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1. The effect of ouabain on the release of [3H]acetylcholine ([3H]ACh) in rat brain cortical slices was investigated. 2. The ouabain-induced release of [3H]ACh was calcium-independent and not blocked by EGTA. 3. BAPTA-AM, a chelator of intracellular calcium, inhibited the ouabain effect suggesting the involvement of intracellular calcium stores. 4. Vesamicol, a drug that blocks the storage of acetylcholine in synaptic vesicles inhibited by 73% the ouabain-induced release of [3H] ACh, suggesting exocytotic release of the neurotransmitter. 5. Dantrolene and tetracaine, inhibitors of ryanodine and InP3 receptors, inhibited by 57 and 66% respectively, the ouabain-elicited release of [3H]ACh in brain cortical slices. 6. Confocal microscopy and calcium imaging showed that ouabain increased the levels of [Ca2+]i in cholinergic SN56 cells and that this increase was concentrated in the cell soma. 7. In conclusion, we suggested that ouabain causes Ca2+ release from intracellular stores that can increase [3H] ACh exocytosis from rat brain cortical slices.

In this paper, the effect of the alpha-scorpion toxin tityustoxin (TsTX) in the release of gamma-[(3)H]aminobutyric acid ([(3)H]GABA) from rat brain cortical slices is described. The TsTX-stimulatory effect on the release of [(3)H]GABA was dependent on incubation time and TsTX concentration, having an EC(50) of 0.33 microM. Tetrodotoxin (TTX) completely inhibited the TsTX action on [(3)H]GABA release. The scorpion toxin effect was calcium-dependent and involves P/Q calcium channels. beta-Alanine also induces the release of [(3)H]GABA that was not inhibited by TTX but was additive in the presence of TsTX. The data suggest a neuronal origin for the release of [(3)H]GABA by TsTX.

The effect of the beta-scorpion toxin, TiTX gamma on the release of [3H]GABA from rat brain cortical slices is described. The stimulatory effect of TiTX gamma on the release of [3H]GABA was dependent on incubation time and TiTX gamma concentration with an EC50 of 0.19 microM. The scorpion toxin effect was calcium dependent and was completely inhibited by tetrodotoxin. beta-Alanine also induced the release of [3H]GABA and this effect was not inhibited by tetrodotoxin but was additive in the presence of TiTX gamma. The data suggest a neuronal origin for the release of [3H]GABA by TiTX gamma.

Volatile general anaesthetics are believed to affect synaptic transmission, but their actions in the central nervous system (CNS) remains unclear. Acetylcholine (ACh) is one of the most important neurotransmitter in the CNS and thus, it is possible that its release could be one of the targets for volatile anaesthetic action. However, the effects of these agents on the release of ACh are not yet fully understood. Rat brain cortical slices were loaded with [(3)H]-choline in order to study the effect of isoflurane on the release of [(3)H]-ACh from this preparation. Isoflurane (28, 43, 54, 95 and 182 nM) significantly increased the basal release of [(3)H]-ACh. This effect was independent of the extracellular sodium and calcium concentration but was decreased by tetracaine and dantrolene, inhibitors of Ca(2+-)release from intracellular stores. These findings indicate that isoflurane may cause a Ca(2+-)release from internal stores that increases [(3)H]-ACh release in rat brain cortical slices.

1. The effect of halothane on the release of [3H]-acetylcholine ([3H]-ACh) in rat brain cortical slices was investigated. 2. Halothane (0.018 mM) did not significantly affect the basal and the electrical field stimulation induced release of [3H]-ACh. However, halothane (0.063 mM) significantly increased the basal release of [3H]-ACh and this effect was additive with the electrical field stimulation induced release of [3H]-ACh. 3. The release of [3H]-ACh induced by 0.063 mM halothane was independent of the extracellular sodium and calcium ion concentration and was decreased by tetracaine, an inhibitor of Ca(2+)-release from intracellular stores or dantrolene, an inhibitor of Ca(2+)-release from ryanodine-sensitive stores 4. Using 2-(4-phenylpiperidino)-cyclohexanol (vesamicol), a drug that blocks the storage of ACh in synaptic vesicles, we investigated whether exocytosis of this neurotransmitter is involved in the effect of halothane. Vesamicol significantly decreased the release of [3H]-ACh evoked by halothane. 5. It is suggested that halothane may cause a Ca2+ release from intracellular stores that increases [3H]-ACh exocytosis in rat brain cortical slices.

