丙泊酚对胎儿型和α7烟碱型乙酰胆碱受体的作用

目的:通过重组受体,观察丙泊酚对胎儿型乙酰胆碱受体和神经型α7乙酰胆碱受体的作用.方法:通过脂质体转染法在HEK293细胞表达胎儿型或α7乙酰胆碱受体.应用全细胞电压-膜片钳技术研究不同浓度(0.1μmol/L-3mmol/L)丙泊酚对2种受体亚型的影响.结果:临床相关浓度丙泊酚对100μmol/L乙酰胆碱激发胎儿型和α7乙酰胆碱受体的电流均有抑制作用.不同浓度丙泊酚对胎儿型或α7乙酰胆碱受体电流抑制作用比较差异无统计学意义(P>0.05).相同浓度丙泊酚对胎儿型乙酰胆碱受体抑制大于对α7乙酰胆碱受体的抑制(P<0.05).结论:丙泊酚对γ-nAChR和a7-nAChR都有抑制作用,抑制程度与丙泊酚的浓度无关.     

酪氨酸激酶抑制剂对hTrp3蛋白介导的α_(1B)-AR引起的Ca~(2+)内流的影响

目的　探讨Trp3(transientreceptorpotential 3)蛋白是否参与α1B AR引起的Ca2 + 内流以及酪氨酸激酶对其调控作用。方法　采用脂质体转染 ,将hTrp3cDNA分别转染到HEK2 93细胞和已有α1B受体稳定表达的HEK2 93细胞 ;Westernblot方法检测Trp3蛋白表达情况 ;Fura 2 /AM荧光分光光度法 ,测定胞浆游离Ca2 + 浓度。结果　HEK2 93细胞上可检测到hTrp3的内源性表达 ,转染后其表达增加。α1B HEK2 93细胞转染hTrp3cDNA后 ,α1B AR引起的Ca2 +内流显著增加 (P <0 0 1) ;转染hTrp3cDNA对thapsigargin诱导的Ca2 + 内流无作用。 5～ 30 μmol·L-1genistein对转染细胞α1B AR诱发的Ca2 + 内流有抑制作用 ,最大抑制率达(75 2± 12 6 ) %。结论　Trp3cDNA转染可能主要通过非CRAC(calciumreleaseactivatedcalcium )途径增加α1B AR引起的Ca2 + 内流 ,这一过程很大程度上依赖酪氨酸激酶的调控     

人淀粉样蛋白前体695基因及人烟碱乙酰胆碱受体α4β2亚单位基因共表达细胞模型的构建

目的为满足寻找可能影响淀粉样β蛋白(Aβ)在阿尔茨海默病过程中的药物实验的需求,建立共表达人淀粉样蛋白前体(APP)695基因和烟碱乙酰胆碱受体(nAChR)α4β2亚单位基因的细胞模型。方法用脂质体转染方法把APP695基因转染进已稳定转染nAChRα4β2亚单位基因的SH-EP1细胞。用500mg.L-1G418加压筛选,并挑选细胞单克隆。用RT-PCR和Western蛋白印迹法对转染后的细胞单克隆进行鉴定,挑取成功转染并高表达APP695的细胞进行克隆。膜片钳检测所挑细胞克隆的α4β2受体功能。结果挑出成功稳定转染人APP695基因及人nAChRα4β2亚单位基因的共表达细胞克隆株。膜片钳方法检测到此细胞克隆株上nAChRα4β2存在活性。结论成功制备了共表达人APP695基因及人nAChRα4β2亚单位基因的细胞模型,为探讨神经元nAChRα4β2亚型对Aβ加工影响的药物实验提供了条件。     

