河蟹眼柄神经内分泌细胞Glu受体研究

作者采用全细胞膜片钳技术测定了中华绒螯蟹 (Eriocheirsinensis)眼柄视神经节端髓X器官 (MTXO)神经内分泌细胞对 0 0 1— 10mmol/L谷氨基酸 (Glu)的反应 ,并结合药理学方法进行了Glu受体研究。结果表明 ,Glu激活A型和B型细胞离子型Cl-通道蛋白受体 ,诱导快速激活、快速失活的配体门控Cl-通道电流 (IGlu)。依据内外液的Cl-浓度比例引发去极化或超极化反应 ,继续施加Glu ,细胞快速出现脱敏反应 ;去除Glu后 ,细胞约需 2 0s恢复对Glu的敏感状态。IGlu幅值呈浓度依赖性 ,量 效关系曲线呈线形 ,激活阈值为 0 0 1mmol/L ,约 5mmol/L达到饱和。河蟹眼柄神经内分泌细胞IGlu明显受到Cl-通道阻断剂picrotoxin(0 5mmol/L)抑制 ;对离子型Glu受体激动剂Quisqualate、Kainate、NMDA、AMPA不敏感。Ibotenicacid(IA)可模拟Glu诱导快速激活、快速脱敏的Cl-电流 ,并与Glu产生交互脱敏作用。Glu和GABA对河蟹眼柄神经内分泌细胞无交叉脱敏和交叉激活作用 ,甘氨酸 (Gly)没有诱导细胞产生任何反应 ,提示中枢神经系统通过Glu和GABA两套系统实现对眼柄神经内分泌系统的精确调控。     

NMDA对GABAA受体的抑制作用由钙-钙调素依赖性蛋白激酶Ⅱ介导

应用制霉菌素(nystatin)穿孔全细胞记录方法,研究了N-甲基-D-门冬氨酸(NMDA)对新鲜分离的大鼠骶髓后连合核(SDCN)神经元γ-氨基丁酸(GABA)激活的全细胞电流的调控.实验结果如下:(ⅰ) 在无Mg2+及含1 μmol/L甘氨酸的标准细胞外液中,当钳制电位为-40 mV时,NMDA(100 μmol/L)可抑制GABA和muscimol (Mus)在SDCN神经元激活的全细胞电流(IGABA和IMus),且NMDA受体的竞争性拮抗剂D-2-amino-5-phosphonovalerate(APV,100 μmol/L)可完全阻断NMDA激活的电流,并可消除NMDA对IGABA的抑制作用.(ⅱ)当细胞外液中无Ca2+及待测神经元被Ca2+螯合剂BAPTA AM预处理2 h后,NMDA对IGABA的抑制作用消失;而用电压依赖性钙通道阻断剂Cd2+(10 μmol/L)或La3+(30μmol/L)预处理后,NMDA对IGABA的抑制作用仍然存在.(ⅲ) NMDA对IGABA的抑制作用可被钙-钙调素依赖性蛋白激酶Ⅱ(CaMKⅡ)的抑制剂KN-62所阻断.提示在大鼠SDCN神经元,NMDA对IGABA的抑制作用为一Ca2+依赖性的过程,这一过程的"下游"机制与细胞内CaMKⅡ有关.揭示了Ca2+和CaMKⅡ分别作为细胞内第二信使和第三信使将NMDA受体和GABAA受体的功能联系起来.     

