钙通道在SIN-1诱导大鼠海马神经元凋亡中的作用

摘要 背景 氯通道参与SIN-1引起的大鼠海马神经元凋亡过程已有报道,而钙通道在这种类型的凋亡中的作用目前未见报道。目的 探讨钙通道在SIN-1诱导大鼠海马神经元凋亡中的作用。方法 方法 离体培养12天的SD大鼠海马神经元,随机分为正常对照组、3-吗啉斯德酮胺(SIN-1)处理组: (SIN-1 1.0 mmol •L-1）、SIN-1处理后加4-4-二异硫氰基苯-2,2’-二磺酸（DIDS,0.1 mmol •L-1）、4-乙酰氨基-4’-异氰酸芪-2,2’-二磺酸(SITS, 1.0 mmol •L-1)、SIN-1处理后加氯化镉（CdCl2）、SIN-1处理后加SITS和CdCl2及SIN-1处理后加DIDS和CdCl2。药物作用时间为18h。DNA荧光染色观察神经元形态及检测凋亡数目的变化,MTT法检测神经元的生存率。结果 SIN-1可以诱导42.32±1.34%的神经元死亡,与正常对照组相比有显著差异（P<0.05）,SIN-1处理的神经元若用氯通道阻断剂SITS或DIDS,生存率分别为87.33±2.03%和83.23±1.01%。SIN-1处理的神经元单独用CdCl2或CdCl2与SITS或DIDS合并使用时,细胞生存率分别为：66.39±2.09%、88.15±1.13%和83.16±2.00%。DNA荧光染色显示,SIN-1处理的神经元若用氯通道阻断剂可以明显减少凋亡百分数,而钙通道阻断剂CdCl2没有明显的抗凋亡作用。结论 氯通道可能参与了SIN-1诱导的大鼠海马神经元凋亡,而钙通道的作用不大。     

ClC-3 chloride channel in hippocampal neuronal apoptosis

Over-production of nitric oxide is pathogenic for neuronal apoptosis around the ischemic area fol- lowing ischemic brain injury. In this study, an apoptotic model in rat hippocampal neurons was es- tablished by 0.5 mmol/L 3-morpholinosyndnomine （SIN-l）, a nitric oxide donor. The models were then cultured with 0.1 mmol/L of 4,4＇-diisothiocyanostilbene-2,2＇-disulfonic acid （DIDS; the chloride channel blocker）for 18 hours. Neuronal survival was detected using the 3-（4,5-dimethylthiazol- 2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay, and apoptosis was assayed by Hoechst 33342-labeled neuronal DNA fluorescence staining. Western blot analysis and immunochemilumi- nescence staining were applied to determine the changes of activated caspase-3 and CIC-3 channel proteins. Real-time PCR was used to detect the mRNA expression of CIC-3. The results showed that SIN-1 reduced the neuronal survival rate, induced neuronal apoptosis, and promoted CIC-3 chloride channel protein and mRNA expression in the apoptotic neurons. DIDS reversed the effect of SIN-I. Our findings indicate that the increased activities of the CIC-3 chloride channel may be involved in hippocampal neuronal apoptosis induced by nitric oxide.     

氯通道在一氧化氮诱导离体大鼠海马神经元凋亡中的作用

目的 观察氯通道阻断剂SITS和DIDS对NO诱导的大鼠离体海马神经元凋亡的效应,探讨氯通道在缺血性脑损伤中的作用。方法 离体培养12天的SD大鼠海马神经元,随机分为正常对照组、NO处理组、NO处理后使用氯通道阻断剂组,对各组神经元分别在相应的时间点进行Hoechst荧光染色观察凋亡细胞数和MTT实验定量检测神经元的存活率,western blot 分析凋亡信号分子caspase-3的变化。结果 SITS和DIDS呈剂量依赖性地抑制NO诱导的神经元损伤,并能抑制损伤所引起的caspase-3的激活,提高神经元的存活率。结论 氯通道阻断剂对NO诱导的大鼠海马神经元凋亡有一定的保护作用。     

Anion channel blockers attenuate delayed neuronal cell death induced by transient forebrain ischemia

