Oxidative stress and apoptotic changes in primary cultures of rat proximal tubular cells exposed to lead

Lead is a known nephrotoxic element. In this study, primary cultures of rat proximal tubular (rPT) cells were treated with different concentrations of lead acetate (0.25, 0.5 and 1 μM) to investigate its cytotoxic mechanism. A progressive loss in cell viability together with a significant increase in the number of apoptotic and necrotic cells and lactate dehydrogenase release were seen in the experiment. Simultaneously, elevation of reactive oxygen species levels and intracellular [Ca²⁺]i, depletion of mitochondrial membrane potential and intracellular glutathione were revealed during the lead exposure. In addition, apoptotic morphological changes induced by lead exposure in rPT cells were demonstrated by Hoechst 33258 staining. The apoptosis was markedly prevented by N-acetyl-l-cysteine, while the necrosis was not affected. Moreover, catalase and superoxide dismutase activities in the living cells rose significantly. In conclusion, exposure of rPT cells to low-concentration lead led to cell death, mediated by an apoptotic and a necrotic mechanism. The apoptotic death induced by oxidative stress was the chief mechanism. Meanwhile, a group of cells survived lead action, mediated by their ability to activate antioxidant defense systems.     

M1 muscarinic receptors mediate intracellular calcium release in NB-OK1 human neuroblastoma cells

Summary Muscarine acetylcholine receptors were characterized in NB-OK1 cells using radioligand (³H-NMS) binding experiments and second messenger (calcium and phosphatidylinositol (PI) turnover) studies. In radioligand binding experiments the displacement curves of pirenzepine (K_I = 1.3 × 10^–8 M), AF-Dx 116 (KI = 8.2 × 10^–7 M), methoctramine (KI = 8.4 × 10^–8 M) and parafluorohexahydrosiladifenidol (pF-HHSiD) (K1 = 1.8 × 10^–8 M) were monophasic and indicated the presence of M 1 muscarinic receptors. Schild analysis with the muscarinic antagonists pirenzepine, AF-Dx 116, methoctramine and pF-HHSiD yielded pA₂ values of 8.40 +- 0.13, 6.48 =- 0.09, 7.61 +- 0.12 and 7.22 +- 0.08 in the calcium experiments and pA₂ values of 8.13 +- 0.30 and 6.26 +- 0.26, 7.65 +- 0.16 and 7.46 +- 0.11, respectively, in the PI turnover experiments. These results indicate that both the carbachol-induced increase in intracellular calcium and the increase in PI turnover are mediated by M1 muscarinic receptors. In calcium free buffer, stimulation with carbachol induced similar responses to those seen under control conditions. From functional and radioligand binding experiments we conclude that the muscarinic receptor expressed in NB-OK1 cells is the M1 subtype. In addition, the M1 receptor-induced calcium response is related to PI turnover and is independent on extracellular calcium. Send offprint requests to H. W. G. M. Boddeke at the above address     

铅对大鼠海马神经元培养细胞ERK活性的影响

目的 观察不同浓度和不同时间染铅对大鼠海马神经元培养细胞细胞质ERK活性的影响。方法 出生4－8d Wistar大鼠海马神经元原代细胞培养,抑制剂PD098059预处理培养细胞,不同浓度及不同时间染铅,应用改良的Takai法测定ERK活性的变化。结果 0．1、0．5、1．0、5．0μmol染铅,ERK活性依浓度依赖方式被激活,比活性倍数分别为2．36、3．16、7．00、3．19（P＜0．05）;PD098059抑制铅激活ERK活性,1．0μmol染铅30min时ERK活性最高,为2．66（P＜0．05）,120min时降至正常,为1．72（P＞0．05）。结论 低浓度染铅可短暂激活大鼠海马神经元原代培养细胞ERK。     

Role of action potentials and calcium influx in ATP- and UDP-induced noradrenaline release from rat cultured sympathetic neurones

