Induced acetylcholine release from active purely cholinergic Torpedo synaptosomes.

Viablse, purely cholinergic synaptosomes were prepared from the electric organ of Torpedo ocellata and partially purified by differential and sucrose density centrifugation. The synaptosomes contain acetylcholine (ACh), synaptic vesicles, cytoplasmic markers and mitochondria. No adherent postsynaptic membranes were detected. K⁺ depolarization as well as the ionophore A23187 mediate Ca²⁺ permeation into the synaptosomes and the consequent release of ACh. Mg²⁺ does not evoke ACh release whereas Sr²⁺ and Ba²⁺ can replace Ca²⁺ in evoking K⁺ depolarization induced ACh secretion. In accordance with the calcium hypothesis of stimulus–secretion coupling, both K⁺ depolarization and the ionophore A23187 seem to mediate the release of the same population of ACh molecules. The mode of action of the ionophore X537A differs from that of A23187. X537A acts independently of Ca²⁺ and induces the release of a larger fraction of the ACh contained in the fractionated nerve terminals. These results demonstrate that the Torpedo synaptosomes contain the neurosecretion apparatus in a functional active state. This preparation extends the utility of synaptosomes for structural and functional biochemical studies of neurotransmission, as it uniquely contains only one neurosecretion system (cholinergic).     

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.     

Efflux of γ-aminobutyric acid from and appearance of free arachidonic acid inside synaptosomes

When synaptosomes were depolarized in the presence of Ca²⁺, or when Ca²⁺ was added to synaptosomes pretreated with Ca²⁺ ionophore (A23187), free arachidonic acid was clearly increased within synaptosomes, and at the same time an efflux of γ-aminobutyric acid from synaptosomes was observed. Moreover, when synaptosomes labelled with [¹⁴C]arachidonic acid were depolarized in the presence of Ca²⁺, there was a significant decrease in the radioactivity of the fatty acid of phosphatidylinositol and phosphatidylcholine. Exogenously added arachidonic acid, but not other fatty acids, stimulated the efflux of γ-aminobutyric acid in the absence of Ca²⁺. These observations suggest that the release of arachidonic acid from phospholipids is an intrinsic part of the biochemical mechanism that modulates the γ-aminobutyric acid efflux.     

Fangchinoline inhibits glutamate release from rat cerebral cortex nerve terminals (synaptosomes)

Fangchinoline, an active component of radix stephaniae tetrandrinea, has been shown to possess neuroprotective properties. It has been reported that excessive glutamate release has been proposed to be involved in the pathogenesis of several neurological diseases. The primary purpose of the present study was to investigate the effect of fangchinoline on glutamate release in rat cerebral cortex nerve terminals and to explore the possible mechanism. Fangchinoline inhibited the release of glutamate evoked by 4-aminopyridine (4-AP) in a concentration-dependent manner, and this phenomenon resulted from a reduction of vesicular exocytosis but not from an inhibition of Ca²⁺-independent efflux via glutamate transporter. Fangchinoline did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization, but significantly reduced depolarization-induced increase in [Ca²⁺]_C. Fangchinoline-mediated inhibition of glutamate release was significantly prevented by the N- and P/Q-type Ca²⁺ channel blocker ω-conotoxin MVIIC, and by the PKC inhibitors, GF109203X and Ro318220. In addition, the glutamate release mediated by direct Ca²⁺ entry with Ca²⁺ ionophore (ionomycin) was unaffected by fangchinoline, which suggests that the inhibitory effect of fangchinoline is not due to directly interfering with the release process at some point subsequent to Ca²⁺ influx. These results suggest that fangchinoline inhibits glutamate release from the rat cortical synaptosomes through the suppression of voltage-dependent Ca²⁺ channel activity and subsequent reduces Ca²⁺ entry into nerve terminals, rather than any upstream effect on nerve terminal excitability. This inhibition appears to involve the suppression of PKC signal transduction pathway. This finding may explain the neuroprotective effects of fangchinoline against neurotoxicity.     

