Participação dos fatores derivados do endotélio no efeito vasodilatador da clonidina

Os mecanismos envolvidos na ação vasodilatadora da clonidina ainda não foram completamente elucidados. Investigamos os mecanismos potencialmente envolvidos utilizando o leito arterial mesentérico (LAM) isolado de rato. No LAM pré-contraído, clonidina (10-300 pmol) induz relaxamento dose-dependente, que foi significativamente inibido pela remoção do endotélio (ácido deoxicólico) e pelos inibidores do receptor α2-adrenérgico, ioimbina (1-3 μM) e rauwolscina (1 μM). A vasodilatação endotélio-dependente induzida pela clonidina foi reduzida pelo inibidor da oxido nítrico sintase (NOS), L-NAME (0.3 mM) e pelo inibidor da guanilato ciclase, ODQ (10 μM), mas não foi alterada pela indometacina (3-10 μM). Na presença do L-NAME, o efeito vasodilatador da clonidina foi adicionalmente reduzido pela solução de potássio elevado (45 mM). Os inibidores dos canais de K⁺ dependentes de cálcio (K_ca), caribdotoxina (ChTx; 0.1 μM) e apamina (0.1 μM) também reduziram a vasodilatação induzida pela clonidina, contudo, esta resposta não foi adicionalmente inibida na presença de L-NAME, como foi observado para acetilcolina (10 pmol). Na presença do bloqueador dos canais de K⁺ dependentes de ATP, glibenclamida (10 μM), o efeito inibitório da associação ChTx, apamina e L-NAME foi aumentado. Em contraste, a vasodilatação induzida pela clonidina não foi afetada pelo inibidor dos canais de potássio dependentes de voltagem (K_v), 4-aminopiridina (4-AP, 1 mM). Concluindo, nossos resultados demonstram que clonidina ativa receptores α2-adrenérgicos no LAM e que a vasodilatação dependente de endotélio é mediada pela ativação da via NO-GMPc e também envolve a ativação de canais de KC_{a e KATP.} Um fator hiperpolarizante derivado do endotélio (EDHF) também parece participar do efeito vasodilatador da clonidina.

