Emi Nakashima

Characterization of the acute effects of alcohol on asymmetry of inferior frontal cortex activity during a Go/No-Go task using functional near-infrared spectroscopy

RationaleSuccessful response inhibition is associated with right-lateralized inferior frontal cortex (IFC) activity, and alcohol impairs this inhibitory control, thereby enhancing false-alarm responses in the Go/No-Go task. However, the neural correlates of effect of alcohol on response inhibition remain unclear.ObjectiveThis study characterized the acute effects of alcohol on IFC activity during Go/No-Go tasks using near-infrared spectroscopy (NIRS).MethodsThirty-two subjects visited our laboratory twice: once for alcohol intake and once for placebo intake. On each visit, subjects performed Go/No-Go tasks immediately before and 10xa0min after intake of the alcohol or placebo. NIRS was used to evaluate IFC activity measured during Go/No-Go tasks.ResultsAlcohol significantly enhanced false-alarm responses in No-Go trials. NIRS analysis showed that IFC activity was greater in the right hemisphere than in the left hemisphere prior to alcohol or placebo intake. This right hemispheric superiority was eliminated in response to alcohol but not in response to placebo. Correlation analysis showed that subjects with right-lateralized IFC activity made fewer false-alarm responses in No-Go trials and that alcohol-induced inhibition of hemispheric IFC asymmetry resulted in higher false-alarm rates.ConclusionThese findings suggest that the right IFC may mediate the acute effects of alcohol on inhibitory control. When the alcohol impairs the right IFC activity, subjects cannot inhibit the pre-potent responses for No-Go trials, resulting in enhanced false-alarm responses. Thus, this study successfully demonstrated the neural correlates of the alcohol effect in the right IFC activity during inhibitory control processes.

Role of OAT4 in Uptake of Estriol Precursor 16α-Hydroxydehydroepiandrosterone Sulfate Into Human Placental Syncytiotrophoblasts From Fetus

Estriol biosynthesis in human placenta requires the uptake of a fetal liver-derived estriol precursor, 16α-hydroxydehydroepiandrosterone sulfate (16α-OH DHEAS), by placental syncytiotrophoblasts at their basal plasma membrane (BM), which faces the fetal circulation. The aim of this work is to identify the transporter(s) mediating 16α-OH DHEAS uptake at the fetal side of syncytiotrophoblasts by using human placental BM-enriched vesicles and to examine the contribution of the putative transporter to estriol synthesis at the cellular level, using choriocarcinoma JEG-3 cells. Organic anion transporter (OAT)-4 and organic anion transporting polypeptide 2B1 proteins were enriched in human placental BM vesicles compared with crude membrane fraction. Uptake of [(3)H]16α-OH DHEAS by BM vesicles was partially inhibited in the absence of sodium but was significantly increased in the absence of chloride and after preloading glutarate. Uptake of [(3)H]16α-OH DHEAS by BM vesicles was significantly inhibited by OAT4 substrates such as dehydroepiandrosterone sulfate, estrone-3-sulfate, and bromosulfophthalein but not by cyclosporin A, tetraethylammonium, p-aminohippuric acid, or cimetidine. These characteristics of vesicular [(3)H]16α-OH DHEAS uptake are in good agreement with those of human OAT4-transfected COS-7 cells as well as forskolin-differentiated JEG-3 cells. Estriol secretion from differentiated JEG-3 cells was detected when the cells were incubated with 16α-OH DHEAS for 8 hours but was inhibited in the presence of 50 μM bromosulfophthalein. Our results indicate that OAT4 at the BM of human placental syncytiotrophoblasts plays a predominant role in the uptake of 16α-OH DHEAS for placental estriol synthesis.

Estrogen Receptor α Induction by Mitoxantrone Increases Abcg2 Expression in Placental Trophoblast Cells

Substrate-induced upregulation of ATP-binding cassette subfamily G member 2 (ABCG2) has been well studied in cancer cells, but it is also important to understand whether ABCG2 is upregulated by its substrates in tissues in which it is constitutively expressed. In the present study, we aimed to clarify the regulatory mechanism of Abcg2 expression by its substrate, mitoxantrone, in placental cells. Abcg2 mRNA expression in rat placental TR-TBT 18d-1 cells treated with 10 μM mitoxantrone for 24 h was increased, compared with that in nontreated cells, whereas 10 μM pheophorbide-a had no effect. Methylated CpG level in the promoter region of the Abcg2 gene was low and was not altered by mitoxantrone. On the contrary, mitoxantrone markedly increased the expression of estrogen receptor (ER) α and progesterone receptor (PR) B. Fulvestrant, an ER antagonist, attenuated the mitoxantrone-induced increase of Abcg2 mRNA expression, whereas mifepristone, a PR antagonist, had little effect. 17β-estradiol, an ER ligand, positively regulated the mitoxantrone-induced increase of Abcg2 expression. DNA demethylation by 5-aza-2-deoxycytidine treatment increased ERα expression, but mitoxantrone failed to facilitate the demethylation of ERα promoter in TR-TBT 18d-1 cells. In conclusion, Abcg2 expression is induced by mitoxantrone via the induction of ERα in TR-TBT 18d-1 cells.

