GABA受体Poly(A)~+mRNA在卵母细胞体内的翻译及其移植受体的生化药理特性

采用SDS/氯仿/苯酚法和oligo(dT)纤维素亲和层析法从胚鸡脑中提取的poly(A)~+mRNA注射到非州爪蟾卵母细胞体内可以成功地被翻译为具有结合功能的GABA受体,并首次用生化方法,包括[~sH]GABA配体结合试验及药物竞争性结合试验证明了卵母细胞膜上移植GABA受体的存在。蛋白质翻译抑制剂三尖杉酯碱可以完全阻断GABA受体的翻译过程。DNA转录抑制剂放线菌素D对这一过程无影响。移植GABA受体具有受体与配体结合的饱和特性,移植GABA受体与[~sH]GABA的结合可以被调变蛋白、蝇蕈醇、双可可碱及异丙基双环磷酸酯所抑制。移植GABA受体与天然鸡脑GABA受体的生化药理学性质基本一致。     

八肽胆囊收缩素对[~3H]埃托啡与大鼠脑阿片受体结合的抑制作用

本实验观察了CCK—8对[~3H]Et与大鼠脑阿片受体结合的影响.含硫CCK—8能抑制[~3H]Et与高亲和位点的结合,使K_D值增大(P<0.001),B_(max)减小(P<0.01),在10 fmol/L～Lμmol/L范围内呈量效关系.但对低亲和位点的结合没有影响.无硫CCK—8仅对[~3H]Et高亲和位点的K_D值有较小程度的增大(P<0.05),不影响B_(max)值.CCK—8(10 nmol/L)抑制[~3H]Et与阿片受体结合的作用能被CCK受体拮抗剂谷丙酰胺(1μmol/L)所阻断.结果提示,CCK—8可能通过激活CCK受体发挥对阿片受体的抑制作用。     

大鼠心肌质膜[~3H]forskolin结合部位的特征(英文)

近年来从植物研究中发现的一种二萜衍生物(forskolin)是一种新型作用于心血管系统的药物。质膜中forskolin结合部位被认为是腺苷酸环化酶(EC4.6.1.1.)的催化亚单位。我们已经证明了它对大鼠心肌质膜腺苷酸环化酶的激活作用呈剂量效应相关。本文用〔1,2-~3H〕forskolin研究了大鼠心肌质膜for-skolin结合部位的特征。实验结果表明:〔~3H〕forskolin结合量和膜蛋白呈线性相关;其特异结合是快速的、可饱和的、可逆的、依赖温度的;结合反应的平衡解离常数(Ka)为0.21±SD 0.08μmol/L,其最大结合浓度为3.3±SD1.2 pmol/mg蛋白,Hill系数为1.07±SD0.05;37℃时缔合速率常数为0.0013(nmol/L)~(-1)·min~(-1),30 min达到平衡状态;心肌质膜结合的〔1,2-~3H〕forskolin被forskolin取代的解离速率常数:37℃时为0.22 min~(-1)(t_(1/2)=3.2 min),0℃时为0.03 min~(-1)(t_(1/2)=25.8min),IC_(50)为1.6μmol/L。本文实验结果提示:心肌质膜〔~3H〕forskolin结合分析可以作为研究激素和药物对心肌腺苷酸环化酶催化亚基作用的良好模型。     

二氢埃托啡对大鼠脑阿片受体的结合特性

本文在大鼠脑匀浆P_2膜上,观察了二氢埃托啡(DHE)对[~3H]纳洛酮,[~3H]DPDPE和[~3H]埃托啡(预先用30nmol/L吗啡和100nmol/L DADLE阻断μ和δ受体)与阿片受体结合的抑制强度。结果表明:DHE对[~3H]纳洛酮与阿片受体结合的抑制强度远远大于对[~3H]DPDPE和[~3H]埃托啡(预先阻断μ和δ受体后)。DHE对μ,δ和κ受体的相对亲和力之比为1951:2:1,提示DHE为μ受体相对选择性配体。     

