Barium Evokes Glutamate Release from Rat Brain Synaptosomes by Membrane Depolarization: Involvement of K+, Na+, and Ca2+ Channels

Abstract: During K⁺ -induced depolarization of isolated rat brain nerve terminals (synaptosomes), 1 m M Ba²⁺ could substitute for 1 m M 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 m M ) 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 n M , but cytosolic [Ba²⁺] increased by more than 1 μ M . 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 Ba²⁺-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⁺.     

Calmodulin Modulates Mitogen-Activated Protein Kinase Activation in Response to Membrane Depolarization in PC12 Cells

Abstract: In the absence of neurotrophic factors, chronic depolarization of plasma membrane has been shown to maintain several populations of primary neurons in culture. We report that in the PC12 cell line, depolarization causes Ca²⁺ influx through voltage-gated Ca²⁺ channels, which is able to stimulate extracellular-regulated kinase (ERK) activity. We studied which mediators were responsible for ERK activation resulting from increased levels of Ca²⁺ in the cytoplasm and found that calmodulin was involved in this process. The addition of W13, a calmodulin inhibitor, to the culture medium, prevented ERK activation when PC12 cells were depolarized. In addition, we show that high K⁺ treatment did not induce Trk A phosphorylation, thus excluding the possibility of Ca²⁺ operating through this receptor to activate the ERK signal transduction pathway. Moreover, although high K⁺ treatment is able to phosphorylate the epidermal growth factor receptor (EGFR) and thus to activate the ERK signal transduction pathway, we demonstrate that W13 did not alter the state of EGFR phosphorylation in conditions that almost completely blocked ERK activation. These data suggest that calmodulin mediates ERK activation induced by increases in intracellular Ca²⁺ concentration in PC12 cells by a mechanism that seems to be independent of Trk A and EGFR activation.     

Depolarization of Cerebellar Granule Cells Increases Phosphorylation of Rabphilin-3A

Abstract: Studies performed over the past several years have provided evidence that phosphorylation of proteins is important in the regulation of neurotransmitter release. In this study, it is shown that rabphilin-3A is present in cerebellar granule cells as a phosphoprotein, by using ³²P-labeling of cerebellar granule cells, immunoprecipitation, phosphoamino acid analysis, and phosphopeptide mapping. The level of phosphorylation was increased (224 ± 13%) (mean ± SEM) on depolarization of the cells with K⁺ (56 m M ) in the presence of external Ca²⁺ (1 m M ). Stimulation of protein kinase C with a phorbol ester (phorbol 12,13-dibutyrate) also enhanced the phosphorylation of rabphilin-3A (217 ± 21%). Inhibitors of Ca²⁺/calmodulin-stimulated protein kinases or protein kinase C reduced the depolarization-enhanced phosphorylation of rabphilin-3A, indicating that rabphilin-3A is one of the targets for Ca²⁺-activated protein kinases in the nerve terminal. Costimulation of cells with phorbol 12,13-dibutyrate and K⁺ depolarization produced an increased level of phosphorylation of rabphilin-3A compared with either stimulus alone (287 ± 61%). Phosphoamino acid analysis showed that serine was the main phosphorylated residue. A slight increase in the threonine phosphorylation could also be detected, whereas tyrosine phosphorylation could not be detected at all. These results suggest that rabphilin-3A is phosphorylated in vivo and undergoes synaptic activity-dependent phosphorylation during Ca²⁺-activated K⁺ depolarization.     

Astroglial Gap Junction Communication Is Increased by Treatment with Either Glutamate or High K+ Concentration

Abstract: Astroglia are extensively coupled by gap junctions and form a functional syncytium. Astroglial gap junctions are thought to be involved in the spatial buffering of K⁺ in vivo and in the Ca²⁺ waves seen on glutamate receptor activation. The conductivity of gap junctions is regulated by several second messengers, with up-regulation by cyclic AMP and down-regulation through activation of protein kinase C, decreases in intracellular pH, or increases in the free cytosolic Ca²⁺ concentration. The results presented here indicate that dye coupling of astroglia is significantly up-regulated by membrane depolarization, both by increases in the extracellular K⁺ concentration and directly by ionophores. Furthermore, glutamate, kainate, and quisqualate, which depolarize astroglial cells through activation of ionotropic receptors, also increase dye coupling in astroglia. The effect of kainate and quisqualate was reversed by 6-cyano-7-nitroquinoxaline-2,3-dione, an inhibitor of the ionotropic glutamate receptor. A dose-dependent decrease in dye coupling was seen when the cells were injected with increasing concentrations of Ca²⁺. However, if the cells were simultaneously depolarized, the inhibitory effect of Ca²⁺ on gap junctional conductance was reversed. Significant increases over basal coupling was attained when the cells were injected with Ca²⁺ if they were treated with kainate or K⁺. These data suggest that ligands that depolarize astroglia enhance gap junction communication between astroglia and that this enhancement may be important in maintaining communication between astroglia in the face of elevated Ca²⁺ levels.     