丙泊酚对胎儿型-和a7-烟碱型乙酰胆碱受体的作用

目的 通过重组受体,观察丙泊酚对胎儿型乙酰胆碱受体和神经型α7乙酰胆碱受体的作用。方法 通过脂质体转染法在HEK293细胞表达胎儿型和α7乙酰胆碱受体。分别研究不同浓度丙泊酚对两种受体亚型的作用。结果 临床相关浓度丙泊酚对100μmol/l乙酰胆碱激发胎儿型和α7乙酰胆碱受体的电流均有抑制作用。不同浓度丙泊酚对胎儿型和α7乙酰胆碱受体电流抑制大小无统计学差异（P>0.05）。相同浓度丙泊酚对胎儿型乙酰胆碱受体抑制大于对α7乙酰胆碱受体的抑制（P<0.05）。结论 从乙酰胆碱受体水平研丙泊酚的作用,结果证明丙泊酚对γ-nAChR和a7-nAChR都有抑制作用,抑制程度与丙泊酚的浓度无关。     

脂质体介导hHCN2基因转染HEK293细胞

目的建立一种研究离子通道的有效模型。方法采用Lipofacta mine2000脂质体将人的超极化激活的环核苷酸门控（HCN）基因转染人胚胎肾（HEK）293细胞,利用全细胞膜片钳技术检测克隆人HCN2基因的表达。结果pcDNA3-hHCN2真核表达载体转染HEK293细胞3d后,全细胞膜片钳技术记录到克隆人HCN2基因编码的通道电流。结论全细胞膜片钳技术稳定、可靠,可为开展克隆离子通道结构和功能关系研究提供基础。     

线性化聚乙烯亚胺介导的高效瞬时转染条件的优化

目的优化线性化聚乙烯亚胺(polyethylenimine linear, PEI)介导的高效瞬时转染条件, 提高重组蛋白在人胚胎肾(human embryonic kidney, HEK)293F悬浮细胞中的瞬时表达效率。方法构建和鉴定重组质粒增强型绿色荧光蛋白(enhanced green fluorescent protein, EGFP)/pcDNA3.1+, 通过瞬时转染的方式将质粒转入HEK293F细胞中进行表达。对转染试剂PEI与重组质粒的比例以及收获时间进行优化, 通过倒置显微镜观察、SDS-PAGE和流式细胞仪检测目标蛋白的表达量, 确定目标蛋白在HEK293F细胞中的最佳表达条件。采用优化后的条件在T500摇瓶(工作体积120 ml)中进行扩大表达。结果构建的重组质粒EGFP/pcDNA3.1+序列正确, 在重组质粒浓度为3.0 μg/ml、DNA∶PEI为2∶1、转染后24 h添加终浓度为2 mmol/L的丙戊酸钠、转染后4 d收获条件下, 目的蛋白的表达量最高。此条件也适用于放大培养体积至T500的瞬时转染。结论优化了一种快速有效的基于PEI的瞬时转染方法来高水...     

趋化因子受体CCR6的克隆及其在HEK293细胞中的表达

目的：构建人趋化因子受体6（CCR6）cDNA序列的真核表达载体，并了解其在HEK293细胞中的表达。方法：提取人淋巴结总RNA，通过逆转录PCR扩增出CCR6基因片段，并构建真核表达载体pcDNA3，1（＋）-CCR6；重组载体通过脂质体转染HEK293细胞，免疫荧光法鉴定CCR6的表达。结果：酶切鉴定和序列分析证实重组质粒含有CCR6编码序列．转染实验表明重组质粒能在HEK293细胞中表达出具有活性的CCR6片段。结论：CCR6真核表达载体构建及表达成功，为下一步CCR6拈抗剂的筛选奠定了基础。     