非洲爪蟾卵母细胞GABA_B和GABA_C受体介导的电流反应

实验应用双电极电压箝技术 ,在具有滤泡膜的非洲爪蟾 (Xenopuslaevis)卵母细胞上记录到γ 氨基丁酸(γ aminobutyricacid ,GABA) 激活电流。此GABA 激活电流的特点及有关GABA受体类型的研究和分析如下 :( 1)在 3 5 5 % ( 5 5 / 15 5 )的受检细胞外加GABA可引起一慢的浓度依赖性的外向电流。 ( 2 )GABAA 受体的选择性拮抗剂bicuculline ( 10 -5mol/L)对GABA ( 10 -5mol/L)引起的外向电流无阻断作用 (n =6)。 ( 3 )GABAB 受体的选择性拮抗剂2 hydroxysaclofen ( 10 -4mol/L)能将GABA ( 10 -5mol/L)引起的外向电流可逆性地转变为内向电流 ,后者又可被GABAC 受体的选择性拮抗剂I4AA ( 10 -5mol/L)所消除 (n =6)。 ( 4 )GABAB 受体的特异性激动剂baclofen可引起部分 ( 2 0 % ,12 / 60 )受检细胞产生一慢的浓度依赖性的外向电流。 3× 10 -6 、3× 10 -5及 3× 10 -4mol/L 2 hydroxysaclofen分别阻断baclofen ( 10 -5mol/L) 激活电流 ( 6 3± 3 2 ) % ,( 4 4 1± 2 2 ) %及 ( 86 0± 1 6) % (n =6)。 ( 5 )baclofen激活电流的I V曲线显示逆转电位在 - 96 8± 7 2mV左右 ,此电流可分别被TEA ( 5mmol/L)和BaCl2 ( 2mmol/L)所阻断。以上结果提示 :在非洲爪蟾的卵母细胞上存在内源性GABAB 和GABAC 受体 ,GA     

非洲爪蟾卵母细胞GABAB和GABAc受体介导的电流反应

实验应用双电极电压箝技术,在具有滤泡膜的非洲爪蟾(Xenopuslaevis)卵母细胞上记录到γ-氨基丁酸(γ-aminobutyricacid,GABA)-激活电流。此GABA-激活电流的特点及有关GABA受体类型的研究和分析如下(1)在35.5%(55/155)的受检细胞外加GABA可引起一慢的浓度依赖性的外向电流。(2)GABAA受体的选择性拮抗剂bicuculline(10     

AMPA对视网膜L型水平细胞所接收的红、绿敏视锥信号的不同调制作用

在离体灌流的鲫鱼视网膜,应用细胞内记录技术,研究了谷氨酸受体激动剂——AMPA对L型水平细胞(LHC)的作用.AMPA压抑LHC由红敏视锥驱动的反应,却增强由绿敏视锥驱动的反应.此效应可为AMPA受体特异的拮抗剂(GYKI53655)所逆转,表明仅有AMPA受体参与其中.印防己毒素(PTX)或双氢红藻氨酸(DHK)存在时,依然可以观察到这种效应,提示自LHC向视锥的负反馈和视锥谷氨酸转运体在此效应中不起显著作用.AMPA的不同调制作用提示,LHC上可能存在不同特性的AMPA受体亚型介导红敏和绿敏视锥的信号传递.     

受体、突触和记忆

大脑中神经元突触间的信号传递是由许多神经递质受体介导的。在过去,Richard L．Huganir实验室一直致力于神经递质受体功能调节的分子机制。而最近,该实验室又聚焦到大脑中一种最主要的兴奋性受体的研究——谷氨酸受体。谷氨酸受体主要可以分为两大类：AMPA受体和NMDA受体。AMPA受体主要介导了快速的兴奋性突触传递;而NMDA受体则在神经可塑性和发育中起到重要作用。实验发现,AMPA受体和NMDA受体都可以被一系列的蛋白激酶磷酸化,而磷酸化的水平则直接影响了这些受体的功能特性,包括通道电导和受体膜定位等。AMPA受体磷酸化的水平同时还在学习和记忆的细胞模型中发生改变,如长时程增强（LTP）和长时程抑制（LTD）。此外,AMPA受体中GluR1亚单位的磷酸化对于各种形式的可塑性以及空间记忆的维持有重要的作用。实验室主要研究突触部位谷氨酸受体在亚细胞水平的定位和聚集的分子机制。最近,一系列可以直接或间接与AMPA和NMDA受体相互作用的蛋白质得以发现,其中包括一个新发现的蛋白家族GRIPs（glutamate receptor interacting proteins）。GRIPs可以直接和AMPA受体的GluR2／3亚单位的C端结合。GRIPs包含7个PDZ结构域,可以介导蛋白与蛋白直接的相互连接,从而把各个AMPA受体交互连接在一起并与其他蛋白相连。另外,GluR2亚单位的c端还可以和兴奋性突触中的蛋白激酶C结合蛋白（PICK1）的PDZ结构域相互作用。另外,GluR2亚单位的C端也可以与一种参与膜融合的蛋白NSF相互作用。这些与AMPA受体相互作用的蛋白质对于受体在膜上的运输以及定位有至关重要的作用。同时,受体与PICK1和GRIP的结合对于小脑运动学习中的LTD有重要作用。总体上说,该实验室发现了一系列可以调节神经递质受体功能的分子机制,这些工作提示受体功能的调节可能是?     

