Aging does not alter cytosolic calcium levels of cortical synaptosomes in Fischer 344 rats

Several lines of experimental evidence support an association between altered Ca²⁺ regulation and aging. It has been supposed that free cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) may decrease or increase in aged animals. In this study, both resting and KCl-stimulated [Ca²⁺]_i were measured in purified cortical synaptosomes from young (3 mo.), middle-aged (12 mo.), and old (24 mo.) Fischer 344 rats. Two additional groups of rats were included, one middle-aged and one old which were trained on a treadmill for 6 months prior to experimentation. The [Ca²⁺]_i was determined using the fluorescent Ca²⁺ chelator fura-2. Net KCl-dependent changes (ΔK) in [Ca²⁺]_i were determined by the difference between stimulatory (100 μM Ca²⁺/60 mM KCl) and resting (100 μM Ca²⁺/5 mM KCl buffer) conditions among the 3 age groups. Significant increases in [Ca²⁺]_i were observed in each age group upon depolarization with 60 mM KCl. However, there were no significant age-dependent differences in either resting [Ca²⁺]_i or KCl-stimulated [Ca²⁺]_i.     

The role of Ca2+ in the generation of spontaneous astrocytic Ca2+ oscillations

Astrocytes in the rat thalamus display spontaneous [Ca²⁺]_i oscillations that are due to intracellular release, but are not dependent on neuronal activity. In this study we have investigated the mechanisms involved in these spontaneous [Ca²⁺]_i oscillations using slices loaded with Fluo-4 AM (5 μM) and confocal microscopy. Bafilomycin A1 incubation had no effect on the number of spontaneous [Ca²⁺]_i oscillations indicating that they were not dependent on vesicular neurotransmitter release. Oscillations were also unaffected by ryanodine. Phospholipase C (PLC) inhibition decreased the number of astrocytes responding to metabotropic glutamate receptor (mGluR) activation but did not reduce the number of spontaneously active astrocytes, indicating that [Ca²⁺]_i increases are not due to membrane-coupled PLC activation. Spontaneous [Ca²⁺]_i increases were abolished by an IP3 receptor antagonist, whilst the protein kinase C (PKC) inhibitor chelerythrine chloride prolonged their duration, indicating a role for PKC and inositol 1,4,5,-triphosphate receptor activation. BayK8644 increased the number of astrocytes exhibiting [Ca²⁺]_i oscillations, and prolonged the responses to mGluR activation, indicating a possible effect on store-operated Ca²⁺ entry. Increasing [Ca²⁺]_o increased the number of spontaneously active astrocytes and the number of transients exhibited by each astrocyte. Inhibition of the endoplasmic reticulum Ca²⁺ ATPase by cyclopiazonic acid also induced [Ca²⁺]_i transients in astrocytes indicating a role for cytoplasmic Ca²⁺ in the induction of spontaneous oscillations. Incubation with 20 μM Fluo-4 reduced the number of astrocytes exhibiting spontaneous increases.

This study indicates that Ca²⁺ has a role in triggering Ca²⁺ release from an inositol 1,4,5,-triphosphate sensitive store in astrocytes during the generation of spontaneous [Ca²⁺]_i oscillations.     