Chloride efflux is known to be involved in the progression of apoptosis in various cell types. We have recently shown that the volume-sensitive outwardly rectifying (VSOR) anion channel serves as the pathway for apoptotic chloride efflux in some cells. In the present study, we tested the neuroprotective effects of drugs that can block the VSOR anion channel, on delayed neuronal death (DND) induced by transient forebrain ischemia. The functional expression of the VSOR anion channel was first examined in hippocampal neurons in both primary culture and hippocampal slice preparations, by the whole-cell patch-clamp technique. We then tested the channel's sensitivity to an anion channel blocker, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and a tyrosine kinase blocker, genistein. By histological examinations and cytochrome c release assessments, the protective effects of these drugs on the DND of hippocampal CA1 neurons in mice subjected to transient ischemia were examined. Drugs were administered via the jugular vein prior to ischemic treatment and into the peritoneal cavity after reperfusion. Hippocampal neurons were found to express the volume-sensitive Cl(-) channel, which exhibits outward rectification and is sensitive to DIDS and genistein. Administration of DIDS or genistein reduced cytochrome c release and the number of damaged neurons in the CA1 region after transient forebrain ischemia. This fact suggests that the DND induction mechanism involves the activity of the VSOR anion channel and that this channel may provide a therapeutic target for the treatment of stroke.     

Disparity between ionic mediators of volume regulation and apoptosis in N1E 115 mouse neuroblastoma cells

Cellular volume loss or shrinkage is a ubiquitous feature of apoptosis and thus may contribute to this form of degeneration. Chloride (Cl(-)) and potassium (K(+)) efflux has been shown to participate in volume regulation and several recent reports have implicated K(+) efflux in apoptotic neuronal death. Here pharmacological inhibitors of various K(+) and Cl(-) channels and transporters were used to decipher the relationship between cellular volume regulation and apoptosis. Following exposure to a hypotonic media, cells swell but over time gradually recover, returning to their original cell volume in a process known as regulatory volume decrease (RVD). RVD in N1E 115 neuroblastoma cells was monitored using time-lapse videomicroscopy, cell size and DNA degradation were followed using flow cytometry and fragmented apoptotic nuclei were visualized using Hoechst staining. RVD was blocked by high K(+), TEA and 4-AP (K(+) channel blockers), DIDS and niflumic acid but not SITS (Cl(-) channel blockers), ethacrynic acid (Cl(-) pump blocker), bumetanide (Na(+)/K(+)/Cl(-) cotransporter blocker) and furosemide (K(+)/Cl(-) cotransport blocker). In contrast, only DIDS and SITS (blockers of the Cl(-)/HCO(3) exchanger) inhibited apoptosis, suggesting that a common mechanistic link between RVD and apoptosis is the Cl(-)/HCO(3) exchanger. Thus, this study does not support the notion that K(+) channels are universal anti-apoptotic targets. Instead, the Cl(-)/HCO(3) exchanger may prove to be a viable target of therapeutic intervention for treating pathological apoptosis and neurodegeneration.     

Chloride channel blocker 4,4-diisothiocyanatostilbene-2,2’-disulfonic acid inhibits nitric oxide-induced apoptosis in cultured rat hippocampal neurons

Apoptosis in cultured rat hippocampal neurons was induced using the nitric oxide donor 3-morpholinosydnonimine, and cells were treated with the chloride channel blocker, 4,4-diisothiocyanatostilbene-2,2’-disulfonic acid. Results showed that the survival rate of neurons was significantly increased after treatment with 4,4-diisothiocyanatostilbene-2,2’-disulfonic acid, and the rate of apoptosis decreased. In addition, the expression of the apoptosis-related proteins poly(adenosine diphosphate-ribose)polymerase-1 and apoptosis-inducing factor were significantly reduced. Our experimental findings indicate that the chloride channel blocker 4,4- diisothiocyanatostilbene-2,2’-disulfonic acid can antagonize apoptotic cell death of hippocampal neurons by inhibiting the expression of the apoptosis-related proteins poly(adenosine diphosphate-ribose)polymerase-1 and apoptosis-inducing factor.     

