Effect of Anticonvulsant Drugs on 7-Hydroxybutyrate Release from Hippocampal Slices: Inhibition by Valproate and Ethosuximide

The effects of some anticonvulsant drugs have been investigated on gamma-hydroxybutyrate release from rat hippocampal and striatal slices. Sodium valproate and ethosuximide inhibited the depolarization-evoked release of gamma-hydroxybutyrate induced by 40 mM K+. The IC50 values for these two drugs are in the concentration range of valproate and ethosuximide that exists in rat brain after administration of anticonvulsant doses to the animals. Trimethadione and pentobarbital are without significant effects. It can be concluded that the inhibition of gamma-hydroxybutyrate release, particularly that observed for hippocampus, might explain the protective effect of valproate and ethosuximide on gamma-hydroxybutyrate-induced seizures and perhaps on other kinds of epileptoid phenomenon.     

Neuroprotection by 7-Nitroindazole Against Iron-Induced Hippocampal Neurotoxicity

(1) Iron plays an important role in maintaining normal brain function. In some neurodegenerative disorders including Parkinson's and Alzheimer's disease, iron levels rise in the brain. It is known that higher iron levels induce neuronal hyperactivity and oxidative stress. A body of evidence indicates a relationship between neuronal death and nitric oxide (NO). The aim of present study was to evaluate the effects of NO produced by neuronal nitric oxide synthase (nNOS) on iron-induced neuronal death. (2) Animals were classified into four groups: control, iron, iron+7-nitroindazole, and iron+vehicle. Rats in iron, iron+7-nitroindazole, and iron+vehicle groups received intracerebroventricular (i.c.v.) FeCl3 injection (200 mM, in 2.5 microl). Rats belonging to control groups received the same amount of saline into the cerebral ventricles. All animals were kept alive for 10 days following the operation. Animals in iron+7-nitroindazole group received intraperitoneal 7-nitroindazole (30 mg/kg/day) injections once a day during this period, while the rats belonging to vehicle group received daily intraperitoneal injection of peanut oil. After 10 days, rats were perfused intracardially under deep urethane anesthesia. Removed brains were processed using the standard histological techniques. (3) The total number of neurons in hippocampus of all rats was estimated with the unbiased stereological techniques. Results of present study show that 7-nitroindazole decreased mean neuron loss from 43% to 11%. Treatment of peanut oil alone did not affect iron-induced hippocampal cell loss with respect to iron group values. (4) Findings of our study suggest that 7-nitroindazole may have neuroprotective effects against iron-induced hippocampal neurotoxicity by inhibiting nNOS.     

动物细胞培养过程中的细胞凋亡

细胞培养过程中的细胞凋亡是细胞受环境因素的影响而发生的现象。随着对细胞凋亡的分子生物学和细胞生物学了解的深入,显示了有效地控制动物细胞培养中细胞凋亡的巨大潜力。包括采用ＤＮＡ重组技术把抗细胞凋亡的基因导入细胞和在培基中加入具有抗细胞凋亡的生存因子或化合物等手段已用于控制细胞培养过程中的细胞凋亡。这些技术将大大延长细胞达到饱和密度后的培养时间,提高细胞培养系统的生产效率。     

Mitochondrial Respiratory Function and Cell Death in Focal Cerebral Ischemia

Pyruvate-supported oxygen uptake was determined as a measure of the functional capacity of mitochondria obtained from rat brain during unilateral middle cerebral artery occlusion and reperfusion. During ischemia, substantial reductions developed in both ADP-stimulated and uncoupled respiration in tissue from the focus of the affected area in the striatum and cortex. A similar pattern of change but with lesser reductions was seen in the adjacent perifocal tissue. Succinate-supported respiration was more affected than that with pyruvate in perifocal tissue at 2 h of ischemia, suggesting additional alterations to mitochondrial components in this tissue. Mitochondrial respiratory activity recovered fully in samples from the cortex, but not the striatum, within the first hour of reperfusion following 2 h of ischemia and remained similar to control values at 3 h of reperfusion. In contrast, impairment of the functional capacity of mitochondria from all three regions was seen in the first 3 h of reperfusion following 3 h of ischemia. Extensive infarction generally affecting the cortical focal tissue with more variable involvement of the perifocal tissue developed following 2 h of focal ischemia. Thus, mitochondrial impairment during the first 3 h of reperfusion was apparently not essential for tissue infarction to develop. Nonetheless, the observed mitochondrial changes could contribute to the damage produced by permanent focal ischemia as well as the larger infarcts produced when reperfusion was initiated following 3 h of ischemia.     

