α1肾上腺素受体亚型在家兔胸主动脉平滑肌细胞生长中的作用

本实验在体外培养的家兔胸主动脉平滑肌细胞上,利用＾３Ｈ－Ｔｈｙｍｉｄｉｎｅ（＾３Ｈ－ＴｄＲ）掺入的方法,观察了α１－肾上腺素受体（ａｄｒｅｎｅｒｇｉｃ ｒｅｃｅｐｔｏｒ,ＡＲ）及其亚型对平滑肌细胞ＤＮＡ合成的影响。结果显示：去甲肾上腺素激动α１－ＡＲ能促进平滑肌细胞ＤＮＡ合成,并有以下特点：（１）α１Ａ激动促进平滑肌细胞ＤＮＡ合成,但α１Ｂ激动抑制这一合成作用;（２）α１Ａ促进ＤＮＡ合成的作用与ｃ     

α1A-肾上腺素受体与骨形成蛋白-1片段在人胚肾293细胞中的结合

用酵母双杂交方法发现骨形成蛋白-1(bone morphogenetic protein-1,BMP-1)的片段可以与α1A-肾上腺素受体(adrenergic receptor,AR)的细胞内游离C末端结合。进一步探讨了二者在哺乳动物表达系统人胚肾293细胞(human embryonic cell 293,HEK293)中的相互作用。采用PCR方法构建含BMP-1片段eDNA的真核表达质粒PCP3HA,将其与含全长人α1A-AR eDNA的质粒PDT-α1A分别或共同转染HEK293细胞,用免疫印迹法检测到α1A-AR和BMP-1在HEK293细胞有相应的蛋白表达。用酶联免疫吸附实验对免疫印迹鉴定过的细胞裂解液进行检测,观察到空白对照组,单独转染PDTα1A-和单独转染PCP3HA的细胞,OD490值分别为0.034±0.027、0.042±0.019、0.030±0.0096,三者之间无显著性差异。共转染PDT-α1A和PCP3HA的细胞,OD490值为0.57±0.12,较其它三组具有显著性差异(均P<0.001)。免疫共沉淀结果显示,单独转染PDT-α1A或PCP3HA的细胞,免疫沉淀产物中均不能检测到BMP-1片段,只有共转染PDT-α1A和PCP3HA的细胞,在其免疫沉淀产物中能检测到BMP-1片段。ELISA和免疫沉淀结果均表明α1A-AR与BMP-1的片段在HEK293细胞中存在蛋白水平的相互作用。     

去甲肾上腺素对蟾蜍背根神经节细胞α-肾上腺素能受体的作用

在蟾蜍离体灌流背根神经节(DRG)标本上,用微电极进行细胞内记录。在51个细胞中A型神经元为46个,C型5个。此两类细胞的静息膜电位为60.06±1.34mV(x±SE)。当灌流液中滴加10~(-4)-10~(-3)mol/L去甲肾上腺素(NA)引起如下的膜电位改变:(1)超极化:幅值8.38±1.12mV(x±SE)(20/48);(2)去极化:幅值9.39±1.24mV(x±SE)(23/48);(3)无反应(5/48)。上述膜电位改变既不能由灌流液中滴加异丙基肾上腺素所拟似,也不能为心得安所阻断,因而排除了β-肾上腺能受体介导的可能性。加苯肾上腺素及可乐宁于灌流液,分别产生膜的去极化和超极化,而应用哌唑唪及育亨宾灌流,则分别阻断NA引起的膜去极化和超极化。因此认为:NA引起的DRG神经元的去极化和超极化反应分别是由胞体膜上之α_1-及α_2-肾上腺素能受体所介导的。     

去甲肾上腺素引起的α1肾上腺素受体三种亚型脱敏过程的差异

将含有α１肾上腺素受体三种亚型的全长ｃＤＮＡ质粒分别转染到人胚肾脏细胞（ＨＥＫ２９３）,α１Ａ－,α１Ｂ－和α１Ｄ－ＡＲ在ＨＥＫ２９３细胞株上得到高水平稳定表达,用＾３Ｈ－ｉｎｏｓｉｔｏｌ标记和柱层析法测定细胞磷酸肌醇积。观察在去甲肾上腺素长期作用下α１三种受体亚型介导磷酸肌醇蓄积敏感性降低的差别。     

