Mode of Action of Palytoxin on the Release of Acetylcholine from Rat Cerebrocortical Synaptosomes

Palytoxin (PTX; 10(-14)-10(-6) M) caused a dose-dependent increase in the release of [3H]acetylcholine ([3H]ACh), cytosolic free Ca2+ concentration ([Ca2+]i), and uptake of 22Na+ and decrease in membrane potential in rat cerebrocortical synaptosomes. The dose-response curves for the PTX-induced increases in [3H]ACh release and in [Ca2+]i were depressed by removing extracellular Ca2+ or by decreasing extracellular Na+ concentrations. The release of [3H]ACh induced by concentrations of PTX less than 10(-10) M was more dependent on the simultaneous presence of both Ca2+ and Na+ than the release induced by higher concentrations of PTX. The PTX-induced increase both in [3H]ACh release and in [Ca2+]i was almost completely abolished by the combination of Ca2+ deprivation and Na+ concentration reduction. All responses to PTX were highly resistant to 10(-6) M tetrodotoxin. These results suggest that low concentrations of PTX cause depolarization as a result of an increase in Na+ permeability through tetrodotoxin-insensitive channels. This, in turn, increases Ca2+ influx and leads to an increase in the release of ACh. It appears that at high concentrations PTX increases the release of [3H]ACh by directly increasing the influx of Ca2+ into synaptosomes and by releasing Ca2+ from intracellular storage sites via an Na(+)-Ca2+ exchange mechanism.     

Glutamate Release from Guinea-Pig Synaptosomes: Stimulation by Reuptake-Induced Depolarization

Glutamate (10-100 microM) reversibly depolarizes guinea-pig cerebral cortical synaptosomes. This does not appear to be because of a conventional autoreceptor. Neither kainate at 1 mM, 100 microM N-methyl-D-aspartate (NMDA), 100 microM L-2-amino-4-phosphonobutanoate (APB), nor 100 microM quisqualate affects the Ca2+-dependent release of glutamate from suboptimally depolarized synaptosomes. However, kainate, quisqualate, and the quisqualate agonists beta-N-oxalylamino-L-alanine and alpha-amino-3-hydroxy-5-methylisoxazole propionate cause a slow Ca2+-independent release of glutamate from polarized synaptosomes. However, unlike kainate, quisqualate does not inhibit the acidic amino acid carrier. APB, NMDA, and the NMDA receptor-mediated neurotoxin beta-N-methylamino-L-alanine do not influence Ca2+-independent release at 100 microM. The depolarization of the plasma membrane by glutamate can be mimicked by D-aspartate, can be blocked by the transport inhibitor dihydrokainate, and is accompanied by the net uptake of acidic amino acids. L-Glutamate or D-aspartate at 100 microM increases the cytoplasmic free Ca2+ concentration. D-aspartate at 100 microM causes a Ca2+-dependent release of endogenous glutamate, superimposed on the Ca2+-independent heteroexchange with glutamate through the acidic amino acid carrier. The results suggest that the glutamatergic subpopulation of synaptosomes can be depolarized by exogenous glutamate.     

Relation of Acetylcholine Release to Ca2+ Uptake and Intraterminal Ca2+ Concentration in Guinea-Pig Cortex Synaptosomes

[14C]Acetylcholine (ACh) release and parallel alterations in 45Ca2+ uptake and intrasynaptosomal free CA2+ concentration ([Ca2+]i) were measured in guinea-pig brain cortex synaptosomes. Depolarization by high K+ concentrations caused a rapid transient increase in Ca2+ uptake, terminating within 60 s (rate constant = 0.060 s-1; t1/2 = 11.6 s). This resulted in a rapid increase (within 1 s) in [Ca2+1]i, which then fell to a maintained but still-elevated plateau level (t1/2 for the decline was 15 s). Peaks of [Ca2+]i showed a sigmoidal dependence on depolarization, contrasting with the simple linear dependence of plateau levels of [Ca2+]i. The K+-evoked ACh release also had two phases: a fast initial increase (t1/2 = 11.3 s), which terminated within 60 s, was followed by a slow additional increase during sustained depolarizations of up to 10 min. Depolarization by veratridine led to a slow gradual increase in Ca2+ uptake (t1/2 = 130 s) over a 10-min incubation period, whereas an elevated plateau level of [Ca2+]i was achieved within 2 min (without a rapid peak elevation). The Ca2+-dependent fraction of the veratridine-evoked ACh release correlated with the increase in [Ca2+]i rather than with Ca2+ uptake. Using two different methods of depolarization partially circumvented the time limitations imposed by a buffering Ca2+ indicator and we suggest that, in the main, ACh is released in bursts associated with [Ca2+]i transients.     

