G-蛋白偶联受体自身抗体与高血压

高血压病发病机制复杂 ,包括神经、内分泌、遗传等诸多方面。最近研究发现 ,高血压病与Grave’s病、系统性红斑狼疮一样 ,免疫异常在其发生发展中发挥重要作用。高血压病免疫异常包括细胞免疫异常 (如T细胞数目、Th1/Th2比值、T细胞功能等 )及体液免疫异常 (如血清Ig升高、自身抗体出现、细胞因子及补体异常等 ) ,特别是自身抗体的出现 ,这些抗体主要有抗核抗体、抗平滑肌抗体、抗心肌抗体及抗G蛋白偶联受体抗体[1] ,其中G蛋白偶联受体自身抗体研究较深入。　　G蛋白偶联受体是与GTP结合蛋白偶联的一组膜表面糖蛋白 ,由 7个多肽链形成…     

G-蛋白偶联受体自身抗体与高血压

高血压病发病机制复杂,包括神经、内分泌、遗传等诸多方面.最近研究发现,高血压病与Grave's病、系统性红斑狼疮一样,免疫异常在其发生发展中发挥重要作用.高血压病免疫异常包括细胞免疫异常(如T细胞数目、Th1/Th2比值、T细胞功能等)及体液免疫异常(如血清Ig升高、自身抗体出现、细胞因子及补体异常等),特别是自身抗体的出现,这些抗体主要有抗核抗体、抗平滑肌抗体、抗心肌抗体及抗G蛋白偶联受体抗体[1],其中G蛋白偶联受体自身抗体研究较深入.     

心房颤动与自身抗体的研究新进展

多项研究显示自身免疫是心律失常的主要机制之一,已有一些证据显示心房颤动(AF)是由自身免疫中的自身抗体介导的。新近的研究提示自身抗体在AF中发挥了重要作用,如抗M2受体自身抗体通过蛋白耦联,减少环磷酸腺苷(cAMP)活性,激活电压依赖性钾通道引发AF;抗β-肾上腺素能受体自身抗体水平升高,使cAMP升高,触发钙离子释放诱发AF;抗热休克蛋白自身抗体通过释放炎症介质,产生免疫炎症应激反应触发AF;抗肌球蛋白重链自身抗体水平升高,可使肿瘤坏死因子(TNF-α)增多,从而对心房肌产生免疫、炎症反应来触发AF;抗钠/钾ATP酶自身抗体水平升高导致Na/K-ATP酶减低,致使Ca离子释放增多而肾上腺素能受体AF发生;综上,自身抗体对AF的发生、发展可能起着重要的作用。     

老年慢性心力衰竭患者血清β1肾上腺素受体自身抗体与心室复极离散度的相关研究

目的研究老年慢性心力衰竭患者血清抗β1肾上腺素受体自身抗体与心室复极离散度的相关性。方法观察2006～2009年老年冠心病慢性心力衰竭患者58例,在强心、利尿治疗的同时,给予酒石酸美托洛尔治疗6月,检测治疗前后心脏超声、脑钠肽(BNP)、动态心电图和抗β1肾上腺素受体自身抗体阳性率的改变,同时检测治疗前后血压、心率、肝肾功能并进行比较。结果与治疗前相比,心力衰竭患者的基础心率和BNP下降,心脏射血分数(EF)升高,心脏功能得到改善;同时发现,酒石酸美托洛尔治疗后,血清中抗β1肾上腺素受体自身抗体的阳性率均明显下降(P0.01),动态心电图检测QTc和QTd也明显下降(P0.01);相关性分析表明,抗β1肾上腺素受体自身抗体阳性与QTd延长有关(P0.01)。结论老年慢性心力衰竭患者中抗β1肾上腺素变体抗体阳性与QTd明显相关,应用酒石酸美托洛尔能有效降低β1肾上腺素受体自身抗体的滴度和QTd,从而有效预防室性心律失常的发生。     