GABA_B自调性受体调节长时增强效应

对长时增强效应(long—term potentiation,LTP)的理解应从细胞分子水平去观察脊椎动物的学习与记忆。一些资料证明,在海马CA_1区诱导的LTP需要有N-methyl-D-aspartate(NMDA)受体系统短暂的激活。在低频传递时,γ-氨基丁酸(GABA)可通过突触抑制来阻断NMDA受体系统的明显活动。这种阻断作用是使神经元超极化时,Mg~(2+)阻断了由NMDA受体所调控的离子通道而引起的。在高频传递时,由于突     

GBE对大鼠急性分离海马神经元NMDA-激活电流的调制作用

目的:观察不同制型的银杏叶提取物(GBE)对N-甲基-D-天门冬氨酸(NMDA)受体激活电流的影响,并比较其作用。方法:应用全细胞膜片钳记录技术记录急性分离大鼠海马神经NMDA激活电流,比较加药前后电流幅度的变化。结果:大部分受检细胞(81.8%,90/110)对外加NMDA敏感,引起一去敏感的内向电流(INMDA)。此电流可被NMDA受体特异阻断剂(MK-801)所阻断。预加不同制型的GBE均能明显抑制NMDA激活电流(P<0.01),但制型不同抑制效应不一,GBE纳米制剂(nGBE)对INMDA的抑制作用明显优于微米型(mGBE组),抑制率分别为64%±15%,40%±17%(n=8),两组比较差异具有统计学意义(P<0.05)。结论:预加GBE能抑制NM-DA-激活电流,从而对抗海马神经元兴奋毒性脑损伤,起神经保护作用。nGBE对NMDA受体的调控作用优于mGBE制剂。     

P物质对大鼠三叉神经节神经元GABA-和5-HT-激活电流的反向调制作用

实验采用全细胞膜片钳技术观察P 物质(SP)对大鼠同一三叉神经节(TG)神经元γ-氨基丁酸激活电流(IGABA)和5-羟色胺激活电流(I5-HT)的调制作用。在受检的47 个 TG 细胞中,多数情况下可在同一细胞记录到IGABA 和 I5-HT 两种电流(63.8%,30/47)。在 30 个同时对 GABA 和 5-HT 敏感的细胞,其中 22 个细胞预加 SP(0.01 μmol/L)后,IGABA 减小(35.7 ± 6.1)%,而I5-HT 增加(65.2 ±8.7)%。此种调制作用可被SP 受体拮抗剂GR82334 及胞内透析GDP-β -S 或GF109203X 所阻断。以上结果表明:SP 受体激活后经G 蛋白耦联,通过相同的PLC-DAG-PKC 转导途径对同一感觉神经元共存的GABAA 受体和5-HT3 受体产生相反的调制效应。     

NMDA受体通道参与大鼠脊髓背角C纤维诱发电位LTP的表达

以往研究表明,激动NMDA受体是引起海马长时程增强(LTP)的必备条件,而LTP的表达主要与AMPA受体的磷酸化及其受体组装到突触后膜有关.但是,近年来有研究表明NMDA受体通道也参与了LTP的表达.为探讨NMDA受体通道是否参与了脊髓背角C纤维诱发电位LTP的表达,诱导LTP后,分别静脉或脊髓局部给予NMDA受体拮抗剂MK801或APV,观察其作用.发现静脉注射非竞争性NMDA受体MK801(0.1mg/kg)对脊髓LTP无影响,注射0.5mg/kg显著抑制LTP,但是当剂量增高到1.0mg/kg时,抑制作用并未进一步增大.脊髓局部给予MK801也能抑制脊髓背角LTP.为验证上述结果,使用了竞争性NMDA受体拮抗剂APⅤ.结果显示,脊髓局部给予50μmol/LAPⅤ对LTP无影响,100μmol/L对LTP有显著的抑制作用,当浓度升至200μmol/L时,抑制作用并未见进一步增强.因此认为,NMDA受体通道部分地参与了脊髓背角C纤维诱发电位LTP的表达.     

