人外周型苯二氮卓受体及其突变受体cDNA克隆和序列分析

用ＲＴ－ＰＣＲ方法扩增并克隆了三种人外周型苯二氮卓受体ＰＢＲｃＤＮＡ,测序表明,４４２ｂｐ片段与文献报道相比缺失８４ｂｐ编码序列,其转录水平高于正常ＰＢＲ．该序列编码一个与ＰＢＲ结构相关但缺失了２８个氨基酸残基的突变受体蛋白．这一异常转录本可能是通过选择性剪接方式转录产生并只存在于中国人肝癌ＢＥＬ７４０２细胞系,表明ＰＢＲ基因表达具有细胞特异性和异质性．突变受体的发现为研究ＰＢＲ的结构和功能提供了理想的分子和细胞模型     

人外周型苯二氮卓受体及其突变受体cDNA克隆和序列分析

用ＲＴ－ＰＣＲ方法扩增并克隆了三种人外周型苯二氮卓受体ＰＢＲｃＤＮＡ,测序表明,４４２ｂｐ片段与献报道相比缺失８４ｂｐ编码序列,其转录水平高于正常ＰＢＲ,该序列编码一个与ＰＢＲ结构相关但缺失了２８个氨基酸残基的突变受体蛋白,这一异常转录本可能是通过选择性剪接方式转录产生并只存在于中国人肝癌ＢＥＬ７４０２细胞系,表明ＰＢＲ基因表达具有细胞特异和异质性,突变受体的发现为研究ＰＢＲ的结构和功能提供了理     

瞬时受体电位香草酸亚型1激活通过抑制线粒体通透性转换孔开放保护急性心肌梗死小鼠的心肌细胞抗凋亡

瞬时受体电位香草酸亚型1（TRPV1）在心肌缺血激活后可传导心绞痛信号,释放神经肽,减轻心肌梗死后的心肌细胞凋亡。目前,TRPV1激活抑制心肌梗死后细胞凋亡的具体机制尚不清楚。线粒体通透性转换孔（MPTP）的开放与心肌细胞缺血再灌注损伤密切相关,抑制其开放可保护心肌缺血后的心肌细胞抗凋亡。本研究证明,TRPV1激活通过抑制MPTP开放而减少心肌细胞凋亡。首先,本研究利用左冠状动脉前降支结扎术建立了TRPV1基因敲除（TRPV1-/-）和野生型（WT）小鼠心肌梗死模型,辅以环孢素A（CSA）预处理抑制 MPTP开放,比较观察TRPV1、MPTP在心肌梗死中的作用。心肌组织切片氯化三苯基四氮唑（TTC）染色显示,心肌缺血24 h,TRPV1-/-小鼠的心肌梗死面积明显大于WT型小鼠。而经CSA预处理的TRPV1-/-小鼠比TRPV1-/-小鼠梗死面积明显减小。TUNEL检测心肌细胞凋亡指数（AI）揭示,WT型心肌梗死小鼠的AI明显低于TRPV1-/- 心肌梗死小鼠,而CSA预处理明显降低TRPV1-/-小鼠心肌细胞的AI。Western印迹检测胱天蛋白酶3、胱天蛋白酶9、Bcl-2、Bax、p53和细胞色素C（Cyt-C）水平。结果证明,TRPV1的激活可抑制MPTP的开放,减少线粒体Cyt-C的外溢,降低胱天蛋白酶9和胱天蛋白酶3的表达。GENMEN光度法检测MPTP开放实验显示,激活的TRPV1明显抑制了MPTP的开放。本研究证实,急性心肌梗死后的TRPV1激活可能通过抑制MPTP开放而抵抗心肌细胞凋亡,对心肌起保护作用。     