八肽胆囊收缩素对抗吗啡抑制大鼠脑突触小体钙摄取的作用

用大鼠脑的膜制备观察吗啡和CCK-8对突触小体摄取~(45)Ca~(2+)的影响。吗啡(10 nmol/L～1μmo1/L)抑制突触小体对~(45)Ca的摄取,该作用能被1μmol/L纳洛酮完全阻断。CCK-8(10nmol/L～1μmol/L)本身能抑制突触小体~(45)Ca摄取,但它在10nmol/L和100 nmol/L时能对抗吗啡对~(45)Ca摄取的抑制作用,浓度提高到1μmol则不能对抗吗啡的这一作用。CCK-8抑制突触小体摄取~(45)Ca,以及对抗吗啡的~(45)Ca摄取抑制的作用,皆能被CCK受体拮抗剂丙谷酰胺(2μmol/L)所阻断.捉示CCK-8是通过激动CCK受体而拮抗吗啡抑制~(45)Ca摄取的,CCK-8的这一拮抗作用可能是其抗阿片作用的机理之一.     

多巴胺D1受体在Sf9昆虫细胞中的表达及左旋氯代斯阔任对重组D1受体的激动作用

目的:在Sf9昆虫细胞中表达D_1受体,并研究左旋氯代斯阔任对重组D_1受体的激动作用。方法:构建含D_1受体cDNA的重组杆状病毒,以其感染Sf9昆虫细胞得到D_1受体表达。[~3H]SCH23390受体结合检测重组D_1受体的药理特性。[~3H]SCH23390受体结合和cAMP测定实验检测左旋氯代斯阔任对重组D_1受体的激动作用。结果:在Sf9昆虫细胞中成功表达D_1受体,[~3H]SCH23390与重组D_1受体最大结合量(B_(max))为(0.94±0.06)nmol/g蛋白,[~3H]SCH23390与重组D_1受体的结合解离常数(K_d)为(1.9±0.3)nmol/L,其药理特性与小牛纹状体脑匀浆所得结果一致。左旋氯代斯阔任对重组D_1受体有高亲和力,解离常数K_i为(6.3±1.4)nmol/L;并剂量依赖地引起胞内cAMP增加,EC_(50)为0.72μmol/L(95％可信限为0.67-0.77μmol/L),表现出D_1激动作用。结论:在杆状病毒/昆虫细胞Sf9中,成功建立了D_1受体异源表达系统。在细胞分子水平,直接证实了左旋氯代斯阔任对D_1受体的激动作用。     

兔血小板膜上血小板活化因子受体的鉴定

本文用放射配基受体结合实验方法研究了兔血小板膜上血小板活化因子（PAF）受体的性质。[^3H]-PAF与膜的特异结合率达70－80％，在25℃测得PAF与膜结合的平衡解离常数（Kd）为40.8nmol/L，最大结合位点为5.4pmol/mg蛋白。特异的PAF受体拮抗剂海风藤酮对结合有较强的抑制作用，IC50为30.0μmol/L。（－）－denudatin B，樟叶素(polysyphorin）和南藤素（wallichinine）是从中草药中分离的化合物，均能抑制这一结合，其IC50分别为0.6μmol/L、10μmol/L和2.5μmol/L。     

可乐定对苯二氮受体激动剂和反相激动剂与大鼠皮层受体结合特性的影响(英文)

本研究旨在探讨α_2受体激动剂可乐定对苯二氮(艹卓)受体激动剂抗焦虑和反相激动剂致焦虑作用影响的可能的分子机理。在10nmol/L至1μmol/L浓度范围内,可乐定对[~3H]氟硝安定与大鼠皮层相应受体低亲和位点结合无显著影响,但非竞争性拮抗其与高亲和位点的结合。在竞争取代实验中,激动剂安定和CL218 872均表现为双位点结合的亲和力,对低亲和位点无显著影响。反相激动剂DMCM竞争结合曲线亦具有双位点结合特性。可乐定可使这种双位点结合转变成三位点结合,出现一个超高亲和位点。可乐定对拮抗剂Ro15-1788竞争结合特性无显著影响。结果提示,α_2受体激动剂可乐定与受体结合可能导致与之相邻的苯二氮(艹卓)受体发生构象变化,这种构象变化有利于激动剂与受体结合,而不利于反相激动剂的结合。     

