Transthyretin-thyroid hormone internalization by trophoblasts.

Normal fetal neurological development depends on a regulated supply of maternal thyroid hormone (TH). We have previously demonstrated that transthyretin (TTR) a TH binding protein, is synthesized, secreted and internalized by trophoblast cells and may provide a route for the transfer of TH from mother to fetus. Our objective was to determine if a member of the low-density lipoprotein receptor family mediates TTR or TTR-TH internalization. TTR-TH internalization by JEG-3 cells is reduced in the presence of receptor associated protein (RAP) or albumin suggesting that TTR-TH is internalized through an LDL-receptor dependent endocytic process.

Transthyretin and the human placenta.

Since its discovery, transthyretin (TTR) has been regarded as an important hepatically derived protein carrier of thyroid hormones and retinol in blood. However, in more recent years it has been shown that TTR has other important functions. TTR is abundant in cerebrospinal fluid, where it may be involved in transport of thyroid hormones into the brain. TTR derived amyloid is associated with diseases such as senile systemic amyloidosis, familial amyloid polyneuropathy and familial amyloid cardiomyopathy. Recently, synthesis, secretion and uptake of TTR by human placenta have been reported. TTR appears to play an important role in the delivery of maternal thyroid hormone to the developing fetus. This review explores the various proposed roles of TTR and more recent findings on TTR synthesis and expression in the placenta.

Ontogenic changes in human placental sodium iodide symporter expression.

The human fetus requires a maternal supply of iodide to synthesize thyroid hormone from 16 weeks gestation. Placental iodide transport is regulated by the sodium iodide symporter (NIS). We studied the ontogeny of NIS in placentas from surgically terminated pregnancies and from normal term pregnancies. NIS mRNA was low at 6 weeks gestation and peaked at 12 weeks gestation. Placental NIS protein levels are significantly correlated with gestational age during early pregnancy and increase with increased placental vascularization. This would lead to increased iodide supply to meet increased fetal requirements for thyroid hormone synthesis as the pregnancy progresses.

Secretion and transfer of the thyroid hormone binding protein transthyretin by human placenta.

CONTEXT: The thyroid hormone and retinol binding protein transthyretin (TTR) is synthesised by human trophoblasts. Polarised JEG-3 choriocarcinoma cells grown in bicameral chambers secrete TTR predominantly apically but also basally and these cells and human trophoblasts also take up TTR suggesting that there may be a placental TTR shuttle that participates in materno-fetal transfer of thyroid hormones and retinol. OBJECTIVES AND METHODS: Our objective was to investigate TTR secretion into the maternal and fetal circuits of the ex vivo dually perfused placental lobule to confirm that placenta secretes TTR into the fetal circulation. We also investigated translocation of Alexa Fluor-594 labelled TTR from incubation medium into the fetal placental capillaries in early (14-15 weeks) and term placental villus explants. RESULTS: The perfused placental lobule secretes TTR into the maternal and fetal circuits. Secretion in both circuits is linear with time and is predominantly into the maternal circuit (mean maternal/fetal ratio 99.4 ± 25.6). The mean data fitted well to a three compartment mathematical model (maternal circuit, placenta and fetal circuit, constant secretion of TTR and return of maternal circuit TTR to the placental compartment). Explants from early (14-15 weeks) and late (38-40 weeks) placentas translocated fluorescently labelled TTR from medium to villus (fetal) capillaries. CONCLUSIONS: Our results confirm that human placenta secretes TTR into maternal and fetal circulations and supports the hypothesis that placental TTR secreted into the maternal placental circulation can be taken up by trophoblasts and translocated to the fetal circulation, forming a TTR shuttle system. This may have important implications for materno-fetal transfer of thyroid hormones, retinol/retinol binding protein and xenobiotics (such as polychlorinated biphenyls) all of which bind to TTR.

Expression and uptake of the thyroxine-binding protein transthyretin is regulated by oxygen in primary trophoblast placental cells.

