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.
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