Adenine and uracil nucleotides release noradrenaline from rat postganglionic sympathetic neurones by activation of P2X-receptors and distinct receptors for uracil nucleotides, respectively. The present study on cultured neurones of rat thoracolumbal paravertebral ganglia has analysed the involvement of action potentials and calcium influx in the nucleotide-induced transmitter release. ATP and UDP (100 μM each) caused a marked release of previously incorporated [³H]noradrenaline. The P2-receptor antagonists suramin (300 μM) and cibacron blue 3GA (3 μM) decreased the ATP-induced but not the UDP-induced release. The response to ATP was decreased by the sodium channel blocker tetrodotoxin (0.5 μM; by 47%), by the N-type calcium channel blocker ω-conotoxin GVIA (100 nM; by 35%), and by the α₂-adrenoceptor agonist UK-14,304 (1 μM; by 45%); it was not changed by the potassium channel blocker tetraethylammonium (10 mM). The response to UDP was abolished by tetrodotoxin, greatly decreased by ω-conotoxin (by 78%), also abolished by UK-14,304, and increased by tetraethylammonium (by 410%). ATP (100 μM) caused a marked increase in intraaxonal free calcium as measured by fura-2 microfluorimetry. Withdrawal of extracellular calcium diminished the calcium response to ATP by 85%, and tetrodotoxin and ω-conotoxin diminished it by about 40%. As studied with the amphotericin B-perforated patch method, ATP (100 μM) induced a large depolarisation and action potential firing. UDP (100 μM) induced only a small depolarisation but it also elicited action potentials. UDP increased the excitability of the neurones. The results indicate that the ATP-induced release of noradrenaline depends on influx of calcium from the extracellular space. Calcium passes through two structures: voltage-gated channels and – probably – the P2X-receptor itself. Only that component of ATP-induced transmitter release which is triggered by opening of voltage-gated calcium channels is sensitive to modulation by α₂-adrenocep-tors. In contrast to ATP, the UDP-induced release of noradrenaline is entirely due to generation of action potentials followed by calcium influx through voltage-gated channels. All of the UDP-induced release is therefore sensitive to α₂-adrenoceptor modulation. Received: 22 September 1998 / Accepted: 25 January 1999     

Effect of glutamate and extracellular calcium on uptake of inorganic lead (Pb2+) in immortalized mouse hypothalamic GT1-7 neuronal cells

We have previously shown that although glutamate alone has no effects on viability of mouse hypothalamic GT1–7 cells, it clearly enhances Pb²⁺-induced cytotoxicity. It is likely that Pb²⁺ must enter cells to exert most of its toxic effects. Pb²⁺ is known to substitute for Ca²⁺ in many cellular processes. Therefore, we studied the uptake mechanisms of Pb²⁺ into GT1–7 neuronal cells with a special focus on the role of extracellular calcium (Ca²⁺), voltage-sensitive calcium channels (VSCCs) and glutamate. Basal uptake of Pb²⁺ (1 μM or 10 μM), i.e. without any external stimulus, clearly increased in nominally Ca²⁺-free buffer and was partially abolished by 13 mM Ca²⁺ when compared to uptake in the presence of a physiological concentration of extracellular Ca²⁺ (1.3 mM). Depolarization by 25 mM K⁺, or antagonists of VSCCs, verapamil (10 μM) or flunarizine (10 μM) had no clear effect on basal Pb²⁺ uptake. Glutamate (1 mM) increased Pb²⁺ uptake, but only when cells were treated with 1 μM Pb²⁺ in the presence of 1.3 mM Ca²⁺. Our data suggest that Pb²⁺ competes for the same cellular uptake pathways with Ca²⁺, although not via VSCCs. In addition, enhancement of Pb²⁺-induced neurotoxicity by glutamate may be due to increased neuronal uptake of Pb²⁺.     