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

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

Inhibition of Voltage-Sensitive Calcium Channels by the A2A Adenosine Receptor in PC12 cells

Abstract: The role of the A_2A adenosine receptor in regulating voltage-sensitive calcium channels (VSCCs) was investigated in PC12 cells. Ca²⁺ influx induced by membrane depolarization with 70 m M K⁺ could be inhibited with CGS21680, an A_2A receptor-specific agonist. Both L- and N-type VSCCs were inhibited by CGS21680 treatment. Effects of adenosine receptor agonists and antagonists indicate that the typical A_2A receptor mediates inhibition of VSCCs. Cholera toxin (CTX) treatment for 24 h completely eliminated the CGS21680 potency. Similar inhibitory effects on VSCCs were obtained by membrane-permeable activators of protein kinase A (PKA). These effects were blocked by Rp -adenosine-3',5'-cyclic monophosphothioate, a PKA inhibitor. The data suggest that activation of the A_2A receptor leads to inhibition of VSCCs via a CTX-sensitive G protein and PKA. ATP pretreatment caused a reduction in subsequent rise in cytosolic free Ca²⁺ concentration induced by 70 m M K⁺, presumably by inactivation of VSCCs. Simultaneous treatment with ATP and CGS21680 produced significantly greater inhibition of VSCCs than treatment with CGS21680 or ATP alone. Furthermore, the CGS21680-induced inhibition of VSCCs was not affected by the presence of reactive blue 2. CGS21680 still significantly inhibited ATP-evoked Ca²⁺ influx without VSCC activity after cobalt or 70 m M K⁺ pretreatment. These data suggest that the A_2A receptor-sensitive VSCCs differ from those activated by ATP treatment. Although A_2A receptors induce inhibition of VSCCs as well as ATP-induced Ca²⁺ influx, the two inhibitory effects are clearly distinct from each other.     

Adenosine A1 Receptor Inhibition of Glutamate Exocytosis and Protein Kinase C-Mediated Decoupling

Abstract: The adenosine modulation of glutamate exoeytosis from guinea pig cerebrocortical synaptosomes is investigated. Endogenously leaked adenosine is sufficient to cause a partial tonic inhibition of 4-aminopyridine-evoked glutamate release, which can be relieved by adenosine deaminase. The adenosine A₁ receptor is equally effective in mediating inhibition of glutamate exocytosis evoked by 4-aminopyridine (where K⁺-channel activation would inhibit release) and by elevated KC1 (where K⁺-channel activation would have no effect), arguing for a central role of Ca²⁺-channel modulation. In support of this, the plateau phase of depolarization-evoked free Ca²⁺ elevation is decreased by adenosine with both depolarization protocols. No effect of adenosine agonists is seen on membrane potential in polarized or KC1- or 4-aminopyridine-stimulated synaptosomes. The interaction of protein kinase C with the A₁ receptormediated inhibition is examined. Activation of protein kinase C by 4β-phorbol dibutyrate has been shown previously by this laboratory to modulate glutamate release via K⁺-channel inhibition, and is shown here to have an additional action of decoupling the adenosine inhibition of glutamate exocytosis.     

Presynaptic Mechanism of Action of 4-Aminopyridine: Changes in Intracellular Free Ca2+ Concentration and Its Relationship to B-50 (GAP-43) Phosphorylation

