Pregnancy-dependent changes in cell signaling underlie changes in differential control of vasodilator production in uterine artery endothelial cells.

During pregnancy, the uterine vasculature shows a marked increase in vasodilator production [prostacyclin (PGI2) and nitric oxide (NO)] in response to a number of agonists including angiotensin II (AII) and ATP. As a consequence vascular resistance is kept low, and uterine blood flow is maximized to meet the needs of the growing fetus. Studies of the molecular basis underlying this change in control of endothelial NO and PGI2 production have been hampered by the lack of availability of a suitable cell model. To that end we have developed and characterized a new ovine uterine artery endothelial cell (UAEC) culture model derived from nonpregnant (NP) or pregnant (P) ewes. Endothelial cells were isolated from pregnant (120-130 days; n = 6) and nonpregnant (n = 4) ewes and maintained in primary culture. Endothelial cells at passage 4 showed uniform expression of endothelial nitric oxide synthase (eNOS; an endothelial marker) as well as AII type 1 receptor and growth factor receptors and uniform uptake of acetylated low density lipoprotein (a property of endothelial cells not shared by fibroblasts or vascular smooth muscle cells), thus demonstrating cell purity. Expressions of eNOS, cyclooxygenase-1, PGI2 synthase, cytosolic phospholipase A2, AII type 1 receptor, and growth factor receptors are also maintained at passage 4. Mitogenesis is maintained in response to basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF) in both NP-UAEC and P-UAEC. The differential production of vasodilators by NP-UAEC and P-UAEC is maintained in a manner similar to that previously reported in vivo. Thus, P-UAEC make NO in response to AII, ATP, bFGF, EGF, and VEGF, whereas NP-UAEC make NO in response to bFGF, EGF, and VEGF only. Similarly, P-UAEC make PGI2 in response to AII, ATP, bFGF, and VEGF, whereas NP-UAEC make PGI2 only in response to ATP and VEGF. As both cytosolic phospholipase A2 and eNOS may be regulated by both Ca2+ and protein kinases, we investigated the effects of these agonists on Ca2+ mobilization and ERK-1/2 phosphorylation. ATP consistently elevates Ca2+ levels in both P-UAEC and NP-UAEC. All other agonists were without acute (0-4 min) effect on Ca2+ in P-UAEC or NP-UAEC. In contrast, all agonists stimulated an acute (10 min) phosphorylation of ERK-1/2 in P-UAEC, whereas only EGF stimulated activation in NP-UAEC. P-UAEC production of PGI2 by agonists of both heptahelical receptors and growth factor receptors correlates closely with ERK-2 phosphorylation alone. For NO, this correlation holds for heptahelical receptor agonists, but additional signaling pathways are also implicated for bFGF and VEGF. In contrast, in NP-UAEC the lack of ERK-2 phosphorylation in response to all agonists other than EGF, and the dissociation between NO or PGI2 production and ERK-2 phosphorylation suggest that alternate pathways play a predominant role.