H型高血压患者单核趋化蛋白-1、白细胞介素-8表达的研究

目的:观察H型高血压患者血清同型半胱氨酸(homocysteine,Hcy)、单核趋化蛋白-1(MCP-1)及白细胞介素-8(IL-8)浓度的变化,并分析Hcy与MCP-1、IL-8之间的相关性。方法:选择佳木斯大学附属第一医院门诊及住院H型高血压患者30例(1组),非H型高血压患者30例(2组),与体检健康者作为正常对照组30例(3组),对3组人群均行Hcy、MCP-1、IL-8水平的测定,并分析H型高血压患者Hcy与MCP-1、IL-8之间的相关性。结果:H型高血压组Hcy、MCP-1、IL-8水平均显著高于非H型高血压组,分别为〔(16.89±2.14)μmol/L vs(8.30±1.43)μmol/L〕(P<0.01)、〔(20.26±3.07)pg/mLvs(13.04±2.30)pg/mL〕(P<0.01)、〔(92.72±9.58)pg/mL vs(77.53±9.26)pg/mL〕,二组均明显高于对照组〔(7.33±1.93)μmol/L〕(分别为P<0.01;P<0.05)、〔(10.88±3.77)pg/mL〕(P<0.01)、〔(66.89±10.23)pg/mL〕(P<0.01),相关分析表明Hcy与MCP-1、IL-8分别呈正相关,相关系数分别为r=0.740,r=0.668(P<0.01)。结论:MCP-1、IL-8在H型高血压中表达明显升高,且Hcy与MCP-1、IL-8分别呈正相关,它们可能参与动脉粥样硬化的发生发展。     

毛地黄黄酮和玄参黄酮抑制[~3H]地西泮和体外大鼠脑膜的结合(英文)

从阿拉伯艾蒿提取到两种苯并二氮杂受体的配基,毛地黄黄酮和玄参黄酮。两种化合物在体外可抑制[~3H]地西泮和大鼠皮层细胞膜的结合,IC_(50)值分别为1.3μmol·L~(-1)和23μmol·L~(-1)。两种化合物GABA比分别为1.1和1.2,都可少量增加[~(35)S]TBPS的结合,提示这种化合物是苯并二氮杂受体的拮抗剂或部分激动剂。     

黄芩苷对γ-氨基丁酸受体和抗焦虑症作用研究

目的观察黄芩苷与γ-氨基丁酸（GABA）受体的关系及其所具有的抗焦虑作用。方法采用同位素标记GABA受体结合实验观察黄芩苷与GABA受体的关系;采用高台十字迷宫实验和四孔箱实验观察动物服用黄芩苷后行为的改变。结果 GABA受体竞争结合试验显示,黄芩苷可竞争性抑制50%GABA受体苯二氮类结合浓度（IC50）为（117.62±8.50）μmol·L-1,抑制结合常数（Ki）为（77.10±4.79）μmol·L-1。黄芩苷可增加实验动物进入十字迷宫开放侧次数,百分率和滞留时间,减少在四孔箱中探头和竖身的次数。这些变化与黄芩苷剂量有明显的关系,可被GABA受体阻断剂Ro 15-1788阻断。结论黄芩苷是GABA结合位点的配体,具有一定的抗焦虑作用。     

Pregnenolone sulfate and dehydroepiandrosterone sulfate inhibit GABA-gated chloride currents in Xenopus oocytes expressing picrotoxin-insensitive GABA(A) receptors

We examined the effects of picrotoxinin, pregnenolone sulfate (PS) and dehydroepiandrosterone sulfate (DHEAS) on gamma-aminobutyric acid (GABA) responses in Xenopus oocytes injected with wild type alpha1, beta2 and gamma2 GABA(A) receptor subunits and in oocytes injected with wild type alpha1 and beta2 subunits and a mutated gamma2 subunit that eliminates picrotoxin sensitivity. All three agents inhibited GABA currents in oocytes injected with wild type subunits. Oocytes injected with the mutated gamma2 subunit showed no inhibition of GABA responses by picrotoxinin at concentrations up to 100 microM. PS and DHEAS inhibited GABA currents at similar concentrations in both sets of oocytes. These results indicate that PS and DHEAS do not require a functional picrotoxin site for inhibition of GABA responses.     