Abstract: Recently we have shown that 4-aminopyridine (4-AP), a drug known to enhance transmitter release, stimulates the phosphorylation of the protein kinase C substrate B-50 (GAP-43) in rat brain synaptosomes and that this effect is dependent on the presence of extracellular Ca²⁺. Hence, we were interested in the relationship between changes induced by 4-AP in the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and B-50 phosphorylation in synaptosomes. 4-AP (100 μ M ) elevates the [Ca²⁺]_i (as determined with fura-2) to approximately the same extent as depolarization with 30 m M K⁺ (from an initial resting level of 240 n M to ∼480 n M after treatment). However, the underlying mechanisms appear to be different: In the presence of 4-AP, depolarization with K⁺ still evoked an increase in [Ca²⁺]_i, which was additive to the elevation caused by 4-AP. Several Ca²⁺ channel antagonists (CdCl₂, LaCl₃, and diphenylhydantoin) inhibited the increase in B-50 phosphorylation by 4-AP. It is interesting that the increase in [Ca²⁺]_i and the increase in B-50 phosphorylation by 4-AP were attenuated by tetrodotoxin, a finding pointing to a possible involvement of Na⁺ channels in this action. These results suggest that 4-AP (indirectly) stimulates both Ca²⁺ influx and B-50 phosphorylation through voltage-dependent channels by a mechanism dependent on Na⁺ channel activity.     

Agents that Promote Protein Phosphorylation Inhibit the Activity of the Na+/Ca2+ Exchanger and Prolong Ca2+ Transients in Bovine Chromaffin Cells

Abstract: The Na⁺/Ca²⁺ exchanger is an important element in the maintenance of calcium homeostasis in bovine chromaffin cells. The Na⁺/Ca²⁺ exchanger from other cell types has been extensively studied, but little is known about its regulation in the cell. We have investigated the role of reversible protein phosphorylation in the activity of the Na⁺/Ca²⁺ exchanger of these cells. Cells treated with 1 m M dibutyryl cyclic AMP (dbcAMP), 1 µ M phorbol 12,13-dibutyrate, 1 µ M okadaic acid, or 100 n M calyculin A showed lowered Na⁺/Ca²⁺ exchange activity and prolonged cytosolic Ca²⁺ transients caused by depolarization. A combination of 10 n M okadaic acid and 1 µ M dbcAMP synergistically inhibited Na⁺/Ca²⁺ exchange activity. Conversely, 50 µ M 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a protein kinase inhibitor, enhanced Na⁺/Ca²⁺ exchange activity. Moreover, we used cyclic AMP-dependent protein kinase and calcium phospholipid-dependent protein kinase catalytic subunits to phosphorylate isolated membrane vesicles and found that the Na⁺/Ca²⁺ exchange activity was inhibited by this treatment. These results indicate that reversible protein phosphorylation modulates the activity of the Na⁺/Ca²⁺ exchanger and suggest that modulation of the exchanger may play a role in the regulation of secretion.     

The fou2 mutation in the major vacuolar cation channel TPC1 confers tolerance to inhibitory luminal calcium

The SV channel encoded by the TPC1 gene represents a Ca²⁺- and voltage-dependent vacuolar cation channel. Point mutation D454N within TPC1 , named fou2 for fatty acid oxygenation upregulated 2 , results in increased synthesis of the stress hormone jasmonate. As wounding causes Ca²⁺ signals and cytosolic Ca²⁺ is required for SV channel function, we here studied the Ca²⁺-dependent properties of this major vacuolar cation channel with Arabidopsis thaliana mesophyll vacuoles. In patch clamp measurements, wild-type and fou2 SV channels did not exhibit differences in cytosolic Ca²⁺ sensitivity and Ca²⁺ impermeability. K⁺ fluxes through wild-type TPC1 were reduced or even completely faded away when vacuolar Ca²⁺ reached the 0.1-m m level. The fou2 protein under these conditions, however, remained active. Thus, D454N seems to be part of a luminal Ca²⁺ recognition site. Thereby the SV channel mutant gains tolerance towards elevated luminal Ca²⁺. A three-fold higher vacuolar Ca/K ratio in the fou2 mutant relative to wild-type plants seems to indicate that fou2 can accumulate higher levels of vacuolar Ca²⁺ before SV channel activity vanishes and K⁺ homeostasis is impaired. In response to wounding fou2 plants might thus elicit strong vacuole-derived cytosolic Ca²⁺ signals resulting in overproduction of jasmonate.     

