Angiotensins, Prostaglandins-Adrenergic Interactions

本提案旨在检验以下假设：细胞色素P450（CYP）1B 1产生的代谢物 雌二醇（E2）（2-甲氧基雌二醇，2-ME）和睾酮（T）（6β-羟基睾酮，6β-OHT） 通过增强血管紧张素（Ang）II对胞浆磷脂酶A2α（cPLA 2 α）的作用， 发挥降压作用的环氧合酶（考克斯）-花生四烯酸（AA）衍生的前列腺素（PG）E2 通过EP 1和EP 3受体，保护女性而不是男性对抗高血压及其发病机制。这 这一假说是基于我们新的初步数据，即：a）卵巢切除（OVX）患者中血管紧张素II诱导的高血压， 或CYP 1B 1基因破坏的（Cyp 1b 1-/-）雌性小鼠;和B）去势（Cas）或Cyp 1b 1-/-雄性小鼠， 通过cPLA 2 α基因破坏（cPLA 2 α-/-），AA代谢抑制剂5，8，11，14- 二十碳四炔酸（ETYA）或COX衍生的PGE 2-EP 1和EP 3受体拮抗剂。更重要的是我们 新的观察表明，脑中CYP 1B 1产生的E2和T代谢产物介导了Ang II的作用 通过反向调节cPLA 2 αAA系统的活性对女性血压（BP）的影响 （抑制性）和雄性（刺激性）小鼠。我们将扩展这些观察，并通过以下方式检验我们的假设： 实现以下具体目标。AIM 1.确定CYP 1B 1与cPLA 2 α/AA系统的相互作用 Ang II诱导的雌性小鼠高血压及其发病机制。次级目标1.为了调查 cPLA 2 α/AA系统和CYP 1B 1在血管紧张素Ⅱ诱导的高血压中的作用及其相互关系 雌性小鼠的发病机制。目标2.研究CYP 1B 1与cPLA 2 α/AA系统在血管紧张素Ⅱ-受体（Ang Ⅱ-受体）中的相互作用。 诱导的雄性小鼠高血压及其发病机制。次级目标2.为了确定中央的贡献， CYP 1B 1及其与cPLA 2 α/AA系统的相互作用在血管紧张素Ⅱ诱导的高血压中的作用及其机制 雄性老鼠为了达到这些目标，我们将使用最先进的技术，其中包括：1）无线电- 用于测量BP和功率谱分析的遥测，以及用于评估心脏的超声心动图 功能; 3）Cyp 1b 1 +/+和Cyp 1b 1-/-和cPLA 2 α+/+和cPLA 2 α-/-小鼠; 2）腺病毒（Ad）CYP 1B 1 shRNA 和Ad CYP 1B 1 DNA，和腺病毒（Ad）cPLA 2 α shRNA和Ad cPLA 2 α DNA，以及针对EP 1和 EP 3和类固醇基因组和非基因组受体，以及它们各自的对照; 性类固醇和类花生酸分析; 5）组织学、免疫组织化学和荧光显微镜 和生物化学技术; 6）流式细胞术以确定血液和组织中的免疫细胞群体。 这项研究将为性别差异的分子机制提供新的见解 通过CYP 1B 1产生的性类固醇代谢产物与cPLA 2 α/AA系统的相互作用确定， 血管紧张素II诱导的高血压及其发病机制此外，这些研究将使我们能够证明 cPLA 2 α作为开发新型选择性抑制剂治疗高血压的潜在靶点 和相关的发病机制在两种性别，和有害的影响，CYP 1B 1抑制剂在女性。

This proposal is aimed to test the hypothesis that: Cytochrome P450 (CYP) 1B1-generated metabolites of estradiol (E2) (2-methoxyestradiol, 2-ME) by inhibiting and testosterone (T) (6β-hydroxytestosterone, 6β-OHT) by enhancing angiotensin (Ang) II effect on cytosolic phospholipase A2α (cPLA2α) and generation of cyclooxygenase (COX)- arachidonic acid (AA)-derived prostaglandin (PG) E2 exerting prohypertensive effect via EP1 and EP3 receptors, protects the female but not males against hypertension and its pathogenesis. This hypothesis is based on our novel preliminary data that Ang II-induced hypertension in a) ovariectomized (OVX) or CYP1B1 gene disrupted (Cyp1b1-/-) female mice; and b) castrated (Cas) or Cyp1b1-/- male mice treated with 6β-OHT, is minimized by cPLA2α gene disruption (cPLA2α-/-), by AA metabolism inhibitor 5,8,11,14- eicosatetraynoic acid (ETYA), or COX-derived PGE2-EP1 and EP3 receptor antagonists. More importantly, our new observation shows that CYP1B1-generated E2 and T metabolites in the brain mediate the effect of Ang II on blood pressure (BP) by modulating the activity of cPLA2αAA system in the opposite direction in female (inhibitory) and male (stimulatory) mice. We will extend these observations and test our hypothesis by addressing the following specific aims. AIM 1. To determine the interaction of CYP1B1 and cPLA2α/AA system in Ang II-induced hypertension and its pathogenesis in female mice. Sub-Aim 1. To investigate the contribution of central cPLA2α/AA system and CYP1B1 and their interaction in Ang II-induced hypertension and its pathogenesis in female mice. Aim 2. To examine the interaction of CYP1B1 with cPLA2α/AA system in Ang II- induced hypertension and its pathogenesis in male mice. Sub-Aim 2. To determine the contribution of central CYP1B1 and its interaction with cPLA2α/AA system in Ang II-induced hypertension and its pathogenesis in male mice. To achieve these objectives, we will use the state-of-the-art techniques which include: 1) Radio- telemetry for measuring BP and for power spectral analysis, and Echocardiography for assessing cardiac function; 3) Cyp1b1+/+ and Cyp1b1-/- and cPLA2α+/+and cPLA2α-/- mice; 2) Adenovirus (Ad) CYP1B1 shRNA and Ad CYP1B1 DNA, and Adenovirus (Ad) cPLA2α shRNA and Ad cPLA2α DNA, and siRNA for EP1 and EP3, and steroid genomic and nongenomic receptors, and their respective controls; 4) UPLC/qTOFMS for the analysis of sex steroids and eicosanoids; 5) histological, immunohistochemical, and fluorescence microscopy and biochemical techniques; 6) Flow cytometry to determine immune cell population in the blood and tissues. The proposed studies should provide novel insights into the molecular mechanisms underlying sex differences as determined by the interaction of CYP1B1-generated metabolites of sex steroids and cPLA2α/AA system in Ang II-induced hypertension and its pathogenesis. Furthermore, these studies would allow us to demonstrate cPLA2α as a potential target for developing novel selective inhibitors of this enzyme for treating hypertension and associated pathogenesis in both sexes, and the detrimental effect of CYP1B1 inhibitors in females.