Kainate receptor-mediated presynaptic inhibition converges with presynaptic inhibition mediated by Group II mGluRs and long-term depression at the hippocampal mossy fiber-CA3 synapse.

Kainate receptors (KARs) effect depression of glutamate release at hippocampal mossy fiber-CA3 (MF-CA3) synapses by a metabotropic action involving adenylyl cyclase (AC) inhibition, cAMP reduction, and diminished protein kinase A (PKA) activation. Using hippocampal slices, we show here that KAR activation interferes with the depression of glutamate release produced by Group II metabotropic glutamate receptor stimulation and low frequency stimulation (LFS)-induced long-term depression (LTD), also expressed through presynaptic AC/cAMP/PKA at MF-CA3 synapses. The mutual occlusion of depression mediated by presynaptic KARs, Group II mGluR and LFS-induced LTD suggests their mechanistic convergence at the MF-CA3 synapse and thus invokes KARs in synaptic plasticity manifest in LTD.

Phosphorylation sites on calcium channel alpha1 and beta subunits regulate ERK-dependent modulation of neuronal N-type calcium channels.

Voltage-dependent calcium channels (VDCCs) in sensory neurones are tonically up-regulated via Ras/extracellular signal regulated kinase (ERK) signalling. The presence of putative ERK consensus sites within the intracellular loop linking domains I and II of neuronal N-type (Ca(v)2.2) calcium channels and all four neuronal calcium channel beta subunits (Ca(v)beta), suggests that Ca(v)2.2 and/or Ca(v)betas may be ERK-phosphorylated. Here we report that GST-Ca(v)2.2 I-II loop, and to a lesser extent Ca(v)beta1b-His(6), are substrates for ERK1/2 phosphorylation. Serine to alanine mutation of Ser-409 and/or Ser-447 on GST-Ca(v)2.2 I-II loop significantly reduced phosphorylation. Loss of Ser-447 reduced phosphorylation to a greater extent than mutation of Ser-409. Patch-clamp recordings from wild-type Ca(v)2.2,beta1b,alpha2delta1 versus mutant Ca(v)2.2(S447A) or Ca(v)2.2(S409A) channels revealed that mutation of either site significantly reduced current inhibition by UO126, a MEK (ERK kinase)-specific inhibitor that down-regulates ERK activity. However, no additive effect was observed by mutating both residues together, suggesting some functional redundancy between these sites. Mutation of both Ser-161 and Ser-348 on Ca(v)beta1b did not significantly reduce phosphorylation but did reduce UO126-induced current inhibition. Crucially, co-expression of Ca(v)2.2(S447A) with Ca(v)beta1b(S161,348A) had an additive effect, abolishing the action of UO126 on channel current, an effect not seen when Ca(v)beta1b(S161,348A) was co-expressed with Ca(v)2.2(S409A). Thus, Ser-447 on Ca(v)2.2 and Ser-161 and Ser-348 of Ca(v)beta1b appear to be both necessary and sufficient for ERK-dependent modulation of these channels. Together, our data strongly suggest that modulation of neuronal N-type VDCCs by ERK involves phosphorylation of Ca(v)2.2alpha1 and to a lesser extent possibly also Ca(v)beta subunits.

Opposing facilitatory and inhibitory modulation of glutamate release elicited by cAMP production in cerebrocortical nerve terminals (synaptosomes).

