Xiang-Qun Hu

Pregnancy Upregulates Large-Conductance Ca 2+ -Activated K + Channel Activity and Attenuates Myogenic Tone in Uterine Arteries

Uterine vascular tone significantly decreases whereas uterine blood flow dramatically increases during pregnancy. However, the complete molecular mechanisms remain elusive. We hypothesized that increased Ca2+-activated K+ (BKCa) channel activity contributes to the decreased myogenic tone of uterine arteries in pregnancy. Resistance-sized uterine arteries were isolated from nonpregnant and near-term pregnant sheep. Electrophysiological studies revealed a greater whole-cell K+ current density in pregnant compared with nonpregnant uterine arteries. Tetraethylammonium and iberiotoxin inhibited K+ currents to the same extent in uterine arterial myocytes. The BKCa channel current density was significantly increased in pregnant uterine arteries. In accordance, tetraethylammonium significantly increased pressure-induced myogenic tone in pregnant uterine arteries and abolished the difference in myogenic responses between pregnant and nonpregnant uterine arteries. Activation of protein kinase C produced a similar effect to tetraethylammonium by inhibiting BKCa channel activity and increasing myogenic tone in pregnant uterine arteries. Chronic treatment of nonpregnant uterine arteries with physiologically relevant concentrations of 17&bgr;-estradiol and progesterone caused a significant increase in the BKCa channel current density. Western blot analyses demonstrated a significant increase of the &bgr;1, but not &agr;, subunit of BKCa channels in pregnant uterine arteries. In accordance, steroid treatment of nonpregnant uterine arteries resulted in an upregulation of the &bgr;1, but not &agr;, subunit expression. The results indicate that the steroid hormone-mediated upregulation of the &bgr;1 subunit and BKCa channel activity may play a key role in attenuating myogenic tone of the uterine artery in pregnancy.

Chronic Hypoxia Suppresses Pregnancy-Induced Upregulation of Large-Conductance Ca2+-Activated K+ Channel Activity in Uterine Arteries

Our previous study demonstrated that increased Ca2+-activated K+ (BKCa) channel activity played a key role in the normal adaptation of reduced myogenic tone of uterine arteries in pregnancy. The present study tested the hypothesis that chronic hypoxia during gestation inhibits pregnancy-induced upregulation of BKCa channel function in uterine arteries. Resistance-sized uterine arteries were isolated from nonpregnant and near-term pregnant sheep maintained at sea level (≈300 m) or exposed to high-altitude (3801 m) hypoxia for 110 days. Hypoxia during gestation significantly inhibited pregnancy-induced upregulation of BKCa channel activity and suppressed BKCa channel current density in pregnant uterine arteries. This was mediated by a selective downregulation of BKCa channel &bgr;1 subunit in the uterine arteries. In accordance, hypoxia abrogated the role of the BKCa channel in regulating pressure-induced myogenic tone of uterine arteries that was significantly elevated in pregnant animals acclimatized to chronic hypoxia. In addition, hypoxia abolished the steroid hormone-mediated increase in the &bgr;1 subunit and BKCa channel current density observed in nonpregnant uterine arteries. Although the activation of protein kinase C inhibited BKCa channel current density in pregnant uterine arteries of normoxic sheep, this effect was ablated in the hypoxic animals. The results demonstrate that selectively targeting BKCa channel &bgr;1 subunit plays a critical role in the maladaption of uteroplacental circulation caused by chronic hypoxia, which contributes to the increased incidence of preeclampsia and fetal intrauterine growth restriction associated with gestational hypoxia.

Chronic hypoxia suppresses pharmacomechanical coupling of the uterine artery in near‐term pregnant sheep.

