(+)-Bicuculline   中文名称: 必扣扣灵

GABAA receptor (IC50 = 2 μM)

BicucuLline（1 和 3 μM）（(+)-BicucuLline；d-BicucuLline）对 GABA 产生最高的反应。 BicucuLline 是 α1β2η2L GABAA 受体的竞争性拮抗剂，因为它平行地将 GABA 剂量反应曲线向右移动，而不降低 GABA 最大反应 [3]。

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在首次发表其作为抑制性神经递质GABA受体拮抗剂的作用40多年后，痉挛性生物碱BicuuLline继续被研究。这一历史视角突出了发现荷包牡丹碱作为GABA拮抗剂的关键方面，以及对这种和其他GABA拮抗剂持续的兴趣。GABA受体的分子生物学、药理学和生理学的令人兴奋的进展为发现新的拮抗剂提供了持续的刺激，这些拮抗剂对无数GABA受体亚类的选择性越来越高。与荷包牡丹碱结构无关的有趣GABA拮抗剂包括加巴嗪、亚水杨基水杨酰肼、RU5135和4-（3-联苯基-5-（4-哌啶基）-3-异恶唑。荷包牡丹碱成为GABAA受体的基准拮抗剂，但并非所有离子型GABA受体都对荷包牡丹素敏感。此外，并非所有GABAA受体拮抗剂都是惊厥药。因此，随着GABA受体研究的发展，仍然会有惊喜。[1]

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小电导钙激活钾通道（SK通道）仅由细胞内钙离子门控，其活性是许多可兴奋细胞中动作电位后缓慢后超极化（AHP）的原因。脑切片研究通常使用GABAA（γ-氨基丁酸）受体拮抗剂BicuuLine（bicuulline-m）的甲基衍生物来减少GABA能突触的强直性抑制作用，或研究这些突触在专门神经网络中的作用。然而，最近的证据表明，荷包牡丹碱-m可能对GABAA受体没有特异性，也可能阻断缓慢的AHP。因此，在爪蟾卵母细胞中表达后，研究了荷包牡丹碱-m对克隆的apamin敏感SK2和apamin不敏感SK1通道的影响。结果表明，在用于切片记录的浓度下，当应用于外向贴片时，荷包牡丹碱-m能有效阻断apamin敏感的SK2电流和apamin不敏感的SK1电流。Apamin不敏感的SK1电流在切除的斑块中下降。补丁切除后，荷包牡丹碱-m阻断的效力也随着时间的推移而降低。定点突变改变SK1孔外前庭中的两个残基，赋予apamin敏感性，也减少了贴片中电流的下降，并赋予了与SK2无法区分的荷包牡丹碱-m稳定的敏感性。因此，在切片记录中使用荷包牡丹碱-m可能会掩盖对apamin敏感的缓慢AHPs，而这些AHPs是神经元兴奋性的重要决定因素。此外，荷包牡丹碱-m不敏感的慢AHPs可能表明潜在的通道已经耗尽。2.

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倍半萜三内酯白果内酯是50:1银杏叶提取物的活性成分之一，广泛用于增强记忆和学习。发现白果内酯可以拮抗γ-氨基丁酸（GABA）对重组α（1）β（2）γ（2L）GABA（A）受体的直接作用。采用双电极电压钳法评估了白果内酯对非洲爪蟾卵母细胞中表达的α（1）β（2）γ（2L）GABA（A）受体GABA直接作用的影响，并将其与经典的GABA（A）受体竞争性拮抗剂BicuuLline和非竞争性拮抗物苦味毒素的影响进行了比较。白果内酯（IC（50）=4.6+/-0.5微M）在α（1）β（2）γ（2L）GABA（A）受体上对40微M GABA（GABA EC（50））的效力几乎与荷包牡丹碱和苦托毒素（IC（50%）分别为2.0+/-0.1和2.4+/-0.5微m）一样强。虽然白果内酯和苦味毒素显然是非竞争性拮抗剂，但白果内酯的效力在高GABA浓度下会降低，这表明存在竞争性拮抗作用[3]。

