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.     

Inhibition of Bradykinin-Induced Calcium Increase by Phosphatase Inhibitors in Neuroblastoma × Glioma Hybrid NG108-15 Cells

Abstract: Prior treatment of NG108-15 cells with phosphatase inhibitors including okadaic acid and calyculin A inhibited the elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) induced by bradykinin by ∼63%. This inhibition was dependent on the concentration of okadaic acid with an IC₅₀ of 0.15 n M . Okadaic acid treatment only lowered the maximal response of [Ca²⁺]_i increase and had no effect on the EC₅₀ value for bradykinin regardless of the presence of extracellular Ca²⁺. Neither the capacity of ⁴⁵Ca²⁺ accumulation within intracellular nonmitochondrial Ca²⁺ stores nor the magnitude of [Ca²⁺]_i increase induced by thapsigargin was reduced by the treatment of okadaic acid. In contrast, the same phosphatase inhibitor treatment inhibited the bradykinin-evoked inositol 1,4,5-trisphosphate (IP₃) generation, the Mn²⁺ influx, and the capacity of mitochondrial Ca²⁺ accumulation. Furthermore, the sensitivity of IP₃ in the Ca²⁺ release was suppressed by okadaic acid pretreatment. Our results suggest that the reduction of bradykinin-induced [Ca²⁺]_i rise by the promotion of protein phosphorylation was attributed to the reduced activity of phospholipase C, the decreased sensitivity to IP₃, and the slowed rate of Ca²⁺ influx. Thus, phosphorylation plays a role in bradykinin-sensitive Ca²⁺ signaling cascade in NG108-15 cells.     

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.     

Involvement of Calcineurin in Ca2+ Paradox-Like Injury of Cultured Rat Astrocytes

Abstract: The Ca²⁺/calmodulin-dependent phosphatase calcineurin may have physiological and pathological roles in neurons, but little is known about the roles of the enzyme in glial cells. We have previously reported that reperfusion of cultured astrocytes in Ca²⁺-containing medium after exposure to Ca²⁺-free medium caused Ca²⁺ influx followed by delayed cell death. In this study, we examined if calcineurin is involved in this Ca²⁺-mediated astrocytic injury. FK506, an inhibitor of calcineurin, protected cultured rat astrocytes against paradoxical Ca²⁺ challenge-induced injury in a dose-dependent manner (10⁻¹⁰–10⁻⁸ M ). Cyclosporin A at 1 µ M mimicked the effect of FK506. Rapamycin (1 µ M ) did not affect astrocyte injury, but it blocked the protective effect of FK506. Deltamethrin (20 n M ), another calcineurin inhibitor, had a similar protective effect, whereas okadaic acid did not. FK506 affected neither paradoxical Ca²⁺ challenge-induced increase in cytosolic Ca²⁺ level nor Na⁺-Ca²⁺ exchange activity in the cells, suggesting that the calcineurin is involved in processes downstream of increased cytosolic Ca²⁺ level. Immunochemical studies showed that both calcineurin A (probably the Aβ2 isoform) and B subunits were expressed in the cells. It is concluded that calcineurin is present in cultured astrocytes and it has a pathological role in the cells.     

Systemin triggers an increase of cytoplasmic calcium in tomato mesophyll cells: Ca2+ mobilization from intra- and extracellular compartments

We show here that, within 1–2 min of application, systemin triggers a transient increase of cytoplasmic free calcium concentration ([Ca²⁺]_c) in cells from Lycopersicon esculentum mesophyll. The systemin-induced Ca²⁺ increase was slightly but not significantly reduced by L-type Ca²⁺ channel blockers (nifedipine, verapamil and diltiazem) and the Ca²⁺ chelator [ethylene glycol tetraacetic acid (EGTA)], whereas inorganic Ca²⁺ channel blockers (LaCl₃, CdCl₂ and GdCl₃) and compounds affecting the release of intracellular Ca²⁺ from the vacuole (ruthenium red, LiCl, neomycin) strongly reduced the systemin-induced [Ca²⁺]_c increase. By contrast, no inhibitory effect was seen with the potassium and chloride channel blockers tested. Unlike systemin, other inducers of proteinase inhibitor (PI) and of wound-induced protein synthesis, such as jasmonic acid (JA) and bestatin, did not trigger an increase of cytoplasmic Ca²⁺. The systemin-induced elevation of cytoplasmic Ca²⁺ which might be an early step in the systemin signalling pathway, appears to involve an influx of extracellular Ca²⁺ simultaneously through several types of Ca²⁺ permeable channels, and a release of Ca²⁺ from intracellular stores sensitive to blockers of inositol 1,4,5-triphosphate (IP₃)- and cyclic adenasine 5'-diphosphoribose (cADPR)-mediated Ca²⁺ release.     