风湿性心脏病患者血清抗β1肾上腺素受体自身抗体的生物学效应

目的探讨风心病患者血清中抗β1肾上腺素能受体（β1AR）自身抗体的生物学效应。方法①应用SA-ELISA方法,检测风心病患者（60例）,正常人（97例）,以及心衰大鼠（33只）和对照大鼠（20只）血清标本中的抗β1AR自身抗体;②亲和层析法纯化风心病患者、心衰大鼠抗体阳性血清中的IgGs;③观察抗β1AR自身抗体IgGs对乳鼠心肌细胞搏动频率的影响;④测定抗β1AR自身抗体IgGs对成年大鼠心肌细胞腺苷酸环化酶活性的影响。结果风心病患者血清中抗β1AR自身抗体的阳性率和抗体滴度分别为43%和1∶（98.3±1.6）,均显著高于正常人的4%和1∶（14.6±2.7）,P<0.01,类似的结果见于心衰大鼠[85%,1∶（67.3±2.4）]与对照大鼠[5%,1∶（17.3±2.1）]（P<0.01）之间;纯化的风心病患者、心衰大鼠抗β1AR抗体IgGs均可增加乳鼠心肌细胞的搏动频率,且在6 h内不脱敏;还可增强成年大鼠心肌细胞的腺苷酸环化酶活性,使cAMP产生量增加。结论风心病患者血清中的抗β1AR自身抗体具有激动剂样刺激效应,即具有正性变时效应（不易脱敏）、可增强腺苷酸环化酶活性。提示抗β1AR自身抗体可能是风心病患者神经体液调节功能紊乱的原因之一。     

血清抗AT1、α1、β1受体自身抗体在高血压合并肾损害患者的初步研究

目的探讨抗血管紧张素Ⅱ受体1型(AT1受体)受体,α1肾上腺素受体(α1受体)、β1肾上腺素受体(β1受体)自身抗体是否与高血压病合并肾损害有关。方法以合成的β1、AT1、α1受体多肽片段为抗原,应用酶联免疫吸附测定(ELISA)技术,检测56例高血压并肾损害患者和58例高血压无肾损患者及40例正常人血清中抗G蛋白偶联型β1、AT1、α1受体自身抗体。结果高血压病并肾损害组抗β1、AT1、α1受体抗体阳性率分别为67.9%(38/56)、46.4%(26/56)、46.4%(26/56),明显高于高血压无肾损害组的19%(11/58)、15.5%(9/58)、12.1%(7/58)和及正常对照组的15%(6/40)、10%(4/40)、12.5%(5/40),比较具有显著性差异(P<0.01)。结论抗G蛋白偶联型受体自身抗体可能与高血压合并肾损害发病有关。     

血管紧张素Ⅱ1型受体、α1和β1肾上腺素能受体自身抗体与甲状腺毒症性心脏病相关性分析

目的 探讨抗G-蛋白耦联型血管紧张素Ⅱ1型受体(AT1R)、α1肾上腺素能受体(α1R)和β1肾上腺素能受体(β1R)自身抗体是否与甲状腺毒症性心脏病(THD)发病相关.方法 以细胞外第二环表位肽段的合成肽作为抗原,应用酶联免疫吸附技术检测277例受试者血清中AT1R、α1R和β1R自身抗体.237例甲状腺毒症(TT)患者分为治疗组(n=148)和恢复组(n=89)、或THD(n=46)和TT无心脏病组(n=191).正常对照组40名.结果 (1)TT组AT1R、α1R和β1R自身抗体阳性率分别为31.6％、27.8％和23.6％,明显高于正常对照组的12.5％ 、10.0％和7.5％ (P＜0.05);TT患者中弥漫性毒性甲状腺肿(GD)组3种受体自身抗体阳性率(36.3％、32.2％和28.1％)明显高于非GD组(19.7％、16.7％和12.1％,均P＜0.05).(2)TT治疗组AT1R和α1R自身抗体阳性率(40.5％和33.1％)明显高于TT恢复组(16.9％和19.1％,均P＜0.05).(3) THD组AT1R和α1R自身抗体阳性率(52.2％和43.5％)明显高于TT无心脏病组(26.7％和24.1％,均P＜0.05).结论 抗G-蛋白耦联型AT1R、α1R和β1R自身抗体可能与甲状腺毒症发病有关,且AT1R和α1R自身抗体在THD病理生理过程中发挥重要作用.     