Transplacental delivery of maternal thyroid hormones to the fetus, in particular thyroxine (T4), is critical in ensuring normal fetal neurological development. The fetus relies on maternal T4 till around 16 weeks gestation, but mechanisms of placental T4 transport are not yet fully elucidated. Placenta produces, secretes and takes up the thyroid hormone-binding protein transthyretin (TTR). Many placental genes are regulated by oxygen levels, which are relatively low (1%) in the early first trimester, rising to 3% in the mid first trimester and 8% in the early second trimester and thereafter. We examined the expression and uptake of TTR in isolated primary human placental cytotrophoblast cells cultured under different oxygen concentrations (1, 3, 8, 21% O2 and 200 µM desferrioxamine (DFO)) for 24 h. We observed sevenfold higher expression of TTR mRNA and protein levels at 1% O2 than at 8 and 21% O2. Significant increases were observed after culture at 3% O2 and following DFO treatment. We observed significantly higher uptake of ¹²5I-TTR and Alexa-594-TTR when cells were cultured at 1 and 3% O2 and in the presence of 200 µM DFO than at 8 and 21% O2. When JEG-3 choriocarcinoma cells were transfected with TTR promoter reporter constructs, increased luciferase activity was measured in cells cultured at 1 and 3% O2 in comparison to 8 and 21% O2. We conclude that placental TTR expression and uptake is increased by the relative hypoxia observed in the first trimester of pregnancy, a time when materno-fetal T4 transfer is the sole source of fetal T4.

Ontogenic changes in placental transthyretin.

OBJECTIVES: Before secretion of fetal thyroid hormone at around 16 weeks gestation normal fetal development depends on a constant supply of maternal thyroid hormone (TH), particularly thyroxine (T(4)). The detailed mechanisms of transplacental delivery of TH are still uncertain. The TH binding protein, transthyretin (TTR), is produced and secreted by placenta and may play a role in this process. The ontogeny of placental TTR is unknown. Our aim was to study changes in placental TTR in early and late pregnancy. STUDY DESIGN: We collected placentas from surgically terminated pregnancies between 6 and 17 weeks gestation (n = 44) and from normal term (38-39 weeks) pregnancies following caesarean section (n = 5). Real time-PCR, western blotting and immunohistochemistry were used to determine TTR mRNA and protein levels. RESULTS: There were highly significant correlations between gestational age and TTR mRNA (r = 0.974; p < 0.0001) and between gestational age and TTR protein (r = 0.901; p < 0.001) levels between weeks 6 and 13 of gestation. TTR expression did not increase between 13 and 17 weeks and was not different at term. Good correlation was observed between TTR mRNA and TTR protein between individual placental samples (r = 0.916; p < 0.0001). A similar trend was observed using immunohistochemical staining of placental paraffin sections. CONCLUSIONS: Our results demonstrate that TTR is expressed in the human placenta from at least 6 weeks gestation. Levels rise during the first trimester at a time when placental oxygen tensions are also rising. We hypothesise that TTR production and secretion by the placenta may facilitate transplacental delivery of TH to the fetus.

Thyroid hormones and fetal neurological development.

The development of fetal thyroid function is dependent on the embryogenesis, differentiation, and maturation of the thyroid gland. This is coupled with evolution of the hypothalamic-pituitary-thyroid axis and thyroid hormone metabolism, resulting in the regulation of thyroid hormone action, production, and secretion. Throughout gestation there is a steady supply of maternal thyroxine (T(4)) which has been observed in embryonic circulation as early as 4 weeks post-implantation. This is essential for normal early fetal neurogenesis. Triiodothyronine concentrations remain very low during gestation due to metabolism via placental and fetal deiodinase type 3. T(4) concentrations are highly regulated to maintain low concentrations, essential for protecting the fetus and reaching key neurological sites such as the cerebral cortex at specific developmental stages. There are many known cell membrane thyroid hormone transporters in fetal brain that play an essential role in regulating thyroid hormone concentrations in key structures. They also provide the route for intracellular thyroid hormone interaction with associated thyroid hormone receptors, which activate their action. There is a growing body of experimental evidence from rats and humans to suggest that even mild maternal hypothyroxinemia may lead to abnormalities in fetal neurological development. Our review will focus on the ontogeny of thyroid hormone in fetal development, with a focus on cell membrane transporters and TR action in the brain.