Modulatory effect of calcium on the influx and binding of cadmium in primary cultures of neonatal rat hepatocytes

Primary cultures of rat hepatocytes were exposed to 3 or 30 microM stable cadmium (Cd) in the presence or absence of 1.8 or 3.6 mM calcium (Ca). The presence of Ca significantly reduced the efflux of lactate dehydrogenase (LDH) from cells regardless of Cd concentration or exposure time. During a 3-h time interval, the influx of Cd into hepatocytes increased from about 5 to 14% of the total extracellular Cd present. The presence of Ca during 30 microM Cd exposures resulted in an 18% reduction (P less than 0.01 or 0.001) in Cd influx during a 3-h exposure. About 11% more Cd was bound to those cells exposed in the absence of Ca following 2-h, but not 0.5-h, exposures. Therefore, binding of Cd to hepatocytes was not related directly to Cd uptake since Cd uptake (but not binding) was elevated at the 0.5-h time interval. Although the presence of Cd did not affect the influx of Ca, the presence of Cd increased the binding of Ca by 557% (P less than 0.001). Interaction between these cations at the same binding and/or entry sites on (or adjacent to) phospholipid head groups could account for the modulatory effect of Ca on Cd-challenged hepatocytes.     

三七总皂甙对大鼠肝细胞钙内流的阻断作用

目的：通过检测三七总皂甙（ＰＮＧＳ）对大鼠单离肝细胞Ｃａ２＋内流的影响，探讨其对肝缺血再灌注损伤保护作用的可能性。方法：采用胶原酶灌注法单离大鼠肝细胞，用４５Ｃａ示踪技术测定三七总皂甙对新鲜分离大鼠肝细胞Ｃａ２＋内流的影响。结果：肝细胞孵育在含４５Ｃａ生理液中，其细胞内４５Ｃａ浓度随孵育时间延长逐渐增高，１５ｍｉｎ时达高峰；２０ｎｍｏｌ·Ｌ－１垂体加压素使４５Ｃａ浓度增高３９％。Ｖｅｒａｐａｍｉｌ、Ｎｉｆｅｄｉｐｉｎｅ和ＰＮＧＳ均能不同程度地阻断静息状态和垂体加压素激动下的肝细胞４５Ｃａ内流，ＰＮＧＳ阻断效果明显优于Ｖｅｒａｐａｍｉｌ和Ｎｉｆｅｄｉｐｉｎｅ，且呈剂量依赖性。结论：ＰＮＧＳ具有特异性阻断肝细胞受体依赖性钙通道（ＲＯＣ）的作用，是一种理想的肝细胞钙通道阻滞剂。     

Copper and lead exposures disturb reproductive features of primary endometrial stromal and epithelial cells

This study investigates if Cu and Pb act as endocrine disruptors affecting endometrial cells. Primary EnSCs and EnECs were exposed to Cu (0, 50, 100 and 200 μM) or Pb (0, 30, 100 and 500 μM) and assessed for viability, decidualization, apoptosis and proliferation on EnSCs, and wound healing and adhesion capabilities on EnECs. Cu exposure decreased significantly cell viability in a dose-dependent manner. Cu and Pb negatively affected in vitro decidualization, showing a significant decrease in PRL secretion. HOXA10 and ERα mRNA levels significantly decreased in decidualized cells (dEnSCs) exposed to Cu. Cu and Pb decreased adhesion and regeneration capability in EnEC. This study reveals that Cu and Pb could negatively affect endometrial functionality, compromising the decidualization process and disrupting endometrial regeneration and embryo adhesion. Therefore, special care should be taken considering heavy metals exposure if pregnancy is being pursued.     