1. The role of inositol 1,4,5‐trisphosphate (InsP3) in the reduced vascular responsiveness to 5‐hydroxytryptamine (5‐HT) caused by chronic hypoxia was examined in uterine arteries obtained from normoxic (control) and chronically hypoxic pregnant sheep (approximately 140 days gestation) maintained at high altitude (3820 m; arterial PO2, 60 mmHg) from 30 days gestation. 2. Chronic hypoxia significantly decreased uterine artery contractile sensitivity in that pD2 (‐logEC50) for the contractile response to 5‐HT was 7.19 +/‐ 0.15 and 6.62 +/‐ 0.12 (P < 0.05) in uterine arteries from normoxic and chronically hypoxic sheep, respectively. The intrinsic efficacy of the agonist was reduced by 75%. Although 5‐HT2A receptor density (Bmax) in the uterine artery was not changed in chronically hypoxic sheep compared with normoxic sheep (32.0 +/‐ 9.8 vs. 31.9 +/‐ 5.9 fmol (mg protein)‐1, respectively) as assessed from the saturation binding of [3H]ketanserin, the agonist binding affinity (pKA, ‐log of dissociation constant) was decreased from 6.25 +/‐ 0.07 in normoxic sheep to 5.85 +/‐ 0.08 in chronically hypoxic sheep (P < 0.05). 3. Chronic hypoxia did not change the time course of 5‐HT‐induced InsP3 synthesis but decreased its potency in inducing InsP3 synthesis, with the pD2 being 6.09 +/‐ 0.11 and 5.51 +/‐ 0.08 (P < 0.05) in uterine arteries from normoxic and chronically hypoxic sheep, respectively. The maximal response of 5‐HT‐induced InsP3 generation in the uterine artery was decreased from 251.3 +/‐ 24.2 pmol (mg protein)‐1 in normoxic sheep to 146.6 +/‐ 11.3 pmol (mg protein)‐1 in chronically hypoxic sheep (P < 0.05). Furthermore, the ability of the activated 5‐HT receptors to couple InsP3 synthesis was significantly decreased in chronically hypoxic compared with normoxic sheep (280 +/‐ 10 vs. 450 +/‐ 20 fmol InsP3 (fmol receptor)‐1, P < 0.01). In addition, for a given amount of InsP3 generated, the contractile force of the uterine artery was significantly less in chronically hypoxic sheep (0.82 +/‐ 0.08 g tension (pmol InsP3)‐1) than that in normoxic sheep (1.28 +/‐ 0.05 g tension (pmol InsP3)‐1) (P < 0.05). 4. These results suggest that chronic hypoxia suppresses pharmacomechanical coupling of the ovine uterine artery by inhibiting the efficiency of receptor‐effector‐contraction coupling. This suppression of the InsP3 pathway may play an important role in the adjustment of vascular tone and uterine blood flow in response to the stress of chronic hypoxia in late pregnancy.

Chronic hypoxia during gestation enhances uterine arterial myogenic tone via heightened oxidative stress.

Chronic hypoxia during gestation has profound adverse effects on the adaptation of uteroplacental circulation in pregnancy. Yet, the underlying mechanisms are not fully understood. The present study tested the hypothesis that enhanced production of reactive oxygen species (ROS) in uterine arteries plays a critical role in the maladaptation of uterine circulation associated with chronic hypoxia. Uterine arteries were isolated from nonpregnant and near-term pregnant sheep maintained at sea level (∼300 m) or exposed to high-altitude (3801 m) hypoxia for 110 days. Hypoxia significantly increased ROS production in uterine arteries of pregnant, but not nonpregnant, sheep. This was associated with a significant increase in NADPH oxidase (Nox) 2, but not Nox1 or Nox4, protein abundance and total Nox activity in uterine arteries of pregnant animals. Chronic hypoxia significantly increased pressure-dependent uterine arterial myogenic tone in pregnant sheep, which was abrogated by a Nox inhibitor apocynin. Additionally, the hypoxia-induced increase in myogenic reactivity of uterine arteries to phorbol 12,13-dibutyrate in pregnant sheep was blocked by apocynin and tempol. In consistence with the myogenic responses, the hypoxia-mediated down-regulation of BKCa channel activity in uterine arteries of pregnant animals was reversed by apocynin. The findings suggest that heightened oxidative stress in uterine arteries plays a key role in suppressing the BKCa channel activity, resulting in increased myogenic reactivity and maladaptation of uteroplacental circulation caused by chronic hypoxia during gestation.

