TMB-8对去甲肾上腺素和BHQ引起新生大鼠单个脑细胞内游离钙增高的影响

应用AR-CM-MIC阳离子测定系统,研究TMB-8对体外新生SD大鼠单个脑细胞内游离钙的抑制作用及其机制。结果表明,在无细胞外钙情况下,静息[Ca²⁺]i为79±13nmol·L^-1。TMB-810,30μmol·L^-1能明显降低静息[Ca²⁺]i。TMB-8100μmol·L^-1对高钾去极化引起的[Ca²⁺]i显著增高无明显影响。在细胞外钙为1.3mmol·L^-1时,去甲肾上腺素诱导的细胞内[Ca²⁺]i升高可部分被TMB-8抑制;TMB-8(30μmol·L^-1)对BHQ引起的[Ca²⁺]i的升高无明显抑制作用。而当细胞外液[Ca²⁺]i为0时,TMB-8几乎完全抑制了去甲肾上腺素和BHQ的作用。提示TMB-8降低脑细胞内游离钙的作用机制是通过促使细胞内钙进入肌浆网以抑制内钙的释放,并通过饱和肌浆网内Ca²⁺间接地阻滞细胞膜钙通道。     

钙拮抗剂TMB-8对培养牛大脑中动脉内皮细胞[Ca2+]i和一氧化氮释放的影响

目的旨在观察TMB-8对血管内皮细胞[Ca²⁺]i水平和NO释放的影响,探讨扩张脑血管的机制。用AR-CM-MIC阳离子测定系统,测量单个细胞内游离钙浓度([Ca²⁺]i),用血红蛋白法测量一氧化氮(NO)的释放。结果表明,在细胞外钙浓度为1.3mmol·L^-1时,TMB-8 12.5及25.0μmol·L^-1对静息[Ca²⁺]i和甲基血红蛋白ΔE无明显影响,而50及100μmol·L^-1时可升高静息[Ca²⁺]i和甲基血红蛋白ΔE。表明TMB-850及100μmol·L^-1升高脑血管内皮[Ca²⁺]i,激活NO合酶,促进NO合成和释放,这可能是其扩张脑血管的重要机制之一。     

AMG-1对突触体内游离钙水平及大鼠尾动脉收缩力的影响

观察了AMG-1对高钾所致大鼠脑突触体内游离钙浓度[Ca²⁺]i升高,及去甲肾上腺素引起的离体大鼠尾动脉环收缩力的影响。结果表明,AMG-110和100μmol·L^-1可对抗高钾(30mmol·L^-1)引起的[Ca²⁺]i升高,使分别降低19±11%及57±12%;在无钙Krebs-Hensleleit液中,AMG-1能抑制去甲肾上腺素引起大鼠尾动脉收缩力的作用。     

四氢原小檗碱类药物对培养大鼠单个心肌细胞内游离Ca2+的影响

用Fura-2/AM技术和AR-CM-MIC阳离子测定系统,直接测定了四氢小檗碱(tetrahy-droberberine,THB)、左旋四氢巴马汀(l-tetrahydropalmatine,THP)和左旋千金藤立定(l-stepholidine,SPO)对培养大鼠单个心室肌细胞内游离钙([Ca²⁺]i)的影响,并与维拉帕米(Ver)做了比较。结果显示,THB,THP,SPD浓度为10～100μmol·L^-1时,均可使静息[Ca²⁺]i轻度升高,河豚毒素不能抑制之;浓度为1～100μmol·L^-1时,可明显抑制高K+引起的[Ca²⁺]i增高;30μmol·L^-1对胞外高Ca²⁺和去甲肾上腺素引起的[Ca²⁺]i增高也有明显的抑制作用,但均较Ver的抑制作用为弱;THPB对哇巴因引起的[ca²⁺]i升高无明显抑制作用。结果提示,THB,THP,SPD在抑制电压依赖性钙通道从而影响细胞膜[ca²⁺]i内流方面与Ver相似,但比Ver弱。     