Involvement of Intracellular Stores in the Ca2+ Responses to N-Methyl-D-Aspartate and Depolarization in Cerebellar Granule Cells

Abstract: The [Ca²⁺]₁ of cerebellar granule cells can be increased in a biphasic manner by addition of NMDA or by depolarization (induced by elevating the extracellular K⁺ level), which both activate Ca²⁺ influx. The possibility that these stimuli activate Ca²⁺-induced Ca²⁺ release was investigated using granule cells loaded with fura 2-AM. Dantrolene, perfused onto groups of cells during the sustained plateau phase of the [Ca²⁺]₁ response to K⁺ or NMDA, was found to reduce the response to both agents in a concentration-dependent manner. Preincubation with thapsigargm (10 μ M ) substantially reduced the plateau phase of the [Ca²⁺], response to K⁺ and both the peak and plateau phases of the NMDA response. Preincubation with ryanodine (10 μ M ) also reduced both the K⁺-evoked plateau response and both phases of the NMDA response. Neither had a consistent effect on the peak response to K⁺. The effects of thapsigargin and ryanodine on the NMDA response were partially additive. These results demonstrate that in cerebellar granule cells a major component of both K⁺- and NMDA-induced elevation of [Ca²⁺]₁ appears to be due to release from intracellular stores. The partial additivity of the effects of thapsigargin and ryanodine suggests that these agents affect two overlapping but nonidentical Ca²⁺ pools.     

A calcium- and phospholipid-dependent protein kinase from rice (Oryza sativa) leaves

Protein kinases in plants have not been examined in detail, but protein phosphorylation has been shown to be essential for regulating plant growth via the signal transduction system. A Ca²⁺- and phospholipid-dependent protein kinase, possibly involved in the intracellular signal transduction system from rice leaves, was partially purified by sequential chromatography on DE52, Phenyl Superose and Superose 12. This protein kinase phosphorylated the substrate, histone III-S, in the presence of Ca²⁺ and phosphatidylserine. The apparent molecular mass of the Ca²⁺- and phosphatidylserine-dependent protein kinase (Ca²⁺/PS PK), determined by phosphorylation in SDS-polyacrylamide gel containing histone III-S, was 50 kDa. The protein kinase differed from Ca²⁺-dependent protein kinase (CDPK) in rice leaves in that Ca²⁺/PS PK showed phospholipid dependency and the molecular mass of Ca²⁺/PS PK exceeded that of CDPK. Investigations were carried out on changes in Ca²⁺/PS PK and CDPK activity in the cytosolic and membrane fractions during germination. The maximum activity of Ca²⁺/PS PK in the cytosolic fraction was observed before imbibition and that of CDPK in the membrane fraction was noted at 6 days following imbibition. Protein kinases are likely to regulate plant growth through protein phosphorylation.     

Phosphorylation status of the NR2B subunit of NMDA receptor regulates its interaction with calcium/calmodulin-dependent protein kinase II

Ca²⁺ influx through NMDA-type glutamate receptor at excitatory synapses causes activation of post-synaptic Ca²⁺/calmodulin-dependent protein kinase type II (CaMKII) and its translocation to the NR2B subunit of NMDA receptor. The major binding site for CaMKII on NR2B undergoes phosphorylation at Ser1303, in vivo . Even though some regulatory effects of this phosphorylation are known, the mode of dephosphorylation of NR2B-Ser1303 is still unclear. We show that phosphorylation status at Ser1303 enables NR2B to distinguish between the Ca²⁺/calmodulin activated form and the autonomously active Thr286-autophosphorylated form of CaMKII. Green fluorescent protein–α-CaMKII co-expressed with NR2B sequence in human embryonic kidney 293 cells was used to study intracellular binding between the two proteins. Binding in vitro was studied by glutathione- S -transferase pull-down assay. Thr286-autophosphorylated α-CaMKII or the autophosphorylation mimicking mutant, T286D-α-CaMKII, binds NR2B sequence independent of Ca²⁺/calmodulin unlike native wild-type α-CaMKII. We show enhancement of this binding by Ca²⁺/calmodulin. Phosphorylation or a phosphorylation mimicking mutation on NR2B (NR2B-S1303D) abolishes the Ca²⁺/calmodulin-independent binding whereas it allows the Ca²⁺/calmodulin-dependent binding of α-CaMKII in vitro . Similarly, the autonomously active mutants, T286D-α-CaMKII and F293E/N294D-α-CaMKII, exhibited Ca²⁺-independent binding to non-phosphorylatable mutant of NR2B under intracellular conditions. We also show for the first time that phosphatases in the brain such as protein phosphatase 1 and protein phosphatase 2A dephosphorylate phospho-Ser1303 on NR2B.     