The role of L-,N-, P- and Q-type voltage-dependent calcium channels in K+-induced release of [3H]adenine nucleotides from rat brain cortical synaptosomes was investigated. Cd2+, a non-specific blocker of calcium channels, inhibited by 69% the release of the nucleotides induced by 33 mM K+. Nifedipine, omega-Conotoxin GVIA and omega-Agatoxin IVA had no effect whereas omega-Conotoxin MVIIC inhibited by 62% the K+ induced release of adenine nucleotides in rat brain cortical synaptosomes. It is concluded that Q-type calcium channels are directly involved in the release of adenine nucleotides in rat brain cortical synaptosomes.

Venoms from spiders are an important source of toxins that can help on the dissection of mechanisms involved in neurotransmission. Among them is the venom of the spider Phoneutria nigriventer that contains several toxic fractions with different targets in mammals and/or insects. We here report that one of these fractions (PhTx2) is able to evoke acetylcholine release from rat cortical synaptosomes and that this effect is dependent on extracellular calcium and is inhibited by the sodium channel blocker tetrodotoxin.

Neurotoxins can help the understanding of mechanisms involved in neurotransmission. We here report that two neurotoxin isoforms, Tx3-3 and Tx3-4 obtained from the venom of the spider Phoneutria nigriventer inhibited the 45Ca2+ influx in rat cortical synaptosomes induced by the scorpion venom tityustoxin. The IC50 for Tx3-3 and Tx3-4 were 0.32 and 7.9 nM, respectively. The neurotoxins Tx3-3 and Tx3-4 are very effective in inhibiting 45Ca2+ influx and they should be useful in studies involving Ca(2+)-dependent processes.

Tityustoxin, a scorpion toxin that alters the Na+ channel activity, induces release of ATP from rat brain cortical synaptosomes. The effect of tityustoxin is dependent on its concentration and incubation time. Continuously or cumulative release of ATP evoked by tityustoxin was calcium-dependent and interestingly only partially inhibited by tetrodotoxin. We suggest that tityustoxin mainly releases ATP from the vesicular pool but other pools may also be involved.

1. The effect of reducing reagents on omega-conotoxin GVIA (omega-CgTX) inhibition of the release of [3H]-acetylcholine ([3H]-ACh) induced by tityustoxin, K+ 50 mM and electrical stimulation was investigated in rat brain cortical slices. 2. In cortical slices the inhibition of tityustoxin or electrically-stimulated [3H]-ACh release by omega-CgTX was dramatically increased by reducing reagents ascorbate or beta-mercaptoethanol. Dehydroascorbic acid did not substitute for ascorbate. 3. Depolarization induced by K+ 50 mM caused [3H]-ACh release from cortical slices which was not inhibited by omega-CgTX, even in the presence of ascorbate. 4. In the guinea-pig myenteric plexus, omega-CgTX inhibition of the tityustoxin induced release of [3H]-ACh was independent of ascorbate. 5. It is suggested that N-type-like calcium channels in guinea-pigs myenteric plexus may have pharmacological/biochemical diversity from similar channels of rat cerebral cortex.

