Human placental taurine transporter in uncomplicated and IUGR pregnancies: cellular localization, protein expression, and regulation

Transplacental transfer is the fetus' primary source of taurine, an essential amino acid during fetal life. In intrauterine growth restriction (IUGR), placental transport capacity of taurine is reduced and fetal taurine levels are decreased. We characterized the protein expression of the taurine transporter (TAUT) in human placenta using immunocytochemistry and Western blotting, tested the hypothesis that placental protein expression of TAUT is reduced in IUGR, and investigated TAUT regulation by measuring the Na+-dependent taurine uptake in primary villous fragments after 1 h of incubation with different effectors. TAUT was primarily localized in the syncytiotrophoblast microvillous plasma membrane (MVM). TAUT was detected as a single 70-kDa band, and MVM TAUT expression was unaltered in IUGR. The PKC activator PMA and the nitric oxide (NO) donor 3-morpholinosydnonimine decreased TAUT activity ( P < 0.05, n = 7–15). However, none of the tested hormones, e.g., leptin and growth hormone, altered TAUT activity significantly. PKC activity measured in MVM from control and IUGR placentas was not different. In conclusion, syncytiotrophoblast TAUT is strongly polarized to the maternal-facing plasma membrane. MVM TAUT expression is unaltered in IUGR, suggesting that the reduced MVM taurine transport in IUGR is due to changes in transporter activity. NO release downregulates placental TAUT activity, and it has previously been shown that IUGR is associated with increased fetoplacental NO levels. NO may therefore play an important role in downregulating MVM TAUT activity in IUGR.

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Involvement of TauT/SLC6A6 in Taurine Transport at the Blood–Testis Barrier

Metabolites ◽

10.3390/metabo12010066 ◽

2022 ◽

Vol 12 (1) ◽

pp. 66

Author(s):

Yoshiyuki Kubo ◽

Sakiko Ishizuka ◽

Takeru Ito ◽

Daisuke Yoneyama ◽

Shin-ichi Akanuma ◽

...

Keyword(s):

Aminobutyric Acid ◽

Seminiferous Tubules ◽

Taurine Transporter ◽

Taurine Transport ◽

Plot Analysis ◽

Influx Transport ◽

Dependent Inhibition ◽

Taurine Uptake ◽

Sertoli Cell Line ◽

Blood Testis Barrier

Taurine transport was investigated at the blood–testis barrier (BTB) formed by Sertoli cells. An integration plot analysis of mice showed the apparent influx permeability clearance of [3H]taurine (27.7 μL/(min·g testis)), which was much higher than that of a non-permeable paracellular marker, suggesting blood-to-testis transport of taurine, which may involve a facilitative taurine transport system at the BTB. A mouse Sertoli cell line, TM4 cells, showed temperature- and concentration-dependent [3H]taurine uptake with a Km of 13.5 μM, suggesting that the influx transport of taurine at the BTB involves a carrier-mediated process. [3H]Taurine uptake by TM4 cells was significantly reduced by the substrates of taurine transporter (TauT/SLC6A6), such as β-alanine, hypotaurine, γ-aminobutyric acid (GABA), and guanidinoacetic acid (GAA), with no significant effect shown by L-alanine, probenecid, and L-leucine. In addition, the concentration-dependent inhibition of [3H]taurine uptake revealed an IC50 of 378 μM for GABA. Protein expression of TauT in the testis, seminiferous tubules, and TM4 cells was confirmed by Western blot analysis and immunohistochemistry by means of anti-TauT antibodies, and knockdown of TauT showed significantly decreased [3H]taurine uptake by TM4 cells. These results suggest the involvement of TauT in the transport of taurine at the BTB.