Inhibition by trimethyl-tin,probenecid and phenylglyoxal of depolarization-induced acetylcholine release in Torpedo synaptosomes

The effects of trimethyl-tin (anion-hydroxyde ionophore, inhibiting oxydative phosphorylation and H⁺-ATPase) probenecid (inhibitor of anion transport in neural cells) and phenylglyoxal (arginine-specific reagent, inhibiting chloride exchanges in erythrocytes) were examined in Torpedo synaptosomes prepared from electric organ. All drugs significantly reduced the stimulated release of acetylcholine triggered by depolarization of nerve endings with high-K⁺ and/or gramicidin D. In contrast, trimethyl-tin, probenecid and phenylglyoxal did not affect the ionophore A23187-induced release of acetylcholine from the synaptosomes. The inhibitory potency of the compound trimethyl-tin was found to be similar to that of probenecid and phenylglyoxal on depolarization-induced acetylcholine release. This leads us to suggest that a relationship exists between modification of anion distribution during depolarization and acetylcholine release process. Moreover, since the release of ACh by calcium-ionophore A23187 was unaffected by trimethyl-tin, probenecid or phenylglyoxal, such compounds may also have an action on voltage-dependent Ca²⁺ flux across presynaptic membrane.     

Perspectives in S-100 protein biology: Review article

The S-100 protein family constitutes a subgroup of Ca²⁺-binding proteins of the EF-hand type comprising three dimeric isoforms, S-100a₀, S-100a and S-100b, plus a number of structurally related proteins displaying 28–55% homology with S-100 subunits. S-100 protein was discovered in 1965; yet, its biological functions have not been fully elucidated. The present report will review the putative biological roles of S-100 protein. Both intracellular and extracellular roles have been proposed for S-100 protein. Within cells, S-100 protein has been reported to regulate protein phosphorylation, ATPase, adenylate cyclase, and aldolase activities and Ca²⁺-induced Ca²⁺ release. Also, cytoskeletal systems, namely microtubules and microfilaments have been reported to be regulated by the protein in the presence of Ca²⁺. Some molecular targets of S-100 protein within cells, have been identified. This is the case with microtubule proteins, caldesmon, and a brain aldolase. S-100 protein has been reported to be secreted; extracellular S-100 protein can stimulate neuronal differentiation, glial proliferation, and prolactin secretion. However, the mechanisms by which S-100 is secreted and stimulates the above processes are largely unknown. Future research should characterize these latter aspects of S-100 biology and find out the linkage between its intracellular effects and its extracellular activities.     

The release of dopamine from synaptosomes from rat striatum by the ionophores X 537A and A 23187

The antibiotics X 537A and A 23187 are negatively charged divalent cation ionophores. X 537A may, in addition, be an ionophore for amines including catecholamines. The effects of these ionophores were examined on the uptake and release of dopamine by synaptosomes prepared from rat corpus striatum. Both X 537A and A 23187, at concentrations less than 0.5 μM, release both endogenous and [³H]-dopamine from synaptosomes. They had virtually no effect on the uptake of exogenous dopamine. These compounds act by different mechanisms. X 537A causes divalent ion-independent release in which a large fraction of the effluent consists of deaminated products. X 537A, in addition, releases [³H]dopamine from rat adrenal medullary chromaffin granules. The results suggest that X 537A causes release of dopamine from intrasynaptosomal storage vesicles and perhaps is acting as a catecholamine carrier across the vesicular membrane. A 23187, on the other hand, causes a Ca²⁺-dependent release in which only a small fraction of the catechol in the effluent is deaminated. A 23187 has little effect on the release of [³H]dopamine from chromaffin granules. These results suggest that A 23187 carries Ca²⁺ into the synaptosomes and thereby initiates exocytotic release.     