胡萝卜素β-和ε-环化酶及辣椒红/辣椒玉红合酶基因的分离及特性研究

利用3’和5' RACE、Uneven PCR等技术成功地从胡萝卜肉质根中分离了茄红素β-环化酶、茄红素ε．环化酶和辣椒红／辣椒玉红素合酶cDNA以及茄红素β一环化酶基因5’端上游的部分序列，并研究了它们在胡萝卜肉质根中的表达模式，对胡萝卜中类胡萝卜素代谢和积累的分子机制进行了探讨。 胡萝卜茄红素β--环化酶cDNA（DCLYC1）长2089bp，包含一个1515bp的开放阅读框架，所编码蛋白长505个氨基酸，其一级结构与番茄、烟草和辣椒等植物的茄红素β--环化酶高度同源。与农杆菌和夏噬孢欧文氏菌等微生物的茄红素环化酶相似性较差，但相互间有3个短小的同源区，且蛋白疏水模式也十分相似。茄红素β--环化酶在胡萝卜肉质根中的表达受品种和组织特异性的调控。在紫色的富含茄红素的“齐头红”胡萝卜肉质根中该基因的表达受到了强烈的抑制，相反，在橙色的富含β--和α--胡萝卜素的“CA201”胡萝卜肉质根中表达十分活跃。茄红素β--环化酶和八氢番茄红素合酶基因的表达在肉质根的韧皮部和木质部之间存在差异，在韧皮部中的表达强于木质部。类胡萝卜素生物合成基因的差异表达是造成不同胡萝卜品种和组织中积累的类胡萝卜素的种类和含量不同的原因。 对紫色品种和橙色品种的茄红素β--环化酶基因组DNA的PCR分析表明两者的基因组中均存在茄红素β一环化酶基因。为了探明茄红素β--环化酶基因在不同胡萝卜品种中差异表达的原因，利用Uneven pCR从胡萝卜基因组DNA中分离克隆了茄红素β--环化酶基因5’端上游部分序列。该DNA片段长1.7kb，3’端286bp区域与DCLYC1的5’端序列交叉重叠，在GenBank中没有找到相似的序列。在1294bp-1336bp位置串连着3个TATA盒，结构十分特殊，在TATA盒上游大约700bβ位置有2个CAAT盒。瞬间表达实验证明它具有启动子活性，可以指导GUS基因在胡萝卜肉质根、叶片和茎等组织中表达。然而，其表达模式却与茄红素B．环化酶基因的Northern杂交结果不同，主要在韧皮部和木质部交界的分生组织中表达，同时在紫色胡萝卜肉质根中其表达并没有受到抑制。这一片段可能还不是完整的胡萝卜茄红素β--环化酶基因启动子，缺少了调控基因进行品种和组织特异性表达的部分序列元件。因此，分离更长的胡萝卜茄红素环化酶基因5’端上游序列，将有助于揭示茄红素β一环化酶基因呈品种和组织特异性表达的分子机制。 所分离的胡萝卜辣椒红／辣椒玉红素合酶cDNA (DCCCS)长1744bp，包含一个长1476bp的开放阅读框架，所编码蛋白长492个氨基酸。与辣椒和柑桔CCS的氨基酸序列同源性分别为为76.6%和75.3%，与DCLYC1等其它植物茄红素β--环化酶的氨基酸序列同源性为63.9-67.4%。DCCCS的表达模式在两个不同颜色的品种之间十分相似，在肉质根韧皮部中强烈表达，而在木质部中表达明显受到了抑制。由于CCS与LYC-B高度同源，有人认为CCS可能具有茄红素环化酶活性，然而本研究结果表明，DCCCS虽然在紫色的齐头红胡萝卜肉质根韧皮部中强烈表达，却没有影响细胞中积累大量的茄红素，因此DCCCS即使具有茄红素环化酶作用，其活性也是极低的。 分离到的胡萝卜茄红素ε--环化酶cDNA片段(DCL YC-E)长1264bp，包含了完整的3’端，5’端尚不完整。按照引物LYCP1上的阅读框架进行翻译得到长385个氨基酸的肽链与莴苣、番茄和拟南芥LYC-E肽链相应区域的氨基酸序列高度同源，达80.5%以上，其中与莴苣茄红素ε--环化酶最为接近。与拟南芥茄红素ε--环化酶第448位基团和莴苣茄红素ε--环化酶第457位基团对应的氨基酸基团为H。这一基团是一个分子开关，决定茄红素ε--环化酶是催化茄红素的一端还是两端形成ε--环，因此，胡萝卜茄红素ε--环化酶可能与莴苣茄红素ε--环化酶具有相同的功能，即可以催化对称的线性茄红素的两端均形成ε--环，生成双ε--环胡萝卜素。DCLYC-E在胡萝卜肉质根中表达模式与DCLYCI不同，在紫色品种齐头红肉质根韧皮部中表达十分强烈，没有受到抑制，而且明显强于木质部;在橙色品种CA201中DCLYCE的表达模式与DCLYCI相似，韧皮部中表达强，而木质部中相对弱得多。DCL YC-E的表达模式在所测试品种间没有差异。在富含茄红素的齐头红胡萝卜肉质根中DCL YC-E强烈表达，可见它并没有将茄红素大量转化为双ε--环胡萝卜素，因此该酶的功能和活性有待进一步研究。

