Endothelial vasodilator production by uterine and systemic arteries. VI. Ovarian and pregnancy effects on eNOS and NOx

Abstract

Normal pregnancy and the follicular phase of the ovarian cycle are both estrogen-dominated physiological states that are characterized by elevations in uterine blood flow and endothelial nitric oxide synthase (eNOS) protein expression in the uterine artery (UA) endothelium. It is unknown if elevations in mRNA level account for the changes in protein or eNOS activity. We tested the hypothesis that pregnancy and the follicular phase are associated with increases in eNOS mRNA and the consequent elevated expression of eNOS protein results in increased circulating nitric oxide (NO) levels. UA were obtained from pregnant (PREG; n = 8; 110–130 days gestation; term = 145 ± 3 days), nonpregnant luteal (LUT; n = 6), nonpregnant follicular (FOL; n= 6), and nonpregnant ovariectomized (OVEX; n = 6) sheep. Circulating NO levels were analyzed as total NO2-NO3 (NOx). Western analysis performed on UA endothelial-isolated proteins demonstrated that eNOS protein levels were OVEX = LUT ≤ FOL < PREG ( P < 0.05), whereas eNOS mRNA expression (RT-PCR) in UA endothelial cells obtained by limited collagenase digestion was OVEX < LUT < FOL < PREG ( P < 0.05). Pregnancy dramatically elevated eNOS protein (4.1- to 6.9-fold) and mRNA (2.4- to 6.9-fold) over LUT controls ( P < 0.01). Circulating NOx levels were not altered by ovariectomy or the ovarian cycle but were elevated from 4.4 ± 1.1 μM in LUT to 12 ± 4, 22 ± 3, and 41 ± 3 μM at 110, 120, and 130 days gestation ( P < 0.01). Systemic NOxlevels in singleton (12.5 ± 1.6 μM) were less ( P < 0.01) than in multiple (twin 27.6 ± 6.5 μM; triplet = 46 ± 10 μM) pregnancies. Therefore, the follicular phase and, to a much greater extent, pregnancy are associated with elevations in UA endothelium-derived eNOS expression, although significant increases in systemic NOx levels were only observed in the PREG group (multiple > singleton). Thus, although UA endothelial increases in eNOS protein and mRNA levels are associated with high estrogen states, increases in local UA NO production may require additional eNOS protein activation to play its important role in the maintenance of uterine blood flow in pregnancy.