Microtubule-Dissociating Drugs and A23187 Reveal Differences in the Inhibition of Synaptosomal Transmitter Release by Botulinum Neurotoxins Types A and B

The inhibitory effects of botulinum neurotoxins types A and B on Ca2(+)-dependent evoked release of [3H]noradrenaline from rat cerebrocortical synaptosomes were compared and their molecular basis investigated. A23187, a Ca2+ ionophore, proved more efficacious in reversing the blockade produced by type A than that by B, whereas the actions of neither were changed by increasing intraterminal cyclic GMP levels using 8-bromo-cyclic GMP of nitroprusside. Disruption of the actin-based cytoskeleton with cytochalasin D did not alter the inhibition seen subsequently with either toxin. However, prior disassembly of microtubules with colchicine, nocodazole, or griseofulvin reduced the potency of type B toxin, but not that of type A toxin; stabilization of the microtubules with taxol counteracted this effect of colchicine. Because colchicine treatment of synaptosomes did not interfere with the measurable binding of type B toxin or its apparent uptake, it appears to act intracellularly. Collectively, these data suggest that botulinum neurotoxins types A and B inactivate transmitter release by interaction at different sites in the process. Based on the consistent results observed with four different drugs known to affect selectively microtubules, their involvement in the action of the type B neurotoxin is proposed.     

Differential Calcium Dependence Between the Release of Endogenous Dopamine and Noradrenaline from Rat Brain Synaptosomes

The characteristics of the release of endogenous dopamine and noradrenaline from rat brain synaptosomes were studied using HPLC with an electrochemical detector. The spontaneous release of dopamine and noradrenaline was inhibited by approximately 50-60% in a Ca2(+)-free medium or a 100 microM La3(+)-containing medium. Also, the high-K+ (30 mM)-evoked release of dopamine and noradrenaline was inhibited by approximately 50-60% in a Ca2(+)-free medium or a 100 microM La3(+)-containing medium. From these results, the ratio of the Ca2(+)-dependent component to the total release of noradrenaline seemed to be similar to that of dopamine. On the other hand, 20 microM La3+ or 1 microM diltiazem inhibited both the spontaneous and 30 mM K(+)-evoked release of dopamine by approximately 50-60% but inhibited neither the spontaneous nor the 30 mM K(+)-evoked release of noradrenaline. The K(+)-evoked rise in intrasynaptosomal Ca2+ concentration was mostly blocked in Ca2(+)-free medium or 100 microM La3(+)-containing medium but was only partially blocked by 20 microM La3+ or 1 microM diltiazem. These data indicate alternative possibilities in that the Ca2(+)-dependent release of noradrenaline might be less sensitive to a change of intracellular Ca2+ concentration than that of dopamine and that the calcium channels directly involved in the noradrenaline release may be more resistant to diltiazem and La3+ than those involved in the dopamine release.     

Characterization of the Inhibitory Action of Botulinum Neurotoxin Type A on the Release of Several Transmitters from Rat Cerebrocortical Synaptosomes

Under optimised conditions for intoxication, botulinum neurotoxin type A was shown to inhibit approximately 90% of Ca2+-dependent K+-evoked release of [3H]acetylcholine, [3H]noradrenaline, and [3H]dopamine from rat cerebrocortical synaptosomes; cholinergic terminals were most susceptible. In each case, the dose-response curve for the neurotoxin was extended, with about 50% of evoked release being inhibited at approximately 10 nM whereas 200 nM was required for the maximal blockade. This may suggest some heterogeneity in the release process. The action of the toxin was time and temperature dependent and appeared to involve binding and sequestration steps prior to blockade of release. The neurotoxin failed to exert any effect on synaptosomal integrity or on Ca2+-independent release of the transmitters tested; it produced only minimal changes in neurotransmitter uptake although small secondary effects were detected with cholinergic terminals. Blockade by the neurotoxin of Ca2+-dependent resting release of transmitter was apparent; Sr2+, Ba2+, or high concentrations of Ca2+ restored the resting release of 3H-catecholamine but not [3H]acetylcholine. Interestingly, none of the latter conditions or 4-aminopyridine could reverse the toxin-induced blockade of evoked release. This lack of specificity in its action on synaptosomes, and other published findings, lead to the conclusion that toxin-sensitive component(s) exist in all nerve terminals that are concerned with transmitter release.     