Persistent Enhancement of Sustained Calcium-Dependent Glutamate Release by Phorbol Esters: Requirement for Localized Calcium Entry

Abstract: Sustained activation of protein kinase C significantly enhanced a secondary (slow) phase in the depolarization-induced release of glutamate from isolated hippocampal nerve endings. The phorbol ester, 4β-phorbol 12,13-dibutyrate, was used to sustain the activation of presynaptic protein kinase C for a prolonged (10-min) period, and then this relatively water-soluble phorbol ester was removed by superfusion before a 2-min stimulus of continuous membrane depolarization. These conditions were used to investigate the persistent effects of sustained protein kinase C activation on the magnitude of the slow phase of evoked glutamate release, in which the efficiency of synaptic vesicle mobilization and recycling may be primary determinants of response magnitude. It is reported here that sustained protein kinase C activation selectively increased the Ca²⁺-dependent component of glutamate release during a prolonged phase of K⁺-induced depolarization. The magnitude of this persistent effect on Ca²⁺-dependent glutamate release was directly related to the dose of 4β-phorbol 12,13-dibutyrate and the duration of exposure that was used to prime the release apparatus, was observed using two alternative synaptosomal preparations, and was evident regardless of the depolarizing stimulus used (elevated [KCl] or 4-aminopyridine). However, 4β-phorbol 12,13-dibutyrate did not alter the release induced by the Ca²⁺ ionophore ionomycin. Thus, the persistent effects of protein kinase C activation on a prolonged phase of glutamate release were dependent on the route of Ca²⁺ influx. The finding that voltage-regulated Ca²⁺ channel blockers were able to neutralize completely the 4β-phorbol 12,13-dibutyrate-dependent facilitation of K⁺-evoked glutamate release provided further support for this conclusion. Thus, 4β-phorbol 12,13-dibutyrate significantly potentiated the sustained release of glutamate without altering the strict requirement that is normally displayed by synaptosomes for localized and voltage-regulated Ca²⁺ entry.     

Carrier-mediated and carrier-independent release of serotonin from isolated central nerve endings

The release of serotonin elicited by Ca²⁺-dependent stimuli (depolarization, ionophore A23187) from rat brain synaptosomes previously labelled with the radioactive indoleamine was not affected by the presence of the serotonin carrier blocker chlorimipramine. In contrast, other releasing stimuli, such as superfusion with a Na⁺-free medium or exposure to various releasing drugs (fenfluramine, p-chloroamphetamine, tryptamine and mianserin, both in normal Krebs-Ringer medium and in low-Na⁺ medium), evoked efflux of serotonin from nerve endings which was prevented by chlorimipramine. The results indicate that serotonin can be released from central nerve endings by two mechanisms, differentially affected by the blockade of the membrane carrier system: the characteristics of the Ca²⁺-dependent release are compatible with an exocytotic mechanism, whereas the release induced by lack of Na⁺ or by phenylethylamines and tryptamine appears to occur by outward transport mediated by the membrane carrier.     

Inhibitory effect of glutamate release from rat cerebrocortical synaptosomes by dextromethorphan and its metabolite 3-hydroxymorphinan