铁离子对人成骨细胞内钙离子转运的影响

目的 研究细胞外铁离子(Fe3 )浓度变化对人成骨细胞(hFOB 1.19)内Ca2 转运的影响.方法 将成骨细胞34℃培养3～4 d(细胞密度90% )并用胰蛋白酶淌化后分种于12孔培养板上.空白组加入双蒸水;实验组加入不同浓度的去铁氨(DFO)和枸橼酸铁铵(FAC).分别干预后立即用激光共聚焦扫描显微镜(CLSM)检测.结果 CLSM显示,随着hFOB 1.19外Fe3 浓度的减少(DFO终浓度为200～300μmol/L),Ca2 向细胞内的转运量增加;而随着hFOB 1.19外Fe3 浓度的增加(FAC终浓度为50～100 μmol/L),Ca2 向细胞内的转运是降低趋势.结论 hFOB 1.19外Fe3 浓度的减少可以增加细胞内的Ca2 ,而胞外Fe3 浓度增加则减少细胞内的Ca2 .     

Methylmercury inhibits gap junctional intercellular communication in primary cultures of rat proximal tubular cells

Methylmercury (MeHg) causes renal injury in addition to central and peripheral neuropathy. To clarify the mechanism of nephrotoxicity by MeHg, we investigated the effect of this compound on intercellular communication through gap junction channels in primary cultures of rat renal proximal tubular cells. Twenty minutes after exposure to 30 μM MeHg, gap junctional intercellular communication (GJIC), which was assessed by dye coupling, was markedly inhibited before appearance of cytotoxicity. When the medium containing MeHg was exchanged with MeHg-free medium, dye coupling recovered abruptly. However, the dye-coupling was abolished again 30 min after replacement with control medium, and the cells were damaged. Intracellular calcium concentration, [Ca²⁺]_i, which modulates the function of gap junctions, significantly increased following exposure of the cells to 30 μM MeHg and returned to control level following replacement with MeHg-free medium. These results suggest that the inhibiting effect of MeHg on GJIC is related to the change in [Ca²⁺]_i, and may be involved in the pathogenesis of renal dysfunction. Received: 22 April 1997 / Accepted: 22 October 1997     

钙、锌对铅致大鼠记忆障碍的保护机制探讨

目的　主要探讨饲料钙、锌干预对脑发育期低水平铅暴露致大鼠仔鼠学习记忆障碍保护作用的可能机制。方法　大鼠孕 14d至仔鼠出生 40d饮 10 0mg L含铅水 ,进食加钙 (1 2 5 % )和加锌 (10 0mg kg)配方饲料组 ,观察仔鼠生长发育情况 ,仔鼠 40d取股动脉血分析全血铅含量 ;取右半侧大脑分析脑铅含量 ;取左侧海马和小脑分析NO含量 ,右侧海马分析蛋白激酶C(PKC)活性。结果　两干预组仔鼠脑铅平均值分别为 3 0 5和 0 62 μg g ,明显低于进食普通配方饲料组 ,而加钙组血铅平均值为 0 2 9μmol L,明显低于加锌和普通配方组 ;两干预组海马和小脑一氧化氮 (NO)平均值分别为 2 7 68和 13 82 μmol g组织蛋白 ,均高于普通配方组 ,而海马细胞胞浆蛋白激酶C活性平均值分别为 0 3 3和 0 3 8pmol (min .μg组织蛋白 ) ,均低于普通配方组。结论　饲料中钙、锌干预对低水平铅暴露致大鼠学习记忆障碍的保护作用可能与降低其体内血、脑中铅蓄积水平 ,升高海马和小脑NO含量以及降低海马细胞胞浆内PKC活性有关     

Protective effect of Vitamin D3 against lead induced hepatotoxicity,oxidative stress,immunosuppressive and calcium homeostasis disorders in rat