Oxygen concentration regulates expression and uptake of transthyretin, a thyroxine binding protein, in JEG-3 choriocarcinoma cells.

Maternal thyroid hormone is provided to the fetus before the onset of fetal thyroid function (at about 16 weeks) and is essential for normal neurologic development. Mechanisms of transport are uncertain but transthyretin (TTR), a thyroxine binding protein produced by the placenta may be involved. Placental oxygen concentrations in early pregnancy are low, about 1% early in the first trimester and rising to 8% over the next 12 weeks. This study investigated the regulation of TTR expression, secretion and uptake in JEG-3 placental cells cultured at different oxygen concentrations. TTR mRNA and protein expression and (125)I-TTR and Alexa-Fluor594-TTR uptake were significantly higher in cells cultured at 1% and 3% O(2), than at 8% O(2). This suggests that increased carrier mediated T(4) transport by placental TTR may be induced by the low oxygen environment of early pregnancy, a time when the fetus has its highest requirement for transport of maternal T(4).

Regulation of hypoxia inducible factors (HIF) in hypoxia and normoxia during placental development.

During the first trimester of pregnancy the human placenta develops in an hypoxic environment caused by the occlusion of uterine spiral arterioles by extravillous trophoblasts (EVT). This period of low oxygen tension is crucial for successful pregnancy. In low oxygen environments, Hypoxia Inducible Factors (HIF) are the main regulators in the transcription of a number of genes. Target genes can induce anaerobic processes, reducing oxygen consumption, or promote angiogenesis, which establishes and enhances the vascular environment. The HIFs can function throughout all stages of placental differentiation and growth both in normal and pathological pregnancies (compromised by hypoxia/ischemia). Interestingly, HIFs respond to a multitude of changes during pregnancy, including 1) low oxygen, 2) renin-angiotensin system (RAS), 3) cytokines, and 4) growth factors, all of which regulate placental function. This review explores oxygen-dependent and oxygen-independent regulation and the role of HIF in placental development and differentiation.

Evaluation of random plasma cortisol and the low dose corticotropin test as indicators of adrenal secretory capacity in critically ill patients: a prospective study.

It is unclear whether a random plasma cortisol measurement and the corticotropin (ACTH) test adequately reflect glucocorticoid secretory capacity in critical illness. This study aimed to determine whether these tests provide information representative of the 24 hour period. Plasma cortisol was measured hourly for 24 hours in 21 critically ill septic patients followed by a corticotropin test with 1 microg dose administered intravenously. Serum and urine were analysed for ACTH and free cortisol respectively. Marked hourly variability in plasma cortisol was evident (coefficient of variation 8-30%) with no demonstrable circadian rhythm. The individual mean plasma cortisol concentrations ranged from 286 +/- 59 nmol/l to 796 +/- 83 nmol/l. The 24 hour mean plasma cortisol was strongly correlated with both random plasma cortisol (r2 0.9, P < 0.0001) and the cortisol response to corticotropin (r2 0.72, P < 0.001). Only nine percent of patients increased their plasma cortisol by 250 nmol/l after corticotropin (euadrenal response). However, 35% of non-responders had spontaneous hourly rises > 250 nmol/l thus highlighting the limitations of a single point corticotropin test. Urinary free cortisol was elevated (865 +/- 937 nmol) in both corticotropin responders and non-responders suggesting elevated plasma free cortisol. No significant relationship was demonstrable between plasma cortisol and ACTH. We conclude that although random cortisol measurements and the low dose corticotropin tests reliably reflect the 24 hour mean cortisol in critical illness, they do not take into account the pulsatile nature of cortisol secretion. Consequently, there is the potential for erroneous conclusions about adrenal function based on a single measurement. We suggest that caution be exercised when drawing conclusions on the adequacy of adrenal function based on a single random plasma cortisol or the corticotropin test.

