In vitro calcium and calmodulin-dependent kinase-mediated phosphorylation of rat brain and spinal cord neurofilament proteins is increased by glycidamide administration

This study was carried out to determine the action of glycidamide (2,3-epoxy-1-propanamide), a neurotoxic metabolite of acrylamide, on Ca²⁺/calmodulin (CaM)-dependent protein kinase phosphorylation of cytoskeletal proteins. Acrylamide has been shown to increase Ca²⁺/CaM-dependent phosphorylation of neurofilament (NF) triplet proteins and autophosphorylation of Ca²⁺/CaM-dependent protein kinase II (CaM kinase II; EC 2.7.1.37). A daily intraperitoneal dose of 0.7 mmol/kg b.wt. of glycidamide or deionized water was administered to male Sprague-Dawley rats. Animals were sacrificed when signs of severe neurotoxicity became apparent at 13–16 days of treatment. Axonal floatation was used to isolate neurofilaments (NFs) and endogenous kinases from brains and spinal cords of treated and control animals. Samples isolated from brain and spinal cord of glycidamide-treated animals showed increased in vitro Ca²⁺/CaM-dependent phosphorylation of endogenous and exogenous NF proteins and increased autophosphorylation of CaM kinase II when compared with controls. CaM binding to the α, β, and β′ subunits of CaM kinase II and antibody binding to the α-subunit of CaM kinase II in brain supernatant isolates was increased as a result of glycidamide treatment. These results suggest that increased Ca^2+/CaM-dependent phosphorylation of cytoskeletal proteins may be involved in the pathogenesis of glycidamide-induced neurotoxicity.     

Inhibitory effect of regucalcin on Ca-dependent protein kinase activity in rat brain cytosol: involvement of endogenous regucalcin

The effect of regucalcin, a Ca²⁺-binding protein, on Ca²⁺-dependent protein kinase activity in the brain cytosol of rats with different ages (5 and 50 weeks old) was investigated. The addition of calmodulin (10 μg/ml) or dioctanoylglycerol (5 μg/ml) in the enzyme reaction mixture caused a significant increase in protein kinase activity in the presence of CaCl₂ (1 mM), indicating that Ca²⁺ calmodulin or protein kinase C is present in the cytosol. Such an increase was completely prevented by the addition of regucalcin (10⁻⁷ M). Moreover, regucalcin (10⁻⁷ M) significantly inhibited cytosolic protein kinase activity without Ca²⁺/calmodulin or dioctanoylglycerol addition. Meanwhile, the presence of anti-regucalcin monoclonal antibody (10–50 ng/ml) in the enzyme reaction mixture caused a significant elevation of protein kinase activity, suggesting an inhibitory effect of endogenous regucalcin. Brain cytosolic protein kinase activity was significantly elevated by increasing age (50-week-old rats). Also, regucalcin (10⁻⁷ M) significantly decreased protein kinase activity without Ca²⁺ addition in the brain cytosol of aged rats. However, the effect of anti-regucalcin monoclonal antibody (50 ng/ml) in elevating protein kinase activity was not seen in the brain cytosol of aged rats. These results suggest that regucalcin has an inhibitory effect on Ca²⁺-dependent protein kinase activity in rat brain cytosol, and that the effect of endogenous regucalcin may be weakened in the brain cytosol of aged rats.     

4-Aminopyridine stimulates B-50 (GAP-43) phosphorylation in rat synaptosomes

Recently, we have shown that stimulation of [³H]-noradrenaline release from hippocampal slices by 4-aminopyridine (4-AP) is accompanied by an enhancement of the phosphorylation of B-50, a major presynaptic substrate of protein kinase C (PKC). PKC has been implicated in the regulation of transmitter release. In this study, we investigated the effects of 4-AP on B-50 phosphorylation in synaptosomes from rat brain and compared the effects of 4-AP with those of depolarization with K⁺, in order to gain more insight into the mechanism of action of 4-AP. B-50 phosphorylation was stimulated by incubation with 4-AP for 2 minutes at concentrations ranging from 10 μM to 5 mM. 4-AP (100 μM) stimulated B-50 phosphorylation already within 15 seconds; longer incubations revealed a sustained increase in the presence of 4-AP. B-50 phosphorylation was also stimulated by depolarization with 30 mM K⁺ for 15 seconds. The effects of both 4-AP or K⁺ depolarization on B-50 phosphorylation were abolished at low extracellular Ca²⁺ concentrations. The increase in B-50 phosphorylation induced by 4-AP seemed to be dependent on the state of depolarization, since the effect of 4-AP was largest under nondepolarizing conditions. Comparing the effects of 4-AP and K⁺ depolarization on B-50 phosphorylation suggests that a different mechanism of action is involved. These results indicate that the stimulation of B-50 phosphorylation by 4-AP in hippocampal slices can be attributed to a direct action of 4-AP on presynaptic terminals. In addition, our results support the hypothesis that B-50 phosphorylation by PKC is involved in Ca²⁺-dependent transmitter release evoked by 4-AP. This research was supported by CLEO-TNO grant A66 of the Dutch Epilepsy Foundation.     