Time-dependence of, and effects of inhibition and cellular aging on, chloride efflux across erythrocyte membranes in Huntington's disease

Previous studies have demonstrated an increased rate of chloride transport across erythrocyte membranes in Huntington's disease, a process regulated at the external side of the major transmembrane protein Band 3. A marked effect of time was noted in 3 Huntington's disease samples that were studied more than 4 h after obtaining the blood. In order to study anion transport more closely in Huntington's disease and the apparent time-dependence of chloride efflux in this disorder, we have performed several sets of experiments. Chloride transport in Huntington's disease erythrocytes was found to be extremely sensitive to time with the efflux rate constant decreasing by approximately 30% over a 24 h period. Chloride transport in control cells was unaffected by time. Inhibition studies with the specific anion transport blocker 4,4'-diisothiocyanto-2,2'-disulfonic acid stilbene (DIDS) demonstrated that the same degree of inhibition of chloride transport could be achieved at a much lower concentration of DIDS in Huntington's disease than in controls. Comparison of chloride efflux in fractions enriched in young and old erythrocytes, respectively, obtained by density centrifugation of fresh blood, demonstrated that only in the young fraction of cells was chloride efflux diminished with time in Huntington's disease. Chloride transport in in vivo aged Huntington's cells or both young and old control cells was essentially not dependent on time. These results are discussed in terms of proposed molecular mechanisms for neuronal loss in this disorder. The alterations in chloride efflux in extraneural erythrocytes are consistent with a proposed cell-surface membrane defect involving a protein in Huntington's disease.     

Mercury compounds disrupt neuronal glutamate transport in cultured mouse cerebellar granule cells

Cerebellar granule cells are targeted selectively by mercury compounds in vivo. Despite the affinity of mercury for thiol groups present in all cells, the molecular determinant(s) of selective cerebellar degeneration remain to be elucidated fully. We studied the effect of mercury compounds on neuronal glutamate transport in primary cultures of mouse cerebellar granule cells. Immunoblots probed with an antibody against the excitatory amino acid transporter (EAAT) neuronal glutamate transporter, EAAT3, revealed the presence of a specific band in control and mercury-treated cultures. Micromolar concentrations of both methylmercury and mercuric chloride increased the release of endogenous glutamate, inhibited glutamate uptake, reduced mitochondrial activity, and decreased ATP levels. All these effects were completely prevented by the nonpermeant reducing agent Tris-(2-carboxyethyl)phosphine (TCEP). Reduction of mitochondrial activity by mercuric chloride, but not by methylmercury, was inhibited significantly by 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) and by reduced extracellular Cl- ion concentration. In addition, DIDS and low extracellular Cl- completely inhibited the release of glutamate induced by mercuric chloride, and produced a partial although significant reduction of that induced by methylmercury. We suggest that a direct inhibition of glutamate uptake triggers an imbalance in cell homeostasis, leading to neuronal failure and Cl(-)-regulated cellular glutamate efflux. Our results demonstrate that neuronal glutamate transport is a novel target to be taken into account when assessing mercury-induced neurotoxicity.     

Selective neuronal uptake of 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS) and other related substituted stilbenes in vivo: a fluorescent whole cell staining technique

The uptake of fluorescent stilbene derivatives including 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) was shown to occur from the blood or cerebrospinal fluid in the adult albino rat. SITS was found to be internalized in select populations of neurons, especially pyramidal neurons in layers V and VI of neocortex, granule, hilar and pyramidal neurons in the hippocampus, Purkinje neurons of the cerebellar cortex and predominantly large multipolar neurons in the oculomotor nuclei, lateral vestibular nucleus, red nucleus and the medial gigantocellular brainstem reticular formation neurons. The extent and quality of labeling of neuronal somata, dendrites and axons can be exploited with this technique to produce selective, 'Golgi' type labeling in the nervous system. The methods utilized are fast, economical, long lasting and compatible with fluorescence tracers and immunofluorescence techniques.     

GABAA receptor-mediated activation of L-type calcium channels induces neuronal excitation in surgically resected human hypothalamic hamartomas.