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.     

Regulation of synaptic vesicle accumulation and axon terminal remodeling during synapse formation by distinct Ca2+ signaling

The synaptic vesicle accumulation and subsequent morphological remodeling of axon terminals are characteristic features of presynaptic differentiation of zebrafish olfactory sensory neurons. The synaptic vesicle accumulation and axon terminal remodeling are regulated by protein kinase A and calcineurin signaling, respectively. To investigate upstream signals of presynaptic differentiation, we focused on Ca²⁺ signaling as Ca²⁺/calmodulin is required for the activation of both calcineurin and some adenylyl cyclases. We here showed that application of Ca²⁺/calmodulin inhibitor or olfactory sensory neuron-specific expression of calmodulin inhibitory peptide suppressed both synaptic vesicle accumulation and axon terminal remodeling. Thus, the trigger of presynaptic differentiation could be Ca²⁺ release from intracellular stores or Ca²⁺ influx. Application of a phospholipase C inhibitor or olfactory sensory neuron-specific expression of inositol 1,4,5-trisphosphate (IP₃) 5-phosphatase suppressed synaptic vesicle accumulation, but not morphological remodeling. In contrast, application of a voltage-gated Ca²⁺ channel blocker or expression of Kir2.1 inward rectifying potassium channel prevented the morphological remodeling. We also provided evidence that IP₃ signaling acted upstream of protein kinase A signaling. Our results suggest that IP₃-mediated Ca²⁺/calmodulin signaling stimulates synaptic vesicle accumulation and subsequent neuronal activity-dependent Ca²⁺/calmodulin signaling induces the morphological remodeling of axon terminals.     

Differential regulation of the Cdk5-dependent phosphorylation sites of inhibitor-1 and DARPP-32 by depolarization

While cyclin-dependent kinase 5 (Cdk5) is of growing importance to neuronal signaling, its regulation remains relatively unexplored. Examination of the mechanism by which NMDA modulates the phosphorylation of protein phosphatase inhibitor-1 at Ser6 and Ser67 and dopamine- and cAMP-regulated phosphoprotein M _r 32 000 at Thr75 revealed that generalized depolarization, rather than specific activation of NMDA receptors, was sufficient to induce decreases in these Cdk5 sites. Although no evidence for the involvement of the Cdk5 cofactors p35 or p39, or for L- and T-type voltage-gated Ca²⁺ channels, was found, evaluation of the role of phosphatases and extracellular cations revealed differential regulation of the three sites. NMDA-induced decreases in the phosphorylation of Thr75 of dopamine- and cAMP-regulated phosphoprotein M _r 32 000 required protein phosphatase 1/2A activity and extracellular Ca²⁺. In contrast, the effects on Ser6 and Ser67 of inhibitor-1 were not cation specific; either Na⁺ or Ca²⁺ sufficed. Furthermore, while the decrease in phosphorylation of Ser6 was partially dependent on protein phosphatase 2B, that of Ser67 was independent of the major protein serine/threonine phosphatases, likely indicating the presence of a pathway by which NMDA inhibits Cdk5 activity. Thus, in the striatum the regulation of phosphorylation of Cdk5-dependent sites by NMDA occurs through multiple distinct pathways.     

Desensitization of CholecystokininB Receptors in GH3 Cells

Abstract: Desensitization of the cholecystokinin (CCK) octapeptide (CCK-8)-induced rise in intracellular free calcium concentration ([Ca²⁺],) was characterized in GH₃ cells, a pituitary tumor cell line, which are known to possess CCK_B receptor subtype. The CCK-8-induced [Ca²⁺], transient was reduced following the initial application of CCK-8. A similar desensitization of the CCK-8-induced response was observed following the first application of thyrotropin-releasing hormone (TRH). By contrast, the TRH- induced response was not desensitized by the preceding application of CCK-8. Desensitization of the CCK-8-induced [Ca²⁺], transient was associated with diminished inositol 1,4,5-trisphosphate formation. The recovery of desensitization of the CCK-8-induced response was delayed by a phosphoserine/phosphothreonine phosphatase inhibitor, calyculin A (100 n M ). The responsiveness to CCK-8 was also reduced by phorbol 12, 13-dibutyrate (PDBu), and this effect of PDBu was completely abolished by preincubation with staurosporine. Staurosporine significantly attenuated the desensitization caused by preincubation with CCK-8, but this effect was too small to attribute the desensitization to the protein kinase C transduction pathway alone. It is likely that desensitization of CCK receptors involves multiple transduction pathways.     