Lead (Pb) is an extremely poisonous, non-essential trace element and toxicity develops in humans following frequent exposure to the heavy metal in polluted environmental and occupational settings. Pb induces hepatic damage through the depletion of the antioxidant system, enhancing cellular oxidative stress and stimulation of proinflammatory cytokines. Although the antioxidant and anti-inflammatory actions of vitamin D₃ (VD₃) are well-established, a minority of studies measured the protective actions of VD₃ against Pb toxicity. Therefore, this work studied the effects of vitamin VD₃ therapy on the fundamental molecular basis underlying hepatic injury induced by chronic Pb toxicity. Twenty-four adult male rats were distributed equally into the negative controls (NC), positive controls (PC) and VD3 groups. While both the PC and VD3 groups received Pb-acetate in drinking water (1000 mg/L) for four weeks, the latter group also received intramuscular VD3 injections (1000 IU/kg; 3 days/week) simultaneously with Pb. The liver enzymes together with the serum and hepatic tissue Pb concentrations increased markedly in the PC group compared with the NC group. Pb toxicity also drastically induced hepatocyte apoptosis/necrosis, increased the hepatic tissue concentrations of malondialdehyde and the pro-inflammatory cytokines (TGF-β, IL-4 & TNF-α) as well as reduced the anti-oxidative enzymes (GSH, GPx & CAT) and the anti-inflammatory cytokine, IL-10, compared with the NC group. Pb also significantly decreased the serum concentrations of VD3 and Ca²⁺. Additionally, the hepatic expressions of VD receptor, Cyp24a1 enzyme, L-type Ca²⁺-channel, calbindin-D_28k & -D_29k, calmodulin and calmodulin-dependent protein kinase II were significantly upregulated, whereas the VD binding protein, CYP2R1 enzyme and T-type Ca²⁺-channel were markedly inhibited at the gene and protein levels following Pb intoxication. VD3 alleviated the hepatic damage, inhibited the oxidative stress and pro-inflammatory molecules as well as upregulated the anti-oxidant and anti-inflammatory markers and restored the expression of the VD/Ca²+ regulatory molecules compared with the PC group. VD3 supplementation discloses promising protective effects against Pb-induced hepatic damage, through its anti-inflammatory and antioxidant actions as well as by modulating the hepatocyte calcium homeostatic molecules.     

Possible mechanism of perfluorooctane sulfonate and perfluorooctanoate on the release of calcium ion from calcium stores in primary cultures of rat hippocampal neurons

Perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) are anthropogenic compounds manufactured since the 1950s and are distributed worldwide. Now, the pollutants are being challenged by entering into the brain and the toxic effect on the central nervous system due to calcium disorder, mainly through channels on cell membrane. However, little is known about the role of calcium store in PFOS- and PFOA-evoked abnormal calcium increase. In the present study, PFOA and PFOS were measured in primary cultures of rat hippocampal neurons by LC/MS/MS analysis. Flow cytometry was used to examine altered calcium patterns in neurons labeled with fluo-3/AM and to disclose the mechanism by which PFOS and PFOA induced calcium increase in cultured neurons. The results indicate that both PFOS and PFOA can accumulate in cultured neurons and elevate calcium concentrations via release of intracellular calcium stores. Furthermore, inositol 1,4,5-trisphosphate receptors (IP₃Rs) and ryanodine receptors (RyRs) were found to take part in PFOS or PFOA inducing calcium release from calcium stores. IP₃Rs seem to serve a predominant role in PFOS-induced calcium release. Calcium release from intracellular stores may partially account for the perturbation of calcium homeostasis caused by PFOS or PFOA.     

谷氨酸对牛大脑中动脉平滑肌细胞胞浆静息Ca^2＋和ATP触发的Ca^2＋内流的影响

目的：观察谷氨酸对牛脑中动脉平滑肌细胞有无直接的激动作以及对ATP触发的Ca^2 内流有无影响？方法：采用Fura-2荧光法测定胞浆内Ca^2 变化技术。在培养的乳牛大脑中动脉平滑肌细胞上观察药物作用。结果：谷氨酸（10－200μmol.L^-1）不能增加或减少细胞胞浆静息游离Ca^2 浓度（[Ca^2 ]i)和ATP触发的Ca^2 内流（谷氨酸：10－400μmol.L^-1）。结论：谷氨酸对大脑中动脉平滑肌细胞信息Ca^2 水平和Ca^2 内流均无直接的影响。谷氨酸可能没有直接与脑血管张力的调节。     

Sulfhydryl modification by 4,4'-dithiodipyridine induces calcium mobilization in human osteoblast-like cells