Tyrosine Hydroxylase Phosphorylation in Digitonin-Permeabilized Bovine Adrenal Chromaffin Cells: The Effect of Protein Kinase and Phosphatase Inhibitors on Ser19 and Ser40 Phosphorylation

Abstract: The protein kinases and protein phosphatases that act on tyrosine hydroxylase in vivo have not been established. Bovine adrenal chromaffin cells were permeabilized with digitonin and incubated with [γ-³²P]ATP, in the presence or absence of 10 µ M Ca²⁺, 1 µ M cyclic AMP, 1 µ M phorbol dibutyrate, or various kinase or phosphatase inhibitors. Ca²⁺ increased the phosphorylation of Ser¹⁹ and Ser⁴⁰. Cyclic AMP, and phorbol dibutyrate in the presence of Ca²⁺, increased the phosphorylation of only Ser⁴⁰. Ser³¹ and Ser⁸ were not phosphorylated. The Ca²⁺-stimulated phosphorylation of Ser¹⁹ was incompletely reduced by inhibitors of calcium/calmodulin-stimulated protein kinase II (46% with KN93 and 68% with CaM-PKII 273–302), suggesting that another protein kinase(s) was contributing to the phosphorylation of this site. The Ca²⁺-stimulated phosphorylation of Ser⁴⁰ was reduced by specific inhibitors of protein kinase A (56% with H89 and 38% with PKAi 5–22 amide) and protein kinase C (70% with Ro 31-8220 and 54% with PKCi 19–31), suggesting that protein kinases A and C contributed to most of the phosphorylation of this site. Results with okadaic acid and microcystin suggested that Ser¹⁹ and Ser⁴⁰ were dephosphorylated by PP2A.     

Ca2+/Calmodulin-Dependent Transcriptional Activation of δ-Opioid Receptor Gene Expression Induced by Membrane Depolarization in NG108-15 Cells

Abstract: Regulation of gene expression is one of the mechanisms by which neuronal activity elicits long-term changes in neuronal phenotype and function. Although activity-dependent induction of immediate-early genes has been extensively studied, much less is known about the late-response genes. We have investigated the activity-dependent regulation of δ-opioid receptor (DOR) mRNA levels in NG108-15 cells. Transsynaptic activation was mimicked by depolarization with 55 m M KCl or veratridine. Both treatments lead to a time-dependent increase of DOR mRNA levels. Ca²⁺ entry through L-type voltage-dependent Ca²⁺ channels activated by depolarization appears to be involved, because L-type channel blockers reduced the induction of DOR expression. Ca²⁺ binding to calmodulin is the next step in the signal transduction pathway, because a calmodulin antagonist, W7, reduced the effect of veratridine. A selective inhibitor of calmodulin kinases (KN-62) and cyclosporin, an inhibitor of calcineurin, also antagonized the depolarization-induced increase in DOR mRNA levels, which indicates that both calcium/calmodulin-dependent enzymes are involved in the activity-dependent induction of DOR gene expression. Induction of DOR gene expression by an activity-dependent increase in intracellular Ca²⁺ concentration may serve as a feedback regulatory mechanism because activation of DOR leads to hyperpolarization and lower excitability of neurons.     

Basic Protein in Brain Myelin Is Phosphorylated by Endogenous Phospholipid-Sensitive Ca2+-Dependent Protein Kinase

Abstract: Phosphorylation of myelin basic protein (MBP) in rat or rabbit brain myelin was markedly stimulated by Ca²⁺, and this reaction was not essentially augmented by exogenous phosphatidylserine or calmodulin or both. Solubilization of myelin with 0.4% Triton X-100 plus 4 m M EGTA, with or without further fractionation, showed that Ca²⁺-dependent phosphorylation of MBP required phosphatidylserine, but not calmodulin. DEAE-cellulose chromatography of solubilized myelin revealed a pronounced peak of protein kinase activity stimulated by a combination of Ca²⁺ and phosphatidylserine; a protein kinase stimulated by Ca²⁺ plus calmodulin was not detected. These findings clearly indicate an involvement of phospholipid-sensitive Ca²⁺-dependent protein kinase in phosphorylation of brain MBP, although a possible role for the calmodulin-sensitive species of Ca²⁺-dependent protein kinase in this reaction could not be excluded or established. Phosphorylation of MBP in solubilized rat myelin catalyzed by the phospholipid-sensitive enzyme was inhibited by adriamycin, palmitoylcarnitine, trifluoperazine, melittin, polymyxin B, and N -(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide (W–7).     

