A transient treatment of hippocampal neurons with alpha-tocopherol induces a long-lasting protection against oxidative damage via a genomic action

Neuroprotection exerted by alpha-tocopherol against oxidative stress was investigated in cultured rat hippocampal neurons. In addition to its direct action as a radical scavenger revealed at concentrations above 10 microM, a transient application of 1 microM alpha-tocopherol phosphate (alpha-TP) to neurons induced a complete delayed long-lasting protection against oxidative insult elicited by exposure to Fe2+ ions, but not against excitotoxicity. A minimal 16-h application of alpha-TP was required to observe the protection against subsequent oxidative stress. This delayed protection could last up to a week after the application of alpha-TP, even when medium was changed after the alpha-TP treatment. Cycloheximide, added either 2 h before or together with alpha-TP, prevented the delayed neuroprotection, but not the acute. However, cycloheximide applied after the 16-h alpha-TP pretreatment did not alter the delayed neuroprotection. Neither Trolox, a cell-permeant analogue of alpha-tocopherol, nor other antioxidants, such as epigallocatechin-gallate and N-acetyl-L-cysteine, elicited a similar long-lasting protection. Only tert-butylhydroquinone could mimic the alpha-TP effect. Depletion of glutathione (GSH) by L-buthionine sulfoximine did not affect the delayed alpha-TP protection. Thus, in addition to its acute anti-radical action, alpha-TP induces a long-lasting protection of neurons against oxidative damage, via a genomic action on antioxidant defenses apparently unrelated to GSH biosynthesis.     

银杏内酯B对谷氨酸诱导海马神经元损伤的影响

目的：观察银杏内酯B（ginkgolide B,GB）在不同给药模式下对谷氨酸所致海马神经元损伤的影响。方法：采用Co2超临界萃取的方法制备GB,建立新生Wistar大鼠原代培养的海马神经元谷氨酸毒性模型,采用台盼蓝染色、程序性细胞死亡检测技术及乳酸脱氢酶测定的方法,观察预处理与急救两种给药模型下不同剂量GB的神经保护作用,并与MK-801急性给药相比较。结果：GB在两种给药模式下均能不同程度地提高细胞存活率,降低凋亡率,减少LDH漏出量,且在一定范围内保护作用呈剂量依赖的方式。其中预处理的效果明显优于急救给药处理。但均弱于MK-801组。结论：GB对谷氨酸细胞毒性损伤有保护作用,预防性用药效果更佳。GB可能不仅仅通过拮抗血小板活化因子（PAF）受体等下游事件实现其神经保护作用。如果我们重视其预处理给药的显著效果。将其用于高危人群的预防干预可能有更大价值。     

Release of calcium from endolysosomes increases calcium influx through N-type calcium channels: Evidence for acidic store-operated calcium entry in neurons

Neurons possess an elaborate system of endolysosomes. Recently, endolysosomes were found to have readily releasable stores of intracellular calcium; however, relatively little is known about how such ‘acidic calcium stores’ affect calcium signaling in neurons. Here we demonstrated in primary cultured neurons that calcium released from acidic calcium stores triggered calcium influx across the plasma membrane, a phenomenon we have termed “acidic store-operated calcium entry (aSOCE)”. aSOCE was functionally distinct from store-operated calcium release and calcium entry involving endoplasmic reticulum. aSOCE appeared to be governed by N-type calcium channels (NTCCs) because aSOCE was attenuated significantly by selectively blocking NTCCs or by siRNA knockdown of NTCCs. Furthermore, we demonstrated that NTCCs co-immunoprecipitated with the lysosome associated membrane protein 1 (LAMP1), and that aSOCE is accompanied by increased cell-surface expression levels of NTCC and LAMP1 proteins. Moreover, we demonstrated that siRNA knockdown of LAMP1 or Rab27a, both of which are key proteins involved in lysosome exocytosis, attenuated significantly aSOCE. Taken together our data suggest that aSOCE occurs in neurons, that aSOCE plays an important role in regulating the levels and actions of intraneuronal calcium, and that aSOCE is regulated at least in part by exocytotic insertion of N-type calcium channels into plasma membranes through LAMP1-dependent lysosome exocytosis.     