神经肽Y可减轻大鼠血管α1肾上腺素受体亚型的脱敏作用

以离体大鼠主动脉与肾动脉对去甲肾上腺素（ＮＥ）的收缩反应分别代表α１Ｂ与α１Ａ亚型肾上腺素受体（ＡＲ）激动时的生物学效应。血管经ＮＥ１０μｍｏｌ／Ｌ温育２ｈ后,α１－ＡＲ介导收缩效应的敏感性显著降低,其中α１Ｂ亚型ＡＲ的脱敏程度显著大于α１Ａ亚型ＡＲ。上述经ＮＥ温育的血管,在神经肽Ｙ（ＮＰＹ）０．５μｍｏｌ／Ｌ存在时,α１Ｂ亚型ＡＲ的脱敏程度显著减轻。ＮＥ收缩主动脉的两时相分析表明,脱敏时主要表现为相性收缩时间延长,收缩幅度降低,而紧张性收缩并无显著改变;在ＮＰＹ存在时上述相性收缩改变消失。提示α１Ｂ亚型ＡＲ脱敏的机制主要与细胞内Ｃａ２＋动员减慢减少有关。     

心功能不全大鼠心脏α_1－肾上腺素受体亚型的变化

本文通过主动脉和左肾动脉缩窄复制心功能不全大鼠模型，并采用放射配基结合实验和半定量逆转录－聚合酶链反应（ＲＴ－ＰＣＲ），从蛋白质和基因转录水平上观察在心功能不全时大鼠心脏α１－肾上腺素受体三种亚型的改变。Ｓｃａｔｃｈａｒｄ分析结果显示，在心功能不全组与对照组，大鼠心脏组织１２５Ｉ－ＢＥ与α１－肾上腺素受体结合的亲和性（２２９±３２与１９５±１５ｐｍｏｌ／Ｌ）和数量（１０２±１２与９６±１７ｆｍｏｌ／ｍｇ）均无显著差异。但ＷＢ４１０１对１２５Ｉ－ＢＥ的竞争性抑制实验显示在心功能不全组高亲和性位点所占百分比较对照组显著增高（５１±７％与２２±５％）。ＲＴ－ＰＣＲ的结果显示，在心功能不全大鼠心脏α１Ａ－ＡＲ的ｍＲＮＡ水平高于对照组，但α１Ｂ－ＡＲ的ｍＲＮＡ水平低于对照组，而α１Ｄ－ＡＲ的ｍＲＮＡ水平在两组之间没有显著差异。综合上述结果可知，在心功能不全大鼠，心脏α１Ａ－ＡＲ增加，α１Ｂ－ＡＲ下降，而α１Ｄ－ＡＲ则没有显著改变。     

糖尿病合并高血压大鼠心脏α1肾上腺素受体的变化

目的:探讨糖尿病合并高血压大鼠心脏α1肾上腺素受体(α1-AR)及其三种亚型的变化规律和可能的意义。方法:用Wistar雄性大鼠建立糖尿病合并高血压模型,放射配体结合实验和离体左心房收缩功能实验等方法观察心脏α1肾上腺素受体(α1-AR)及其三种亚型的改变。结果:与正常对照大鼠相比,糖尿病合并高血压大鼠心脏α1-AR最大结合容量(Bmax)显著增加(P<0.05),且α1A-AR和α1D-AR均增加。糖尿病合并高血压大鼠左心房α1-AR介导的最大收缩反应较对照组降低41%(P<0.05),pD2值不变。α1-AR亚型选择性拮抗剂5-MU、spiperon和BMY7378拮抗NE正性变力效应的pA2值不变。结论:糖尿病合并高血压大鼠心脏α1-AR介导的最大收缩反应的降低,其主要与受体后信号转导效应减弱有关,其中以α1A-AR和α1D-AR尤为显著。     

α1Β-肾上腺素受体的磷酸化与减敏

α1Β-肾上腺素受体(α1B-AR)的减敏是一个复杂的过程,它包括同源性减敏和异源性减敏.同源性减敏主要是由G蛋白偶联受体激酶(GRKs)和arrestins介导的快速过程,同时可伴随异源性减敏发生.许多途径都可引起异源性减敏,在此简要综述了四种引起异源性减敏的途径:(1)非肾上腺素受体引起的蛋白激酶C激活引起α1Β-AR磷酸化或减敏; (2)激活与Gq偶联的受体,如ETA受体,使α1Β-AR磷酸化或减敏; (3) 激活与Gi偶联的受体,如溶血磷脂酸受体, 使α1Β-AR磷酸化或减敏; (4)受体通过内源性酪氨酸激酶活化引起α1Β-AR磷酸化或减敏. 所有这些减敏机制都只是初步探索,确切机制和意义有待进一步研究.     