The effect of oxidants on Ca²⁺ movement in osteoblasts is unclear. In this study, we show that 4,4-dithiodipyridine (4,4-DTDP), a reactive disulphide that mobilizes Ca²⁺ in muscle, induces an increase in cytoplasmic free-Ca²⁺ concentrations ([Ca²⁺]_i) in MG63 human osteosarcoma cells loaded with the Ca²⁺-sensitive dye fura-2. 4,4-DTDP acted in a concentration-dependent manner with an EC₅₀ of 10 M. Removing extracellular Ca²⁺ reduced the Ca²⁺ signal by 35%. In Ca²⁺-free medium, the 4,4-DTDP-induced [Ca²⁺]_i increase was not changed by depleting store Ca²⁺ with 50 M brefeldin A (a Golgi apparatus permeabilizer), by 2 M carbonylcyanide m-chlorophenylhydrazone (CCCP, a mitochondrial uncoupler), by 1 M thapsigargin (an inhibitor of the endoplasmic reticulum Ca²⁺ pump) or by 5 M ryanodine. Ca²⁺ signals induced by 4,4-DTDP in Ca²⁺-containing medium were not affected by modulation of protein kinase C activity or suppression of phospholipase C activity. However, 4,4-DTDP-induced Ca²⁺ release was inhibited by a thiol-selective reducing reagent, dithiothreitol (0.05-2.5 mM), in a concentration-dependent manner. Collectively, this study shows that 4,4-DTDP induced [Ca²⁺]_i increases in human osteosarcoma cells via releasing store Ca²⁺ from multiple stores in a manner independent of protein kinase C or phospholipase C activity. The store Ca²⁺ release induced by 4,4-DTDP appears to be associated with thiol oxidation. Furthermore, overnight incubation with 4,4-DTDP inhibited cell activity in a concentration-dependent manner.     

铅对大鼠C6细胞GRP78表达的影响

目的观察不用时间点暴露于不同剂量醋酸铅后大鼠神经胶质瘤C6细胞葡萄糖调节蛋白78(glucoseregulated protein 78,GRP78)表达量的影响,探讨不同时间不同剂量铅暴露对内质网应激反应的影响。方法Wistar大鼠神经胶质瘤C6细胞培养于含醋酸铅的培养液中,分别于不同时间终止染铅,用Western印迹法检测内质网GRP78表达量。结果(1)0.2μmol/L染铅组:7和30 d GRP78蛋白表达显著增高,其余各个时间点均无显著性变化;(2)1.0μmol/L染铅组:1 d后GRP78蛋白表达量开始显著增高,染铅30 d时已达到染铅前的6.3倍;(3)2.0μmol/L染铅组:0.5 h起GRP78表达量即显著增高,染铅7 d时达到高峰,是染铅前的4.3倍,到30 d时表达量又下降为染铅前的2.6倍。结论铅可以使Wistar大鼠神经胶质瘤细胞内质网上的GRP78蛋白应激性表达增加,内质网上的GRP78是铅的重要蓄积库。     

Functional characterisation of P2 purinoceptors in PC12 cells by measurement of radiolabelled calcium influx