Inhibition of noradrenaline stimulated increase in [Ca]i in cultured astrocytes by chronic treatment with a therapeutically relevant lithium concentration

Chronic treatment of mouse astrocytes in primary cultures with 1 mM lithium chloride for 7–14 days decreased the basal level of free cytosolic calcium concentration ([Ca²⁺]_i) from 50–70 nM to 70% of this value and reduced the increase in [Ca²⁺]_i caused by exposure to 1 μM noradrenaline (normally to 500–700 nM) by almost one half. A similar, but much smaller, response to serotonin was unaffected by chronic treatment with lithium. Acute exposure to lithium (30 min) had no effect on either basal or noradrenaline stimulated [Ca²⁺]_i The dependence on chronic, versus acute treatment suggests that this effect may be related to the therapeutic effect of lithium as a mood-stabilizing drug, which likewise requires chronic treatment. Since good evidence is found that noradrenaline increases [Ca²⁺]_i by activation of the phosphoinositol second messenger system the present findings are also consistent with literature data that lithium acts by interfering with this system.     

ATP-induced arachidonic acid release in cultured astrocytes is mediated by Gi protein coupled P2Y1 and P2Y2 receptors

ATP-induced arachidonic acid (AA) release was studied in [³H]AA-prelabeled cultured astrocytes. To characterize the P₂ purinoceptor-mediated effect of ATP, the subtype-specific agonists 2-methylthio ATP (2-MeSATP) and UTP were compared. ATP, UTP, or 2-MeSATP induced a dose-dependent increase of [³H]AA release, with EC₅₀ values of 22.7 μM, 29.4 μM, and 1.68 μM, respectively; α,β-methylene ATP and adenosine had no effect. The order of potency was ATP = UTP ≥ 2-MeSATP, indicating that ATP interacted with both P2Y₁ and P2Y₂ receptors to mediate AA release in astrocytes. The effect of ATP, UTP, or 2-MeSATP was markedly inhibited by pretreatment of cells with pertussis toxin. Ca²⁺ ionophore-A23187 and PKC activator-TPA mimicked the effects of these three agonists to stimulate AA release. ATP, UTP, and 2-MeSATP induced a rapidly initial rise of [Ca²⁺]_i and a sustained [Ca²⁺]_i increase. The AA release was blocked in the external Ca²⁺ free in condition the sustained [Ca²⁺]_i increase was abolished. Both A23187- and TPA-induced AA release were also blocked in this condition. Furthermore, inorganic Ca²⁺ channel blocker Co²⁺ inhibited ATP, UTP, or 2-MeSATP induced AA release as well. Long-term (24 h) treatment of cells with TPA resulted in an attenuation of three agonists, TPA or A23187 response. Similarly, ATP or TPA promoted AA release was inhibited by the mitogen-activated protein kinase (MAPK) cascade inhibitor PD 98059. ATP, TPA, or A23187 induced an increase in the activity and tyrosine phosphorylation of p42 MAPK, as well as a molecular weight shift, consistent with phosphorylation, of cytosolic phospholipase A₂ (cPLA₂). ATP- and TPA-stimulated activation of p42 MAPK activity and tyrosine phosphorylation were inhibited by long-term TPA treatment, while A23187-stimulated effects were completely blocked. Furthermore, tyrosine phosphorylation and activation of p42 MAPK and mobility shift of cPLA₂ induced by A23187 were reversed in the absence of external Ca²⁺, suggesting the involvement of PKCα in MAPK activation and mobility shift of cPLA₂. Taken together, ATP-stimulated AA release was secondary to the activation of P2Y₁ and P2Y₂ receptors/PLC pathway. Ca²⁺ and PKC interact to regulate this response. Elevation of intracellular Ca²⁺, the mechanism involving extracellular Ca²⁺ influx, might act partly through PKCα activation and in turn MAPK might be activated, leading to cPLA₂ phosphorylation and AA release. GLIA 22:360–370, 1998. © 1998 Wiley-Liss, Inc.     