拟南芥生育酚环化酶基因（VTE1）在烟草中的表达及功能研究

维生素E（V.E.）在动物细胞内具有抗氧化等重要作用，但在植物体内的功能却鲜为人知。本研究以烟草为材料，利用根癌农杆菌（Agrobacterium tumefaciens）介导法在烟草中过量表达拟南芥来源的VTE1。通过外源VTE1基因的过量表达提高内源V.E.的含量, 进而研究转VTE1基因植株对胁迫的耐受性反应，以探讨植物体内V.E.含量与植物胁迫耐受性的关系，为植物抗逆机理的研究和利用奠定基础。 本实验利用CaMV35s启动子与拟南芥来源的生育酚环化酶基因（VTE1）构建的嵌合表达载体，以根癌农杆菌介导的叶盘法转化烟草W38。实验结果表明: 1. 具有卡那霉素抗性的再生植株经PCR检测，得到了与阳性对照一致的495bp的目标片段;经RT-PCR检测，其中90%有外源基因表达。 2. 转基因植株的V.E.含量比对照植株高2倍左右，个别株系高达10.16倍。 3. VTE1基因的表达受环境胁迫的影响，不同程度的冷冻、热激、PEG处理均可影响VTE1基因的表达。经过冷冻处理60分钟、热处理20小时、以及PEG处理6小时，该基因表达量均有提高。冷冻处理条件下该基因的表达量是未处理的3倍，热处理条件下是未处理的2倍左右，PEG处理是未处理的3.5倍。在冷冻、热激、PEG胁迫处理过程中，转化苗的V.E.含量变化与外源VTE1基因的表达相对应，表明转化苗的V.E.合成主要由外源VTE1基因的终产物VTE1催化;在冷冻、热激、PEG胁迫处理过程中，V.E.含量与APX、CAT、SOD等抗氧化酶活性之间存在一定程度的正相关性，表明V.E.与这些抗氧化酶共同组成了植物体内的抗氧化网络，保护植株免受氧化损伤;V.E.的变化与MDA之间存在一定程度的负相关性，减轻植物的过氧化损伤; 4. V.E.可提高植物的抗旱性，我们检测了11个转化烟草株系的叶片相对含水量（RWC），在大多数转化烟草植株中，干旱胁迫24小时的RWC都比野生型高，高出0.16-45%（p<0.001）。表明转基因烟草比野生型更抗旱; 5. 在耐盐性实验中转基因植株对盐的抗性明显高于野生型烟草;同时，在不同盐浓度（150、250mM）胁迫下转基因植株V.E.含量比未转化植株增加了1.3-1.8倍。 这些研究结果表明，在植物体内转入V.E.代谢途径中的单个外源基因，可有效提高内源V.E.合成，提高植株对环境胁迫的抗性。

水稻2,3-氧化鲨烯环化酶基因的功能研究

植物通过异戊二烯代谢途径合成多种具有生物活性和功能的三萜及甾醇类化合物，它们在调节植物生长发育、维持膜的完整和功能、抵抗病原微生物侵染中发挥着重要的作用。2,3-氧化鲨烯为三萜和甾醇合成途径的分枝点，参与这一关键步骤的酶被通称为2,3-氧化鲨烯环化酶（OSCs）。本研究系统分了水稻基因组中全部11个OSC基因序列，发现其中四个可能为假基因。亚种间非同义替换率Ka和同义替换率Ks的比值（Ka/Ks）以及进化树的分析表明OsOSC8是单子叶植物特有的功能保守基因，而OsOSC9在水稻两个亚种间发生了功能快速进化，这种快速进化的基因往往参与植物和病原菌相互作用的代谢途径。 根据基因结构、表达谱以及与其它植物已知功能的OSC酶氨基酸序列的比对推测OsOSC3可能具有环阿屯醇合成酶的功能，参与植物甾醇的合成，而OsOSC7、OsOSC10和OsOSC11可能具有β-香树素合成酶的功能，其余OSCs可能参与合成其它三萜化合物。为了进一步分析和验证OSCs酶的功能，将水稻7个OSC基因的开放阅读框（ORF）构建到酵母表达载体并在pichia酵母中表达，发现仅有OsOSC9和OsOSC12能够将酵母内源的2,3-氧化鲨烯分别环化为四环三萜化合物Parkeol和植物中稀有的五环三萜化合物Isoarborinol，目前还未在其它植物中发现参与这两种三萜化合物的基因。另外，水稻所有的OSC基因均不能互补酵母羊毛甾醇缺陷型菌株，表明水稻OSCs不具有合成羊毛甾醇的功能。 RNAi沉默以及启动子融合GUS的表达实验发现OsOSC8可能参与花粉的发育，该基因的下调影响水稻的育性，暗示水稻中存在一个可能与雄性不育有关的三萜代谢途径。水稻其它OSC基因RNAi植株可能在逆境环境和病原菌侵染下才会显现出表型。