The BeWo choriocarcinoma cell line as a model of iodide transport by placenta.

Cultured human choriocarcinoma cells of the BeWo line exhibited saturable accumulation of radioiodide. Inhibition by competing anions followed the affinity series perchlorate >> iodide > or = thiocyanate, consistent with uptake through the thyroid iodide transporter, NIS, whose messenger RNA was found in BeWo cells, and whose protein was distributed towards the apical pole of the cells. Efflux obeyed first order kinetics and was inhibited by DIDS, an antagonist of anion exchangers including pendrin, whose messenger RNA was also present. In cultures where iodide uptake through NIS was blocked with excess perchlorate, radioiodide accumulation was stimulated by exposure to medium in which physiological anions were replaced by 2-morpholinoethanesulfonic acid (MES), consistent with the operation of an anion exchange mechanism taking up iodide. Chloride in the medium was more effective than sulfate at inhibiting this uptake, matching the ionic specificity of pendrin. These studies provide evidence that the trophoblast accumulates iodide through NIS and releases it to the fetal compartment through pendrin.

Thyroid hormone export from cells: contribution of P-glycoprotein.

Verapamil inhibits tri-iodothyronine (T3) efflux from several cell types, suggesting the involvement of multidrug resistance-associated (MDR) proteins in T3 transport. The direct involvement of P-glycoprotein (P-gp) has not, however, been investigated. We compared the transport of 125I-T3 in MDCKII cells that had been transfected with mdr1 cDNA (MDCKII-MDR) versus wild-type MDCKII cells (MDCKII), and examined the effect of conventional (verapamil and nitrendipine) and specific MDR inhibitors (VX 853 and VX 710) on 125I-T3 efflux. We confirmed by Western blotting the enhanced expression of P-gp in MDCKII-MDR cells. The calculated rate of 125I-T3 efflux from MDCKII-MDR cells (around 0.30/min) was increased twofold compared with MDCKII cells (around 0.15/min). Overall, cellular accumulation of 125I-T3 was reduced by 26% in MDCKII-MDR cells compared with MDCKII cells, probably reflecting enhanced export of T3 from MDCKII-MDR cells rather than reduced cellular uptake, as P-gp typically exports substances from cells. Verapamil lowered the rate of 125I-T3 efflux from both MDCKII and MDCKII-MDR cells by 42% and 66% respectively, while nitrendipine reduced 125I-T3 efflux rate by 36% and 48% respectively, suggesting that both substances inhibited other cellular T3 transporters in addition to P-gp. The specific MDR inhibitors VX 853 and VX 710 had no effect of 125I-T3 efflux rate from wild-type MDCKII cells but reduced 125I-T3 export in MDCKII-MDR cells by 50% and 53% respectively. These results have provided the first direct evidence that P-gp exports thyroid hormone from cells.

Assessing quality of life in Australian patients with Graves' ophthalmopathy.

AIMS: To determine quality of life and adequacy of education and counselling in Australian patients with Graves' ophthalmopathy during the course of their illness. METHODS: A cross sectional study was conducted at the orbital and endocrinology clinics of Royal Brisbane Hospital on 162 consecutive patients with Graves' ophthalmopathy who were managed between the 1992 and 2000. The Graves' ophthalmopathy quality of life (GO-QOL) survey modified for Australian conditions was distributed to study participants. Of the 19 questions asked, nine questions related to visual functioning, eight questions were about the psychosocial consequences of changed appearance, and two questions referred to education and counselling. Additionally, clinical data on the severity of illness were collected retrospectively from the medical notes of these patients. RESULTS: Completed questionnaires were received from 128 patients. The majority of patients reported limitations in daily activities such as hobbies, driving, watching television and reading, as well as impaired self confidence. The mean GO-QOL scores in this study were (100 representing maximum QOL): visual functioning 59.0 (SD 28.0), psychosocial consequences of changed appearance 54.5 (28.4), and education and counselling 59.1 (38.8). Only about a quarter of patients indicated that education and counselling were adequate and helpful. CONCLUSION: Graves' ophthalmopathy profoundly affects QOL and adequate education and counselling are essential for helping patients to cope with their illness. The GO-QOL survey is a simple, practical tool that can be used easily in a clinic to determine the QOL issues in subjects with Graves' ophthalmopathy.