Protective effect of hypotaurine against oxidative stress-induced cytotoxicity in rat placental trophoblasts

INTRODUCTIONnHypotaurine is a precursor of taurine and an antioxidant, and is concentrated in fetal plasma compared to maternal plasma. Hypotaurine is significantly decreased in fetal plasma of ezrin (Vil2) knock-out mice, and fetuses show intrauterine growth retardation. The aim of this study was to characterize the mechanism through which cellular hypotaurine level is maintained in placental trophoblasts, and the effect of hypotaurine on oxidative stress induced by hydrogen peroxide (H2O2).nnnMETHODSnHypotaurine transfer from extracellular fluid and antioxidant effect of hypotaurine were analyzed in rat placental trophoblast TR-TBT 18d-1 cells.nnnRESULTSnWe found that hypotaurine is concentrated into rat placental trophoblast TR-TBT 18d-1 cells, and the level of hypotaurine was markedly reduced by culture in medium supplemented with dialyzed fetal bovine serum (FBS) instead of normal FBS. The hypotaurine level recovered almost completely when hypotaurine was added to the culture medium, indicating that intracellular hypotaurine is predominantly supplied by transport across the plasma membrane from extracellular fluid rather than by biosynthesis. Hypotaurine showed a cytoprotective effect against H2O2-induced oxidative damage in TR-TBT 18d-1 cells. Hypotaurine treatment of TR-TBT 18d-1 cells increased antioxidant capacity against hydroxyl radical and peroxyl radical. The concentration of intracellular hydroxyl radical induced by H2O2 in TR-TBT 18d-1 cells was significantly reduced by hypotaurine treatment.nnnDISCUSSIONnThese results indicate that intracellular hypotaurine is mainly supplied to placental trophoblasts by transfer from extracellular fluid across the plasma membrane, and may play a role in cell protection by scavenging reactive oxygen species.

Organic Anion Transporter 4-Mediated Transport of Olmesartan at Basal Plasma Membrane of Human Placental Barrier

Mechanisms regulating fetal transfer of olmesartan, an angiotensin-II receptor type 1 antagonist, are important as potential determinants of life-threatening adverse fetal effects. The purpose of this study was to examine the olmesartan transport mechanism through the basal plasma membrane (BM) of human syncytiotrophoblasts forming the placental barrier. Uptake of olmesartan by human placental BM vesicles was potently inhibited by dehydroepiandrosterone sulfate (DHEAS), estrone 3-sulfate, and bromosulfophthalein, which are all typical substrates of organic anion transporter (OAT) 4 localized at the BM of syncytiotrophoblasts, and was increased in the absence of chloride. In tetracycline-inducible OAT4-expressing cells, [(3) H]olmesartan uptake was increased by tetracycline treatment. Olmesartan uptake via OAT4 was concentration dependent with a Km of 20 μM, and was increased in the absence of chloride. [(3) H]Olmesartan efflux via OAT4 was also observed and was trans-stimulated by extracellular chloride and DHEAS. Thus, OAT4 mediates bidirectional transport of olmesartan and appears to regulate fetal transfer of olmesartan at the BM of syncytiotrophoblasts. Efflux transport of olmesartan via OAT4 from syncytiotrophoblasts to the fetal circulation might be facilitated in the presence of an inwardly directed physiological chloride gradient and extracellular DHEAS.

System A amino acid transporter SNAT2 shows subtype-specific affinity for betaine and hyperosmotic inducibility in placental trophoblasts

Betaine uptake is induced by hypertonic stress in a placental trophoblast cell line, and involvement of amino acid transport system A was proposed. Here, we aimed to identify the subtype(s) of system A that mediates hypertonicity-induced betaine uptake. Measurement of [(14)C]betaine uptake by HEK293 cells transiently transfected with human or rat sodium-coupled neutral amino acid transporters (SNATs), SNAT1, SNAT2 and SNAT4 revealed that only human and rat SNAT2 have betaine uptake activity. The Michaelis constants (Km) of betaine uptake by human and rat SNAT2 were estimated to be 5.3 mM and 4.6 mM, respectively. Betaine exclusively inhibited the uptake activity of SNAT2 among the rat system A subtypes. We found that rat SNAT1, SNAT2 and SNAT4 were expressed at the mRNA level under isotonic conditions, while expression of SNAT2 and SNAT4 was induced by hypertonicity in TR-TBT 18d-1 cells. Western blot analyses revealed that SNAT2 expression on plasma membrane of TR-TBT 18d-1 cells was more potently induced by hypertonicity than that in total cell lysate. Immunocytochemistry confirmed the induction of SNAT2 expression in TR-TBT 18d-1 cells exposed to hypertonic conditions and indicated that SNAT2 was localized on the plasma membrane in these cells. Our results indicate that SNAT2 transports betaine, and that tonicity-sensitive SNAT2 expression may be involved in regulation of betaine concentration in placental trophoblasts.

Role of protein kinase A in regulating steroid sulfate uptake for estrogen production in human placental choriocarcinoma cells.