细胞外ATP通过刺激NADPH氧化酶缓解水杨酸诱导的细胞死亡

水杨酸（SA）是植物重要的信号分子,低浓度的SA能够诱导植物的抗病反应,而高浓度的SA导致植物细胞死亡。本文采用500μmol·L-1的SA处理烟草悬浮细胞BY-2,研究了细胞外ATP在SA诱导的细胞死亡中的作用及可能的机制。结果显示,外源ATP可缓解SA诱导的细胞死亡水平的上升。另外,SA导致NADPH氧化酶活性下降,而外源ATP则刺激其活性上升。外源ATP能缓解SA对NADPH氧化酶活性的抑制,且这种缓解作用可被NADPH氧化酶的抑制剂——二亚苯基碘（DPI）所消除。DPI还可部分消除外源ATP对SA所诱导的细胞死亡的缓解作用。上述结果表明,胞外ATP通过刺激NADPH氧化酶活性缓解SA诱导的细胞死亡。     

Role of Potassium Channels in Amyloid-Induced Cell Death

Abstract: Basal forebrain cholinergic neurons are severely depleted early in Alzheimer's disease and appear particularly susceptible to amyloid β-peptide (Aβ) toxicity in vivo. To model this effect in vitro, a cholinergic septal cell line (SN56) was exposed to Aβ. SN56 cells exhibited a tetraethylammonium (TEA)-sensitive outward K⁺ current with delayed rectifier characteristics. Increases of 64% (±19; p < 0.02) and 44% (±12; p < 0.02) in K⁺ current density were noted 6–12 and 12–18 h following the addition of Aβ to SN56 cell cultures, respectively. Morphological observation and staining for cell viability showed that 25 ± 4 and 39 ± 4% of SN56 cells were dead after 48- and 96-h exposures to Aβ, respectively. Perfusion of SN56 cells with 10–20 mM TEA blocked 71 ± 6 to 92 ± 2% of the outward currents, widened action potentials, elevated [Ca²⁺]_i, and inhibited 89 ± 14 and 68 ± 14% of the Aβ toxicity. High [K⁺]_o, which depolarizes cell membranes and increases [Ca²⁺]_i, also protected SN56 cells from Aβ toxicity. This effect appeared specific since glucose deprivation of SN56 cells did not alter K⁺ current density and TEA did not protect these cells from hypoglycemic cell death. Furthermore, Aβ was toxic to a dopaminergic cell line (MES23.5) that expressed a K⁺ current with delayed rectifier characteristics; K⁺ current density was not altered by Aβ and MES23.5 cells were not protected by TEA from Aβ toxicity. In contrast, a noncholinergic septal cell line (SN48) that shows minimal outward K⁺ currents was resistant to the toxicity of Aβ. These data suggest that a K⁺ channel with delayed rectifier characteristics may play an important role in Aβ-mediated toxicity for septal cholinergic cells.     

Cotton GhBAK1 Mediates Verticillium Wilt Resistance and Cell Death

Virus-induced gene silencing (VIGS) offers a powerful approach for functional analysis of individual genes by knocking down their expression. We have adopted this approach to dissect gene functions in cotton resistant to Verticillium wilt, one of the most devastating diseases worldwide. We showed here that highly efficient VIGS was obtained in a cotton breeding line (CA4002) with partial resistance to Verticillium wilt, and GhMKK2 and GhVe1 are required for its resistance to Verticillium wilt. Arabidopsis AtBAK1/SERK3, a central regulator in plant disease resistance, belongs to a subfamily of somatic embryogenesis receptor kinases (SERKs) with five members, AtSERK1 to AtSERK5. Two BAK1 orthologs and one SERK1 ortholog were identified in the cotton genome. Importantly, GhBAK1 is required for CA4002 resistance to Verticillium wilt. Surprisingly, silencing of GhBAK1 is sufficient to trigger cell death accompanied with production of reactive oxygen species in cotton. This result is distinct from Arabidopsis in which AtBAK1 and AtSERK4 play redundant functions in cell death control. Apparently, cotton has only evolved SERK1 and BAK1 whereas AtSERK4/5 are newly evolved genes in Arabidopsis. Our studies indicate the functional importance of BAK1 in Verticillium wilt resistance and suggest the dynamic evolution of SERK family members in different plant species.     