A late phase of exocytosis from synaptosomes induced by elevated [Ca2+]i is not blocked by Clostridial neurotoxins

Treatment of rat cerebrocortical synaptosomes with botulinum toxin types E and C1 or tetanus toxin removed the majority of intact SNAP-25, syntaxin 1A/1B, and synaptobrevin and diminished Ca(2+)-dependent K+ depolarization-induced noradrenaline secretion. With botulinum toxin type E, <10% of intact SNAP-25 remained and K(+)-evoked release of glutamate and GABA was inhibited. The large component of noradrenaline release evoked within 120 s by inclusion of the Ca2+ ionophore A23187 with the K+ stimulus was also attenuated by these toxins; additionally, botulinium neurotoxin type E blocked the first 60 s of ionophore-induced GABA and glutamate exocytosis. However, exposure to A23187 for longer periods induced a phase of secretion nonsusceptible to any of these toxins (>120 s for noradrenaline; >60 s for glutamate or GABA). Most of this late phase of release represented exocytosis because of its Ca2+ dependency, ATP requirement, and sensitivity to a phosphatidylinositol 4-kinase inhibitor. Based on these collective findings, we suggest that the ionophore-induced elevation of [Ca2+]i culminates in the disassembly of complexes containing nonproteolyzed SNAP receptors protected from the toxins that can then contribute to neuroexocytosis.     

Clostridium neurotoxins influence serotonin uptake and release differently in rat brain synaptosomes

Clostridium neurotoxins produce inhibition of both basal and K(+)-evoked serotonin release in rat brain synaptosomes. To produce these effects, tetanus toxin (TeTx), as well as botulinum neurotoxin type A (BoNT/A), added to brain synaptosomes, must be incubated at 37 degrees C over a long interval (hours). This serotonin exocytosis inhibition was abolished with previous treatment with specific Zn2(+)-metalloprotease inhibitors. Nevertheless, a short incubation time produces different behavior of the indicated neurotoxins: TeTx significantly blocks the sodium-dependent, high-affinity serotonin uptake, whereas a small increase of this uptake was found with BoNT/A. Both Zn2(+)-metalloprotease active fragments, light chains of TeTx and BoNT/A, are unable to reproduce the block of the serotonin uptake, whereas the C-terminal portion of the TeTx heavy chain (Hc-TeTx), which binds specifically to the target tissue, inhibited the serotonin uptake in a dose-dependent manner. The IC50 of Hc-TeTx ranges from 0.62 to 2.08 nM. Binding of [3H]imipramine and [3H]serotonin did not change after toxin treatments, which indicates that these clostridium neurotoxins do not act on the serotonin high-affinity site at the serotonin transporter or at other serotonin high-affinity sites. These results could indicate that TeTx and Hc-TeTx bind to different targets than BoNT/A in the plasma membrane.     

Effects of Antidepressant Drug Treatments on α2-Adrenoceptor Control of [3H]Noradrenaline Release from Hypothalamic Synaptosomes

KCl (16 mM) stimulated the release of [3H]noradrenaline ([3H]NA) from rat hypothalamic synaptosomes in a Ca2+-dependent manner; this release was attenuated by clonidine (0.01-100 microM). Changes in the release of [3H]NA and the functional status of alpha 2-adrenoceptors in the medial hypothalamus of rats treated acutely and chronically with clorgyline (1 mg/kg/day) or desipramine (DMI, 10 mg/kg/day) were assessed using superfused synaptosomes in which the attenuating effects of clonidine (1 microM) or the potentiating effects of yohimbine (1 microM) on K+-evoked release of [3H]NA were measured. After acute administration of DMI, significantly less [3H]NA was accumulated into synaptosomes. Although total (spontaneous + K+-evoked) [3H]NA release from these synaptosomes was unchanged, a significant reduction was apparent in the K+-evoked release from the DMI-treated tissue. Attenuation of K+-evoked release by clonidine was abolished in both these acute treatment groups. Following the chronic antidepressant drug regimens, [3H]NA uptake into DMI-treated tissue remained significantly reduced although total percent and K+-evoked [3H]NA release were unchanged. The K+-evoked release of [3H]NA in S1 was significantly enhanced (by 22%) in the clorgyline treatment group. Attenuation of K+-evoked [3H]NA release by clonidine in both chronic antidepressant-treated tissues was not significantly changed. It is concluded that the functional sensitivity of alpha 2-adrenoceptors on nerve endings in the medial hypothalamus is unchanged by these chronic antidepressant drug regimens. In synaptosomes from untreated tissue, yohimbine significantly potentiated K+-evoked release of [3H]NA; this effect was unchanged after acute regimens and reduced after chronic administration of both the antidepressants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitric oxide inhibits depolarization-evoked glutamate release from rat cerebellar granule cells.