Inhibition of Glutamate Release by Arachidonic Acid in Rat Cerebrocortical Synaptosomes

The action of arachidonic acid on glutamate release in rat cerebrocortical synaptosomes was investigated. The Ca(2+)-dependent release of glutamate evoked by 4-aminopyridine (4-AP) was inhibited by arachidonic acid (0.5-10 microM), but the KCl-evoked release was not modified. The Ca(2+)-independent release of glutamate was insensitive to low concentrations of arachidonic acid, but higher concentrations of this free fatty acid (30 microM) induced a slow efflux of cytoplasmic glutamate. The decrease in the Ca(2+)-dependent release of glutamate by arachidonic acid was consistent with a reduction in both the depolarization and the subsequent rise in the cytoplasmic free Ca2+ concentration induced by 4-AP in the nerve terminal. The inhibitory action by arachidonic acid observed in glutamate release was reversed in the presence of the K(+)-channel blocker tetraethylammonium.     

Inhibition by Quinacrine of Depolarization-Induced Acetylcholine Release and Calcium Influx in Rat Brain Cortical Synaptosomes

The effects of quinacrine on depolarization-induced [³H]acetylcholine (ACh) release and ⁴⁵Ca²⁺ influx were examined in rat brain cortical synaptosomes. Quinacrine significantly reduced the stimulated release of [³H]ACh by high K⁺ and veratridine without affecting the spontaneous efflux from the preloaded synaptosomes. Quinacrine had no effect on ionophore A23187-induced release of [³H]ACh from the synaptosomes. Quinacrine (100 μM) markedly diminished the stimulated Ca²⁺ influx by veratridine and high K⁺ but not that by “Na⁺-free.” Trifluoperazine, a potent calmodulin antagonist, inhibited both Ca²⁺ influx and ACh release induced by the depolarizing agents. Inhibitory potencies of the two drugs on ACh release and Ca²⁺ influx were compared with the antagonism of calmodulin by two drugs, suggesting that the inhibition of depolarization-induced Ca²⁺ influx and ACh release by these drugs could not be explained by the antagonism of calmodulin.     

Possible Regulation of Caffeine-Induced Intracellular Ca2+ Mobilization by Intracellular Free Na+

To gain some understanding of the regulatory mechanism involved in caffeine-induced Ca2+ release in adrenal chromaffin cells, we took advantage of the paradoxical observation that removal of divalent cations potentiated the secretory response to caffeine. We measured the concentration of cytosolic free Ca2+ ([Ca]in) in isolated cat chromaffin cells, by fura-2 microfluorometry, to see whether there was any correlation between the secretory response and the rise in [Ca]in. The caffeine-induced [Ca]in rise and catecholamine secretion were increased by treatment of cells with a divalent cation-deficient solution. These potentiated responses were strongly inhibited either by pretreatment with ryanodine, by the reduction of the external Na+ concentration, or by the addition of Ca2+ channel blockers. Removal of divalent cations caused a large rise in the cytosolic free Na+ concentration ([Na]in), which was measured using SBFI microfluorometry. This rise in [Na]in was reduced either by adding Ca2+ channel blockers or by reducing the external Na+ concentration. These results show a good correlation between caffeine-induced Ca2+ release and [Na]in at the time of stimulation, suggesting that caffeine-induced Ca2+ release is regulated by [Na]in.     

