Cultured Rat Cerebellar Granule Cells Research Articles

Developmental Pb2+-exposure alters KCC₂ and VSCC-β3 subunit expression patterns in the postnatal rat brain and cerebellar granule cell cultures: Implications for disrupted GABA-shifts resulting from neurotoxicant exposures.

Developmental Pb2+-exposure alters KCC₂ and VSCC-β3 subunit expression patterns in the postnatal rat brain and cerebellar granule cell cultures: Implications for disrupted GABA-shifts resulting from neurotoxicant exposures.

Antisecretory Factor Modulates GABAA Receptor Activity in Neurons.

The antisecretory factor is an endogenous protein found in all mammalian tissues investigated so far. It acts by counteracting intestinal hypersecretion and various forms of inflammation, but the detailed mechanism of antisecretory factor (AF) action is unknown. We tested neuronal GABAA receptors by means of AF-16, a potent AF peptide derived from amino acids 36-51 from the NH2 part of AF. Cultured rat cerebellar granule cells were used, and the effects on the GABA-mediated chloride currents were determined by whole-cell patch clamp. Both the neurotransmitter GABA and AF-16 were added by perfusion of the experimental system. A 3-min AF-16 preincubation was more efficacious than 30s in significantly elevating the rapidly desensitizing GABA-activated chloride current. No effect was found on the tonic, slowly desensitizing current. The GABA-activated current increase by AF-16 demonstrated a low k of 41pM with a maximal increase of 37% persisting for some minutes after AF washout, independent from GABA concentration. This indicates an effect on the maximal stimulation (E%Max) excluding an altered affinity between GABA and its receptor. An immunocytochemical fluorescence approach with anti γ2 subunit antibodies demonstrated an increased expression of GABAA receptors. Thus, both the electrophysiological and the immunofluorescence approach indicate an increased appearance of GABAA receptors on the neuronal membrane. The rationale of the experiments was to test the effect of AF on a defined neuronal population of GABAA receptors. The implications of the results on the impact of AF on the enteric nervous system or on brain function are discussed.

Altered expression of genes involved in programmed cell death in primary cultured rat cerebellar granule cells acutely challenged with tetrabromobisphenol A

In the present study, primary cultures of rat cerebellar granule cells (CGC) and the RT2 Profiler PCR array were used to examine the effect of acutely applied brominated flame retardant tetrabromobisphenol A (TBBPA) on the expression of 84 genes related to the main modes of programmed cell death. CGC, at the 7th day of culture, were exposed to 10 or 25μM TBBPA for 30min. Then, 3, 6, and 24h later, the viability of the cells was examined by the staining with propidium iodide (PI) or using the calcein/ethidium homodimer (CA/ET) live/dead kit, and RNA was extracted for the evaluation of gene expression by RT-PCR. At 3, 6 and 24h after the treatment, the number of viable neurons decreased, according to the PI staining method, to 75%, 58% and 41%, respectively, and with the CA/ET method to 65%, 58% and 28%, respectively. In CGC analyzed 3h after the treatment with 25μM TBBPA or 6h after 10μM TBBPA, the only change in the gene expression was a reduction in the expression of Tnf, which is associated with autophagy and may activate some pro-apoptotic proteins. Six hours after 25μM TBBPA, only 2 genes were over-expressed, a pro-apoptotic Tnfrsf10b and Irgm, which is related to autophagy, and the genes that were suppressed included the anti-apoptotic gene Xiap, the necrosis-related Commd4, pro-apoptotic Abl1, 5 genes involved in autophagy (App, Atg3, Mapk8, Pten, and Snca) and 2 genes that participate in two metabolic pathways: Atp6v1g2 (pro-apoptotic and necrosis) and Tnf (pro-apoptotic, autophagy). Autophagy-related Snca and Tnf remained under-expressed 24h after treatment with 25μM TBBPA, which was accompanied by the over-expression of the pro-apoptotic Casp6, the anti-apoptotic Birc3, 2 genes related to autophagy (Htt and Irgm) and 2 genes (Fas and Tp53) that are involved in both apoptosis (pro-apoptotic) and autophagy. These results show a complex pattern of TBBPA-evoked changes in the expression of the genes involved in the programmed neuronal death, indicating no induction of programmed necrosis, an early suppression of the autophagy and anti-apoptotic genes, followed by a delayed activation of genes associated with apoptosis.

