一氧化氮合酶激活参与烧伤后Ca2+介导的心肌线粒体损伤

目的:探讨线粒体一氧化氮合酶(mtNOS)在严重烧伤早期心肌线粒体损害中的作用。方法:复制30%TBSAⅢ°烧伤大鼠模型,取正常及伤后1、3、6、12、24h大鼠心肌分离线粒体,测其呼吸功能、Ca²⁺浓度(_m)及细胞色素c氧化酶、mtNOS活性。结果:①伤后1h心肌线粒体呼吸控制率(RCR)显著高于正常组,但3、6、12、24h明显低于正常组,Ⅲ态呼吸速率(ST₃)变化与RCR平行,ST₄仅于伤后3h明显升高;②伤后各时点_m均明显高于正常组,尤以3、6h为甚,而mtNOS活性于伤后3、6、12、24h显著高于对照组,细胞色素c氧化酶活性于伤后3、6、12、24h显著低于正常组;③伤后mtNOS活性与_m呈明显正相关,相关系数为0.8945(P＜0.05),RCR与mtNOS活性呈显著负相关,相关系数为-0.9347(P＜0.05)。结论:伤后_m升高激活mtNOS,可能参与严重烧伤早期心肌线粒体损害。     

氢氟酸烧伤中毒对兔外周血单个核细胞凋亡与胞内钙浓度的影响

目的: 研究氢氟酸烧伤中毒对兔外周血单个核细胞(PBMC)早期凋亡百分率和胞内游离钙浓度([Ca²⁺]_i)的影响。方法: 用流式细胞仪检测兔烧伤染毒前后外周血单个核细胞的Annexin V变化, 以观察其早期凋亡百分率。用Fluo-3/Am荧光探针观察烧伤染毒前后外周血单个核细胞内Ca²⁺平均荧光强度值的变化, 以观察细胞内[Ca²⁺]_i的变化。 结果: 12只烧伤中毒兔外周血单个核细胞早期凋亡百分率显著增加, 烧伤染毒前后比较P<0.01。而12只烧伤中毒兔中8只染毒后1 h外周血单个核细胞内[Ca²⁺]_i显著降低, 烧伤染毒前后比较P<0.05。其余4只却表现[Ca²⁺]_i染毒前后比较P>0.05。 结论: 本实验氢氟酸烧伤中毒使兔外周血单个核细胞早期凋亡百分率显著增加, 外周血单个核细胞内[Ca²⁺]_i却显著降低。提示氢氟酸烧伤中毒引导的细胞凋亡并非细胞内[Ca²⁺]_i增加所引发。     

心肌肽素对缺氧-再给氧损伤心肌

目的:研究多肽类物质心肌肽素对缺氧-再给氧损伤心肌细胞的保护作用。方法:复制培养心肌细胞缺氧-再给氧损伤模型,缺氧60min,再给氧30min,观察心肌肽素对细胞超微结构的影响。以ACAS570进行细胞内游离Ca^2＋的定性检测;以荧光染料Fura-2-AM定量测定细胞内游离Ca^2＋浓度;以荧光偏振法测定细胞膜脂质流动性。结果:心肌肽素能使心肌细胞线粒体超微结构的损伤减轻;可剂量依赖性地降低[Ca^2＋]_i和提高细胞膜脂质流动性;可明显减少Ca^2＋伪彩色三维图色彩值。结论:心肌肽素对缺氧-再给氧损伤心肌细胞有明显保护作用,可能与其降低[Ca^2＋]sub>i和提高细胞膜脂质流动性有关。     