PURPOSE: The human hypothalamic hamartoma (HH) is a rare, intrinsically epileptogenic lesion associated with gelastic seizures, but the underlying mechanisms remain unclear. Here, we examined the role of GABAA receptors in surgically resected HH tissue. METHODS: HH tissue slices (350 microm) were studied using cellular electrophysiological, calcium imaging, and immunocytochemical techniques. RESULTS: Two neuronal cell types were seen: small (10-16 microm) spontaneously firing GABAergic neurons and large (20-28 microm) quiescent neurons. In gramicidin-perforated patch recordings, muscimol (30 microM) induced membrane depolarization in 70% of large (but not small) neurons and a concomitant rise in intracellular calcium. These responses were blocked by bicuculline methiodide (50 microM). Depolarizing neurons also exhibited more positive reversal potentials (Emuscimol) and significantly higher intracellular chloride concentrations compared to those that hyperpolarized. The cation chloride co-transporters NKCC1 and KCC2 were coexpressed in the majority of large neurons, but fluorometric measurements revealed that 84% of large HH neurons expressed solely or relatively more NKCC1. Bumetanide (20 microM), a NKCC1 antagonist, partially suppressed muscimol-induced excitation in large neurons. Concordant with robust expression of CaV1.2 and CaV1.3 subunits in HH neurons, the L-type calcium channel blocker nifedipine (100 microM) prevented muscimol-induced neuronal excitation. CONCLUSIONS: GABAA receptor-mediated excitation, due in part to differential expression of NKCC1 and KCC2 and subsequent activation of L-type calcium channels, may contribute to seizure genesis in HH tissue. Given the ready availability of L-type calcium channel blockers, our results have clinical ramifications for the treatment of seizures associated with HH lesions.     

Role of potassium channels in Abeta(1-40)-activated apoptotic pathway in cultured cortical neurons

Potassium channel dysfunction has been implicated in Alzheimer's disease (AD). In the present study, by using potassium channel blocker tetraethylammonium (TEA), we investigated the relationship between the enhancement of potassium currents and the alteration of apoptotic cascade in the neuronal apoptotic model induced by beta-amyloid peptide 1-40(Abeta(1-40)). Cortical neurons exposed to Abeta(1-40) 5 muM developed a specific increase in the delayed rectifier potassium current (I(K)), but not the transient outward potassium currents (I(A)), before the appearance of neuronal apoptosis. Abeta(1-40) induced various apoptotic features such as chromatin condensation, a decrease in the amount of Bcl-2 protein, an increase in the amount of Bax protein, cytochrome c release from mitochondria, and caspase-3 activation. Potassium channel blocker 5 mM TEA attenuated Abeta(1-40)-induced neuronal death and prevented the alterations of all above mentioned apoptotic indicators. The study indicates that I(K) enhancement might play an important role in certain form of programmed cell death induced by beta-amyloid peptide (Abeta). Increased potassium channel activity might trigger the activation of apoptosis cascade in Abeta(1-40)-treated rat cortical neurons.     

Porcine pancreatic group IB secretory phospholipase A2 potentiates Ca2+ influx through L-type voltage-sensitive Ca2+ channels

Secretory phospholipase A₂ (sPLA₂) exhibits neurotoxicity in the central nervous system. There are high-affinity binding sites of the porcine pancreatic group IB sPLA₂ (sPLA₂-IB) in the brain. sPLA₂-IB causes neuronal cell death via apoptosis in the rat cerebral cortex. Although apoptosis is triggered by an influx of Ca²⁺ into neurons, it has not yet been ascertained whether the Ca²⁺ influx is associated with the neurotoxicity of sPLA₂-IB. We thus examined the possible involvement of Ca²⁺ in the neurotoxicity of sPLA₂-IB in the primary culture of rat cortical neurons. sPLA₂-IB induced neuronal cell death in a concentration- and time-dependent manner. This death was accompanied by condensed chromatin and fragmented DNA, exhibiting apoptotic features. Before apoptosis, sPLA₂-IB markedly enhanced the influx of Ca²⁺ into neurons. A calcium chelator suppressed neurons from sPLA₂-IB-induced neuronal cell death in a concentration-dependent manner. An L-type voltage-sensitive Ca²⁺ channel (L-VSCC) blocker significantly protected the sPLA₂-IB-potentiated influx of Ca²⁺. On the other hand, blockers of N-VSCC and P/Q-VSCC did not. An L-VSCC blocker protected neurons from sPLA₂-IB-induced neuronal cell death. In addition, the L-VSCC blocker ameliorated the apoptotic features of sPLA₂-IB-treated neurons. Neither an N-VSCC blocker nor P/Q-VSCC blockers affected the neurotoxicity of the enzyme. In conclusion, these findings demonstrate that the influx of Ca²⁺ into neurons play an important role in the neurotoxicity of sPLA₂-IB. Furthermore, the present study suggests that L-VSCC contribute to the sPLA₂-IB-potentiated influx of Ca²⁺ into neurons.     