Vip Modulates Intracellular Calcium Oscillations in Human Lymphoblasts

Vasoactive intestinal polypeptide (VIP) has been shown to stimulate adenylate cyclase in a human lymphoblast cell line (MOLT 4). In the present study, we monitored fluorescence in cell suspensions and in single fura-2 loaded MOLT 4 lymphoblasts to determine if VIP modulates intracellular calcium concentrations ([Ca²⁺]_i), and if this modulation is mediated by adenylate cyclase. The distribution of [Ca²⁺]_i in resting and stimulated cells was non-homogeneous, with gradients of high [Ca²⁺]_i present in the subplasmalemmal space. In a subset of cells (10-30% of all cells studied), [Ca²⁺]_i showed La³⁺-sensitive, temporal changes in the form of [Ca²⁺]_i oscillations with a baseline [Ca²⁺]_i value of 115±10 nM, an oscillation amplitude of 150±18 nM and a mean period of 9.2±2s. The remaining non-oscillating cells showed a constant [Ca²⁺]_i level of 75±5 nM (n=65 cells from 4 experiments). In the subset of cells with spontaneous [Ca²⁺]_i oscillations, VIP dose-dependendy (10^-12 to 10^-8M) increased the amplitude of oscillations but did not stimulate their frequency. The stimulatory effect of VIP was correlated with baseline [Ca²⁺]_i in these cells, was attenuated in the presence of La³⁺ (25 μM), but was unaffected by cell depolarization (126 mM KC1). Dibutyryl cyclic AMP (10^-4 to 10^-3 M) and forskolin (10^-4M) had no effect on [Ca²⁺]_i oscillations, or on [Ca²⁺]_i in cells without oscillations. In cell suspensions, baseline [Ca²⁺]_i was found to be 55. 1±11.2 nM (meanS.E.M., n=11); VIP, cyclic AMP analogues or forskolin had no significant effect on [Ca²⁺]_i. These findings suggest that: a) VIP modulates the amplitude of [Ca²⁺]_i oscillations generated by a cytosolic [Ca²⁺] oscillator in a subset of cells at a concentration of 10^-12M, a thousand-fold below the K_D for the VIP receptor; b) baseline [Ca²⁺] values may be related to both the ability of cells to generate spontaneous [Ca²⁺] oscillations and of oscillating cells to respond to VIP; c) due to the small number of responding cells, VIP-induced [Ca²⁺]_i changes are not detectable when studied in cell suspensions.     

Measurements of intracellular calcium in sensory neurons of adult and old rats

Cytosolic free calcium ([Ca²⁺]_i) was measured using fluorescent digital imaging microscopy in rat dorsal root ganglion neurons isolated from animals of two age groups (adult: seven months; and old: 30 months). Neurons were enzymatically isolated and maintained in primary culture for 14 days. Cultured neurons were loaded with the fluorescent dye, Fura-2. The spatial distribution of resting [Ca²⁺]_i was even in both adult and old rats, but the value of cytoplasmic free calcium in old neurons was significantly higher (207 ± 37 nmol/l vs96 ± 23 nmol/l) in comparison with adult ones. Depolarization with 50 mmol/l K⁺ produced a rapid increase in [Ca²⁺]_i in all neurons, but the values of depolarization-induced increase of [Ca²⁺]_i in old neurons were significantly lower (423 ± 54 nmol/l) compared with cells isolated from adult rats (1011 ± 91 nmol/l). The time of the complete restoration of [Ca²⁺]_i to the resting level was 10-times longer in old neurons. The caffeine-induced rise of intracellular calcium was somewhat higher in neurons from old animals, and its restoration to normal level was delayed.

The findings indicate a substantial alteration of the mechanisms of regulation of intracellular calcium homeostasis with neuronal ageing.     

Regulation of intracellular Ca concentration by interleukin-1β in rat cortical synaptosomes: an age-related study

The pro-inflammatory cytokine interleukin-1β (IL-1β) is released by cells during injury and stress, and increased neuronal expression of IL-1β is a feature of age-related neurodegeneration. We have recently reported that IL-1β has a biphasic effect on the K⁺-induced rise in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cortical synaptosomes, exerting an inhibitory effect on the K⁺-induced rise in [Ca²⁺]_i at lower (3.5 ng/mL) concentrations and a stimulatory effect on the K⁺-induced rise in [Ca²⁺]_i at higher (100 ng/mL) concentrations. In the present study, we observed that the K⁺-induced rise in [Ca²⁺]_i was inhibited to a similar extent by the lower concentration of IL-1β in cortical synaptosomes prepared from young (3-month-old), middle-aged (12-month-old) and aged (24-month-old) rats. In contrast, cortical synaptosomes prepared from the aged rats exhibited an increased susceptibility to the higher concentration of IL-1β, resulting in a marked elevation in [Ca²⁺]_i. We propose that the age-related increase in neuronal concentration of IL-1β promotes a dramatic elevation in [Ca²⁺]_i following membrane depolarization, thereby altering Ca²⁺ homeostasis and exacerbating neuronal vulnerability to excitotoxicity.     

