Local Effects of Pregnancy on Connexin Proteins That Mediate Ca ²⁺ -Associated Uterine Endothelial NO Synthesis

Abstract

Uterine artery adaptations during gestation facilitate increases in uterine blood flow and fetal growth. Hypothesis: Local expression and distribution of uterine artery connexins play roles in mediating in vivo gestational eNOS activation and Nitric Oxide production. We established an ovine model restricting pregnancy to a single uterine horn and measured uterine blood flow, uterine artery shear stress, connexins 37/43 and P635eNOS protein levels in uterine artery and systemic artery [Omental and Renal] endothelium and connexins in vascular smooth muscle. Uterine blood flow and shear stress were locally (unilaterally) and substantially elevated by gestation. During pregnancy uterine artery endothelial gap junction proteins connexins 37/43 were locally regulated in the gravid horn and elevated 10.3- and 25.6-fold; uterine artery endothelial P635eNOS and total eNOS were elevated 3.3- and 2.9-fold; whereas uterine artery vascular smooth muscle connexins 37/43 were locally elevated 12.5- and 5.9-fold, respectively. Less pronounced changes were observed in systemic vasculature except for significant pregnancy-associated increases in omental artery vascular smooth muscle connexin 43 and omental artery endothelial P635eNOS and total eNOS. Gap junction blockade using connexin 43, but not connexin 37 specific GAP peptides abrogated uterine artery endothelial ATP-induced Ca2+-mediated nitric oxide production. Thus uterine artery endothelial connexin43, but not connexin 37 regulates Ca2+-mediated nitric oxide production required for the vasodilation to accommodate increases in uterine blood flow and shear stress during healthy pregnancies.

Uterine artery leptin receptors during the ovarian cycle and pregnancy regulate angiogenesis in ovine uterine artery endothelial cells†

“…This model restricts pregnancy to only one horn (gravid) throughout gestation allowing us to study local pregnancy-specific maternal-fetal vascular function and angiogenesis [25][26][27]. The control pregnant group comprised of sham operated (n = 15) and nonsurgical controls (n = 22).…”

“…For the ex vivo western analysis of the LEPR in UA, OA, and RA that were obtained from synchronized nonpregnant (luteal; n = 8 and follicular; n = 8), unilateral pregnant (UA nongravid versus gravid sides; n = 15), and control bilateral pregnant sheep (120-130 days/gestation; n = 23) by reprobing the same western blots available from our previous work [26]. Western analysis were performed using methods we previously described [26,30,35,38] and detection was performed using enhanced chemiluminescence (ECL) and HyperFilm ECL. Validation and loading controls for these western blots are described in these previous publications [26,30] and the on line supplement.…”

“…Western analysis were performed using methods we previously described [26,30,35,38] and detection was performed using enhanced chemiluminescence (ECL) and HyperFilm ECL. Validation and loading controls for these western blots are described in these previous publications [26,30] and the on line supplement.…”

“…UAECs were isolated and fully validated from nonpregnant (luteal, n = 4; follicular, n = 4), and late pregnant (n = 4; 120-130 days/gestation) sheep as previously described and studied in culture at passage 4 UAECs [36,38] western analysis were performed using methods we previously described and validated [26,35,38]. Protein detection was performed using enhanced ECL and HyperFilm ECL with β-actin as loading control.…”

“…During pregnancy, these uterine vascular adaptations lead to the rises in UBF which are required to provide for an adequate maternal-fetal vascular interface to meet the nutrient and oxygen demands of the rapidly growing fetus [21]. Using a preconception surgical model, we established a cohort of unilateral ovine pregnancies and demonstrated that rises in UBF as well as uterine vascular adaptations are seen only ipsilateral, but not contralateral, to the fetus and placental unit [25][26][27]. Additionally, we previously reported experimental analyses of the ex vivo uterine artery endothelium (UAendo) and uterine artery vascular smooth muscle (UAvsm) from pregnant versus nonpregnant ewes.…”