Effects of blockade of voltage-sensitive Ca/Na channels by a novel phenylpyrimidine derivative, NS-7, on CREB phosphorylation in focal cerebral ischemia in the rat

NS-7 is a novel blocker of voltage-sensitive Ca²⁺ and Na⁺ channels, and it significantly reduces infarct size after occlusion of the middle cerebral artery. Persistent activation of cyclic AMP response element binding protein (CREB), which can be induced by increase in intracellular Ca²⁺ concentrations or other second messengers, has recently been found to be closely associated with neuronal survival in cerebral ischemia. The present study was therefore undertaken to evaluate the neuroprotective effects of NS-7 by analyzing changes in CREB phosphorylation in a focal cerebral ischemia model. CREB phosphorylation in the brain of rats was investigated immunohistochemically at 3.5–48-h recirculation after 1.5-h occlusion of the middle cerebral artery. NS-7 (1 mg/kg; NS-7 group) or saline (saline group) was intravenously injected 5 min after the start of recirculation. The NS-7 group showed significantly milder activation of CREB phosphorylation in various cortical regions after 3.5 h of recirculation than the saline group. The inner border zone of ischemia in the NS-7 group subsequently exhibited a moderate, but persistent, increase in number of phosphorylated CREB-positive neurons with no apparent histological damage. By contrast, the saline group displayed a marked, but only transient, increase in number of immunopositive neurons in this border zone after 3.5 h of recirculation, and this was followed by clear suppression of CREB phosphorylation and subsequent loss of normal neurons. These findings suggest that: (1) the marked enhancement of CREB phosphorylation in the acute post-ischemic phase may be triggered largely by an influx of calcium ions as a result of activation of the voltage-sensitive Ca²⁺ and Na⁺ channels; and that (2) the neuroprotective effects of NS-7 may be accompanied by persistent activation of CREB phosphorylation in the inner border zone of ischemia.     

Decreased agonist-stimulated Ca2+ response in neutrophils from patients under chronic lithium therapy

Summary The agonist-stimulated increase in the intracellular concentration of free Ca²⁺ ([Ca²⁺]_i) was determined in neutrophils from patients under chronic lithium therapy and a control group of age- and sex-matched healthy drug-free subjects. Cells were stimulated with the chemotactic peptide formylmethionylleucylphenylalanin (fMLP) and the Ca²⁺ concentrations measured with the fluorescent Ca²⁺ indicator Fura-2. The Ca²⁺ response to stimulation with fMLP was significantly attenuated in neutrophils from patients chronically treated with lithium. The data suggest that lithium treatment inhibits the inositol phospholipid second messenger generating system in human cells and support the results of earlier inositol phosphate measurements in fMLP-stimulated neutrophils.     

Stimulation of L-type Ca2+ channel in growth cones activates two independent signaling pathways

Although growth cones respond to various modulators of neurite outgrowth, such as neurotrophins, neurotransmitters, and cell adhesion molecules, the signal-transducing mechanisms for these modulators in growth cones are unclear. Since recent studies have suggested that the signals of these modulators are mediated by Ca²⁺ influx through L-type voltage-sensitive Ca²⁺ channels (VSCCs) in the growth cone, we examined L-type VSCC-dependent signaling pathways, using isolated growth cones (IGCs) from developing rat forebrains. Binding assays revealed that L-type VSCC is enriched in growth cone membrane and gradually decreased in amount developmentally, while N-type VSCC has the opposite tendency. In intact IGCs, Bay K 8644 (BK, an L-type agonist) induced much more rapid elevation of [Ca²⁺]_i than that in adult synaptosomes. Ca²⁺-dependent phosphorylation of GAP-43 and MARCKS protein by protein kinase C (PKC) was enhanced in the IGC by BK, resulting in the release of these proteins from the membrane, which is consistent with our recent report. In addition, the Ca²⁺-dependent degradation of brain spectrin (fodrin) by calpain was also enhanced by BK or GABA, consequently inducing the release of α-actinin from the membrane skeleton of the growth cones. The activities of PKC and calpain were not inhibited by inhibitors of the other, indicating that these reactions occur independently. Our results suggest that Ca²⁺ influx through L-type VSCCs activates two distinct signaling branches, probably in the different domains of the growth cone, i.e., Ca²⁺-dependent phosphorylation of GAP-43 and MARCKS protein, and Ca²⁺-dependent degradation of brain spectrin and the release of α-actinin by calpain. J. Neurosci. Res. 51:682–696, 1998. © 1998 Wiley-Liss, Inc.     

