Osthole and imperatorin,the active constituents of Cnidium monnieri (L.) Cusson,facilitate glutamate release from rat hippocampal nerve terminals

We examined the effects of osthole and imperatorin, two active compounds of Cnidium monnieri (L.) Cusson, on the release of glutamate from rat hippocampal synaptosomes and investigated the possible mechanism. The results showed that osthole or imperatorin significantly facilitated 4-aminopridine (4-AP)-evoked glutamate release in a concentration-dependent manner. The facilitatory action of osthole or imperatorin was blocked by the vesicular transporter inhibitor bafilomycin A1, not by the glutamate transporter inhibitor l-transpyrrolidine-2,4-dicarboxylic acid (l-trans-PDC), indicating that the release facilitation by osthole or imperatorin results from a enhancement of vesicular exocytosis and not from an increase of Ca²⁺-independent efflux via glutamate transporter. Examination of the effect of osthole and imperatorin on cytosolic [Ca²⁺] revealed that the facilitation of glutamate release could be attributed to an increase in voltage-dependent Ca²⁺ influx. Consistent with this, ω-conotoxin MVIIC, a wide-spectrum blocker of the N- and P/Q-type Ca²⁺ channels, significantly suppressed the osthole or imperatorin-mediated facilitation of glutamate release, but intracellular Ca²⁺ release inhibitor dantrolene had no effect. Osthole or imperatorin did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization; thus, the facilitation of 4-AP-evoked Ca²⁺ influx and glutamate release produced by osthole or imperatorin was not due to it decreasing synaptosomal excitability. In addition, osthole or imperatorin-mediated inhibition of 4-AP-evoked release was prevented by protein kinase C (PKC) inhibitors. Furthermore, osthole or imperatorin increased 4-AP-induced phosphorylation of PKC. Together, these results suggest that osthole or imperatorin effects a facilitation of glutamate release from nerve terminals by positively modulating N-and P/Q-type Ca²⁺ channel activation through a signaling cascade involving PKC.     

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.     

Facilitatory effect of glutamate exocytosis from rat cerebrocortical nerve terminals by alpha-tocopherol, a major vitamin E component

The effect of alpha-tocopherol, the major vitamin E component, on the release of endogenous glutamate has been investigated using rat cerebrocortical nerve terminals. Results showed that alpha-tocopherol facilitated the Ca2+-dependent but not the Ca2+-independent glutamate release evoked by 4-aminopyridine (4AP). This release facilitation was insensitive to glutamate transporter inhibitor L-trans-PDC or DL-TBOA, and blocked by the exocytotic neurotransmitter release inhibitor tetanus neurotoxin, indicating that alpha-tocopherol affects specifically the physiological exocytotic vesicular release without affecting the non-vesicular release. Facilitation of glutamate exocytosis by alpha-tocopherol was not due to its increasing synaptosomal excitability, because alpha-tocopherol did not alter the 4AP-evoked depolarization of the synaptosomal plasma membrane potential. Rather, examination of the effect of alpha-tocopherol on cytoplasmic free Ca2+ concentration revealed that the facilitation of glutamate release could be attributed to an increase in voltage-dependent Ca2+ influx. Consistent with this, the alpha-tocopherol-mediated facilitation of glutamate release was significantly reduced in synaptosomes pretreated with omega-CgTX MVIIC, a wide spectrum blocker of N- and P/Q-type Ca2+ channels. In addition, alpha-tocopherol modulation of glutamate release appeared to involve a protein kinase C (PKC) signalling cascade, insofar as pretreatment of synaptosomes with the PKC inhibitor GF109203X effectively suppressed the facilitatory effect of alpha-tocopherol on 4AP- or ionomycin-evoked glutamate release. Furthermore, alpha-tocopherol increased the phosphorylation of MARCKS, the major presynapic substrate for PKC, and this effect was also significantly attenuated by PKC inhibition. Together, these results suggest that alpha-tocopherol exerts an increase in PKC activation, which subsequently enhances voltage-dependent Ca2+ influx and vesicular release machinery to cause an increase in evoked glutamate release from rat cerebrocortical glutamatergic terminals. This finding might provide important information regarding to the action of vitamin E in the central nervous system.     

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.     

