α1—肾上腺素受体的信号转导

α１－肾上腺受体可激活细胞内多种信号转导途径,除经典的磷酸肌醇－钙信号系统与二酰基甘油－蛋白激酶Ｃ信号系统外,还包括磷脂酶Ａ２－花生四烯酸信号系统,酪氨酸激酶磷酸化系统与腺苷酸环化酶－ｃＡＭＰ信号系统等。α１－ＡＲ所介导的诸多瀑布式的链级信号系统,呈网络状连接在一起,一种信号系统的激活可引发、加强或阻抑另一信号系统。这些信号系统参与α１－ＡＲ介导的一系列生理和病理生理过程。不同的α１－ＡＲ亚型所介     

老年大鼠血管α1肾上腺素受体及其亚型的改变

本工作用离体与整体实验方法,研究了老年(18月龄)与年轻(3月龄)大鼠血管中 α_1肾上腺素受体储备和 α_1(?)与 α_(1b)亚型比值的差别。在离体实验中用机械方法分离血管,用 Krebs 溶液灌流,在灌流液中加入 α_2和 β肾上腺素受体拮抗剂,然后用去甲肾上腺素(NE)激动 α_1受体。结果显示;老年大鼠主动脉、肾动脉和肠系膜动脉由去甲肾上腺素(NE)引起的最大收缩反应与年轻大鼠无显著差别,但浓度-效应曲线显著右移,功能性解离常数 K_A 值不变,而 K_A 与EC_(50)的比值减小。此外,在老年大鼠主动脉和肠系膜动脉血管,选择性 α_(1b)亚型拮抗剂 CEC对 NE 引起的缩血管效应的阻断作用显著减弱,硝苯吡啶(选择性阻断 α_(1a) 亚型的效应)对 NE 缩血管效应的阻断作用显著增强。整体实验显示老年大鼠硝苯吡啶的降血压作用比年轻大鼠增强,在用硝苯吡啶的基础上给予苯肾上腺素升血压作用减弱。上述结果提示:与年轻大鼠相比较,老年大鼠 α_(1-) 肾上腺素受体储备减少,α_(1a) 亚型相对 α_(1b)亚型的比率增高。     

自发性高血压大鼠血管α1肾上腺素受体亚型的改变

本工作在离体与整体条件下比较易卒中型自发性高血压(SHRSP)大鼠与WKY大鼠血管中α_1受体的两种亚型。在离体灌流的主动脉、肾动脉与肠系膜动脉,50μmol/L氯甲基可乐定(CEC)预温育30min可使α_1受体激动时引起的最大收缩张力在SHRSP与WKY大鼠分别降为对照时的31.4±8.3％与35.2±2.9％,68.4±8.2％与80.1±7.3％,68.4±6.3％与55.4±7.0％,两者间均无显著性差别。但10μmol/L硝苯吡啶对α_1受体收缩效应的阻断作用则在SHRSP大鼠大大超过WKY大鼠,最大收缩张力分别降为对照时的3.1±1.5％与56.5±4.8％(P<0.01),9.0±4.1％与23.6±3.5％(P<0.05),5.9±2.5％与28.0±0.8％(P<0.01)。整体动物实验也显示硝苯吡啶的降血压作用及对苯肾上腺素升血压效应的阻断作用在SHRSP大鼠都较WKY大鼠显著增强。离体主动脉a_1受体激动时的快速相与持续相收缩均主要由α_(1B)亚型激动引起,硝苯吡啶对快速相收缩的阻断作用在SHRSP与WKY大鼠无显著性差别,但对持续相收缩的阻断作用则在SHRSP大鼠显著强于WKY大鼠。上述结果提示SHRSP大鼠血管α_1受体两种亚型的分布没有显著改变,但α_(1B)受体激动时继发性细胞外Ca~(2+)进入的途径由非双氢吡啶敏感性钙通道转变为双氢吡啶敏感性钙通道。     

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。     

氧自由基对大鼠心肌α1肾上腺素受体及其亚型的影响

采用放射配体结合方法观察外源性氧自由基对大鼠心肌α_１肾上腺素受体（α_１受体）及其亚型α_（１Ａ）与α_（１Ｂ）的影响和对β肾上腺素受体（β受体）的影响。发现：（１）·ＯＨ,使α_１受体与~（１２５）ＩＢＥ２２５４最大结合量（Ｂ_（ｍａｘ））分别下降５５％与３６％。（２）·ＯＨ可使α_１受体与~（１２５）ＩＢＥ２２５４结合的Ｋ_ｄ值增高１７１％,而对Ｋ_ｄ值无影响。（３）·ＯＨ,均使α_１受体亚型α_（１Ａ）与α_（１Ｂ）之间的比例增加。（４）·ＯＨ,对β受体与~（１２５）ＩＰＩＮ最大结合量Ｂ_（ｍａｘ）和Ｋ_ｄ值均无影响。（５）氧自由基清除剂甘露醇或超氧化物歧化酶均可对抗氧自由基引起α_１受体及其亚型的改变。上述结果提示,氧自由基可直接作用于心肌膜,引起α_１受体数量和亲和性下降,α_１受体亚型α_（１Ａ）与α_（１Ｂ）之间的比例改变。     