白细胞介素-2对缺氧/复氧心肌细胞[Ca2+]i的作用

目的:观察白细胞介素-2(IL-2)对心肌细胞在缺氧/复氧过程中电刺激诱导的[Ca²⁺]i的作用。方法:采用酶解分离成年大鼠心室肌细胞化学缺氧模型, 以Fura-2/AM为钙探针, 用细胞内双波长钙荧光系统检测心肌[Ca²⁺]i的变化。结果:①缺氧/复氧过程中, 缺氧5min时, 心肌[Ca²⁺]i幅度降低、舒张末期[Ca²⁺]i升高, [Ca²⁺]i达峰时间(TTP)延长, 恢复时间(RT)延长。复氧10min后, 心肌[Ca²⁺]i幅度、舒张末期[Ca²⁺]i、TTP及RT逐渐回复, 但不能完全恢复到对照水平;②在缺氧期间加入IL-2(2×10⁵U/L), 复氧期间[Ca²⁺]i各参数回复减慢;③用κ-阿片受体拮抗剂nor-BNI(10^-8mol/L)预处理后, 缺氧+IL-2对复氧时[Ca²⁺]i作用的影响被减弱, 而δ-阿片受体拮抗剂纳曲吲哚(10^-6mol/L)预处理则无此作用。结论:缺氧时同时存在IL-2, 可加剧复氧时心肌[Ca²⁺]i的变化, 其机制可能是IL-2通过心肌κ-阿片受体而发挥作用。     

脂多糖对大鼠肺微血管内皮细胞[Ca2+]i和Gq蛋白的影响及山莨菪碱的干预

目的:观察脂多糖对大鼠肺微血管内皮细胞(RPMVECs)[Ca²⁺]_i和Gq蛋白的影响及山莨菪碱的干预作用。方法:分离、培养并鉴定Wistar大鼠RPMVECs;应用Fura-2/AM法测定RPMVECs[Ca²⁺]_i;流式细胞仪技术测定RPMVECsGq蛋白。结果:①LPS作用于RPMVECs30min和90min后,[Ca²⁺]_i显著高于对照组;Gq蛋白显著低于对照组。②山莨菪碱可抑制LPS的上述作用。结论:①LPS致RPMVECs[Ca²⁺]_i增加和Gq蛋白下降;②山莨菪碱通过抑制LPS诱导RPMVECs[Ca²⁺]_i增加和Gq蛋白下降的作用而保护其内皮屏障功能。     

噻庚啶和山莨菪碱拮抗TNFα诱导的内皮细胞[Ca2+]i增高

目的：研究噻庚啶（Cyp）和山莨菪碱（Ani）对肿瘤坏死因子（TNFα）诱导单个内皮细胞内Ca²⁺浓度（[Ca²⁺]_i）变化的影响，以探TNFα介导休克和Cyp、Ani的抗休克的机制。方法：人脐静脉内皮细胞株（ECV304）接种于35 mm含2 mL DMEM培养基的组织培养盘中培养。Fluo-3/AM负载细胞，激光扫描共聚焦显微技术（LSCM）测定单个内皮细胞[Ca²⁺]_i。结果：TNFα使单个内皮细胞[Ca²⁺]_i呈剂量依赖性升高，在60 s内达到峰值，然后下降并保持在基础水平之上。共聚焦扫描图像显示细胞核区[Ca²⁺]_i升高比胞浆区明显，下降比胞浆区慢。Cyp（3×10^-5 mol/L或6×10^-5 mol/L）、Ani（2×10^-5 mol/L或4×10^-5 mol/L）均能显著抑制由TNFα（1.2×10^-9 mol/L）诱导的单个内皮细胞[Ca²⁺]_i升高。结论：TNFα诱导内皮细胞[Ca²⁺]_i升高可能是TNFα介导休克的重要机制；Cyp和Ani抑制TNFα诱导的[Ca²⁺]_i升高可能是其抗休克作用的机制之一。     