Internalization of Voltage-Dependent Sodium Channels in Fetal Rat Brain Neurons: A Study of the Regulation of Endocytosis

Abstract: In fetal rat brain neurons, activation of voltage-dependent Na⁺ channels induced their own internalization, probably triggered by an increase in intracellular Na⁺ level. To investigate the role of phosphorylation in internalization, neurons were exposed to either activators or inhibitors of cyclic AMP- and cyclic GMP-dependent protein kinases, protein kinase C, and tyrosine kinase. None of the tested compounds mimicked or inhibited the effect of Na⁺ channel activation. An increase in intracellular Ca²⁺ concentration induced either by thapsigargin, a Ca²⁺-ATPase blocker, or by A23187, a Ca²⁺ ionophore, was unable to provoke Na⁺ channel internalization. However, Ca²⁺ seems to be necessary because both neurotoxin- and amphotericin B-induced Na⁺ channel internalizations were partially inhibited by BAPTA-AM. The selective inhibitor of Ca²⁺/calmodulin-dependent protein kinase II, KN-62, caused a dose-dependent inhibition of neurotoxin-induced internalization due to a blockade of channel activity but did not prevent amphotericin B-induced internalization. The rate of increase in Na⁺ channel density at the neuronal cell surface was similar before and after channel internalization, suggesting that recycling of internalized Na⁺ channels back to the cell surface was almost negligible. Pretreatment of the cells with an acidotropic agent such as chloroquine prevented Na⁺ channel internalization, indicating that an acidic endosomal/lysosomal compartment is involved in Na⁺ channel internalization in neurons.     

The possible involvement of protein phosphatase 1 in thrombin-induced Ca2+ influx of human platelets

Protein phosphatase 1 is considered to be involved in thrombin-induced platelet activation (Murata et al., Biochem Int 26:327–334, 1992). To clarify the mechanism, we examined the effects of protein phosphatase 1 and 2A inhibitors (calyculin A, tautomycin, okadaic acid) on Ca²⁺ influx. In the presence of 1 mM Ca²⁺, thrombin- (0.1 U/ml) induced platelet aggregation and ATP release were inhibited by calyculin A, while this inhibitory effect was abolished in the absence of Ca²⁺ (EGTA 1 mM). Furthermore, thrombin-induced Mn²⁺ influx but not intracellular Ca²⁺ mobilization was inhibited by calyculin A in a dose-related manner. Calyculin A also blocked the ongoing Ca²⁺ influx when added 3 min after thrombin stimulation. Similar inhibitory effects were observed with okadaic acid and tautomycin in the same potency sequence as the reported one for protein phosphatase 1 (calyculin A > tautomycin > okadaic acid). These results suggest that the anti-platelet effects of phosphatase inhibitors are due to the inhibition of Ca²⁺ influx and that protein phosphatase 1 plays a key role in the regulation of receptor operated Ca²⁺ channel of human platelets.     

Modulation of Ion Gradients and Glutamate Release in Cultured Cerebellar Granule Cells by Ouabain

Abstract: Upon addition of the cardiac glycoside ouabain to cultured cerebellar granule cells, an immediate increase in intracellular free sodium is evoked mediated by two pathways, a voltage-sensitive channel blocked by tetrodotoxin and a channel sensitive to flunarizine. Ouabain induces a steady plasma membrane depolarization in low Ca²⁺ medium; whereas in the presence of Ca²⁺, a distinct discontinuity is observed always preceded by a large increase in intracellular free Ca²⁺ ([Ca²⁺]_c). The plateau component of the increase can be inhibited additively by the L-type Ca²⁺ channel antagonist nifedipine, the spider toxin Aga-Gl, and the NMDA receptor antagonist MK-801. Single-cell imaging reveals that the [Ca²⁺]_c increase occurs asynchronously in the cell population and is not dependent on a critical level of extracellular glutamate or synaptic transmission between the cells. A prolonged release of glutamate is also observed that is predominantly Ca²⁺ dependent for the first 6–10 min after the evoked increase in [Ca²⁺]_c. This release is four times as large as that observed with 50 m M KCl and is predominantly exocytotic because release was inhibited by tetanus toxin, the V-type ATPase inhibitor bafilomycin, and Aga-Gl. It is proposed, therefore, that ouabain induces a period of membrane excitability culminating in a sustained exocytosis above that observed upon permanent depolarization with KCl.     