肾上腺髓质素降低培养海马神经元胞内游离钙离子浓度

经荧光探针Fluo 3-AM标记细胞内游离钙后,用激光共聚焦显微镜检测肾上腺髓质素(adrenomedullin,ADM)对原代培养大鼠海马神经元内游离钙浓度([Ca^2 ]1)的影响。实验结果如下：(1)ADM(0．01-1．0μmol／L)浓度依赖性地降低细胞内钙浓度。(2)降钙素基因相关肽受体阻断剂(calcitonin gene-related peptide,CGRP8-37)预处理可部分抑制ADM的效应。(3)ADM可显著抑制高钾引起的[Ca^2 ]1增加。(4)ADM可显著抑制三磷酸肌醇(inositol 1,4,5-trisphosphate,IP3)引起的内钙释放,而对兰尼定(ryanodine)引起的内钙释放无显著影响。以上结果提示,ADM降低培养海马神经元内游离钙浓度,此作用与其抑制IP,引起的内钙释放有关,ADM对静息状态下的Ca^2 内流无影响,但可显著抑制高钾引起的Ca^2 内流,CGRP受体介导了ADM的上述效应。     

Orexin A-induced extracellular calcium influx in prefrontal cortex neurons involves L-type calcium channels

Orexins, novel excitatory neuropeptides from the lateral hypothalamus, have been strongly implicated in the regulation of sleep and wakefulness. In this study, we explored the effects and mechanisms of orexin A on intracellular free Ca²⁺ concentration ([Ca²⁺]_i) of freshly dissociated neurons from layers V and VI in prefrontal cortex (PFC). Changes in [Ca²⁺]_i were measured with fluo-4/AM using confocal laser scanning microscopy. The results revealed that application of orexin A (0.1 ≈1 μM) induced increase of [Ca²⁺]_i in a dose-dependent manner. This elevation of [Ca²⁺]_i was completely blocked by pretreatment with selective orexin receptor 1 antagonist SB 334867. While depletion of intracellular Ca²⁺ stores by the endoplasmic reticulum inhibitor thapsigargin (2 μM), [Ca²⁺]_i in PFC neurons showed no increase in response to orexin A. Under extracellular Ca²⁺-free condition, orexin A failed to induce any changes of Ca²⁺ fluorescence intensity in these acutely dissociated cells. Our data further demonstrated that the orexin A-induced increase of [Ca²⁺]_i was completely abolished by the inhibition of intracellular protein kinase C or phospholipase C activities using specific inhibitors, BIS II (1 μM) and D609 (10 μM), respectively. Selective blockade of L-type Ca²⁺ channels by nifedipine (5 μM) significantly suppressed the elevation of [Ca²⁺]_i induced by orexin A. Therefore, these findings suggest that exposure to orexin A could induce increase of [Ca²⁺]_i in neurons from deep layers of PFC, which depends on extracellular Ca²⁺ influx via L-type Ca²⁺ channels through activation of intracellular PLC-PKC signaling pathway by binding orexin receptor 1.     

Regulation of Kv4.2 channels by glutamate in cultured hippocampal neurons

Somatodendritic voltage-dependent K⁺ currents (Kv4.2) channels mediate transient A-type K⁺ currents and play critical roles in controlling neuronal excitability. Accumulating evidence has indicated that Kv4.2 channels are key regulatory components of the signaling pathways that lead to synaptic plasticity. In contrast to the extensive studies of glutamate-induced AMPA [(±) α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrate] receptors redistribution, less is known about the regulation of Kv4.2 by glutamate. In this study, we report that brief treatment with glutamate rapidly reduced total Kv4.2 levels in cultured hippocampal neurons. The glutamate effect was mimicked by NMDA, but not by AMPA. The effect of glutamate on Kv4.2 was dramatically attenuated by pre-treatment of NMDA receptors antagonist MK-801 [(5 S ,10 R )-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate] or removal of extracellular Ca²⁺. Immunocytochemical analysis showed a loss of Kv4.2 clusters on the neuronal soma and dendrites following glutamate treatment, which was also dependent on the activation of NMDA receptors and the influx of Ca²⁺. Furthermore, whole-cell patch-clamp recordings revealed that glutamate caused a hyperpolarized shift in the inactivation curve of A-type K⁺ currents, while the activation curve remained unchanged. These results demonstrate a glutamate-induced alteration of Kv4.2 channels in cultured hippocampal neurons, which might be involved in activity-dependent changes of neuronal excitability and synaptic plasticity.     