Chronic Hypoxia Inhibits Pregnancy-Induced Upregulation of SKCa Channel Expression and Function in Uterine Arteries

Small-conductance Ca2+-activated K+ (SKCa) channels are crucial in regulating vascular tone and blood pressure. The present study tested the hypothesis that SKCa channels play an important role in uterine vascular adaptation in pregnancy, which is inhibited by chronic hypoxia during gestation. Uterine arteries were isolated from nonpregnant and near-term pregnant sheep maintained at sea level (≈300 m) or exposed to high-altitude (3801 m) hypoxia for 110 days. Immunohistochemistry revealed the presence of SKCa channels type 2 (SK2) and type 3 (SK3) in both smooth muscles and endothelium of uterine arteries. The expression of SK2 and SK3 channels was significantly increased during pregnancy, which was inhibited by chronic hypoxia. In normoxic animals, both SKCa channel opener NS309 and a large-conductance (BKCa) channel opener NS1619 relaxed norepinephrine-contracted uterine arteries in pregnant but not nonpregnant sheep. These relaxations were inhibited by selective SKCa and BKCa channel blockers, respectively. NS309-induced relaxation was largely endothelium-independent. In high-altitude hypoxic animals, neither NS1691 nor NS309 produced significant relaxation of uterine arteries in either nonpregnant or pregnant sheep. Similarly, the role of SKCa channels in regulating the myogenic reactivity of uterine arteries in pregnant animals was abrogated by chronic hypoxia. Accordingly, the enhanced SKCa channel activity in uterine arterial myocytes of pregnant animals was ablated by chronic hypoxia. The findings suggest a novel mechanism of SKCa channels in regulating myogenic adaptation of uterine arteries in pregnancy and in the maladaptation of uteroplacental circulation caused by chronic hypoxia during gestation.

Gestational Hypoxia Increases Reactive Oxygen Species and Inhibits Steroid Hormone–Mediated Upregulation of Ca 2+ -Activated K + Channel Function in Uterine Arteries

Gestational hypoxia inhibits steroid hormone–induced upregulation of Ca2+-activated K+ (KCa) channel activities in uterine arteries. We tested the hypothesis that increased reactive oxygen species play an important role in hypoxia-mediated inhibition of KCa channel activities. Uterine arteries were isolated from nonpregnant (nonpregnant uterine artery) and near-term (≈142–145 day) pregnant (pregnant uterine artery) sheep maintained at either sea level or high altitude (3820 m, PaO2: 60 mm Hg) for 110 days. In pregnant uterine arteries, hypoxia significantly decreased large conductance channel opener NS1619- and small conductance channel opener NS309-induced relaxations, which were partially restored by reactive oxygen species inhibitor N-acetylcysteine (NAC). NAC significantly increased large conductance KCa but not small conductance KCa current densities in uterine arterial smooth muscle cells in pregnant animals acclimatized to high altitude. The NAC-sensitive component of small conductance KCa–induced relaxations was diminished in endothelium-denuded arteries. In nonpregnant uterine arteries, NS1619- and NS309-induced relaxations were diminished compared with those in pregnant uterine arteries. Treatment of nonpregnant uterine arteries with 17&bgr;-estradiol and progesterone for 48 hours increased small conductance KCa type 3 protein abundance and NS1619- and NS309-induced relaxations, which were inhibited by hypoxia. This hypoxia-mediated inhibition was reversed by NAC. Consistently, steroid hormone treatment had no significant effects on large conductance KCa current density in nonpregnant uterine arteries of hypoxic animals in the absence of NAC but significantly increased it in the presence of NAC. These results suggest an important role of hypoxia-mediated reactive oxygen species in negatively regulating steroid hormone–mediated upregulation of KCa channel activity and adaptation of uterine vascular reactivity in pregnancy, which may contribute to the increased incidence of preeclampsia and fetal intrauterine growth restriction associated with gestational hypoxia.