N-Methyl-D-aspartate receptor blockade induces neuronal apoptosis in cortical culture.

Whereas excessive activation of the NMDA receptor may contribute to ischemic neuronal injury, physiologic activation may promote neuronal survival under certain conditions. Consistently, it has recently been shown that NMDA antagonists induce apoptosis of central neurons in immature rats. In the present study, we have examined whether NMDA antagonists induce neuronal apoptosis also in a culture condition. Exposure of cortical cultures (DIV 10-13) to MK-801 (1-10 microM) for 48 h resulted in death of about 30-40% of neurons. Similar neuronal death was induced by exposure to other NMDA antagonists, D-AP5 and dextromethorphan. The neuronal death was dependent on the culture age; MK-801 induced much less neuronal death in younger (DIV 7) and older (DIV 16-19) cultures. The NMDA antagonist-induced neuronal death was accompanied by cell body shrinkage, nuclear fragmentation, and cleavage/activation of caspase-3. Furthermore, it was attenuated by cycloheximide and zVAD-fmk, indicating that the death occurred mainly by the apoptosis mechanism. As in several other apoptosis models, high-potassium medium blocked the NMDA antagonist-induced apoptosis, which was reversed by voltage-gated calcium channel blockers. The present results demonstrate that NMDA antagonists induce neuronal apoptosis in cortical culture, consistent with the findings obtained in immature rats. Since the activation of the voltage-gated calcium channels attenuated the NMDA antagonist-induced apoptosis, it may be another example of the "calcium set point hypothesis." Copyright 1999 Academic Press.     

Prevention of delayed neuronal death in gerbil hippocampus by ion channel blockers

We used a gerbil model of cerebral ischemia to study the effects of ion channel blockers on neuronal death resulting from enhanced glutamate release and calcium ion influx. The common carotid arteries of gerbils were occluded for 5 minutes and injected intraperitoneally immediately after ischemia with an alkylene iminopropylene derivative (glutamate blocker) or a piperazinyl ethanol derivative (calcium blocker) given at high or low doses. Two vehicle groups received saline or 0.2% methyl cellulose solution. Seven days later, the gerbils were perfusion-fixed and their brains were processed for histologic study. The number of neurons per millimeter (neuronal density) of the CA1 region was calculated, and the neuronal density in each group was statistically compared using the Mann-Whitney U test. Compared with a control group not subjected to carotid ligation, neurons of the two vehicle groups and the low-dose calcium blocker group were almost nonexistent in the CA1 region. Neuronal densities of the glutamate blocker group and the high-dose calcium blocker group were similar and were found to be within normal limits by statistical analysis. Our study shows that detrimental membrane phenomena and the incidence of delayed neuronal death may be counteracted by the systemic administration of these ion channel blockers after ischemic insult.     

Effects of the anion channel blocker DIDS on ouabain- and high K(+)-induced release of amino acids from the rat cerebral cortex

Amino acid release from the rat cerebral cortex was analyzed using an in vivo cortical cup perfusion model. Topical applications of ouabain or high extracellular K(+) were used to mimic two dimensions of ischemic conditions which promote cell swelling and amino acid release. Ouabain (30 microM) induced significant releases of taurine, gamma-aminobutyric acid (GABA), aspartate, glutamate and phosphoethanolamine. The anion channel blocker, 4, 4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS; 1 mM), inhibited ouabain-induced release of all these amino acids except for glutamate. Exposure to high extracellular K(+) (75 mM) induced a delayed rise in the levels of taurine in the superfusates and an immediate increase in GABA levels. There were no significant releases of other amino acids. The release of taurine and GABA was sensitive to the blocking of anion channels with DIDS. Both ouabain- and high K(+)-induced taurine release is likely to be mediated by DIDS sensitive anion channels. The extracellular accumulation of the other amino acids, where insensitive to DIDS, may be mediated by mechanisms other than swelling-induced anion channels.     