Berberine Inhibits the Release of Glutamate in Nerve Terminals from Rat Cerebral Cortex

Berberine, an isoquinoline plant alkaloid, protects neurons against neurotoxicity. An excessive release of glutamate is considered to be one of the molecular mechanisms of neuronal damage in several neurological diseases. In this study, we investigated whether berberine could affect endogenous glutamate release in nerve terminals of rat cerebral cortex (synaptosomes) and explored the possible mechanism. Berberine inhibited the release of glutamate evoked by the K⁺ channel blocker 4-aminopyridine (4-AP), and this phenomenon was prevented by the chelating extracellular Ca²⁺ ions and the vesicular transporter inhibitor bafilomycin A1, but was insensitive to the glutamate transporter inhibitor DL-threo-beta-benzyl-oxyaspartate. Inhibition of glutamate release by berberine was not due to it decreasing synaptosomal excitability, because berberine did not alter 4-AP-mediated depolarization. The inhibitory effect of berberine on glutamate release was associated with a reduction in the depolarization-induced increase in cytosolic free Ca²⁺ concentration. Involvement of the Ca_v2.1 (P/Q-type) channels in the berberine action was confirmed by blockade of the berberine-mediated inhibition of glutamate release by the Ca_v2.1 (P/Q-type) channel blocker ω-agatoxin IVA. In addition, the inhibitory effect of berberine on evoked glutamate release was prevented by the mitogen-activated/extracellular signal-regulated kinase kinase (MEK) inhibitors. Berberine decreased the 4-AP-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and synapsin I, the main presynaptic target of ERK; this decrease was also blocked by the MEK inhibition. Moreover, the inhibitory effect of berberine on evoked glutamate release was prevented in nerve terminals from mice lacking synapsin I. Together, these results indicated that berberine inhibits glutamate release from rats cortical synaptosomes, through the suppression of presynaptic Cav2.1 channels and ERK/synapsin I signaling cascade. This finding may provide further understanding of the mode of berberine action in the brain and highlights the therapeutic potential of this compound in the treatment of a wide range of neurological disorders.     

Facilitatory effect of aspirin on glutamate release from rat hippocampal nerve terminals: involvement of protein kinase C pathway

The effect of aspirin on glutamate release from isolated nerve terminals (synaptosomes) from rat hippocampus was examined. The Ca(2+)-dependent release of glutamate evoked by 4-aminopyridine (4AP) was facilitated by aspirin in a concentration-dependent manner, but the 4AP-evoked Ca(2+)-independent release was not modified. Also, aspirin-mediated facilitation of glutamate release was completely inhibited by bafilomycin A1, which depletes vesicle content by inhibiting the synaptic vesicle H(+)-ATPase that drives glutamate uptake, not by l-trans-pyrrolidine-2,4-dicarboxylic acid (l-trans-PDC), a excitatory amino acid (EAA) transporter inhibitor, suggesting that the facilitation of glutamate release produced by aspirin originates from synaptic vesicle exocytosis rather than reversal of the plasma membrane glutamate transporter. In addition, aspirin did not alter either 4AP-evoked depolarization of the synaptosomal plasma membrane potential or Ca(2+) ionophore ionomycin-induced glutamate release, but significantly increased in 4AP-evoked Ca(2+) influx. A possible effect of aspirin on synaptosomal Ca(2+) channels was confirmed in experiments where synaptosomes pretreated with a combination of the N- and P/Q-type Ca(2+) channel blockers, which abolished the aspirin-mediated facilitation of glutamate release. The facilitatory action by aspirin observed in glutamate release was mimicked and occluded by arachidonic acid (AA) and eicosatetraynoic acid (ETYA), an analogue of AA that mimics the effect of AA but cannot be metabolized. Furthermore, this aspirin-mediated facilitation of glutamate release may depend on activation of protein kinase C (PKC), because PKC activator and PKC inhibitor, respectively, superseding or suppressing the facilitatory effect of aspirin. Together, these results suggest that aspirin exerts their presynaptic facilitatory effect, likely through AA directly to induce the activation of PKC, which subsequently enhances the Ca(2+) influx through voltage-dependent N- and P/Q-type Ca(2+) channels to cause an increase in evoked glutamate release from rat hippocampal nerve terminals.     