The Role of Ca2+ Imbalance in the Induction of Acute Oxidative Stress and Cytotoxicity in Cultured Rat Cerebellar Granule Cells Challenged with Tetrabromobisphenol A

Using primary cultures of rat cerebellar granule cells (CGC) we examined the role of calcium transients induced by tetrabromobisphenol A (TBBPA) in triggering oxidative stress and cytotoxicity. CGC were exposed for 30 min to 10 or 25 µM TBBPA. Changes in intracellular calcium concentration ([Ca2+]i), in the production of reactive oxygen species (ROS), and in the potential of mitochondria (∆Ψm) were measured fluorometrically during the exposure. The intracellular glutathione (GSH) and catalase activity were determined after the incubation; cell viability was evaluated 24 h later. TBBPA concentration-dependently increased [Ca2+]i and ROS production, and reduced GSH content, catalase activity, ∆Ψm and neuronal viability. The combination of NMDA and ryanodine receptor antagonists, MK-801 and bastadin 12 with ryanodine, respectively, prevented Ca2+ transients and partially reduced cytotoxicity induced by TBBPA at both concentrations. The antagonists also completely inhibited oxidative stress and depolarization of mitochondria evoked by 10 µM TBBPA, whereas these effects were only partially reduced in the 25 µM TBBPA treatment. Free radical scavengers prevented TBBPA-induced development of oxidative stress and improved CGC viability without having any effect on the rises in Ca2+ and drop in ∆Ψm. The co-administration of scavengers with NMDA and ryanodine receptor antagonists provided almost complete neuroprotection. These results indicate that Ca2+ imbalance and oxidative stress both mediate acute toxicity of TBBPA in CGC. At 10 µM TBBPA Ca2+ imbalance is a primary event, inducing oxidative stress, depolarization of mitochondria and cytotoxicity, whilst at a concentration of 25 µM TBBPA an additional Ca2+-independent portion of oxidative stress and cytotoxicity emerges.

Cholesterol modulates open probability and desensitization of NMDA receptors.

NMDA receptors (NMDARs) are glutamate-gated ion channels that mediate excitatory neurotransmission in the CNS. Although these receptors are in direct contact with plasma membrane, lipid-NMDAR interactions are little understood. In the present study, we aimed at characterizing the effect of cholesterol on the ionotropic glutamate receptors. Whole-cell current responses induced by fast application of NMDA in cultured rat cerebellar granule cells (CGCs) were almost abolished (reduced to 3%) and the relative degree of receptor desensitization was increased (by seven-fold) after acute cholesterol depletion by methyl-β-cyclodextrin. Both of these effects were fully reversible by cholesterol repletion. By contrast, the responses mediated by AMPA/kainate receptors were not affected by cholesterol depletion. Similar results were obtained in CGCs after chronic inhibition of cholesterol biosynthesis by simvastatin and acute enzymatic cholesterol degradation to 4-cholesten-3-one by cholesterol oxidase. Fluorescence anisotropy measurements showed that membrane fluidity increased after methyl-β-cyclodextrin pretreatment. However, no change in fluidity was observed after cholesterol enzymatic degradation, suggesting that the effect of cholesterol on NMDARs is not mediated by changes in membrane fluidity. Our data show that diminution of NMDAR responses by cholesterol depletion is the result of a reduction of the open probability, whereas the increase in receptor desensitization is the result of an increase in the rate constant of entry into the desensitized state. Surface NMDAR population, agonist affinity, single-channel conductance and open time were not altered in cholesterol-depleted CGCs. The results of our experiments show that cholesterol is a strong endogenous modulator of NMDARs.

Small Aβ1-42 oligomer-induced membrane depolarization of neuronal and microglial cells: role of N-methyl-D-aspartate receptors.