Calcium-dependent serine phosphorylation of synaptophysin

The phosphorylation of synaptophysin, a major integral membrane protein of small synaptic vesicles, was found to be regulated in a Ca²⁺ -dependent manner in rat cerebrocortical slices, synaptosome preparations, and highly purified synaptic vesicles isolated from rat forebrain. K⁺-induced depolarization of slices and synaptosomes prelabeled with ³²P-orthophosphate produced a rapid, transient increase in serine phosphorylation of synaptophysin. In synaptosomes, the depolarization-dependent increase in synaptophysin phosphorylation required the presence of external Ca²⁺ in the incubation medium. The addition of Ca²⁺ plus calmodulin to purified synaptic vesicles resulted in a 4-fold increase in serine phosphorylation of synaptophysin, and this phosphorylation was antagonized by a peptide inhibitor of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II). Purified rat forebrain CaM kinase II phosphorylated both purified synaptophysin and endogenous, vesicle-associated synaptophysin, and the resulting 2-dimensional chymotryptic phosphopeptide maps were similar to those derived from synaptophysin phosphorylated in cerebrocortical slices. These data demonstrate that Ca²⁺-dependent phosphorylation of synaptophysin, mediated by CaM kinase II, occurs under physiological conditions. © 1993 Wiley-Liss, Inc.     

Increase of calmodulin-stimulated striatal particulate phosphorylation response in chronic haloperidol-treated rats

Calcium- and calmodulin-dependent protein kinase and phosphatase activities were studied in rat striatal particulate preparations. The effect of Ca²⁺ (0.1–0.5 mM) on phosphorylation was completely abolished in the preparation which had been washed 3 times with a buffer containing ethylene glycol bis-(β-aminoethyl ether)-N,N′-tetraacetic acid (EGTA, 1.2 mM). Ca²⁺-stimulated phosphorylation was restored in a dose-dependent manner after calmodulin (1 μg) was added to the assays. Ca²⁺ and calmodulin promoted the phosphate incorporation into two major striatal protein bands with estimated M_r at 52 and 40 kdaltons. The presence of phosphatase in the EGTA-pretreated preparations was negligible. Chronic treatment in rats with haloperidol (1 mg/kg, 20 days) produced a significant decrease in the Ca²⁺-independent phosphorylation but an increase in the extent of Ca²⁺ and calmodulin-dependent phosphorylation in the striatum. The chronic haloperidol treatment did not alter the striatal [¹²⁵I]calmodulin binding curve. In vitro, haloperidol (even at 10⁻⁴ M) had no effect on calmodulin-dependent phosphorylation. Haloperidol (10⁻⁴ M) did not reduce the number but decreased the rate of [¹²⁵I]calmodulin binding to the striatal particulates. These data suggest that the link between dopamine receptors and calmodulin-dependent enzyme is dissociated in vitro. On the other hand, the potential sensitivity of calmodulin-dependent protein kinase in the chronic haloperidol-treated rats correlated with the supersensitive dopamine receptor responses occurred in these rats. Therefore, calmodulin-dependent protein kinase may biochemically regulate dopamine receptor function in the striatum in vivo.     

Phosphorylation promotes the desensitization of the opioid-induced Ca increase in NG108-15 cells