植物细胞程序死亡的机理及其与发育的关系

细胞程序死亡（ＰＣＤ）是在植物体发育过程中普遍存在的,在发育的特定阶段发生的自然的细胞死亡过程,这一死亡过程是由某些特定基因编码的“死亡程序”控制的。ＰＣＤ的细胞分化的最后阶段。细胞分化的临界期就牌死亡程序执行中的某个阶段。ＰＣＤ包含启动期和清除期三个阶段,其间ＣＡＳＰＡＳＥ家族起着重要作用。ＰＣＤ在细胞和组织的平衡、特化,以及组织分化、器官建成和对病原体的反应等植物发育过程中起着重要作用。ＰＣＤ     

CHP调节NHE1活性影响细胞生长和死亡

钠氢离子交换蛋白(NHE)定位于细胞膜,它的重要功能是调节细胞内pH值。钙调磷酸酶B同源蛋白(CHP)是NHE必要的活性调节亚单位。研究了NHE1结合CHP与否对细胞生长和死亡的影响。结果显示,CHP结合于NHE1细胞质调节区域之中靠近细胞膜部位,二者以疏水键结合而形成蛋白IV级结构。在细胞内pH5.4的非生理条件下,表达没有CHP结合能力的突变体NHE1-4R细胞只有表达野生型NHE细胞7.6%的最大摄取钠活性;在细胞内pH7.2的生理条件下,这个比例降至1.2%的摄取钠活性。与野生型NHE1比较,有血清时表达突变体NHE1-4R的细胞生长速度减慢;在血清饥饿时这些细胞因自身的胞浆酸性化而死亡数增加。实验结果证明,CHP是NHE1生理活性的必要调节因子,它能影响细胞生长和死亡。     

Cell Death and Growth Recovery of Barley after Transient Salt Stress

3 H-thymidine) in old roots were partially recycled for new tissue formation. This result indicates that cell death may have some physiological roles under transient salt stress. Received 11 January 2000/ Accepted in revised form 29 June 2000     

N-Methyl-D-Aspartate Receptor Activation and Ca2+ Account for Poor Pyramidal Cell Structure in Hippocampal Slices

The CA1 pyramidal cells appear damaged in micrographs of guinea pig hippocampal slices incubated in normal physiological buffer at 36–37°C. This is remedied if slices are incubated in modified buffers for the first 45 min. Cell morphology is improved if this buffer is devoid of added Ca²⁺ and much improved if it contains N-methyl-D-aspartate (NMDA) receptor antagonists or 0 mM Ca²⁺ and 10 mM Mg²⁺. The cells then appear similar to CA1 pyramidal cells in situ. These findings support the notion that NMDA receptor activation and Ca²⁺, acting in the period immediately after slice preparation, permanently damage CA1 pyramidal cells in vitro.     

Transfection of N-Methyl-d-Aspartate Receptors in a Nonneuronal Cell Line Leads to Cell Death

Abstract: Neurons grown in culture die when they are exposed to high concentrations (0.1–1 m M ) of the neurotransmitter l -glutamate. A similar phenomenon may occur in the mammalian brain during ischemia and other injuries that cause excessive glutamate release. Activation of N -methyl- d -aspartate (NMDA) receptors and the consequent Ca²⁺ influx are thought to play a critical role in the process of neuronal toxicity. Events subsequent to the Ca²⁺ influx are not well understood. We have discovered that nonneuronal kidney cells expressing NMDA receptors after DNA transfection undergo cell death unless they are protected by drugs that block the NMDA receptor ion channel. Furthermore, transfected cells expressing a mutated NMDA receptor that conducts less Ca²⁺ are less vulnerable to cell death. In addition, we find that even though several active forms of NMDA receptors can be synthesized in these cells after transfection with different cloned subunits, not all receptor types are equally toxic. These experiments suggest that Ca²⁺ influx through NMDA channels may be toxic to nonneuronal cells and that the NMDA receptor expression may be the major neuron-specific component of excitotoxicity.     

Depletion of the Central Metabolite NAD Leads to Oncosis-mediated Cell Death

Depletion of the central metabolite NAD in cells results in broad metabolic defects leading to cell death and is a proposed novel therapeutic strategy in oncology. There is, however, a limited understanding of the underlying mechanisms that connect disruption of this central metabolite with cell death. Here we utilize GNE-617, a small molecule inhibitor of NAMPT, a rate-limiting enzyme required for NAD generation, to probe the pathways leading to cell death following NAD depletion. In all cell lines examined, NAD was rapidly depleted (average t_½ of 8.1 h) following NAMPT inhibition. Concurrent with NAD depletion, there was a decrease in both cell proliferation and motility, which we attribute to reduced activity of NAD-dependent deacetylases because cells fail to deacetylate α-tubulin-K40 and histone H3-K9. Following depletion of NAD by >95%, cells lose the ability to regenerate ATP. Cell lines with a slower rate of ATP depletion (average t_½ of 45 h) activate caspase-3 and show evidence of apoptosis and autophagy, whereas cell lines with rapid depletion ATP (average t_½ of 32 h) do not activate caspase-3 or show signs of apoptosis or autophagy. However, the predominant form of cell death in all lines is oncosis, which is driven by the loss of plasma membrane homeostasis once ATP levels are depleted by >20-fold. Thus, our work illustrates the sequence of events that occurs in cells following depletion of a key metabolite and reveals that cell death caused by a loss of NAD is primarily driven by the inability of cells to regenerate ATP.     