12月与10周龄大鼠主动脉功能性α1肾上腺素受体亚型比较

本文采用离体血管收缩功能实验方法,比较１２月与１０周龄大鼠主动脉α１肾上腺素受体及其亚型的分布。结果显示;１２月龄大鼠与１０周龄大鼠相比,去甲肾上腺素引起的最大收缩反应显著降低,而半效激动浓度值无显著性改变;氯乙基可乐定对ＮＥ引起血管收缩的抑制作用明显减弱;ＷＢ４１０１拮抗ＮＥ缩血管效应的ＰＡ２值没有改变;ＢＭＹ７３７８的半效抑制浓度值明显降低;Ｓｅｒｔｉｎｄｏｌｅ的ｐＡ２值显著增大。     

心力衰竭时β肾上腺素受体亚型对心脏调节作用的变化及其临床意义

在运动或应激状况下,β肾上腺素受体(β AR)的激活可有力地增加心输出量.然而,心衰时持续的β AR激活可导致心肌肥大、心肌细胞凋亡等病理性心肌重塑过程.目前认为,心肌细胞表面存在结构、效应特异的三种β AR亚型:β1、β2及β3 AR.β1AR可激活经典的Gs-AC-cAMP-PKA信号通路;β2AR同时激活Gs-AC-cAMP-PKA及Giα-Giβγ-PI3K-Akt信号通路;而β3 AR则通过Gi-eNOS-NO-cGMP介导负性变力效应.目前研究表明:心衰时长期的β1AR激活可通过GsCa2+-CaMKⅡ通路导致心肌肥大、心肌细胞凋亡等病理性心肌重塑过程;而持续的β2AR刺激则通过Giα-Giβγ-PI3K-Akt通路产生抗心肌肥大、心肌细胞凋亡效应.对心衰时上述βAR亚型的信号转导、效应的深入认识不仅对βAR阻滞剂治疗慢性心衰提供了分子和细胞机制的依据,而且为我们带来了一些治疗慢性心衰的新思路.     

α2-肾上腺素受体介导大鼠主动脉收缩的特性

为阐明功能性α2-肾上腺素受体(α2-AR)的特性及其与α2-AR的相互关系,以离体大鼠主动脉为模型,进行收缩功能实验.发现在大鼠主动脉中,α2-AR和α2-AR均可介导其收缩效应并以α1-AR的作用为主.α1-AR可增强α2-AR介导的收缩效应,而α2-AR则对α1-AR介导的收缩效应无影响.在不可逆阻断α1-AR而保留α2-AR的条件下,α2-AR介导的缩血管效应消失,仅在阈值浓度的KCl在下才能显示,且收缩幅度较对照显著降低.结果表明,在离体大鼠主动脉中存在功能性α2-AR,α2-AR的缩血管作用依赖于α1-AR的激动,其最大收缩反应远远小于α1-AR.     

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     

激动剂作用下α1-肾上腺素受体亚型在HEK293A细胞中的分布变化

为了明确α1-肾上腺素受体（α1-adrenergic receptor,α1-AR）三种亚型在人胚胎肾（human embryonic kidney,HEK）293A细胞株中的分布特点,及其在激动剂作用下在细胞内的定位改变,本研究采用放射配体结合实验、实时荧光共聚焦成像和Western blot方法检测α1-AR三种亚型在细胞中的定位及蛋白质表达的变化。结果发现：（1）α1-AR三种亚型在HEK293A细胞株转染效率相同,均达90％以上。三株细胞的粗制膜上α1B-AR表达量最高,α1D-AR最低,α1A-AR居中,但三者的解离常数（配）相等;（2）在无激动剂作用时,α1A-AR均匀地分布在HEK293A细胞的胞膜和胞浆,α1B-AR主要位于胞膜,而α1D-AR则主要分布在胞浆中：（3）用α1-AR激动剂苯‘肾上腺素（phenylephrine,PE）刺激细胞1h后,α1A-和α1B-AR在胞膜上分布明显减少,而在胞浆中分布增加,其中α1B-AR变化更为显著,α1D-AR的分布在PE作用下无明显变化。以上结果提示,在激动剂作用下,α1-AR二种亚型在HEK293A细胞中的定位特点和分布变化各有不同。     

