Expression of Endothelial and Inducible Nitric Oxide Synthases and Nitric Oxide Production in Ovine Placental and Uterine Tissues During Late Pregnancya

Abstract

We evaluated the expression of endothelial (eNOS) and inducible (iNOS) nitric oxide (NO) synthases, NO production, and the role of angiotensin II (ANG II) in regulating NO production during late ovine pregnancy (day 110–142). Samples of the following tissues were obtained: fetal [cotyledonary (COT)] and maternal [caruncular (CAR)] portions of the placentoma, intercotyledonary fetal chorioallantoic membrane (ICOT) and intercaruncular maternal endometrium (ICAR). Using immunohistochemistry, eNOS positive staining was detected in all four tissues, primarily in the endothelium, chorioallantoic membrane, and luminal and glandular epithelium. For iNOS, the positive staining was observed primarily in stromal cells in ICOT and ICAR. Expression of eNOS and iNOS proteins was confirmed in COT using Western immunoblot. eNOS protein levels increased (P< 0.05) approximately 3.5-fold from day 110 to 130 and then declined at term, whereas no change in iNOS protein levels was observed throughout the days studied. The tissue explants of COT, CAR, ICOT and ICAR were cultured in media in the absence or presence of ANG II (10−9or 10−7m) for 24h. Total NO (nitrate and nitrite) levels in the explant-conditioned media were determined by chemiluminesence. In fetal COT, total NO levels increased (P< 0.05) 3.5-fold from day 110 to 130 and then declined (P< 0.05) at term. In ICOT, total NO levels exhibited a gradually increasing trend (r2=0.96, P< 0.01) from day 110 to days 130 and 142. In maternal CAR, total NO levels were higher (P< 0.05) on day 130 than those on days 120 and 142, whereas no change in total NO levels was observed in ICAR. ANG II at 10−7m treatment decreased (P< 0.05) total NO levels in COT on day 130. Thus, during late ovine pregnancy: (1) eNOS is expressed in COT, CAR, ICOT and ICAR while iNOS is primarily seen in stromal cells of ICOT and ICAR; (2) NO production by COT exhibits a biphasic pattern and parallels the changes in eNOS, but not iNOS protein levels, suggesting that eNOS is a predominant NOS isoform for the NO production; and (3) ANG II may contribute partially to decreases in NO production by COT at term.