Nitric oxide (NO) modulates the release of various neurotransmitters, some of these are considered to be involved in neuronal plasticity that includes long-term depression in the cerebellum. To date, there have been no reports on the modulation of the exocytotic release of neurotransmitters in the cerebellar granule cells (CGCs) by NO. The aim of this study was to investigate the effects of NO on the exocytotic release of glutamate from rat CGCs. Treatment with NO-related reagents revealed that NO inhibited high-K(+)-evoked glutamate release. Clostridium botulinum type B neurotoxin (BoNT/B) attenuated the enhancement of glutamate release caused by NO synthase (NOS) inhibition; this indicates that NO acts on the high-K(+)-evoked exocytotic pathway. cGMP-related reagents did not affect the high-K(+)-evoked glutamate release. NO-related reagents did not affect Ca(2+) ionophore-induced glutamate release, suggesting that NO inhibits Ca(2+) entry through voltage-dependent Ca(2+) channels (VDCC). Monitoring of intracellular Ca(2+) revealed that NO inhibited high-K(+)-evoked Ca(2+) entry. L-type VDCC blockers inhibited glutamate release and NO did not have an additive effect on the inhibition produced by the L-type VDCC blocker. The inhibition of the high-K(+)-evoked glutamate release by NO was abolished by a reducing reagent; this suggested that NO regulates the high-K(+)-evoked glutamate release from CGCs by redox modulation.     

The sensitivity of catecholamine release to botulinum toxin C1 and E suggests selective targeting of vesicles set into the readily releasable pool

The impact of syntaxin and SNAP-25 cleavage on [3H]noradrenaline ([3H]NA) and [3H]dopamine ([3H]DA) exocytotic release evoked by different stimuli was studied in superfused rat synaptosomes. The external Ca2+-dependent K+-induced [3H]catecholamine overflows were almost totally abolished by botulinum toxin C1 (BoNT/C1), which hydrolyses syntaxin and SNAP-25, or by botulinum toxin E (BoNT/E), selective for SNAP-25. BoNT/C1 cleaved 25% of total syntaxin and 40% of SNAP-25; BoNT/E cleaved 40% of SNAP-25 but left syntaxin intact. The GABA uptake-induced releases of [3H]NA and [3H]DA were differentially affected: both toxins blocked the former, dependent on external Ca2+, but not the latter, internal Ca2+-dependent. BoNT/C1 or BoNT/E only slightly reduced the ionomycin-evoked [3H]catecholamine release. More precisely, [3H]NA exocytosis induced by ionomycin was sensitive to toxins in the early phase of release but not later. The Ca2+-independent [3H]NA exocytosis evoked by hypertonic sucrose, thought to release from the readily releasable pool (RRP) of vesicles, was significantly reduced by BoNT/C1. Pre-treating synaptosomes with phorbol-12-myristate-13-acetate, to increase the RRP, enhanced the sensitivity to BoNT/C1 of [3H]NA release elicited by sucrose or ionomycin. Accordingly, cleavage of syntaxin was augmented by the phorbol-ester. To conclude, our results suggest that clostridial toxins selectively target exocytosis involving vesicles set into the RRP.     

Ca2+-Dependent Regulation of Presynaptic Stimulus-Secretion Coupling

In the present study, we have investigated the role of Ca2+ in the coupling of membrane depolarization to neurotransmitter secretion. We have measured (a) intracellular free Ca2+ concentration ([Ca2+]i) changes, (b) rapid 45Ca2+ uptake, and (c) Ca2+-dependent and -independent release of endogenous glutamate (Glu) and gamma-aminobutyric acid (GABA) as a function of stimulus intensity by elevating the extracellular [K+] to different levels in purified nerve terminals (synaptosomes) from rat hippocampus. During stimulation, Percoll-purified synaptosomes show an increased 45Ca2+ uptake, an elevated [Ca2+]i, and a Ca2+-dependent as well as a Ca2+-independent release of both Glu and GABA. With respect to both amino acids, synaptosomes respond on stimulation essentially in the same way, with maximally a fourfold increase in Ca2+-dependent (exocytotic) release. Ca2+-dependent transmitter release as well as [Ca2+]i elevations show maximal stimulation at moderate depolarizations (30 mM K+). A correlation exists between Ca2+-dependent release of both Glu and GABA and elevation of [Ca2+]i. Ca2+-dependent release is maximally stimulated with an elevation of [Ca2+]i of 60% above steady-state levels, corresponding with an intracellular concentration of approximately 400 nM, whereas elevations to 350 nM are ineffective in stimulating Ca2+-dependent release of both Glu and GABA. In contrast, Ca2+-independent release of both Glu and GABA shows roughly a linear rise with stimulus intensity up to 50 mM K+. 45Ca2+ uptake on stimulation also shows a continuous increase with stimulus intensity, although the relationship appears to be biphasic, with a plateau between 20 and 40 mM K+.(ABSTRACT TRUNCATED AT 250 WORDS)     