Differential Calcium Dependence of γ-Aminobutyric Acid and Acetylcholine Release in Mouse Brain Synaptosomes

The dependence of gamma-aminobutyric acid (GABA) and acetylcholine (ACh) release on Ca2+ was comparatively studied in synaptosomes from mouse brain, by correlating the influx of 45Ca2+ with the release of the transmitters. It was observed that exposure of synaptosomes to a Na+-free medium notably increases Ca2+ entry, and this condition was used, in addition to K+ depolarization and the Ca2+ ionophore A23187, to stimulate the influx of Ca2+ and the release of labeled GABA and ACh. The effect of ruthenium red (RuR) on these parameters was also investigated. Of the three experimental conditions used, the absence of Na+ in the medium proved to be the most efficient in increasing Ca2+ entry. RuR inhibited by 60-70% the influx of Ca2+ stimulated by K+ depolarization but did not affect its basal influx or its influx stimulated by the absence of Na+ or by A23187. The release of ACh was stimulated by K+ depolarization, absence of Na+ in the medium, and A23187 in a strictly Ca2+-dependent manner, whereas the release of GABA was only partially dependent on the presence of Ca2+ in the medium. The extent of stimulation of ACh release was related to the extent of Ca2+ entry, whereas no such correlation was observed for GABA. In the presence of Na+, RuR did not affect the release of the transmitters induced by A23187. In the absence of Na+, paradoxically RuR notably enhanced the release of both ACh and GABA induced by A23187, in a Ca2+-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of Calcium Channels in Depolarization-Evoked Release of Adenosine from Spinal Cord Synaptosomes

Abstract: The potential involvement of L- and N-type voltage-sensitive calcium (Ca²⁺) channels and a voltage-independent receptor-operated Ca²⁺ channel in the release of adenosine from dorsal spinal cord synaptosomes induced by depolarization with K⁺ and capsaicin was examined. Bay K 8644 (10 n M ) augmented release of adenosine in the presence of a partial depolarization with K⁺ (addition of 6 m M ) but not capsaicin (1 and 10 μ M ). This augmentation was dose dependent from 1 to 10 n M and was followed by inhibition of release from 30 to 100 n M . Nifedipine and nitrendipine inhibited the augmenting effect of Bay K 8644 in a dose-dependent manner, but neither antagonist had any effect on release of adenosine produced by K⁺ (24 m M ) or capsaicin (1 and 10 μ M ) ω-Conotoxin inhibited K⁺-evoked release of adenosine in a dose-dependent manner but had no effect on capsaicin-evoked release. Ruthenium red blocked capsaicin-induced release of adenosine but had no effect on K⁺-evoked release. Although L-type voltage-sensitive Ca²⁺ channels can modulate release of adenosine when synaptosomes are partially depolarized with K⁺, N-type voltage-sensitive Ca²⁺ channels are primarily involved in K⁺-evoked release of adenosine. Capsaicin-evoked release of adenosine does not involve either L- or N-type Ca²⁺ channels, but is dependent on a mechanism that is sensitive to ruthenium red.     

Effect of α-Latrotoxin on Acetylcholine Release and Intracellular Ca2+ Concentration in Synaptosomes: Na+-Dependent and Na+-Independent Components

Abstract: We studied the effect of α-latrotoxin (αLTX) on [¹⁴C]acetylcholine ([¹⁴C]ACh) release, intracellular Ca²⁺ concentration ([Ca²⁺]_i), plasma membrane potential, and high-affinity choline uptake of synaptosomes isolated from guinea pig cortex. αLTX (10^?10-10^?8M) caused an elevation of the [Ca²⁺]_i as detected by Fura 2 fluorescence and evoked [¹⁴C]ACh efflux. Two components in the action of the toxin were distinguished: one that required the presence of Na⁺ in the external medium and another that did not. Displacement of Na⁺ by sucrose or N-methylglucamine in the medium considerably decreased the elevation of [Ca²⁺]_i and [¹⁴C]ACh release by αLTX. The Na⁺-dependent component of the αLTX action was obvious in the inhibition of the high-affinity choline uptake of synaptosomes. Some of the toxin action on both [Ca²⁺]_i and [¹⁴C]ACh release remained in the absence of Na⁺. Both the Na⁺-dependent and the Na⁺-independent components of the αLTX-evoked [¹⁴C]ACh release partly required the presence of either Mg²⁺ or Ca²⁺. The nonneurotransmitter [¹⁴C]choline was released along with [¹⁴C]ACh, but this release did not depend on the presence of either Na⁺ or Ca²⁺, indicating nonspecific leakage through the plasma membrane. We conclude that there are two factors in the release of ACh from synaptosomes caused by the toxin: (1) cation-dependent ACh release, which is related to (a) Na⁺-dependent divalent cation entry and (b) Na⁺-independent divalent cation entry, and (2) nonspecific Na⁺- and divalent cation-independent leakage.     