Substituted phenyl as a steroid A-ring mimetic: Providing agonist activity to a class of arylsulfonamide nonsteroidal glucocorticoid ligands

A class of arylsulfonamide glucocorticoid receptor agonists that contains a substituted phenyl group as a steroid A-ring mimetic is reported. The structural design and SAR that provide the functional switching of a GR antagonist to an agonist is described. A combination of specific hydrogen bonding and lipophilic elements on the A-ring moiety is required to achieve potent GR agonist activity. This study culminated in the identification of compound 23 as a potent GR agonist with selectivity over the PR and MR nuclear hormone receptors.     

雄激素和雌激素受体药物筛选方法的研究进展

雄激素和雌激素受体通过与相应激素特异性结合促进细胞分化和组织生长,发挥重要的生理功能,其功能失调可诱发多种疾病。雄激素和雌激素受体的选择性调节剂是治疗相关疾病的重要药物。基于基因组学、分子生物学、细胞生物学和生物信息学等最新研究成果而发展形成的实验技术或方法被用于新型雄激素和雌激素受体调节剂的筛选,显著加快了新药开发的进程。     

Analysis of 3D models of octopus estrogen receptor with estradiol: evidence for steric clashes that prevent estrogen binding

Relatives of the vertebrate estrogen receptor (ER) are found in Aplysia californica, Octopus vulgaris, Thais clavigera, and Marisa cornuarietis. Unlike vertebrate ERs, invertebrate ERs are constitutively active and do not bind estradiol. To investigate the molecular basis of the absence of estrogen binding, we constructed a 3D model of the putative steroid-binding domain on octopus ER. Our 3D model indicates that binding of estradiol to octopus ER is prevented by steric clashes between estradiol and amino acids in the steroid-binding pocket. In this respect, octopus ER resembles vertebrate estrogen-related receptors (ERR), which have a ligand-binding pocket that cannot accommodate estradiol. Like ERR, octopus ER also may have the activation function 2 domain (AF2) in a configuration that can bind to coactivators in the absence of estrogens, which would explain constitutive activity of octopus ER.     

The ecdysteroid receptor

Summary

The steroid molting hormone of insects and other arthropods regulates gene activity in target tissues through its association with a specific, high affinity receptor protein. In this review we summarize recent advances in several areas of ecdysteroid receptor research, including efforts to characterize and purify the receptor protein, cytochemical studies of its tissue distribution and subcellular localization during development, and current molecular genetic studies ecdysteroid action.     

Proteinase-activated receptors, nucleotide P2Y receptors, and μ-opioid receptor-1B are under the control of the type I transmembrane proteins p23 and p24A in post-Golgi trafficking

We recently characterized the proteinase-activated receptor (PAR)-2, a G protein-coupled receptor (GPCR), as the first cargo protein recognized by p24A. Here, we demonstrate that p24A binds to several other GPCRs, including PAR-1, the nucleotide receptors P2Y(1), P2Y(2), P2Y(4), and P2Y(11), as well as the μ-opioid receptor 1B. The acidic amino acid residues Glu and Asp at the second extracellular loop of GPCRs are essential for interaction with p24A. p23, another member of the p24 family, also interacts with GPCRs, similar to p24A. However, p23 shows a delayed dissociation from PAR-2 after activation of PAR-2, compared to the dissociation between PAR-2 and p24A. p24A and p23 arrest both P2Y(4) receptor and μ-opioid receptor 1B at the intracellular compartments, as observed for PAR-2. A comparable result was obtained when we studied primary rat astrocytes in culture. Over-expression of the N-terminal p24A fragment impairs PAR-2 resensitization in astrocytes that extends our findings to a native system. In summary, we demonstrate that p24A and p23 are specific cargo receptors of GPCRs and differentially control GPCR trafficking in the biosynthetic pathway, and thereby, p24A and p23 regulate GPCR signaling in astrocytes.     

降钙素基因相关肽家族受体及其受体活性修饰蛋白

降钙素基因相关肽家族是一类多功能的激素家族 ,参与人体的多种生物学功能 ,与多种疾病有关。降钙素基因相关肽受体包括降钙素受体 (CTR)和降钙素受体样受体 (CRLR) ,CTR可以独自与降钙素结合 ,而CRLR必须与一组称作受体活性修饰蛋白 (RAMPs)的蛋白质共同作用才能发挥生物学功能。综述CTR的研究概况及CRLR与RAMPs相互作用的机制和表达调控 ,以期为人们设计新型药物提供参考。     

The insulin-binding domain of insulin receptor is encoded by exon 2 and exon 3.

