During pregnancy (P), the uterine artery (UA) becomes more refractory to vasoconstrictors. There is also a dramatic enhancement of agonist induced NO production and/or angiogenesis by both the UA and placental artery (PA), so leading to a reduction in vascular resistance and coordinated increases in blood flow on both the maternal and fetal side of the placenta which is so necessary to support the growing fetus. We have coordinated increases in blood flow on both the maternal and fetal side of the placenta which is so necessary to support the growing fetus. We have recently shown in both UA endothelial cells (UAEC) and PA endothelial cells (OFPAEC) that both activation of eNOS and mitogenesis may be mediated more through Extracellular signal Regulated Protein Kinase 1 and 2 (ERK-1/2, members of the MAPK family) rather than elevations in Ca2+, but that additional pathways may also be involved. In addition this signaling pathway may be a convergence point of vascular control by both classical growth factors bFGF and VEGF as well as heptahelical receptor agonists such as Angiotensin II (AII). Therefore we propose that a greater understanding of the signaling events by which bFGF and VEGF control UAEC and OFPAEC function would be of value, as would be an understanding of the signaling events by which bFGF and VEGF as well as heptahelical receptor agonists such as Angiotensin II (AII). Therefore we propose that a greater understanding of the signaling events by which bFGF and VEGF control UAEC and OFPAEC function would be of value, as would be an understanding of how these events integrate with the signaling pathways initiated through heptahelical receptors. To this end we propose Specific Aim 1: to establish if the action of bFGF or VEGF, but not AII or ATP on MEK mediated activation of ERK-1/2 occurs via growth factor receptor autophosphorylation and subsequent phosphotyrosine recruitment of adaptor proteins (Shc/Grb2/SOS) leading to Ras/Raf activation of MEK in P-UAEC and OFPAEC and at what level this is uncoupled in NP-UAEC. Specific Aim 2: to establish if AII and ATP mediated activation of MEK occurs through alternative pathways independent of growth factor receptor phosphorylation and adaptor protein recruitment, namely via the Scr/RAF/MEK cascade in P0UAEC and OFPAEC and at what level this is uncoupled in NP-UAEC. Specific Aim 3: to further delineate the changes which occur in cell signaling in progression from the non-pregnant to pregnant state and so further clarify the mechanistic basis for these changes. This will be investigated by: (3A) further comparing the time course of activation of the Shc/Grb2/SOS/Ras/Raf pathway and the Src/Raf/MEK pathway in response to bFGF, VEGF, AII, ATP and phorbol ester (TPA-control) in PO-UAEC vs. NP-UAEC., and (3B) to further investigate possible MEK independent signaling via PI3-kinase/AKT in NP-UAEC, P-UAEC vs. NP-UAEC, AND (3B) to further investigate possible MEK independent signaling via PI3-kinase/AKT in NP-UAEC, P-UAEC and OFPAEC. Specific Aim 4: To establish the importance of these signaling intermediates so identified in Aims 1-3 at the level of agonist-stimulated NO production in NP-UAEC, P-UAEC and OFPAEC by observing the effects of selective inhibitors on activity of signaling intermediates and relating any such effects to changes in NO production, eNOS phosphorylation and mitogenesis respectively. In this way not only will we address the state hypothesis but also provide additional information necessary for the completion of project 2 as well as a greater mechanistic understanding of the control of mitogenesis through growth factor secretion, as investigated in projects 3 and 4. Specific Aim 5: to relate these observed changes in vitro back to the pregnancy induced changes in vivo. Where signaling pathway intermediates have been shown to be lost or gained at the level of function we will perform western analysis on UA endothelium and Immunohistochemistry on UA secretions from OVEX, follicular phase, luteal phase and pregnant ewes to determine if change in expression occurs in vivo. We believe these studies will provide a significant advice in both the understanding of normal controls and coordination of UA and PA endothelial function and the mechanism underlying pregnancy-induction of increased UA refractoriness to a variety of vasoconstrictors, and may also provide a new model of endothelial cell function on which future studies searching for the maternal and feto-placental vascular dysfunction leading to preeclampsia and IUGR may be based.