扩张型心肌病与抗G-蛋白偶联受体的自身抗体

目的　探讨抗G 蛋白偶联型 β2 、α1和血管紧张素Ⅱ受体 1型 (AT1受体 )的自身抗体是否与扩张型心肌病的发病有关。方法　以细胞外第二环表位肽段的合成肽作为抗原 ,应用酶联免疫吸附测定 (ELISA)技术 ,检测 86例扩张型心肌病患者和 6 1例正常人血清中抗G 蛋白偶联型 β2 、α1和AT1受体的自身抗体。结果　 (1)扩张型心肌病组抗G 蛋白偶联型 β2 、α1和AT1受体自身抗体的阳性率分别为 4 4 2 %、4 5 3%和 4 0 7% ,明显高于正常组的 11 5 %、9 8%和 14 8% (P <0 0 1) ;(2 )扩张型心肌病组自身抗体阳性患者的抗体滴度分别为 1∶136、1∶12 4、1∶12 6 ,明显高于正常组的 1∶4 6、1∶32、1∶2 8(P <0 0 1) ;(3)扩张型心肌病组 β2 受体自身抗体阳性患者血清中 ,6 5 8%的患者同时具有α1受体的自身抗体 ;71 1%的患者同时具有AT1受体的自身抗体 ;4 7 4 %的患者同时具有上述 3种受体的自身抗体。结论　扩张型心肌病患者血清中不仅存在抗G 蛋白偶联型 β2 、α1和AT1受体的自身抗体 ,而且抗体滴度也明显高于正常对照组。约 5 0 %的扩张型心肌病患者同时具有上述 3种受体的自身抗体。提示免疫学机制可能参与扩张型心肌病的病理生理过程 ,参与机理有待进一步深入研究     

胆碱能受体和肾上腺素受体在肺部的分布、功能与相互作用

肺主要由交感和副交感双重神经支配，交感和副交感神经则分别依赖于其肾上腺素能和M受体的活性。肾上腺素能受体和M受体都是G蛋白耦联受体，具有相似的信号转导分子。这些受体在肺内广泛表达，不同物种表达不一。在正常气道功能的调节中受体表达的定位和亚型很重要。副交感纤维释放的乙酰胆碱激活气道平滑肌上的M3受体，引起支气管收缩。与此相反，位于副交感神经上的M2受体则抑制乙酰胆碱释放。β2肾上腺素受体表达于气道平滑肌，其激活可引起支气管扩张。自主神经中也存在肾上腺素受体，调节神经递质的释放。这些G蛋白耦联受体的交互作用及其下游信息传递能确保正常的气道功能。突触前后的M受体和肾上腺素能受体能调控气道张力，其任何失衡或受体的选择性阻断都会引起这种调控减弱，导致气道高反应性的发生。在设计、研发和使用治疗气道疾病药物时，必须考虑到肾上腺素受体和M受体的定位、功能及交互作用。     

扩张型心肌病抗心肌细胞膜自身抗体的初步研究

应用免疫转印技术检测21例DCM患者血清中抗MCM抗体,并与对照组19例作比较,结果发现,DCM患者血清中可检出抗MCM52KD(阳性率57.14％)、87kD(33.33％)、59kD(23.81％)和48kD(19.05％)各组份的自身抗体;而对照组血清则均为阴性。自身抗体所针对的靶抗原可能包括心肌细胞膜钙通道和β_1-肾上腺素能受体。本研究提示,自身免疫反应可能参与DCM的发病机制,检测抗MCM自身抗体可作为DCM的重要诊断指标。     