蓝藻应答高 pH 胁迫的亚细胞蛋白质组学研究

蓝藻是迄今地球上发现的最古老、分布最广和最具多样性的光合自养原核生物，其细胞结构简单，具有类似于植物的光合作用，是研究光合作用及其它代谢过程重要的模式生物。由于这类生物起源于远古前寒武纪，但至今依然繁多，在极端寒冷的南北极冰湖和近于沸腾温度的温泉，以及高盐、强碱的极端环境中均有存在,它们在漫长的进化过程中如何应对灾难性环境、针对随时可能遭遇的不同胁迫环境因子形成了怎样的分子适应机制，是近年来倍受关注但仍未诠释的问题之一。由于蓝藻与高等植物叶绿体在进化上密切相关，搞清楚这类生物适应不同胁迫环境因子的分子基础及其作用机制，对从进化的角度理解光合生物与环境相互作用、通过同源性发现作物抗逆育种新靶标，有重要的理论和实践意义。 逆境应答蛋白的表达是细胞对逆境胁迫的主要适应机制之一。在特定的逆境条件下，细胞通常会表达一组蛋白质，用于识别与传递环境胁迫信号、稳定细胞内环境、消除并修复逆境造成的损伤等。因此，逆境应答蛋白的系统鉴定和功能确认，是揭示逆境条件下细胞代谢网络及抗逆性分子机制的关键。单细胞模式蓝藻基因组序列的确定，极大地推动了蓝藻细胞蛋白质组成模式研究，也为系统发掘蓝藻逆境应答蛋白、理解和揭示分子适应机制提供了新的切入点。Synechocystis 6803是第一个完成基因组测序的放氧光合模式生物。由于其具有易培养、可转化、对环境条件变化反应快等优点，以该藻种为材料所展开的逆境应答特别是盐胁迫蛋白质组研究方面已经取得了重要的进展，而对高pH胁迫的蛋白质组研究还鲜有报道。因此，本论文以Synechocystis 6803为材料，从分离纯化的亚细胞组分入手，采用蛋白质组学研究手段，对蓝藻细胞应答高pH胁迫的蛋白质代谢网络进行探讨。利用蔗糖密度离心和水溶性两相分离法相结合的方法，分别获得了对照（pH7.5）和处理（pH11）细胞的质膜、外膜和类囊体膜，并分别构建了包括可溶性蛋白和膜组分的一维和二维蛋白质凝胶电泳图谱。分析结果表明，高pH胁迫下质膜和可溶性蛋白蛋白组分的变化较外膜和类囊体膜蛋白组分更为明显。在考马斯亮兰染色胶上共发现有近110个蛋白点上调或下调表达，其中有82个蛋白点来源于质膜。对质膜蛋白进行的差异荧光标记双向电泳（2-D DIGE）分析结果与考马斯亮兰染色结果基本一致。对质膜上的82个蛋白点进行胶内消化和MALDI-TOF和MALDI-TOF/TOF质谱鉴定，得到了39个不同基因产物，其中25个是上调蛋白，14个是下调蛋白。在这些发生变化的蛋白中，近1/3是ABC型转运蛋白，如3个磷转运蛋白（Sll0679，Sll0683，Sll0684）均在高pH胁迫下明显上调。其它高pH响应蛋白包括参与光合作用（PsaF，Sll0819；CpcA，Sll1578）、呼吸作用（CoxB，Sll0813）以及细胞分裂过程的蛋白（MinD，Sll0289）。还有LexA repressor (Sll1626)和Guanylyl cyclase(Cya2，Sll0646)等起调控作用的蛋白质。此外发现8个高pH胁迫响应蛋白为功能未知的新蛋白。生物信息学预测结果显示，在已鉴定的质膜响应蛋白中有17个蛋白具有信号肽。6个蛋白为具有跨膜域的膜蛋白，其中的3个膜蛋白是首次被证明定位于质膜上，且其表达与高pH胁迫有关。这些研究结果对从分子水平理解蓝藻细胞主动应对高pH胁迫、维护细胞内pH相对稳定机制有重要启示。