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+.     

Differential contribution of syntaxin 1 and SNAP-25 to secretion in noradrenergic and adrenergic chromaffin cells

We used botulinum neurotoxins (BoNT) to examine whether differences in the secretory activity of noradrenergic and adrenergic chromaffin cells are related to differences in the exocytotic machinery of these two types of bovine adrenal medulla cells. Cleavage of syntaxin and SNAP-25 by BoNT/C1 decreased in a dose-dependent way the release of both noradrenaline and adrenaline, but noradrenaline release was more sensitive to BoNT/C1. Cleavage of SNAP-25 by BoNT/A also had a larger inhibitory effect on noradrenaline release than on adrenaline release. Neither BoNT/C1 nor BoNT/A affected the intracellular Ca2+ responses induced by K+-depolarisation, and the extent of the inhibition of K+-evoked catecholamine release by selective blockers of voltage-gated Ca2+ channels was not affected by BoNT/C1. Therefore, our data do not support the hypothesis of a regulatory effect of syntaxin or SNAP-25 on the activity of Ca2+ channels. The lower sensitivity of adrenaline release to BoNT was not due to a reduced ability of the toxins to enter or to cleave their protein targets in adrenergic cells, since immunoblot analysis showed the cleavage of a larger fraction of syntaxin 1A in adrenergic cells, as compared to the cleavage in noradrenergic cells. The immunoblot analysis also showed larger amounts of syntaxin 1A in noradrenergic chromaffin cells than in adrenergic cells. Thus, in spite of a greater cleavage of syntaxin 1A in adrenergic cells by BoNT/C1, adrenaline release was less sensitive to BoNT/C1, suggesting that the release process in noradrenergic cells might be more dependent on syntaxin 1A and SNAP-25, as compared to adrenergic cells.     

An Ion Channel Locus for the Protein Kinase C Potentiation of Transmitter Glutamate Release from Guinea Pig Cerebrocortical Synaptosomes

The mechanism by which protein kinase C (PKC) activates transmitter release from guinea pig cerebrocortical synaptosomes was investigated by employing parallel fluorescent assays of glutamate release, cytoplasmic free Ca2+, and plasma membrane potential. 4 beta-Phorbol dibutyrate (4 beta-PDBu) enhances the Ca(2+)-dependent, 4-aminopyridine (4AP)-evoked release of glutamate from synaptosomes, the 4AP-evoked elevation of cytoplasmic free Ca2+, and the 4AP-evoked depolarization of the plasma membrane. 4 beta-PDBu itself causes a slow depolarization, which may underlie the small effect of 4 beta-PDBu on spontaneous, KCl-evoked, and Ca(2+)-independent/4AP-evoked glutamate release. Because 4AP (but not KCl) generates spontaneous, tetrodotoxin-sensitive action potentials in synaptosomes, a major locus of presynaptic PKC action is to enhance these action potentials, perhaps by inhibiting delayed rectifier K+ channels.     

Activation of Protein Kinase C Suppresses the γ-Aminobutyric AcidB Receptor-Mediated Inhibition of the Vesicular Release of Noradrenaline and Acetylcholine