小鼠腹腔巨噬细胞基态游离钙离子浓度的不均一性及其和细胞反应性的关系

目的:在单细胞水平上研究小鼠腹腔巨噬细胞(PM)基态游离钙离子浓度([Ca²⁺]_i)的不均一性及其和细胞反应性的关系。方法:用荧光指示剂Fura-2/AM结合荧光显微镜成像系统检测单个PM基态的及用激动剂刺激细胞后的[Ca²⁺]_i;同时结合NBT染色法定量检测单个PM产超氧阴离子(O₂^-)水平。结果:对7只正常小鼠共392个PM基态[Ca²⁺]_i的研究表明小鼠PM的基态[Ca²⁺]_i呈正态分布[(54±24)nmol/L,n=392],但波动范围较大(从10nmol/L到高于100nmol/L),以[Ca²⁺]_i在40-60nmol/L的细胞数量最多(约占50%)。用PMA、fMLP刺激后PM[Ca²⁺]_i升高,且受刺激后[Ca²⁺]_i升高的峰值和基态[Ca²⁺]_i之间呈正相关(PMA刺激组:r=052,P<0.01,n=58;fMLP刺激组:r=0.59,P<0.01,n=44。此两组实验均以不同的小鼠重复3次,其它两只小鼠的结果与上同。下面的表述方法同此)。另外小鼠PM的基态[Ca²⁺]_i与其受PMA刺激后产生O₂^-的量也呈显著正相关(r=0.42,P<0.01,n=43,重复4次)。结论:小鼠PM的基态[Ca²⁺]_i是不均一的,且基态[Ca²⁺]_i的高低和该细胞对致炎因子的反应性密切相关。     

丹参对抗缺氧/复氧引起的大鼠心室肌细胞收缩和细胞内钙的改变

目的:观察丹参在常氧和缺氧/复氧过程中对心肌细胞收缩和电刺激诱导的细胞内钙([Ca²⁺]_i)瞬态的影响。方法: 采用酶解分离成年大鼠心室肌细胞化学缺氧模型, 用视频跟踪计算机系统和细胞内双波长钙荧光系统分别观察心肌细胞收缩力学和[Ca²⁺]_i等指标。结果:丹参(1-9 g/L)处理后降低心肌细胞最大收缩和舒张速率、收缩幅度以及电刺激诱导的[Ca²⁺]_i幅度, 且呈剂量依赖性。缺氧后, 与对照相比细胞收缩力和钙瞬态幅度降低、舒张末细胞长度缩短、舒张末钙水平增高;复氧后细胞收缩力、钙瞬态幅度和舒张末钙水平有所回复, 但不能达对照水平。用3 g/L的丹参处理后, 缺氧/复氧引起的心肌细胞最大收缩和舒张速率、收缩幅度和电刺激诱导的[Ca^2＋]_i幅度高于单纯缺氧组, 舒张末[Ca²⁺]_i水平低于单纯缺氧组。结论:丹参可对抗缺氧/复氧引起的大鼠心室肌细胞收缩力降低和细胞内动态和静态钙的变化。     

阿魏酸钠拮抗氧化型脂蛋白(a)对人动脉平滑肌细胞的作用

目的:研究脂蛋白(a)氧化前后致人动脉平滑肌细胞(SMC)增殖及细胞内游离钙浓度([Ca²⁺]_i)的变化,观察阿魏酸钠(SF)对其的影响。方法:Lp(a)经体外Cu²⁺氧化法氧化,硫代巴比妥酸(TBARS)比色法检测氧化程度,培养的人动脉SMC中分别加入不同浓度SF,作用12h后再与天然和氧化型Lp(a)共同孵育,以MTT比色法、流式细胞仪检测细胞增殖状况,采用荧光探针Fura-2/AM检测细胞[Ca²⁺]_i。结果:氧化型Lp(a)促人动脉SMC增殖的同时亦明显增加了[Ca²⁺]_i水平,作用较天然Lp(a)明显,SF(40,80mg/L)可显著抑制氧化型Lp(a)所致的细胞增殖和[Ca²⁺]_i增加,并呈剂量效应关系,而对天然Lp(a)所致的细胞增殖和[Ca²⁺]_i增加无明显影响。结论:氧化型Lp(a)通过升高[Ca²⁺]_i而显著促动脉SMC增殖可能是其致动脉粥样硬化的机制之一,SF拮抗这种作用可能与其抗氧化能力有关。     

Carboxyeosin decreases the rate of decay of the [Ca2+]i transient in uterine smooth muscle cells isolated from pregnant rats