Although it is well documented that soluble beta amyloid (Aβ) oligomers are critical factors in the pathogenesis of Alzheimer's disease (AD) by causing synaptic dysfunction and neuronal death, the primary mechanisms by which Aβ oligomers trigger neurodegeneration are not entirely understood. We sought to investigate whether toxic small Aβ(1-42) oligomers induce changes in plasma membrane potential of cultured neurons and glial cells in rat cerebellar granule cell cultures leading to neuronal death and whether these effects are sensitive to the N-methyl-D-aspartate receptor (NMDA-R) antagonist MK801. We found that small Aβ(1-42) oligomers induced rapid, protracted membrane depolarization of both neurons and microglia, whereas there was no change in membrane potential of astrocytes. MK801 did not modulate Aβ-induced neuronal depolarization. In contrast, Aβ1(-42) oligomer-induced decrease in plasma membrane potential of microglia was prevented by MK801. Small Aβ(1-42) oligomers significantly elevated extracellular glutamate and caused neuronal necrosis, and both were prevented by MK801. Also, small Aβ(1-42) oligomers decreased resistance of isolated brain mitochondria to calcium-induced opening of mitochondrial permeability transition pore. In conclusion, the results suggest that the primary effect of toxic small Aβ oligomers on neurons is rapid, NMDA-R-independent plasma membrane depolarization, which leads to neuronal death. Aβ oligomers-induced depolarization of microglial cells is NMDA-R dependent.

Different ataxin-3 amyloid aggregates induce intracellular Ca2 + deregulation by different mechanisms in cerebellar granule cells

This work aims at elucidating the relation between morphological and physicochemical properties of different ataxin-3 (ATX3) aggregates and their cytotoxicity. We investigated a non-pathological ATX3 form (ATX3Q24), a pathological expanded form (ATX3Q55), and an ATX3 variant truncated at residue 291 lacking the polyQ expansion (ATX3/291Δ). Solubility, morphology and hydrophobic exposure of oligomeric aggregates were characterized. Then we monitored the changes in the intracellular Ca2+ levels and the abnormal Ca2+ signaling resulting from aggregate interaction with cultured rat cerebellar granule cells. ATX3Q55, ATX3/291Δ and, to a lesser extent, ATX3Q24 oligomers displayed similar morphological and physicochemical features and induced qualitatively comparable time-dependent intracellular Ca2+ responses. However, only the pre-fibrillar aggregates of expanded ATX3 (the only variant which forms bundles of mature fibrils) triggered a characteristic Ca2+ response at a later stage that correlated with a larger hydrophobic exposure relative to the two other variants. Cell interaction with early oligomers involved glutamatergic receptors, voltage-gated channels and monosialotetrahexosylganglioside (GM1)-rich membrane domains, whereas cell interaction with more aged ATX3Q55 pre-fibrillar aggregates resulted in membrane disassembly by a mechanism involving only GM1-rich areas. Exposure to ATX3Q55 and ATX3/291Δ aggregates resulted in cell apoptosis, while ATX3Q24 was substantially innocuous. Our findings provide insight into the mechanisms of ATX3 aggregation, aggregate cytotoxicity and calcium level modifications in exposed cerebellar cells.

Substance P activates ADAM9 mRNA expression and induces α-secretase-mediated amyloid precursor protein cleavage

Altered levels of Substance P (SP), a neuropeptide endowed with neuroprotective and anti-apoptotic properties, were found in brain areas and spinal fluid of Alzheimer's disease (AD) patients. One of the hallmarks of AD is the abnormal extracellular deposition of neurotoxic beta amyloid (Aβ) peptides, derived from the proteolytic processing of amyloid precursor protein (APP). In the present study, we confirmed, the neurotrophic action of SP in cultured rat cerebellar granule cells (CGCs) and investigated its effects on APP metabolism. Incubation with low (5 mM) potassium induced apoptotic cell death of CGCs and amyloidogenic processing of APP, whereas treatment with SP (200 nM) reverted these effects via NK1 receptors. The non-amyloidogenic effect of SP consisted of reduction of Aβ1–42, increase of sAPPα and enhanced α-secretase activity, without a significant change in steady-state levels of cellular APP. The intracellular mechanisms whereby SP alters APP metabolism were further investigated by measuring mRNA and/or steady-state protein levels of key enzymes involved with α-, β- and γ-secretase activity. Among them, Adam9, both at the mRNA and protein level, was the only enzyme to be significantly down-regulated following the induction of apoptosis (K5) and up-regulated after SP treatment. In addition to its neuroprotective properties, this study shows that SP is able to stimulate non-amyloidogenic APP processing, thereby reducing the possibility of generation of toxic Aβ peptides in brain.

