Uterine blood flow (UBF) increases during the follicular phase of the estrous cycle when the systemic and local estrogen/progesterone ratio is elevated. In the nonpregnant sheep. acute estradiol-17beta )E2beta) administration increases UBF and cardiac output and chronic administration mimics nearly all of the cardiovascular adaptations to pregnancy (another high estrogen state). Although the acute (1-3 hour) E2beta-induced increases in and maintenance of UBF are inhibited by L- NAME, a nitric oxide synthase (NOS) inhibitor, little is known about the tissue/cell-specific regulation of microvascular blood flow distribution during the normal ovarian cycle or with steroid treatment. Long-term goals of this project are to determine if tissue/cellular-specific expression of endothelial nitric oxide synthase (ecNOS) is responsible for estrogen (+/- progesterone) modulation of uterine and systemic flows. Overall Hypothesis: There is a cause and effect relationship wherein local exposure of the uterus directly to ovarian estrogen, with or without progesterone priming, will specifically increase expression of the endothelial cell isoform of NOS (ecNOS) within the uterine vasculature via an estrogen receptor action, and that altered uterine and systemic flow distribution during estrogen treatment is indicative of specific tissue endothelial cell changes in ecNOS expression.
Specific Aims :
AIM I. To determine, using radioactive microspheres, the distribution of both uterine perfusion (endometrium, myometrium and caruncles) and cardiac output (systemic flows) during the normal ovarian cycle and the administration of E2beta, and whether L-NAME inhibits these changes in microvascular perfusion. This will confirm the physiologic involvement of NOS.
AIM II. To determine if endogenous ovarian steroids or exogenous E2beta treatment will also increase the expression of NOS at the level of specific activity, protein (Westerns) and mRNA (Northern/PCR), and to immunocytochemically evaluate the specificity of ecNOS, nNOS (neuronal) or macNOS (macrophage-inducible) expression, as well as the tissue distribution of these NOS isoforms. We will perform comparisons with systemic vasculature, based on the results of the microsphere experiments, to establish the specificity of uterine responses and which systemic vascular beds are estrogen responsive.
AIM III. To determine if the direct in vitro exposure of uterine and systemic artery explants in culture to E2beta and/or progesterone will increase ecNOS activity, immunodetectable protein and mRNA levels. These studies will provide a physiologic, biochemical, and molecular foundation for understanding how estrogen and/or progesterone normally modulate the tissue/cell-specific expression of NOS in order control to uterine and/or systemic tissue perfusion. Clinically, these mechanisms are relevant in uterine and systemic tissues, and have significance for both oral contraceptive use and steroid replacement therapy in post-menopausal women, as well as during pregnancy, a natural state of high estrogen and progesterone levels.
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