Ursodeoxycholic acid inhibits uptake and vasoconstrictor effects of taurocholate in human placenta.

Intrahepatic cholestasis of pregnancy (ICP) causes increased transfer of maternal bile acids to the fetus and an increased incidence of sudden fetal death. Treatment includes ursodeoxycholic acid (UDCA), but it is not clear if UDCA protects the fetus. This study explores the placental transport of the bile acid taurocholate (TC) by the organic anion-transporting polypeptide, (OATP)4A1, its effects on the placental proteome and vascular function, and how these are modified by UDCA. Various methodological approaches including placental villous fragments and Xenopus laevis oocytes were used to investigate UDCA transport. Placental perfusions and myography investigated the effect of TC on vasculature. The effects of acute TC exposure on placental tissue were investigated using quantitative proteomics. UDCA inhibited OATP4A1 activity in placental villous fragments and oocytes. TC induced vasoconstriction in placental and rat vasculature, which was attenuated by UDCA. Quantitative proteomic analysis of villous fragments showed direct effects of TC on multiple placental pathways, including oxidative stress and autophagy. The effects of TC on the placental proteome and vasculature demonstrate how bile acids may cause fetal distress in ICP. UDCA inhibition of OATP4A1 suggests it will protect the mother and fetus against the vascular effects of TC by inhibiting its cellular uptake. UDCA may protect the fetus in ICP by inhibiting OATP4A1-mediated bile acid transfer and TC-induced placental vasoconstriction. Understanding the physiologic mechanisms of UDCA may allow better therapeutic interventions to be designed specifically for the fetus in the future.-Lofthouse, E. M., Torrens, C., Manousopoulou, A., Nahar, M., Cleal, J. K., O'Kelly, I. M., Sengers, B. G., Garbis, S. D., Lewis, R. M. Ursodeoxycholic acid inhibits uptake and vasoconstrictor effects of taurocholate in human placenta.

Estrone sulphate uptake by the microvillous membrane of placental syncytiotrophoblast is coupled to glutamate efflux.

Organic anion transporters (OATs) and organic anion transporting polypeptides (OATPs) are transport proteins that mediate exchange of metabolites, hormones and waste products. Directional transport by these transporters can occur when exchange is coupled to the gradients of other substrates. This study investigates whether the activity of OATP4A1 and OATP2A1 on the maternal facing microvillus membrane of the placental syncytiotrophoblast is coupled to the glutamate gradient. OAT and OATP transporter proteins were over expressed in Xenopus oocytes to study their transport characteristics. Further transport studies were performed in term human placental villous fragments. Xenopus oocytes expressing OATP4A1 mediated glutamate uptake. No glutamate transport was observed in oocytes expressing OAT1, OAT3, OAT7 or OATP2A1. In oocytes expressing OATP4A1, uptake of estrone sulphate, thyroid hormones T3 and T4 and the bile acid taurocholate stimulated glutamate efflux. In term placental villous fragments addition of estrone sulphate and taurocholate trans-stimulated glutamate efflux. Coupling of OATP4A1 to the glutamate gradient may drive placental uptake of estrone-sulphate and thyroid hormone while also facilitating uptake of potentially harmful bile acids. In contrast, if OATP2A1 is not coupled to a similar gradient, it may function more effectively as an efflux transporter, potentially mediating efflux of prostaglandins to the mother. This study provides further evidence for glutamate as an important counter-ion driving transport into the placenta.

Rare variants in NR2F2 cause congenital heart defects in humans.

Congenital heart defects (CHDs) are the most common birth defect worldwide and are a leading cause of neonatal mortality. Nonsyndromic atrioventricular septal defects (AVSDs) are an important subtype of CHDs for which the genetic architecture is poorly understood. We performed exome sequencing in 13 parent-offspring trios and 112 unrelated individuals with nonsyndromic AVSDs and identified five rare missense variants (two of which arose de novo) in the highly conserved gene NR2F2, a very significant enrichment (p = 7.7 × 10(-7)) compared to 5,194 control subjects. We identified three additional CHD-affected families with other variants in NR2F2 including a de novo balanced chromosomal translocation, a de novo substitution disrupting a splice donor site, and a 3 bp duplication that cosegregated in a multiplex family. NR2F2 encodes a pleiotropic developmental transcription factor, and decreased dosage of NR2F2 in mice has been shown to result in abnormal development of atrioventricular septa. Via luciferase assays, we showed that all six coding sequence variants observed in individuals significantly alter the activity of NR2F2 on target promoters.

Glutamate cycling may drive organic anion transport on the basal membrane of human placental syncytiotrophoblast.

The organic anion transporter OAT4 (SLC22A11) and organic anion transporting polypeptide OATP2B1 (SLCO2B1) are expressed in the basal membrane of the placental syncytiotrophoblast. These transporters mediate exchange whereby uptake of one organic anion is coupled to efflux of a counter-ion. In placenta, these exchangers mediate placental uptake of substrates for oestrogen synthesis as well as clearing waste products and xenobiotics from the fetal circulation. However, the identity of the counter-ion driving this transport in the placenta, and in other tissues, is unclear. While glutamate is not a known OAT4 or OATP2B1 substrate, we propose that its high intracellular concentration has the potential to drive accumulation of substrates from the fetal circulation. In the isolated perfused placenta, glutamate exchange was observed between the placenta and the fetal circulation. This exchange could not be explained by known glutamate exchangers. However, glutamate efflux was trans-stimulated by an OAT4 and OATP2B1 substrate (bromosulphothalein). Exchange of glutamate for bromosulphothalein was only observed when glutamate reuptake was inhibited (by addition of aspartate). To determine if OAT4 and/or OATP2B1 mediate glutamate exchange, uptake and efflux of glutamate were investigated in Xenopus laevis oocytes. Our data demonstrate that in Xenopus oocytes expressing either OAT4 or OATP2B1 efflux of intracellular [(14)C]glutamate could be stimulated by conditions including extracellular glutamate (OAT4), estrone-sulphate and bromosulphothalein (both OAT4 and OATP2B1) or pravastatin (OATP2B1). Cycling of glutamate across the placenta involving efflux via OAT4 and OATP2B1 and subsequent reuptake will drive placental uptake of organic anions from the fetal circulation.

