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Submitted on November 4, 2002
Accepted on April 15, 2003
1 Perinatal Research Laboratories, Depts Ob/Gyn & Meat/Animal Sci, Univ Wisconsin-Madison, Madison WI 53715.
* To whom correspondence should be addressed. E-mail: imbird{at}facstaff.wisc.edu.
Ovine uterine artery endothelial cells maintained in culture to passage 4 (UAEC) retain pregnancy-specific changes in vasodilator production, which in turn is associated with differences in Ca2+ and ERK 1/2 signaling. The question remains whether this is an accurate portrayal of the situation in vivo, or more simply if these same signaling responses seen at passage 4 accurately reflect those functioning in the cells in vivo. Small groups of eNOS positive cells from both pregnant and nonpregnant ewes were freshly isolated and used to image changes in the intracellular free calcium concentration ([Ca2+]i) 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 [Ca2+]i elevation in response to AII, EGF, bFGF, or VEGF but did respond to ATP in a dose-dependent (1-300 µM) manner. At higher doses of ATP [Ca2+]i elevation was sustained longer and showed a high incidence of regular oscillations in cells from pregnant compared with nonpregnant ewes. Also ATP and bFGF 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 Ca2+ 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 is both programed at the level of cell signaling and retained in culture.
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