粉防己碱对胎鼠大脑细胞游离钙含量的影响

结果表明,粉防己碱(Tet)非常明显地阻断K+去极化导致的胎鼠大脑[Ca²⁺]i含量的增加。经不同浓度Tet处理的大脑细胞加入KCl后,[Ca²⁺]i含量仍基本维持在静息状态下水平。对[Ca²⁺]i的阻断程度与Tet浓度无关。Tet对L-谷氨酸(L-Glu)所致大脑[Ca²⁺]i升高亦有明显抑制作用。10^-7mol·L^-1浓度的Tet还降低BayK8644引起的[Ca²⁺]i上升高。Tet能抑制高K⁺,二氢吡啶类钙激动剂及兴奋性神经递质导致的胎鼠大脑[Ca²⁺]i含量增加,提示Tet对神经细胞损伤可能有一定保护作用。     

小檗碱对培养大鼠神经细胞内游离Ca2+的影响

以Fura-2/AM为细胞内钙离子的荧光指示剂,用AR-CM-MIC阳离子测定系统,直接测定了体外培养的新生大鼠神经细胞内游离钙([Ca²⁺]i)值,并观察了小檗碱(Ber)的影响。结果表明,Ber对神经细胞静息[Ca²⁺]i无明显影响,Ber1～100μmol·L^-1能剂量依赖地抑制去甲肾上腺素和H₂O₂引起的[Ca²⁺]i升高,其IC₅₀分别为39.9和17.9μmol·L^-1。高剂量Ber(10～100μmol·L^-1)能抑制高K+引起的[Ca²⁺]i升高。姐果提示,Ber对去甲肾上腺素,高K⁺及H₂O₂引起的[Ca²⁺]i升高的抑制作用可能是其抗脑缺血作用机制之一。     

四肽FMRFa对大鼠心室肌Na+/Ca2+交换的抑制

目的　研究四肽FMRFa对大鼠单个心室肌细胞Na⁺/Ca²⁺交换的作用。方法　用膜片钳全细胞记录法测定成年大鼠心室肌细胞Na⁺/Ca²⁺交换电流(I_Na⁺/Ca²⁺)和其他离子通道电流。结果　FMRFa对大鼠心室肌细胞I_Na⁺/Ca²⁺呈浓度依赖性抑制,100μmol·L^-1浓度时抑制内向和外向I_Na⁺/Ca²⁺密度分别达60.1%和56.5%,对内向电流及外向电流的IC₅₀分别为20μmol·L^-1和34μmol·L^-1。FMRFa5μmol·L^-1抑制I_Na⁺/Ca²⁺内向和外向电流密度分别为38.7%和34.9%,但FMRFa5μmol·L^-1及20μmol·L^-1对L型钙电流、钠电流、瞬时外向电流和内向整流钾电流均无显著抑制作用。结论　FMRFa对大鼠心室肌细胞是一个特异性Na⁺/Ca²⁺交换抑制剂。     

阿米洛利对压力超负荷心肌肥厚大鼠心肌细胞内游离钙的影响

目的:观察阿米洛利预防性给药对压力超负荷心肌肥厚大鼠心肌细胞[Ca²⁺]i 影响。方法:以Fura-2/AM为指示剂,测定单细胞[Ca²⁺]i。结果:压力超负荷大鼠左室心肌细胞[Ca²⁺]i 升高,阿米洛利和依那普利组则[Ca²⁺]i 明显降低。给予KCl,NE刺激后,给药组左室心肌细胞[Ca²⁺]i 的增加值明显低于左室肥厚组,百日咳毒素(PTX,100 ng·L^-1) 处理5 h 后,其静息[Ca²⁺]i 无显著变化,但抑制NE诱导左室心肌细胞[Ca²⁺]i 升高,该作用在左室肥厚组更明显,对KCl 刺激引起的[Ca²⁺]i 升高,给药组无影响,左室肥厚组的升高值略有增加。结论:NE所致心肌细胞[Ca₂₊]i 升高与PTX敏感的G 蛋白有关,肥厚时此途径亢进。     