Action of hypoxia on different types of calcium channels in hippocampal neurons

Whole-cell patch clamp and polarographic oxygen partial pressure (pO2) measurements were used to establish the sensitivity of high-voltage-activated (HVA) Ca2+ channel subtypes of CA1 hippocampal neurons of rats to hypoxic conditions. Decrease of pO2 to 15-30 mm Hg induced a potentiation of HVA Ca2+ currents by 94%. Using selective blockers of N- and L-types of calcium channels, we found that inhibition of L-type channels decreased the effect by 54%, whereas N-type blocker attenuated the effect by 30%. Taking into account the ratio of currents mediated by these channel subtypes in CA1 hippocampal neurons, we concluded that both types of HVA Ca2+ channels are sensitive to hypoxia, however, L-type was about 3.5 times more sensitive to oxygen reduction.     

吗啡对培养海马神经元钙离子作用的机制研究

目的：研究吗啡对海马神经元[Ca^2 ]i影响的机制,为探索吗啡成瘾的神经生物学机制与可能的治疗途径。方法：荧光探针Fluo-4标记细胞内游离钙后,用激光共聚焦显微镜检测吗啡对大鼠原代培养海马神经元[Ca^2 ]i的影响。结果：吗啡急性刺激引起海马神经元[Ca^2 ]i升高,CTOP不能阻断吗啡引起的细胞内[Ca^2 ]i增加,而naltrindole能阻断吗啡引起的细胞内[Ca^2 ]i反应;Thapsigargin预处理阻断吗啡诱导的细胞内[Ca^2 ]i增加,Verapamil预处理不能完全抑制吗啡引起的细胞内[Ca^2 ]i增加;吗啡长时程作用后,海马神经元[Ca^2 ]i升高,加入纳络酮急性戒断后,不能阻断吗啡引起的细胞内[Ca^2 ]i升高,反而引起[Ca^2 ]i异常升高。结论：吗啡急性刺激引起的海马神经元内游离钙增加主要来源于δ2阿片受体介导的IP3敏感的钙库释放。     

Phenylalanine intraperitoneal injection effect on high-voltage calcium channels in rat hippocampal neurons

Neurons from the hippocampus of one-day-old (controls) rats intraperitoneally injected with phenylalanine were isolated and cultured [3, 8]. The potential-dependent calcium input channel (I_Ca) in hippocampal neurons of controls after five-seven days of culture was made up of two components, low and high voltage, when the membrane potential was fixed under intracellular perfusion. The high-voltage channel was 69±13% of the total calcium channel at Vt = –10 mV. After rats were injected with phenylalanine, the high-voltage I_Ca significantly decreased to 32±14% of the total channel in the neurons at the same Vt. The low-voltage I_Ca did not change significantly.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 1, pp. 98–104, January–February, 1991.     

Muscarinic inhibition of high-voltage-activated calcium channels in excised membranes of rat hippocampal neurons

The effect of muscarine on voltage-gated calcium channels was investigated in outside-out patches from rat hippocampal neurons in culture. By clamping the excised patches at –60 mV holding potential, single and multiple Ca channel currents were recorded, and these displayed features similar to the high-voltage-activated Ca current, with unitary conductance of 6.4 pS in 50 mM external Ca²⁺. These channels turned out to be insensitive both to Bay K 8644 and to -conotoxin. In excised patches muscarine caused a marked reduction in the probability of opening of this class of Ca channels without significant changes in the unitary current amplitude. Interestingly, the degree of current inhibition was reduced by depolarization, thus suggesting a voltage-dependent inhibitory action of the agonist. We conclude that in hippocampal neurons one of the possible ways of HVA Ca channel modulation by muscarine occurs through activation of a substratum localized within the plasma membrane of the cell, independent of the involvement of diffusible intracellular messengers. Correspondence to: M. Toselli     