Activation of cAMP-protein kinase A (PKA) is widely reported to facilitate synaptic transmission. Here, we examined the presynaptic loci of PKA action using isolated nerve terminals (synaptosoms). The adenylyl cyclase (AC) activator, forskolin, failed to have any effect on 4-aminopyridine (4-AP)-evoked glutamate release, when added alone. However, in the presence of the alkylxanthine, IBMX, forskolin strongly facilitated glutamate release. This potentiation of release was blocked by the PKA inhibitors Rp-cAMPS and H7. Given that IBMX has dual activity, antagonizing adenosine receptors as well as inhibiting cAMP phosphodiesterase, we examined the effect of a selective adenosine A(1) receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and RO20-1724, a specific phosphodiesterase inhibitor. Both unmasked the forskolin-mediated modulation of glutamate release. Conversely, the adenosine analogue, N(6)-cyclohexyladenosine (CHA), reversed the facilitation induced by forskolin+RO20-1724. Adenosine A(1) receptor activation, therefore, appears to curtail cAMP/PKA-induced potentiation of glutamate release. Looking at the targets for cAMP/PKA-mediated potentiation of glutamate release, while synaptosomal excitability was only marginally increased, basal and 4-AP-evoked-increases in [Ca(2+)](c) were substantially enhanced by forskolin+IBMX. Moreover, glutamate release elicited by Ca(2+)-ionophore (ionomycin)-induced Ca(2+)-entry was facilitated by forskolin+IBMX. cAMP/PKA-mediated facilitation of glutamate release may therefore involve modulation of Ca(2+)-entry, as well as downstream events controlling synaptic vesicle recruitment and exocytosis.

Opposing changes in phosphorylation of specific sites in synapsin I during Ca2+-dependent glutamate release in isolated nerve terminals.

Synapsins are major neuronal phosphoproteins involved in regulation of neurotransmitter release. Synapsins are well established targets for multiple protein kinases within the nerve terminal, yet little is known about dephosphorylation processes involved in regulation of synapsin function. Here, we observed a reciprocal relationship in the phosphorylation-dephosphorylation of the established phosphorylation sites on synapsin I. We demonstrate that, in vitro, phosphorylation sites 1, 2, and 3 of synapsin I (P-site 1 phosphorylated by cAMP-dependent protein kinase; P-sites 2 and 3 phosphorylated by Ca(2+)-calmodulin-dependent protein kinase II) were excellent substrates for protein phosphatase 2A, whereas P-sites 4, 5, and 6 (phosphorylated by mitogen-activated protein kinase) were efficiently dephosphorylated only by Ca(2+)-calmodulin-dependent protein phosphatase 2B-calcineurin. In isolated nerve terminals, rapid changes in synapsin I phosphorylation were observed after Ca(2+) entry, namely, a Ca(2+)-dependent phosphorylation of P-sites 1, 2, and 3 and a Ca(2+)-dependent dephosphorylation of P-sites 4, 5, and 6. Inhibition of calcineurin activity by cyclosporin A resulted in a complete block of Ca(2+)-dependent dephosphorylation of P-sites 4, 5, and 6 and correlated with a prominent increase in ionomycin-evoked glutamate release. These two opposing, rapid, Ca(2+)-dependent processes may play a crucial role in the modulation of synaptic vesicle trafficking within the presynaptic terminal.

Lamotrigine inhibition of glutamate release from isolated cerebrocortical nerve terminals (synaptosomes) by suppression of voltage-activated calcium channel activity.

Lamotrigine (LAG) is an antiepileptic drug which is believed to suppress seizures by inhibiting the release of excitatory neurotransmitters. The present study was aimed at investigating the effect of LAG on the 4-aminopyridine (4AP)-evoked glutamate release in cerebrocortical nerve terminals (synaptosomes). LAG inhibited the release of glutamate evoked by 4AP in a concentration-dependent manner. This inhibitory effect was associated with a reduction in the depolarization-evoked increase in the cytoplasmic free Ca2+ concentration ([Ca2+]C). In addition, LAG did not alter the resting synaptosomal membrane potential or 4AP-evoked depolarization. Furthermore, ionomycin-evoked glutamate release was not affected by LAG. Based on these results, we suggest that presynaptic calcium influx blockade and inhibition of glutamate release may underlie the mechanism of action of LAG. These action may also contribute to their neuroprotective properties in excitotoxic injury.

A modulatory role for protein phosphatase 2B (calcineurin) in the regulation of Ca2+ entry.