Dextromethorphan (DM), a widely used antitussive, has demonstrated an effective neuroprotective effect. Excessive release of glutamate is considered to be an underlying cause of neuronal damage in several neurological diseases. In the present study, we investigated whether DM or its metabolite 3-hydroxymorphinan (3-HM) could affect glutamate release in rat cerebral cortex nerve terminals (synaptosomes). DM or 3-HM inhibited the Ca²⁺-dependent release of glutamate that was evoked by exposing synaptosomes to the K⁺ channel blocker 4-aminopyridine (4-AP), and this presynaptic inhibition was concentration-dependent. Inhibition of glutamate release by DM or 3-HM was resulted from a reduction of vesicular exocytosis, because the vesicular transporter inhibitor bafilomycin A1 completely blocked DM or 3-HM-mediated inhibition of 4-AP-evoked glutamate release. DM or 3-HM did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization, but significantly reduced depolarization-induced increase in [Ca²⁺]_C. DM or 3-HM-mediated inhibition of 4-AP-evoked glutamate release was blocked by ω-conotoxin MVIIC, an antagonist of N- and P/Q-type Ca²⁺ channel, not by dantrolene, an intracellular Ca²⁺ release inhibitor. DM or 3-HM modulation of 4-AP-evoked glutamate release appeared to involve a protein kinase C (PKC) signaling cascade, insofar as pretreatment of synaptosomes with the PKC inhibitors GF109203X or Ro318220 all effectively occluded the inhibitory effect of DM or 3-HM. Furthermore, 4-AP-induced phosphorylation of PKC was reduced by DM or 3-HM. These results suggest that DM or 3-HM inhibits glutamate release from rat cortical synaptosomes through the suppression of presynaptic voltage-dependent Ca²⁺ entry and PKC activity. This may explain the neuroprotective effects of DM against neurotoxicity.     

Sodium-Dependent Efflux of [3H]GABA from Synaptosomes Probably Related to Mitochondrial Calcium Mobilization

It has been suggested that mitochondria might modify transmitter release through the control of intracellular Ca²⁺levels. Treatments known to inhibit Ca²⁺retention by mitochondria lead to an increased transmitter liberation in the absence of external Ca²⁺, both at the frog neuromuscular junction and from isolated nerve endings. Sodium ions stimulate Ca²⁺efflux from mitochondria isolated from excitable tissues. In the present study, the effect of increasing internal Na⁺ levels on [³H]γ-aminobutyric acid ([³H]GABa) release from isolated nerve endings is reported. Results show that the efflux of [³H]GABA from prelabeled synaptosomes is stimulated by ouabain, veratrine, gramicidin D, and K⁺-free medium, which increase the internal sodium concentration. This effect was not observed when Na⁺ was omitted from the incubation medium and it was independent of external Ca²⁺, the experiments having been performed in a Ca²⁺-free, EGTA-containing medium. Since preincubation of synaptosomes with 2,4-diaminobutyric acid did not prevent the stimulatory effect of increased internal Na⁺ levels on [³H]GABA efflux, it appears to be unrelated to an enhanced activity of the outward carrier-mediated GABA transport. These results suggest that the augmented release of [³H]GABA may be due to an increased Ca²⁺efflux from mitochondria eiicited by the accumulation of Na⁺ at the nerve endings. Sandoval M. E. Sodium-dependent efflux of [³H]GABA from synaptosomes probably related to mitochondrial calcium mobilization. J. Neurochem. 35 , 915–921 (1980).     

A rapid separation of bovine brain S-100a and S-100b proteins and related conformation studies

S-100 protein absorbs to the calmodulin antagonist W-7 coupled to epoxy-activated Sepharose 6B in the presence of Ca²⁺ and is eluted by ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid buffer. S-100a and S-100b were separated and isolated by Ca²⁺-dependent affinity chromatography on W-7 Sepharose. The Ca²⁺-induced conformational changes of S-100a and S-100b were examined using circular dichroism, ultraviolet difference spectra, and a fluorescence probe. Differences in Ca²⁺-dependent conformational changes between S-100a and S-100b became apparent. Circular dichroism studies revealed that both S-100a and S-100b undergo a conformational change upon binding of Ca²⁺ in the aromatic and far-uv range. In the presence or absence of Ca²⁺, the aromatic CD spectrum of S-100a differed completely from that of S-100b, possibly due to the single tryptophan residue of S-100a. Far-uv studies indicate that α-helical contents of both S-100a and S-100b decreased with addition of Ca²⁺. Ca²⁺-induced conformational changes of S-100a and S-100b were also detected by uv difference spectra. The spectrum of S-100a also differed from that of S-100b. Fluorescence studies using 2-p-toluidinylnaphthalene-6-sulfonate (TNS), a hydrophobic probe for protein, revealed a slight difference in conformational changes of these two components. The interaction of TNS and S-100b was observed with concentrations above 3 μm Ca²⁺; on the other hand, S-100a required concentrations above 8 μm. This finding was supported by the difference in the binding affinities of S-100a and S-100b to the W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide)-Sepharose column; both S-100a and S-100b bound the column in the presence of Ca²⁺ but S-100a was eluted prior to S-100b. These results suggest that S-100a and S-100b differ in their dependence on Ca²⁺ and that the affinity-chromatographic separation of S-100a from S-100b on the W-7-Sepharose column makes feasible a rapid purification of these two components.     