The purpose of this study is to assess the role of the protein kinase A (PKA) in regulating uptake of dehydroepiandrosterone sulfate (DHEAS), an estrogen precursor, by syncytiotrophoblasts. Forskolin, a PKA activator, significantly increased [(3)H]DHEAS uptake and the mRNA expression levels of organic anion transporter (OAT) 4 and CYP19A1 in choriocarcinoma JEG-3 cells, while other steroid sulfate transporters present in the placenta showed no change in expression level. KT5720, a PKA inhibitor, attenuated these effects of forskolin. Accordingly, the PKA pathway appears to play an important role in estrogen synthesis by cooperatively regulating OAT4 and steroidogenic enzymes in syncytiotrophoblasts.

Fetal Growth Retardation and Lack of Hypotaurine in Ezrin Knockout Mice

Ezrin is a membrane-associated cytoplasmic protein that serves to link cell-membrane proteins with the actin-based cytoskeleton, and also plays a role in regulation of the functional activities of some transmembrane proteins. It is expressed in placental trophoblasts. We hypothesized that placental ezrin is involved in the supply of nutrients from mother to fetus, thereby influencing fetal growth. The aim of this study was firstly to clarify the effect of ezrin on fetal growth and secondly to determine whether knockout of ezrin is associated with decreased concentrations of serum and placental nutrients. Ezrin knockout mice (Ez−/−) were confirmed to exhibit fetal growth retardation. Metabolome analysis of fetal serum and placental extract of ezrin knockout mice by means of capillary electrophoresis–time-of-flight mass spectrometry revealed a markedly decreased concentration of hypotaurine, a precursor of taurine. However, placental levels of cysteine and cysteine sulfinic acid (precursors of hypotaurine) and taurine were not affected. Lack of hypotaurine in Ez−/− mice was confirmed by liquid chromatography with tandem mass spectrometry. Administration of hypotaurine to heterogenous dams significantly decreased the placenta-to-maternal plasma ratio of hypotaurine in wild-type fetuses but only slightly decreased it in ezrin knockout fetuses, indicating that the uptake of hypotaurine from mother to placenta is saturable and that disruption of ezrin impairs the uptake of hypotaurine by placental trophoblasts. These results indicate that ezrin is required for uptake of hypotaurine from maternal serum by placental trophoblasts, and plays an important role in fetal growth.

Contributions of system A subtypes to α-methylaminoisobutyric acid uptake by placental microvillous membranes of human and rat

System A consists of three subtypes, sodium-coupled neutral amino acid transporter 1 (SNAT1), SNAT2, and SNAT4, which are all expressed in the placenta. The aim of this study was to evaluate the contributions of each of the three subtypes to total system A-mediated uptake in placental MVM of human and rat, using betaine and l-arginine as subtype-selective inhibitors of SNAT2 and SNAT4, respectively. Appropriate concentrations of betaine and l-arginine for subtype-selective inhibition in SNAT-overexpressing cells were identified. It was found that 10xa0mM betaine specifically and almost completely inhibited human and rat SNAT2-mediated [14C]α-methylaminoisobutyric acid ([14C]MeAIB) uptake, while 5xa0mM l-arginine specifically and completely inhibited [3H]glycine uptake via human SNAT4, as well as [14C]MeAIB uptake via rat SNAT4. In both human and rat placental MVM vesicles, sodium-dependent uptake of [14C]MeAIB was almost completely inhibited by 20xa0mM unlabeled MeAIB. l-Arginine (5xa0mM) partly inhibited the uptake in humans, but hardly affected that in rats. Betaine (10xa0mM) partly inhibited the uptake in rats, but hardly affected it in humans. These results suggest that SNAT1 is most likely the major contributor to system A-mediated MeAIB uptake by human and rat MVM vesicles and that the remaining uptake is mainly mediated by SNAT4 in humans and SNAT2 in rats. Thus, inhibition studies using betaine and l-arginine are useful to characterize the molecular mechanisms of system A-mediated transport.

Layer II of placental syncytiotrophoblasts expresses MDR1 and BCRP at the apical membrane in rodents

The purpose of this study is to clarify the subcellular localizations of multidrug resistance protein 1 (MDR1)/ABCB1 and breast cancer resistance protein (BCRP)/ABCG2 in the rodent placental SynT bilayer, i.e., a maternal-facing (SynT-I) layer and a fetal-facing (SynT-II) layer. In double immunofluorescence staining, the signals of MDR1 and BCRP appeared midway between the signals of glucose transporter 1 on the apical membrane of SynT-I and the basal plasma membrane of SynT-II, and mostly overlapped with signals of connexin 26, which forms gap junctions between SynT-I and SynT-II. In detail, median intensities (pixels) of the MDR1 and BCRP signals were significantly closer to the fetal circulation as compared to the location of connexin 26 signals. In double in situ hybridization studies, the signals of Mdr1b mRNA mostly overlapped with those of Syncytin-B, a SynT-II marker. In conclusion, MDR1 and BCRP are expressed on apical membranes of the rodent placental SynT-II layer.