 In myometrial smooth muscle cells the rate of decline of intracellular calcium ([Ca²⁺]_i) is determined by Ca²⁺ extrusion from the cell and uptake into intracellular stores. The relative quantitative contribution of these processes however, has not been established. We therefore examined the effect of the sarcolemmal Ca²⁺ pump inhibitor, carboxyeosin, on the rate of the [Ca²⁺]_i transient decline in myocytes isolated from pregnant rat uterus. Indo-1 was used in conjunction with the whole-cell patch-clamp technique to measure [Ca²⁺]_i simultaneously with transmembrane calcium current (I _Ca). [Ca²⁺]_i transients were elicited by repetitive membrane depolarization to simulate the natural pattern of uterine electrical activity. The rate of [Ca²⁺]_i removal was calculated from the falling phase of the [Ca²⁺]_i transient. Pre-treatment of the cells with 2 μM carboxyeosin led to a marked decrease in the rate of [Ca²⁺]_i transient decay, suggesting that the sarcolemmal Ca²⁺ pump is involved in the calcium extrusion process. Removal of the extracellular Na also decreased the rate of [Ca²⁺]_i decay, indicating an important role for the Na⁺/Ca²⁺ exchange. When both the sarcolemmal Ca²⁺ pump and Na⁺/Ca²⁺ exchange were inhibited the cell failed to restore [Ca²⁺]_i after the stimulation. Comparison of the rate constants of [Ca²⁺]_i decay in control conditions and after carboxyeosin treatment shows that approximately 30% of [Ca²⁺]_i decay is due to the sarcolemmal calcium pump activity. The remaining 70% can be attributed to the activity of Na⁺/Ca²⁺ exchanger and the intracellular calcium stores. Received: 17 July 1998 / Received after revision: 23 September 1998 / Accepted: 25 September 1998     

Mechanisms of reduced mitochondrial Ca2+ accumulation in failing hamster heart

Mitochondrial Ca²⁺ plays important roles in the regulation of energy metabolism and cellular Ca²⁺ homeostasis. In this study, we characterized mitochondrial Ca²⁺ accumulation in Syrian hamster hearts with hereditary cardiomyopathy (strain BIO 14.6). Exposure of isolated mitochondria from 70 nM to 30 μM Ca²⁺ ([Ca²⁺]_o) caused a concentration-dependent increase in intramitochondrial Ca²⁺ concentrations ([Ca²⁺]_m). The [Ca²⁺]_m was significantly lower in cardiomyopathic (CMP) hamsters than in healthy hamsters when [Ca²⁺]_o was higher than 1 μM and a decrease of about 52% was detected at [Ca²⁺]_o of 30 μM (916 ± 67 nM vs 1,932 ± 132 nM in control). A possible mechanism responsible for the decreased mitochondrial Ca²⁺ uptake in CMP hamsters is the depolarization of mitochondrial membrane potential (Δψ _m). Using a tetraphenylphosphonium (TPP⁺) electrode, the measured Δψ _m in failing heart mitochondria was −136 ± 1.5 mV compared with −159 ± 1.3 mV in controls. Analyses of mitochondrial respiratory chain demonstrated a significant impairment of complex I and complex IV activities in failing heart mitochondria. In summary, a less negative Δψ _m resulting from defects in the respiratory chain may lead to attenuated mitochondrial Ca²⁺ accumulation, which in turn may contribute to the depressed energy production and myocardial contractility in this model of heart failure. In addition to other known impairments of ion transport in sarcoplasmic reticulum and plasma membrane, results from this paper on mitochondrial dysfunctions expand our understanding of the molecular mechanisms leading to heart failure.     

