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 vasodi lator production [prostacyclin (PGI(2)) and nitric oxide (NO)] in response to a number of agonists including angiotensin II (AII) and ATP. As a conseq uence vascular resistance is kept low, and uterine blood flow is maximized to meet the needs of the growing fetus. Studies of the molecular basis unde rlying this change in control of endothelial NO and PGI(2) 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 endothe lial 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. Endothelia l cells at passage 4 showed uniform expression of endothelial nitric oxide synthase (eNOS; an endothelial marker) as well as AII type 1 receptor and g rowth factor receptors and uniform uptake of acetylated low density lipopro tein (a property of endothelial cells not shared by fibroblasts or vascular smooth muscle cells), thus demonstrating cell purity. Expressions of eNOS, cyclooxygenase-1, PGI(2) synthase, cytosolic phospholipase A(2), AII type 1 receptor, and growth factor receptors are also maintained at passage 4. M itogenesis is maintained in response to basic fibroblast growth factor (bFG F), epidermal growth factor (EGF), and vascular endothelial growth factor ( VEGF) in both NP-UAEC and P-UAEC. The differential production of vasodilato rs by NP-UAEC and P-UAEC is maintained in a manner similar to that previous ly reported in vivo. Thus, P-UAEC make NO in response to AII, ATP, bFGF, EG F, and VEGF, whereas NP-UAEC make NO in response to bFGF, EGF, and VEGF onl y. Similarly, P-UAEC make PGI(2) in response to AII, ATP, bFGF, and VEGF, w hereas NP-UAEC make PGI(2) only in response to ATP and VEGF. As both cytoso lic phospholipase A(2) and eNOS may be regulated by both Ca2+ and protein k inases, 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 PGI(2) by agonists of both hep tahelical receptors and growth factor receptors correlates closely with ERK -2 phosphorylation alone. For NO, this correlation holds for heptahelical r eceptor agonists, but additional signaling pathways are also implicated for bFGF and VEGF. In contrast, in NP-UAEC the lack of ERK-2 phosphorylation i n response to all agonists other than EGF, and the dissociation between NO or PGI(2) production and ERK-2 phosphorylation suggest that alternate pathw ays play a predominant role.