Presynaptic mechanisms underlying the alpha-lipoic acid facilitation of glutamate exocytosis in rat cerebral cortex nerve terminals

The antioxidant alpha-lipoic acid has been reported to prevent and reverse age-related impairments in learning and memory. However, it is unclear how alpha-lipoic acid improves cognitive function. In this study, the effect of alpha-lipoic acid on the release of endogenous glutamate from rat cerebrocortical nerve terminals (synaptosomes) was examined. We found that alpha-lipoic acid potently facilitated 4-aminopyridine (4AP)-evoked glutamate release, and this release facilitation results from an enhancement of vesicular exocytosis and not from an increase of non-vesicular release. Examination of the effect of alpha-lipoic acid on cytosolic [Ca(2+)] revealed that the facilitation of glutamate release was associated with an increase in voltage-dependent Ca(2+) influx. Consistent with this, alpha-lipoic acid-mediated facilitation of glutamate release was completely prevented in synaptosomes pretreated with a wide spectrum blocker of the N- and P/Q-type Ca(2+) channels, omega-conotoxin MVIIC. The facilitatory effect of alpha-lipoic acid on Ca(2+) influx was not due to an increase of synaptosomal excitability because alpha-lipoic acid did not alter the 4AP-evoked depolarization of the synaptosomal plasma membrane potential. In addition, both ionomycin and hypertonic sucrose-induced glutamate release were enhanced by alpha-lipoic acid. Furthermore, disruption of cytoskeleton organization with cytochalasin D occluded the facilitatory effect of alpha-lipoic acid on 4AP or ionomycin-evoked glutamate release. These results suggest that the antioxidant alpha-lipoic acid enhances the Ca(2+) entry through presynaptic N- and P/Q-type Ca(2+) channels as well as the vesicular release machinery to cause an increase in evoked glutamate release from rat cerebrocortical synaptosomes. Also, activation of PKA and PKC may underlie, at least in part, the alpha-lipoic acid-mediated facilitation of glutamate release observed here as alpha-lipoic acid-enhanced 4AP and ionomycin-evoked glutamate release were significantly attenuated by PKA and PKC inhibitors. This finding may provide some information regarding the mechanism of action of alpha-lipoic acid in the central nervous system (CNS).     

Tamoxifen depresses glutamate release through inhibition of voltage-dependent Ca2+ entry and protein kinase Cα in rat cerebral cortex nerve terminals

This study was aimed at examining the effect of tamoxifen, a selective estrogen receptor modulator, on the release of endogenous glutamate in rat cerebral cortex nerve terminals (synaptosomes) and exploring the possible mechanism. Tamoxifen inhibited the release of glutamate that was evoked by the K(+) channel blocker 4-aminopyridine (4-AP), and this phenomenon was concentration-dependent and insensitive to the estrogen receptor antagonist. The effect of tamoxifen on the evoked glutamate release was prevented by the chelating extracellular Ca(2+) ions, and by the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor dl-threo-beta-benzyloxyaspartate did not have any effect on the action of tamoxifen. Tamoxifen did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization whereas it decreased the 4-AP-induced increase in cytosolic [Ca(2+)]. Furthermore, the inhibitory effect of tamoxifen on the evoked glutamate release was abolished by the Ca(v)2.2 (N-type) and Ca(v)2.1 (P/Q-type) channel blocker ω-conotoxin MVIIC, but not by the ryanodine receptor blocker dantrolene, or the mitochondrial Na(+)/Ca(2+) exchanger blocker CGP37157. In addition, the protein kinase C (PKC) inhibitors GF109203X or Ro318220 prevented tamoxifen from inhibiting glutamate release. Western blotting showed that tamoxifen significantly decreased the 4-AP-induced phosphorylation of PKC and PKCα. Together, these results suggest that tamoxifen inhibits glutamate release from rat cortical synaptosomes, through the suppression of presynaptic voltage-dependent Ca(2+) entry and PKC activity.     

Persistent Enhancement of Sustained Calcium-Dependent Glutamate Release by Phorbol Esters: Role of Calmodulin-Independent Serine/Threonine Phosphorylation and Actin Disassembly

Abstract: The phorbol ester 4β-phorbol 12,13-dibutyrate increases the final extent of Ca²⁺-dependent glutamate release during the continuous depolarization of the synaptosomal plasma membrane. Based on this finding, we suggested that the sustained activation of protein kinase C has a positive influence on the efficiency of synaptic vesicle recycling in the presence of saturating concentrations of Ca²⁺. Previous work from our laboratory demonstrated that this 4β-phorbol 12,13-dibutyrate-dependent enhancement of synaptic vesicle recycling persists following the removal of 4β-phorbol 12,13-dibutyrate, requires localized Ca²⁺ entry through voltage-regulated channels, and is insensitive to the protein kinase inhibitor staurosporine. In the present study, we examined the possibility that the facilitation of glutamate release may be propagated through interactions between the protein kinase C- and multifunctional Ca²⁺/calmodulin-dependent protein kinase pathways. However, our data argue strongly against the involvement of such a mechanism in the persistent enhancement of sustained glutamate release. We observed that 4β-phorbol 12,13-dibutyrate did not increase the availability of cytosolic free calmodulin or the level of autonomous Ca²⁺/calmodulin-dependent protein kinase activity. In addition, we determined the effects of various serine/threonine kinase and phosphatase inhibitors on the phorbol ester-dependent enhancement of sustained glutamate release and found that protein kinase C increased the extent, but not the duration, of Ca²⁺-dependent glutamate release through a kinase-independent mechanism. Given our finding that the actin-depolymerizing agent cytochalasin D totally occluded the effect of 4β-phorbol 12,13-dibutyrate on release, we postulate that protein kinase C signals may be transduced through direct interactions between protein kinase C isoforms and cytoskeletal protein kinase C binding proteins.     