Direct Observation of Serotonin 5-HT3 Receptor-Induced Increases in Calcium Levels in Individual Brain Nerve Terminals

Abstract: Confocal microscopy was used to assess internal calcium level changes in response to presynaptic receptor activation in individual, isolated nerve terminals (synaptosomes) from rat corpus striatum, focusing, in particular, on the serotonin 5-HT₃ receptor, a ligand-gated ion channel. The 5-HT₃ receptor agonist-induced calcium level changes in individual synaptosomes were compared with responses evoked by K⁺ depolarization. Using the fluorescent dye fluo-3 to measure relative changes in internal free Ca²⁺ concentration ([Ca²⁺]_i), K⁺-induced depolarization resulted in variable but rapid increases in apparent [Ca²⁺]_i among the individual terminals, with some synaptosomes displaying large transient [Ca²⁺]_i peaks of varying size (two- to 12-fold over basal levels) followed by an apparent plateau phase, whereas others displayed only a rise to a sustained plateau level of [Ca²⁺]_i (two- to 2.5-fold over basal levels). Agonist activation of 5-HT₃ receptors induced slow increases in [Ca²⁺]_i (rise time, 15–20 s) in a subset (∼5%) of corpus striatal synaptosomes, with the increases (averaging 2.2-fold over basal) being dependent on Ca²⁺ entry and inhibited by millimolar external Mg²⁺. We conclude that significant increases in brain nerve terminal Ca²⁺, rivaling that found in response to excitation by depolarization but having distinct kinetic properties, can therefore result from the activation of presynaptic ligand-gated ion channels.     

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

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

Glutamate Induces a Calcineurin-Mediated Dephosphorylation of Na+,K+-ATPase that Results in Its Activation in Cerebellar Neurons in Culture

Abstract: In primary cultures of cerebellar neurons glutamate neurotoxicity is mainly mediated by activation of the NMDA receptor, which allows the entry of Ca²⁺ and Na⁺ into the neuron. To maintain Na⁺ homeostasis, the excess Na⁺ entering through the ion channel should be removed by Na⁺,K⁺-ATPase. It is shown that incubation of primary cultured cerebellar neurons with glutamate resulted in activation of the Na⁺,K⁺-ATPase. The effect was rapid, peaking between 5 and 15 min (85% activation), and was maintained for at least 2 h. Glutamate-induced activation of Na⁺,K⁺-ATPase was dose dependent: It was appreciable (37%) at 0.1 µ M and peaked (85%) at 100 µ M . The increase in Na⁺,K⁺-ATPase activity by glutamate was prevented by MK-801, indicating that it is mediated by activation of the NMDA receptor. Activation of the ATPase was reversed by phorbol 12-myristate 13-acetate, an activator of protein kinase C, indicating that activation of Na⁺,K⁺-ATPase is due to decreased phosphorylation by protein kinase C. W-7 or cyclosporin, both inhibitors of calcineurin, prevented the activation of Na⁺,K⁺-ATPase by glutamate. These results suggest that activation of NMDA receptors leads to activation of calcineurin, which dephosphorylates an amino acid residue of the Na⁺,K⁺-ATPase that was previously phosphorylated by protein kinase C. This dephosphorylation leads to activation of Na⁺,K⁺-ATPase.     

N-Terminal-Specific Anti-B-50 (GAP-43) Antibodies Inhibit Ca2+-Induced Noradrenaline Release, B-50 Phosphorylation and Dephosphorylation, and Calmodulin Binding

Abstract: B-50 (GAP-43) is a presynaptic protein kinase C (PKC) substrate implicated in the molecular mechanism of noradrenaline release. To evaluate the importance of the PKC phosphorylation site and calmodulin-binding domain of B-50 in the regulation of neurotransmitter release, we introduced two monoclonal antibodies to B-50 into streptolysin O-permeated synaptosomes isolated from rat cerebral cortex. NM2 antibodies directed to the N-terminal residues 39–43 of rat B-50 dose-dependently inhibited Ca²⁺-induced radiolabeled and endogenous noradrenaline release from permeated synaptosomes. NM6 C-terminal-directed (residues 132–213) anti-B-50 antibodies were without effect in the same dose range. NM2 inhibited PKC-mediated B-50 phosphorylation at Ser⁴¹ in synaptosomal plasma membranes and permeated synaptosomes, inhibited ³²P-B-50 dephosphorylation by endogenous synaptosomal phosphatases, and inhibited the binding of calmodulin to synaptosomal B-50 in the absence of Ca²⁺. Similar concentrations of NM6 did not affect B-50 phosphorylation or dephosphorylation or B-50/calmodulin binding. We conclude that the N-terminal residues 39–43 of the rat B-50 protein play an important role in the process of Ca²⁺-induced noradrenaline release, presumably by serving as a local calmodulin store that is regulated in a Ca²⁺- and phosphorylation-dependent fashion.