The Ca2+/calmodulin-dependent protein phosphatase 2B (PP2B) also known as calcineurin (CN) has been implicated in the Ca2+-dependent inactivation of Ca2+ channels in several cell types. To study the role of calcineurin in the regulation of Ca2+-channel activity, phosphatase expression was altered in NG108-15 cells by transfection of sense and antisense plasmid constructs carrying the catalytic subunit of human PP2Bbeta3. Relative to mock-transfected (wild-type) controls, cells overexpressing calcineurin showed dramatically reduced high-voltage-activated Ca2+ currents which were recoverable by the inclusion of 1 microM FK506 in the patch pipette. Conversely, in cells with reduced calcineurin expression, high-voltage-activated Ca2+ currents were larger relative to controls. Additionally in these cells, low-voltage-activated currents were significantly reduced. Analysis of high-voltage-activated Ca2+ currents revealed that the kinetics of inactivation were significantly accelerated in cells overexpressing calcineurin. Following the delivery of a train of depolarizing pulses in experiments designed to produce large-scale Ca2+ influx across the cell membrane, Ca2+-dependent inactivation of high-voltage-activated Ca2+ currents was increased in sense cells, and this increase could be reduced by intracellular application of 1 mM BAPTA or 1 microM FK506. These data support a role of calcineurin in the negative feedback regulation of Ca2+ entry through voltage-operated Ca2+ channels.

Synapsins as mediators of BDNF-enhanced neurotransmitter release.

We examined enhancement of synaptic transmission by neurotrophins at the presynaptic level. In a synaptosomal preparation, brain-derived neurotrophic factor (BDNF) increased mitogen-activated protein (MAP) kinase-dependent synapsin I phosphorylation and acutely facilitated evoked glutamate release. PD98059, used to inhibit MAP kinase activity, markedly decreased synapsin I phosphorylation and concomitantly reduced neurotransmitter release. The stimulation of glutamate release by BDNF was strongly attenuated in mice lacking synapsin I and/or synapsin II. These results indicate a causal link of synapsin phosphorylation via BDNF, TrkB receptors and MAP kinase with downstream facilitation of neurotransmitter release.

Ca(2+)-permeable AMPA receptors induce phosphorylation of cAMP response element-binding protein through a phosphatidylinositol 3-kinase-dependent stimulation of the mitogen-activated protein kinase signaling cascade in neurons.

Ca(2+)-permeable AMPA receptors may play a key role during developmental neuroplasticity, learning and memory, and neuronal loss in a number of neuropathologies. However, the intracellular signaling pathways used by AMPA receptors during such processes are not fully understood. The mitogen-activated protein kinase (MAPK) cascade is an attractive target because it has been shown to be involved in gene expression, synaptic plasticity, and neuronal stress. Using primary cultures of mouse striatal neurons and a phosphospecific MAPK antibody we addressed whether AMPA receptors can activate the MAPK cascade. We found that in the presence of cyclothiazide, AMPA caused a robust and direct (no involvement of NMDA receptors or L-type voltage-sensitive Ca(2+) channels) Ca(2+)-dependent activation of MAPK through MAPK kinase (MEK). This activation was blocked by GYKI 53655, a noncompetitive selective antagonist of AMPA receptors. Probing the mechanism of this activation revealed an essential role for phosphatidylinositol 3-kinase (PI 3-kinase) and the involvement of a pertussis toxin (PTX)-sensitive G-protein, a Src family protein tyrosine kinase, and Ca(2+)/calmodulin-dependent kinase II. Similarly, kainate activated MAPK in a PI 3-kinase-dependent manner. AMPA receptor-evoked neuronal death and arachidonic acid mobilization did not appear to involve signaling through the MAPK pathway. However, AMPA receptor stimulation led to a Ca(2+)-dependent phosphorylation of the nuclear transcription factor CREB, which could be prevented by inhibitors of MEK or PI 3-kinase. Our results indicate that Ca(2+)-permeable AMPA receptors transduce signals from the cell surface to the nucleus of neurons through a PI 3-kinase-dependent activation of MAPK. This novel pathway may play a pivotal role in regulating synaptic plasticity in the striatum.

A high-affinity presynaptic kainate-type glutamate receptor facilitates glutamate exocytosis from cerebral cortex nerve terminals (synaptosomes).