Modulation Of [35S]-tert-butylbicyclophosphorothionate binding by somatostatin in rat hypothalamus

1. The present study was designed to assess the effect of the tetradecapeptide somatostatin on the GABA(A) receptor complex in the rat hypothalamus. 2. GABA(A) receptors were labelled with [35S]-tert-butylbicyclophosphorothionate (TBPS), which binds in or near the chloride channel, and binding as assessed by in vitro quantitative autoradiography using a computer-assisted image analysis system. 3. Somatostatin inhibited the binding of [35S]-TBPS to the convulsant site of the hypothalamic GABA(A) receptor complex of rat slide-mounted hypothalamic structures in a concentration-dependent manner with an affinity in the micromolar range (10(-6) to 3 x 10(-6) mol/L). Somatostatin appeared to mimic the effects of the neurosteroid 5alpha-pregnane-3alpha ol-one (5alpha3alphaP), GABA and picrotoxin on [35S]-TBPS binding in the rat hypothalamus in all structures examined. Furthermore, GABA or muscimol (a GABA(A) receptor agonist), when added to the incubation medium, enhanced the capacity of somatostatin to inhibit [35S]-TBPS binding, with an IC50 of 10(-7) mol/L. However, incubation with bicuculline (a GABA(A) receptor antagonist) led to the abolition of the inhibitory effect of somatostatin on [35S]-TBPS specific binding in rat hypothalamus. 4. The present results demonstrate the presence of a modulatory effect of somatostatin on the GABA(A) receptor complex in rat hypothalamic structures. Furthermore, the data suggest that somatostatin allosterically modifies [35S]-TBPS binding through a mechanism similar to that of GABA. Taken together, these results provide evidence for the presence of somatostatin- GABA interactions in rat hypothalamus.     

3H-muscimol binding sites within guinea pig ovary: a histoautoradiographic study

The distribution of 3H-muscimol within guinea pig ovary was studied using combined radioreceptor binding studies and histoautoradiographic technique performed on frozen sections of ovary mounted on microscope slides. 3H-muscimol was bound by sections of ovary in a manner consistent with the existence of specific GABA receptors. The binding was reversible, saturable, of high affinity (KD was 38 +/- 6 nmol/l and Bmax was about 378 +/- 61 fmol/mg protein, and inhibited by GABA receptor interfering drugs. 3H-muscimol binding sites were found in the blood vessel wall, in the follicle and in the oocytes. The number of oocyte and follicular receptors gradually decreases during the development of ovarian follicles.     

Mechanisms underlying the renoprotective effect of GABA against ischaemia/reperfusion‐induced renal injury in rats

Excitation of the renal sympathetic nervous system is important for the development of ischaemic acute kidney injury (AKI) in rats. We reported that intravenous treatment with GABA has preventive effects against ischaemia/reperfusion (I/R)‐induced renal dysfunction with histological damage in rats; however, the mechanisms underlying these effects on renal injury remain unknown. Thus, the aim of the present study was to clarify how GABA mechanistically affects ischaemic AKI in rats. Ischaemic AKI was induced in rats by clamping the left renal artery and vein for 45 min and then reperfusing the kidney to produce I/R‐induced injury. Treatment with the GABA_B receptor antagonist CGP52432 (100 nmol/kg, i.v., or 1 nmol/kg, i.c.v.) abolished the suppressive effects of 50 μmol/kg, i.v., GABA on enhanced renal sympathetic nerve activity (RSNA) during ischaemia, leading to elimination of the renoprotective effects of GABA. Intracerebroventricular treatment with 0.5 μmol/kg GABA or i.v. treatment with 1 μmol/kg baclofen, a selective GABA_B receptor agonist, prevented the I/R‐induced renal injury equivalent to i.v. treatment with GABA. Conversely, i.v. treatment with 10 μmol/kg bicuculline, a GABA_A receptor antagonist, failed to affect the preventive effects of GABA against ischaemic AKI. We therefore concluded that GABA_B receptor stimulation in the central nervous system, rather than peripheral GABA_B receptor stimulation, mediates the preventive effect of GABA against ischaemic AKI by suppressing the enhanced RSNA induced by renal ischaemia.     