Hydrogen Peroxide Induces a Long-Lasting Inhibition of the Ca2+-Dependent Glutamate Release in Cerebrocortical Synaptosomes Without Interfering with Cytosolic Ca2+

Abstract: We studied the action of H₂O₂ on the exocytosis of glutamate by cerebrocortical synaptosomes. The treatment of synaptosomes with H₂O₂ (50–150 µ M ) for a few minutes results in a long-lasting depression of the Ca²⁺-dependent exocytosis of glutamate, induced by KCl or by the K⁺-channel inhibitor 4-aminopyridine. The energy state of synaptosomes, as judged by the level of phosphocreatine and the ATP/ADP ratio, was not affected by H₂O₂, although a transient decrease was observed after the treatment. H₂O₂ did not promote peroxidation, as judged by the formation of malondialdehyde. In indo-1-loaded synaptosomes, the treatment with H₂O₂ did not modify significantly the KCl-induced increase of [Ca²⁺]_i. H₂O₂ inhibited exocytosis also when the latter was induced by increasing [Ca²⁺]_i with the Ca²⁺ ionophore ionomycin. The effects of H₂O₂ were unchanged in the presence of superoxide dismutase and the presence of the Fe³⁺ chelator deferoxamine. These results appear to indicate that H₂O₂, apparently without damaging the synaptosomes, induces a long-lasting inhibition of the exocytosis of glutamate by acting directly on the exocytotic process.     

Acetylcholine Incorporation by Cholinergic Synaptic Vesicles from Torpedo marmorata

[3H]Acetylcholine efflux and Na+-K+ ATPase ion pump activity were measured concomitantly in rat cortical synaptosomes. Ouabain (500 microM), strophanthidin (500 microM), and parachloromercuribenzene sulfonate (500 microM) each inhibited ouabain-sensitive 86Rb uptake and elevated [3H]acetylcholine release independently of the external calcium concentration. Veratridine (10 microM), electrical field stimulation (60 V, 60 Hz, 5-ms pulse duration), or the calcium ionophore A23187 (10 micrograms/ml) also inhibited ouabain-sensitive 86Rb uptake and released [3H]acetylcholine, but via a calcium-dependent process. Veratridine-induced [3H]acetylcholine release and ion pump inhibition were correlated over a wide range of drug concentrations and both effects were blocked by pre-treatment with tetrodotoxin (1 microM). The rate of [3H]acetylcholine efflux from superfused synaptosomes was increased within 15 s of exposure to ouabain, strophanthidin, veratridine, A23187, or field stimulation, while ouabain-sensitive 86Rb uptake was significantly decreased within a similar interval. These results suggest that [3H]acetylcholine release is due at least in part to inhibition of Na+-K+ ATPase.     

Effect of Liposomes Containing Various Divalent Cations on the Release of Acetylcholine from Synaptosomes

Abstract: The effect of increasing the cytoplasmic levels of various divalent cations on the release of [³H]acetylcholine ([³H]ACh) from synaptosomes was investigated. Synaptosomes prepared from rat brain and prelabeled with [³H]choline were incubated with liposomes containing Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺, Sr²⁺, or Ba²⁺. This treatment allows the transfer of the aqueous contents of the liposomes to the cytoplasm of the synaptosomes. The efflux of radioactivity subsequent to this treatment was measured, and the relative proportions of [³H]ACh and [³H]choline were determined. The release of radioactivity from synaptosomes incubated with liposomes containing Mg²⁺, Mn²⁺, or Co²⁺ was not altered when compared with synaptosomes incubated either without liposomes or with liposomes containing isotonic K⁺/Na⁺. Synaptosomes incubated with liposomes containing Ca²⁺, Sr²⁺, or Ba²⁺, however, released significantly more radioactivity than did controls. Moreover, the released radioactivity consisted almost entirely of [³H]ACh. Liposomes containing either Ca²⁺ or Sr²⁺ were equally effective in promoting the release of [³H]ACh from synaptosomes, whereas liposomes containing Ba²⁺ were 2.5 times more effective in promoting the release of [³H]ACh than were liposomes containing either Ca²⁺ or Sr²⁺. Since liposomes introduce their aqueous contents into cytoplasm via a mechanism not involving plasma membrane channels, the increased release of [³H]ACh caused by liposomes containing Ca²⁺, Sr²⁺, or Ba²⁺ is attributable to an increase in the intrasynaptosomal concentration of these ions, and not to their passage through calcium channels.     