Insulin receptors are disulfide-linked oligotetramers composed of two heterodimers each containing a 130-kDa alpha subunit and a 90-kDa beta subunit. Insulin binds to the extracellular alpha subunit, and in the process stimulates the autophosphorylation of the beta subunit and the expression of tyrosine kinase activity. Studies combining the use of photoaffinity labeling and immunoprecipitation with anti-peptide antibody have directly demonstrated that the cysteine-rich domain, encoded by exon 3, in the alpha subunit is part of the insulin-binding site of the receptor. Experiments with chimeric insulin receptors and chimeric insulin-like growth factor I receptors have confirmed that the cysteine-rich domain constitutes a part of the insulin-binding site. In addition, results from these experiments suggest that the N-terminal sequence, encoded by exon 2, in the alpha subunit also participates in insulin binding. In this review it is proposed that, assuming two insulin-binding sites per each holoreceptor oligotetramer, each insulin-binding domain may contain respectively two sub-domains for hydrophobic and charge contact with insulin, and that high-affinity binding would require the interaction of both subunits with the possibility of each subunit reciprocally contributing one of the sub-domains.     

Cloning and characterization of a family C orphan G-protein coupled receptor

Members of the family C receptors within the G-protein coupled receptor superfamily include the metabotropic glutamate receptors, GABA(B) receptors, the calcium-sensing receptor (CaSR), the V2R pheromone receptors, the T1R taste receptors, and a small group of uncharacterized orphan receptors. We have cloned and studied the mouse GPRC6A family C orphan receptor. The open reading frame codes for a protein with highest sequence identity to the fish 5.24 odorant receptor and the mammalian CaSR. The gene structure shows a striking resemblance to that of the CaSR. Results from RT-PCR analyses showed that mouse GPRC6A mRNA is expressed in mouse brain, skeletal muscle, heart, lung, spleen, kidney, liver, and in the early stage mouse embryo. Immunocytochemical analysis of the cloned mouse GPRC6A cDNA expressed in human embryonic kidney 293 cells demonstrated that GPRC6A was present on the plasma membrane, as well as in the endoplasmic reticulum and nuclear envelope membranes of transfected cells. A chimeric cDNA construct in which the extracellular ligand binding domain of the fish 5.24 amino acid-activated odorant receptor was ligated to the complementary downstream sequence of the mouse GPRC6A receptor indicated that GPRC6A is coupled to phosphoinositol turnover and release of intracellular calcium. Further studies with mouse GPRC6A expressed in Xenopus laevis oocytes demonstrated that this receptor possesses a pharmacological profile resembling that of the fish 5.24 odorant receptor. These findings suggest that GPRC6A may function as the receptor component of a novel cellular transmitter system in mammals.     

Ago-Allosteric Modulation and Other Types of Allostery in Dimeric 7TM Receptors

Conventionally, an allosteric modulator is neutral in respect of efficacy and binds to a receptor site distant from the orthosteric site of the endogenous agonist. However, recently compounds being ago-allosteric modulators have been described i.e., compounds acting both as agonists on their own and as enhancers for the endogenous agonists in both increasing agonist potency and providing additive efficacy—superagonism. The additive efficacy can also be observed with agonists, which are neutral or even negative modulators of the potency of the endogenous ligand. Based on the prevailing dimeric concept for 7TM receptors, it is proposed that the ago-allosteric modulators bind in the orthosteric binding site, but–importantly–in the “other” or allosteric protomer of the dimer. Hereby, they can act both as additive co-agonists, and through intermolecular cooperative effects between the protomers, they may influence the potency of the endogenous agonist. It is of interest that at least some endogenous agonists can only occupy one protomer of a dimeric 7TM receptor complex at a time and thereby they leave the orthosteric binding site in the allosteric protomer free, potentially for binding of exogenous, allosteric modulators. If the allosteric modulator is an agonist, it is an ago-allosteric modulator; if it is neutral, it is a classical enhancer. Molecular mapping in hetero-dimeric class-C receptors, where the endogenous agonist clearly binds only in one protomer, supports the notion that allosteric modulators can act through binding in the “other” protomer. It is suggested that for the in vivo, clinical setting a positive ago-allosteric modulator should be the preferred agonist drug.