Ionotropic glutamate receptor agonists, kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and domoate, all facilitated 4-aminopyridine-evoked glutamate release from rat cerebrocortical nerve terminals (synaptosomes). The non-selective, non-N-methyl-D-aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione blocked kainate facilitation of glutamate release. AMPA responses were non-desensitizing and insensitive to the AMPA receptor desensitization inhibitor, cyclothiazide. The AMPA receptor antagonist GYKI 52466 failed to block ionotropic glutamate receptor-mediated facilitation, but the ionotropic glutamate receptor 6 kainate receptor subunit antagonist NS-102 was a potent blocker. Furthermore, kainate and AMPA responses were not additive. Taken together, our results indicate that, in the cerebral cortex, both kainate and AMPA may be facilitating glutamate release through the activation of a high-affinity kainate receptor containing glutamate receptor 6 kainate subunits. Kainate enhanced 4-aminopyridine-evoked depolarization of the synaptosomal plasma membrane potential, indicating that a ligand-gated ion channel that conducts cations may underlie the mechanism by which kainate mediates facilitation of glutamate release. While the facilitatory effect of kainate on glutamate release is consistent with a classical ionotropic action of ionotropic glutamate receptors, our observation that kainate inhibits GABA release suggests that alternative presynaptic mechanisms may operate in cerebrocortical nerve terminals to mediate the ionotropic glutamate receptor modulation of glutamate and GABA release. We conclude that high-affinity kainate-type glutamate autoreceptors represent a positive feed-forward system for potentiating the release of glutamate from cerebrocortical nerve terminals.

Calcineurin involvement in the regulation of high-threshold Ca2+ channels in NG108-15 (rodent neuroblastoma x glioma hybrid) cells.

1. We examined the relationship between calcineurin (protein phosphatase 2B (PP2B) and voltage-operated Ca2+ channels (VOCCs) in NG108-15 cells. PP2B expression in NG108-15 cells was altered by transfection with plasmid constructs containing a full length cDNA of human PP2B beta(3) in sense (CN-15) and antisense (CN-21) orientation. 2. Confocal immunocytochemical localization showed that in wild-type cells, PP2B immunoreactivity is uniformly distributed in undifferentiated cells and located at the inner surface of soma membrane and neurites in differentiated cells. 3. To test the Ca2+ dependence of the VOCC, we used high-frequency stimulation (HFS). The L- and N-type VOCCs decreased by 37 and 52%, respectively, whereas the T-type current was only marginally sensitive to this procedure. FK-506 (2 microM), a specific blocker of PP2B, reduced the inhibition of L- and N-type VOCCs induced by HFS by 30 and 33%, respectively. 4. In CN-15-transfected cells overexpressing PP2B, total high-voltage-activated (HVA) VOCCs were suppressed by about 60% at a test potential of +20 mV. Intracellular addition of EGTA or FK-506 into CN-15-transfected cells induced an up to 5-fold increase of HVA VOCCs. 5. These findings indicate that PP2B activity does not influence the expression of HVA Ca2+ channels, but modulates their function by Ca(2+)-dependent dephosphorylation. Thus HVA VOCCs, in a phosphorylated state under control conditions, are downregulated by PP2B upon stimulation, with the major effect on N-type VOCCs.

An essential role for a small synaptic vesicle-associated phosphatidylinositol 4-kinase in neurotransmitter release.

Glutamate release from nerve terminals is the consequence of Ca2+-triggered fusion of small synaptic vesicles with the presynaptic plasma membrane. ATP dependence of neurotransmitter release has been suggested to be founded, in part, on phosphorylation steps preceding membrane fusion. Here we present evidence for an essential role of phosphatidylinositol phosphorylation in stimulated release of neurotransmitter glutamate from isolated nerve terminals (synaptosomes). Specifically, we show that a phosphatidylinositol 4-kinase (PtdIns 4-kinase) activity resides on nerve terminal-derived small synaptic vesicles (SSVs) and that inhibition of the PtdIns 4-kinase activity in intact synaptosomes leads to attenuation of the evoked release of glutamate. The attenuation of transmitter release is reversible and correlates with respective changes in intrasynaptosomal PtdIns 4-kinase activity. Because only the Ca2+-dependent release of glutamate is affected, regulation appears to be at the level of exocytosis. Taken together, our data imply a mandatory role for PtdIns 4-kinase and phosphoinositide products in the regulated exocytosis of SSV in mammalian nerve terminals.