瞬时受体电位香草酸亚型1激活通过抑制线粒体通透性转换孔开放保护急性心肌梗死小鼠的心肌细胞抗凋亡北大核心CSCD

瞬时受体电位香草酸亚型1(TRPV1)在心肌缺血激活后可传导心绞痛信号,释放神经肽,减轻心肌梗死后的心肌细胞凋亡。目前,TRPV1激活抑制心肌梗死后细胞凋亡的具体机制尚不清楚。线粒体通透性转换孔(MPTP)的开放与心肌细胞缺血再灌注损伤密切相关,抑制其开放可保护心肌缺血后的心肌细胞抗凋亡。本研究证明,TRPV1激活通过抑制MPTP开放而减少心肌细胞凋亡。首先,本研究利用左冠状动脉前降支结扎术建立了TRPV1基因敲除(TRPV1-/-)和野生型(WT)小鼠心肌梗死模型,辅以环孢素A(CSA)预处理抑制MPTP开放,比较观察TRPV1、MPTP在心肌梗死中的作用。心肌组织切片氯化三苯基四氮唑(TTC)染色显示,心肌缺血24 h,TRPV1-/-小鼠的心肌梗死面积明显大于WT型小鼠。而经CSA预处理的TRPV1-/-小鼠比TRPV1-/-小鼠梗死面积明显减小。TUNEL检测心肌细胞凋亡指数(AI)揭示,WT型心肌梗死小鼠的AI明显低于TRPV1-/-心肌梗死小鼠,而CSA预处理明显降低TRPV1-/-小鼠心肌细胞的AI。Western印迹检测胱天蛋白酶3、胱天蛋白酶9、Bcl-2、Bax、p53和细胞色素C(Cyt-C)水平。结果证明,TRPV1的激活可抑制MPTP的开放,减少线粒体Cyt-C的外溢,降低胱天蛋白酶9和胱天蛋白酶3的表达。GENMEN光度法检测MPTP开放实验显示,激活的TRPV1明显抑制了MPTP的开放。本研究证实,急性心肌梗死后的TRPV1激活可能通过抑制MPTP开放而抵抗心肌细胞凋亡,对心肌起保护作用。     

中枢去甲肾上腺素和内阿片肽系统对苯二氮受体反相激动剂DMCM致焦虑作用的影响

在大鼠条件反射性惊恐反应致焦虑模型上,中枢苯二氮(艹卓)(BZD)受体激动剂安定,可使冲突惊恐反应期(PP)的操作数目显著增加,BZD受体反相激动剂DMCM则显著减少PP的操作数目;DMCM的这一作用,可分别为中枢BZD受体拮抗剂Ro 15-1788,GABA A型受体激动剂蝇蕈醇、阿片受体拮抗剂环丙甲羟二氢吗啡酮和中枢去甲肾上腺素能(NA)突触前α_2受体激动剂可乐定所阻断。结果表明,中枢阿片肽和NA系统共同参与BZD受体反相激动剂DMCM的致焦虑作用。     

安定对兔心乳头肌动作电位和收缩的作用

本文观察了安定、戊脉安和PK11195对兔心乳头肌动作电位和收缩的作用,并比较了安定对兔与豚鼠心肌收缩作用的差异。安定(0.7×10~(-6)-22.4×10~(-6)mol/L)使兔乙乳头肌动作电位时程(APD)及钙依赖动作电位(Ca-AP)时程缩短,并使Ca-AP的最大上升速率(V_(max))和幅值减小;安定还抑制兔与豚鼠心室乳头肌的收缩,相比之下对豚鼠的作用更明显。在戊脉安(2×10~(-6)mol/L)作用下,兔心乳头肌Ga-AP逐渐消失。提高溶液中Ca~(2 )的浓度可拮抗安定和戊脉安的作用。PK11195(1×10~(-6)mol/L)可使安定对兔心乳头肌APD作用的剂量-反应曲线平行右移,且拮抗安定对Ca-AP的作用。预先加入PK11195(3×10~(-6)mol/L),则戊脉安不能取消Ca-AP。上述结果提示。(1)兔心乳头肌存在的苯二氮(艹卓)结合位点具有外周型苯二氮(艹卓)受体(BZR)的性质;(2)这种受体的激活可导致钙内流减少;(3)BZR的密度及反应性可能存在种属差异。     