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SUN-267 Dissecting Type 2 CRH Receptor Signaling Characteristics in the Hypothalamic Cell Line MHYPOA-2/30

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.1555 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

Viridiana Alcantara-Alonso ◽

Patricia de Gortari ◽

Robert Dallmann ◽

Dimitris Grammatopoulos

Keyword(s):

Gene Expression ◽

Plasma Membrane ◽

Protein Expression ◽

Cell Line ◽

Cellular Localization ◽

Luciferase Reporter ◽

Mrna Levels ◽

Luciferase Reporter Gene

Abstract The stress peptides coticotropin-releasing hormone (CRH) and urocortins (Ucns) exert anorectic effects acting mainly through the type 2 CRH receptor (CRH-R2) in the hypothalamus. Impairment of CRH-R2 signaling in chronically stressed rats has been linked with the development of hyperphagia (Alcantara-Alonso et al. Neuropeptides, 2017) however the exact mechanisms and molecular defects are unknown. In the present study we used the mHypoA-2/30, a hypothalamic immortalized cell line derived from adult mice (Belsham et al. FASEB J, 2009) to further explore the signaling molecules mediating the anorexigenic effect of the CRH-R2 cognate agonist urocortin 2 (Ucn2). Specifically, we investigated mRNA, protein expression and cellular localization of CRH-R2 in the mHypoA-2/30 neurons. Additionally, we examined the effects of Ucn2 on the phosphorylation of CREB and AMPK, as well as its transcriptional effects on genes of feeding-related peptides and molecules involved in modulation of circadian rhythms. Both CRH-R2 mRNA and protein expression were detected in mHypoA-2/30; indirect immunoflourescence experiments using a specific CRH-R2 antibody demonstrated widespread localization in the plasma membrane and cytoplasm. Moreover, the receptor sub-cellular localization was redistributed in response to activation by Ucn2 (100 nM), as the plasma membrane immunofluorescent signal was decreased after 4h of agonist treatment, suggesting CRH-R2 homologous internalization. We also observed a 50% increase in the phosphorylation of CREB associated with a concomitant decrease in AMPK phosphorylation after 30 min of Ucn2 treatment. Among the panel of hypothalamic genes analyzed, we identified after 24h of Ucn2 treatment increases in the gene expression of the anorexigenic peptides neurotensin and proopiomelanocortin. Interestingly, sustained CRH-R2 activation also led to an increase in the mRNA levels of Aryl Hydrocarbon Receptor Nuclear Translocator Like (ARNTL), a protein involved in the control of circadian rhythm. A luciferase reporter gene analysis of ARNTL showed that the mHypoA-2/30 cells also exhibit circadian patterns of expression and that the treatment with Ucn2 enhanced circadian amplitude of ARNTL reporter on these cells, which in turn may be involved in glucocorticoid release in circadian cycles and stimulating appetite during the activity phase of the animals. In conclusion, we found that the mHypoA-2/30 cell line expresses endogenous functional CRH-R2 that is linked to downstream regulation of anorexigenic gene expression. This cell line appears to be a useful in vitro tool to study hypothalamic CRH-R2 signaling machinery involved in central control of food intake and circadian cycles.

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Identification of a high affinity taurine transporter which is not dependent on chloride

Bioscience Reports ◽

10.1007/bf01540457 ◽

1995 ◽

Vol 15 (4) ◽

pp. 231-239 ◽

Cited By ~ 7

Author(s):

D. B. Shennan

Keyword(s):

Amino Acids ◽

Transport System ◽

Niflumic Acid ◽

Cell Swelling ◽

Mammary Tissue ◽

Taurine Transporter ◽

Taurine Transport ◽

High Affinity ◽

Taurine Uptake

Taurine transport by lactating gerbil mammary tissue has been examined. Taurine uptake is, mediated by a high-affinity system which is specific for β-amino acids. The uptake of taurine is Na+-dependent but appears not to be obligatorly dependent upon Cl−. Thus, replacing Na+ with choline almost abolished taurine uptake. Substituting Cl− with NO3− had no effect whereas SCN− induced a small but significant increase in taurine influx. Taurine uptake was Na+-dependent under conditions where Cl− had been replaced with NO3−. However, it is apparent that the Na+-dependent taurine transport system requires the presence of a permeable anion because replacing Cl− with gluconate markedly reduced taurine uptake. Cell-swelling, induced by a hyposmotic challenge, increased the efflux of taurine from gerbil mammary tissue via a pathway sensitive to niflumic acid.