NO synthase and xanthine oxidase activities of rabbit brain synaptosomes: Peroxynitrite formation as a causative factor of neurotoxicity

In the present study we demonstrated that synaptosomes isolated from rabbit brain cortex contain NO synthase and xanthine oxidase that can be activated by ultraviolet B radiation and Ca²⁺ accumulation to produce nitric oxide and superoxide which react together to form peroxynitrite. Irradiation of synaptosomes with ultraviolet B (up to 100 mJ/cm²), or increase the intrasynaptosomal calcium concentration using various doses (up to 100 μM) of the calcium ionophore A 23187, a gradual increase in both nitric oxide and peroxynitrite release that was inhibited by N-monomethyl-L-arginine (100 μM) was observed. The rate of nitric oxide release and cyclic GMP production by NO synthase and soluble guanylate cyclase, both located in the soluble fraction of synaptosomes (synaptosol), were increased approximately eight fold after treatment of synaptosomes with Ultraviolet B radiation (100 mJ/cm²). In reconstitution experiments, when purified NO synthase isolated from synaptosol was added to xanthine oxidase, in the presence of the appropriate cofactors and substrates, a ten fold increase in peroxynitrite production at various doses (up to 20 mJ/cm²) of UVB radiation was observed. Ultraviolet B irradiated synaptosomes promptly increased malondialdehyde production with subsequent decrease of synaptosomal plasma membrane fluidity estimated by fluorescence anisotropy of 1-4-(trimethyl-amino-phenyl)-6-phenyl-hexa-1,3,5-triene. Desferrioxamine (100 μM) tested in Ultraviolet B-irradiated synaptosomes showed a decrease (approximately 80%) in malondialdehyde production with subsequent restoration of the membrane fluidity to that of non-irradiated (control) synaptosomes. Ca²⁺-stimulated ATPase activity was decreased after Ultraviolet B (100 mJ/cm²) radiation of synaptosomes indicating that the subsequent increase of intrasynaptosomal calcium promoted peroxynitrite production by a calmodulin-dependent increase of NO synthase and xanthine oxidase activities. Furthermore, it was shown that UVB-irradiated synaptosomes were subjected to higher oxidative stress by exogenous peroxynitrite (100 μM) compared to non-irradiated (control) synaptosomes. In summary, the present results indicate that activation of NO synthase and xanthine oxidase of brain cells lead to the formation of peroxynitrite providing important clues in the role of peroxynitrite as a causative factor in neurotoxicity.     

Acute Restraint Stress Enhances Calcium Mobilization and Glutamate Exocytosis in Cerebrocortical Synaptosomes from Mice

Acute stress is known to enhance the memory of events that are potentially threatening to the organisms. Glutamate, the most abundant excitatory neurotransmitter in the mammalian central nervous system, plays a critical role in learning and memory formation and calcium (Ca²⁺) plays an essential role in transmitter release from nerve terminals (synaptosomes). In the present study, we investigated the effects of acute restraint stress on cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and glutamate release in cerebrocortical synaptosomes from mice. Acute restraint stress caused a significant increase in resting [Ca²⁺]_i and significantly enhanced the ability of the depolarizing agents K⁺ and 4-aminopyridine (4-AP) to increase [Ca²⁺]_i. It also brought about a significant increase in spontaneous (unstimulated) glutamate release and significantly enhanced K⁺- and 4-AP-induced Ca²⁺-dependent glutamate release. The pretreatment of synaptosomes with a combination of ω-agatoxin IVA (a P-type Ca²⁺ channel blocker) and ω-conotoxin GVIA (an N-type Ca²⁺ channel blocker) completely suppressed the enhancements of [Ca²⁺]_i and Ca²⁺-dependent glutamate release in acute restraint-stressed mice. These results indicate that acute restraint stress enhances K⁺- or 4-AP-induced glutamate release by increasing [Ca²⁺]_i via stimulation of Ca²⁺ entry through P- and N-type Ca²⁺ channels.     