Hypoxia Represses ER-α Expression and Inhibits Estrogen-Induced Regulation of Ca2+-Activated K+ Channel Activity and Myogenic Tone in Ovine Uterine Arteries Causal Role of DNA Methylation

Previous in vivo study demonstrated that chronic hypoxia during gestation was associated with estrogen receptor-&agr; (ER-&agr;) gene repression in ovine uterine arteries. Yet, it remains undetermined whether hypoxia had a direct effect and if DNA methylation played a causal role in hypoxia-mediated ER-&agr; gene repression. Thus, this study tested the hypothesis that prolonged hypoxia has a direct effect and increases promoter methylation resulting in ER-&agr; gene repression and inhibition of estrogen-mediated adaptation of uterine vascular tone. Uterine arteries isolated from nonpregnant and pregnant sheep were treated ex vivo with 21.0% O2 and 10.5% O2 for 48 hours. Hypoxia significantly increased ER-&agr; promoter methylation at both specificity protein-1 and upstream stimulatory factor binding sites, decreased specificity protein-1 and upstream stimulatory factor binding to the promoter, and suppressed ER-&agr; expression in uterine arteries of pregnant animals. Of importance, the effects of hypoxia were blocked by a methylation inhibitor 5-aza-2′-deoxycytidine. In addition, hypoxia abrogated steroid hormone–mediated increase in ER-&agr; expression and inhibited the hormone-induced increase in large-conductance Ca2+-activated K+ channel activity and decrease in myogenic tone in uterine arteries of nonpregnant animals, which were reversed by 5-aza-2′-deoxycytidine. The results provide novel evidence of a direct effect of hypoxia on heightened promoter methylation that plays a causal role in ER-&agr; gene repression and ablation of steroid hormone–mediated adaptation of uterine arterial large conductance Ca2+-activated K+ channel activity and myogenic tone in pregnancy.

Direct effect of chronic hypoxia in suppressing large conductance Ca2+‐activated K+ channel activity in ovine uterine arteries via increasing oxidative stress

Chronic hypoxia has a direct effect in down‐regulating the BKCa channel β1 subunit and inhibiting the BKCa channel activity in uterine arteries of pregnant sheep. Oxidative stress plays a causal role in hypoxia‐mediated suppression of BKCa channel function. The steroid hormone‐induced effect on BKCa channels is a target of hypoxia‐mediated oxidative stress. Inhibition of oxidative stress ameliorates the adverse effect of hypoxia both ex vivo and in vivo in pregnant sheep exposed to long‐term high‐altitude hypoxia. Our findings provide novel evidence of a causative role of oxidative stress in hypoxia‐mediated inhibition of the BKCa channel activity in uterine arteries and new insights in understanding and alleviating pregnancy complications associated with gestational hypoxia such as pre‐eclampsia and fetal growth restriction.

Pregnancy Reprograms Large-Conductance Ca2+-Activated K+ Channel in Uterine ArteriesNovelty and Significance: Roles of Ten-Eleven Translocation Methylcytosine Dioxygenase 1–Mediated Active Demethylation