Synaptic Acidification Enhances GABAASignaling

To determine the role of cellularly generated protons in synaptic signaling, we recorded GABA miniature IPSCs (mIPSCs) from cultured rat cerebellar granule cells (CGCs) while varying the extracellular pH buffering capacity. Consistent with previous reports, we found that increasing pH from 7.4 to 8.0 sped mIPSC rise time and suppressed both amplitude of the current and total charge transferred. Conversely, acidification (from pH 7.4 to 6.8) slowed the rise time and increased current amplitude and total charge transferred. In a manner consistent with alkalinization, increasing the buffering capacity from 3 to 24 mm HEPES at pH 7.4 resulted in faster mIPSC rise time, a 37% reduction in amplitude, and a 48% reduction in charge transferred. Supplementing the normal physiological buffers (24 mm HCO(3)(-)/5%CO(2)) with 10 mm HEPES similarly diminished mIPSCs in a manner consistent with alkalinization, resulting in faster rise time, a 39% reduction in amplitude, and a 51% reduction in charge transferred. These findings suggest the existence of an acidifying synaptic force that is overcome by commonly used concentrations (10 mm) of HEPES buffer. Here we show that Na(+)/H(+) exchanger (NHE) activity appears to, in part, contribute to this synaptic acidification because inhibition of NHE by amiloride or lithium under physiological or weak buffering conditions alters mIPSCs in a manner consistent with alkalinization. These results suggest that acidification of the synaptic cleft occurs physiologically during GABAergic transmission and that NHE plays a critical role in generating the acidic nano-environment at the synapse.

Adverse Effects of 2,4-Dichlorophenoxyacetic Acid on Rat Cerebellar Granule Cell Cultures Were Attenuated by Amphetamine

2,4-Dichlorophenoxyacetic acid (2,4-D), a worldwide-used herbicide, has been shown to produce a wide range of adverse effects in the health--from embryotoxicity and teratogenicity to neurotoxicity--of animals and humans. In this study, neuronal morphology and biochemical events in rat cerebellar granule cell (CGC) cultures have been analyzed to define some of the possible mechanisms involved in 2,4-D-induced cell death. For that purpose, amphetamine (AMPH) that has been shown to accelerate the recovery of several functions in animals with brain injury has been used as a pharmacologycal tool and was also investigated as a possible protecting agent. Addition of 2,4-D to CGC cultures produced a drastic decrease in cell viability, in association with an increased incidence of necrosis and apoptosis, and an increased level of reactive oxygen species, a decrease in glutathione content, and an abnormal activity of some enzymes with respect to the control group. The adverse effects of 2,4-D were partly attenuated in presence of AMPH. Some deleterious effects on several ultrastructural features of the cells, as well as the enhanced incidence of apoptosis, were partially preserved in AMPH-protected cultures as compared with those which were exposed to 2,4-D alone. The collected evidences (1) confirms the previously observed, deleterious effects of 2.4D on the same or a similar model; (2) suggests that the 2,4-D-induced apoptosis could have been mediated by or associated to an oxidative imbalance in the affected cells, and (3) shows some evidence of a protective effect of AMPH on 2,4-D-induced cell death, which could have been exerted through a reduction in the oxidative stress.

Evaluation of the protective effects of alkaloids isolated from the hooks and stems of Uncaria sinensis on glutamate-induced neuronal death in cultured cerebellar granule cells from rats.