对(艹孟)烷-3,8-二醇的合成与应用研究进展

对(艹孟)烷-3,8-二醇是一种具有生物活性的化合物,主要来源于柠檬桉精油,可直接用于香水、杀虫剂和药品等.通常采用化学和生物化学的方法合成对(艹孟)烷-3,8-二醇,以替代从柠檬桉叶油分离得到的天然产品.以香茅醛为原料通过Prins环缩合和水解,或者通过Solanum aviculare悬浮培养方式进行生物转化得到对(艹孟)烷-3,8-二醇;通过薄荷醇的氧化,或者以2-丁烯醛为原料与甲基锂、NaClO2等试剂经过较长步骤也可合成对(艹孟)烷-3,8-二醇.对(艹孟)烷-3,8-二醇可应用于制造驱虫剂、凉味剂、药物或者化妆品的添加剂以及海水防护涂料添加剂等.     

苯二氮(艹卓)受体的内源性配体——次黄嘌呤核苷和乙烯3-羧基β-咔啉

次黄嘌呤核苷和乙烯3-羧基β-咔啉(B-CCE)是哺乳动物和人脑内存在的化合物,体外实验均表现竞争性抑制~3H-安定同苯二氮(艹卓)(BDZ)受体结合的特性,而对其它已知的神经递质受体无影响。次黄嘌呤核苷具有BDZ的药理作用,在腹膜注射戊四唑前一分钟,将次黄嘌呤核苷注入脑室,可使成四唑引起惊厥的潜伏期延长三倍。β-CCE可拮抗BDZ的部分药理作用,降低荷包牡丹碱诱发鼠痉挛的阈值,抵消安定降低小脑cGMP的作用。     

3,6-(二甲氨基)-二苯并碘杂六环葡萄糖酸盐对损伤心肌细胞的保护作用

目的：观察3,6-（二甲氨基）-二苯并碘杂六环葡萄糖酸盐（IHC-93）对培养心肌细胞损伤的影响。方法：在培养的心肌细胞建立低氧／复氧损伤模型和过氧化氢损伤模型,观察心肌细胞存活率、乳酸脱氢酶（LDH活性、超氧化物歧化酶（SOD）活性、丙二醛（MDA含量,以研究IHC-93对培养心肌细胞损伤的影响。结果：10、20、40μmol／LIHC-93呈剂量依赖地提高损伤心肌细胞存活率和SOD的活性,减少LDH的释放,降低MDA含量。结论：IHC-93对损伤心肌细胞具有直接保护作用。     

Implication of glucocorticoid in anti-inflammatory effects of Ro5-4864 in mouse pleurisy induced by carrageenan

Mouse pleurisy induced by carrageenan is used to determine the mechanism of anti-inflammatory action of 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2-H-1,4-benzodiazepin-2 (Ro5-4864). Pre-treatment with Ro5-4864 inhibits different inflammatory parameters, such as neutrophil influx, MPO activity and NO levels in the early phase (4 h), as well as mononuclear cells and ADA activity in the late phase (48 h) of pleurisy. dl-Aminoglutethimide, inhibitor of steroidal synthesis, reverted the effect of Ro5-4864 on these different inflammatory parameters. Our results suggest that anti-inflammatory action of Ro5-4864 may be partly due to its capacity to inhibit leukocyte migration, as well as leukocyte activation and formation of NO by a mechanism dependent on glucocorticoids.     

Characterization of the Binding of [3H]Ro 5-4864, a Convulsant Benzodiazepine, to Guinea Pig Brain

The density of high affinity binding sites for [3H]4'-chlorodiazepam [( 3H]Ro 5-4864) in guinea pig cerebral cortex is significantly higher (3.8-fold) than the density reported in the rat, and is nearly equal to the density of binding sites for other [3H]benzodiazepines (e.g., diazepam, flunitrazepam). The density of these [3H]Ro 5-4864 binding sites was generally higher in guinea pig brain than in rat brain, with the exception of olfactory bulb. Both the subcellular distribution and pharmacologic profile of these sites in guinea pig brain appears qualitatively similar to observations previously reported in the rat. The high density of binding sites for [3H]Ro 5-4864, coupled with the potency of this compound as a convulsant in the guinea pig, suggest this species will be a valuable model for elucidating putative pharmacologic and physiologic functions of these sites in brain.     