α1-肾上腺素受体对胶原诱导性关节炎小鼠Treg细胞功能的影响*

目的: 利用胶原诱导性关节炎 (CIA) 模型小鼠,探讨去甲肾上腺素 (NE) 及其α1-肾上腺素受体 (AR ) 对CIA小鼠Treg细胞的作用。方法: 雄性DBA/1小鼠 32 只,随机分为对照组 (n=8) 和CIA模型组 (n=24)。II型胶原 (CII) 乳剂100 μl 尾根部注射DBA/1小鼠制备CIA小鼠模型,在初次免疫后第 41 日,用免疫荧光法检测小鼠脾脏中CD4⁺T与α1-AR的共定位情况;用Western blot法检测小鼠踝关节和脾脏中α1-AR的蛋白表达。分离纯化CIA小鼠脾脏中CD4⁺ T细胞,用抗CD3和抗CD28的单克隆抗体刺激CD4⁺T细胞,进行细胞培养,分为未加药组和加药组,加药组用NE或α1-AR激动剂苯肾上腺素 (phenylephrine) 处理细胞,用流式细胞术检测CIA小鼠CD4⁺T细胞中Treg的细胞数;用Western blot法检测CIA小鼠CD4⁺T中转化生长因子-β (TGF-β) 和IL-10的蛋白表达。结果: CD4⁺ T细胞能够表达α1-AR;与对照组相比,CIA小鼠踝关节和脾脏中α1-AR的蛋白表达显著降低(P＜0.01);与未加药的CIA小鼠的CD4⁺ T细胞相比,NE加入后的CIA小鼠CD4⁺ T细胞中Treg细胞的功能显著增强(P＜0.01);α1-AR激动剂phenylephrine加入后的CIA小鼠CD4⁺ T细胞中Treg细胞的功能显著增强(P＜ 0.01)。结论: 激活CIA小鼠CD4⁺ T细胞上的α1-AR可增强Treg细胞的功能,促进CD4⁺ T细胞向Treg细胞方向分化,发挥抗炎作用。     

缺氧大鼠心肌α1,β肾上腺素能受体的变化

为了探讨α_1、β肾上腺素能受体在大鼠缺氧性心脏肥大进程中的作用,本研究应用放射配基结合法观察了不同缺氧时间大鼠心室α_1、β肾上腺素能受体变化的动态过程,同时也观察了α_1、β受体阻断剂在预防缺氧性心肌肥大发生中的作用。缺氧10d后,测定右心室重量及组织学检查未见右心室肥大,但此时心室肌α_1受体数量从对照组的27.49±1.25增加到33.80±0.90fmol/mg蛋白(P<0.05);β受体从对照组的51.80±7.60下降到25.10±2.30fmol/mg蛋白(P<0.01)。缺氧20和30d后α_1受体进一步增加到40.70±1.43和32.30±1.96fmol/mg蛋白(P<0.05);β受体分别为27.90±2.30和42.80±1.70fmol/mg蛋白(P<0.05)。缺氧20和30d后右心室重量指数明显高于对照组,在整个缺氧过程中α_1、β受体的亲和性(K_d)未见明显变化,未见左心室肥大。缺氧同时应用α_1受体阻断剂(哌唑嗪4mg·kg~(-1)·d~(-1))30d,可明显预防缺氧所致的右心肥大;而β受体阻断剂(心得安10mg·kg~(-1)·d~(-1))没有此种作用。由此可见,在缺氧所致右心肥大之前,心肌α_1受体数量即明显升高,α_1受体阻断剂可以预防缺氧引起的右心肥大。     

Pharmacology of the agonist binding sites of rat neuronal nicotinic receptor subtypes expressed in HEK 293 cells

The binding affinities of agonists at heteromeric nicotinic receptors composed of rat alpha2, alpha3 and alpha4 subunits in combination with beta2 or beta4 subunits were examined in stably transfected HEK 293 cells. In most cases, the affinities of agonists were higher at receptors composed of an alpha subunit in combination with the beta2 subunit than the beta4 subunit, and in some cases this difference was quite large (>250 times), suggesting the possibility of developing subtype-selective ligands and therapeutically useful drugs.     