Using the fluorescent Ca²⁺ indicator fura-2, we demonstrated that, in a single NG108-15 cell, acute repetitive challenge with leucine-enkephalin (EK) results in a gradual reduction of the increase of the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) at agonist exposure times of 90 s or less; increasing the EK exposure time of each challenge from 30 to 90 s results in greater desensitization, with complete desensitization occurring at 90 s exposure. Similar results are seen with ATP. In opioid-desensitized cells, bradykinin can still induce a marked [Ca²⁺]_i increase, while exposure of desensitized cell to 50 mM K⁺ restores the response EK-induced, suggesting a role of intracellular Ca²⁺ stores in the desensitization process. Pretreatment of cells with certain protein kinase inhibitors, including N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004) and staurosporine, prevented desensitization, while others, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) and {1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl- -tyrosyl]-4-phenyl-piperazine (KN-62), had no effect. In contrast, activation of protein kinase C by phorbol 12-myristate 13-acetate promoted desensitization. Thus, the desensitization is dependent on protein phosphorylation. HA1004 alone did not alter EK- or bradykinin-induced inositol 1,4,5-trisphosphate (IP₃) generation; however, the inhibitory effect of calyculin A on EK- or bradykinin-induced IP₃ generation was reversed by HA1004. In addition, in the presence of HA1004, the blockade of Ca²⁺ influx by either verapamil or removal of extracellular Ca²⁺ or the depletion of Ca²⁺ pools by thapsigargin still led to desensitization, suggesting that phosphorylation does not alter the activity of the Ca²⁺ transporters involved in Ca²⁺ influx and Ca²⁺ release. Our results imply that emptying of intracellular Ca²⁺ stores and protein phosphorylation in the phospholipase C signaling pathway play roles in the process of desensitization.     

Action potential broadening induced by lithium may cause a presynaptic enhancement of excitatory synaptic transmission in neonatal rat hippocampus

Lithium enhances excitatory synaptic transmission in CA1 pyramidal cells, but the mechanisms remain unclear. The present study demonstrates that lithium enhances the N-methyl-d -aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-isoxazole propionic acid (AMPA) receptor-mediated components of the excitatory postsynaptic current (EPSC). Lithium decreased the magnitude of paired-pulse facilitation and presented an inverse correlation between the lithium-induced enhancement of synaptic transmission and initial paired-pulse facilitation, which is consistent with a presynaptic mode of action. The enhancement of synaptic strength is likely to act, at least in part, by increasing the amplitude of the presynaptic Ca²⁺ transient. One mechanism which could account for this change of the presynaptic Ca²⁺ transient is an increase in the duration of the action potential. We investigated action potential in hippocampal pyramidal neurons and found that lithium (0.5–6 mm ) increased the half-amplitude duration and reduced the rate of repolarization, whereas the rate of depolarization remained similar. To find out whether the lithium synaptic effects might be explained by spike broadening, we investigated the field recording of the excitatory postsynaptic potential (EPSP) in hippocampal slices and found three lines of evidence. First, the prolongation of the presynaptic action potential with 4-aminopyridine and tetraethylammonium blocked or reduced the synaptic effects of lithium. Second, the lithium-induced synaptic enhancement was modulated when presynaptic Ca²⁺ influx was varied by changing the external Ca²⁺ concentration. Finally, both effects, the synaptic transmission increment and the action potential broadening, were independent of inositol depletion. These results suggest that lithium enhances synaptic transmission in the hippocampus via a presynaptic site of action: the mechanism underlying the potentiating effect may be attributable to an increased Ca²⁺ influx consequent to the broadening effect of lithium on the action potential.     

Tau-Induced Ca<Superscript>2+</Superscript>/Calmodulin-Dependent Protein Kinase-IV Activation Aggravates Nuclear Tau Hyperphosphorylation

Hyperphosphorylated tau is the major protein component of neurofibrillary tangles in the brains of patients with Alzheimer’s disease (AD). However, the mechanism underlying tau hyperphosphorylation is not fully understood. Here, we demonstrated that exogenously expressed wild-type human tau40 was detectable in the phosphorylated form at multiple AD-associated sites in cytoplasmic and nuclear fractions from HEK293 cells. Among these sites, tau phosphorylated at Thr205 and Ser214 was almost exclusively found in the nuclear fraction at the conditions used in the present study. With the intracellular tau accumulation, the Ca²⁺ concentration was significantly increased in both cytoplasmic and nuclear fractions. Further studies using site-specific mutagenesis and pharmacological treatment demonstrated that phosphorylation of tau at Thr205 increased nuclear Ca²⁺ concentration with a simultaneous increase in the phosphorylation of Ca²⁺/calmodulin-dependent protein kinase IV (CaMKIV) at Ser196. On the other hand, phosphorylation of tau at Ser214 did not significantly change the nuclear Ca²⁺/CaMKIV signaling. Finally, expressing calmodulin-binding protein-4 that disrupts formation of the Ca²⁺/calmodulin complex abolished the okadaic acid-induced tau hyperphosphorylation in the nuclear fraction. We conclude that the intracellular accumulation of phosphorylated tau, as detected in the brains of AD patients, can trigger nuclear Ca²⁺/CaMKIV signaling, which in turn aggravates tau hyperphosphorylation. Our findings provide new insights for tauopathies: hyperphosphorylation of intracellular tau and an increased Ca²⁺ concentration may induce a self-perpetuating harmful loop to promote neurodegeneration.     