Functional characterization of sll0659 from Synechocystis sp PCC 6803

Synchocystis sp. PCC 6803 lacks a gene for the any known types of lycopene cyclase. Recently, we reported that sll0659 (unknown for its function) from Synechocystis sp. PCC6803 shows similarity in sequence to a lycopene cyclase gene-CruA from Chlorobium tepidum. To test, whether Sll0659 encoded protein serves as lycopene cyclase, in this study, we investigated the carotenoids of the wild types ans mutants, In the sll0659 deleted mutant, there is no blockage at the lycopene cyclization step. Our results demonstrate that sll0659 does not affect lycopene cyclization. However, the ultrastructure of mutants suggests the involvement or necessity of sll0659 in the cell division.

Lineage-specific domain fusion in the evolution of purine nucleotide cyclases in cyanobacteria

Cyclic nucleotides (both cAMP and cGMP) play extremely important roles in cyanobacteria, such as regulating heterocyst formation, respiration, or gliding. Catalyzing the formation of cAMP and cGMP from ATP and GTP is a group of functionally important enzymes named adenylate cyclases and guanylate cyclases, respectively. To understand their evolutionary patterns, in this study, we presented a systematic analysis of all the cyclases in cyanobacterial genomes. We found that different cyanobacteria had various numbers of cyclases in view of their remarkable diversities in genome size and physiology. Most of these cyclases exhibited distinct domain architectures, which implies the versatile functions of cyanobacterial cyclases. Mapping the whole set of cyclase domain architectures from diverse prokaryotic organisms to their phylogenetic tree and detailed phylogenetic analysis of cyclase catalytic domains revealed that lineage-specific domain recruitment appeared to be the most prevailing pattern contributing to the great variability of cyanobacterial cyclase domain architectures. However, other scenarios, such as gene duplication, also occurred during the evolution of cyanobacterial cyclases. Sequence divergence seemed to contribute to the origin of putative guanylate cyclases which were found only in cyanobacteria. In conclusion, the comprehensive survey of cyclases in cyanobacteria provides novel insight into their potential evolutionary mechanisms and further functional implications.

Premature ovarian aging in mice deficient for Gpr3

After becoming competent for resuming meiosis, fully developed mammalian oocytes are maintained arrested in prophase I until ovulation is triggered by the luteotropin surge. Meiotic pause has been shown to depend critically on maintenance of cAMP level in the oocyte and was recently attributed to the constitutive Gs (the heterotrimeric GTP-binding protein that activates adenylyl cyclase) signaling activity of the G protein-coupled receptor GPR3. Here we show that mice deficient for Gpr3 are unexpectedly fertile but display progressive reduction in litter size despite stable age-independent alteration of meiotic pause. Detailed analysis of the phenotype confirms premature resumption of meiosis, in vivo, in about one-third of antral follicles from Gpr3-/- females, independently of their age. In contrast, in aging mice, absence of GPR3 leads to severe reduction of fertility, which manifests by production of an increasing number of nondeveloping early embryos upon spontaneous ovulation and massive amounts of fragmented oocytes after superovulation. Severe worsening of the phenotype in older animals points to an additional role of GPR3 related to protection (or rescue) of oocytes from aging. Gpr3-defective mice may constitute a relevant model of premature ovarian failure due to early oocyte aging.