大鼠颈上神经节培养神经元烟碱电流及美加明的使用依赖性阻滞

比较几种烟碱型乙酰胆碱受体(nAChR)激动剂及拮抗剂对颈上神经节(SCG)培养神经元nAChR的作用和SCG培养神经元nAChR的药理特征.方法:用全细胞膜片钳技术结合局部压力喷射给药观察培养的新生大鼠SCG神经元对胆碱能药物的反应.结果:所测试的交感神经元均可被nAChR激动剂所激动,激动剂在相同浓度下诱发的电流峰值分别为:乙酰胆碱(ACh),443±183 pA;烟碱,1175±377 pA;碘化1,1-二甲基-4-苯基哌嗪(DMPP),2946±358 pA.在几种nAChR拮抗剂中,美加明(Mec),六烃季胺,箭毒在相同浓度下拮抗DMPP引起的全细胞电流的峰值分别为:435±154 pA,725±320 pA,887±214 pA,但α-银环蛇毒对之无影响.Mec对DMPP电流的拮抗具有使用依赖性.当Mec 1 μmol·L~(-1)与DMPP 100 μmol·L~(-1)混合向SCG神经元压力喷射时,前6次电流分别为第一次的％:100,64±3,50±3,41±4,36±4,32±3％.结论:SCG神经元的nAChR与骨胳肌及中枢nAChR有不同的药理特征.     

阿片κ-受体和ORL1受体二聚化的初步研究

为探讨κ-受体(κ-opioid receptor,KOR)和阿片受体样受体(opioid receptor like-1 receptor,ORL1 receptor)是否能够形成异源性受体二聚体,在原代培养的大鼠神经元细胞和用带有HA(hemagglutinin,血细胞凝集素)、Myc或Flag标签的KOR和ORL1质粒共同转染的中国仓鼠卵巢(CHO)细胞、人胚肾上皮(HEK293)细胞上,采用免疫荧光和免疫共沉淀的方法,研究KOR和ORL1之间的共定位以及是否存在相互作用。结果表明:在原代培养的海马和皮质神经元上,KOR和ORL1的免疫荧光在细胞膜上有重叠。同样,在HA-KOR和Myc-ORL1共同瞬时转染的CHO和HEK293细胞上也有类似的发现。另外,在共同表达Flag-KOR和Myc-ORL1的CHO细胞裂解液中,KOR与ORL1的受体蛋白能够被相互免疫共沉淀。这些研究结果提示,作为阿片受体不同亚型的KOR和ORL1受体之间有可能存在着异源二聚体,这也为进一步解释阿片受体结构的多样性和功能的复杂性提供了新的实验依据。     

α7烟碱型乙酰胆碱受体点突变体的制备及其功能研究

目的 利用氨基酸定点突变技术以大鼠野生型α7烟碱型乙酰胆碱受体(nAChR)为模板制备α7 nAChR突变体。方法 采用体外转录、定点突变、凝胶电泳、双电极电压钳等技术对α7 nAChR进行点突变,在非洲爪蟾卵母细胞上表达α7 nAChR及其突变体,并研究其受体活性功能。结果 将α7 nAChR第111位的丙氨酸突变为丝氨酸,制备了α7 nAChR的点突变体,测定了突变体对激动剂乙酰胆碱(ACh)的半数效应浓度(EC₅₀)为165.6 μmol/L。结论 该结果不仅为受体定点突变提供了一种方法,同时为药物筛选和研究α7 nAChR结构与功能关系提供了模型。     

七氟醚和异氟醚对不同浓度罗库溴铵抑制乙酰胆碱受体内向电流的影响

目的探讨挥发性麻醉药七氟醚(sevoflurane)、异氟醚(isoflurane)对不同浓度罗库溴铵(rocuronium)抑制骨骼肌成人型乙酰胆碱受体(ε-nAChR)内向电流的影响。方法通过脂质体转染建立表达ε-nAChR的HEK293细胞,用全细胞膜片钳检测乙酰胆碱(ACh)激动受体峰电流。拟合七氟醚、异氟醚、罗库溴铵抑制受体的浓度效应关系(IC50)。用0.5IC50浓度的七氟醚、异氟醚预处理ε-nAChR,记录浓度为IC5、0.5 IC50、IC50的罗库溴铵对ACh诱发峰电流的抑制率。各组以单独使用相应浓度罗库溴铵作为对照。结果七氟醚、异氟醚、罗库溴铵的IC50值分别为:(824.27±14.73)μmol.L-1、(1031.53±62.91)μmol.L-1、(150.45±12.5)μmol.L-1(P<0.01)。0.5 IC50浓度的异氟醚增强IC5浓度罗库溴铵拮抗受体的作用强于七氟醚(P<0.01)。结论七氟醚、异氟醚增强罗库溴铵对ε-nAChR的阻滞作用;两种吸入麻醉药对低浓度的罗库溴铵具有更明显的协同效应,且异氟醚作用强于七氟醚。     