钙敏感受体通过G蛋白-PLC-IP_3信号途径调节肺动脉张力

目的:探讨钙敏感受体(calcium-sensing receptor,Ca SR)在肺血管张力调节中的作用及信号途径。方法:采用Ⅱ型胶原酶消化法提取大鼠肺动脉平滑肌细胞(pulmonary artery smooth muscle cells,PASMCs),激光共聚焦扫描显微镜技术观察不同条件下PASMCs中钙离子浓度的变化,组织浴槽血管环技术观察血管张力的变化。结果:Ca SR激动剂(钙、钆)引起剂量依赖性的细胞内钙增加和血管张力增大,U73122和D609(PLC抑制剂)以及2-APB和肝素(IP3受体抑制剂)可减弱Ca SR的作用(P0.05),而U73343(U73122的无生物活性类似物)则无此作用(P0.05)。结论:Ca SR活化导致细胞内钙增加,进而引起肺动脉环收缩,这些过程是通过G蛋白-PLC-IP3信号转导通路实现的。     

The aminoglycoside G418 suppresses muscarinic receptor-activated calcium release in stably transfected murine N1E-115 neuroblastoma cells

The aminoglycoside G418 inhibited the release of calcium (Ca²⁺) from internal stores coupled to muscarinic receptors in murine N1E-115 neuroblastoma cells carrying the aminoglycoside resistance gene neomycin phosphotransferase (NPT). No significant effect was observed on responses coupled to histamine or bradykinin receptors. Cells were transfected using the eukaryotic expression vector pHβAPr-1-neo and selected using G418. Two groups were differentiated either in the continued presence of G418 or in the absence of G418. Carbachol (1 mM), histamine (200 μM) and bradykinin (100 nM) were administered to cells for thirty seconds and changes in [Ca²⁺]_i were measured with fluorescence video microscopy of single cells loaded with the Ca²⁺ indicator fura-2. The effects of G418 on carbachol evoked Ca²⁺ release included a 73% reduction in the number of cells responding, a two fold increase in the time to reach half-maximal response, a 35% reduction of the peak [Ca²⁺]_i in response to agonist and an elevation of resting [Ca²⁺]_i from 99± 14 nM (mean ± S.E.M.) to 155 ± 27 nM. Acute application (20 min) of G418 to transfected cells differentiated without G418 also reduced the percentage of cells responding to carbachol. This effect was less pronounced in non-transfected parent cells. Thus, the mechanism might involve a metabolite of G418 produced in cells expressing NPT. These results indicate that G418 attenuates Ca²⁺ release coupled to muscarinic receptors.     

Spatial and dynamic changes in intracellular Ca measured by confocal laser-scanning microscopy in bullfrog sympathetic ganglion cells