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荷包牡丹碱、苦味毒素和白果内酯剂量依赖性地抑制了40μM GABA产生的Cl-电导（图2A-C）。当这些化合物在100μM下单独使用时，没有观察到任何影响。图3A–E分别显示了苦毒毒素和白果内酯对10μM GABA（EC10）、40μM GABA和100μM GABA的抑制剂量-反应曲线。图3B还包括荷包牡丹碱对40μM GABA的抑制剂量-反应曲线（EC50）。表1列出了每种化合物的IC50和nH值。[3]

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在1和3μM荷包牡丹碱存在下的GABA浓度-效应曲线（图5A和B）显示了平行的右移，并达到了GABA的最大反应。在1和3μM荷包牡丹碱存在下，GABA反应分别为GABA最大反应的99.5%（P=0.9415）和101.0%（P=0.0702）（表2）。1和3μM的荷包牡丹碱增加了GABA EC50值：分别为1.6倍（41.0-67.0μM）和3.6倍（36.1-129.0μM）（表2）。Bicuulline似乎使GABA的剂量-反应曲线平行向右移动，而不会降低GABA的最大反应，这表明它是α1β2γ2L GABAA受体的竞争性拮抗剂[3]。

荷包牡丹碱/BicucuLline可用于动物建模中创建惊厥模型。[5]

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在258只出生后第3天至第22天的未成熟大鼠中研究了γ-氨基丁酸（GABA）拮抗剂BicucuLline/荷包牡丹碱的作用。行为和皮层电图事件相关。早在出生后第三天，BicucuLline/荷包牡丹碱就诱导了行为和心电图癫痫发作，此时荷包牡丹素的CD50最低，因此对它的敏感性最高。因此，对于出生后第一周涉及大鼠的癫痫研究，荷包牡丹碱可能是一种合适的惊厥药[4]。

电生理记录[3]
注射后2-8天，用双电极电压钳技术测量受体活性。记录微电极用微量移液器制造，并填充3 M KCl溶液。将卵母细胞置于细胞浴中，电压钳制在-60 mV。细胞持续用ND96缓冲液超灌。在Macintosh Quadra 605计算机上，使用Geneclamp 500放大器、Mac Lab 2e记录器和Chart 3.5.2版程序记录了药物应用引起的电流。测试了药物对GABAA受体GABA的直接激活。为了测量药物对受体激活的抑制作用，将药物加入含有GABA的缓冲溶液中，其浓度在受体处产生10%、50%、75%、90%和100%的效果（GABA EC10、EC50、EC75、EC90和EC100），以构建GABA抑制剂量-反应曲线。采用相同的程序，但拮抗剂浓度固定，GABA浓度增加，构建GABA剂量-反应曲线。每次用药之间允许3-5分钟的洗脱期，以防止受体脱敏。

Expression of α1β2γ2L GABAA receptors in Xenopus laevis oocytes [3]
Female X. laevis were anaesthetised with 0.17% ethyl 3-aminobenzoate in saline and a lobe of the ovaries surgically removed. The lobe of ovaries was rinsed with OR-2 buffer that contained 82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2·6H2O, 5 mM HEPES, pH 7.4, and suspended in a solution of collagenase A (2 mg/ml in OR-2) for 2 h to separate oocytes from connective tissues and follicular cells. Released oocytes were then thoroughly rinsed in ND96 buffer supplemented with 2.5 mM sodium pyruvate, 0.5 mM theophylline and 50 μg/ml gentamycin, and stage V to VI oocytes were collected. Human α1, β2 and γ2L cDNAs subcloned in pcDM8 were linearised using the restriction enzyme NOT1. Linearised plasmids containing α1, β2 and γ2L cDNAs were transcribed using T7 RNA Polymerase and capped with 5,7-methylguanosine using the “mMESSAGE mMACHINE” kit. Ten nanograms per 50 nl of a 1:1:1 mixture of α1, β2 and γ2L cRNAs were injected using a 15–20 μm diameter tip micropipette into the cytoplasm of individual defolliculated oocytes by using a Nanoject injector. The oocytes were incubated in ND96 buffer at 16 °C in an orbital shaker with a twice-daily change of buffer.