Characterization of [3H]Ro 5-4864 Binding Sites in Rat Vas Deferens

The presence of benzodiazepine binding sites in rat vas deferens was detected using [3H]Ro 5-4864 as a radioligand. The binding of [3H]Ro 5-4864 to the mitochondrial sites is saturable, reversible, and temperature and time dependent. The association rate constant (k1) was 8.7 +/- 0.7 x 10(7) M-1 min-1, and the dissociation rate constant (k-1) was 0.031 +/- 0.003 min-1. The dissociation constant (KD) determined by saturation binding was 5.22 +/- 0.56 nM. The density of binding was 4,926 +/- 565 fmol/mg of protein. The Hill coefficient of binding was 0.99 +/- 0.01, an indication that [3H]Ro 5-4864 binds to a single site. The [3H]Ro 5-4864 binding was inhibited competitively by Ro 5-4864 and 2-hydroxy-5-nitrobenzyl-6-thioguanosine and noncompetitively by PK 11195, nitrendipine, alpha,beta-methylene-ATP, and carboxyatractyloside and was not affected by clonazepam, dicyclohexylcarbodiimide, or protoporphyrin IX. Our data indicate that [3H]Ro 5-4864 binding sites are not identical to those labeled by PK 11195. These binding sites are modulated by the ADP/ATP mitochondrial carrier, and an interaction of dihydropyridines and [3H]Ro 5-4864 binding sites in rat vas deferens is suggested.     

"Peripheral-Type"Binding Sites for Benzodiazepines in Brain: Relationship to the Convulsant Actions of Ro 5–4864

Previous studies have shown that Ro 5-4864 is a potent convulsant and increases the firing rate of substantia nigra zona reticulata neurons. The pharmacologic profile of compounds that antagonize these actions suggested that the effects of Ro 5-4864 were not mediated by "brain-type" benzodiazepine receptors. We examined a number of compounds that are structurally related to Ro 5-4864 for their capacities to displace [3H]Ro 5-4864 from "peripheral-type" binding sites and their potencies as convulsants (or as antagonists of Ro 5-4864-induced convulsions). It was observed that compounds such as KW 3600 (the N-desmethyl analog of Ro 5-4864), which have very low affinities for "peripheral-type" sites, are convulsants with a potency nearly equal to that of Ro 5-4864. In contrast, compounds such as Ro 5-6900 and PK 11195, which bind with very high affinities to "peripheral-type" binding sites, are neither convulsants nor do they antagonize the convulsant actions of Ro 5-4864. Within a series of compounds that are structurally related to Ro 5-4864 there is a good correlation (r = 0.93; p less than 0.01) between their potencies as convulsants and their capacities to displace [35S]t-butylbicyclophosphorothionate from sites that may be associated with the chloride ionophore. Thus, it appears that occupation of "peripheral-type" binding sites by high-affinity ligands may not be directly involved in the convulsant actions of Ro 5-4864 and related compounds.     

Differential Binding Properties of the Peripheral-Type Benzodiazepine Ligands [3H]PK 11195 and [3H]Ro 5-4864 in Trout and Mouse Brain Membranes

High-affinity binding sites for [3H]PK 11195 have been detected in brain membranes of rainbow trout (Salmo gairdneri) and mouse forebrain, where the densities of receptors were 1,030 and 445 fmol/mg of protein, respectively. Ro 5-4864 (4'-chlorodiazepam) was 2,200-fold less potent as a competitor of [3H]PK 11195 binding in the piscine than the murine membranes. Investigation of the regional distribution of these sites in trout yielded a rank order of density of spinal cord greater than olfactory bulb = optic tectum = rhombencephalon greater than cerebellum greater than telencephalon. This site in trout shared some of the characteristics of the peripheral-type benzodiazepine receptor (PTBR) (also known as the mitochondrial benzodiazepine receptor) in rodents, i.e., high affinity for PK 11195 and the endogenous ligand protoporphyrin IX, but was unique in the low affinity of Ro 5-4864 (41 microM) and diazepam and the relatively high affinity of the calcium channel ligand diltiazem and two central benzodiazepine ligands, CGS 8216 and CGS 9896. The differential affinity for the two prototypic PTBR ligands in trout is similar to that previously observed in calf and human brain membranes. Structural differences for the trout sites are indicated by the relative inability of diethyl pyrocarbonate to modify histidine residues of the binding site in trout as compared with mouse membranes. Heterogeneity of binding of the two prototypic PTBR ligands in mouse brain membranes was indicated by additivity studies, equilibrium competition experiments, and saturation isotherms, which together support the hypothesis that Ro 5-4864 discriminates between two [3H]PK 11195 binding sites having high (nanomolar) and low (micromolar) affinity, respectively.     