We have previously shown that an aqueous extract of the hooks and stems of Uncaria sinensis (Oliv.) Havil., Uncariae Uncus Cum Ramulusis, protects against glutamate-induced neuronal death in cultured cerebellar granule cells by inhibition of Ca2+ influx. Because it is not known which components of Uncaria sinensis are active, in this study we have evaluated, by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) staining, the neuroprotective effects of the oxyindole alkaloids corynoxeine, rhynchophylline, isorhynchophylline and isocorynoxeine, and the indole alkaloids geissoschizine methyl ether, hirsuteine and hirsutine, isolated from the hooks and stems of Uncaria sinensis, on glutamate-induced cell death. We also investigated the inhibitory effects of the compounds on 45Ca2+ influx in cultured rat cerebellar granule cells. Cell viability evaluated by the MTT assay was significantly increased by application of rhynchophylline (10(-3) M), isorhynchophylline (10(-4)-10(-3) M), isocorynoxeine (10(-4)-10(-3) M), hirsuteine (10(-4)-3 x 10(-4) M) or hirsutine (10(-4)-3 x 10(-4) M) compared with exposure to glutamate only, with the effect of isorhynchophylline being the strongest. The increased 45Ca2+ influx into cells induced by glutamate was significantly inhibited by administration of rhynchophylline (10(-3) M), isorhynchophylline (3 x 10(-4)-10(-3) M), isocorynoxeine (3 x 10(-4)-10(-3) M), geissoschizine methyl ether (10(-3) M), hirsuteine (3 x 10(-4)-10(-3) M) or hirsutine (3 x 10(-4)-10(-3) M). These results suggest that oxyindole alkaloids such as isorhynchophylline, isocorynoxeine and rhynchophylline and indole alkaloids such as hirsuteine and hirsutine are the active components of the hooks and stems of Uncaria sinensis which protect against glutamate-induced neuronal death in cultured cerebellar granule cells by inhibition of Ca2+ influx.

P.1.d.002 Absence of tolerance to the antiaggressive action of the mGlul R antagonist JNJ16259685 after repeated administration to mice

Our results demonstrate that acute exposure of primary rat cerebellar granule cell cultures to homocysteine at millimolar concentrations induces a glutamate receptor-mediated decrease in tau protein phosphorylation, which is accompanied by excitotoxic neuronal damage. This contrasts with the previously reported hyperphosphorylation of tau in homocysteine-treated neurons, and with the assumption that hyperhomocysteinemia is one of the risk factors in Alzheimer disease, in which abnormal hyperphosphorylation of tau protein leads to neurofibrillary degeneration. The mechanisms of homocysteine neurotoxicity have been explained mainly either by disturbances in methylation processes, that may also lead to the accumulation of phosphorylated tau due to reduced activity of tau-selective protein phosphatase 2A, or by excitotoxicity. Since the relationships between homocysteine excitotoxicity and tau phosphorylation are unclear, the aim of this study was to characterize these processes in neurons acutely treated with homocysteine at neurotoxic concentrations, and to link them to the activities of glutamate receptors and protein phosphatase 2A. Within 24 h following a 30 min exposure of neuronal cultures to 20 mM d,l-homocysteine, significant neurotoxicity was induced. This could be reduced by treatment with an uncompetitive NMDA receptor antagonist, MK-801 (0.5 μM), or by mGlu1 and mGlu5 receptor antagonists, LY367385 and MPEP, respectively (both at 25 μM). Western blot analysis showed a rapid decrease in immunostaining of phospho-tau, 2 h after incubation of cell cultures with 15 mM d,l-homocysteine, which persisted for 6 h after the insult. Application of MK-801, LY367385 or okadaic acid (100 nM), an inhibitor of protein phosphatases 1 and 2A, significantly prevented dephosphorylation of tau, implying a role for the activation of glutamate receptors and protein phosphatase 2A. The phosphorylation of tau may be increased or reduced by treatment with homocysteine, and the nature of the cellular response to this sulfur-containing amino acid depends on the neuronal phenotype.