Distinct Muscarinic Receptor Subtypes Differentially Modulate Acetylcholine Release from Corticocerebral Synaptosomes

The effect of McN-A-343 and oxotremorine on acetylcholine (ACh) release and choline (Ch) transport was studied in corticocerebral synaptosomes of the guinea pig. The synaptosomes were preloaded with [3H]Ch after treatment with the irreversible cholinesterase inhibitor, diisopropyl fluorophosphate, and then tested for their ability to release isotope-labeled ACh and Ch in the presence and absence of these agents. The kinetics of release were determined at the resting state (basal release) and in the presence of 50 mM K+. Under either condition, McN-A-343 enhanced the release of isotope-labeled ACh, whereas oxotremorine inhibited the K(+)-evoked release but had no effect on the basal release. The enhancing effect of McN-A-343 on basal ACh release was fully blocked by the selective M1 muscarinic antagonist, pirenzepine (100 nM). In contrast to its enhancing effect on ACh release, McN-A-343 potently inhibited Ch efflux as well as Ch influx. These effects were not blocked by atropine, a nonselective muscarinic antagonist. Oxotremorine had no effect on Ch transport. Binding studies showed that McN-A-343 was 3.6-fold more potent in displacing radiolabeled quinuclidinyl benzilate from cerebral cortex muscarinic receptors (mostly M1 subtype) than from cerebellar receptors (mostly M2 subtype), whereas oxotremorine was 2.6-fold more potent in the cerebellum. The displacements of radio-labeled pirenzepine and cis-dioxolane confirmed the M1 subtype preference of McN-A-343 and the M2 subtype preference of oxotremorine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aging Decreases the Sensitivity of Rat Cortical Synaptosomes to Calcium Ionophore-Induced Acetylcholine Release

The capacity of calcium ions to trigger acetylcholine release was studied in cerebral cortical synaptosomes from adult (6-month-old) and senescent (24-month-old) rats, using a calcium ionophore, A23187, that bypasses voltage-sensitive calcium channels. The potency but not the efficacy of the A23187 was reduced with respect to releasing acetylcholine (ACh) in the aged animals. There was no age-related difference in the synthesis of ACh or potency of the ionophore with respect to increasing 45calcium uptake. These results suggest that aging reduces the sensitivity of cerebral cortical nerve terminals to calcium-triggered ACh-release.     

Ca2+-Surrogate Action of Pb2+ on Acetylcholine Release from Rat Brain Synaptosomes

The effect of lead ions on the release of acetylcholine (ACh) was investigated in intact and digitonin-permeabilized rat cerebrocortical synaptosomes that had been prelabeled with [3H]choline. Release of ACh was inferred from the release of total 3H label or by determination of [3H]ACh. Application of 1 microM Pb2+ to intact synaptosomes in Ca2(+)-deficient medium induced 3H release, which was enhanced by K+ depolarization. This suggests that entry of Pb2+ into synaptosomes and Pb2(+)-induced ACh release can be augmented by activation of the voltage-gated Ca2+ channels in nerve terminals. The lead-induced release of [3H]ACh was blocked by treatment of synaptosomes with vesamicol, which prevents uptake of ACh into synaptic vesicles without affecting its synthesis in the synaptoplasm. This indicates that Pb2+ selectively activates the release of a vesicular fraction of the transmitter with little or no effect on the leakage of cytoplasmic ACh. Application of 1-50 nM (EC50 congruent to 4 nM) free Pb2+ to digitonin-permeabilized synaptosomes elicited release of 3H label that was comparable with the release induced by 0.2-5 microM (EC50 congruent to 0.5 microM) free Ca2+. This suggests that Pb2+ triggers transmitter exocytosis directly and that it is a some 100 times more effective activator of exocytosis than is the natural agonist Ca2+.     