Confocal laser scanning microscopy (CLSM) was used to record spatial and dynamic changes in the intracellular Ca²⁺ ([Ca²⁺]_i) of bullfrog sympathetic ganglion cells in excised tissue or in culture. A CLSM utilizing Ar ion laser (488 nm) and recording fluo-3 fluorescence yielded the sliced image of ganglion cells, while conventional epifluorescence microscopy provided the cell image of a convex structure. A high K⁺ (50 mM) solution, caffeine (3–10 mM) and electrical stimulation (10–20 Hz, 0.5–10 s) caused a homogeneous increase in fluo-3 fluorescence with or without regional differences, possibly due to intracellular organelles and other constituents. Scanning a single horizontal line across the cytoplasm with He-Cd laser (325 nm) and recording indo-1 fluorescence demonstrated that the rate of rise in [Ca²⁺]_i following action potentials depends on the distance from the cell membrane and on the cytoplasmic constituents, showing an inward spread of ‘Ca²⁺-wave’ at variable speeds of 17–219 μm/s. These results suggest that heterogeneity of the cytoplasmic structures and constituents affects dynamic and spatial changes of [Ca²⁺]_i in response to stimuli in neurones. Such heterogenic changes in [Ca²⁺]_i would better be studied by CLMS.     

PACAP deficient mice display reduced carbohydrate intake and PACAP activates NPY-containing neurons in the rat hypothalamic arcuate nucleus

Pituitary adenylate cyclase-activating polypeptide (PACAP) potentiates both insulin release from islets and insulin action in adipocytes. Therefore, this peptide is considered a regulator of glucose homeostasis. PACAP and its receptors are localized not only in the peripheral tissues but in the central nervous system. The present study examined whether PACAP regulates the feeding behavior and the activity of neurons in the hypothalamic arcuate nucleus (ARC), a feeding center. Food intake was measured in the PACAP knock-out mice. Cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in single neurons isolated from the ARC of rats was measured by fura-2 microfluorometry, followed by immunocytochemical staining with anti-NPY antiserum. PACAP knock-out mice showed a decrease in the intake of high carbohydrate, but not high fat, food. PACAP increased [Ca²⁺]_i in NPY neurons of the ARC that are implicated in the feeding, particularly the carbohydrate ingestion. Agonists of PACAP receptors, PAC1-R and VPAC2-R, also increased [Ca²⁺]_i. The present study, by demonstrating that PACAP directly reacts with the ARC NPY neurons to increase [Ca²⁺]_i and that ingestion of the carbohydrate-rich food is reduced in PACAP-deficiency, suggests a facilitative role for PACAP in the carbohydrate intake.     

活性氧簇介导血管紧张素Ⅱ对延髓神经元胞内游离Ca~(2+)水平的调节作用

目的:探讨活性氧簇(reactive oxygen species,ROS)介导血管紧张素Ⅱ(angiotensin Ⅱ,Ang Ⅱ)对延髓神经元胞内游离Ca~(2+)的调节作用及其机制。方法:原代培养延髓神经元;免疫荧光双标法鉴定培养神经元的特征;给予Ang Ⅱ处理后,用二氢乙啶荧光探针法测定神经元ROS水平;同时或单独给予Ang Ⅱ和NADPH氧化酶抑制剂apocynin或自由基清除剂TEMPOL后,Fura-2/AM钙瞬变法记录神经元胞内游离Ca~(2+)的水平;CCK-8法检测神经元活性。结果:原代培养的延髓神经元多数为谷氨酸阳性的神经元;Ang Ⅱ(5μmol/L)可在10 min内显著升高神经元ROS水平(P0.01);给予Ang Ⅱ处理后延髓神经元胞内Ca~(2+)水平显著升高(P0.01);给予apocynin/TEMPOL预处理后,Ang Ⅱ引起的延髓神经元胞内Ca~(2+)的升高则被抑制(P0.05)。实验浓度的Ang Ⅱ对神经元无毒性作用。结论:ROS介导Ang Ⅱ诱导的延髓神经元胞内Ca~(2+)的升高作用,可能是Ang Ⅱ在中枢诱导氧化应激作用的潜在细胞内信号机制。     