In vivo ventricular gene delivery of a beta-adrenergic receptor kinase inhibitor to the failing heart reverses cardiac dysfunction.

BACKGROUND: Genetic manipulation to reverse molecular abnormalities associated with dysfunctional myocardium may provide novel treatment. This study aimed to determine the feasibility and functional consequences of in vivo beta-adrenergic receptor kinase (betaARK1) inhibition in a model of chronic left ventricular (LV) dysfunction after myocardial infarction (MI). METHODS AND RESULTS: Rabbits underwent ligation of the left circumflex (LCx) marginal artery and implantation of sonomicrometric crystals. Baseline cardiac physiology was studied 3 weeks after MI; 5x10(11) viral particles of adenovirus was percutaneously delivered through the LCx. Animals received transgenes encoding a peptide inhibitor of betaARK1 (Adeno-betaARKct) or an empty virus (EV) as control. One week after gene delivery, global LV and regional systolic function were measured again to assess gene treatment. Adeno-betaARKct delivery to the failing heart through the LCx resulted in chamber-specific expression of the betaARKct. Baseline in vivo LV systolic performance was improved in Adeno-betaARKct-treated animals compared with their individual pre-gene delivery values and compared with EV-treated rabbits. Total beta-AR density and betaARK1 levels were unchanged between treatment groups; however, beta-AR-stimulated adenylyl cyclase activity in the LV was significantly higher in Adeno-betaARKct-treated rabbits compared with EV-treated animals. CONCLUSIONS: In vivo delivery of Adeno-betaARKct is feasible in the infarcted/failing heart by coronary catheterization; expression of betaARKct results in marked reversal of ventricular dysfunction. Thus, inhibition of betaARK1 provides a novel treatment strategy for improving the cardiac performance of the post-MI heart.

Augmentation of cardiac contractility mediated by the human beta(3)-adrenergic receptor overexpressed in the hearts of transgenic mice.

BACKGROUND: Stimulation of beta(1)- and beta(2)-adrenergic receptors (ARs) in the heart results in positive inotropy. In contrast, it has been reported that the beta(3)AR is also expressed in the human heart and that its stimulation leads to negative inotropic effects. METHODS AND RESULTS: To better understand the role of beta(3)ARs in cardiac function, we generated transgenic mice with cardiac-specific overexpression of 330 fmol/mg protein of the human beta(3)AR (TGbeta(3) mice). Hemodynamic characterization was performed by cardiac catheterization in closed-chest anesthetized mice, by pressure-volume-loop analysis, and by echocardiography in conscious mice. After propranolol blockade of endogenous beta(1)- and beta(2)ARs, isoproterenol resulted in an increase in contractility in the TGbeta(3) mice (30%), with no effect in wild-type mice. Similarly, stimulation with the selective human beta(3)AR agonist L-755,507 significantly increased contractility in the TGbeta(3) mice (160%), with no effect in wild-type mice, as determined by hemodynamic measurements and by end-systolic pressure-volume relations. The underlying mechanism of the positive inotropy incurred with L-755,507 in the TGbeta(3) mice was investigated in terms of beta(3)AR-G-protein coupling and adenylyl cyclase activation. Stimulation of cardiac membranes from TGbeta(3) mice with L-755,507 resulted in a pertussis toxin-insensitive 1.33-fold increase in [(35)S]GTPgammaS loading and a 1.6-fold increase in adenylyl cyclase activity. CONCLUSIONS: Cardiac overexpression of human beta(3)ARs results in positive inotropy only on stimulation with a beta(3)AR agonist. Overexpressed beta(3)ARs couple to G(s) and activate adenylyl cyclase on agonist stimulation.