The influx of Ca and the release of noradrenaline evoked by the stimulation of presynaptic nicotinic receptors of chick sympathetic neurons in culture are not mediated via L-, N-, or P-type calcium channels

We have shown earlier that nicotinic agonists induce the release of noradrenaline from chick sympathetic neurons in culture in two ways: (a) by activating the postsynaptic nicotinic receptors on nerve cell bodies, giving rise to spreading electrical activity and opening of voltage operated calcium channels in neuronal processes; (b) by activating the presynaptic nicotinic receptors on neuronal processes. In the present work, we investigated the contribution of various pathways to the observed Ca²⁺ influx and subsequent noradrenaline release. Sympathetic neurons in culture were stimulated either by the nicotinic agonist dimethylphenylpiperazinium or electrically, in the presence or absence of tetrodotoxin and of specific blockers of calcium or nicotinic channels, and the effects on [Ca²⁺]_i in the area of neuronal processes and on noradrenaline release were measured. Under control conditions, the N-type channel blocker ω-conotoxin (0.1 μmol/1) diminished the release of noradrenaline and the increase of intraterminal Ca²⁺ by 48% and 55%, respectively, whereas the L-type channel blocker (+)Bay k 8644 (1 μmol/1) diminished the release of noradrenaline by 25% and the increase of [Ca²⁺]_i by 39%. The P-type channel blocker ω-agatoxin (0.3 μmol/1) had no effect. The effects of the L-type channel ligands were complex and could only be explained on the assumption that, at high concentrations, these drugs also act as nicotinic antagonists. Tetrodotoxin blocked the Ca²⁺ response evoked by electrical stimulation whereas DMPP applied in the presence of tetrodotoxin still evoked an increase of [Ca²⁺]_i and the release of noradrenaline (27% and 30% of control without tetrodotoxin, respectively). These residual responses were not blocked by any of the calcium channel blockers used or by their combination. Apparently, a substantial part of the influx of Ca²⁺ induced by the activation of presynaptic nicotinic receptors is not carried by the N-, L- or P-type channels and probably occurs directly via the open channels of nicotinic receptors.     

Differential connexin expression,gap junction intercellular coupling,and hemichannel formation in NT2/D1 human neural progenitors and terminally differentiated hNT neurons

Connexin-mediated gap junctions and open hemichannels in nonjunctional membranes represent two biologically relevant mechanisms by which neural progenitors can coordinate their response to changes in the extracellular environment. NT2/D1 cells are a teratocarcinoma progenitor line that can be induced to differentiate terminally into functional hNT neurons and NT-G nonneuronal cells. Clinical transplants of hNT neurons and experimental grafts of NT2/D1 progenitors or hNT neurons have been used in cell-replacement therapy in vivo. Previous studies have shown that NT2/D1 cells express connexin 43 (Cx43) and that NT2/D1 progenitors are capable of dye transfer. To determine whether NT2/D1 progenitors and differentiated hNT cultures express other connexins, Cx26, Cx30, Cx32, Cx36, Cx37, Cx43, and Cx46.6 mRNA and protein were analyzed. NT2/D1 progenitors express Cx30, Cx36, Cx37, and Cx43. hNT/NT-G cultures express Cx36, Cx37, and de novo Cx46.6. Cx26 and Cx32 were not expressed in NT2/D1 or hNT/NT-G cells. NT2/D1 progenitors formed functional gap junctions as assessed by dye coupling as well as open hemichannels in nonjunctional membranes as assessed by dye-uptake studies. Dye coupling was inhibited by the gap junction blocker 18alpha-glycyrrhetinic acid. Hemichannel activity was inhibited by the dual-specificity chloride channel/connexin hemichannel inhibitor flufenamic acid but not by the chloride channel inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Both dye coupling and dye uptake were substantially reduced following differentiation of NT2/D1 progenitors. We conclude that the pattern of connexin expression in NT2/D1 cells changes over the course of differentiation corresponding with a reduction in biochemical coupling and hemichannel activity in differentiated cells.     