Signaling Pathway Downstream of GABAA Receptor in the Growth Cone

Abstract: The growth cone is responsible for axonal elongation and pathfinding by responding to various modulators for neurite growth, including neurotransmitters, although the sensor mechanisms are not fully understood. Among neurotransmitters, GABA is most likely to demonstrate activity in vivo because GABA and the GABA_A receptor appear even in early stages of CNS development. We investigated the GABA_A receptor-mediated signaling pathway in the growth cone using isolated growth cones (IGCs). Both the GABA_A binding site and the benzodiazepine modulatory site were enriched in the growth cone membrane. In the intact IGC, GABA induced picrotoxin-sensitive Cl⁻ flux (not influx but efflux) and increased the intracellular Ca²⁺ concentration in a picrotoxin- and verapamil-sensitive manner. Protein kinase C (PKC)-dependent phosphorylation of two proteins identified as GAP-43 and MARCKS protein was enhanced in the intact IGC stimulated by GABA, resulting in the release of MARCKS protein and GAP-43 from the membrane. Collectively, our results suggest the following scheme: activation of the functional GABA_A receptor localized in the growth cone membrane → Cl⁻ efflux induction through the GABA_A-associated Cl⁻ channel → Ca²⁺ influx through an L-type voltage-sensitive Ca²⁺ channel → Ca²⁺-dependent phosphorylation of GAP-43 and MARCKS protein by PKC.     

5'-(N-Ethylcarboxamido)adenosine Inhibits Ca2+ Influx and Activates a Protein Phosphatase in Bovine Adrenal Chromaffin Cells

Abstract: We investigated the effect of the adenosine receptor agonist 5'-( N -ethylcarboxamido)adenosine (NECA) in catecholamine secretion from adrenal chromaffin cells that exhibit only the A_2b subtype adenosine receptor. NECA reduced catecholamine release evoked by the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) in a time-dependent manner. Inhibition reached 25% after 30–40-min exposure to NECA. This effect on DMPP-evoked catecholamine secretion was mirrored by a similar (27.7 ± 3.3%), slowly developing inhibition of [Ca²⁺]_i transients induced by DMPP that peaked at 30-min preincubation with NECA. The capacity of the chromaffin cells to buffer Ca²⁺ load was not affected by the treatment with NECA. Short-term treatment with NECA failed both to modify [Ca²⁺]_i levels and to increase endogenous diacylglycerol production, showing that NECA does not activate the intracellular Ca²⁺/protein kinase C signaling pathway. The inhibitory effects of NECA were accompanied by a 30% increase of protein phosphatase activity in chromaffin cell cytosol. We suggest that dephosphorylation of a protein involved in DMPP-evoked Ca²⁺ influx pathway (e.g., L-type Ca²⁺ channels) could be the mechanism of the inhibitory action of adenosine receptor stimulation on catecholamine secretion from adrenal chromaffin cells.     

Cav1 L-type Ca2+ channel signaling complexes in neurons

Ca_v1 L-type Ca²⁺ channels play crucial and diverse roles in the nervous system. The pre- and post-synaptic functions of Ca_v1 channels not only depend on their intrinsic biophysical properties but also their dynamic regulation by a host of cellular influences. These include protein kinases and phosphatases, G-protein coupled receptors, scaffolding proteins, and Ca²⁺-binding proteins. The cytoplasmic domains of the main pore forming α₁ subunit of Ca_v1 offer a number of binding sites for these modulators, permitting fast and localized regulation of Ca²⁺ entry. Through effects on Ca_v1 gating, localization, and coupling to effectors, protein modulators are efficiently positioned to adjust Ca_v1 Ca²⁺ signals that control neuronal excitability, synaptic plasticity, and gene expression.     

Induced Ca2+ uptake and callose synthesis in suspension-cultured cells of Catharanthus roseus are decreased by the protein phosphatase inhibitor okadaic acid

Subtoxic concentrations of the saponin digitonin. the polyene antibiotic amphotericin B and the bacterial phytotoxin syringomycin induce increased uptake of ⁴⁵Ca²⁺ into suspension-cultured plant cells and a rapid Ca²⁺-dependent defense response, callose synthesis. Both reactions were inhibited by preincubation of the cells with okadaic acid, a specific inhibitor of type 1 and type 2A protein phosphatases. These results suggest that Ca²⁺ uptake induced by the above agents does not occur due to unspecific perturbation of plasma membrane permeability but involves transport proteins which are controlled by protein phosphorylation/dephosphorylation. Phosphoproteins appear also to be involved in the regulation of callose synthesis, although it remains open whether this control is effected at the level of Ca²⁺ transport or at the 1,3-ß-glucan synthase involved in deposition of the polymer.     