Modulation of the gamma-aminobutyric acidB (GABAB) receptor-mediated response by protein kinase C (PKC) was examined with regard to inhibition by stimulation of the GABAB receptor of stimulation-evoked release of noradrenaline (NA) from slices of cerebellar cortex and of acetylcholine (ACh) from strips of ileum. 12-O-Tetradecanoylphorbol 13-acetate (TPA) potentiated the high K(+)-evoked Ca2+-dependent release of NA and ACh, but not the ouabain-evoked release, even in the presence of external Ca2+. The potentiating effect was antagonized by sphingosine, thereby suggesting that PKC participates in the exocytotic-vesicular release of neurotransmitters, but does not do so in case of a nonvesicular release. GABA inhibited the high K(+)-evoked release of NA and ACh, but not the ouabain-evoked Ca(2+)-independent release. The effect of GABA was mimicked by baclofen and was antagonized by phaclofen, thereby suggesting that stimulation of the GABAB receptor inhibits the vesicular but not the nonvesicular release of neurotransmitters. TPA suppressed the GABAB receptor-mediated inhibition of high K(+)-evoked release of NA and ACh. The effect of TPA was antagonized by sphingosine. These results indicate that stimulation of the GABAB receptor inhibits the stimulation-evoked Ca(2+)-dependent release of neurotransmitters and that activation of PKC suppresses the GABAB receptor-mediated response.     

Synaptosomal Protein Kinase C Subspecies: B. Down-Regulation Promoted by Phorbol Ester and Its Effect on Evoked Norepinephrine Release

The effect of phorbol esters was investigated on the down-regulation of protein kinase C (PKC) and on the release of [3H]norepinephrine (NE) in synaptosomes from the rat cerebrum. Treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) promoted the translocation of PKC activity in a P2 fraction from the cytosol to the membrane fraction and then its down-regulation, in a dose-dependent manner. TPA induced a rapid down-regulation of the type II(beta) and type III(alpha) subspecies, but did not change the activity of the type I(gamma) subspecies in the cytosolic fraction for at least 15 min. The gamma-subspecies was subsequently decreased at a slower rate. In the synaptosomes thus having only the gamma-subspecies, a subsequent dose of TPA could not enhance K(+)-evoked NE release, although, in the original synaptosomes, TPA was able to enhance K(+)-evoked NE release. Pretreatment with TPA did not alter the K(+)-evoked NE release itself. TPA was also found to enhance the K(+)-evoked NE release from synaptosomes prepared from both hippocampus, which express the gamma-subspecies of PKC at a negligible level, and cerebral cortex, which have a significant level of the gamma-subspecies, to the same degree. These results suggest that the gamma-subspecies of PKC does not participate in the TPA-enhanced K(+)-evoked NE release from synaptosomes.     

Presynaptic Glutamate Receptors Regulate Noradrenaline Release from Isolated Nerve Terminals

The wide-ranging neuronal actions of excitatory amino acids, such as glutamate, are thought to be mediated mainly by postsynaptic N-methyl-D-aspartate (NMDA) and non-NMDA receptors. We now report the existence of presynaptic glutamate receptors in isolated nerve terminals (synaptosomes) prepared from hippocampus, olfactory bulb, and cerebral cortex. Activation of these receptors by NMDA or non-NMDA agonists, in a concentration-dependent manner, resulted in Ca(2+)-dependent release of noradrenaline from vesicular transmitter stores. The NMDA-stimulated release was potentiated by glycine and was blocked by Mg2+ and selective NMDA antagonists. In contrast, release stimulated by selective non-NMDA agonists was blocked by 6-cyano-7-nitroquinoxaline-2,3- dione, but not by Mg2+ or NMDA antagonists. Our data suggest that the presynaptic glutamate receptors can be classified pharmacologically as both the NMDA and non-NMDA types. These receptors, localized on nerve terminals of the locus ceruleus noradrenergic neurons, may play an important role in interactions between noradrenaline and glutamate.     

Inhibition by Cyclic AMP of Phorbol Ester-Potentiated Norepinephrine Release from Guinea Pig Brain Cortical Synaptosomes

The involvement of Ca2+/phospholipid-dependent protein kinase (protein kinase C, PKC) and cyclic AMP-dependent protein kinase in the K+-evoked release of norepinephrine (NE) was studied using guinea pig brain cortical synaptosomes preloaded with [3H]NE. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a potent activator of PKC, enhanced the K+-evoked release of [3H]NE, in a concentration-dependent manner, but with no effect on the spontaneous outflow and uptake of [3H]NE in the synaptosomes. The apparent affinity of the evoked release for added calcium but not the maximally evoked release was increased by TPA (10(-7) M). Inhibitors of PKC, polymyxin B, and a more potent inhibitor, staurosporine, counteracted the TPA-induced potentiation of the evoked release. Both forskolin and dibutyryl cyclic AMP (DBcAMP) enhanced the evoked release, but reduced the TPA-potentiated NE release. A novel inhibitor of cyclic AMP-dependent protein kinase, KT5720, blocked both the forskolin-induced increase in the evoked release and its inhibition of TPA-induced potentiation in the evoked release, thereby suggesting that forskolin or DBcAMP counteracts the Ca2+-dependent release of NE by activating cyclic AMP-dependent protein kinase. These results suggest that the activation of PKC potentiates the evoked release of NE and that the activation of cyclic AMP-dependent protein kinase acts negatively on the PKC-activated exocytotic neurotransmitter release process in brain synaptosomes of the guinea pig.     

