Expression of endothelin 1 and its receptors in the hypoxic pregnant rat

Abstract

Endothelin 1 (EDN1) plays a primary role in the pathophysiology of hypoxia-induced fetal growth restriction in the rat. In this study we evaluated the effects of chronic maternal hypoxia on the expression of endothelin and its receptors and on receptor binding activity in the uterus and placenta of the rat, in order to elucidate their roles in hypoxia-induced fetal growth restriction. Timed-pregnant Sprague-Dawley rats were maintained in either a normoxic or a normobaric hypoxic (12% O(2)) atmosphere from Gestational Days 18-21. Uterine and placental tissues collected on Gestational Day 21 were assayed for Edn1, Ednra, and Ednrb (endothelin receptors) mRNA expression by real-time quantitative RT-PCR, for localization of EDN1 and its receptors by immunohistochemistry, for EDNRA and EDNRB protein expression by Western blot, and for receptor binding activity by homologous competitive binding assays. EDN1 mRNA expression was significantly increased in the hypoxic placenta, but not in the uterus, compared with normoxic controls. Immunohistochemistry revealed increased EDN1 specifically in the labyrinth of the placenta. Receptor mRNA levels were not significantly affected by hypoxia, but EDNRA protein expression was significantly decreased specifically in the uterine placental beds. Receptor binding decreased significantly in response to hypoxia in all tissues investigated, compared with controls. These results suggest that chronic maternal hypoxia results in increased expression of EDN1 in the placenta but not in the uterus, and that reduced binding activity, rather than regulation of receptor expression, is a mechanism by which these tissues regulate the local hemodynamic response to increased endogenous placental EDN1 in the setting of hypoxia.

FIG. 1

Expression of Edn1, as well as Ednra and Ednrb receptor mRNA in rat placentas (P), uterine placental beds (UPB), and uterine free wall (UFW). Comparative mRNA expression was determined by real-time quantitative RT-PCR in tissues from rats maintained in normoxic conditions (n = 6) or in 12% O₂ for 3 days (Gestational Days 18–21, n = 6). Results are expressed as percent of an internal control gene, cytoplasmic beta actin (Actb), and each value represents the mean ± SEM. *P < 0.05 and **P < 0.01 vs. normoxic control.

FIG. 2

Protein expression of EDNRA (48 M_r × 10⁻³, left) and EDNRB (49 M_r × 10⁻³, right) receptors in pregnant rats. Western blots were performed on membranes isolated from placental (P), uterine placental bed (UPB), and uterine free wall (UFW) homogenates from normoxic (N; n = 6) and hypoxic (H; Gestational Days 18–21, 12% O₂, n = 6) pregnant rats. Gels included control membranes (CM) from rat brain and the densitometric results are expressed as the mean ± SEM of the ratio of reproductive tissue and control receptor band densities. Na⁺/K⁺ transporting ATPase alpha 1 polypeptide (ATP1A1; 112 M_r × 10⁻³) was used as a control membrane protein to verify loading of equal amounts of protein into each well. **P < 0.01 vs. normoxic control.

FIG. 3

Immunohistochemistry of the rat placental labyrinth from normoxic rats (A, C, E, and G) and hypoxic rats (B, D, F, and H). Localization is shown for EDNRA (A and B), EDNRB (C and D), EDN1 (E and F), PBS control (G), and EDN1-adsorbed control (H). All photomicrographs are at the same magnification. Bar = 100 μm.

FIG. 4

Homologous competitive binding of EDN1 and EDN3 by EDNRA and EDNRB receptors in rat placenta (P), uterine placental bed (UPB), and uterine free wall (UFW). Placental membranes were prepared from normoxic (n = 6) and hypoxic (3 days, Gestational Days 18–21, 12% O₂, n = 6) pregnant rats. A, C, and E) Representative binding curves for normoxic placental, uterine placental bed, and uterine free wall membrane preparations (A, C, and E, respectively). B, D, and F) Representative binding curves for hypoxic placental, uterine placental bed, and uterine free wall membrane preparations (B, D, and F, respectively). Each experiment was performed in triplicate. G) The maximum binding coefficient, B_max, for both receptors is expressed as fmol endothelin/μg protein and each value represents the mean ± SEM. EDN in captions and axis labels refers to both EDN1 and EDN3. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. normoxic control.