SP protects cerebellar granule cells against β-amyloid-induced apoptosis by down-regulation and reduced activity of Kv4 potassium channels

The tachykinin endecapeptide substance P (SP) has been demonstrated to exert a functional role in neurodegenerative disorders, including Alzheimer's disease (AD). Aim of the present study was to evaluate the SP neuroprotective potential against apoptosis induced by the neurotoxic beta-amyloid peptide (Aβ) in cultured rat cerebellar granule cells (CGCs). We found that SP protects CGCs against both Aβ 25–35- and Aβ 1–42-induced apoptotic CGCs death as revealed by live/dead cell assay, Hoechst staining and caspase(s)-induced PARP-1 cleavage, through an Akt-dependent mechanism. Since in CGCs the fast inactivating or A-type K + current ( I KA) was potentiated by Aβ treatment through up-regulation of Kv4 subunits, we investigated whether I KA and the related potassium channel subunits could be involved in the SP anti-apoptotic activity. Patch-clamp experiments showed that the Aβ-induced increase of I KA current amplitude was reversed by SP treatment. In addition, as revealed by Western blot analysis and immunofluorescence studies, SP prevented the up-regulation of Kv4.2 and Kv4.3 channel subunits expression. These results indicate that SP plays a role in the regulation of voltage-gated potassium channels in Aβ-mediated neuronal death and may represent a new approach in the understanding and treatment of AD.

Homocysteine-induced acute excitotoxicity in cerebellar granule cells in vitro is accompanied by PP2A-mediated dephosphorylation of tau

Our results demonstrate that acute exposure of primary rat cerebellar granule cell cultures to homocysteine at millimolar concentrations induces a glutamate receptor-mediated decrease in tau protein phosphorylation, which is accompanied by excitotoxic neuronal damage. This contrasts with the previously reported hyperphosphorylation of tau in homocysteine-treated neurons, and with the assumption that hyperhomocysteinemia is one of the risk factors in Alzheimer disease, in which abnormal hyperphosphorylation of tau protein leads to neurofibrillary degeneration. The mechanisms of homocysteine neurotoxicity have been explained mainly either by disturbances in methylation processes, that may also lead to the accumulation of phosphorylated tau due to reduced activity of tau-selective protein phosphatase 2A, or by excitotoxicity. Since the relationships between homocysteine excitotoxicity and tau phosphorylation are unclear, the aim of this study was to characterize these processes in neurons acutely treated with homocysteine at neurotoxic concentrations, and to link them to the activities of glutamate receptors and protein phosphatase 2A. Within 24 h following a 30 min exposure of neuronal cultures to 20 mM d, l-homocysteine, significant neurotoxicity was induced. This could be reduced by treatment with an uncompetitive NMDA receptor antagonist, MK-801 (0.5 μM), or by mGlu1 and mGlu5 receptor antagonists, LY367385 and MPEP, respectively (both at 25 μM). Western blot analysis showed a rapid decrease in immunostaining of phospho-tau, 2 h after incubation of cell cultures with 15 mM d, l-homocysteine, which persisted for 6 h after the insult. Application of MK-801, LY367385 or okadaic acid (100 nM), an inhibitor of protein phosphatases 1 and 2A, significantly prevented dephosphorylation of tau, implying a role for the activation of glutamate receptors and protein phosphatase 2A. The phosphorylation of tau may be increased or reduced by treatment with homocysteine, and the nature of the cellular response to this sulfur-containing amino acid depends on the neuronal phenotype.

PLC‐dependent intracellular Ca2+ release was associated with C6‐ceramide‐induced inhibition of Na+ current in rat granule cells