Protein kinase A is central for forward transport of two-pore domain potassium channels K2P3.1 and K2P9.1.

Acid-sensitive two-pore domain potassium channels (K2P3.1 and K2P9.1) play key roles in both physiological and pathophysiological mechanisms, the most fundamental of which is control of resting membrane potential of cells in which they are expressed. These background "leak" channels are constitutively active once expressed at the plasma membrane, and hence tight control of their targeting and surface expression is fundamental to the regulation of K(+) flux and cell excitability. The chaperone protein, 14-3-3, binds to a critical phosphorylated serine in the channel C termini of K2P3.1 and K2P9.1 (Ser(393) and Ser(373), respectively) and overcomes retention in the endoplasmic reticulum by ßCOP. We sought to identify the kinase responsible for phosphorylation of the terminal serine in human and rat variants of K2P3.1 and K2P9.1. Adopting a bioinformatic approach, three candidate protein kinases were identified: cAMP-dependent protein kinase, ribosomal S6 kinase, and protein kinase C. In vitro phosphorylation assays were utilized to determine the ability of the candidate kinases to phosphorylate the channel C termini. Electrophysiological measurements of human K2P3.1 transiently expressed in HEK293 cells and cell surface assays of GFP-tagged K2P3.1 and K2P9.1 enabled the determination of the functional implications of phosphorylation by specific kinases. All of our findings support the conclusion that cAMP-dependent protein kinase is responsible for the phosphorylation of the terminal serine in both K2P3.1 and K2P9.1.

N-acetylcysteine, xCT and suppression of Maxi-chloride channel activity in human placenta.

INTRODUCTION: Placental oxidative stress features in pregnancy pathologies but in clinical trials antioxidant supplementation has not improved outcomes. N-acetylcysteine (NAC) stimulates glutathione production and is proposed as a therapeutic agent in pregnancy. However, key elements of N-acetylcysteine biology, including its cellular uptake mechanism, remains unclear. This study explores how the cystine/glutamate transporter xCT may mediate N-acetylcysteine uptake and how N-acetylcysteine alters placental redox status. METHODS: The involvement of xCT in NAC uptake by the human placenta was studied in perfused placenta and Xenopus oocytes. The effect of short-term N-acetylcysteine exposure on the placental villous proteome was determined using LC-MS. The effect of N-acetylcysteine on Maxi-chloride channel activity was investigated in perfused placenta, villous fragments and cell culture. RESULTS: Maternoplacental N-acetylcysteine administration stimulated intracellular glutamate efflux suggesting a role of the exchange transporter xCT, which was localised to the microvillous membrane of the placental syncytiotrophoblast. Placental exposure to a bolus of N-acetylcysteine inhibited subsequent activation of the redox sensitive Maxi-chloride channel independently of glutathione synthesis. Stable isotope quantitative proteomics of placental villi treated with N-acetylcysteine demonstrated changes in pathways associated with oxidative stress, apoptosis and the acute phase response. DISCUSSION: This study suggests that xCT mediates N-acetylcysteine uptake into the placenta and that N-acetylcysteine treatment of placental tissue alters the placental proteome while regulating the redox sensitive Maxi-chloride channel. Interestingly N-acetylcysteine had antioxidant effects independent of the glutathione pathway. Effective placental antioxidant therapy in pregnancy may require maintaining the balance between normalising redox status without inhibiting physiological redox signalling.

System-specific O2 sensitivity of the tandem pore domain K+ channel TASK-1.

Hypoxic inhibition of TASK-1, a tandem pore domain background K+ channel, provides a critical link between reduced O2 levels and physiological responses in various cell types. Here, we examined the expression and O2 sensitivity of TASK-1 in immortalized adrenomedullary chromaffin (MAH) cells. In physiological (asymmetrical) K+ solutions, 3 microM anandamide or 300 microM Zn2+ inhibited a strongly pH-sensitive current. Under symmetrical K+ conditions, the anandamide- and Zn2+-sensitive K+ currents were voltage independent. These data demonstrate the functional expression of TASK-1, and cellular expression of this channel was confirmed by RT-PCR and Western blotting. At concentrations that selectively inhibit TASK-1, anandamide and Zn2+ were without effect on the magnitude of the O2-sensitive current or the hypoxic depolarization. Thus TASK-1 does not contribute to O2 sensing in MAH cells, demonstrating the failure of a known O2-sensitive K+ channel to respond to hypoxia in an O2-sensing cell. These data demonstrate that, ultimately, the sensitivity of a particular K+ channel to hypoxia is determined by the cell, and we propose that this is achieved by coupling distinct hypoxia signaling systems to individual channels. Importantly, these data also reiterate the indirect O2 sensitivity of TASK-1, which appears to require the presence of an intracellular mediator.