The large-conductance Ca2+-activated K+ (BKCa) channel is of critical importance in pregnancy-mediated increase in uterine artery vasodilation and blood flow. The present study tested the hypothesis that active DNA demethylation plays a key role in pregnancy-induced reprogramming and upregulation of BKCa channel &bgr;1 subunit (BK&bgr;1) in uterine arteries. Uterine arteries were isolated from nonpregnant and near-term pregnant sheep. Pregnancy significantly increased the expression of ten-eleven translocation methylcytosine dioxygenase 1 (TET1) in uterine arteries. A half-palindromic estrogen response element was identified at the TET1 promoter, and estrogen treatment increased TET1 promoter activity and TET1 expression in uterine arteries. In accordance, pregnancy and steroid hormone treatment resulted in demethylation of BK&bgr;1 promoter by increasing 5-hydroxymethylcytosine and decreasing 5-methylcytosine at the CpG in the Sp1-380 binding site that is of critical importance in the regulation of the promoter activity and BK&bgr;1 expression. Inhibition of TET1 with fumarate significantly decreased BK&bgr;1 expression in uterine arteries of pregnant animals and blocked steroid hormone–induced upregulation of BK&bgr;1. Functionally, fumarate treatment inhibited pregnancy and steroid hormone–induced increases in BKCa channel current density and BKCa channel–mediated relaxations. In addition, fumarate blocked pregnancy and steroid hormone–induced decrease in pressure-dependent myogenic tone of the uterine artery. The results demonstrate a novel mechanism of estrogen-mediated active DNA demethylation in reprogramming of BKCa channel expression and function in the adaption of uterine circulation during pregnancy.The large-conductance Ca2+-activated K+ (BKCa) channel is of critical importance in pregnancy-mediated increase in uterine artery vasodilation and blood flow. The present study tested the hypothesis that active DNA demethylation plays a key role in pregnancy-induced reprogramming and upregulation of BKCa channel β1 subunit (BKβ1) in uterine arteries. Uterine arteries were isolated from nonpregnant and near-term pregnant sheep. Pregnancy significantly increased the expression of ten-eleven translocation methylcytosine dioxygenase 1 (TET1) in uterine arteries. A half-palindromic estrogen response element was identified at the TET1 promoter, and estrogen treatment increased TET1 promoter activity and TET1 expression in uterine arteries. In accordance, pregnancy and steroid hormone treatment resulted in demethylation of BKβ1 promoter by increasing 5-hydroxymethylcytosine and decreasing 5-methylcytosine at the CpG in the Sp1-380 binding site that is of critical importance in the regulation of the promoter activity and BKβ1 expression. Inhibition of TET1 with fumarate significantly decreased BKβ1 expression in uterine arteries of pregnant animals and blocked steroid hormone–induced upregulation of BKβ1. Functionally, fumarate treatment inhibited pregnancy and steroid hormone–induced increases in BKCa channel current density and BKCa channel–mediated relaxations. In addition, fumarate blocked pregnancy and steroid hormone–induced decrease in pressure-dependent myogenic tone of the uterine artery. The results demonstrate a novel mechanism of estrogen-mediated active DNA demethylation in reprogramming of BKCa channel expression and function in the adaption of uterine circulation during pregnancy. # Novelty and Significance {#article-title-68}

Chronic hypoxia upregulates DNA methyltransferase and represses large conductance Ca2+-activated K+ channel function in ovine uterine arteries†

Abstract Chronic hypoxia during gestation suppresses large-conductance Ca2+-activated K+ (BKCa) channel function and impedes uterine arterial adaptation to pregnancy. This study tested the hypothesis that chronic hypoxia has a direct effect in upregulating DNA methyltransferase (DNMT) and epigenetically repressing BKCa channel beta-1 subunit (KCNMB1) expression in uterine arteries. Resistance-sized uterine arteries were isolated from near-term pregnant sheep maintained at ∼300 m above sea level or animals acclimatized to high-altitude (3,801 m) hypoxia for 110 days during gestation. For ex vivo hypoxia treatment, uterine arteries from normoxic animals were treated with 21.0% O2 or 10.5% O2 for 48 h. High-altitude hypoxia significantly upregulated DNMT3b expression and enzyme activity in uterine arteries. Similarly, ex vivo hypoxia treatment upregulated DNMT3b expression and enzyme activity that was blocked by a DNMT inhibitor 5-aza-2′-deoxycytidine (5- Aza). Of importance, 5-Aza inhibited hypoxia-induced hypermethylation of specificity protein (SP) 1 binding site at the KCNMB1 promoter and restored transcription factor binding to the KCNMB1 promoter, resulting in the recovery of KCNMB1 gene expression in uterine arteries. Furthermore, 5-Aza blocked the effect of hypoxia and rescued BKCa channel activity and reversed hypoxia-induced decrease in BKCa channel-mediated relaxations and increase in myogenic tone of uterine arteries. Collectively, these results suggest that chronic hypoxia during gestation upregulates DNMT expression and activity, resulting in hypermethylation and repression of KCNMB1 gene and BKCa channel function, impeding uterine arterial adaptation to pregnancy. Summary Sentence Gestational hypoxia promotes hypermethylation and repression of KCNMB1 gene and BKCa channel function in uterine arteries by upregulating DNMTexpression and activity, leading tomaladaptation of uterine circulation during pregnancy.