Level of beta-adrenergic receptor kinase 1 inhibition determines degree of cardiac dysfunction after chronic pressure overload-induced heart failure.

BACKGROUND: Heart failure is characterized by abnormalities in beta-adrenergic receptor (betaAR) signaling, including increased level of myocardial betaAR kinase 1 (betaARK1). Our previous studies have shown that inhibition of betaARK1 with the use of the Gbetagamma sequestering peptide of betaARK1 (betaARKct) can prevent cardiac dysfunction in models of heart failure. Because inhibition of betaARK activity is pivotal for amelioration of cardiac dysfunction, we investigated whether the level of betaARK1 inhibition correlates with the degree of heart failure. METHODS AND RESULTS: Transgenic (TG) mice with varying degrees of cardiac-specific expression of betaARKct peptide underwent transverse aortic constriction (TAC) for 12 weeks. Cardiac function was assessed by serial echocardiography in conscious mice, and the level of myocardial betaARKct protein was quantified at termination of the study. TG mice showed a positive linear relationship between the level of betaARKct protein expression and fractional shortening at 12 weeks after TAC. TG mice with low betaARKct expression developed severe heart failure, whereas mice with high betaARKct expression showed significantly less cardiac deterioration than wild-type (WT) mice. Importantly, mice with a high level of betaARKct expression had preserved isoproterenol-stimulated adenylyl cyclase activity and normal betaAR densities in the cardiac membranes. In contrast, mice with low expression of the transgene had marked abnormalities in betaAR function, similar to the WT mice. CONCLUSIONS: These data show that the level of betaARK1 inhibition determines the degree to which cardiac function can be preserved in response to pressure overload and has important therapeutic implications when betaARK1 inhibition is considered as a molecular target.

The G-protein-coupled receptor kinases beta ARK1 and beta ARK2 are widely distributed at synapses in rat brain.

The beta-adrenergic receptor kinase (beta ARK) phosphorylates the agonist-occupied beta-adrenergic receptor to promote rapid receptor uncoupling from Gs, thereby attenuating adenylyl cyclase activity. Beta ARK-mediated receptor desensitization may reflect a general molecular mechanism operative on many G-protein-coupled receptor systems and, particularly, synaptic neurotransmitter receptors. Two distinct cDNAs encoding beta ARK isozymes were isolated from rat brain and sequenced. The regional and cellular distributions of these two gene products, termed beta ARK1 and beta ARK2, were determined in brain by in situ hybridization and by immunohistochemistry at the light and electron microscopic levels. The beta ARK isozymes were found to be expressed primarily in neurons distributed throughout the CNS. Ultrastructurally, beta ARK1 and beta ARK2 immunoreactivities were present both in association with postsynaptic densities and, presynaptically, with axon terminals. The beta ARK isozymes have a regional and subcellular distribution consistent with a general role in the desensitization of synaptic receptors.

Beta-arrestin-mediated beta1-adrenergic receptor transactivation of the EGFR confers cardioprotection.

Deleterious effects on the heart from chronic stimulation of beta-adrenergic receptors (betaARs), members of the 7 transmembrane receptor family, have classically been shown to result from Gs-dependent adenylyl cyclase activation. Here, we identify a new signaling mechanism using both in vitro and in vivo systems whereby beta-arrestins mediate beta1AR signaling to the EGFR. This beta-arrestin-dependent transactivation of the EGFR, which is independent of G protein activation, requires the G protein-coupled receptor kinases 5 and 6. In mice undergoing chronic sympathetic stimulation, this novel signaling pathway is shown to promote activation of cardioprotective pathways that counteract the effects of catecholamine toxicity. These findings suggest that drugs that act as classical antagonists for G protein signaling, but also stimulate signaling via beta-arrestin-mediated cytoprotective pathways, would represent a novel class of agents that could be developed for multiple members of the 7 transmembrane receptor family.