Crystallization scale purification of α7 nicotinic acetylcholine receptor from mammalian cells using a BacMam expression system

Aim:

To report our methods for expression and purification of α7 nicotinic acetylcholine receptor (α7-nAChR), a ligand-gated pentameric ion channel and an important drug target.

Methods:

α7-nAChRs of 10 different species were cloned into an inducible BacMam vector with an N-terminal tag of a tandem maltose-binding protein (MBP) and a TEV cleavage site. This α7-nAChR fusion receptor was expressed in mammalian HEK293F cells and detected by Western blot. The expression was scaled up to liters. The receptor was purified using amylose resin and size-exclusion chromatography. The quality of the purified receptor was assessed using SDS-PAGE gels, thermal stability analysis, and negative stain electron microscopy (EM). The expression construct was optimized through terminal truncations and site-directed mutagenesis.

Results:

Expression screening revealed that α7-nAChR from Taeniopygia guttata had the highest expression levels. The fusion receptor was expressed mostly on the cell surface, and it could be efficiently purified using one-step amylose affinity chromatography. One to two milligrams of the optimized α7-nAChR expression construct were purified from one liter of cell culture. The purified α7-nAChR samples displayed high thermal stability with a Tm of 60 °C, which was further enhanced by antagonist binding but decreased in the presence of agonist. EM analysis revealed ring-like structures with a central hydrophilic hole, which was consistent with the pentameric assembly of the α7-nAChR channel.

Conclusion:

We have established methods for crystallization scale expression and purification of α7-nAChR, which lays a foundation for high-resolution structural studies using X-ray crystallography or single particle cryo-EM analysis.     

罗库溴铵复合维库溴铵或阿曲库铵对骨骼肌成人型和胎儿型乙酰胆碱受体的作用

目的:应用全细胞膜片钳研究罗库溴铵复合维库溴铵或阿曲库铵对骨骼肌成人型(ε-nAChR)和胎儿型(γ-nAChR)乙酰胆碱受体的作用.方法:通过脂质体转染法在HEK293细胞表达ε-nAChR和γ-nAChR.分别研究罗库溴铵、维库溴铵和阿曲库铵各药对两种受体亚型的作用.再以各药等效浓度混合,观察罗库溴铵复合维库溴铵或阿曲库铵混和液对两种受体亚型的作用.结果:罗库溴铵、维库溴铵和阿曲库铵呈浓度依赖性抑制乙酰胆碱对ε-nAChR和γ-nAChR的激动作用.应用等效线图分析发现罗库溴铵复合维库溴铵对ε-nAChR呈现协同作用,对γ-nAChR呈现相加作用;罗库溴铵复合阿曲库铵对ε-nAChR和γ-nAChR均为协同作用.结论:不同化学结构肌松药对ε-nAChR和γ-nAChR的抑制程度不同,而ε-nAChR和γ-nAChR对一种肌松药的亲和力也有差异.不同化学结构的罗库溴铵复合阿曲库铵对ε-nAChR和γ-nAChR均为协同作用;相同化学结构的罗库溴铵复合维库溴铵对ε-nAChR呈现协同作用,对γ-nAChR呈现相加作用.     

Magnesium sulfate enhances non-depolarizing muscle relaxant vecuronium action at adult muscle-type nicotinic acetylcholine receptor in vitro

Aim:

To investigate the effect of magnesium sulfate and its interaction with the non-depolarizing muscle relaxant vecuronium at adult muscle-type acetylcholine receptors in vitro.