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Regulation of taurine transporter activity in LLC-PK1 cells: role of protein synthesis and protein kinase C activation.

Journal of the American Society of Nephrology ◽

10.1681/asn.v251021 ◽

1991 ◽

Vol 2 (5) ◽

pp. 1021-1029 ◽

Cited By ~ 1

Author(s):

D P Jones ◽

L A Miller ◽

C Dowling ◽

R W Chesney

Keyword(s):

Protein Synthesis ◽

Protein Kinase C ◽

Protein Kinase ◽

Free Medium ◽

Taurine Transporter ◽

Taurine Transport ◽

Kinase C ◽

Taurine Uptake ◽

Protein Kinase C Activation

Taurine transporter activity increases after exposure of cultured renal epithelial cells to taurine-free medium for 24 h and decreases after incubation in high (500 microM) taurine. This adaptive response mimics that observed in rat kidney after manipulation of dietary taurine. In order to elucidate potential mechanisms involved in the regulation of beta-amino acid transporter activity, the role of RNA transcription, protein synthesis, and protein import (trafficking), as well as protein kinase C activation, on the control of taurine transport was examined in the continuous proximally derived LLC-PK1 renal cell line. Inhibition of RNA transcription with actinomycin D did not alter the up-regulatory and down-regulatory adaptive responses. Inhibition of protein synthesis with cycloheximide prevented the increased taurine transport in response to taurine-free medium as well as the decrease in taurine transport after exposure to high taurine. Colchicine prevented the response to taurine-free medium but had no effect on the response to high-taurine medium. Exposure of confluent cell monolayers to the active phorbol esters, phorbol 12-myristate 13-acetate and phorbol 12,13 dibutyrate, resulted in a reduction in taurine uptake. The effect was seen within minutes of exposure but was not observed in the presence of the inactive phorbol 4-alpha. This inhibitory action was blocked by staurosporin, an inhibitor of protein kinase C (PKC). Treatment of cells with the diacylglycerol kinase inhibitor R59022, which results in increased intracellular diacylglycerol, a natural stimulant of PKC, also inhibited taurine uptake, providing further evidence for a specific effect of PKC activation.(ABSTRACT TRUNCATED AT 250 WORDS)

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Fasting enhances taurine transport by rat liver plasma membrane vesicles

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1994.267.5.g932 ◽

1994 ◽

Vol 267 (5) ◽

pp. G932-G937

Author(s):

D. Berkowitz ◽

P. Hug ◽

R. G. Sleight ◽

J. C. Bucuvalas

Keyword(s):

Amino Acids ◽

Plasma Membrane ◽

Membrane Vesicles ◽

Male Rats ◽

Control Group ◽

Plasma Membrane Vesicles ◽

Hepatic Transport ◽

Taurine Transport ◽

Liver Plasma Membrane ◽

Taurine Uptake

Hepatic taurine stores are maintained by biosynthesis from the sulfur-containing amino acids, methionine and cysteine, and by uptake via a Na(+)- and Cl(-)-dependent transport system, which is specific for beta-amino acids. We hypothesized that liver stores of taurine are maintained by enhanced hepatic transport during fasting when dietary sources for taurine and its precursors are diminished. Liver plasma membrane vesicles, enriched for the basolateral domain, were prepared from adult male rats fasted for 72 h and from control rats. The maximum velocity for Na(+)-dependent taurine uptake was twofold greater for the fasted group compared with the control group (0.87 +/- 0.09 vs. 0.31 +/- 0.03 nmol.mg protein-1.min-1). The apparent Michaelis constant for taurine was also greater for fasted compared with control (154.0 +/- 0.5 vs. 80.0 +/- 2.0 microM). gamma-Aminobutyric acid, but not alanine or glutamine, abolished the effect of fasting on hepatic taurine transport. To determine the effect of fasting independent of changes in the lipid microenvironment, taurine uptake was measured in proteoliposomes reconstituted by inserting detergent-solubilized membrane proteins into asolectin vesicles. Taurine uptake by proteoliposomes reconstituted from membranes prepared from the fasted group was significantly greater than from the control group. We conclude that Na(+)-dependent taurine transport is enhanced in liver plasma membranes prepared from fasted rats. Our findings imply that enhanced taurine uptake with fasting is due to either an increased number of functional carriers or activation of existing transporters.