Solubilization and characterization of benzodiazepine receptors in frontal cortex of rabbit brain

[3H]Flunitrazepam binding to benzodiazepine receptors solubilized by the detergent 3-[(3-cholamidopropyl)-dimethyl-ammonio]-l-propanesulfonate (CHAPS) was saturable and showed non-linear Scatchard plot with KD1 0.31 nmol/L and KD2 6.7 nmol/L. The affinities of soluble receptors to benzodiazepine were consistent with P2 membrane. One radioactive zone was found by SDS-PAGE after photoaffinity labelling of soluble membrane and the apparent molecular weight of 55,000 was reported. [3H]Flunitrazepam binding to soluble receptors was enhanced by GABA, NaCl or KCl and barbiturates, but inhibited by bicuculline and picrotoxinin. The enhancement of GABA on [3H]flunitrazepam binding was amplified by NaCl or KCl and antagonized by bicuculline and picrotoxinin. These results suggest that the benzodiazepine receptors solubilized by CHAPS have their pharmacological properties and are still associated with GABA receptors and chloride channel.     

CR1409拮抗缩胆囊素升高细胞内Ca~(2+)的作用

CR1409是一种新合成的缩胆囊素(CCK)受体拮抗剂。本文使用荧光指示剂Fura-2作为细胞内钙离子的探测物,在离体的大白鼠胰腺细胞研究OR1409对CCK引起细胞内钙离子浓度升高的拮抗作用。结果表明:10μmol/L CR1409完全抑制1nmol/L CCK的作用,半效抑制浓度为0.37μmol/L,比另一种拮抗剂双丁酰环鸟嘌呤核苷(db cGMP)强100倍。随着CR1409浓度的递增,使CCK升高细胞内钙离子浓度的量效曲线右移,根据Schild方法计算,pA_2值为6.93(r=0.992)。CR1409对氯氨甲酰胆碱和蛙皮素无拮抗作用。实验结果证明CR1409是一种效力较强的、特异性的和竞争性的CCK受体拮抗剂。     

Mechanisms of U46619‐induced contraction in mouse intrarenal artery

Thromboxane A₂ (TXA₂) has been implicated in the pathogenesis of vascular complications, but the underlying mechanism remains unclear. The contraction of renal arterial rings in mice was measured by a Multi Myograph System. The intracellular calcium concentration ([Ca²⁺]_i) in vascular smooth muscle cells (VSMCs) was obtained by using a fluo‐4/AM dye and a confocal laser scanning microscopy. The results show that the U46619‐induced vasoconstriction of renal artery was completely blocked by a TXA₂ receptor antagonist GR32191, significantly inhibited by a selective phospholipase C (PI‐PLC) inhibitor U73122 at 10 μmol/L and partially inhibited by a Phosphatidylcholine ‐ specific phospholipase C (PC‐PLC) inhibitor D609 at 50 μmol/L. Moreover, the U46619‐induced vasoconstriction was inhibited by a general protein kinase C (PKC) inhibitor chelerythrine at 10 μmol/L, and a selective PKCδ inhibitor rottlerin at 10 μmol/L. In addition, the PKC‐induced vasoconstriction was partially inhibited by a Rho‐kinase inhibitor Y‐27632 at 10 μmol/L and was further completely inhibited together with a putative IP₃ receptor antagonist and store‐operated Ca²⁺ (SOC) entry inhibitor 2‐APB at 100 μmol/L. On the other hand, U46619‐induced vasoconstriction was partially inhibited by L‐type calcium channel (Cav1.2) inhibitor nifedipine at 1 μmol/L and 2‐APB at 50 and 100 μmol/L. Last, U46619‐induced vasoconstriction was partially inhibited by a cell membrane Ca²⁺ activated C1^? channel blocker 5‐Nitro‐2‐(3‐phenylpropylamino) benzoic acid (NPPB) at 50 and 100 μmol/L. Our results suggest that the U46619‐induced contraction of mouse intrarenal arteries is mediated by Cav1.2 and SOC channel, through the activation of thromboxane‐prostanoid receptors and its downstream signaling pathway.