Blockage of Na+ currents through poorly selective cation channels in the apical membrane of frog skin and toad urinary bladder

The blockage of Na⁺ movements through the poorly selective cation channels in the apical membrane of frog skin (Rana temporaria) and toad urinary bladder (Bufo marinus) was investigated with noise, impedance analysis and microelectrode techniques. Na⁺ currents through this pathway were studied with NaCl Ringer solutions on both sides. After removal of Ca²⁺ and other divalent cations from the mucosal compartment, a considerable part of I _SC became insensitive to amiloride. In frog skin, the inhibitory effect of amiloride in mucosal Ca²⁺-free solutions was highly variable. In some experiments a complete lack of inhibition was observed. Similarly, in the absence of amiloride, the inhibitory effect of mucosal Ca²⁺ varied strongly among frogs. In the absence of mucosal Ca²⁺, analysis of the fluctuation in I _SC revealed a Lorentzian component in the power density spectrum. The corner frequency (f _c) of this spontaneous Lorentzian was 12.3 Hz in frog skin and 347 Hz in the toad urinary bladder. In frog skin, nanomolar concentrations of mucosal Ca²⁺ induced an additional Lorentzian noise component. Its corner frequency shifted upwards with increasing mucosal Ca²⁺ concentration ([Ca²⁺]_m). The relation between 2f_c and [Ca²⁺]_m was linear at small [Ca²⁺]_m whereas a parabolic increase of f _c was observed at the highest [Ca²⁺]_m. In the bladder, nanomolar concentrations of mucosal Ca²⁺ did not induce an additional noise component but modified the spontaneous Lorentzian noise by increasing f _c proportionally with [Ca²⁺]_m. Microelectrode recordings demonstrated that at least part of the Ca²⁺-blockable current passes through the granulosum cells and confirmed the apical localization of the poorly selective cation channel. The lack of the inhibitory effect of amiloride in Ca²⁺-free solutions seems to originate from the parallel arrangement of the amiloride- and Ca²⁺-blockable pathways and from influences of the blockage of apical channels on the basolateral membrane conductances. The latter cross-talk seems to find its origin in the voltage dependence of the basolateral membrane conductance Garty H (1984) J Membr Biol 77:213–222; Nagel W (1985) Pflügers Arch 405 [Suppl 1]:S39–S43}.     

A physiological role for protoporphyrin IX photodynamic action in the rat Harderian gland?

Aims: The lipid‐secreting exocrine Harderian gland contains a large amount of porphyrins (mainly protoporphyrin IX, PPIX) in the glandular cells, the physiological significance of which is rather poorly understood. Methods: In the present study, the possibility of using Fura‐2 to measure intracellular calcium ([Ca²⁺]_c) changes in these cells was assessed. Results: It was found that when Fura‐2‐loaded cells were excited by light at 340/380 nm, [Ca²⁺]_c increased spontaneously, indicating a photodynamic action powered by light at 340/380 nm. In contrast, with the visible spectrum calcium probe Fluo‐3 (λ_ex = 475 nm), carbachol at 10 μm induced [Ca²⁺]_c increase; [Ca²⁺]_c did not change without carbachol stimulation. Brief illumination with light at 340/380 nm induced a large [Ca²⁺]_c increase in Fluo‐3‐loaded cells. Photodynamic stimulation of [Ca²⁺]_c increase was confirmed with an exogenous photosensitizer sulphonated aluminium phthalocyanine (SALPC) and visible light (>580 nm). The wavelength‐dependence of the [Ca²⁺]_c increase correlates well with the excitation spectrum of the isolated Harderian glandular cells. Conclusion: These data suggest that PPIX present in rat Harderian glandular cells plays the role of a photosensitizer which upon activation by UVA and blue components of daylight and subsequent singlet oxygen generation, triggers [Ca²⁺]_c increase and secretory response. The PPIX photodynamic action may also play a potential role in photic entrainment of the central circadian clock.     