Ca2+-Dependent Enhancement of [3H]Dopamine Uptake in Rat Striatum: Possible Involvement of Calmodulin-Dependent Kinases

Abstract: We studied effects of Ca²⁺ in the incubation medium on [³H]dopamine ([³H]DA) uptake by rat striatal synaptosomes. Both the duration of the preincubation period with Ca²⁺ (0–30 min) and Ca²⁺ concentration (0–10 m M ) in Krebs-Ringer medium affected [³H]DA uptake by the synaptosomes. The increase was maximal at a concentration of 1 m M Ca²⁺ after a 10-min preincubation (2.4 times larger than the uptake measured without preincubation), which reflected an increase in V _max of the [³H]DA uptake process. On the other hand, [³H]DA uptake decreased rapidly after addition of ionomycin in the presence of 1 m M Ca²⁺. The Ca²⁺-dependent enhancement of the uptake was still maintained after washing synaptosomes with Ca²⁺-free medium following preincubation with 1 m M Ca²⁺. Protein kinase C inhibitors did not affect apparently Ca²⁺-dependent enhancement of the uptake, whereas 1-[ N,O -bis(1,5-isoquinolinesulfonyl)- N -methyl- l -tyrosyl]-4-phenylpiperazine (KN-62; a Ca²⁺/calmodulin-dependent kinase II inhibitor) and wortmannin (a myosin light chain kinase inhibitor) significantly reduced it. Inhibitory effects of KN-62 and wortmannin appeared to be additive. N -(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7; a calmodulin antagonist) also remarkably inhibited the enhancement. These results suggest that Ca²⁺-dependent enhancement of [³H]DA uptake is mediated by activation of calmodulin-dependent protein kinases.     

Contributions of Ca2+ and Zn2+ to spreading depression-like events and neuronal injury

The phenomenon of spreading depression (SD) involves waves of profound neuronal and glial depolarization that spread throughout brain tissue. Under many conditions, tissue recovers full function after SD has occurred, but SD-like events are also associated with spread of injury following ischemia or trauma. Initial large cytosolic Ca²⁺ increases accompany all forms of SD, but persistently elevated Ca²⁺ loading is likely responsible for neuronal injury following SD in tissues where metabolic capacity is insufficient to restore ionic gradients. Ca²⁺ channels are also involved in the propagation of SD, but the channel subtypes and cation fluxes differ significantly when SD is triggered by different types of stimuli. Ca²⁺ influx via P/Q type channels is important for SD generated by localized application of high K⁺ solutions. In contrast, SD-like events recorded in in vitro ischemia models are not usually prevented by Ca²⁺ removal, but under some conditions, Zn²⁺ influx via L-type channels contributes to SD initiation. This review addresses different roles of Ca²⁺ in the initiation and consequences of SD, and discusses recent evidence that selective chelation of Zn²⁺ can be sufficient to prevent SD under circumstances that may have relevance for ischemic injury.     

Involvement of Protein Kinase C in Ca2+-Signaling Pathways to Activation of AP-1 DNA-Binding Activity Evoked via NMDA- and Voltage-Gated Ca2+ Channels

Abstract: Stimulation of cultured cerebellar granule cells with N -methyl- d -aspartate (NMDA) or kainic acid (KA) leads to activation of activator protein-1 (AP-1) DNA-binding activity, which can be monitored by an increase in 12- O -tetradecanoylphorbol 13-acetate (TPA)-responsive element (TRE)-binding activity, in concert with c- fos induction. For this increase in TRE-binding activity, Ca²⁺ influx across the plasma membrane is essential. Treatment of cells with an intracellular Ca²⁺ chelator, BAPTA-AM, abolished this increase. Close correspondence between the dose-response curves of ⁴⁵Ca²⁺ uptake and TRE-binding activity by NMDA or KA suggested that Ca²⁺ influx not only triggered sequential activation of Ca²⁺-signaling processes leading to the increase in TRE-binding activity, but also controlled its increased level. Stimulation of non-NMDA receptors by KA mainly caused Ca²⁺ influx through voltage-gated Ca²⁺ channels, whereas stimulation of NMDA receptors caused Ca²⁺ influx through NMDA-gated ion channels. The protein kinase C (PKC) inhibitors staurosporine and calphostin C inhibited the increase in TRE-binding activity caused by NMDA and KA at the same concentration at which they inhibited that caused by TPA. Furthermore, down-regulation of PKC inhibited the increase in TRE-binding activity by NMDA and KA. Thus, a common pathway that includes PKC could, at least in part, be involved in the Ca²⁺-signaling pathways for the increase in TRE-binding activity coupled with the activation of NMDA- and non-NMDA receptors.