RhoGDI2的研究进展

Rho鸟苷酸解离抑制因子-2（RhoGDI2）是小G蛋白调节因子家族成员之-,调控多种小G蛋白的生物学活性。它对Rho蛋白有双重调节作用,并从上游调节鸟苷酸交换因子、GTP酶活化蛋白的活性。目前研究表明RhoGDI2影响肿瘤侵袭转移,在多种恶性肿瘤中的异常表达将参与肿瘤的发生发展。研究RhoGDI2将有助于加深对肿瘤侵袭转移机制的了解,为预防肿瘤转移提供特异性、多靶位的治疗方法。另外,RhoGDI2在β2肾上腺素能受体（β2AR）减敏的哮喘小鼠肺组织中高表达,研究RhoGDIz和β2AR的关系为临床防治肚AR减敏提供-个新的切入点和治疗方向。     

A macromolecular complex of beta 2 adrenergic receptor,CFTR, and ezrin/radixin/moesin-binding phosphoprotein 50 is regulated by PKA

It has been demonstrated previously that both the cystic fibrosis transmembrane conductance regulator (CFTR) and beta(2) adrenergic receptor (beta(2)AR) can bind ezrinradixinmoesin-binding phosphoprotein 50 (EBP50, also referred to as NHERF) through their PDZ motifs. Here, we show that beta(2) is the major adrenergic receptor isoform expressed in airway epithelia and that it colocalizes with CFTR at the apical membrane. beta(2)AR stimulation increases CFTR activity, in airway epithelial cells, that is glybenclamide sensitive. Deletion of the PDZ motif from CFTR uncouples the channel from the receptor both physically and functionally. This uncoupling is specific to the beta(2)AR receptor and does not affect CFTR coupling to other receptors (e.g., adenosine receptor pathway). Biochemical studies demonstrate the existence of a macromolecular complex involving CFTR-EBP50-beta(2)AR through PDZ-based interactions. Assembly of the complex is regulated by PKA-dependent phosphorylation. Deleting the regulatory domain of CFTR abolishes PKA regulation of complex assembly. This report summarizes a macromolecular signaling complex involving CFTR, the implications of which may be relevant to CFTR-dysfunction diseases.     

Protein kinase cross-talk: membrane targeting of the beta-adrenergic receptor kinase by protein kinase C.

The beta-adrenergic receptor kinase (betaARK) is the prototypical member of the family of cytosolic kinases that phosphorylate guanine nucleotide binding-protein-coupled receptors and thereby trigger uncoupling between receptors and guanine nucleotide binding proteins. Herein we show that this kinase is subject to phosphorylation and regulation by protein kinase C (PKC). In cell lines stably expressing alpha1B- adrenergic receptors, activation of these receptors by epinephrine resulted in an activation of cytosolic betaARK. Similar data were obtained in 293 cells transiently coexpressing alpha1B- adrenergic receptors and betaARK-1. Direct activation of PKC with phorbol esters in these cells caused not only an activation of cytosolic betaARK-1 but also a translocation of betaARK immunoreactivity from the cytosol to the membrane fraction. A PKC preparation purified from rat brain phospborylated purified recombinant betaARK-1 to a stoichiometry of 0.86 phosphate per betaARK-1. This phosphorylation resulted in an increased activity of betaARK-1 when membrane-bound rhodopsin served as its substrate but in no increase of its activity toward a soluble peptide substrate. The site of phosphorylation was mapped to the C terminus of betaARK-1. We conclude that PKC activates betaARK by enhancing its translocation to the plasma membrane.     

A small region of the beta-adrenergic receptor is selectively involved in its rapid regulation.