Studies on the Role of B-50 (GAP-43) in the Mechanism of Ca2+-Induced Noradrenaline Release: Lack of Involvement of Protein Kinase C After the Ca2+ Trigger

Abstract: The involvement of B-50, protein kinase C (PKC), and PKC-mediated B-50 phosphorylation in the mechanism of Ca²⁺-induced noradrenaline (NA) release was studied in highly purified rat cerebrocortical synaptosomes permeated with streptolysin-O. Under optimal permeation conditions, 12% of the total NA content (8.9 pmol of NA/mg of synaptosomal protein) was released in a largely (>60%) ATP-dependent manner as a result of an elevation of the free Ca²⁺ concentration from 10^?8 to 10^?5M Ca²⁺ The Ca²⁺ sensitivity in the micromolar range is identical for [³H]NA and endogenous NA release, indicating that Ca²⁺-induced [³H]NA release originates from vesicular pools in noradrenergic synaptosomes. Ca²⁺-induced NA release was inhibited by either N- or C-terminal-directed anti-B-50 antibodies, confirming a role of B-50 in the process of exocytosis. In addition, both anti-B-50 antibodies inhibited PKC-mediated B-50 phosphorylation with a similar difference in inhibitory potency as observed for NA release. However, in a number of experiments, evidence was obtained challenging a direct role of PKC and PKC-mediated B-50 phosphorylation in Ca²⁺-induced NA release. PKC pseudosubstrate PKC_19-36, which inhibited B-50 phosphorylation (IC₅₀ value, 10^?5M), failed to inhibit Ca²⁺-induced NA release, even when added before the Ca²⁺ trigger. Similar results were obtained with PKC inhibitor H-7, whereas polymyxin B inhibited B-50 phosphorylation as well as Ca²⁺-induced NA release. Concerning the Ca²⁺ sensitivity, we demonstrate that PKC-mediated B-50 phosphorylation is initiated at a slightly higher Ca²⁺ concentration than NA release. Moreover, phorbol ester-induced PKC down-regulation was not paralleled by a decrease in Ca²⁺-induced NA release from streptolysin-O-permeated synaptosomes. Finally, the Ca²⁺- and phorbol ester-induced NA release was found to be additive, suggesting that they stimulate release through different mechanisms. In summary, we show that B-50 is involved in Ca²⁺-induced NA release from streptolysin-O-permeated synaptosomes. Evidence is presented challenging a role of PKC-mediated B-50 phosphorylation in the mechanism of NA exocytosis after Ca²⁺ influx. An involvement of PKC or PKC-mediated B-50 phosphorylation before the Ca²⁺ trigger is not ruled out. We suggest that the degree of B-50 phosphorylation, rather than its phosphorylation after PKC activation itself, is important in the molecular cascade after the Ca²⁺ influx resulting in exocytosis of NA.     