Biphasic effects of nitric oxide on calcium influx in human platelets

In this study the effects of nitric oxide (NO) donors on intracellular free calcium ([Ca²⁺]_i) in human platelets was examined. Inhibition of guanylyl cyclase (GC) with either methylene blue or ODQ slightly inhibited the ability of submaximal concentrations of thrombin to increase [Ca²⁺]_i which suggests that a small portion of the thrombin mediated increase in [Ca²⁺]_i was due to an increase in NO and subsequent increase in cGMP and activation of cGMP dependent protein kinase (cGPK). Thrombin predominantly increases [Ca²⁺]_i by stimulating store-operated Ca²⁺ entry (SOCE). The NO donor GEA3162 was previously shown to stimulate SOCE in some cells. In platelets GEA3162 had no effect to increase [Ca²⁺]_i however it inhibited the ability of thrombin to increase [Ca²⁺]_i and this effect was reversed by ODQ. The addition of low concentrations (2.0 - 20 nM) of the NO donor sodium nitroprusside (SNP) slightly potentiated the ability of thrombin to increase [Ca²⁺]_i whereas higher concentrations (> 200 nM) of SNP inhibited thrombin induced increases in [Ca²⁺]_i. Both of these effects of SNP were reversed by ODQ which implies that they were both mediated by cGPK. Ba²⁺ influx was stimulated by low concentrations (2.0 nM) of SNP and inhibited by high concentrations (> 200 nM) of SNP and both effects were inhibited by ODQ. Previous studies showed that Ba²⁺ influx was blocked by the SOCE inhibitors 2-aminoethoxydipheny borate and diethylstilbestrol. It was concluded that low levels of SNP can stimulate SOCE in platelets and this effect may account for the increased aggregation and secretion previously observed with low concentrations of NO donors. Of the proteins known to be involved in SOCE (e.g. stromal interaction molecule 1 (Stim1), Stim2 and Orai1) only Stim2 has cGPK phosphorylation sites. The possibility that Stim2 phosphorylation regulates SOCE in platelets is discussed.     

Modification of hippocampal excitability in brain slices pretreated with a low nanomolar concentration of Zn2+

Synaptic Zn²⁺ homeostasis may be changed during brain slice preparation. However, much less attention has been paid to Zn²⁺ in artificial cerebrospinal fluid (ACSF) used for slice experiments than has been paid to Ca²⁺. The present study assesses addition of Zn²⁺ to ACSF, focused on hippocampal excitability after acute brain slice preparation. When the static levels of intracellular Zn²⁺ and Ca²⁺ were compared between brain slices prepared with conventional ACSF without Zn²⁺ and those pretreated with ACSF containing 20 nM ZnCl₂ for 1 hr, both levels were almost the same. On the other hand, intracellular Ca²⁺ levels were significantly increased in the stratum lucidum of the control brain slices after stimulation with high K⁺, although the increase was significantly suppressed by the pretreatment with ACSF containing Zn²⁺, suggesting that neuronal excitation is enhanced in brain slices prepared with ACSF without Zn²⁺. The increase in extracellular Zn²⁺ level, an index of glutamate release, after stimulation with high K⁺ was also significantly suppressed by pretreatment with ACSF containing Zn²⁺. When mossy fiber excitation was assessed in brain slices with FM4‐64, an indicator of presynaptic activity, attenuation of FM 4‐64 fluorescence based on presynaptic activity was suppressed in the stratum lucidum of brain slices pretreated with ACSF containing Zn²⁺. The present study indicates that hippocampal excitability is enhanced in brain slices prepared with ACSF without Zn²⁺. It is likely that a low nanomolar concentration of Zn²⁺ is necessary for ACSF. © 2015 Wiley Periodicals, Inc.     