P2 receptors in satellite glial cells in trigeminal ganglia of mice

There is strong evidence for the presence of nucleotide (P2) receptors in sensory neurons, which might play a role in the transmission of pain signals. In contrast, virtually nothing is known about P2 receptors in satellite glial cells (SGCs), which are the main glial cells in sensory ganglia. We investigated the possibility that P2 receptors exist in SGCs in murine trigeminal ganglia, using Ca²⁺ imaging, patch-clamp recordings, and immunohistochemistry. We found that ATP caused an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in SGCs. As adenosine had no effect on [Ca²⁺]_i, and the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid largely blocked the response to ATP we conclude that P1 receptors did not contribute to the responses. We obtained the following evidence that the responses to ATP were mediated by metabotropic P2Y receptors: (i) persistence of the responses in Ca²⁺-free solution, (ii) inhibition of the response by cyclopiazonic acid, (iii) [Ca²⁺]_i increases in response to the P2Y agonists uridine triphosphate, adenosine thiodiphosphate, and 2-methylthio ADP, and (iv) failure of the P2X agonist ,β-methylene ATP to elicit a response. Agonists of P2Y₁ receptors and uridine triphosphate, an agonist at P2Y₂ and P2Y₄ receptors, induced [Ca²⁺]_i increases suggesting that at least these P2Y receptor subtypes are present on SGCs. Using an antibody against the P2Y₄ receptor, we found immunopositive SGCs. Patch-clamp recordings of SGCs did not reveal any inward current due to ATP. Therefore, there was no evidence for the activation of ionotropic P2X receptors under the present conditions.

The results indicate the presence of functional nucleotide (P2Y) receptors in SGCs.     

Modulation of Ca channels by activation of adenosine A1 receptors in rat striatal glutamatergic nerve terminals

We determined that activation of adenosine A₁ receptors in striatal synaptosomes with 100 nM N⁶-cyclopentyladenosine (CPA) inhibited both the release of endogenous glutamate and the increase of intracellular free Ca²⁺ concentration ([Ca²⁺]_i), due to 4-aminopyridine (4-AP) stimulation, by 28 and 19%, respectively. Furthermore, CPA enhanced the inhibition of endogenous glutamate release due to ω-conotoxin GVIA (ω-Cgtx GVIA), ω-Cgtx MVIIC or ω-Cgtx GVIA plus ω-Cgtx MVIIC. Similar effects were observed in the [Ca²⁺]_i signal. The inhibitory effects of CPA and ω-Cgtx GVIA were additive, but the effects of CPA and ω-Cgtx MVIIC were only partially additive. These results suggest that P/Q-type Ca²⁺ channels and other type(s) of Ca²⁺ channel(s), coupled to glutamate release, are inhibited subsequently to activation of adenosine A₁ receptors.     

Modulation of the dihydropyridine-insensitive Ca influx by 8-bromo-guanosine-3′:5′-monophosphate, cyclic (8-Br-cGMP) in bovine adrenal chromaffin cells

Pretreatment of chromaffin cells with the permeable analogue of cGMP, 8-Br-cGMP (100 μM), leads to a reduction (35%) of depolarization-evoked intracellular calcium concentration ([Ca²⁺]_i) increases. There is evidence that bovine adrenal chromaffin cells are provided with both dihydropyridine-sensitive and -resistant voltage-sensitive Ca²⁺ influx pathways. Combined incubations with nifedipine 10 μM and 8-Br-cGMP reduced KCl-evoked intracellular Ca²⁺ concentration to a greater extent that each compound separately. Moreover, 8-Br-cGMP failed to affect the [Ca²⁺]_i transient induced by the L-type Ca²⁺ channel agonist Bay K 8644 (1μM) under conditions of low depolarization. Neomycin (0.2 mM) and θ-Aga Toxin-IVA (AgTx) (1μM) inhibited the calcium transient to a similar extent, and this inhibition was not enhanced by the presence of 8-Br-cGMP. It is concluded that 8-Br-cGMP modulated the dihydropyridine-insensitive Ca²⁺ influx pathway in the chromaffin cell.     