Glucose Metabolism Assessed with 2-Deoxyglucose and the Effect of Glutamate in Subdivisions of Rat Hippocampal Slices

A new approach to the study of glucose phosphorylation in brain slices is described. It is based on timed incubation with nonradioactive 2-deoxyglucose (DG), after which the tissue levels of DG and 2-deoxyglucose-6-phosphate (DG6P) are measured separately with sensitive enzymatic methods applied to specific small subregions. The smallest samples had dry weights of approximately 0.5 microgram. Direct measurements in different regions of hippocampal slices showed that within 6 min after exposure to DG, the ratios of DG to glucose in the tissue were almost the same as in the incubation medium, which simplifies the calculation of glucose phosphorylation rates and increases their reliability. Data are given for ATP, phosphocreatine, sucrose space, and K+ in specific subregions of the slices. DG6P accumulation proceeded at a constant rate for at least 10 min, even when stimulated by 10 mM glutamate in the medium. The calculated control rate of glucose phosphorylation was 2 mmol/kg (dry weight)/min. In the presence of 10 mM glutamate it was twice as great. The response to 10 mM glutamate of different regions of the slice was not uniform, ranging from 164% of control values in the molecular layer of CA1 to 256% in the stratum radiatum of CA1. There was a profound fall in phosphocreatine levels (75%) in response to 10 mM glutamate despite a 2.4-fold increase in glucose phosphorylation. Even in the presence of 1 mM glutamate, the increase in glucose phosphorylation (50%) was not great enough to prevent a significant drop in phosphocreatine content.     

Mitochondrial Genome Mutation in Cell Death and Aging

This article reviews the concept, molecular genetics, and pathology of cell death and agingin relation to mitochondrial genome mutation. Accumulating evidence emphasizes the role ofgenetic factors in the development of naturally occurring cell death and aging. The ATPrequired for a cell's biological activity is almost exclusively produced by mitochondria. Eachmitochondrion possesses its own DNA (mtDNA) that codes essential subunits of themitochondrial energy-transducing system. Recent studies confirm that mtDNA is unexpectedly fragileto hydroxyl radical damage, hence to the oxygen stress. Cellular mtDNA easily fragmentsinto over a hundred-types of deleted mtDNA during the life of an individual. Cumulativeaccumulation of these oxygen damages and deletions in mtDNA results in a defective energytransducing system and in bioenergetic crisis. The crisis leads cells to the collapse ofmitochondrial trans-membrane potential, to the release of the apoptotic protease activating factors intocytosol, to uncontrolled cell death, to tissue degeneration and atrophy, and to aging. Thetotal base sequencing of mtDNA among individuals revealed that germ-line point mutationstransmitted from ancestors accelerate the somatic oxygen damages and mutations in mtDNAleading to phenotypic expression of premature aging and degenerative diseases. A practicalsurvey of point mutations will be useful for genetic diagnosis in predicting the life-span ofan individual.     

Hypoxia Ischemia-Mediated Cell Death in Neonatal Rat Brain

The examination of Bcl-2-associated X protein (Bax) protein’s role in the activation of cognate nuclear, mitochondrial and ER cell death signaling cascades and the resulting effects on cell death phenotype in the brain after neonatal hypoxia-ischemia (HI) requires an understanding of neonatal HI insult and progression, as well as, its dysfunctional outcomes. In addition, knowledge of key concepts of oxidative stress, a major injurious component of HI, and the different cell death phenotypes (i.e. apoptosis and necrosis) will aid the design of appropriate useful experimental paradigms. Here we discuss organelle cell death signaling cascades in the context of the different cell death phenotypes associated with animal models of neonatal hypoxia ischemia and tissue culture models used in the study of hypoxia ischemia, focusing on the intracellular shifts of the Bcl-2 associated X protein (Bax) in the hypoxic brain. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

Lithium and hormesis: Enhancement of adaptive responses and biological performance via hormetic mechanisms

Biomedical and consumer interest in the health-promoting properties of pure single entities of known or unknown chemical constituents and mixtures has never been greater. Since its “rediscovery” in the 1950s, lithium is an example of such a constituent that represents an array of scientific and public health challenges and medical potentials that may now be understood best when seen through the lens of the dose-response paradigm known as hormesis. The present paper represents the first review of the capacity of lithium to induce hormetic dose responses in a broad range of biological models, organ systems, and endpoints. Of significance is that the numerous hormetic findings occur with extensive concentration/dose response evaluations with the optimal dosing being similar across multiple organ systems. The particular focus of these hormetic dose-response findings was targeted to research with a broad spectrum of stem cell types and neuroprotective effects. These findings suggest that lithium may have critically valuable systemic effects with respect to those therapeutically treated with lithium as well as for exposures that may be achieved via dietary intervention.