Gifford Shannon M, Cale Jackie M, Tsoi Stephen, Magness Ronald R, Bird Ian M
Departments of Obstetrics/Gynecology, University of Wisconsin-Madison, Madison, Wisconsin 53715, USA.
Endocrinology. 2003 Aug;144(8):3639-50. doi: 10.1210/en.2002-0006.
Ovine uterine artery (UA) endothelial cells (UAEC) maintained in culture to passage 4 retain pregnancy-specific changes in vasodilator production, which in turn is associated with differences in Ca(2+) and ERK 1/2 signaling. The question remains whether this is an accurate portrayal of the situation in vivo, or more simply whether these same signaling responses seen at passage 4 accurately reflect those functioning in the cells in vivo. Small groups of endothelial nitric oxide synthase-positive cells from both pregnant and nonpregnant ewes were freshly isolated and used to image changes in the intracellular free calcium concentration (Ca(2+)) using fura 2 and to detect ERK 1/2 phosphorylation by immunocytochemistry. Furthermore, detailed comparisons of mRNA species were made between freshly isolated and cultured (passage 4) cells using cDNA microarray analysis and verified, where possible, using PowerBlot analysis. Freshly isolated cells showed no detectable Ca(2+) elevation in response to angiotensin II, epidermal growth factor, basic fibroblast growth factor, or vascular endothelial growth factor but did respond to ATP in a dose-dependent (1-300 microM) manner. At higher doses of ATP, Ca(2+) elevation was sustained longer and showed a high incidence of regular oscillations in cells from pregnant compared with nonpregnant ewes. Also, ATP and basic fibroblast growth factor treatment caused activation of ERK 1/2 in significantly greater numbers of freshly isolated cells from pregnant than from nonpregnant ewes. cDNA microarray analysis showed results consistent with endothelium but revealed few differences in mRNA species and levels between freshly isolated and passage 4 cells or between the pregnant and nonpregnant ewes. In conclusion, our data show for the first time that pregnancy-specific changes in Ca(2+) and ERK 1/2 signaling are indeed observed in freshly isolated UA endothelium. This suggests in turn that such pregnancy-specific changes in UA endothelial function in vivo in response to a variety of agonists during pregnancy are both programmed at the level of cell signaling and retained in culture.
培养至第4代的绵羊子宫动脉（UA）内皮细胞（UAEC）保留了血管舒张剂产生方面的妊娠特异性变化，而这又与Ca(2+)和ERK 1/2信号传导的差异相关。问题仍然存在，这是否准确描绘了体内的情况，或者更简单地说，在第4代观察到的这些相同的信号反应是否准确反映了体内细胞中的功能。从怀孕和未怀孕的母羊中新鲜分离出小群内皮型一氧化氮合酶阳性细胞，用于使用fura 2成像细胞内游离钙浓度（[Ca(2+)]i）的变化，并通过免疫细胞化学检测ERK 1/2磷酸化。此外，使用cDNA微阵列分析对新鲜分离的细胞和培养（第4代）细胞之间的mRNA种类进行了详细比较，并在可能的情况下使用PowerBlot分析进行了验证。新鲜分离的细胞对血管紧张素II、表皮生长因子、碱性成纤维细胞生长因子或血管内皮生长因子无明显的[Ca(2+)]i升高反应，但对ATP有剂量依赖性（1 - 300 microM）反应。在较高剂量的ATP作用下，与未怀孕母羊相比，怀孕母羊细胞中的[Ca(2+)]i升高持续时间更长，且出现规律性振荡的发生率更高。此外，ATP和碱性成纤维细胞生长因子处理导致怀孕母羊新鲜分离细胞中ERK 1/2的激活数量明显多于未怀孕母羊。cDNA微阵列分析结果与内皮细胞一致，但新鲜分离细胞与第4代细胞之间或怀孕与未怀孕母羊之间的mRNA种类和水平差异很少。总之，我们的数据首次表明，在新鲜分离的UA内皮中确实观察到了Ca(2+)和ERK 1/2信号传导的妊娠特异性变化。这进而表明，怀孕期间UA内皮功能在体内对多种激动剂的这种妊娠特异性变化在细胞信号传导水平上是预先编程的，并在培养中得以保留。