Peripheral benzodiazepine receptors in isolated human pancreatic islets

Peripheral benzodiazepine receptors have been shown in some endocrine tissues, namely the testis, the adrenal gland, and the pituitary gland. In this work we evaluated whether peripheral benzodiazepine receptors can be found in the purified human pancreatic islets and whether they may have a role in insulin release. Binding of the isoquinoline compound [³H]1-(2-chlorophenyl-N-methyl-1-methyl-propyl)-3-isoquinolinecarboxamide ([³H]PK-11195), a specific ligand of peripheral benzodiazepine receptors, to cellular membranes was saturable, and Scatchard's analysis of the saturation curve demonstrated the presence of a single population of binding sites, with an affinity constant value of 9.20 ± 0.80 nM and a maximum number of binding sites value of 8913 ± 750 fmol/mg of proteins. PK-11195 and 7-chloro-1,3-dihydro-1-methyl-5-(p-chlorophenyl)-2H-1,4-benzodiazepin-2-on (Ro 5-4864) significantly potentiated insulin secretion from freshly isolated human islets at 3.3 mM glucose. These results show the presence of peripheral benzodiazepine receptors in purified human pancreatic islets and suggest their role in the mechanisms of insulin release. J. Cell. Biochem. 64:273–277. © 1997 Wiley-Liss, Inc.     

Modulation of cholinephosphotransferase activity in breast cancer cell lines by Ro5-4864, a peripheral benzodiazepine receptor agonist

Changes in phospholipid and fatty acid profile are hallmarks of cancer progression. Increase in peripheral benzodiazepine receptor expression has been implicated in breast cancer. The benzodiazepine, Ro5-4864, increases cell proliferation in some breast cancer cell lines. Biosynthesis of phosphatidylcholine (PC) has been identified as a marker for cells proliferating at high rates. Cholinephosphotransferase (CPT) is the terminal enzyme for the de novo biosynthesis of PC. We have addressed here whether Ro5-4864 facilitates some cancer causing mechanisms in breast cancer. We report that cell proliferation increases exponentially in aggressive breast cancer cell lines 11-9-1-4 and BT-549 when treated with nanomolar concentrations of Ro5-4864. This increase is seen within 24 h of treatment, consistent with the cell doubling time in these cells. Ro5-4864 also upregulates c-fos expression in breast cancer cell lines 11-9-1-4 and BT-549, while expression in non-tumorigenic cell line MCF-12A was either basal or slightly downregulated. We further examined the expression of the CPT gene in breast cancer (11-9-1-4, BT-549) and non-tumorigenic cell lines (MCF-12A, MCF-12F). We found that the CPT gene is overexpressed in breast cancer cell lines compared to the non-tumorigenic cell lines. Furthermore, the activity of CPT in forming PC is increased in the breast cancer cell lines cultured for 24 h. Additionally, we examined the CPT activity in the presence of nanomolar concentrations of Ro5-4864. Biosynthesis of PC was increased in breast cancer cell lines upon treatment. We therefore propose that Ro5-4864 facilitates PC formation, a process important in membrane biogenesis for proliferating cells.     