Comparison of neuronal and endothelial isoforms of nitric oxide synthase in stably transfected HEK 293 cells

The neuronal and endothelial isoforms of nitric oxide (NO) synthase (nNOS and eNOS, respectively) both catalyze the production of NO but are regulated differently. Stably transfected HEK 293 cell lines containing nNOS, eNOS, and a soluble mutant of eNOS were therefore established to compare their activity in a common cellular environment. NOS activity was determined by measuring L-[3H]citrulline production in homogenates and intact cells, the conversion of oxyhemoglobin to methemoglobin, and the production of cGMP. The results indicate that nNOS is more active than eNOS, both in unstimulated as well as calcium-stimulated cells. Under basal conditions, the soluble mutant of eNOS appeared to be slightly more active than wild-type eNOS in terms of NO and cGMP formation, suggesting that membrane association may be crucial for inhibition of basal NO release but is not required for stimulation by Ca2+-mobilizing agents. The maximal activity of soluble guanylate cyclase was significantly reduced by transfection with wild-type eNOS due to downregulation of mRNA expression. These results demonstrate that nNOS and eNOS behave differently even in an identical cellular environment.     

α1-肾上腺素受体亚型介导细胞内游离Ca2+增高的信号转导途径

本文探讨了α１ａ,α１ｂ,α１ｄ三种亚型肾上腺素受体激动时细胞内Ｃａ６２＋浓度升高的信号转导途径。在稳定表达三亚型α１－ＡＲ的ＨＥＫ２９３细胞２系中,用ｆｕｒａ－２方法细胞内Ｃａ＾２＋信号强弱的变化。结果显示,百日咳毒素对去甲肾上腺素激动三亚型α１－ＡＲ而引起的「Ｃａ＾２＋」ｉ升高无影响,Ｕ－７３１２２和ＰＭＡ明显抑制「Ｃａ＾２＋」ｉ升高．     

Large-scale purification and characterization of human parathyroid hormone-1 receptor stably expressed in HEK293S GnTI- cells

Human parathyroid hormone-1 receptor (hPTHR1) belongs to class II of the G protein-coupled receptor (GPCR) family, whose members all contain a seven-transmembrane helix domain. The receptor regulates bone metabolism through interactions with its ligand, human parathyroid hormone (hPTH). For structural studies of the hPTHR1/hPTH complex, we constructed a mammalian cell line to stably express recombinant hPTHR1 in large-scale. The receptor was solubilized with dodecyl maltoside and purified with affinity chromatography. The purified receptor displayed restricted N-glycosylation as expected. Functionality was demonstrated: the hPTHR1 retained affinity for bPTH-(1-34) and specifically cross-linked to a radioiodinated bPTH-(1-34) analog. This work describes an approach for preparing milligram-scale quantities of receptor for elucidation of the structural biology of this seven-transmembrane GPCR.     

Properties of HERG channels stably expressed in HEK 293 cells studied at physiological temperature.

We have established stably transfected HEK 293 cell lines expressing high levels of functional human ether-a go-go-related gene (HERG) channels. We used these cells to study biochemical characteristics of HERG protein, and to study electrophysiological and pharmacological properties of HERG channel current at 35 degrees C. HERG-transfected cells expressed an mRNA band at 4.0 kb. Western blot analysis showed two protein bands (155 and 135 kDa) slightly larger than the predicted molecular mass (127 kDa). Treatment with N-glycosidase F converted both bands to smaller molecular mass, suggesting that both are glycosylated, but at different levels. HERG current activated at voltages positive to -50 mV, maximum current was reached with depolarizing steps to -10 mV, and the current amplitude declined at more positive voltages, similar to HERG channel current expressed in other heterologous systems. Current density at 35 degrees C, compared with 23 degrees C, was increased by more than twofold to a maximum of 53.4 +/- 6.5 pA/pF. Activation, inactivation, recovery from inactivation, and deactivation kinetics were rapid at 35 degrees C, and more closely resemble values reported for the rapidly activating delayed rectifier K+ current (I(Kr)) at physiological temperatures. HERG channels were highly selective for K+. When we used an action potential clamp technique, HERG current activation began shortly after the upstroke of the action potential waveform. HERG current increased during repolarization to reach a maximum amplitude during phases 2 and 3 of the cardiac action potential. HERG contributed current throughout the return of the membrane to the resting potential, and deactivation of HERG current could participate in phase 4 depolarization. HERG current was blocked by low concentrations of E-4031 (IC50 7.7 nM), a value close to that reported for I(Kr) in native cardiac myocytes. Our data support the postulate that HERG encodes a major constituent of I(Kr) and suggest that at physiological temperatures HERG contributes current throughout most of the action potential and into the postrepolarization period.     

