonic stimulation of the cat's superior cervical ganglion in the presence of hemicholinium-3 (HC-3) produced the expected depletion of acetylcholine (ACh) stores, but failed to cause a corresponding reduction in the choline content. These results suggest that either HC-3 possesses an intracellular site of action or that in lower doses it selectively inhibits a specialized choline transport system in cholinergic nerves. At a dose of 2 mg/kg, HC-3 probably blocked ACh synthesis completely in ganglia stimulated at 20 Hz. Under these conditions, there was a rapid depletion of ACh to about 50% of control levels during the first 5 min of stimulation and thereafter the rate of decline in ACh levels proceeded at a much slower pace. Since the 2 mg/kg dose of HC-3 did not raise plasma choline concentrations, it may be assumed that non-specialized choline transport systems in other tissues were not significantly inhibited by this dose of HC-3. However, when the dose of HC-3 was increased to 4 mg/kg, plasma choline levels increased by 58%.     

Post-ischemic regional changes in acetylcholine synthesis following transient forebrain ischemia in gerbils

The synthesis rate of brain acetylcholine (ACh) was estimated 30 min and 5 days following transient forebrain ischemia performed by 10 min bilateral carotid occlusion in gerbils. ACh synthesis was evaluated from the conversion of radiolabeled choline (Ch) into ACh after an i.v. administration of [methyl-³H]Ch. Endogenous and labeled Ch and ACh were quantified by HPLC. The synthesis rate of ACh was significantly decreased following 30 min of recirculation. The reductions reached 55.4% in the hippocampus, 51.2% in the cerebral cortex and 44.4% in the striatum. Five days after ischemia, the values returned to normal in the cerebral cortex and in the striatum, while ACh synthesis remained selectively lowered (–30.4%, p<0.01) in the hippocampus. These cholinergic alterations may account for both early and delayed post-ischemic behavioral and mnesic deficits.     

Inhibition of the synthesis of acetylcholine in rat brain slices by (-)-hydroxycitrate and citrate

Abstract: Slices of rat caudate nucleus were incubated in a solution of 123 mM-NaCl, 5 mM-KCl, 1.2 mM-MgCl₂, 1.2 mM-NaH₂PO₄, 25 mM-NaHCO₃, 0.2 mM-choline chloride, 0.058 mM-paraoxon, 1 mM-EGTA, and oxidizable substrates. (−)-Hydroxycitrate, a specific inhibitor of ATP-citrate lyase (EC 4.1.3.8), used at a concentration of 2.5 mM, inhibited the synthesis of acetylcholine (ACh) from [1,5-¹⁴C]citrate by 82–86%, but that from [U-¹⁴C]glucose by only 33%, from [2-¹⁴C]pyruvate by 24% and from [1-¹⁴C-acetyl]carnitine by 8%; the production of ¹⁴CO₂ from these substrates was not substantially changed. The synthesis of ACh from glucose and pyruvate was in hibited also by citrate; 2.5 mM- and 5 mM-citrate diminished it by 43% and 66%, respectively; the production of from [U-¹⁴C]glucose and from [1-¹⁴C]pyruvate was not affected. The mechanism of the inhibitory effect of citrate on the synthesis of ACh is not clear; the possibility is discussed that citrate alters the intracellular milieu in cholinergic neurons by chelating the intracellular Ca²⁺ and decreases the supply of mitochondrial acetyl-CoA to the cytosol. The results with (−)-hydroxycitrate indicate that the cleavage of citrate by ATP-citrate lyase is not responsible for the supply of more than about one-third of the acetyl-CoA which is used for the synthesis of ACh when glucose or pyruvate are the main oxidizable substrates. This proportion may be even smaller, since (−)-hydroxycitrate possibly affects the synthesis of ACh from glucose and pyruvate by a mechanism (unknown) similar to that of citrate, rather than by the inhibition of ATP-citrate lyase.     

Monoamine storage sites in the rat superior cervical ganglion following synthesis inhibition

Summary Monoamine storage sites in paraganglionic (PG-)cells of the rat superior cervical ganglion were investigated by electron and fluorescence microscopy following treatment with p-chlorophenylalanine (pCPA), disulfiram or guanethidine respectively.Dense core vesicles in PG-cells are significantly decreased (p< 0.001) in number following pCPA, and in the majority of these cells following disulfiram and guanethidine. However in a minor portion of PG-cells the latter agents cause an increase in number and in size of dense core vesicles, in parallel with structural alterations. In agreement with these electron microscopic findings fluorescence microscopic and cytophotometric evaluations reveal a general decrease in catecholamine content with few cells showing an increase.The findings provide a morphological basis for the assumption, that monoamine storage sites in PG-cells can be decreased by inhibition of monoamine synthesis, following administration of pCPA, disulfiram and guanethidine. However the two types of responses of PG-cells which occur after disulfiram and guanethidine demonstrate a functional heterogeneity of this cell system in the rat superior cervical ganglion which is discussed.Supported by Deutsche Forschungsgemeinschaft — Grant He 919/1.I like to thank Prof. Arnold, Tübingen, for the kind disposal of cytophotometric equipment.     

Acetylcholine Synthesis and Release by a Sympathetic Ganglion in the Presence of 2-(4-Phenylpiperidino) Cyclohexanol (AH5183)

These experiments measured the release and the synthesis of acetylcholine (ACh) by cat sympathetic ganglia in the presence of 2-(4-phenylpiperidino) cyclohexanol (AH5183), an agent that blocks the uptake of ACh into synaptic vesicles. Evoked transmitter release during short periods of preganglionic nerve stimulation was not affected by AH5183, but release during prolonged stimulation was not maintained in the drug's presence, whereas it was in the drug's absence. The amount of ACh releasable by nerve impulses in the presence of AH5183 was 194 +/- 10 pmol, which represented 14 +/- 1% of the tissue ACh store. The effect of AH5183 on ACh release was not well antagonized by 4-aminopyridine (4-AP), and not associated with inhibition of stimulation-induced calcium accumulation by nerve terminals. It is concluded that AH5183 blocks ACh release indirectly, and that the proportion of stored ACh releasable in the compound's presence represents transmitter in synaptic vesicles available to the release mechanism. The synthesis of ACh during 30 min preganglionic stimulation in the presence of AH5183 was 2,448 +/- 51 pmol and in its absence it was 2,547 +/- 273 pmol. Thus, as the drug decreased ACh release it increased tissue content. The increase in tissue content of ACh in the presence of AH5183 was not evident in resting ganglia; it was evident in stimulated ganglia whether or not tissue cholinesterase was inhibited; it was increased by 4-AP and reduced by divalent cation changes expected to decrease calcium influx during nerve terminal depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)