Evidence that the Peripheral-Type Benzodiazepine Receptor Ligand Ro 5–4864 Inhibits β-Endorphin Release from AtT-20 Cells by Blockade of Voltage-Dependent Calcium Channels

The demonstrations that Ro 5-4864, a ligand selective for the peripheral-type benzodiazepine (BZD) binding site, inhibited cellular differentiation and proliferation and that occupancy of the peripheral-type BZD binding site likely mediated the observed BZD effects on diverse endocrine tissues suggested that Ro 5-4864 disrupted a common cellular regulatory event. Using a well-characterized anterior pituitary-derived tumor cell line (AtT-20 cells), which synthesizes and secretes adrenocorticotropic hormone (ACTH), beta-lipotropin hormone (beta-LPH), and beta-endorphin (BE), we have investigated the molecular mechanism of action of Ro 5-4864's capacity to alter BE secretion. Ro 5-4864 inhibits basal and induced BE release from AtT-20 cells, through a cyclic AMP-independent mechanism. Ro 5-4864 completely blocked the corticotropin-releasing hormone and forskolin-induced release of BE without altering the concomitant production of cyclic AMP. The addition to AtT-20 cells of CGP 28392, a dihydropyridine that has been demonstrated in other systems to specifically activate voltage-dependent Ca2+ channels, resulted in a cyclic AMP-independent, dose-related increase in BE secretion. This CGP-induced BE release was blocked by increasing concentrations of Ro 5-4864. In contrast to the capacity of Ro 5-4864 to block CGP-induced BE release, Ro 5-4864 lacked the capacity to block enhanced BE secretion due to the calcium ionophore A23187, which increases intracellular Ca2+ levels independent of the voltage-dependent Ca2+ channels. Our findings suggest that Ro 5-4864 inhibits BE secretion from AtT-20 cells through a blockade of the voltage-dependent membrane Ca2+ channels and this mechanism of action may be responsible for Ro 5-4864's diverse effects observed on other cell types.     

Binding of [3H]Ro 5–4864 and [3H]PK 11195 to Cerebral Cortex and Peripheral Tissues of Various Species: Species Differences and Heterogeneity in Peripheral Benzodiazepine Binding Sites

The binding of [3H]PK 11195 and [3H]Ro 5-4864 to membrane preparations from cerebral cortex and peripheral tissues of various species was studied. [3H]PK 11195 (0.05-10 nM) bound with high affinity to rat and calf cerebral cortical and kidney membranes. [3H]Ro 5-4864 (0.05-30 nM) also successfully labeled rat cerebral cortical and kidney membranes, but in calf cerebral cortical and kidney membranes, its binding capacity was only 3 and 4%, respectively, of that of [3H]PK 11195. Displacement studies showed that unlabeled Ro 5-4864, diazepam, and flunitrazepam were much more potent in displacing [3H]PK 11195 from rat cerebral cortex and kidney membranes than from calf tissues. The potency of unlabeled Ro 5-4864 in displacing [3H]PK 11195 from the cerebral cortex of various other species was also tested, and the rank order of potency was rat = guinea pig greater than cat = dog greater than rabbit greater than calf. Analysis of these displacement curves revealed that Ro 5-4864 bound to two populations of binding sites from rat and calf kidney and from rat, guinea pig, rabbit, and calf cerebral cortex but to a single population of binding sites from cat and dog cerebral cortex. Using [3H]PK 11195 as a ligand, the rank order of binding capacity in cerebral cortex of various species was cat greater than calf greater than guinea pig greater than rabbit greater than dog greater than rat, whereas when [3H]Ro 5-4864 was used, the rank order of binding capacity was cat greater than guinea pig greater than rat greater than rabbit greater than calf greater than dog.     

Molecular mechanisms of peripheral benzodiazepine receptors

Peripheral-type benzodiazepine receptors were identified initially as binding sites in peripheral tissues with markedly different drug specificity than the central type receptors. The density of peripheral receptors varies greatly among tissue with selective localization within organs. Steroid producing areas of glands, such as the adrenal, testes and ovary, are highly enriched in these receptors. Intracellular localizations provide further insight into function with peripheral receptors largely if not exclusively associated with outer membranes of mitochondria. Purification of the peripheral receptor protein from rat kidney mitochondria reveals two apparent subunits with molecular weights of about 30 and 18 kD respectively. This complex is functionally intact as determined by its ability to reversibly bink PK-11195 Ro5-4864, and PK-14105 with high affinity and specificity.Acknowledgements: Supported by USPHS grant DA-00266, Research Scientist Award DA-00074 to S.H.S. and a gift of the Bristol Myers Company.Special issue dedicated to Dr. Erminio Costa.