Synaptosomes and synaptosome membrane vesicles from the brain of Mamestra configurata: Application to voltage-dependent and ATP-dependent Ca2+ ion transport studies

High yields of relatively pure, morphologically well-preserved, functionally competent synaptosomes were prepared from brains of moths of Mamestra configurata using a modified microscale Ficoll flotation technique. Typical preparations yielded 10 mg of synaptosomal protein per gram of moth brains. The moth brain synaptosomes were virtually free of endoplasmic reticulum and mitochondrial contaminants as judged from marker enzyme studies and electron microscopy.Voltage-dependent Ca²⁺ ion transport was studied using the moth brain synaptosome preparations. Synaptosomes took up radioactive ⁴⁵Ca²⁺ from the incubation medium. The rate of uptake was increased up to three-fold when the synaptosomes were incubated in a depolarizing, high [K⁺] medium. Time course studies indicated that voltage-dependent Ca²⁺ uptake was composed of an early (<2 sec) fast phase and a late (>10 sec) slow phase.ATP-dependent Ca²⁺ ion transport was studied in moth brain synaptosome membrane vesicles prepared from synaptosomes by osmotic shock and purified on a second Ficoll gradient. The inside-out synaptosome membrane vesicles contained an ATP-dependent calcium ion pump which transported ⁴⁵Ca²⁺ from the incuation medium into the interior of the vesicle in the presence of ATP. The calcium ionophore A23187 rapidly released accumulated ⁴⁵Ca²⁺ from the vesicles. The maximal rate of ATP-dependent Ca²⁺ transport occurred at a [Ca²⁺ free] of 0.1 to 0.2 nM, indicating that the transport process has a very high affinity for Ca²⁺ ions.     

Mechanism of interaction of myelin basic protein and S-100 protein: metal binding and fluorescence studies.

Abstract— It has been reported that myelin basic protein (MBP) forms a specific complex with S-100 protein in the presence of either Ca²⁺ or Mn²⁺, as detected by Immunoelectrophoresis. We have now studied the binding of Ca²⁺ and Mn²⁺ to these two proteins. We find that MBP binds 1 mol of Mn²⁺/mol of protein, and this binding produces an increment in its fluorescence, indicating a conformational change. Ca²⁺ does not bind to MBP nor does it affect the fluorescence of MBP. S-100 protein, as has been reported, binds about 10 mol of Ca²⁺/mol and this binding produces a conformational change. S-100 protein also has 25 binding sites for Mn²⁺, but this binding does not alter fluorescence and does not appear to affect conformation. Competitive binding experiments demonstrate that the binding sites of S-100 protein for Ca²⁺ and Mn²⁺ are independent. The alteration of electrophoretic migration in gels of S-100 protein produced by Ca²⁺ and of MBP produced by Mn²⁺ are in accord with the observations based on fluorescence. Mn²⁺ does not affect the electrophoretic mobility of S-100. These results indicate that the formation of the complex between MBP and S-100 protein in the presence of either Ca²⁺ or Mn²⁺ is due to the conformational change induced by these ions in S-100 protein, MBP, or both.     

Concomitant protein phosphorylation and endogenous acetylcholine release induced by nicotine: Dependency on neuronal nicotinic receptors and desensitization

Summary 1. Nicotine stimulated two Ca²⁺-dependent processes in rat frontal cortex synaptosomes: the phosphorylation of an 80-kDa protein band and the release of endogenous ACh.³ Both effects were mediated by neuronal nAChRs and coincided with depolarization of the synaptosomal plasma membrane induced by the drug. Changes in the state of phosphorylation of the 80-kDa band (presumed to contain synapsin I) were correlated with changes in the release of ACh as follows, from 2 to 4.2. Blockade of predominant, nerve terminal P-type Ca²⁺ channels with -agatoxin-IVA, did not prevent nicotine from stimulating ACh release. In contrast, exposure to the toxin partially inhibited the release promoted by the depolarizing agent veratridine and attenuated protein phosphorylation induced by either nicotine or veratridine. Taken together, these data suggest that, upon nicotine stimulation, Ca²⁺ enters nerve terminals through two distinct pathways. The first, via Ca²⁺ channels, is necessary (but not sufficient) for both nicotine-induced phosphorylation and ACh release. The second, both necessary and sufficient for nicotine-induced phosphorylation and release, is the neuronal nAChR itself.3. Preincubation of the synaptosomes with a subeffective concentration of nicotine inactivated both nicotine-induced ACh liberation and phosphorylation. This shows that diminished release is associated to decreased phosphorylation of the 80-kDa protein band, most likely as a consequence of nicotine-promoted nAChR desensitization.4. Augmented ACh release and phosphorylation of the 80-kDa protein band were achieved by using the protein phosphatase inhibitor okadaic acid. However, okadaic acid did not summate with either nicotine or veratridine to increase ACh release further. This is probably because okadaic acid, as in other neurons, increases intracellular Ca²⁺ (Cholewinskiet al., 1993), thus promoting desensitization of ACh release.     