Apolipoprotein E (ApoE) peptide regulates tau phosphorylation via two different signaling pathways

Previous studies have shown that treating rat cortical neurons in primary culture with apolipoprotein E (apoE) peptide increased cytoplasmic Ca²⁺ by 2 mechanisms: 1) an influx of extracellular Ca²⁺ resulting from the activation of a cell surface Ca²⁺ channel; and 2) release of Ca²⁺ from internal Ca²⁺ stores via a G-protein-coupled pathway (Wang and Gruenstein, 1997). These studies employed a biologically active apoE synthetic peptide (apoE_dp) derived from the receptor binding domain of apoE. In the present study we examined whether activation of these 2 signal transduction pathways affects phosphorylation of microtubule-associated protein tau. The levels of tau phosphorylation at thr231, ser235, and ser396 were quantified by ELISA employing monoclonal antibodies PHF-6, SMI33, and PHF-1. ApoE_dp treatment resulted in a concentration- and time-dependent dephosphorylation of tau at all 3 phosphorylation sites. The apoE_dp-induced dephosphorylation of tau at thr231, and ser235 was dependent on the influx of extracellular Ca²⁺, while dephosphorylation at ser396 was mediated by a pertusis toxin-sensitive G-protein pathway. The involvement of protein phosphatases in mediating the apoE_dp-induced dephosphorylation of tau was examined. Pretreatment with the protein phosphatase 2B inhibitor cyclosporin A blocked the apoE_dp-induced dephosphorylation of tau at thr231 and ser235 but not at ser396. Pretreatment with the protein phosophatase 2A/1 inhibitor okadaic acid blocked the apoE_dp-induced dephosphorylation of tau at all 3 sites, while pretreatment with the protein phosphates 1 inhibitor tautomycin was without effect. The present study suggests that apoE may affect several Ca²⁺-associated signal transduction pathways that increase the activity of protein phosphatases 2A and 2B, which in turn dephosphorylate tau. J. Neurosci. Res. 51:658–665, 1998. © 1998 Wiley-Liss, Inc.     

Different characteristics of cell volume and intracellular calcium ion concentration dynamics between the hippocampal CA1 and lateral cerebral cortex of male mouse brain slices during exposure to hypotonic stress

The mechanism of brain edema is complex and still remains unclear. Our aim was to investigate the regional differences of cell volume and intracellular Ca²⁺ concentration ([Ca²⁺]_i) dynamics during hypotonic stress in male mouse hemi‐brain slices. Brain slices were loaded with the fluorescence Ca²⁺ indicator fura‐2, and cell volume and [Ca²⁺]_i in the lateral cerebral cortex (LCC) and hippocampal CA1 (CA1) region were measured simultaneously during exposure to hypotonic stress using Ca²⁺ insensitive (F360) and Ca²⁺ sensitive fluorescence (F380), respectively. Brain cell swelling induced by hypotonic stress was followed by a regulatory volume change that coincided with an increase in [Ca²⁺]_i. The degrees of change in cell volume and [Ca²⁺]_i were significantly different between the LCC and CA1. The increase in cell volume and [Ca²⁺]_i in the LCC, but not in the CA1, was decreased by the transient receptor potential channel blockers LaCl₃ and GdCl₃. The increase in [Ca²⁺]_i in both the LCC and CA1, was significantly decreased by the intracellular Ca²⁺ modulators thapsigargin and xestospongin C. The K⁺ channel activator isoflurane and Cl^‐ channel blocker NPPB significantly decreased [Ca²⁺]_i in the LCC. This study demonstrated that, between cells located in the LCC and in the CA1, the characteristics of brain edema induced by hypotonic stress are different. This can be ascribed to the different contribution of volume sensitive G‐protein coupled receptor and stretch sensitive Ca²⁺ channels.     

Heat shock protects cultured rat astrocytes in a model of reperfusion injury

We have previously found that incubation of cultured rat astrocytes in Ca²⁺-containing medium after exposure to Ca²⁺-free medium caused an increase in intracellular Ca²⁺ ([Ca²⁺_i) followed by delayed cell death. Here, we examined whether thermal stress protects astrocytes from cell death in this model system of reperfusion injury. Cultured astrocytes were preincubated at 40–44°C for 10–20 min in fetal calf serum-free medium, incubated at 37°C for 24 h in serum-containing medium, and subjected to the in vitro reperfusion experiment. Thermal stress attenuated reperfusion-induced cell toxicity. Furthermore, the stress increased cell viability after incubation with serum-free medium containing Ca²⁺. These effects of heat shock required incubation in serum-containing medium for at least 12 h after heat shock, and it was blocked by the protein synthesis inhibitor cycloheximide. Thermal stress increased synthesis of several proteins, and one of the inducible proteins was identified as the 72-kDa heat shock protein by an immunoblot analysis. Neither the increase in [Ca²⁺]_i nor the Na⁺-Ca²⁺ exchange activity in astrocytes induced in this model were affected by thermal stress. These findings suggest that heat shock proteins protect astrocytes from cell death in a model of reperfusion injury and they may affect processes down stream of the increase in [Ca²⁺]_i.     