Characterization of the Effect of Monensin on γ-Amino-n-Butyric Acid Release from Isolated Nerve Terminals

The action of the polyether antibiotic monensin on the release of gamma-[3H]amino-n-butyric acid [( 3H]GABA) from mouse brain synaptosomes is characterized. Monensin enhances the release of this amino acid transmitter in a dose-dependent manner and does not modify the efflux of the nontransmitter amino acid alpha-[3H]aminoisobutyrate. The absence of external Ca2+ fails to prevent the stimulatory effect of monensin on [3H]GABA release. Furthermore, monensin is less effective in stimulating [3H]GABA release in the presence of Ca2+. The releasing response to monensin is absolutely dependent on external Na+. The blockade of voltage-sensitive Na+ or Ca2+ channels does not modify monensin-induced release of the transmitter. Also, the blockade of the GABA uptake pathway fails to prevent the stimulatory effect of monensin on [3H]GABA release. Although monensin markedly increases Na+ permeability in synaptosomes, these data indicate that the Ca2+-independent monensin-stimulated transmitter release is not mediated by the Na+-dependent uptake pathway. It is concluded that the entrance of Na+ through monensin molecules inserted in the presynaptic membrane might be sufficient to initiate the intraterminal molecular events underlying transmitter release.     

Uptake and K+-evoked release of 3H-norepinephrine in the mesodiencephalic synaptosomes of albino rats during aging. Role of N-acetyl-L-aspartic acid

The uptake and K(+)-evoked (40 mM) release of 3H-norepinephrine (3H-NE) in mesodiencephalic synaptosomes of adult and senescent rats and the effect of N-acetylaspartic acid (NAA) on these processes have been studied. It has been shown that the uptake of 3H-NE by old rats is reduced considerably. The K(+)-evoked release of 3H-NE from rats synaptosomes is significantly decreased in aged rats. In the presence of 10(-4)-3.10(-3) M NAA the uptake of 3H-NE by adult and senescent rats synaptosomes remains unchanged. In these concentrations NAA inhibits the K(+)-evoked release of 3H-NE from synaptosomes of adult rats, but it exerts no effect on this process in senescent rats.     

External pH changes affect NMDA-evoked and spontaneous release of cholecystokinin, somatostatin and noradrenaline from rat cerebrocortical nerve endings

It was previously reported that the K+-evoked release of somatostatin-like immunoreactivity (SRIF-LI) and of cholecystokinin-like immunoreactivity (CCK-LI) from superfused rat cerebrocortical synaptosomes can be enhanced by NMDA or D-serine alone. We here studied the effects of extraterminal pH changes on SRIF-LI and CCK-LI release. Lowering pH from 7.4 to 6.9 or 6.4 abolished the effects of NMDA or D-serine on the K+-evoked peptide release. Identical results were obtained when external pH was raised to 8 or 8.7. Sudden alkalinization of the superfusion medium, in absence of K+-depolarization, induced SRIF-LI or CCK-LI release which was insensitive to NMDA. Based on experiments in Ca2+-free medium and with voltage-sensitive Ca2+ channel (VSCC) blockers, the pH 8.7-induced release of SRIF-LI and CCK-LI was only in part (30-50%) dependent on external Ca2+ and Ca2+ channel activation. In contrast, the alkalinization-evoked release of [3H]noradrenaline was highly sensitive to external Ca2+ removal and to blockade of Ca2+ channels with omega-conotoxins. The pH 8.7-evoked SRIF-LI and CCK-LI was about halved in synaptosomes intoxicated with botulinum toxin C1. The results suggest that the pH-sensitive NMDA receptors mediating somatostatin and cholecystokinin release contain NR1 subunits lacking the exon-5 cassette. Alkalinization represents a novel releasing stimulus which elicits neuropeptide release in part by conventional exocytosis and largely by an external Ca2+-independent mechanism. Differently, the release of noradrenaline provoked by alkalinization occurs entirely by conventional exocytosis.