Evidence that Somatostatin Enhances Endogenous Acetylcholine Release in the Rat Hippocampus

The present experiments show that somatostatin (SS)-like immunoreactive material is present in the hippocampus and that its release can be increased by K+ stimulation of rat hippocampal slices, suggesting that SS-like peptides may be of significance to neurotransmission in the hippocampus. Exogenous SS-28 and SS-14 enhanced the K(+)-evoked release of endogenous acetylcholine (ACh) from rat hippocampal slices, whereas amino-terminal fragments of SS-28 did not. The increased ACh release in the presence of either peptide appeared to be mediated by an interaction with SS receptors because cyclo-SS, a putative SS antagonist, abolished the effects of both SS-28 and SS-14. In addition, the increase in ACh release induced by SS-14 or SS-28 was antagonized by the calcium channel antagonists omega-conotoxin GVIA, nifedipine, and cinnarizine, implicating voltage-sensitive calcium channels in this effect. Moreover, the effect was sensitive to tetrodotoxin, suggesting an indirect action of the peptides at a site distal to cholinergic nerve terminals. Cysteamine, which has been reported to deplete SS content and to increase SS release in brain, augmented the basal and evoked release of ACh from hippocampal slices, without affecting SS-like content and release. Finally, neuropeptide Y, which is colocalized with SS in many neurons of the hippocampal formation, did not alter ACh release, nor did it facilitate the SS-induced increase. The results suggest that in the rat hippocampus, both SS-28 and SS-14 interact with SS receptors to regulate ACh release indirectly by a mechanism that involves alterations of calcium influx during depolarization.     

Release of Acetylcholine from Rat Brain Synaptosomes Stimulated with Leptinotarsin, A New Neurotoxin

Abstract: Leptinotarsin is a neurotoxic protein found in the hemolymph of potato beetles of the genus Leptinotarsa. In order to study the action of leptinotarsin from two species, L. haldemani and L. decemlineata , synaptosomes were prelabeled with [³H]choline in order to synthesize [³H] acetylcholine (ACh). These synaptosomes were then immobilized on Millipore filters and used for assay. Toxins from both species induce the release of radioactivity in this system. Fractionation of the released radioactivity indicated that ACh was released in preference to choline. The toxin that caused release was heat-labile and was partially dependent on Ca²⁺ in the perfusing medium. Release followed apparent first order kinetics when stimulation was effected with leptinotarsin from L. haldemani (leptinotarsin-h), but was more complex when using leptinotarsin from L. decemlineata (leptinotarsin-d). Increasing the concentration of toxin increased the rate of release, but the shapes of the dose-release curves elicited by the leptinotarsins from the two species were different. While leptinotarsin-h exhibited a simple, saturating dose-release curve, leptinotarsin-d was characterized by a sigmoid function, which was well described, with a Hill coefficient of 1.8. Antibodies directed toward black widow spider venom glands had no effect upon the releasing activity of leptinotarsin-h but could partially neutralize that of leptinotarsin-d. Toxins from both species have been partially purified and do not appear to be identical. The purified toxins should be useful tools with which to study the release of acetylcholine.     

Effect of N,N-Dicyclohexylcarbodiimide on Acetylcholine Release from Torpedo Synaptosomes and Proteoliposomes Reconstituted with the Proteolipid Mediatophore

The mediatophore is a presynaptic membrane protein that has been shown to translocate acetylcholine (ACh) under calcium stimulation when reconstituted into artificial membranes. The mediatophore subunit, a 15-kDa proteolipid, presents a very high sequence homology with the N,N'-dicyclohexylcarbodiimide (DCCD)-binding proteolipid subunit of the vacuolar-type H(+)-ATPase. This prompted us to study the effect of DCCD, a potent blocker of proton translocation, on calcium-dependent ACh release. The present work shows that DCCD has no effect on ACh translocation either from Torpedo synaptosomes or from proteoliposomes reconstituted with purified mediatophore. However, using [14C]DCCD, we were able to demonstrate that the drug does bind to the 15-kDa proteolipid subunit of the mediatophore. These results suggest that although the 15-kDa proteolipid subunits of the mediatophore and the vacuolar H(+)-ATPase may be identical, different domains of these proteins are involved in proton translocation and calcium-dependent ACh release and that the two proteins have a different membrane organization.     