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Pharmacokinetic study [6]
Male Sprague-Dawley rats (200–220 g) were used. Diet was prohibited for 12 h before the experiment but water was freely available. Blood samples (0.3 mL) were collected from the tail vein into heparinized 1.5 mL polythene tubes at 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6 h after given BicucuLline (15 mg/kg) by manual gavage. The samples were immediately centrifuged at 3000g for 10 min. The plasma obtained (100 μL) was stored at −20 °C until analysis. Plasma BicucuLline concentration versus time for each rat was analyzed by DAS (Drug and statistics) software.

Bicuculline, a phthalide isoquinoline alkaloid is of current interest as an antagonist of gamma-aminobutyric acid (GABA). A simple and sensitive liquid chromatography mass spectrometry method for determination of bicuculline in rat plasma was developed over the range of 5-500ng/mL. After addition of midazolam as internal standard, protein precipitation with acetonitrile-methanol (9:1, v/v) was used as sample preparation. Chromatographic separation was achieved on a Zorbax SB-C18 (2.1mm×150mm, 5μm) column with acetonitrile -0.1% formic acid in water as mobile phase with gradient elution. Electrospray ionization (ESI) source was applied and operated in positive ion mode; selective ion monitoring (SIM) mode was used for quantification using target fragment ions m/z 368 for bicuculline and m/z 326 for the IS. Linear calibration was obtained with correlation coefficients r>0.99. The CV of the precision measurements was less than 13%. The accuracy of the method ranged from 93.6% to 100.5%. Mean recoveries of bicuculline in plasma were in the range of 80.5-91.8%. The method was successfully applied to the pharmacokinetic study after gavage administration of 15mg/kg bicuculline in rats.[6]

Toxicity Summary
The action of bicuculline is primarily on the ionotropic GABAA receptors, which are ligand-gated ion channels concerned chiefly with the passing of chloride ions across the cell membrane, thus promoting an inhibitory influence on the target neuron. These receptors are the major targets for benzodiazepines and related anxiolytic drugs. The half-maximal inhibitory concentration (IC50) of bicuculline on GABAA receptors is 3 μM. In addition to being a potent GABAA receptor antagonist, bicuculline can be used to block Ca2+-activated potassium channels. Sensitivity to bicuculline is defined by IUPHAR as a major criterion in the definition of GABAA receptors.

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mouse LD50 intraperitoneal 8480 ug/kg Current Toxicology., 1(199), 1993

[1]. Johnston GA. Advantages of an antagonist: bicuculline and other GABA antagonists. Br J Pharmacol. 2013;169(2):328-336.

[2]. Bicuculline block of small-conductance calcium-activated potassium channels. Pflugers Arch. 1999;438(3):314-321.

[3]. Bilobalide, a sesquiterpene trilactone from Ginkgo biloba, is an antagonist at recombinant alpha1beta2gamma2L GABA(A) receptors. Eur J Pharmacol. 2003;464(1):1-8.

[4]. Bicuculline induced seizures in infant rats: ontogeny of behavioral and electrocortical phenomena. Brain Res Dev Brain Res. 1990 Dec 15;57(2):291-5.

[5]. Induction of immediate early gene encoded proteins in the rat hippocampus after bicuculline-induced seizures: differential expression of KROX-24, FOS and JUN proteins. Neuroscience. 1992;48(2):315-24.

[6]. Determination of bicuculline in rat plasma by liquid chromatography mass spectrometry and its application in a pharmacokinetic study. J Chromatogr B Analyt Technol Biomed Life Sci. 2014 Mar 15:953-954:143-6.

Bicuculline is a benzylisoquinoline alkaloid that is 6-methyl-5,6,7,8-tetrahydro[1,3]dioxolo[4,5-g]isoquinoline which is substituted at the 5-pro-S position by a (6R)-8-oxo-6,8-dihydrofuro[3,4-e][1,3]benzodioxol-6-yl group. A light-sensitive competitive antagonist of GABAA receptors. It was originally identified in 1932 in plant alkaloid extracts and has been isolated from Dicentra cucullaria, Adlumia fungosa, Fumariaceae, and several Corydalis species. It has a role as an agrochemical, a central nervous system stimulant, a GABA-gated chloride channel antagonist, a neurotoxin and a GABAA receptor antagonist. It is an isoquinoline alkaloid, a member of isoquinolines and a benzylisoquinoline alkaloid.
Bicuculline is a light-sensitive competitive antagonist of GABAA receptors. It was originally identified in 1932 in plant alkaloid extracts and has been isolated from Dicentra cucullaria, Adlumia fungosa, Fumariaceae, and several Corydalis species.
Bicuculline has been reported in Corydalis repens, Corydalis decumbens, and other organisms with data available.
Bicuculline is a light-sensitive competitive antagonist of GABAA receptors. It was originally identified in 1932 in plant alkaloid extracts and has been isolated from Dicentra cucullaria, Adlumia fungosa, Fumariaceae, and several Corydalis species. Since it blocks the inhibitory action of GABA receptors, the action of bicuculline mimics epilepsy. This property is utilized in laboratories across the world in the in vitro study of epilepsy, generally in hippocampal or cortical neurons in prepared brain slices from rodents. This compound is also routinely used to isolate glutamatergic (excitatory amino acid) receptor function.
An isoquinoline alkaloid obtained from Dicentra cucullaria and other plants. It is a competitive antagonist for GABA-A receptors.