Presynaptic GABA(B) receptor modulation of glutamate exocytosis from rat cerebrocortical nerve terminals: receptor decoupling by protein kinase C.

GABA and the GABA(B) receptor agonist (-)-baclofen inhibited 4-aminopyridine (4AP)- and KCl-evoked, Ca2+-dependent glutamate release from rat cerebrocortical synaptosomes. The GABA(B) receptor antagonist CGP 35348, prevented this inhibition of glutamate release, but phaclofen had no effect. (-)-Baclofen-mediated inhibition of glutamate release was insensitive to 2 microg/ml pertussis toxin. As determined by examining the mechanism of GABA(B) receptor modulation of glutamate release, (-)-baclofen caused a significant reduction in 4AP-evoked Ca2+ influx into synaptosomes. The agonist did not alter the resting synaptosomal membrane potential or 4AP-mediated depolarization; thus, the inhibition of Ca2+ influx could not be attributed to GABA(B) receptor activation causing a decrease in synaptosomal excitability. Ionomycin-mediated glutamate release was not affected by (-)-baclofen, indicating that GABA(B) receptors in this preparation are not coupled directly to the exocytotic machinery. Instead, the data invoke a direct coupling of GABA(B) receptors to voltage-dependent Ca2+ channels linked to glutamate release. This coupling was subject to regulation by protein kinase C (PKC), because (-)-baclofen-mediated inhibition of 4AP-evoked glutamate release was reversed when PKC was stimulated with phorbol ester. This may therefore represent a mechanism by which inhibitory and facilitatory presynaptic receptor inputs interplay to fine-tune transmitter release.

Neurotrophins stimulate phosphorylation of synapsin I by MAP kinase and regulate synapsin I-actin interactions.

The ability of neurotrophins to modulate the survival and differentiation of neuronal populations involves the Trk/MAP (mitogen-activated protein kinase) kinase signaling pathway. More recently, neurotrophins have also been shown to regulate synaptic transmission. The synapsins are a family of neuron-specific phosphoproteins that play a role in regulation of neurotransmitter release, in axonal elongation, and in formation and maintenance of synaptic contacts. We report here that synapsin I is a downstream effector for the neurotrophin/Trk/MAP kinase cascade. Using purified components, we show that MAP kinase stoichiometrically phosphorylated synapsin I at three sites (Ser-62, Ser-67, and Ser-549). Phosphorylation of these sites was detected in rat brain homogenates, in cultured cerebrocortical neurons, and in isolated presynaptic terminals. Brain-derived neurotrophic factor and nerve growth factor upregulated phosphorylation of synapsin I at MAP kinase-dependent sites in intact cerebrocortical neurons and PC12 cells, respectively, while KCl- induced depolarization of cultured neurons decreased the phosphorylation state at these sites. MAP kinase-dependent phosphorylation of synapsin I significantly reduced its ability to promote G-actin polymerization and to bundle actin filaments. The results suggest that MAP kinase-dependent phosphorylation of synapsin I may contribute to the modulation of synaptic plasticity by neurotrophins and by other signaling pathways that converge at the level of MAP kinase activation.

Impairment of synaptic vesicle clustering and of synaptic transmission, and increased seizure propensity, in synapsin I-deficient mice.

Synapsin I has been proposed to be involved in the modulation of neurotransmitter release by controlling the availability of synaptic vesicles for exocytosis. To further understand the role of synapsin I in the function of adult nerve terminals, we studied synapsin I-deficient mice generated by homologous recombination. The organization of synaptic vesicles at presynaptic terminals of synapsin I-deficient mice was markedly altered: densely packed vesicles were only present in a narrow rim at active zones, whereas the majority of vesicles were dispersed throughout the terminal area. This was in contrast to the organized vesicle clusters present in terminals of wild-type animals. Release of glutamate from nerve endings, induced by K+,4-aminopyridine, or a Ca2+ ionophore, was markedly decreased in synapsin I mutant mice. The recovery of synaptic transmission after depletion of neurotransmitter by high-frequency stimulation was greatly delayed. Finally, synapsin I-deficient mice exhibited a strikingly increased response to electrical stimulation, as measured by electrographic and behavioral seizures. These results provide strong support for the hypothesis that synapsin I plays a key role in the regulation of nerve terminal function in mature synapses.