TMB-8抑制5-HT和KCl引起大鼠脑血流量减少

目的研究TMB-8对5-HT和KCl引起的大鼠脑血流量(CBF)减少的作用。方法用激光多普勒血流仪测量大鼠CBF,在人工脑脊液灌流液中加入TMB-8和工具药进行干预。结果12.5, 25和50 μmol·L^-1 TMB-8对大鼠CBF无明显影响,TMB-8可抑制5-HT引起的大鼠CBF减少。在1 μmol·L^-1 5-HT引起大鼠CBF持续下降的状态下,TMB-8可浓度依赖的增加大鼠CBF。TMB-8也可抑制KCl引起的大鼠CBF减少。在20 mmol·L^-1 KCl引起大鼠CBF持续下降的状态下,TMB-8可浓度依赖的增加大鼠CBF。结论TMB-8可抑制5-HT和KCl引起的大鼠CBF减少,也可浓度依赖的增加被5-HT和KCl所减少的大鼠CBF,改善大鼠缺血区脑血供。     

灯盏花素对谷氨酸诱导大鼠海马神经细胞毒性的保护作用

选用培养的海马神经细胞研究灯盏花素(breviscapine，Bre)对谷氨酸(glutamate，Glu)诱导神经细胞毒性的保护作用及其机制。新生大鼠海马神经细胞体外培养8 d后， 用L-谷氨酸(0.1， 0.5及1.0 mmol·L^-1)处理30 min； 灯盏花素处理组在加入L-谷氨酸的同时给予不同剂量的灯盏花素(10， 20及40 μmol·L^-1)； 继续正常培养24 h后， 用Annexin V联合流式细胞仪检测细胞的凋亡和坏死率； RT-PCR分析凋亡蛋白抑制剂XIAP mRNA的表达。结果表明， L-谷氨酸浓度依赖性地诱导神经细胞发生凋亡和坏死， 并使细胞XIAP mRNA表达发生浓度依赖性的双向变化： 0.1 mmol·L^-1 L-谷氨酸使神经细胞XIAP mRNA表达增强， 而较高浓度使XIAP mRNA表达下调。灯盏花素(20和40 μmol·L^-1)可有效抑制谷氨酸诱导的神经细胞死亡， 分别使细胞凋亡率下降30.4%和40.1%， 坏死率下降32.5%和38.8%； 并上调XIAP mRNA表达45.1%和54.9%。激光共聚焦显微技术联合Fluo-3荧光标记检测表明，L-谷氨酸处理过程中海马神经细胞内Ca²⁺水平显著升高， 而灯盏花素可抑制谷氨酸引起的细胞内Ca²⁺超载(P<0.01)。以上结果提示， 灯盏花素可能通过抑制细胞Ca²⁺超载， 调节凋亡抑制因子XIAP的表达而有效保护谷氨酸对神经细胞的兴奋性毒性作用。     

TMB—8抑制神经递质引起的单个脑细胞内游离钙的升高

AIM: To study the effects of TMB-8 on [Ca2+]i elevation induced by neurotransmitters in dissociated brain cells. METHODS: The brain cell suspension was made using a gentle trituration for 1 min with a polished pipette. The changes of [Ca2+]i were detected by the fluorescent indicator, Fura 2-AM. RESULTS: In the presence of extracellular Ca2+ 1.3 mmol.L-1, sodium glutamate (Glu), histamine (His), and serotonin (5-HT) markedly increased the [Ca2+]i which were reduced by TMB-8 30 mumol.L-1. TMB-8 3 mumol.L-1 produced inhibitory effects on the increase of [Ca2+]i by His and 5-HT in a Ca(2+)-free Hanks' solution. The increase of [Ca2+]i by His and 5-HT was reduced to control level by TMB-8 10 mumol.L-1. CONCLUSION: TMB-8 inhibited the [Ca2+]i elevation induced by Glu, 5-HT, and His in brain cells.     