Evidence for a Role of Protein Kinase C Substrate B-50 (GAP-43) in Ca2+-Induced Neuropeptide Cholecystokinin-8 Release from Permeated Synaptosomes

Abstract: To study the involvement of the protein kinase C (PKC) substrate B-50 [also known as growth-associated protein-43 (GAP-43), neuromodulin, and F1] in presynaptic cholecystokinin-8 (CCK-8) release, highly purified synaptosomes from rat cerebral cortex were permeated with the bacterial toxin streptolysin O (SL-O). CCK-8 release from permeated synaptosomes, determined quantitatively by radioimmunoassay, could be induced by Ca²⁺ in a concentration-dependent manner (EC₅₀ of ～10^-5M). Ca²⁺-induced CCK-8 release was maximal at 10⁴M Ca²⁺, amounting to ～10% of the initial 6,000 ± 550 fmol of CCK-8 content/mg of synaptosomal protein. Only 30% of the Ca^a+-induced CCK-8 release was dependent on the presence of exogenously added ATP. Two different monoclonal anti-B-50 antibodies were introduced into permeated synaptosomes to study their effect on Ca²⁺-induced CCK-8 release. The N-terminally directed antibodies (NM2), which inhibited PKC-mediated B-50 phosphorylation, inhibited Ca²⁺-induced CCK-8 release in a dose-dependent manner, whereas the C-terminally directed antibodies (NM6) affected neither B-50 phosphorylation nor CCK-8 release. The PKC inhibitors PKC_19–36 and 1 ?(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), which inhibited B-50 phosphorylation in permeated synaptosomes, had no effect on Ca²⁺-induced CCK-8 release. Our data strongly indicate that B-50 is involved in the mechanism of presynaptic CCK-8 release, at a step downstream of the Ca²⁺ trigger. As CCK-8 is stored in large densecored vesicles, we conclude that B-50 is an essential factor in the exocytosis from this type of neuropeptide-containing vesicle. The differential effects of the monoclonal antibodies indicate that this B-50 property is localized in the N-terminal region of the B-50 molecule, which contains the PKC phosphorylation site and calmodulin-binding domain.     

CALCIUM-DEPENDENT RELEASE OF EXOGENOUSLY LOADED γ-AMINO-[U-14C]BUTYRATE FROM SYNAPTOSOMES: TIME COURSE OF STIMULATION BY POTASSIUM, VERATRIDINE, AND THE CALCIUM IONOPHORE, A23187

—Synaptosomes which had taken up [¹⁴C]GABA were applied to a filter and rapidly perfused with various solutions in order to study the time course of release of this putative transmitter and the characteristics of its release. Depolarization of the synaptosomes with veratridine or 56mM-K⁺ or pretreatment with the Ca²⁺ ionophore, A23187, increased the calcium-dependent efflux of [¹⁴C]GABA. Release of [¹⁴C]GABA was increased by Ca²⁺ within 0.3 s of exposure, and the maximal release rate was not maintained for longer than 0.6 s. The reduction in the rate of release was not attributable to a decrease in calcium influx, but rather appeared to reflect fatigue at some subsequent stage in release. Stimulation by 56mM-K⁺ also elicited a calcium-independent increase in the efflux of radioactive GABA, which appeared to arise in part from subcellular particles other than synaptosomes.