钙离子感受蛋白STIM1与Orai1在钙敏感受体介导钙内流及NO生成中的相互作用

目的:研究Ca~(2+)感受蛋白[基质相互作用分子1(STIM1)与钙释放激活钙通道蛋白1(Orai1)]在人脐静脉内皮细胞(HUVECs)钙敏感受体(Ca SR)介导的钙内流和一氧化氮(NO)生成中的相互作用。方法:将Ca SR激动剂精胺[钙池操纵性钙通道(SOC)和受体操纵性钙通道(ROC)均激活]单用或与ROC模拟剂12-O-十四烷酰佛波醇-13-醋酸酯(TPA)+Ca SR负性变构调节剂Calhex 231(激活ROC、阻断SOC)、蛋白激酶C(PKC)抑制剂Ro 31-8220和PKCα/β1选择性抑制剂Go 6976(激活SOC、阻断ROC)联合孵育HUVECs;利用免疫荧光技术检测HUVECs中STIM1和Orai1的蛋白表达和共定位;免疫共沉淀法检测STIM1和Orai1之间的相互作用;取2~3代HUVECs随机分为特异性的质粒转染组(sh STIM1+sh Orai1组)、空质粒组(vehicle-STIM1+vehicle-Orai1组)和未转染组(control组),将3组细胞分别加入上述4组不同药物刺激,采用荧光探针Fura-2/AM检测HUVECs中Ca~(2+)浓度的变化,NO荧光探针DAF-FM DA负载方法同步检测HUVECs中NO生成的变化。结果:STIM1和Orai1蛋白表达共定位于胞浆,与control组相比,加入Calhex 231+TPA、Ro 31-8220和Go 6976刺激后,STIM1和Orai1在胞浆中的定位均减少,且二者的相互作用均减弱;在4种不同处理因素作用下,sh STIM1+sh Orai1组细胞内Ca~(2+)浓度和NO净荧光强度均明显降低(P0.05)。结论:STIM1与Orai1以二元复合物的形式共同调节Ca SR,并通过激活SOC和ROC介导钙内流及NO生成。     

Role of intracellular calcium in fatigue in single skeletal muscle fibers isolated from the rat

The aim of the present study was to demonstrate the role of intracellular calcium ([Ca²⁺]_i) in the performance of fatigued muscle fibers isolated from the skeletal muscle of the rat. We measured developed tension of a single myocyte during short tetanic electrical stimulation of various intensities along with [Ca²⁺]_i dynamics by fura-2. The performance of individual muscle fiber was assessed by developed tension during 100 Hz tetanic stimulation (‘100 Hz force’). We regarded the muscle fiber fatigued when, after repeated tetanic stimulations, the developed tension declined to 50% of the initial level. When fatigue was induced by maximal stimulation (100 Hz tetani), the 100 Hz force measured immediately following completion of fatigue was considerably decreased (48% of control). This change in the muscle performance was associated with significant increase in the resting [Ca²⁺]_i (280% of control) and decrease in Ca²⁺ transient (54% of control). The 50% relaxation time after cessation of tetanic stimulation (RT₅₀) was also prolonged. In contrast, when fatigue was induced by low frequency electrical stimulation (30 Hz tetani), neither the 100 Hz force, RT₅₀, nor Ca²⁺ transient in fatigue were significantly different from the controls, while the resting [Ca²⁺]_i increased only slightly. These findings suggest a tight relationship between [Ca²⁺]_i and the performance of fatigued single isolated skeletal muscles. Also, the results show that performance of the fatigued muscle fiber may in part depend on the protocol used to produce muscle fatigue.     