Plasma membrane receptors that couple to guanine nucleotide-binding regulatory proteins (G proteins) undergo a variety of rapid (minutes) and longer term (hours) regulatory processes induced by ligands. For the beta 2-adrenergic receptor (beta 2AR), the rapid processes include functional desensitization, mediated by phosphorylation of the receptor by the cAMP-dependent protein kinase and the beta-adrenergic receptor kinase, as well as a loss of hydrophilic ligand binding proposed to represent sequestration of receptors into a cellular compartment distinct from the plasma membrane. The slower processes include beta 2AR down-regulation (i.e., a decrease in the total cellular complement of receptors). It is not yet known whether beta 2AR phosphorylation and/or sequestration are prerequisites for down-regulation of the receptor. Like other G protein-coupled receptors, the beta 2AR molecule spans the plasma membrane seven times, and the cytoplasmic carboxyl-terminal domain has been proposed to contain molecular determinants for each of these regulatory processes. We replaced four serine and threonine residues located within a 10-amino acid segment of this domain of beta 2AR and thereby prevented agonist-promoted phosphorylation, sequestration, and rapid desensitization of the adenylyl cyclase response. In contrast, these mutations did not affect functional coupling to the stimulatory G protein Gs or long-term down-regulation. These findings thus define a small, hitherto unappreciated region of the receptor molecule that may selectively subserve its rapid regulation. In addition, with the demonstration that beta 2AR does not have to be phosphorylated or sequestered in order to enter the down-regulation pathway, the results suggest that the classical receptor endocytosis model may not be appropriate for beta 2AR regulation.     

beta-Arrestin-mediated PDE4 cAMP phosphodiesterase recruitment regulates beta-adrenoceptor switching from Gs to Gi

Phosphorylation of the beta(2) adrenoreceptor (beta(2)AR) by cAMP-activated protein kinase A (PKA) switches its predominant coupling from stimulatory guanine nucleotide regulatory protein (G(s)) to inhibitory guanine nucleotide regulatory protein (G(i)). beta-Arrestins recruit the cAMP-degrading PDE4 phosphodiesterases to the beta(2)AR, thus controlling PKA activity at the membrane. Here we investigate a role for PDE4 recruitment in regulating G protein switching by the beta(2)AR. In human embryonic kidney 293 cells overexpressing a recombinant beta(2)AR, stimulation with isoprenaline recruits beta-arrestins 1 and 2 as well as both PDE4D3 and PDE4D5 to the receptor and stimulates receptor phosphorylation by PKA. The PKA phosphorylation status of the beta(2)AR is enhanced markedly when cells are treated with the selective PDE4-inhibitor rolipram or when they are transfected with a catalytically inactive PDE4D mutant (PDE4D5-D556A) that competitively inhibits isoprenaline-stimulated recruitment of native PDE4 to the beta(2)AR. Rolipram and PDE4D5-D556A also enhance beta(2)AR-mediated activation of extracellular signal-regulated kinases ERK12. This is consistent with a switch in coupling of the receptor from G(s) to G(i), because the ERK12 activation is sensitive to both inhibitors of PKA (H89) and G(i) (pertussis toxin). In cardiac myocytes, the beta(2)AR also switches from G(s) to G(i) coupling. Treating primary cardiac myocytes with isoprenaline induces recruitment of PDE4D3 and PDE4D5 to membranes and activates ERK12. Rolipram robustly enhances this activation in a manner sensitive to both pertussis toxin and H89. Adenovirus-mediated expression of PDE4D5-D556A also potentiates ERK12 activation. Thus, receptor-stimulated beta-arrestin-mediated recruitment of PDE4 plays a central role in the regulation of G protein switching by the beta(2)AR in a physiological system, the cardiac myocyte.     

Lentivirus vector-mediated Rho guanine nucleotide dissociation inhibitor 2 overexpression induces beta-2 adrenergic receptor desensitization in airway smooth muscle cells

Background

Beta-2 adrenergic receptor (β₂AR) downregulation is critical to asthma rescue therapy; however, tolerance, also known as β₂AR or bronchodilator desensitization, mechanisms potentially resulting in life-threatening rescue treatment failure remain poorly understood.