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.     

Pre‐synaptic kainate receptor‐mediated facilitation of glutamate release involves PKA and Ca2+‐calmodulin at thalamocortical synapses

We have investigated the mechanisms underlying the facilitatory modulation mediated by kainate receptor (KAR) activation in the cortex, using isolated nerve terminals (synaptosomes) and slice preparations. In cortical nerve terminals, kainate (KA, 100 μM) produced an increase in 4‐aminopyridine (4‐AP)‐evoked glutamate release. In thalamocortical slices, KA (1 μM) produced an increase in the amplitude of evoked excitatory post‐synaptic currents (eEPSCs) at synapses established between thalamic axon terminals from the ventrobasal nucleus onto stellate neurons of L4 of the somatosensory cortex. In both, synaptosomes and slices, the effect of KA was antagonized by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione, and persisted after pre‐treatment with a cocktail of antagonists of other receptors whose activation could potentially have produced facilitation of release indirectly. Mechanistically, the observed effects of KA appear to be congruent in synaptosomal and slice preparations. Thus, the facilitation by KA of synaptosomal glutamate release and thalamocortical synaptic transmission were suppressed by the inhibition of protein kinase A and occluded by the stimulation of adenylyl cyclase. Dissecting this G‐protein‐independent regulation further in thalamocortical slices, the KAR‐mediated facilitation of synaptic transmission was found to be sensitive to the block of Ca²⁺ permeant KARs by philanthotoxin. Intriguingly, the synaptic facilitation was abrogated by depletion of intracellular Ca²⁺ stores by thapsigargin, or inhibition of Ca²⁺‐induced Ca²⁺‐release by ryanodine. Thus, the KA‐mediated modulation was contingent on both Ca²⁺ entry through Ca²⁺‐permeable KARs and liberation of intracellular Ca²⁺ stores. Finally, sensitivity to W‐7 indicated that the increased cytosolic [Ca²⁺] underpinning KAR‐mediated regulation of synaptic transmission at thalamocortical synapses, requires downstream activation of calmodulin. We conclude that neocortical pre‐synaptic KARs mediate the facilitation of glutamate release and synaptic transmission by a Ca²⁺‐calmodulin dependent activation of an adenylyl cyclase/cAMP/protein kinase A signalling cascade, independent of G‐protein involvement.

     

Glutamate Receptor Desensitization Block Potentiates the Stimulated GABA Release Through External Ca2+-Independent Mechanisms from Granule Cells of Olfactory Bulb

Glutamate stimulated release of [³H]GABA was studied, during receptor desensitization block and its modulation by voltage gated Ca²⁺ channels, internal Ca²⁺ mobilization and GABA transport inhibitors from olfactory bulb slices. Under control conditions, glutamate and agonists induced release was strongly inhibited by Mg/0 Ca²⁺ Krebs and Cd²⁺ and partially inhibited by Ni²⁺ and nifedipine. Cyclothiazide, which blocks desensitization of glutamate receptors, potentiated glutamate, kainate, AMPA and quisqualate induced release. This effect was less dependent of entry of external Ca²⁺, but was inhibited by trifluoperazine and thapsigargin, inhibitors of Ca²⁺-calmodulin and endoplasmatic Ca²⁺ ATPase respectively. Nipecotic acid and NO-711, inhibitors of the GABA transporter, were also able to reduce cyclothiazide potentiated release induced by the 4 secretagogues. Under control conditions, glutamate stimulates the release of GABA in cooperation with VDCC. However, during receptor desensitization block, glutamate stimulated GABA release is mainly modulated through mechanisms dependent on internal Ca²⁺ mobilization and reversal of the GABA transporter.     