Enhanced myocardial relaxation in vivo in transgenic mice overexpressing the beta2-adrenergic receptor is associated with reduced phospholamban protein.

To assess the effect of targeted myocardial beta-adrenergic receptor (AR) stimulation on relaxation and phospholamban regulation, we studied the physiological and biochemical alterations associated with overexpression of the human beta2-AR gene in transgenic mice. These mice have an approximately 200-fold increase in beta-AR density and a 2-fold increase in basal adenylyl cyclase activity relative to negative littermate controls. Mice were catheterized with a high fidelity micromanometer and hemodynamic recordings were obtained in vivo. Overexpression of the beta2-AR altered parameters of relaxation. At baseline, LV dP/dt(min) and the time constant of LV pressure isovolumic decay (Tau) in the transgenic mice were significantly shorter compared with controls, indicating markedly enhanced myocardial relaxation. Isoproterenol stimulation resulted in shortening of relaxation velocity in control mice but not in the transgenic mice, indicating maximal relaxation in these animals. Immunoblotting analysis revealed a selective decrease in the amount of phospholamban protein, without a significant change in the content for either sarcoplasmic reticulum Ca2+ ATPase or calsequestrin, in the transgenic hearts compared with controls. This study indicates that myocardial relaxation is both markedly enhanced and maximal in these mice and that conditions associated with chronic beta-AR stimulation can result in a selective reduction of phospholamban protein.

Identification of beta-adrenergic receptors in human lymphocytes by (-) (3H) alprenolol binding.

Human lymphocytes are known to posessess a catecholamine-responsive adenylate cyclase which has typical beta-adrenergic specificity. To identify directly and to quantitate these beta-adenergic receptors in human lymphocytes, (-) [3H] alprenolol, a potent beta-adrenergic antagonist, was used to label binding sites in homogenates of human mononuclear leukocytes. Binding of (-) [3H] alprenolol to these sites demonstrated the kinetics, affinity, and stereospecificity expected of binding to adenylate cyclase-coupled beta-adrenergic receptors. Binding was rapid (t1/2 less than 30 s) and rapidly reversible (t1/2 less than 3 min) at 37 degrees C. Binding was a saturable process with 75 +/- 12 fmol (-) [3H] alprenolol bound/mg protein (mean +/- SEM) at saturation, corresponding to about 2,000 sites/cell. Half-maximal saturation occurred at 10 nM (-) [3H] alprenolol, which provides an estimate of the dissociation constant of (-) [3H] alprenolol for the beta-adrenergic receptor. The beta-adrenergic antagonist, (-) propranolol, potently competed for the binding sites, causing half-maximal inhibition of binding at 9 nM. beta-Adrenergic agonists also competed for the binding sites. The order of potency was (-) isoproterenol greater than (-) epinephrine greater than (-)-norepinephrine which agreed with the order of potency of these agents in stimulating leukocyte adenylate cyclase. Dissociation constants computed from binding experiments were virtually identical to those obtained from adenylate cyclase activation studies. Marked stereospecificity was observed for both binding and activation of adenylate cyclase. (-)Stereoisomers of beta-adrenergic agonists and antagonists were 9- to 300-fold more potent than their corresponding (+) stereoisomers. Structurally related compounds devoid of beta-adrenergic activity such as dopamine, dihydroxymandelic acid, normetanephrine, pyrocatechol, and phentolamine did not effectively compete for the binding sites. (-) [3H] alprenolol binding to human mononuclear leukocyte preparations was almost entirely accounted for by binding to small lymphocytes, the predominant cell type in the preparations. No binding was detectable to human erythrocytes. These results demonstrate the feasibility of using direct binding methods to study beta-adrenergic receptors in a human tissue. They also provide an experimental approach to the study of states of altered sensitivity to catecholamines at the receptor level in man.