Mechanism of histamine-induced calcium efflux from cultured bovine adrenal chromaffin cells: possible involvement of an Na/Ca exchange mechanism

The effect of stimulation of the histamine receptor on Ca²⁺ mobilization in cultured bovine adrenal chromaffin cells was examined. Histamine (10⁻⁵ M) increased the intracellular free Ca²⁺ ([Ca²⁺]_i) to a peak in the presence or absence of extracellular Ca²⁺, followed by decrease with time. Histamine (10⁻⁸–10⁻⁵ M) also stimulated ⁴⁵Ca²⁺ efflux from cultured bovine adrenal chromaffin cells in a concentration dependent manner. Its stimulatory effect on ⁴⁵Ca²⁺ efflux was inhibited by the specific histamine H₁ receptor antagonist mepyramine. The increase in histamine-stimulated ⁴⁵Ca²⁺ efflux was inhibited by deprivation of extracellular Na⁺ and by the Na⁺/Ca²⁺ exchange inhibitor amiloride. In addition, histamine stimulated ²²Na⁺ influx into the cells, and this action was inhibited by amiloride. These results suggest that stimulation of the histamine H₁ receptor regulates Na⁺/Ca²⁺ exchange in cultured bovine adrenal chromaffin cells.     

H_2S抑制ATP诱导的大鼠小胶质细胞活化及IL-1β的释放

目的:观察硫化氢(H_2S)供体硫氢化钠(Na HS)对三磷酸腺苷(ATP)损伤的大鼠小胶质细胞通透性、胞内Ca~(2+)浓度([Ca~(2+)]_i)及IL-1β释放的影响,探讨H_2S对ATP-P2X嘌呤信号通路的作用及其神经保护的分子机制。方法:取对数期形态结构及生长分化良好的大鼠小胶质细胞用于实验。Fura-2/AM荧光染料检测各组[Ca~(2+)]_i,细胞内YO-PRO-1荧光强度检测以反映膜的通透性,ELISA检测ATP-细菌脂多糖(LPS)激活的大鼠小胶质细胞IL-1β的释放水平。结果:随着ATP浓度的增加,大鼠小胶质细胞内YO-PRO-1的荧光强度显著增加,而给予Na HS预处理后细胞膜通透性明显降低,对YO-PRO-1的摄取明显减少(P0.05),胞内荧光强度明显减弱(P0.05)。ATP处理大鼠小胶质细胞引起[Ca~(2+)]_i迅速升高后缓慢下降形成持续时间较长的平台期。Na HS预处理虽不能改变[Ca~(2+)]_i的峰值,但可抑制平台期的形成(P0.05)。ATP与LPS联合作用可促进大鼠小胶质细胞IL-1β的生成和分泌,而Na HS预处理可明显逆转这一效应(P0.05)。结论:Na HS可降低ATP诱导的大鼠小胶质细胞膜通透性、Ca~(2+)内流和IL-1β释放,抑制该细胞的激活,提示嘌呤信号通路可能介导H_2S的细胞保护作用。     

硫化氢保护被ATP损伤的PC12细胞

目的:观察硫化氢的供体硫氢化钠(Na HS)对三磷酸腺苷(ATP)诱导的PC12细胞活力、胞内Ca2+浓度([Ca2+]i)及膜通透性的变化,探讨硫化氢神经保护作用的嘌呤信号机制。方法:将对数生长期高分化的PC12细胞,随机分为4组,分别为(1)正常对照组:常规培养,不进行ATP处理;(2)ATP组:接种细胞24 h后ATP处理;(3)Na HS+ATP组:Na HS预先孵育30 min后再用ATP处理,并且Na HS始终存在于反应体系中;(4)KN-62(P2X7受体阻断剂)+ATP组:KN-62预先孵育30 min,其余同Na HS+ATP组。MTT检测各组细胞活力,Fura-2/AM荧光染料检测各组[Ca2+]i,检测荧光染料YO-PRO-1的相对荧光单位以反映膜的通透性。结果:(1)0.3mmol/L ATP对细胞活力无影响,但1、3、5、10 mmol/L ATP则呈浓度依赖式明显降低细胞活力,200μmol/L Na HS干预可明显逆转ATP引起的细胞活力下降(P0.05),而800μmol/L Na HS预处理则加剧ATP对PC12细胞的损伤(P0.05)。(2)ATP处理PC12细胞会引起[Ca2+]i迅速升高并且呈浓度依赖性,Na HS预处理能对抗ATP引起的[Ca2+]i升高(P0.05)。(3)随着ATP浓度的增加及作用时间的延长,PC12细胞内YO-PRO-1的荧光强度显著增加,Na HS预处理可明显减少细胞对YO-PRO-1的摄取(P0.05)。结论:硫化氢可保护ATP损伤的PC12细胞,可能与其抑制[Ca2+]i升高和YO-PRO-1荧光增强有关。     