TMB—8抑制组胺,5—羟色胺和谷氨酸引起的培养乳牛基底动脉平滑肌细 …

AIM: To study the effects of 8-(N,N-diethylamino)-n-octyl-3,4,5- trimethoxybenzoate (TMB-8) on intracellular free calcium ([Ca2+]i) in cultured calf basilar artery smooth muscle cells. METHODS: [Ca2+]i was examined by a system of measurement of AR-CM-MIC, using Fura 2-AM as a fluorescent indicator. RESULTS: In the presence of extracellular Ca2+ 1.3 mmol.L-1, histamine (His), serotonin (5-HT), and sodium glutamate (Glu) markedly increased the [Ca2+]i which was attenuated by TMB-8. In Ca2+ free Hanks' solution containing egtazic acid 0.1 mmol.L-1, TMB-8 not only reduced the resting [Ca2+]i, but also inhibited the elevation of [Ca2+]i evoked by His and 5-HT. CONCLUSION: TMB-8 reduced the resting [Ca2+]i and attenuated His-, 5-HT-, and Glu-induced increases of [Ca2+]i in basilar artery smooth muscle cells.     

小檗胺对高钾,去甲肾上腺素及咖啡因引起大鼠心肌细胞内钙动拮？…

AIM: To study the effects of berbamine (Ber) on intracellular calcium concentration ([Ca2+]i) mobilized by KCl depolarization, norepinephrine (NE), and caffeine. METHODS: [Ca2+]i was measured with fluorescent intensity (FI) by confocal microscope in single cultured cardiomyocytes of newborn rats loaded with Fluo 3-AM 2 mumol.L-1. RESULTS: FI value of [Ca2+]i in control level was 248 +/- 70 in the presence of extracellular calcium 1.5 mmol.L-1 and was not changed by Ber 3-30 mumol.L-1. KCl (60 mmol.L-1)- and NE (30 mumol.L-1)-induced [Ca2+]i mobilizations were inhibited (P < 0.01) by Ber 30 mumol.L-1, similar to that of verapamil (Ver). The inhibitory effect of Ber on [Ca2+]i induced by KCl was further increased (P < 0.05) in the presence of egtazic acid 3 mmol.L-1, but that on [Ca2+]i induced by NE was not changed. The [Ca2+]i mobilized by caffeine 80 and 160 mumol.L-1 in D-Hanks' solution was not affected (P > 0.05) by Ber and Ver. CONCLUSION: Ber possessed the antagonistic effects on [Ca2+]i increases via voltage-dependent Ca2+ channel and receptor-operated Ca2+ channel in newborn rat cardiomyocytes, but without effect on intracellular Ca2+ release.     

小檗碱对培养大鼠心肌细胞胞内游离Ca2+的作用

利用Fura-2技术和AR-CM-MIC阳离子测定系统,直接观察了小檗碱对培养大鼠心肌细胞胞内[Ca²⁺]i的影响。结果显示:小檗碱可明显升高心肌细胞静息[Ca²⁺]i且具饱合性,维拉帕米和CoCl₂对其有一定的抑制作用;小檗碱与高K⁺,高Ca²⁺,去甲肾上腺素,哇巴因合用比单用上述激动剂更能明显增高[Ca²⁺]i;维拉帕米对其有抑制作用;在胞外无外Ca²⁺和无外Ca²⁺,外K⁺,外Na⁺时,小檗碱30～200μmol·L^-1仍能升高静息[Ca²⁺]i,维拉帕米只对前者有一定抑制作用。结果提示:小檗碱可能通过促胞外Ca²⁺内流和胞内Ca²⁺释放等途径有限度地增高心肌细胞内游离Ca²⁺浓度,显示强心作用。     

前胡丙素对高血压大鼠血管肥厚、细胞内钙、胶原及NO的影响

目的　研究前胡丙素对自发性高血压大鼠SHR及肾型高血压大鼠RHR的血管肥厚、细胞内钙、胶原、NO及血管收缩的反应性影响。方法用显微测微仪测定血管中膜层厚度,细胞大小,用Fura-2/AM为荧光指示剂,测定单细胞内［Ca²⁺］i,以测定羟脯氨酸含量反映胶原含量,用Griess法测定NO含量,以血管环观察收缩反应。结果　前胡丙素抑制血管中膜层增厚,维持细胞内［Ca²⁺］i稳态。减少胶原形成,增加SMCs释放NO。抑制血管环高反应状态。结论　前胡丙素抑制高血压血管肥厚,降低胶原含量及血管异常反应。     

