Pregnancy-dependent changes in cell signaling underlie changes in differential control of vasodilator production in uterine artery endothelial cells
Pregnancy-dependent changes in cell signaling underlie changes in differential control of vasodilator production in uterine artery endothelial cells
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DOI：
10.1210/en.141.3.1107
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发表时间：
2000-03-01
期刊：
影响因子：
4.8
通讯作者：
Magness, RR
中科院分区：
文献类型：
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作者：
Bird, IM;Sullivan, JA;Magness, RR
怀孕期间，子宫脉管系统对包括血管紧张素 II (AII) 和 ATP 在内的多种激动剂作出反应，血管舒张剂 [前列环素 (PGI(2)) 和一氧化氮 (NO)] 的产生显着增加。因此，血管阻力保持在较低水平，子宫血流量最大化，以满足胎儿生长的需要。由于缺乏合适的细胞模型，对内皮 NO 和 PGI(2) 产生控制变化的分子基础的研究受到了阻碍。为此，我们开发并表征了一种源自非怀孕（NP）或怀孕（P）母羊的新绵羊子宫动脉内皮细胞（UAEC）培养模型。从怀孕（120-130 天；n = 6）和非怀孕（n = 4）母羊中分离内皮细胞，并维持在原代培养物中。第 4 代的内皮细胞表现出内皮一氧化氮合酶（eNOS；一种内皮标记物）以及 AII 1 型受体和生长因子受体的均匀表达，并均匀摄取乙酰化低密度脂蛋白（成纤维细胞或血管平滑肌细胞不具有的内皮细胞特性），从而证明了细胞纯度。 eNOS、环氧合酶-1、PGI(2) 合酶、胞质磷脂酶 A(2)、AII 1 型受体和生长因子受体的表达也维持在第 4 代。在碱性成纤维细胞生长因子 (bFGF)、表皮生长因子 (EGF) 和血管内皮生长因子 (VEGF) 的作用下，有丝分裂得以维持。 NP-UAEC 和 P-UAEC。 NP-UAEC 和 P-UAEC 血管舒张剂的差异产生以与先前报道的体内相似的方式维持。因此，P-UAEC响应AII、ATP、bFGF、EGF和VEGF而产生NO，而NP-UAEC仅响应bFGF、EGF和VEGF而产生NO。类似地，P-UAEC 响应 AII、ATP、bFGF 和 VEGF 产生 PGI(2)，而 NP-UAEC 仅响应 ATP 和 VEGF 产生 PGI(2)。由于胞质磷脂酶 A(2) 和 eNOS 都可能受到 Ca2+ 和蛋白激酶的调节，因此我们研究了这些激动剂对 Ca2+ 动员和 ERK-1/2 磷酸化的影响。 ATP 持续升高 P-UAEC 和 NP-UAEC 中的 Ca2+ 水平。所有其他激动剂对 P-UAEC 或 NP-UAEC 中的 Ca2+ 没有急性（0-4 分钟）作用。相反，所有激动剂均刺激 P-UAEC 中 ERK-1/2 的急性（10 分钟）磷酸化，而只有 EGF 刺激 NP-UAEC 中的激活。七螺旋受体和生长因子受体激动剂产生的 PGI(2) P-UAEC 与单独的 ERK-2 磷酸化密切相关。对于 NO，七螺旋受体激动剂也存在这种相关性，但 bFGF 和 VEGF 也涉及其他信号通路。相反，在 NP-UAEC 中，ERK-2 磷酸化缺乏对 EGF 以外的所有激动剂的反应，并且 NO 或 PGI(2) 产生与 ERK-2 磷酸化之间的分离表明替代途径发挥着主导作用。
During pregnancy, the uterine vasculature shows a marked increase in vasodilator production [prostacyclin (PGI(2)) 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 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 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, PGI(2) synthase, cytosolic phospholipase A(2), 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 PGI(2) in response to AII, ATP, bFGF, and VEGF, whereas NP-UAEC make PGI(2) only in response to ATP and VEGF. As both cytosolic phospholipase A(2) 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 PGI(2) 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 PGI(2) production and ERK-2 phosphorylation suggest that alternate pathways play a predominant role.