Gestational intermittent hypoxia induces endothelial dysfunction and hypertension in pregnant rats: role of endothelin type B receptor†

Abstract

Obstructive sleep apnea is a recognized risk factor for gestational hypertension, yet the exact mechanism behind this association remains unclear. Here, we tested the hypothesis that intermittent hypoxia, a hallmark of obstructive sleep apnea, induces gestational hypertension through perturbed endothelin-1 signaling. Pregnant Sprague-Dawley rats were subjected to normoxia (control), mild intermittent hypoxia (10.5% O2), or severe intermittent hypoxia (6.5% O2) from gestational days 10-21. Blood pressure was monitored. Plasma was collected and mesenteric arteries were isolated for myograph and protein analyses. The mild and severe intermittent hypoxia groups demonstrated elevated blood pressure, reduced plasma nitrate/nitrite, and unchanged endothelin-1 levels compared to the control group. Western blot analysis revealed decreased expression of endothelin type B receptor and phosphorylated endothelial nitric oxide synthase, while the levels of endothelin type A receptor and total endothelial nitric oxide synthase remained unchanged following intermittent hypoxia exposure. The contractile responses to potassium chloride, phenylephrine, and endothelin-1 were unaffected in endothelium-denuded arteries from mild and severe intermittent hypoxia rats. However, mild and severe intermittent hypoxia rats exhibited impaired endothelium-dependent vasorelaxation responses to endothelin type B receptor agonist IRL-1620 and acetylcholine compared to controls. Endothelium denudation abolished IRL-1620-induced vasorelaxation, supporting the involvement of endothelium in endothelin type B receptor-mediated relaxation. Treatment with IRL-1620 during intermittent hypoxia exposure significantly attenuated intermittent hypoxia-induced hypertension in pregnant rats. This was associated with elevated circulating nitrate/nitrite levels, enhanced endothelin type B receptor expression, increased endothelial nitric oxide synthase activation, and improved vasodilation responses. Our data suggested that intermittent hypoxia exposure during gestation increases blood pressure in pregnant rats by suppressing endothelin type B receptor-mediated signaling, providing a molecular mechanism linking intermittent hypoxia and gestational hypertension.

Keywords: blood pressure; eNOS; endothelin type B receptor; endothelium; intermittent hypoxia; pregnancy.

Graphical Abstract

Figure 1

Effect of IH on maternal mean blood pressure, and circulating endothelin-1 and nitrate/nitrite (NO_x) levels. Pregnant rats were exposed to control, mild intermittent hypoxia (mIH), or severe intermittent hypoxia (sIH) from GD 10 to 21 during their sleep cycle. (A) Blood pressures were monitored non-invasively from GD 17 to 20. n = 6 per group. Repeated measures ANOVA and Dunnett post hoc test. ^#P < 0.05 for mIH versus Control. ^*P < 0.05 for sIH versus Control. (B) Plasma endothelin-1 and NO_x levels were measured using enzyme immunoassay. n of 6 in each group. One-way ANOVA and Dunnett post hoc test. All data are expressed as mean ± SEM.

Figure 2

Effect of IH on protein expression levels of endothelin type A receptor (ET_AR), endothelin type B receptor (ET_BR), endothelial nitric oxide synthase (eNOS), and phosphorylated eNOS (p-eNOS) at Ser¹¹⁷⁷ in the mesenteric artery. Mesenteric arteries were collected on GD 21, processed for protein isolation, and probed for endothelin receptors and eNOS. Representative blots are shown in the top panel. Blot density obtained from densitometric scanning of ET_AR, ET_BR, and eNOS normalized to GAPDH and of p-eNOS normalized to total eNOS is shown in the bottom panel; n of 6 in each group. Data are shown as mean ± SEM (one-way ANOVA and Dunnett post hoc test).

Figure 3

Effect of IH on vascular relaxation function in the mesenteric artery. Mesenteric arterial rings mounted in a wire myograph were pre-contracted with submaximal phenylephrine (PE) concentration and tested for endothelium-dependent relaxation responses to incremental doses of (A) endothelin type B receptor (ET_BR) selective agonist IRL-1620 and (B) acetylcholine (ACh), and endothelium-independent relaxation to (C) sodium nitroprusside (SNP). n of 6 in each group. Data are expressed as mean ± SEM. ^#P < 0.05 for mIH versus Control. ^*P < 0.05 for sIH versus Control (two-way ANOVA and Sidak post hoc test).

Figure 4

Effect of IRL-1620 treatment on maternal mean blood pressure and plasma nitrate/nitrite (NO_x) levels. Pregnant rats exposed to control or severe intermittent hypoxia (sIH) were implanted with osmotic pumps delivering endothelin B receptor (ET_BR) selective agonist IRL-1620 on GD 14. (A) Blood pressures were measured from GD 17 to 20. (B) Plasma NO_x levels were assessed using enzyme immunoassay. n of 6 in each group. Data are expressed as mean ± SEM. Repeated measures ANOVA and Sidak post hoc test in (A), and two-way ANOVA and Sidak post hoc test in (B). ^# were noted for 0.05 < P < 0.1. ^* were noted for P < 0.05.

Figure 5

Effect of IRL-1620 treatment on relaxation function in the mesenteric artery of control and severe intermittent hypoxia (sIH) exposed rats. Mesenteric arterial rings were isolated for wire myograph. (A) Endothelium-dependent relaxation to ET_BR selective agonist IRL-1620, (B) endothelium-dependent relaxation to acetylcholine (ACh), and (C) endothelium-independent relaxation to sodium nitroprusside (SNP) were tested following pre-contraction with submaximal phenylephrine (PE) concentration. n of 6 in each group. Data are expressed as mean ± SEM. ^* were noted for P < 0.05 compared to sIH + Vehicle group (two-way ANOVA and Sidak post hoc test).

Figure 6

Effect of IRL-1620 treatment on protein expression levels of endothelin type A receptor (ET_AR), endothelin type B receptor (ET_BR), endothelial nitric oxide synthase (eNOS), and phosphorylated eNOS (p-eNOS) at Ser¹¹⁷⁷ in the mesenteric artery. Protein isolated from mesenteric arteries was probed for endothelin receptors and eNOS. Representative blots are shown in the top panel. Blot density obtained from densitometric scanning of ET_AR, ET_BR, and eNOS normalized to GAPDH and of p-eNOS normalized to total eNOS is shown in the bottom panel. n of 6 in each group. Data are expressed as mean ± SEM (two-way ANOVA and Sidak post hoc test).