红细胞生成素抑制活性氧诱导的红细胞衰亡

目的:观察红细胞生成素(erythropoietin,EPO)对过氧化氢(H_2O_2)刺激后红细胞衰亡(eryptosis)和红细胞中活性氧簇(reactive oxygen species,ROS)生成的影响,并探讨其可能机制。方法:将1%健康人红细胞悬液在以下3组不同的体外培养液中孵育:对照组(C组,培养基为PBS液)、H_2O_2组(H组,培养基为H_2O_2终浓度100μmol/L的PBS液)和EPO组(E组,培养基为H_2O_2终浓度100μmol/L、EPO终浓度2×10~4U/L的PBS液)。分别在孵育24 h和60 h时,留取红细胞以备检测。使用流式细胞术检测红细胞的衰亡率、红细胞内ROS和红细胞内钙离子浓度(_i~([Ca2+])),观察各检测指标的变化并分析其相关性。结果:红细胞衰亡率在C组随孵育时间延长而增加,在相同观察时点,H组较C组明显增加(P0.01),E组较H组明显降低(P0.01)。H组红细胞的ROS生成较C组明显增多,_i~([Ca2+])较C组明显升高(P0.01);E组红细胞的ROS生成较H组明显减少,_i~([Ca2+])较H组明显降低(P0.05或P0.01)。结论:H_2O_2诱导健康红细胞加速衰亡,而EPO可以抑制H_2O_2诱导的红细胞衰亡,其机制可能与抗氧化及_i~([Ca2+])的改变有关。     

Presence and functional significance of presynaptic ryanodine receptors

Ca²⁺-induced Ca²⁺ release (CICR) mediated by sarcoplasmic reticulum resident ryanodine receptors (RyRs) has been well described in cardiac, skeletal and smooth muscle. In brain, RyRs are localised primarily to endoplasmic reticulum (ER) and have been demonstrated in postsynaptic entities, astrocytes and oligodendrocytes where they regulate intracellular Ca²⁺ concentration ([Ca²⁺]_i), membrane potential and the activity of a variety of second messenger systems. Recently, the contribution of presynaptic RyRs and CICR to functions of central and peripheral presynaptic terminals, including neurotransmitter release, has received increased attention. However, there is no general agreement that RyRs are localised to presynaptic terminals, nor is it clear that RyRs regulate a large enough pool of intracellular Ca²⁺ to be physiologically significant. Here, we review direct and indirect evidence that on balance favours the notion that ER and RyRs are found in presynaptic terminals and are physiologically significant. In so doing, it became obvious that some of the controversy originates from issues related to (i) the ability to demonstrate conclusively the physical presence of ER and RyRs, (ii) whether the biophysical properties of RyRs are such that they can contribute physiologically to regulation of presynaptic [Ca²⁺]_i, (iii) how ER Ca²⁺ load and feedback gain of CICR contributes to the ability to detect functionally relevant RyRs, (iv) the distance that Ca²⁺ diffuses from plasma membranes to RyRs to trigger CICR and from RyRs to the Active Zone to enhance vesicle release, and (v) the experimental conditions used. The recognition that ER Ca²⁺ stores are able to modulate local Ca²⁺ levels and neurotransmitter release in presynaptic terminals will aid in the understanding of the cellular mechanisms controlling neuronal function.