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Immunocytochemistry with specific antisera was used to assess regional levels of six immediate early gene encoded proteins (KROX-24, c-FOS, FOS B, c-JUN, JUN B and JUN D) in the rat hippocampus after 15 min of bicuculline-induced seizures. Serial sections of the dorsal hippocampus were examined at various postictal recovery periods up to 24 h. The results demonstrate a complex temporal and spatial pattern of immediate early gene synthesis and accumulation. Three major categories of immediate early gene products could best be distinguished in the dentate gyrus: KROX-24 and c-FOS showed a concurrent rapid rise with peak levels at 2 h and a return to baseline levels within 8 h after seizure termination. FOS B, c-JUN and JUN B levels increased more gradually with peak intensities in the dentate gyrus reached at 4 h. These immediate early gene products showed above normal levels in various hippocampal subpopulations up to 24 h. JUN D exhibited the most delayed onset combined with a prolonged increase of seizure-induced immunoreactivity. Irrespective of this differential temporal expression profile of individual transcription factors, the sequence of induction in the hippocampal subpopulations was identical for all immediate early gene-encoded proteins examined: first in the dentate gyrus granule cells followed by CA1 and CA3 neurons, respectively. Our data indicate an asynchronous synthesis of several immediate early gene-encoded proteins in the brain after status epilepticus. FOS and JUN proteins act via homo- or heterodimer complexes at the AP-1 and other DNA binding sites. The different time-courses for individual immediate early gene products strongly suggest, that at different time-points after status epilepticus, different AP-1 complexes are effective. In vitro studies have shown that different AP-1 complexes possess different DNA binding affinities as well as different transcriptional regulatory effects. Our results suggest that these molecular mechanisms are also effective in vivo. [5]

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Bicuculline is a competitive antagonist of GABAA receptors (Akaike et al., 1985). The competitive antagonism of bicuculline and noncompetitive antagonism of picrotoxinin at GABAA receptors are also exemplified at the human α1β2γ2L subunit combination. At α1β2γ2L GABAA receptors, bicuculline displayed the general property of the competitive antagonist, producing a parallel shift of GABA concentration–effect curves and having no effect on the maximal response of GABA. [3]

C20H17NO6

367.35

367.105

C, 65.39; H, 4.66; N, 3.81; O, 26.13

485-49-4

38641-83-7；66016-70-4

10237

Off-white to yellow solid powder

1.5±0.1 g/cm3

542.3±50.0 °C at 760 mmHg

196-198 ºC

281.8±30.1 °C

0.0±1.4 mmHg at 25°C

1.665

2.88

66.46

0

7

1

27

615

2

CN1CCC2=CC3=C(C=C2[C@H]1[C@H]4C5=C(C6=C(C=C5)OCO6)C(=O)O4)OCO3

IYGYMKDQCDOMRE-ZWKOTPCHSA-N

InChI=1S/C20H17NO6/c1-21-5-4-10-6-14-15(25-8-24-14)7-12(10)17(21)18-11-2-3-13-19(26-9-23-13)16(11)20(22)27-18/h2-3,6-7,17-18H,4-5,8-9H2,1H3/t17-,18+/m0/s1

(R)-6-((S)-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-[1,3]dioxolo[4,5-e]isobenzofuran-8(6H)-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 本产品在运输和储存过程中需避光。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.81 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (6.81 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (6.81 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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