Methods:

Adult muscle-type acetylcholine receptors were expressed in HEK293 cells. Drug-containing solution was applied via a gravity-driven perfusion system. The inward currents were activated by brief application of acetylcholine (ACh), and recorded using whole-cell voltage-clamp technique.

Results:

Magnesium sulfate (1–100 mmol/L) inhibited the inward currents induced ACh (10 μmol/L) in a concentration-dependent manner (IC₅₀=29.2 mmol/L). The inhibition of magnesium sulfate was non-competitive. In contrast, vecuronium produced a potent inhibition on the adult muscle-type acetylcholine receptor (IC₅₀=8.7 nmol/L) by competitive antagonism. Magnesium sulfate at the concentrations of 1, 3, and 6 mmol/L markedly enhanced the inhibition of vecuronium (10 nmol/L) on adult muscle-type acetylcholine receptors.

Conclusion:

Clinical enhancement of vecuronium-induced muscle relaxation by magnesium sulfate can be attributed partly to synergism between magnesium sulfate and non-depolarizing muscle relaxants at adult muscle-type acetylcholine receptors.     

Characterization of human alpha 4 beta 2-nicotinic acetylcholine receptors stably and heterologously expressed in native nicotinic receptor-null SH-EP1 human epithelial cells

Naturally expressed nicotinic acetylcholine receptors composed of alpha4 and beta2 subunits (alpha4beta2-nAChR) are the predominant form of high affinity nicotine binding site in the brain implicated in nicotine reward, mediation of nicotinic cholinergic transmission, modulation of signaling through other chemical messages, and a number of neuropsychiatric disorders. To develop a model system for studies of human alpha4beta2-nAChR allowing protein chemical, functional, pharmacological, and regulation of expression studies, human alpha4 and beta2 subunits were stably introduced into the native nAChR-null human epithelial cell line SHEP1. Heterologously expressed alpha4beta2-nAChR engage in high-affinity, specific binding of 3H-labeled epibatidine (H-EBDN; macroscopic KD = 10 pM; kon = 0.74/min/nM, koff = 0.013/min). Immunofluorescence studies show alpha4 and beta2 subunit protein expression in virtually every transfected cell, and microautoradiographic studies show expression of 125I-labeled iodo-deschloroepibatidine binding sites in most cells. H-EBDN binding competition studies reveal high affinity for nicotinic agonists and lower affinity for nicotinic antagonists. Heterologously expressed alpha4beta2-nAChR functional studies using 86Rb+ efflux assays indicate full efficacy of epibatidine, nicotine, and acetylcholine; partial efficacy for 1,1-dimethyl-4-phenyl-piperazinium, cytisine, and suberyldicholine; competitive antagonism by dihydro-beta-erythroidine, decamethonium, and methyllycaconitine; noncompetitive antagonism by mecamylamine and eserine; and mixed antagonism by pancuronium, hexamethonium, and d-tubocurarine. These results demonstrate utility of transfected SH-EP1 cells as models for studies of human alpha4beta2-nAChR, and they also reveal complex relationships between apparent affinities of drugs for radioligand binding and functional sites on human alpha4beta2-nAChR.     

Cell-type specific calcium signaling by corticotropin-releasing factor type 1 (CRF1) and 2a (CRF2(a)) receptors: phospholipase C-mediated responses in human embryonic kidney 293 but not SK-N-MC neuroblastoma cells