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.     

Ca2+-dependent and Ca2+-independent somatic release from trigeminal neurons

Besides the nerve endings, the soma of trigeminal neurons also respond to membrane depolarizations with the release of neurotransmitters and neuromodulators in the extracellular space within the ganglion, a process potentially important for the cross-communication between neighboring sensory neurons. In this study, we addressed the dependence of somatic release on Ca²⁺ influx in trigeminal neurons and the involvement of the different types of voltage-gated Ca²⁺ (Cav) channels in the process. Similar to the closely related dorsal root ganglion neurons, we found two kinetically distinct components of somatic release, a faster component stimulated by voltage but independent of the Ca²⁺ influx, and a slower component triggered by Ca²⁺ influx. The Ca²⁺-dependent component was inhibited 80% by ω-conotoxin-MVIIC, an inhibitor of both N- and P/Q-type Cav channels, and 55% by the P/Q-type selective inhibitor ω-agatoxin-IVA. The selective L-type Ca²⁺ channel inhibitor nimodipine was instead without effect. These results suggest a major involvement of N- and P/Q-, but not L-type Cav channels in the somatic release of trigeminal neurons. Thus antinociceptive Cav channel antagonists acting on the N- and P/Q-type channels may exert their function by also modulating the somatic release and cross-communication between sensory neurons.     

Synaptopathy under conditions of altered gravity: Changes in synaptic vesicle fusion and glutamate release

Glutamate release and synaptic vesicle heterotypic/homotypic fusion were characterized in brain synaptosomes of rats exposed to hypergravity (10 G, 1 h). Stimulated vesicular exocytosis determined as KCl-evoked fluorescence spike of pH-sensitive dye acridine orange (AO) was decreased twice in synaptosomes under hypergravity conditions as compared to control. Sets of measurements demonstrated reduced ability of synaptic vesicles to accumulate AO (10% higher steady-state baseline level of AO fluorescence). Experiments with preloaded l-[¹⁴C]glutamate exhibited similar amount of total glutamate accumulated by synaptosomes, equal concentration of ambient glutamate, but the enlarged level of cytoplasmic glutamate measuring as leakage from digitonin-permeabilized synaptosomes in hypergravity. Thus, it may be suggested that +G-induced changes in stimulated vesicular exocytosis were a result of the redistribution of intracellular pool of glutamate, i.e. a decrease in glutamate content of synaptic vesicles and an enrichment of the cytoplasmic glutamate level. To investigate the effect of hypergravity on the last step of exocytosis, i.e. membrane fusion, a cell-free system consisted of synaptic vesicles, plasma membrane vesicles, cytosolic proteins isolated from rat brain synaptosomes was used. It was found that hypergravity reduced the fusion competence of synaptic vesicles and plasma membrane vesicles, whereas synaptosomal cytosolic proteins became more active to promote membrane fusion. The total rate of homo- and heterotypic fusion reaction initiated by Ca²⁺ or Mg²⁺/ATP remained unchanged under hypergravity conditions. Thus, hypergravity could induce synaptopathy that was associated with incomplete filling of synaptic vesicles with the neuromediator and changes in exocytotic release.     

Inhibition by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide,a calmodulin inhibitor,of dopamine release from rat brain synaptosomes

The release of preloaded [³H]dopamine by the synaptosomal fraction prepared from rat forebrain was examined in the presence and absence of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin inhibitor. The release induced by high K⁺ was blocked by W-7 in a concentration-dependent manner after the pretreatment with and in the presence of the inhibitor. The inhibition by W-7 may specifically involve calmodulin, because little effects were seen with N-(6-aminohexyl)-naphthalenesulfonamide, an analog of W-7 with only a low affinity for calmodulin. W-7 may not affect the voltage-dependent Ca²⁺ channel of synaptosomal plasmalemma, since the inhibitor produced no change in the synaptosomal ⁴⁵Ca²⁺ uptake induced by high K⁺ depolarization. Thus, calmodulin may play a role in transmitter release and may function at the step(s) after the increase of free Ca²⁺ concentration in the cytosol of the nerve terminal. W-7 affected only to a small extent [³H]dopamine release in the presence of A23187 plus Ca²⁺.     

