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）和中间肽（IMD）在调节女性血管功能中的作用，并检查这些肽在子宫胎盘功能中的参与。在本申请中，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参与。据报道，这两种相关肽均通过单个GPCR、降钙素受体样受体（CRLR）与受体活性修饰蛋白（RAMP）的异二聚体复合物起作用。根据我们新的初步数据，我们假设，它们的表达，以及肠系膜和子宫血管舒张反应AM和IMD，在怀孕期间上调，AM和IMD拮抗剂的输液将减少胎儿胎盘重量。拟定的具体目标是：具体目标1：描述整个妊娠期间AM和IMD合成和对AM和IMD的血管舒张反应的变化，以及性类固醇激素对其的调节。我们将检查血清AM和IMD以及对外源性AM、IMD或其拮抗剂的血压（BP）变化的反应：1）在妊娠期间和用性类固醇激素拮抗剂治疗的大鼠中; 2）在用雌二醇（E2）、孕酮（P4）或E2 + P4治疗的卵巢切除（OVX）大鼠中。具体目标二：研究AM和IMD对妊娠期大鼠肠系膜动脉血管舒张作用、受体水平和作用机制的变化以及性类固醇激素对其的调节作用。我们将测量血管舒张反应AM和IMD，第二信使效应机制，和CRLR，RAMP，RAMP 2和RAMP 3在整个妊娠肠系膜动脉和性类固醇激素治疗OVX大鼠的mRNA和蛋白水平。具体目标3：研究AM和IMD对大鼠妊娠期子宫动脉血管舒张的影响、其受体水平和作用机制的变化以及性类固醇激素对其的调节。我们将评估血管舒张反应AM和IMD的变化，第二信使效应机制，CRLR，RAMP，RAMP 2和RAMP 3的mRNA和蛋白质水平在整个妊娠子宫动脉，并在性类固醇激素治疗OVX大鼠。具体目标4：使用特异性RAMPs N-末端结构域的抗体确定特异性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RAMP 2和RAMP 3蛋白的N-末端结构域，并产生多克隆抗体。这些抗体将用于血管组织浴;将在妊娠和类固醇激素治疗大鼠的肠系膜和子宫动脉中评估对AM和IMD的舒张反应。具体目的5：评估AM和IMD拮抗剂输注妊娠大鼠对胎盘的影响！和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AM 22.52和IMD 17.47，并测量血压、胎仔和胎盘重量。我们将评估胎盘中的凋亡变化。

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.