Protection against heat stress-induced oxidative damage in Arabidopsis involves calcium, abscisic acid, ethylene, and salicylic acid

Plants, in common with all organisms, have evolved mechanisms to cope with the problems caused by high temperatures. We examined specifically the involvement of calcium, abscisic acid (ABA), ethylene, and salicylic acid (SA) in the protection against heat-induced oxidative damage in Arabidopsis. Heat caused increased thiobarbituric acid reactive substance levels (an indicator of oxidative damage to membranes) and reduced survival. Both effects required light and were reduced in plants that had acquired thermotolerance through a mild heat pretreatment. Calcium channel blockers and calmodulin inhibitors increased these effects of heating and added calcium reversed them, implying that protection against heat-induced oxidative damage in Arabidopsis requires calcium and calmodulin. Similar to calcium, SA, 1-aminocyclopropane-1-carboxylic acid (a precursor to ethylene), and ABA added to plants protected them from heat-induced oxidative damage. In addition, the ethylene-insensitive mutant etr-1, the ABA-insensitive mutant abi-1, and a transgenic line expressing nahG (consequently inhibited in SA production) showed increased susceptibility to heat. These data suggest that protection against heat-induced oxidative damage in Arabidopsis also involves ethylene, ABA, and SA. Real time measurements of cytosolic calcium levels during heating in Arabidopsis detected no increases in response to heat per se, but showed transient elevations in response to recovery from heating. The magnitude of these calcium peaks was greater in thermotolerant plants, implying that these calcium signals might play a role in mediating the effects of acquired thermotolerance. Calcium channel blockers and calmodulin inhibitors added solely during the recovery phase suggest that this role for calcium is in protecting against oxidative damage specifically during/after recovery.     

Haloperidol induces calcium ion influx via L-type calcium channels in hippocampal HN33 cells and renders the neurons more susceptible to oxidative stress

Haloperidol is a classical neuroleptic drug that is still in clinical use and can lead to abnormal motor activity following repeated administration. However, there is little knowledge of how it triggers neuronal impairment. In this study, we report that it induced calcium ion influx via L-type calcium channels and that the elevation of calcium ions induced by haloperidol appeared to render hippocampal cells more susceptible to oxidative stress. Indeed, the level of cytotoxic reactive oxygen species (ROS) and the expression of pro-apoptotic Bax increased in response to oxidative stress in haloperidol-treated cells, and these effects were inhibited by verapamil, a specific L-type calcium channel blocker, but not by the T-type calcium channel blocker, mibefradil. These findings indicate that haloperidol induces calcium ion influx via L-type calcium channels and that this calcium influx influences neuronal fate.     

Lipid peroxidation reduction and hippocampal and cortical neurons protection against ischemic damage in animal model using Stellaria media

This study was aimed to determine the neuroprotective influence of Stellaria media in terms of restoring normal state of the rat’s hippocampus and cortex after oxidative insult caused by in vitro ischemia and reperfusion. Cell viability and membrane integrity were assessed using MTT and lactate dehydrogenase (LDH) assay, respectively. Ischemic insult was introduced in the rat brain’s hippocampal and cortical slivers by exposing oxygen and glucose deficiency (OGD) for 2 h, followed by 1 h of re-perfusion. Cellular oxidative stress levels were quantified by incorporating 2?,7?-dichlorofluorescein diacetate fluorescent probes. Additionally, the lipid peroxidation was assessed using TBARS assay. Findings revealed significant neuroprotection against OGD-induced mitochondrial impairment at 40 µg/mL of S. media in rat’s hippocampal and cortical slices. The LDH levels were decreased significantly (P < 0.001) during pre-incubation and reoxygenation periods using varied concentrations of S. media extract. Cellular oxidative stress levels results showed significant (P < 0.001) reduction in dichlorofluorescein fluorescence in slices homogenate of hippocampus and cortex using S. media extract. The lipid peroxidation assay results showed decreased (P < 0.01) levels of malondialdehyde in liver tissues of treated rats treated (200 mg/kg body weight) when compared to the ischemic animal. In summary, findings clearly indicated the neuroprotective effects of extract against in vitro ischemia in brain hippocampal and cortex slivers. S. media could undoubtedly be utilized as a healing agent in preventing neuronal cells’ loss during is chemic-reperfusion process.     