Ca/calmodulin-dependent kinase II inhibitor KN62 attenuates glutamate release by inhibiting voltage-dependent Ca(2+)-channels.

The effect of KN62 (1-[N,O-bis(5-isoquinolinesulphonyl)-N -methyl-L-tyrosyl]-4-phenylpiperazine), a putative inhibitor of Ca/calmodulin-dependent kinase II (Ca/CaM-K II), on glutamate release from isolated nerve-terminals (synaptosomes) was examined. The drug caused a potent inhibition of KCl- and 4-aminopyridine-evoked glutamate release from isolated nerve-terminals (synaptosomes). Examination of the effect of the inhibitor on Ca(2+)-influx revealed that the diminution of glutamate release could be attributed to a decrease in cytosolic Ca. A direct effect of KN62 on synaptosomal Ca(2+)-channels was confirmed in experiments where Ba, which does not support CaM-dependent processes, was used in place of Ca. Additionally, whole-cell patch-clamping of cerebellar granule neurones directly demonstrated inhibition of Ca-currents by KN62. We therefore suggest that, in cellular systems, conclusions based on the use of KN62 as a Ca/CaM-K II blocker may be ambiguous and should be viewed with caution unless the effect of the drug on Ca-influx has also been quantified. The effect of KN62 on Ca(2+)-influx appears to be specific to slowly-or non-inactivating conductances, and therefore presents KN62 as a potentially useful tool in this context.

A role for calcineurin (protein phosphatase-2B) in the regulation of glutamate release.

Previous studies have shown that 4-aminopyridine (4AP) induced Ca-influx effects the release of glutamate from nerve terminals (synaptosomes) isolated from rat cerebral cortex. We now show that the Ca-dependent component of this release is potentiated by preincubation of the synaptosomes with the immunosuppressant, FK506, an inhibitor of protein phosphatase-2B (calcineurin). FK506 did not inhibit the Ca-independent release of glutamate from a cytosolic pool. Examination of the effect of FK506 on the influx of Ca elicited by 4AP indicated that inhibition of calcineurin activity resulted in an increase of voltage-dependent Ca-influx. Based on these results, we suggest that protein dephosphorylation effected by calcineurin may suppress voltage-dependent Ca-channel activity and in so doing inhibits evoked glutamate release. Activation of calcineurin produced by initial Ca-entry may represent a negative feedback to limit the activity of Ca-channels coupled to the release of glutamate.

Phosphorylation of synapsin I and MARCKS in nerve terminals is mediated by Ca2+ entry via an Aga-GI sensitive Ca2+ channel which is coupled to glutamate exocytosis.

Ca2+ entry is a prerequisite for both exocytosis and the phosphorylation of synapsin I and MARCKS proteins in mammalian cerebrocortical synaptosomes. The novel spider toxin Aga-GI completely blocks KCl-evoked glutamate exocytosis but only partially inhibits KCl-evoked cytoplasmic Ca2+ elevations, thus revealing at least two pathways for KCl-induced Ca2+ entry. Aga-GI completely attenuates KCl-induced phosphorylation of synapsin I and MARCKS proteins. We therefore conclude that both exocytosis and the phosphorylation of synapsin I and MARCKS proteins are specifically coupled to Ca2+ entry via a subset of voltage dependent Ca2+ channels at the nerve terminal which are sensitive to Aga-GI.

Glutamate exocytosis and MARCKS phosphorylation are enhanced by a metabotropic glutamate receptor coupled to a protein kinase C synergistically activated by diacylglycerol and arachidonic acid.

