Simultaneous imaging of [Ca2+]iand intracellular NO production in freshly isolated uterine artery endothelial cells: effects of ovarian cycle and pregnancy

Abstract

Pregnancy and the follicular phase of the ovarian cycle show elevation of uterine blood flow and associated increases in uterine artery endothelium (UAE) endothelial nitric oxide (NO) synthase (eNOS) expression. Nonetheless, a role for increased NO production during pregnancy and the follicular phase has only been inferred by indirect measures. The recent development of a uterine artery endothelial cell model further suggests that pregnancy is associated with reprogramming of cell signaling, such that eNOS may become more Ca2+sensitive and be subject to regulation by Ca2+-independent kinases. This study describes for the first time the direct and simultaneous monitoring of NO production and intracellular free Ca2+concentration ([Ca2+]i) in freshly isolated UAE from pregnant, follicular, and luteal sheep. The pharmacological agonists ionomycin (calcium ionophore) and thapsigargin (TG; endoplasmic reticulum Ca2+pump inhibitor) were used to maximally elevate [Ca2+]iand fully activate eNOS as a measure of eNOS expression. NO production stimulated by ionomycin (5 μM) and TG (10 μM) were 1.95- and 2.05-fold, respectively, in pregnant-UAE and 1.34- and 1.37-fold in follicular-UAE compared with luteal-UAE. In contrast, the physiological agonist ATP (100 μM) stimulated a 3.43-fold increase in NO in pregnant-UAE and a 1.90-fold increase in follicular-UAE compared with luteal-UAE, suggesting that pregnancy and follicular phase enhance eNOS activation beyond changes in expression in vivo. 2-aminoethoxydiphenyl borate (APB; an inositol 1,4,5-trisphosphate receptor blocker) totally prevented the ATP-induced [Ca2+]iresponse but only partially inhibited NO production. Thus pregnancy-enhanced eNOS activation in UAE is mediated through [Ca2+]i-insensitive pathways as well as through a greater eNOS sensitivity to [Ca2+]i.