Cytoplasmic and mitochondrial Ca2+ levels in brown adipocytes

Aim: We elucidated the mitochondrial functions of brown adipocytes in intracellular signalling, paying attention to mitochondrial activity and noradrenaline‐ and forskolin‐induced Ca²⁺ mobilizations in cold‐acclimated rats. Methods: A confocal laser‐scanning microscope of brown adipocytes from warm‐ or cold‐acclimated rats was employed using probes rhodamine 123 which is a mitochondria‐specific cationic dye, and the cytoplasmic and mitochondrial Ca²⁺ probes fluo‐3 and rhod‐2. X‐ray microanalysis was also studied. Results: The signal of rhodamine 123 in the cells was decreased by antimycin A which effect was less in cold‐acclimated cells than warm‐acclimated cells. Cytoplasmic and mitochondrial Ca²⁺ in cold‐acclimated brown adipocytes double‐loaded with fluo‐3 and rhod‐2 were measured. Noradrenaline induced the rise in cytoplasmic Ca²⁺ ([Ca²⁺]_cyto) followed by mitochondrial Ca²⁺ ([Ca²⁺]_mito), the effect being transformed into an increase in [Ca²⁺]_cyto whereas a decrease in [Ca²⁺]_mito by antimycin A or carbonyl cyanide m‐chlorophenylhydrazone (CCCP). Antimycin A induced small Ca²⁺ release from mitochondria. CCCP induced Ca²⁺ release from mitochondria only after the cells were stimulated with noradrenaline. Further, forskolin also elicited an elevation in [Ca²⁺]_cyto followed by [Ca²⁺]_mito in the cells. The Ca measured by X‐ray microanalysis was higher both in the cytoplasm and mitochondria whereas K was higher in the mitochondria of cold‐acclimated cells in comparison to warm‐acclimated cells. Conclusions: These results suggest that noradrenaline and forskolin evoked an elevation in [Ca²⁺]_cyto followed by [Ca²⁺]_mito, in which H⁺ gradient across the inner membrane is responsible for the accumulation of calcium on mitochondria. Moreover, cAMP also plays a role in intracellular and mitochondrial Ca²⁺ signalling in cold‐acclimated brown adipocytes.     

Intracellular calcium in mammalian brain cells: fluorescence measurements with quin2

Summary Dispersed brain cells from 12–14 day old mouse embryos were loaded with the Ca²⁺-sensitive fluorescent probe, quin2 and shown to have a resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) of 158 nM (SE ± 5) in the presence of 1 mM [Ca²⁺]_o. When external [Ca²⁺] was raised from 0 to 1 mM there was an increase of [Ca²⁺]_i of 70 nM; with further additions of Ca to >10 mM [Ca²⁺]_o the level of [Ca²⁺]_i increased by <25 nM. Releasable intracellular Ca²⁺ stores, estimated from the increase in [Ca²⁺] produced by 4Br A23187 in the absence of extracellular Ca²⁺, were 24 fmol/10⁶ cells. A small increase in [Ca²⁺]_i could be produced by the mitochondrial inhibitor, carbonyl cyanide m-chlorophenylhydrazone (CCCP). When extracellular K⁺ was raised by 10–20 mM, intracellular Ca²⁺ levels increased from 152 (SE ± 7) to 204 nM (SE ± 10). These K⁺-induced increases in [Ca²⁺]_i were blocked by verapamil, did not occur in the absence of extracellular Ca²⁺, and presumably reflect the activation of voltage-dependent Ca²⁺ channels. N-methyl-D-aspartic acid (NMDA) evoked an increase in [Ca²⁺]_i, while the kainate-like lathyrus sativus neurotoxin, L-3-oxalyl-amino-2aminopropionic acid (L-3,2-OAP) did not; this is consistent with previous observations of different and respectively Ca²⁺-dependent and -independent mechanisms of action of these excitatory amino acids.     