4-Aminopyridine evokes repetitive firing of synaptosomes and exocytosis of glutamate by inhibiting a dendrotoxin-sensitive K+ channel responsible for stabilizing the membrane potential. We have shown previously that activation of protein kinase C (PKC) by high concentrations of phorbol ester (4 beta-phorbol dibutyrate) can increase release by inhibiting a dendrotoxin-insensitive ion channel, whereas the metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate [(1S,3R)-ACPD] mimics the action of 4 beta-phorbol dibutyrate, but only in the presence of 2 microM arachidonic acid (AA). In this article, we investigate the role of AA. AA plus (1S,3R)-ACPD is without effect on KCl-induced glutamate exocytosis, indicating that the regulatory pathway acts upstream of the release-coupled Ca2+ channel or Ca(2+)-secretion coupling. Diacylglycerol concentrations are greatly enhanced by (1S,3R)-ACPD alone, independently of AA, indicating that AA acts downstream of phospholipase C. Myristoylated alanine-rich C kinase substrate (MARCKS) is the major presynaptic substrate for PKC. mGluR activation by (1S,3R)-ACPD enhances phosphorylation of MARCKS, but only in the presence of AA. These results strongly suggest that AA acts on presynaptic PKC synergistically with diacylglycerol generated by the phospholipase-coupled mGluR, consistent with the known behaviour of certain purified PKC isoforms. The magnitude of the effects observed in a population of rat cerebrocortical synaptosomes suggests that this is a major mechanism regulating the release of the brain's dominant excitatory neurotransmitter and supports the concept that AA, or a related compound with a similar locus of action, may in certain circumstances play a role in synaptic plasticity.

Barium evokes glutamate release from rat brain synaptosomes by membrane depolarization: involvement of K+, Na+, and Ca2+ channels.

During K(+)-induced depolarization of isolated rat brain nerve terminals (synaptosomes), 1 mM Ba2+ could substitute for 1 mM Ca2+ in evoking the release of endogenous glutamate. In addition, Ba2+ was found to evoke glutamate release in the absence of K(+)-induced depolarization. Ba2+ (1-10 mM) depolarized synaptosomes, as measured by voltage-sensitive dye fluorescence and [3H]-tetraphenylphosphonium cation distribution. Ba2+ partially inhibited the increase in synaptosomal K+ efflux produced by depolarization, as reflected by the redistribution of radiolabeled 86Rb+. The release evoked by Ba2+ was inhibited by tetrodotoxin (TTX). Using the divalent cation indicator fura-2, cytosolic [Ca2+] increased during stimulation by approximately 200 nM, but cytosolic [Ba2+] increased by more than 1 microM. Taken together, our results indicate that Ba2+ initially depolarizes synaptosomes most likely by blocking a K+ channel, which then activates TTX-sensitive Na+ channels, causing further depolarization, and finally enters synaptosomes through voltage-sensitive Ca2+ channels to evoke neurotransmitter release directly. Though Ba(2+)-evoked glutamate release was comparable in level to that obtained with K(+)-induced depolarization in the presence of Ca2+, the apparent intrasynaptosomal level of Ba2+ required for a given amount of glutamate release was found to be several-fold higher than that required of Ca2+.

Protein kinase C and the regulation of glutamate exocytosis from cerebrocortical synaptosomes.

The role of protein kinase C (PKC) in the regulation of transmitter glutamate release from rat cerebral cortical synaptosomes is investigated. Two depolarization protocols are used: first, elevated KCl, which produces a clamped depolarization, and second, 4-aminopyridine, which evokes spontaneous "action potentials" allowing any potential modulation of Na+ or K+ channels to influence release. Although the PKC inhibitor Ro 31-8220 prevents both the depolarization-evoked and phorbol dibutyrate (PDBu)-evoked phosphorylation of the major presynaptic PKC substrate, myristoylated alanine-rich C kinase substrate, it is without effect on KCl-evoked Ca(2+)-dependent glutamate release. Ro 31-8220 totally inhibits the Ca(2+)-dependent 4-aminopyridine-evoked release of glutamate in the presence and absence of PDBu and again decreases the phosphorylation of myristoylated alanine-rich C kinase substrate. Ro 31-8220 strongly inhibits the 4-aminopyridine-evoked increase in [Ca2+] both in the presence and absence of PDBu and antagonizes the PDBu enhancement of depolarization. This indicates that PKC isoforms activatable by PDBu and sensitive to Ro 31-8220 play no discernable role in Ca(2+)-secretion coupling per se in cerebral cortical glutamatergic nerve terminals, but that the kinase plays a major role in regulating the depolarization of the terminal.