Methods

Airway smooth muscle cells (ASMCs) from BALB/c mice were primarily cultured. The full-length Rho guanine nucleotide dissociation inhibitor 2 (RhoGDI₂) gene from ASMCs was amplified by RT-PCR, and RhoGDI₂ gene was subcloned into the digested PWPXL plasmid. The recombinant lentivirus PWPXL-RhoGDI₂ expression plasmid was packaged into mature lentivirus by 293T cells and used to infect ASMCs. Fluorescent quantitation RT-PCR and Western Blot were used to detect the level of mRNA and protein expression of RhoGDI₂, β₂AR, guanine nucleotide exchange factor (GEF), GTPase-activating protein (GAP) and G protein-coupled receptor kinases (GRKs) in overexpression RhoGDI₂-ASMCs group, negative GFP control ASMCs group and normal control ASMCs group. Membrane receptor numbers of β₂AR was observed by radioligand receptor binding assay in overexpression RhoGDI₂-ASMCs group, negative GFP control ASMCs group and normal control ASMCs group.

Results

RhoGDI₂ vector successfully transfected ASMCs, with infection efficiency (the percentage of GFP-positive cells) >80%. RhoGDI_2, GEF and G-protein-coupled receptor kinase 2 (GRK₂) expressions significantly increased in the RhoGDI₂ overexpression group compared to control and negative control groups (all P<0.05). Conversely, β₂AR and GAP expressions were significantly lower in the RhoGDI₂ overexpression group (both P<0.05), exhibiting an inverse correlation with RhoGDI₂ expression. Control and negative control groups exhibiting β₂AR density more than 2-fold higher than that observed in the RhoGDI₂ overexpression group.

Conclusions

RhoGDI₂ reduces β₂AR density, potentially by reducing β₂AR and GAP expressions and increase GEF and GRK₂ expressions. Thus, RhoGDI₂ is central in cellular β₂AR desensitization, though this full mechanism and intermediates merit further investigation.KEYWORDS : Beta-2 adrenergic receptor (β₂AR), desensitization, Rho guanine nucleotide dissociation inhibitor 2 (RhoGDI₂), guanine nucleotide exchange factor (GEF), GTPase-activating protein (GAP), G-protein-coupled receptor kinase 2 (GRK₂)     

Small GTP-binding proteins and their regulators in cardiac hypertrophy

Small GTP-binding proteins (small G proteins) act as GDP-GTP-regulated molecular switches and are activated by guanine nucleotide exchange factors (GEFs) in response to diverse extracellular stimuli. During this last decade, numerous molecular and cellular studies, as well as genetically-modified animal models, have highlighted the role of small G proteins in the regulation of cardiac hypertrophy. The growing interest in small G protein signalling comes from the fact that chronic hypertrophic response is considered maladaptive and predisposes individuals to heart failure. Although some of the hypertrophic signalling pathways involving small G proteins have now been identified, a central question deals with the identity of the GEFs that modulate small G protein activation in the context of cardiac hypertrophy. Here, we discuss the precise regulation of Ras and Rho subfamilies of GTPases by GEFs and other regulatory proteins during cardiac hypertrophy. In addition, we summarize recent published data, mainly those describing the role of small G proteins in the development of myocardial hypertrophy and we further present the importance of their downstream effectors in myocardial remodelling.     

Patterns of guanine nucleotide exchange reflecting disparate neutrophil activation pathways by opsonized and unopsonized Candida albicans hyphae

Through guanosine triphosphate (GTP) regulatory proteins are crucial components in signal transduction by most soluble and opsonized particulate stimuli, previous data suggest that neutrophil (PMNL) activation by unopsonized hyphae differs. Most of the PMNL superoxide response evoked by unopsonized hyphae was independent of both Ca++ ions and pertussis toxin-sensitive guanine nucleotide regulatory proteins. To determine whether related regulatory proteins were involved in PMNL activation by unopsonized hyphae, separated PMNL plasma membranes were incubated with GTP and a poorly hydrolyzed, radiolabeled GTP analogue, 5'-guanylylimido-diphosphate, then stimulated. Particulate Candida albicans hyphae and soluble chemotactic peptide induced comparable guanine nucleotide release. In contrast, while unopsonized hyphae caused release, it was considerably delayed, though opsonization discernibly affected neither PMNL attachment nor spreading over hyphal surfaces. This paralleled earlier observations of other delayed responses by intact PMNL to unopsonized hyphae: phospholipase C activation, the rise in cytosolic free Ca++ ions, and actin polymerization.