Direct coupling between arachidonic acid-induced Ca2+ release and Ca2+ entry in HEK293 cells

Arachidonic acid (AA) modulates intracellular Ca2+ signaling via Ca2+ release or/and Ca2+ entry. However, the mechanism underlies either process is unknown; nor is it clear as to whether the two processes are mechanistically linked. By using Fura2/AM, we found that AA induced mobilization of internal Ca2+ store and an increment in Ca2+, Mn2+ and Ba2+ influx in HEK293 cells. The AA-mediated Ca2+ signaling was not due to AA metabolites, and insensitive to capacitative Ca2+ entry inhibitors. Interestingly, isotetrandrine and Gd3+ inhibited both AA-induced Ca2+ release and Ca2+ entry in a concentration-dependent manner without affecting Ca2+ discharge caused by carbachol, caffeine, or thapsigargin. Additionally, similar pattern of inhibition was observed with tetracaine treatment. More importantly, the three compounds exhibited almost equal potent inhibition of AA-initiated Ca2+ release as well as Ca2+ influx. Therefore, this study, for the first time, provides evidence for a direct coupling between AA-mediated Ca2+ release and Ca2+ entry.     

The biochemical activation of T-type Ca2+ channels in HEK293 cells stably expressing alpha1G and Kir2.1 subunits

In order to investigate the currently unknown cellular signaling pathways of T-type Ca(2+) channels, we decided to construct a new cell line which would stably express alpha(1G) and Kir2.1 subunits in HEK293 cells (HEK293/alpha(1G)/Kir2.1). Compared to cells which only expressed alpha(1G) (HEK293/alpha(1G)), HEK293/alpha(1G)/Kir2.1 cells produced an enormous inward rectifying current which was blocked by external Ba(2+) and Cs(+) in a concentration-dependent manner. The expression of Kir2.1 channels contributed significantly to the shift of membrane potential from -12.2+/-2.8 to -57.3+/-3.7mV. However, biophysical and pharmacological properties of alpha(1G)-mediated Ca(2+) channels remained unaffected by the expression of Kir2.1 subunits, except for the enlarging of the window current region. Biochemical activation of alpha(1G) channels using 150mM KCl brought about an increase in [Ca(2+)](i), which was blocked by mibefradil, the T-type Ca(2+) channel blocker. These data suggest that the HEK293/alpha(1G)/Kir2.1 cell line would have potential uses in the study of T-type Ca(2)(+) channel-mediated signaling pathways and possibly useful in the development of new therapeutic drugs associated with T-type Ca(2)(+) channels.     

Activation of muscarinic receptors reduces store-operated Ca2+ entry in HEK293 cells

In many cell types membrane receptors for hormones or neurotransmitters activate a signal transduction pathway which releases Ca2+ from intracellular Ca2+ stores by the second messenger inositol 1,4,5-trisphosphate. As a consequence store-operated Ca2+ entry (SOCE) becomes activated. In the present study we addressed the question if receptor/agonist binding can modulate Ca2+ entry by mechanisms different from the store-operated one. Therefore SOCE was examined in HEK293 cells microscopically with the fura-2 technique and with patch clamp. We found that maximally preactivated SOCE could, concentration dependently, be reduced up to 80% by the muscarinic agonist acetylcholine when the cytoplasmic Ca2+ concentration was used as a measure. Muscarinic receptors seem to mediate this decrease since atropine blocked the effect completely and cell types without muscarinic receptors (BHK21, CHO) did not show acetylcholine-induced decrease of Ca2+ entry. Moreover expression of muscarinic receptor subtypes M1 and M3 in BHK21 cells established the muscarinic decrease of SOCE. Electrical measurements revealed that the membrane potential of HEK293 cells did not show any response to ACh, excluding that changes of driving forces are responsible for the block of Ca2+ entry. In contrast the electrical current which is responsible for SOCE in HEK293 cells (Ca2+ release-activated Ca2+ current (I(CRAC)) was inhibited (maximally 55%) by 10 microM ACh. From these data we conclude that in HEK293 cells a muscarinic signal transduction pathway exists which decreases the cytoplasmic Ca2+ concentration by an inhibition of I(CRAC). This mechanism may serve as a modulator of Ca2+ entry preventing a Ca2+ overload of the cytoplasm after Ca2+ store depletion.     