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Taurine transporter in fetal T lymphocytes and platelets: differential expression and functional activity

AJP Cell Physiology ◽

10.1152/ajpcell.00634.2005 ◽

2007 ◽

Vol 292 (1) ◽

pp. C332-C341 ◽

Cited By ~ 8

Author(s):

C. G. Iruloh ◽

S. W. D'Souza ◽

P. F. Speake ◽

I. Crocker ◽

W. Fergusson ◽

...

Keyword(s):

Amino Acid ◽

T Lymphocytes ◽

Protein Expression ◽

Primary Source ◽

Cell Types ◽

Amino Acid Transporters ◽

Fetal Cell ◽

Taurine Transporter ◽

Transporter Protein ◽

Gene And Protein Expression

Transplacental transfer of taurine, a β-amino acid essential for fetal and neonatal development, constitutes the primary source of taurine for the fetus. Placental transport of taurine is compromised in pregnancies complicated by intrauterine growth restriction, resulting in a reduced concentration of taurine in cord plasma. This could impact on fetal cellular metabolism as taurine represents the most abundant intracellular amino acid in many fetal cell types. In the present study, we have used pure isolates of fetal platelets and T lymphocytes from cord blood of placentas, from normal, term pregnancies, as fetal cell types to examine the cellular uptake mechanisms for taurine by the system β transporter and have compared gene and protein expression for the taurine transporter protein (TAUT) in these two cell types. System β activity in fetal platelets was 15-fold higher compared with fetal T lymphocytes ( P < 0.005), mirroring greater TAUT mRNA expression in platelets than T lymphocytes ( P < 0.005). Cell-specific differences in TAUT protein moieties were detected with a doublet of 75 and 80 kDa in fetal platelets compared with 114 and 120 kDa in fetal T lymphocytes, with relatively higher expression in platelets. We conclude that greater system β activity in fetal platelets compared with T lymphocytes is the result of relatively greater TAUT mRNA and protein expression. This study represents the first characterization of amino acid transporters in fetal T lymphocytes.

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Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

Biochemical Journal ◽

10.1042/bj3390299 ◽

1999 ◽

Vol 339 (2) ◽

pp. 299-307 ◽

Cited By ~ 33

Author(s):

Arthur L. KRUCKEBERG ◽

Ling YE ◽

Jan A. BERDEN ◽

Karel van DAM

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Green Fluorescent Protein ◽

Glucose Transport ◽

Cellular Localization ◽

Fluorescent Protein ◽

Hexose Transporter ◽

High Concentrations ◽

Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

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Claudin-19 mediates the effects of NO on the paracellular pathway in thick ascending limbs

AJP Renal Physiology ◽

10.1152/ajprenal.00065.2019 ◽

2019 ◽

Vol 317 (2) ◽

pp. F411-F418

Author(s):

Casandra M. Monzon ◽

Jeffrey L. Garvin

Keyword(s):