Intracellular calcium and calmodulin link brain‐derived neurotrophic factor to p70S6 kinase phosphorylation and dendritic protein synthesis

The mammalian target of rapamycin (mTOR)/p70S6 kinase (S6K) pathway plays an important role in brain‐derived neurotrophic factor (BDNF)‐mediated protein synthesis and neuroplasticity. Although many aspects of neuronal function are regulated by intracellular calcium ([Ca²⁺]_i) and calmodulin (CaM), their functions in BDNF‐induced phosphorylation of p70S6K and protein synthesis are largely unknown. Here, we report that BDNF, via TrkB‐dependent activation of mTOR, induces sustained phosphorylation of p70S6K at Thr389 and Thr421/Ser424. BDNF‐induced phosphorylation at Thr389 was dependent on PI3 kinase but independent of ERK‐MAPK. The previously identified MAPK phosphorylation site at Thr421/Ser424 required both PI3K and MAPK in BDNF‐stimulated neurons. Furthermore, we found that the reduction in [Ca²⁺]_i, but not extracellular calcium, blocked the BDNF‐induced phosphorylation of p70S6K at both sites. Inhibition of CaM by W13 also blocked p70S6K phosphorylation. In correlation, W13 inhibited BDNF‐induced local dendritic protein synthesis. Interestingly, sustained elevation of [Ca²⁺]_i by membrane depolarization antagonized the BDNF‐induced p70S6K phosphorylation. Finally, the BDNF‐induced p70S6K phosphorylation did not require the increase of calcium level through either extracellular influx or PLC‐mediated intracellular calcium release. Collectively, these results indicate that the basal level of intracellular calcium gates BDNF‐induced activation of p70S6K and protein synthesis through CaM. © 2009 Wiley‐Liss, Inc.     

Ca(2+)-independent syntaxin binding to the C(2)B effector region of synaptotagmin

Although synaptotagmin I, which is a calcium (Ca²⁺)-binding synaptic vesicle protein, may trigger soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated synaptic vesicle exocytosis, the mechanisms underlying the interaction between these proteins remain controversial, especially with respect to the identity of the protein(s) in the SNARE complex that bind(s) to synaptotagmin and whether Ca²⁺ is required for their highly effective binding. To address these questions, native proteins were solubilized, immunoprecipitated from rat brain extracts, and analyzed by immunoblotting. SNARE complexes comprising syntaxin 1, 25-kDa synaptosomal-associated protein (SNAP-25), and synaptobrevin 2 were coprecipitated with synaptotagmin I in the presence of ethylene glycol tetraacetic acid. The amount of coprecipitated proteins was significantly unaltered by the addition of Ca²⁺ to the brain extract. To identify the component of the SNARE complex that bound to synaptotagmin, SNARE was coexpressed with synaptotagmin in HEK293 cells and immunoprecipitated. Syntaxin, but not SNAP-25 and synaptobrevin, bound to synaptotagmin in a Ca²⁺-independent manner, and the binding was abolished in the presence of 1 M NaCl. Synaptotagmin contains 2 Ca²⁺-binding domains (C₂A, C₂B). Mutating the positively charged lysine residues in the putative effector-binding region of the C₂B domain, which are critical for transmitter release, markedly inhibited synaptotagmin-syntaxin binding, while similar mutations in the C₂A domain had no effect on binding. Synaptotagmin-syntaxin binding was reduced by mutating multiple negatively charged glutamate residues in the amino-terminal half of the syntaxin SNARE motif. These results indicate that synaptotagmin I binds to syntaxin 1 electrostatically through its C₂B domain effector region in a Ca²⁺-independent fashion, providing biochemical evidence that synaptotagmin I binds SNARE complexes before Ca²⁺ influx into presynaptic nerve terminals.