Depolarization-evoked GABA release from myenteric plexus is partially coupled to L-, N-, and P/Q-type calcium channels

1. There are many evidences suggesting that -aminobutyrate (GABA) is an important neurotransmitter and/or neuromodulator in the gut.2. Using the myenteric plexus-longitudinal muscle preparation from the guinea pig ileum, we investigated the evoked release of [³H] GABA from enteric neurons by electrical pulses or high KCl, which occurs in a calcium-dependent and -independent way. In addition, using selective calcium channel blockers, we report the participation of distinct subtypes of calcium channels in the evoked release, showing a minor participation of L- and Q-type calcium channels, while N- and P-type have a participation of approximately 15%, each. However, regardless of the combination of Ca²⁺ channel blockers, we did not observe an inhibition greater than 50% of the calcium-dependent component of [³H] GABA release.3. Thus, while the observed Ca²⁺-independent release mostly probable occur via reversal of the membrane GABA transporter, in our conditions, a considerable portion of the Ca²⁺-dependent evoked release of [³H] GABA is not coupled to L-, N-, or P/Q-type calcium channels, suggesting the involvement of intracellular calcium stores or other ways of getting calcium across the membrane.     

Lipid-Dependent Modulation of Ca2+ Availability in Isolated Mossy Fiber Nerve Endings

An enhancement of glutamate release from hippocampal neurons has been implicated in long-term potentiation, which is thought to be a cellular correlate of learning and memory. This phenomenom appears to be involved the activation of protein kinase C and lipid second messengers have been implicated in this process. The purpose of this study was to examine how lipid-derived second messengers, which are known to potentiate glutamate release, influence the accumulation of intraterminal free Ca²⁺, since exocytosis requires Ca²⁺ and a potentiation of Ca²⁺ accumulation may provide a molecular mechanism for enhancing glutamate release. The activation of protein kinase C with phorbol esters potentiates the depolarization-evoked release of glutamate from mossy fiber and other hippocampal nerve terminals. Here we show that the activation of protein kinase C also enhances evoked presynaptic Ca²⁺ accumulation and this effect is attenuated by the protein kinase C inhibitor staurosporine. In addition, the protein kinase C-dependent increase in evoked Ca²⁺ accumulation was reduced by inhibitors of phospholipase A₂ and voltage-sensitive Ca²⁺ channels, as well as by a lipoxygenase product of arachidonic acid metabolism. That some of the effects of protein kinase C activation were mediated through phospholipase A₂ was also indicated by the ability of staurosporine to reduce the Ca²⁺ accumulation induced by arachidonic acid or the phospholipase A₂ activator melittin. Similarly, the synergistic facilitation of evoked Ca²⁺ accumulation induced by a combination of arachidonic acid and diacylglycerol analogs was attenuated by staurosporine. We suggest, therefore, that the protein kinase C-dependent potentiation of evoked glutamate release is reflected by increases in presynaptic Ca²⁺ and that the lipid second messengers play a central role in this enhancement of chemical transmission processes.     

Regulatory sites and effectors ofd-[3H]aspartate release from rat cerebral cortex

To study the effect of agents interfering with the biosynthesis and/or the K⁺-evoked Ca²⁺-dependent release of neurotransmitter glutamate, rat cerebral slices were preincubated with Krebs-Ringer-HEPES-glucose-glutamine buffer (KRH buffer), loaded withd-[³H]aspartate and superfused with the preincubation medium in the presence or in the absence of Ca²⁺. The difference in radioactivity release divided by the basal release per min under the two conditions represented the K⁺-evoked Ca²⁺-dependent release. The agents used were: 1) Aminooxyacetic acid (AOAA), the inhibitor of transaminases, 2) Leucine (Leu), the inhibitor of phosphate activated glutaminase (PAG), 3) NH₄ ⁺, the inhibitor of PAG, 4) Phenylsuccinic acid (Phs), the inhibitor of the mitochondrial ketodicarboxylate carrier, 5) ketone bodies, the inhibitors of glycolysis, 6) the absence of glutamine, the substrate of PAG. The results show that Leu, NH₄ ⁺, Phs and the absence of Gln significantly increase the K⁺-evoked Ca²⁺-dependent release of radioactivity by 64%, 200%, 95% and 147% respectively, indicating that these agents are inhibitors of the K⁺-evoked Ca²⁺-dependent release of glutamate. Ketone bodies and AOAA had no effect. These results indicate that the major if not the exclusive biosynthetic pathway of neurotransmitter glutamate in rat cerebral cortex is through the PAG reaction and support a model for the pathway followed by neurotransmitter glutamate i.e. glutamate formed outside the inner mitochondrial membrane has to enter the mitochondrial matrix or is formed within it from where it can be extruded to supply the transmitter pool in exchange of GABA.