Effect of cytosolic Mg2+ on mitochondrial Ca2+ signaling

Cytosolic Ca²⁺ signals are followed by mitochondrial Ca²⁺ uptake, which, in turn, modifies several biological processes. Mg²⁺ is known to inhibit Ca²⁺ uptake by isolated mitochondria, but its significance in intact cells has not been elucidated. In HEK293T cells, activation of purinergic receptors with extracellular ATP caused cytosolic Ca²⁺ signals associated with parallel changes in cytosolic [Mg²⁺]. Neither signals were affected by omitting bivalent cations from the extracellular medium. The effect of store-operated Ca²⁺ influx on cytosolic Mg²⁺ concentration ([Mg²⁺]_c) was negligible. Uncaged Ca²⁺ displaced Mg²⁺ from cytosolic binding sites, but for an equivalent Ca²⁺ signal, the change in [Mg²⁺] was significantly smaller than that measured after adding extracellular ATP. Inositol 1,4,5-trisphosphate mobilized Ca²⁺ and Mg²⁺ from internal stores in permeabilized cells. The increase of [Mg²⁺] in the range that occurred in ATP-stimulated cells inhibited mitochondrial Ca²⁺ uptake in permeabilized cells without affecting mitochondrial Ca²⁺ efflux. Therefore, the Mg²⁺ signal generated by Ca²⁺ mobilizing agonists may attenuate mitochondrial Ca²⁺ uptake.     

Nonselective cation channels are essential for maintaining intracellular Ca2+ levels and spontaneous firing activity in the midbrain dopamine neurons

Intracellular Ca²⁺ and Ca²⁺-permeable ion channels are important in regulating the firing activity and pattern of midbrain dopamine neurons, but the role of Ca²⁺-permeable nonselective cation channels (NSCCs) on spontaneous firing activity is unclear. Therefore, we investigated how Ca²⁺-permeable NSCCs modulate spontaneous firing activity and cytosolic Ca²⁺ concentration ([Ca²⁺]_c) in acutely isolated midbrain dopamine neurons of the rat. Applications of voltage-dependent Ca²⁺ channels antagonists failed to abolish spontaneous firing activity completely, but they decreased firing rate and [Ca²⁺]_c. However, a blockade of NSCCs by 2-APB or SKF96365 more potently suppressed spontaneous firings with a depolarization of membrane potential and strong decreases in basal [Ca²⁺]_c levels. The depolarization of membrane potentials was attenuated by intracellular dialysis with 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). NSCCs blockers inhibited oscillatory potentials and decreased basal [Ca²⁺]_c in the presence of tetrodotoxin. Apamin, a small-conductance Ca²⁺-activated K⁺ channel inhibitor, depolarized membrane potentials and enhanced firing rates. From these data, we conclude that NSCCs not only make up the tonic Ca²⁺ entry pathways to uphold basal [Ca²⁺]_c levels but also contribute to generation of spontaneous firings, thereby regulating spontaneous firing activities of the midbrain dopamine neurons.     

Glutamine-mediated protection from neuronal cell death depends on mitochondrial activity

The specific aim of this study was to elucidate the role of mitochondria in a neuronal death caused by different metabolic effectors and possible role of intracellular calcium ions ([Ca²⁺]_i) and glutamine in mitochondria- and non-mitochondria-mediated cell death. Inhibition of mitochondrial complex I by rotenone was found to cause intensive death of cultured cerebellar granule neurons (CGNs) that was preceded by an increase in intracellular calcium concentration ([Ca²⁺]_i). The neuronal death induced by rotenone was significantly potentiated by glutamine. In addition, inhibition of Na/K-ATPase by ouabain also caused [Ca²⁺]_i increase, but it induced neuronal cell death only in the absence of glucose. Treatment with glutamine prevented the toxic effect of ouabain and decreased [Ca²⁺]_i. Blockade of ionotropic glutamate receptors prevented neuronal death and significantly decreased [Ca²⁺]_i, demonstrating that toxicity of rotenone and ouabain was at least partially mediated by activation of these receptors. Activation of glutamate receptors by NMDA increased [Ca²⁺]_i and decreased mitochondrial membrane potential leading to markedly decreased neuronal survival under glucose deprivation. Glutamine treatment under these conditions prevented cell death and significantly decreased the disturbances of [Ca²⁺]_i and changes in mitochondrial membrane potential caused by NMDA during hypoglycemia. Our results indicate that glutamine stimulates glutamate-dependent neuronal damage when mitochondrial respiration is impaired. However, when mitochondria are functionally active, glutamine can be used by mitochondria as an alternative substrate to maintain cellular energy levels and promote cell survival.