The human corticotropin-releasing factor (hCRF) receptors CRF1 and CRF2(a) couple to the Gs protein. It has been postulated that CRF receptors may also signal through phospholipase C (PLC). To test this hypothesis, binding and signaling properties were determined for both receptor subtypes stably expressed in human embryonic kidney 293 (HEK293) and human SK-N-MC neuroblastoma cells. CRF receptors were highly expressed and strongly coupled to Gs in HEK293 and SK-N-MC cells. However, when the calcium mobilization pathway was investigated, marked differences were observed. In SK-N-MC cells, neither CRF receptor stimulated calcium mobilization in the fluorometric imaging plate reader (FLIPR) assay, whereas activation of orexin type 1 and 2 receptors stably expressed in SK-N-MC cells revealed robust calcium responses. In contrast, intracellular calcium was strongly mobilized by agonist stimulation of hCRF1 and hCRF2(a) receptors in HEK293 cells. In HEK293 cells, potency rank orders for calcium and cAMP responses were identical for both receptors, despite a rightward shift of the dose-response curves. Complete inhibition of calcium signaling of both hCRF1 and hCRF2(a) receptors was observed in the presence of the PLC inhibitor U-73,122 whereas ryanodine, an inhibitor of calcium release channels and the protein kinase A inhibitor Rp-cAMPS were ineffective. Finally, CRF agonists produced a small but significant stimulation of inositol 1,4,5-triphosphate (IP3) accumulation in hCRF1-and hCRF2(a)-transfected HEK293 cells. These data clearly show that phospholipase C-mediated signaling of CRF receptors is dependent upon the cellular background and that in HEK293 cells human CRF receptors robustly respond in the FLIPR format.     

Oral administration of MA-2029, a novel selective and competitive motilin receptor antagonist, inhibits motilin-induced intestinal contractions and visceral pain in rabbits

The pharmacological properties of MA-2029, a novel motilin receptor antagonist, were investigated. In vitro, MA-2029 (1 to 30 nM) competitively inhibited motilin-induced contractions in isolated rabbit duodenal longitudinal muscle strips, with a pA2 value of 9.17+/-0.01 (n=5). However, contractile responses to acetylcholine and substance P were unaffected even at 1 microM of MA-2029. MA-2029 concentration-dependently inhibited the binding of [125 I]motilin to motilin receptors in a homogenate of rabbit colon smooth muscle tissue and membranes of HEK 293 cells expressing human motilin receptors. The pKi of MA-2029 was 8.58+/-0.04 in the rabbit colon homogenate (n=4) and 8.39 in the HEK 293 cells (mean of duplicate experiments). In vivo, orally-administered MA-2029 (3 to 30 mg/kg) dose-dependently inhibited colonic contractions induced by motilin (3 microg/kg, i.v.) in conscious rabbits. Inhibition was caused by all doses at 30 min after administration and by 10 mg/kg or more at 4 h after administration. The plasma concentration of MA-2029 correlated with its inhibitory effect. Furthermore, the oral administration of MA-2029 (0.3 to 3 mg/kg) also inhibited abdominal muscle contractions (an index of the visceral pain) induced by intravenous infusion of motilin (3 microg/kg/h) during colorectal distension in conscious rabbits. These results indicate that MA-2029 is an orally active, selective and competitive motilin receptor antagonist. It is suggested that this compound may be useful for gastrointestinal disorders associated with disturbed gastrointestinal motility such as irritable bowel syndrome.     

The M-channel blocker linopirdine is an agonist of the capsaicin receptor TRPV1

Linopirdine is a well known blocker of voltage-gated potassium channels from the Kv7 (or KCNQ) family that generate the so called M current in mammalian neurons. Kv7 subunits are also expressed in pain-sensing neurons in dorsal root ganglia, in which they modulate neuronal excitability. In this study we demonstrate that linopirdine acts as an agonist of TRPV1 (transient receptor potential vanilloid type 1), another ion channel expressed in nociceptors and involved in pain signaling. Linopirdine induces increases in intracellular calcium concentration in human embryonic kidney 293 (HEK293) cells expressing TRPV1, but not TRPA1 and TRPM8 or in wild-type HEK293 cells. Linopirdine also activates an inward current in TRPV1-expressing HEK293 cells that is almost completely blocked by the selective TRPV1 antagonist capsazepine. At low concentrations linopirdine sensitizes both recombinant and native TRPV1 channels to heat, in a manner that is not prevented by the Kv7-channel opener flupirtine. Taken together, these results indicate that linopirdine exerts an excitatory action on mammalian nociceptors not only through inhibition of the M current but also through activation of the capsaicin receptor TRPV1.