小檗碱对培养大鼠神经细胞内游离Ca^2＋的影响

以Ｆｕｒａ２／ＡＭ为细胞内钙离子的荧光指示剂，用ＡＲＣＭＭＩＣ阳离子测定系统，直接测定了体外培养的新生大鼠神经细胞内游离钙（［Ｃａ２＋］ｉ）值，并观察了小檗碱（Ｂｅｒ）的影响。结果表明，Ｂｅｒ对神经细胞静息［Ｃａ２＋］ｉ无明显影响，Ｂｅｒ１～１００μｍｏｌ·Ｌ－１能剂量依赖地抑制去甲肾上腺素和Ｈ２Ｏ２引起的［Ｃａ２＋］ｉ升高，其ＩＣ５０分别为３９．９和１７．９μｍｏｌ·Ｌ－１。高剂量Ｂｅｒ（１０～１００μｍｏｌ·Ｌ－１）能抑制高Ｋ＋引起的［Ｃａ２＋］ｉ升高。姐果提示，Ｂｅｒ对去甲肾上腺素，高Ｋ＋及Ｈ２Ｏ２引起的［Ｃａ２＋］ｉ升高的抑制作用可能是其抗脑缺血作用机制之一。     

金丝桃苷对分离的新生大鼠脑细胞内游离钙浓度的影响

AIM: To study the effects of hyperin (Hyp) on free intracellular calcium concentration ([Ca2+]i) of brain cells. METHODS: The neonatal rat brain cells were dissociated. [Ca2+]i in presence and absence of extracellular high K+, L-glutamic acid (Glu), 5-hydroxytryptamine (5-HT), and norepinephrine (NE) were assayed with Fura 2-AM. RESULTS: The resting [Ca2+]i in Hanks' solution (CaCl2 1.3 mmol.L-1) was (208 +/- 12) nmol.L-1 (n = 17). Hyp had no significant effects on the resting [Ca2+]i. Hyp 1.0, 4.0, and 16.0 mumol.L-1 markedly inhibited the increase of [Ca2+]i evoked by K+ 50 mmol.L-1 in a concentration-dependent manner. Hyp 16.0 mumol.L-1 inhibited the increases of [Ca2+]i induced by NE 1, 2, 4, and 8 mumol.L-1. Hyp (16.0 mumol.L-1) also markedly attenuated 5-HT and Glu-induced increase of [Ca2+]i. CONCLUSION: Hyp possessed inhibitory effects on influx of Ca2+ in the neonatal rat brain cells.     

小檗胺对培养的HeLa细胞内游离钙浓度的作用

AIM: To study the involvement of Ca2+ signaling and the effects of berbamine (Ber) on intracellular calcium concentration ([Ca2+]i) elevated in cultured HeLa cells. METHODS: [Ca2+]i was measured by confocal microscopy in single HeLa cell loaded with Fluo 3-AM. The change of [Ca2+]i was represented by fluorescent intensity (FI). RESULTS: (1) In the presence of extracellular Ca2+ 1.3 mmol.L-1, the resting level of FI was 186 +/- 44, n = 49 cells from all control experiments, and KCl, NE, caffeine, and calcimycin (Cal) all induced [Ca2+]i elevations in cultured HeLa cells. (2) The resting level of FI was not affected by pretreatment with Ber. The FI increased by KCl 60 mmol.L-1, NE 100 micromol.L-1, and Cal 30 micromol.L-1 were attenuated (P < 0.05 or P < 0.01), the slope and the time to peak of FI increase were decreased and prolonged. (3) In the absence of extracellular Ca2+, caffeine 80 mmol.L-1-induced [Ca2+]i mobilization was not inhibited by Ber 100 micromol.L-1 pretreatment. (4) These effects of Ber were similar to those of verapamil (Ver) 10 mumol.L-1. CONCLUSION: Although it was derived from cervical cancer, the HeLa cells which were belong to the nonexcitable cell possessed the similar biological properties with excitable cells, and Ca2+ also played a crucial role in signal transduction processes.