Effects of cannabinoids on endogenous K+ and Ca2+ currents in HEK293 cells

Effects of cannabinoids on endogenous potassium and calcium currents in HEK293 cells were studied using the whole-cell variant of the patch-clamp technique. The cannabinoid agonists WIN 55,212-2, methanandamide, and anandamide (1 microM) decreased the calcium current by 53.1 +/- 2.6, 47.5 +/- 1.2, and 38.8 +/- 3.1%, respectively, after transfection of human CB1 cannabinoid receptor (hCB1) cDNA into HEK293 cells. The delayed rectifier-like current was not changed after application of these agonists, but the inward rectifier was increased by 94.0 +/- 3.6, 83.7 +/- 5.1, and 63.0 +/- 2.5% after application of WIN 55,212-2, methanandamide, and anandamide, respectively. The effects of the cannabinoid antagonists (AM251, AM281, and AM630) on the inward rectifier and calcium currents were the opposite of those seen with cannabinoid agonists; thus, these compounds act as inverse agonists in this preparation. These results suggest that endogenous inward rectifier and calcium currents are modulated by cannabinoids in HEK293 cells, and that some expressed receptors may be constitutively active.     

Over-expression of GFP-FEZ1 causes generation of multi-lobulated nuclei mediated by microtubules in HEK293 cells

FEZ1 (Fasciculation and elongation protein zeta 1) is an ortholog of the Caenorhabditis elegans protein UNC-76, involved in neuronal development and axon outgrowth, in that worm. Mammalian FEZ1 has already been reported to cooperate with PKC-zeta in the differentiation and polarization of PC12 neuronal cells. Furthermore, FEZ1 is associated with kinesin 1 and JIP1 to form a cargo-complex responsible for microtubule based transport of mitochondria along axons. FEZ1 can also be classified as a hub protein, since it was reported to interact with over 40 different proteins in yeast two-hybrid screens, including at least nine nuclear proteins. Here, we transiently over-expressed GFP-FEZ1full in human HEK293 and HeLa cells in order to study the sub-cellular localization of GFP-FEZ1. We observed that over 40% of transiently transfected cells at 3 days post-transfection develop multi-lobulated nuclei, which are also called flower-like nuclei. We further demonstrated that GFP-FEZ1 localizes either to the cytoplasm or the nuclear fraction, and that the appearance of the flower-like nuclei depends on intact microtubule function. Finally, we show that FEZ1 co-localizes with both, α- and especially with γ-tubulin, which localizes as a centrosome like structure at the center of the multiple lobules. In summary, our data suggest that FEZ1 has an important centrosomal function and supply new mechanistic insights to the formation of flower-like nuclei, which are a phenotypical hallmark of human leukemia cells.     

Influence of chloride ions on alpha1-adrenoceptor mediated contraction and Ca2+ influx in rat caudal artery.

The objective of the present investigation was to compare and contrast the effects of 8-bromoguanosine 3':5'-cyclic monophosphate (8-Bromo-cyclic GMP), an analogue of guanosine 3':5'-cyclic monophosphate, felodipine, a dihydropyridine Ca2+ channel antagonist, and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), a putative chloride channel antagonist on alpha1-adrenoceptor mediated contraction and Ca2+ influx in rat caudal artery, in normal physiological salt solution and in chloride-free solution. Isometric contractions and 45Ca2+ influx were measured in isolated rat caudal arterial rings. Phenylephrine induced concentration-dependent contractions were inhibited by 8-Bromo-cyclic GMP (10 microM), felodipine (10 nM) and NPPB (3.0 microM). Removal of chloride ions also impaired phenylephrine-induced contractions. In chloride-free buffer, phenylephrine-induced contractions were partially inhibited by the presence of 8-Bromo-cGMP or felodipine, while NPPB had no effect. Phenylephrine induced 45Ca2+ influx was inhibited by the presence of 8-Bromo-cyclic GMP, felodipine and NPPB. Moreover, removal of chloride ions also inhibited phenylephrine-induced 45Ca2+ influx. The results of our study demonstrate that in the rat caudal artery the inhibitory effects of 8-Bromo-cyclic GMP, felodipine and NPPB, are mediated through a reduction of Ca2+ influx. In addition, chloride ions, in part, play a role in alpha1-adrenoceptor-mediated Ca2+ influx. However, the influence of removal of chloride ions on phenylephrine stimulated contraction is limited. Moreover, 8-Bromo-cyclic GMP and felodipine, but not NPBB, impair phenylephrine-induced contractions in the absence of chloride ions.     

Ca2+及其信号转导途径与长时程增强的关系

Ca2 作为信号转导过程中的第二信使,参与机体的各种反应,尤其在神经突触可塑性方面,突触前后Ca2 浓度的变化发挥了重要的信息传递作用.Ca2 在长时程增强(long-term potentiation,LTP)过程中也发挥重要作用.它不仅是LTP产生的触发器,而且能通过激活下游的蛋白激酶、磷酸化ERK,以及活化即刻早期基因(IEGs)等促进基因转录和蛋白质合成,最终参与LTP的维持.