The aim of this study was to determine whether ⁴⁵Ca²⁺ influx could be used as a quantitative measure of channel activation for functional characterisation of P2X purinoceptors in cell lines. In undifferentiated PC12 cells, grown in suspension, ATP (EC₅₀ = 45 M), ATPTS (EC₅₀ = 50 M) and 2-meSATP (EC₅₀ = 81 M) but not meATP (I mM) stimulated ⁴⁵Ca²⁺ influx 2–5 fold. This effect did not appear to be due to activation of P2U or P2Y purinoceptors since 1 mM UTP, ADP or ADPS did not produce any significant effect. Similarly, the effects of ATP were not apparently mediated through activation of P2Z purinoceptors since dibenzylATP behaved as a weak (EC₅₀ = 191 M) partial agonist (Maximal effect 29.5% of ATP maximum) and there was no detectable ATP-stimulated ethidium bromide uptake in the PC12 cells.ATP-stimulated ⁴⁵Ca²⁺ influx was not affected by nifedipine suggesting that it was not secondary to activation of L-type calcium channels and rather reflected influx through a P2X purinoceptor present in these cells. The ATP-stimulated ⁴⁵Ca²⁺ influx could be reduced by monovalent cations, presumably as a result of direct competition for influx through the cation channel, with the following rank order of potency :- guanidinium (EC₅₀ = 16 mM) > sodium > Tris > choline > N-methyl-D-glucamine = sucrose.A number of P2 purinoceptor antagonists inhibited ATP-stimulated ⁴⁵Ca²⁺ influx. Pyridoxalphosphate-6-azophenyl-2,4-disulphonic acid (3–300 M), pyridoxal 5phosphate (3–300 M) and d-tubocurarine (30–300 M) produced an insurmountable antagonism of responses to ATP, with no marked change in agonist EC₅₀. Suramin (100–300 M) and cibacron blue (30–300 M) produced a surmountable antagonism while DIDS (4,4-diisothiocyana-tostilbene-2,2disulfonic acid) only antagonised responses to ATP at concentrations in excess of 300 M. The general properties of the P2X purinoceptor population identified in these cells were consistent with them being P2X₂ purinoceptors. These findings suggest that ATP-stimulated ⁴⁵Ca²⁺ influx may be used as a reliable and quantitative functional assay for characterisation of P2X purinoceptor subtypes in cell lines.     

The high-affinity immunoglobulin E receptor (FcepsilonRI) regulates mitochondrial calcium uptake and a dihydropyridine receptor-mediated calcium influx in mast cells: Role of the FcepsilonRIbeta chain immunoreceptor tyrosine-based activation motif

A growing body of evidence suggests that mitochondria take up calcium upon receptor (agonist) stimulation and that this contributes to the dynamics of spatiotemporal calcium signaling. We have previously shown that engagement of the high-affinity receptor for immunoglobulin E (FcepsilonRI) stimulates mitochondrial calcium ([Ca2+]m) uptake in mast cells. The present study was undertaken to investigate the mechanisms and biological significance of FcepsilonRI regulation of [Ca2+]m. Antigen stimulated [Ca2+]m uptake in a dose-dependent manner with a minimal effective dose of 0.03-3 ng/ml. This [Ca2+]m uptake took place immediately, reaching its peak within minutes and was inhibited by the src family kinase inhibitor PP1 and phosphatidylinositol-3-kinase inhibitor wortmannin. Analyses using mast cells expressing the wild-type or the mutated type of the FcepsilonRIbeta immunoreceptor tyrosine-based activation motif (ITAM) in which all tyrosine residues were replaced by phenylalanine revealed that the FcepsilonRIbeta ITAM is essential for a sustained [Ca2+]m uptake. The FcepsilonRIbeta ITAM was essential for overall calcium response upon weak FcepsilonRI stimulation (at low antigen concentration), while upon strong stimulation (at high antigen concentration) it appeared necessary selectively to an immediate calcium response that was sensitive to the dihydropyridine receptor (DHPR) antagonist nifedipine and wortmannin but not to the store-operated calcium entry (SOCE) antagonists such as 2-aminoethoxyphenyl borate and SK&F96365. These data demonstrate that the FcepsilonRIbeta regulates [Ca2+]m uptake in mast cells via the ITAM and suggest that this plays a key role in regulating calcium influx especially that induced via a DHPR-mediated calcium channel.     

芍药苷下调PC12细胞酸敏感离子通道1a的表达拮抗酸诱导的钙内流

<正>酸敏感离子通道(acid sensing ion channels,ASICs)是一类能被细胞外H+激活的阳离子通道,其中的ASIC1a亚基能介导钙的内流,参与了脑缺血缺氧、帕金森病(Parkinson’s