The present experiments investigated the effect of some of the toxic components present in the venom of the spider Phoneutria nigriventer on the release of neurotransmitter. The toxic fraction, Phoneutria nigriventer toxin-3 (PhTx3), abolished Ca2+-dependent glutamate release, but did not alter Ca2+-independent secretion of glutamate when rat brain cortical synaptosomes were depolarized with 33 mM KCl. This effect was most likely due to interference with the entry of calcium through voltage-gated calcium channels, because PhTx3 reduced by 50% the increase in intrasynaptosomal free calcium induced by membrane depolarization, and did not affect the release of glutamate evoked by a calcium ionophore (ionomycin). A polypeptide (Tx3-3) present in the PhTx3 fraction reproduced the effects of the PhTx3 fraction on transmitter release and intrasynaptosomal free calcium in the low nanomolar range. We compared the alterations produced by the Tx3-3 with the actions of toxins known to block calcium channels coupled to exocytosis: the results indicated that the Tx3-3 inhibition of glutamate release and intrasynaptosomal calcium resemble that observed with omega-conotoxin MVIIC. We suggest that the Tx3-3 is a calcium-channel antagonist that blocked glutamate exocytosis.

The aim of the present experiments was to test whether vesamicol alters the evoked release of ATP from nerve terminals. Continuous or cumulative release of ATP evoked by 33 mM KC1 from rat cerebrocortical synaptosomes was largely calcium-dependent. Vesamicol interfered with release of ATP from synaptosomes depolarized with KCl (33 mM) in a dose-dependent and stereoselective way. The (-)-vesamicol decreased the output of ATP in doses much lower than (+)-vesamicol. The release of the major excitatory neurotransmitter glutamate from depolarized nerve endings was not impaired by vesamicol. We suggest that vesamicol may alter the release of ATP specifically, probably by interacting with a protein similar to the vesamicol receptor found in cholinergic synaptic vesicles.

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-aminobenzovesamicol (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.

The venom of the Brazilian spider Phoneutria nigriventer possesses several neurotoxic polypeptidic fractions. Previous work has established that one of the toxic components, PhTx3, inhibited Ca(2+)-dependent glutamate release and the increase in cytosolic free Ca2+ in response to membrane depolarization. In the present work, we investigated the effect of PhTx3 on the release of acetylcholine (ACh) from brain and peripheral neurons. PhTx3 decreased the release of [3H]-ACh induced by tityustoxin and KCl in brain cortical slices and myenteric plexus. The inhibitory effect of myenteric plexus had the same magnitude as that obtained in the absence of extracellular Ca2+. However, in brain PhTx3 was less efficient at decreasing the evoked release of ACh. These experiments suggest that the target of PhTx3 is coupled to the process of release of ACh in brain and autonomic nervous system.

In this paper, the effects of tityustoxin and ouabain on the release and mobilization of ACh from cytoplasmic and vesicular stores of slices of cortex from the brain of the rat are described. Tityustoxin induced an increase of release of ACh which was time-dependent and this effect was accompanied by a concomitant decrease of the ACh, measured in cytoplasmic and crude vesicular fractions. Unlike tityustoxin, ouabain did not increase the release and synthesis of ACh during a 2 min period of stimulation but later, at 5, 10 and 15 min, ouabain increased the release and synthesis of ACh from incubated slices of cortex. After 10 min, ouabain caused a reduction of the content of ACh from cytoplasmic and crude vesicular fractions.

The effect of vesicular acetylcholine (ACh) transport blocker 2-(4-phenylpiperidino)cyclohexanol (AH-5183) on the subcellular storage and release of ACh was studied in rat brain cortical slices. AH-5183 reduced the release of ACh from cortical slices stimulated by tityustoxin and ouabain. Tissue stimulated in the presence of AH-5183 contained more ACh in both the nerve terminal synaptic vesicles and cytoplasmic fraction than did tissue stimulated in drug's absence. Thus, AH-5183 blocked the tityustoxin and ouabain induced release of ACh from both cytoplasmic and vesicular pools. AH-5183 also depressed the spontaneous release of ACh from incubated slices and, in this condition, the drug had no effect in the subcellular distribution of ACh. It is suggested that AH-5183 interferes with the process of ACh release independent of its blocking action on ACh transport into the synaptic vesicles.