Plasma Membrane ◽

Control Period ◽

Paracellular Pathway ◽

Membrane Expression ◽

No Donor ◽

Charge Selectivity ◽

Plasma Membrane Expression ◽

Cgmp Signaling ◽

Blocking Antibodies ◽

Dilution Potential

Claudins are a family of tight junction proteins that provide size and charge selectivity to solutes traversing the paracellular space. Thick ascending limbs (TALs) express numerous claudins, including claudin-19. Nitric oxide (NO), via cGMP, reduces dilution potentials in perfused TALs, a measure of paracellular permeability, but the role of claudin-19 is unknown. We hypothesized that claudin-19 mediates the effects of NO/cGMP on the paracellular pathway in TALs via increases in plasma membrane expression of this protein. We measured the effect of the NO donor spermine NONOate (SPM) on dilution potentials with and without blocking antibodies and plasma membrane expression of claudin-19. During the control period, the dilution potential was −18.2 ± 1.8 mV. After treatment with 200 μmol/l SPM, it was −14.7 ± 2.0 mV ( P < 0.04). In the presence of claudin-19 antibody, the dilution potential was −12.7 ± 2.1 mV. After SPM, it was −12.9 ± 2.4 mV, not significantly different. Claudin-19 antibody alone had no effect on dilution potentials. In the presence of Tamm-Horsfall protein antibody, SPM reduced the dilution potential from −9.7 ± 1.0 to −6.3 ± 1.1 mV ( P < 0.006). Dibutyryl-cGMP (500 µmol/l) reduced the dilution potential from −19.6 ± 2.6 to −17.2 ± 2.3 mV ( P < 0.002). Dibutyryl-cGMP increased expression of claudin-19 in the plasma membrane from 29.9 ± 3.8% to 65.9 ± 10.1% of total ( P < 0.011) but did not change total expression. We conclude that claudin-19 mediates the effects of the NO/cGMP signaling cascade on the paracellular pathway.

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Effects of prenatal glucocorticoid exposure on cardiac calreticulin and calsequestrin protein expression during early development and in adulthood

Biochemical Journal ◽

10.1042/bj20021771 ◽

2003 ◽

Vol 371 (1) ◽

pp. 61-69 ◽

Cited By ~ 21

Author(s):

Maria L. LANGDOWN ◽

Mark J. HOLNESS ◽

Mary C. SUGDEN

Keyword(s):

Cardiac Function ◽

Protein Expression ◽

Growth Retardation ◽

Premature Death ◽

Fetal Life ◽

Dexamethasone Treatment ◽

Increased Risk ◽

Uterine Growth ◽

Intra Uterine Growth Retardation ◽

Glucocorticoid Action

Overexpression of the conserved Ca2+-binding proteins calreticulin and calsequestrin impairs cardiac function, leading to premature death. Calreticulin is vital for embryonic development, but also impairs glucocorticoid action. Glucocorticoid overexposure during late fetal life causes intra-uterine growth retardation and programmed hypertension in adulthood. To determine whether intra-uterine growth retardation or programmed hypertension was associated with altered calreticulin or calsequestrin expression, effects of prenatal glucocorticoid overexposure (maternal dexamethasone treatment on days 15—21 of pregnancy) were examined during fetal life and postnatal development until adulthood (24 weeks). Dexamethasone (100 or 200μg/kg of maternal body weight) was administered via osmotic pump. Calreticulin was detected as a 55kDa band and calsequestrin as 55 and 63kDa bands in 21 day fetal hearts. Only the 55kDa calsequestrin band was detected postnatally. Prenatal glucocorticoid overexposure at the higher dose decreased calreticulin protein expression (26%; P<0.05) but increased calsequestrin protein expression, both 55 and 63kDa bands, by 87% (P<0.01) and 78% (P<0.01); only the 55kDa calsequestrin band was increased at the lower dose (66%; P<0.05). Offspring of dams treated at the lower dexamethasone dose were studied further. In control offspring, cardiac calreticulin protein expression declined between 2 and 3 weeks of age, and remained suppressed until adulthood. Cardiac calsequestrin protein expression increased 2-fold between fetal day 21 and postnatal day 1 and continued to increase until adulthood, at which time it was 3.4-fold higher (P<0.001). Prenatal dexamethasone exposure minimally affected postnatal calsequestrin protein expression, but the postnatal decline in calreticulin protein expression was abrogated and calreticulin protein expression in adulthood was 2.2-fold increased (P<0.001) compared with adult controls. In view of the known associations between cardiac calreticulin overexpression and impaired cardiac function, targeted up-regulation of calreticulin may contribute to the increased risk of adult heart disease introduced as a result of prenatal overexposure to glucocorticoids.

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