Contribution of Kv channel subunits to glutamate‐induced apoptosis in cultured rat hippocampal neurons

Potassium channel dysfunction has been implicated in apoptosis in many pathological conditions. However, which Kv channel subunit is involved in glutamate‐induced apoptosis remains unknown. In this study, the contributions of nine Kvα and three Kvβ subunits to glutamate‐induced hippocampal neuronal apoptosis were investigated. Results showed that neuronal apoptosis was not obvious with 12 hr incubation of glutamate but increased markedly after 18 hr, which was attenuated by the Kv channel blocker TEA. Among all the Kv subunits investigated, gene and protein expression of Kv2.1 increased significantly before the appearance of neuronal apoptosis, whereas the Kv1.1 mRNA level decreased quickly, and protein expression was reduced gradually after the insult. Seven other Kvα subunits and three Kvβ subunits were not obviously affected over time. In addition, Kv1.1 overexpression could reduce glutamate‐induced hippocampal neuronal apoptosis. Therefore, the alterations of Kv1.1 and Kv2.1 might contribute to glutamate‐induced toxicity in hippocampal neurons. These findings suggest that these two Kv channel subunits may represent potential therapeutic targets for neuropathological conditions in which glutamate‐induced toxicity is thought to contribute to neuronal dysfunction. © 2009 Wiley‐Liss, Inc.     

A chloride-dependent component of the release of labeled GABA and taurine from the chick retina

The characteristics and ionic dependence of the release of [3H]gamma-aminobutyric acid ([3H]GABA) and [3H]taurine from the chick retina, stimulated by kainic acid (KA) and by depolarizing concentrations of potassium was examined and compared to those of [3H]dopamine. KA (100 microM) highly stimulated the release of [3H]GABA (25-fold over resting efflux), induced a moderate increase in [3H]taurine and did not increase the efflux of [3H]dopamine. The efflux of [3H]GABA stimulated by KA was totally calcium-independent but it was markedly sodium and chloride dependent. Chloride dependence was assessed by replacing chloride with the impermeant anion gluconate, or by addition of the anion transport blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Depolarizing concentrations of KCl (56 mM) stimulated the release of [3H]GABA, [3H]taurine and [3H]dopamine to about the same extent. The release of [3H]GABA and [3H]taurine was only partially calcium dependent, in contrast to the highly calcium-dependent efflux of [3H]dopamine. A sodium-free medium increased the resting efflux and decreased the potassium-stimulated release of [3H]GABA and [3H]taurine; the resting efflux of [3H]dopamine was unaffected and the potassium-induced efflux was somewhat increased. The potassium-stimulated efflux of [3H]GABA and [3H]taurine showed a chloride-dependent component which was higher for taurine whereas the resting efflux was not modified. The stimulated release of [3H]dopamine was increased in a chloride-free medium. The ionic dependence of KA and potassium stimulated efflux of [3H]GABA and [3H]taurine showed properties similar to those of the homoexchange-activated efflux of amino acids which was also found sodium and chloride dependent and clearly different from the calcium-coupled neurotransmitter release process. Exposure of retinas to KA and potassium produced retinal cell swelling which is prevented in a chloride-free medium. Results are discussed in terms of a particular efflux mechanism for [3H]GABA and [3H]taurine in the retina in response to stimulation associated with changes in ionic gradients and retinal cell swelling.     

刺五加皂甙对谷氨酸毒性神经元凋亡的保护作用

目的观察神经元在谷氨酸毒性损伤时一氧化氮(NO)的动态变化及其与凋亡的关系,探讨刺五加皂甙(ASS)的有效保护浓度。方法采用谷氨酸(Glu)诱导的皮质神经元凋亡模型。随机分成Glu组、正常对照组及ASS3组;用流式细胞仪检测神经元凋亡率,用硝酸还原酶法测定细胞培养上清液中NO的含量,用MTT法测定神经元存活率并在电镜下观察细胞形态学变化。结果(1)Glu呈剂量和时间依赖性增加神经元培养液中NO含量,ASS能不同程度地减少NO含量;(2)与Glu共培养的神经元,其存活率呈剂量和时间依赖性下降,ASS能增加神经元存活率;(3)经Glu处理的神经元发生凋亡,细胞超微结构呈现凋亡样改变,其凋亡率与正常对照组比较有显著性差异(P<0.01)。ASS能减少Glu毒性神经元凋亡。结论NO介导了Glu毒性神经元凋亡,ASS可能通过抑制NO的释放及其神经毒性作用,拮抗Glu引起的神经元凋亡。