Sex Steroid Hormones and Calcitonin Gene-Related Peptide

描述（由申请人提供）：我们的长期目标是确定降钙素基因相关肽（CGRP）家族肽、肾上腺髓质素（AM）和中间素（IMD）在女性血管功能调节中的作用，并研究这些肽在子宫胎盘功能中的作用。在这项应用中，我们计划确定AM和IMD在妊娠期间调节血管功能和胎儿生长中的作用。据报道，这两种相关肽通过单一GPCR的异二聚体复合物（降钙素受体样受体（CRLR））与受体活性修饰蛋白（RAMPs）结合发挥作用。根据我们新的初步数据，我们假设它们的表达以及对AM和IMD的肠系膜和子宫血管松弛反应在妊娠期间上调，并且输注AM和IMD拮抗剂会降低胎儿胎盘重量。目的1：研究AM和IMD合成的变化，AM和IMD对妊娠期血管的舒张反应，以及性类固醇激素对AM和IMD的调节。我们将检查血清AM和IMD以及血压（BP）对外源性AM、IMD或其拮抗剂的反应：1)在怀孕期间和接受性类固醇激素拮抗剂治疗的大鼠；2)雌二醇（E2）、黄体酮（P4）或E2 + P4治疗去卵巢大鼠。特异性目的2：研究AM和IMD对妊娠大鼠肠系膜动脉血管舒张的影响、受体水平和作用机制的变化，以及性类固醇激素对它们的调节作用。我们将测量妊娠期间和性类固醇激素治疗的OVX大鼠肠系膜动脉血管对AM和IMD的松弛反应、第二信使效应机制以及CRLR、RAMP、RAMP2和RAMP3的mRNA和蛋白水平。特异性目的3：研究AM和IMD对妊娠大鼠子宫动脉血管舒张的影响、受体水平和作用机制的变化以及性类固醇激素对它们的调节作用。我们将评估AM和IMD对血管松弛反应的变化，第二信使效应机制，以及妊娠期间子宫动脉中CRLR、RAMP、RAMP2和RAMP3 mRNA和蛋白的水平，以及在性类固醇激素治疗的OVX大鼠中。特异性目的4：利用特异性RAMPs的n端结构域抗体，确定特异性RAMPs参与AM-或imd诱导的肠系膜和子宫动脉舒张。我们将克隆和表达RAMP2和RAMP3蛋白的n端结构域，并生成多克隆抗体。这些抗体将用于血管组织浴；将评估妊娠大鼠和类固醇激素治疗大鼠的肠系膜和子宫动脉对AM和IMD的松弛反应。专项目的5：评价AM和IMD拮抗剂对妊娠大鼠胎盘的影响！还有胎儿生长。我们将在妊娠第8 - 15天或第14 - 22天对妊娠大鼠皮下注射不同剂量的am22.52和imd17.47，并测量血压、胎儿和胎盘重量。我们将评估胎盘细胞凋亡的变化。

DESCRIPTION (provided by applicant): Our long-term goal is to define the role of calcitonin gene-related peptide (CGRP) family peptides, adrenomedullin (AM), and intermedin (IMD) in the regulation of female vascular functions, and to examine the involvement of these peptides in uteroplacental function. In this application, we plan to determine the involvement of AM and IMD in regulating vascular functions and fetal growth during pregnancy. Both these related peptides were reported to function through a heterodimeric complex of a single GPCR, calcitonin receptor-like receptor (CRLR), in combination with the receptor activity modifying proteins (RAMPs). Based upon our new preliminary data, we hypothesize that the expression of them, as well as mesenteric and uterine vascular relaxation responses to AM and IMD, are upregulated during pregnancy, and infusion of AM and IMD antagonists will decrease fetoplacental weight. The proposed Specific Aims are: Specific Aim 1: To characterize changes in AM and IMD synthesis and vasodilatory responses to AM and IMD throughout gestation, and their regulation by sex steroid hormones. We will examine serum AM and IMD and changes in blood pressure (BP) in response to exogenous AM, IMD, or their antagonists: 1) in rats during pregnancy and upon treatment with sex-steroid-hormone antagonists and; 2) in ovariectomized (OVX) rats treated with estradiol (E2), progesterone (P4), or E2 + P4. Specific Aim 2: To investigate changes in the effects of AM and IMD on vasodilation, their receptor levels, and mechanism(s) of action in mesenteric arteries in rats throughout gestation and their regulation by sex steroid hormones. We will measure vasorelaxation responses to AM and IMD, second messenger effector mechanisms, and the mRNA and protein levels for CRLR, RAMP, RAMP2, and RAMP3 in mesenteric arteries throughout gestation and in sex-steroid-hormone-treated OVX rats. Specific Aim 3: To examine changes in AM and IMD effects on vasodilation, their receptor levels and mechanisms of action in uterine arteries in rats throughout gestation and their regulation by sex steroid hormones. We will assess changes in the vasorelaxation responses to AM and IMD, second messenger effector mechanisms, and the levels of mRNA and protein for CRLR, RAMP, RAMP2, and RAMP3 in uterine arteries throughout gestation, and in sex-steroid-hormone-treated OVX rats. Specific Aim 4: To determine the involvement of specific RAMPs in AM- or IMD-induced mesenteric and uterine artery relaxations using antibodies to the N-terminal domain of specific RAMPs. We will clone and express N-terminal domains of both RAMP2 and RAMP3 proteins and generate polyclonal antibodies. These antibodies will be used in vascular tissue baths; relaxation responses to AM and IMD will be assessed in both mesenteric and uterine arteries from pregnant and steroid-hormone-treated rats. Specific Aim 5: To assess the effects of infusion of AM and IMD antagonists to pregnant rats on placenta! and fetal growth. We will subcutaneously infuse varying doses of AM22.52and IMD17.47to pregnant rats from either day 8 to 15 or day 14 to 22 of gestation and measure BP, fetal, and placental weights. We will assess the apoptotic changes in the placenta.