Functional characterization of mouse alpha4beta2 nicotinic acetylcholine receptors stably expressed in HEK293T cells

Mouse alpha4beta2 nicotinic acetylcholine receptors (nAchRs) were stably expressed in HEK293T cells. The function of this stable cell line, termed mmalpha4beta2, was assessed using an aequorin-based luminescence method that measures agonist-evoked changes in intracellular calcium. Agonist-elicited changes in intracellular calcium were due primarily to direct entry of calcium through the alpha4beta2 channel, although release of calcium from intracellular stores contributed approximately 28% of the agonist-evoked response. Agonist pharmacologies were very similar between the mmalpha4beta2 cells and most cell lines that stably express human alpha4beta2 nAchRs. Based on agonist profiles and sensitivity to the antagonist dihydro-beta-erythroidine (DHbetaE), the predominant alpha4beta2 nAchR expressed in the mmalpha4beta2 cells exhibits a pharmacology that most resembles the DHbetaE-sensitive component of 86Rb+ efflux from mouse brain synaptosomes. However, when evaluated with the aequorin assay, the mmalpha4beta2 nAchR was found to be atypically sensitive to blockade by the presumed alpha7-selective antagonist methyllycaconitine (MLA), exhibiting an IC50 value of 31 +/- 0.1 nm. Similar IC50 values have been reported for the MLA inhibition of nicotine-stimulated dopamine release, a response that is mediated by beta2-subunit-containing nAchRs and not alpha7-subunit-containing nAchRs. Consequently, at low nanomolar concentrations, MLA may not be as selective for alpha7-containing nAchRs as previously thought.     

cAMP and IP3 signaling pathways in HEK293 cells transfected with canine olfactory receptor genes

Olfactory receptors (ORs) expressed at the cell surface of olfactory sensory neurons lining the olfactory epithelium are the first actors of events leading to odor perception and recognition. As for other mammalian ORs, few dog OR have been deorphanized, mainly because of the absence of good methodology and the difficulties encountered to express ORs at the cell surface. Within this work, our aim was 1) to deorphanize a large subset of dog OR and 2) to compare the implication of the 2 main pathways, namely the cAMP and inositol 1,4,5-triphosphate (IP3) pathways, in the transduction of the olfactory message. For this, we used 2 independent tests to assess the importance of each of these 2 pathways and analyzed the responses of 47 canine family 6 ORs to a number of aliphatic compounds. We found these ORs globally capable of inducing intracellular calcium elevation through the IP3 pathway as confirmed by the use of specific inhibitors and/or a cAMP increase in response to aldehyde exposure. We showed that the implication of the cAMP or/and IP3 pathway was dependent upon the ligand-receptor combination rather than on one or the other partner. Finally, by exposing OR-expressing cells to the 21 possible pairs of C6-C12 aliphatic aldehydes, we confirmed that some odorant pairs may have an inhibitory or additive effect. Altogether, these results reinforce the notion that odorant receptor subfamilies may constitute functional units and call for a more systematic use of 2 complementary tests interrogating the cAMP and IP3 pathways when deorphanizing ORs.     

The preparation of HEK293 alpha1A or HEK293 alpha1B cell membrane stationary phase and the chromatographic affinity study of ligands of alpha1 adrenoceptor

As a novel bioaffinity chromatographic technique, cell membrane chromatography (CMC) originated in 1996. The cell membrane stationary phase (CMSP) consists of porous silica coated with active cell membranes. By immersing silica into a suspension of cell membranes, the whole surface of silica was covered by the cell membranes. The present study repeatedly investigated the interaction between ligands and receptors by employing the system of CMC and especially evaluated the accuracy and feasibility of the CMC model in the study of subtype receptors. The cDNA encoding alpha1A or alpha1B adrenergic receptors (ARs) was transfected into human embryonic kidney 293 (HEK293) cell lines; cell lines stably overexpressing subtype receptors were obtained. HEK293 alpha1A or HEK293 alpha1B ARs CMSP were prepared by immobilizing relevant cell membranes on silica. In the described chromatography-based CMSP, the retention times of nine alpha1 adrenoceptor ligands and calculated capacity factors, as chromatographic parameters, were recorded carefully. These results showed a good correlation with the affinity of the same compounds for the corresponding cloned alpha1 adrenoceptor subtype. The rank order of capacity factors was consistent with the affinity rank order obtained from radioligand binding assays. The immobilized subtype-selective CMSP was stable and reproducible. The study demonstrates that the HEK293 alpha1A and HEK293 alpha1B CMSP can be utilized for initial screening of drug candidates.