In this report, the effects of C(6)-ceramide on the voltage-gated inward Na(+) currents (I(Na)), two types of main K(+) current [outward rectifier delayed K(+) current (I(K)) and outward transient K(+) current (I(A))], and cell death in cultured rat cerebellar granule cells were investigated. At concentrations of 0.01-100 microM, ceramide produced a dose-dependent and reversible inhibition of I(Na) without alteration of the steady-state activation and inactivation properties. Treatment with C(2)-ceramide caused a similar inhibitory effect on I(Na). However, dihydro-C(6)-ceramide failed to modulate I(Na). The effect of C(6)-ceramide on I(Na) was abolished by intracellular infusion of the Ca(2+)-chelating agent, 1,2-bis (2-aminophenoxy) ethane-N, N, N9, N9-tetraacetic acid, but was mimicked by application of caffeine. Blocking the release of Ca(2+) from the sarcoplasmic reticulum with ryanodine receptor blocker induced a gradual increase in I(Na) amplitude and eliminated the effect of ceramide on I(Na). In contrast, the blocker of the inositol 1,4,5-trisphosphate-sensitive Ca(2+) receptor did not affect the action of C(6)-ceramide. Intracellular application of GTPgammaS also induced a gradual decrease in I(Na) amplitude, while GDPbetaS eliminated the effect of C(6)-ceramide on I(Na). Furthermore, the C(6)-ceramide effect on I(Na) was abolished after application of the phospholipase C (PLC) blockers and was greatly reduced by the calmodulin inhibitors. Fluorescence staining showed that C(6)-ceramide decreased cell viability and blocking I(Na) by tetrodotoxin did not mimic the effect of C(6)-ceramide, and inhibiting intracellular Ca(2+) release by dantrolene could not decrease the C(6)-ceramide-induced cell death. We therefore suggest that increased PLC-dependent Ca(2+) release through the ryanodine-sensitive Ca(2+) receptor may be responsible for the C(6)-ceramide-induced inhibition of I(Na), which does not seem to be associated with C(6)-ceramide-induced granule neuron death.

Protective Effects of Sanjoinine A against N-Methyl-D-aspartate-Induced Seizure

Sanjoinine A is a component of the alkaloid fraction of Zizyphi Spinosi Semen. This experiment was performed to investigate whether sanjoinine A acts as an anticonvulsive in the N-methyl-D-aspartate (NMDA)-induced experimental seizure model. We also examined whether it protects against seizure-form electroencephalogram (EEG) alterations induced by NMDA in vivo and/or cell killing due to NMDA in cultured cerebellar granule cells. Administration of sanjoinine A increased the survival rate and the latency of seizure onset, and decreased the seizure scores and the weight-loss induced by NMDA in mice, in a dose-dependent manner. In addition, sanjoinine A blocked seizure-form EEG alterations induced by NMDA and inhibited NMDA-induced cell killing in cultured rat cerebellar granule cells, measured by both the trypan blue exclusion test and the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Moreover, sanjoinine A inhibited the elevation of intracellular calcium influx induced by NMDA, which was measured using a fluorescent dye, Furo 3-AM. It is suggested that sanjoinine A protects against NMDA-induced seizures by inhibiting intracellular calcium influx.

Mefenamic acid bi-directionally modulates the transient outward K + current in rat cerebellar granule cells

The effect of non-steroidal anti-inflammatory drugs (NSAIDs) on ion channels has been widely studied in several cell models, but less is known about their modulatory mechanisms. In this report, the effect of mefenamic acid on voltage-activated transient outward K + current ( I A) in cultured rat cerebellar granule cells was investigated. At a concentration of 5 μM to 100 μM, mefenamic acid reversibly inhibited I A in a dose-dependent manner. However, mefenamic acid at a concentration of 1 μM significantly increased the amplitude of I A to 113 ± 1.5% of the control. At more than 10 μM, mefenamic acid inhibited the amplitude of I A without any effect on activation or inactivation. In addition, a higher concentration of mefenamic acid induced a significant acceleration of recovery from inactivation with an increase of the peak amplitude elicited by the second test pulse. Intracellular application of mefenamic acid could significantly increase the amplitude of I A, but had no effect on the inhibition induced by extracellular mefenamic acid, implying that mefenamic acid may exert its effect from both inside and outside the ion channel. Furthermore, the activation of current induced by intracellular application of mefenamic acid was mimicked by other cyclooxygenase inhibitors and arachidonic acid. Our data demonstrate that mefenamic acid is able to bi-directionally modulate I A channels in neurons at different concentrations and by different methods of application, and two different mechanisms may be involved.