Calcium Buffering and Free Ca2+ in Rat Brain Synaptosomes

Abstract: The effects of K⁺ depolarization and of stimulation by veratridine on apparent cytosolic free Ca²⁺ ([Ca²⁺]_cyt) and net Ca²⁺ accumulation were measured in isolated rat brain presynaptic nerve terminals (synaptosomes). [Ca²⁺]_cyt was determined with fura-2, and Ca²⁺ accumulation was measured with tracer ⁴⁵Ca. [Ca²⁺]_cyt was ～ 325 nM in synaptosomes incubated in the normal physiological salt solution under resting conditions. When [K⁺]₀, was increased from the normal 5 mM to 30 or 50 mM, ⁴⁵Ca uptake and [Ca²⁺]_cyt both increased within 1 s. Both increases were directly related to [Ca²⁺]₀ for [Ca²⁺]₀= 0.02–1.2 mM; however, the increase in ⁴⁵Ca uptake greatly exceeded the increase in [Ca²⁺]_cyt. With small Ca²⁺ loads ≤100 μmol/L of cell water, equivalent to the Ca²⁺ entry during a train of ≤60 impulses), the ⁴⁵Ca uptake exceeded the increase in [Ca²⁺]_cyt by a factor of nearly 1,000. This indicates that ～99.9% of the entering Ca²⁺ was buffered and/or sequestered within ～ 1 s. With larger Ca²⁺ loads, a larger fraction of the entering Ca²⁺ was buffered; ～99.97% of the load was buffered with loads of 250–425 μmol/L of cell water. The ratio between the total Ca²⁺ entry and the increase in [Ca²⁺]_cyt, the “calcium buffer ratio”β, was therefore ～ 3,500:1. This ratio was somewhat lower than the ratio of total intraterminal calcium: [Ca²⁺]_cyt, which ranged between ～7,300:1 and 12,800:1. When the synaptosomes were activated with 10 μM veratridine ([Ca²⁺]₀= 0.2–0.6 mM), ⁴⁵Ca influx and [Ca²⁺]_cyt increased progressively for ～10 s (β= 2,700:13,050:1) and then leveled off. Application of 10 μM tetrodotoxin before the introduction of veratridine prevented the increases in ⁴⁵Ca influx and [Ca²⁺]_cyt. Application of 10 μM tetrodotoxin after 5–10 s of exposure to veratridine caused both the [Ca²⁺]_cyt and the veratridine-stimulated ⁴⁵Ca within the terminals to decline, thereby demonstrating that the Ca²⁺ loading is reversible in the presence of extracellular Ca²⁺. These data show that synaptosomes are capable of buffering and metabolizing Ca²⁺ in a manner expected for intact neurons.     

Opiates Inhibit Acetylcholine Release from Torpedo Nerve Terminals by Blocking Ca2+ Influx

Abstract: In the present communication we report that Ca²⁺-dependent acetylcholine release from K⁺-depolarized Torpedo electric organ synaptosomes is inhibited by morphine, and that this effect is blocked by the opiate antagonist naloxone. This finding suggests that the purely cholinergic Torpedo electric organ neurons contain pre-synaptic opiate receptors whose activation inhibits acetylcholine release. The mechanisms underlying this opiate inhibition were investigated by comparing the effects of morphine on acetylcholine release induced by K⁺ depolarization and by the Ca²⁺ ionophore A23187 and by examining the effect of morphine on ⁴⁵Ca²⁺ influx into Torpedo nerve terminals. These experiments revealed that morphine inhibits ⁴⁵Ca²⁺ influx into K⁺-depolarized Torpedo synaptosomes and that this effect is blocked by naloxone. The effects of morphine on K⁺ depolarization-mediated ⁴⁵Ca²⁺ influx and on acetylcholine release have similar dose dependencies (half-maximal inhibition at 0.5–1 μ M ), suggesting that opiate inhibition of release is due to blockage of the presynaptic voltage-dependent Ca²⁺ channel. This conclusion is supported by the finding that morphine does not inhibit acetylcholine release when the Ca²⁺ channel is bypassed by introducing Ca²⁺ into the Torpedo nerve terminals via the Ca²⁺ ionophore.