Progesterone potentiates calcium release through IP3 receptors by an Akt-mediated mechanism in hippocampal neurons

Progesterone (P4) is a steroid hormone that plays multiple roles in the central nervous system (CNS) including promoting neuroprotection. However, the precise mechanisms involved in its neuroprotective effects are still unknown. Given that the regulation of the intracellular calcium (Ca²⁺) concentration is critical for cell survival, we determined if inositol 1, 4, 5-trisphosphate receptors (IP₃Rs) are relevant targets of P4. Using primary hippocampal neurons, we tested the hypothesis that P4 controls the gain of IP3R-mediated intracellular Ca²⁺ signaling in neurons and characterized the subcellular distribution and phosphorylation of potential signaling intermediates involved in P4s actions. Our results reveal that P4 treatment altered the intensity and distribution of IP3R immunoreactivity and induced the nuclear translocation of phosphorylated Akt. Further, P4 potentiated IP₃R-mediated intracellular Ca²⁺ responses. These results suggest a potential involvement of P4 in particular and of steroid hormone signaling pathways in general in the control of intracellular Ca²⁺ signaling and its related functions.     

Regulation of N-type calcium channels by G-proteins: Multiple pathways to control calcium entry into neurons

Review from: Alexandra E. Kisilevsky and Gerald W. Zamponi. D2 dopamine receptors interact directly with N-type calcium channels and regulate surface expression levels. Channels 2008; 2(4)1-8.     

NGF and bFGF protect rat hippocampal and human cortical neurons against hypoglycemic damage by stabilizing calcium homeostasis.

NGF and bFGF have recently been shown to have biological activity in central neurons, but their normal functions and mechanisms of action are unknown. Since central neurons are particularly vulnerable to hypoglycemia that occurs with ischemia or insulin overdose, we tested the hypothesis that growth factors can protect neurons against hypoglycemic damage. NGF and bFGF each prevented glucose deprivation-induced neuronal damage in human cerebral cortical and rat hippocampal cell cultures (EGF was ineffective). Protection was afforded when the growth factors were administered before (NGF and bFGF) or up to 12 hr following (NGF) the onset of hypoglycemia. Direct measurements of intracellular calcium levels and manipulations of calcium influx demonstrated that sustained elevations in intracellular calcium levels mediated the hypoglycemic damage. NGF and bFGF each prevented the hypoglycemia-induced elevations of intracellular calcium. These findings indicate that growth factors can stabilize neuronal calcium homeostasis in central neurons and thereby protect them against environmental insults.     

Role of calcium ion in protection against heat and high irradiance stress-induced oxidative damage to photosynthesis of wheat leaves

Cross stress of heat and high irradiance (HI) resulted in the accumulation of active oxygen species and photo-oxidative damage to photosynthetic apparatus of wheat leaves during grain development. Pre-treatment with calcium ion protected the photosynthetic system from oxidative damage by reducing O^-. ₂ production, inhibiting lipid peroxidation, and retarding electrolyte leakage from cell. Therefore, high F_v/F_m [maximal photochemical efficiency of photosystem 2 (PS2) while all PS2 reaction centres are open], F_m/F₀ (another expression for the maximal photochemical efficiency of PS2), Φ_PS2 (actual quantum yield of PS2 under actinic irradiation), q_P (photochemical quenching coefficient), and P _N (net photosynthetic rate) were maintained, and lower q_NP (non-photochemical quenching coefficient) of the leaves was kept under heat and HI stress. EGTA (a chelant of calcium ion) and LaCl₃ (a blocker of Ca²⁺ channel in cytoplasmic membrane) had the opposite effect. Thus Ca ion may help protect the photosynthetic system of wheat leaves from oxidative damage induced by the cross stress of heat and HI.