Nitric oxide from neuronal nitric oxide synthase sensitises neurons to hypoxia‐induced death via competitive inhibition of cytochrome oxidase

Hypoxia/ischaemia is known to trigger neuronal death, but the role of neuronal nitric oxide synthase (nNOS) in this process is controversial. Nitric oxide (NO) inhibits cytochrome oxidase in competition with oxygen. We tested whether NO derived from nNOS synergises with hypoxia to induce neuronal death by inhibiting mitochondrial cytochrome oxidase. Sixteen hours of hypoxia (2% oxygen) plus deoxyglucose (an inhibitor of glycolysis) caused extensive, excitotoxic death of neurons in rat cerebellar granule cell cultures. Three different nNOS inhibitors (including the selective inhibitor N-4S-4-amino-5-2-aminoethyl-aminopentyl-N'-nitroguanidine) decreased this neuronal death by half, indicating a contribution of nNOS to hypoxic death. The selective nNOS inhibitor did not, however, block neuronal death induced either by added glutamate or by added azide (an uncompetitive inhibitor of cytochrome oxidase), indicating that nNOS does not act downstream of glutamate or cytochrome oxidase. Hypoxia plus deoxyglucose-induced glutamate release and neuronal depolarisation, and the nNOS inhibitor decreased this. Hypoxia inhibited cytochrome oxidase activity in the cultures, but a selective nNOS inhibitor prevented this inhibition, indicating NO from nNOS was inhibiting cytochrome oxidase in competition with oxygen. These data indicate that hypoxia synergises with NO from nNOS to induce neuronal death via cytochrome oxidase inhibition causing neuronal depolarisation. This mechanism might contribute to ischaemia/stroke-induced neuronal death in vivo.

Flufenamic Acid Bi-Directionally Modulates the Transient Outward K+ Current in Rat Cerebellar Granule Cells

In this report, the effect of flufenamic acid on voltage-activated transient outward K(+) current (I(A)) in cultured rat cerebellar granule cells was investigated. At a concentration of 20 microM to 1 mM, flufenamic acid reversibly inhibited I(A) in a dose-dependent manner. However, flufenamic acid at a concentration of 0.1 to 10 microM significantly increased the current amplitude of I(A). In addition to the current amplitude of I(A), a higher concentration of flufenamic acid had a significant effect on the kinetic parameters of the steady-state activation and inactivation process, suggesting that the binding affinity of flufenamic acid to I(A) channels may be state-dependent. Silencing the K(v)4.2, K(v)4.3, and K(v)1.1 genes of I(A) channels using small interfering RNA did not change the inhibitory effect of flufenamic on I(A), indicating that flufenamic acid did not act specifically on any of the subunits of the I(A)-channel protein. Intracellular application of flufenamic acid could significantly increase the I(A) amplitude but did not alter the inhibited effect induced by extracellular application of flufenamic acid, implying that flufenamic acid may exert its effect from both the inside and outside sites of the channel. Furthermore, the activation of current induced by intracellular application of flufenamic acid could mimic other cyclooxygenase inhibitors and arachidonic acid. Our data are the first that demonstrate how flufenamic acid is able to bidirectionally modulate I(A) channels in neurons at different concentrations and by different methods of application and that two different mechanisms may be involved.

Changes in the composition of detergent‐resistant membrane domains of cultured neurons following protein kinase C activation

Changes in the composition of cell fractions, and in particular of detergent-resistant membranes (DRM) isolated from cultured rat cerebellar granule cells, were taken as possible changes in lipid raft composition during a signal transduction event. After activation of protein kinase C (PKC) with phorbol esters (PMA) or glutamate, the content of PKC and of proteins highly enriched (GAP43, Fyn, and PrP(c)) or not (MARCKS) in DRM was followed. PKC activation strongly increased its association with membranes (from 2% to 75%), causing its enrichment within DRM; the substrate GAP43, enriched in DRM, remained membrane associated, but its proportion in DRM dramatically decreased (from about 40% to 2.5%), suggesting its shift from raft to nonraft membranes, possibly as a consequence of phosphorylation by PKC. The distribution of Fyn and PrP(c) (DRM-enriched) and of MARCKS (present mainly outside DRM) did not change. PKC activation was followed by an increase of GAP43 and MARCKS phosphorylation (about 7- and 8-fold, respectively). Noteworthy was that, after cell treatment with the lipid raft-disrupting drug methyl-beta-cyclodextrin, PKC activation occurred normally, followed by MARCKS phosphorylation, but GAP43 phosphorylation did not occur. Taken altogether, these data suggest that the integrity of lipid rafts is necessary for PKC to affect GAP43 and catalyze its phosphorylation.