Sodium iodide symporter (NIS) gene expression in human placenta.

The placenta must allow the passage of iodide from the maternal to the fetal circulation for synthesis of thyroxine by the fetal thyroid. The thyroid sodium iodide symporter (NIS) was cloned in 1996 and, although widely distributed among epithelial tissues, early studies failed to detect it in placenta. We demonstrated NIS mRNA in human placenta and in the human choriocarcinoma cell line, JAr. NIS protein was localized to trophoblasts, with a tendency to apical distribution, in sections of human placenta immunostained with a monoclonal antibody against hNIS. We conclude that NIS is expressed in placenta and may mediate placental iodide transport.

Different transporters for tri-iodothyronine (T(3)) and thyroxine (T(4)) in the human choriocarcinoma cell line, JAR.

We investigated transport systems for tri-iodothyronine (T(3)) and thyroxine (T(4)) in the human choriocarcinoma cell line, JAR, using a range of structurally similar compounds to determine whether these thyroid hormones are transported by common or different mechanisms. Saturable T(3) but not saturable T(4) uptake was inhibited by a wide range of aromatic compounds (nitrendipine, nifedipine, verapamil, meclofenamic acid, mefenamic acid, diazepam, phenytoin). Nitrendipine and diazepam were the most effective inhibitors of saturable thyroid hormone uptake. Nitrendipine decreased the K(m) for T(4) uptake from a control value of around 500 nM to around 300 nM (n=6). In contrast, the K(m) for T(3) uptake was increased from a control value of around 300 nM to around 750 nM (n=4). Diazepam had similar effects. This divergent shift in affinity for the uptake of T(3) and T(4) suggested that separate uptake systems exist for these two thyroid hormones. This provides evidence for at least two transporters mediating uptake of T(3) and T(4) in JAR cells: a specific T(4) transporter that does not interact with T(3) or structurally similar compounds; and a shared iodothyronine transporter that interacts with T(3), T(4), nitrendipine and diazepam.

Lack of membrane transport of l-thyroxine sulphate in the human choriocarcinoma cell line, JAr.

We examined uptake of l -thyroxine sulphate (T(4)S) and possible interactions between T(4)S and thyroxine (T(4)) uptake in the choriocarcinoma cell line JAr. Cells were incubated with 50 p m(125)I-T(4)S in the absence (total uptake) and in the presence (non-specific uptake) of 10 microm T(4)S. Cells were also incubated at 37 degrees C for 2 min with 50 p m(125)I-T(4)in the presence of an increasing amount of unlabelled T(4)(0-10 microm) or T(4)S (0-30 microm). There was negligible total uptake of(125)I-T(4)S (1.14+/-0. 05 fmol/mg cellular protein, mean+/-sem) and no specific uptake after 120 min incubation. Minor inhibition of(125)I-T(4)uptake by T(4)S could be explained entirely by a low level of residual T(4)(0. 2 per cent) in the T(4)S preparation. These findings indicate that T(4)S does not share the T(4)membrane transporter.

Comparison of mechanisms mediating uptake and efflux of thyroid hormones in the human choriocarcinoma cell line, JAR.

We compared the specificities of transport mechanisms for uptake and efflux of thyroid hormones in cells of the human choriocarcinoma cell line, JAR, to determine whether triiodothyronine (T3), thyroxine (T4) and reverse T3 (rT3) are carried by the same transport mechanism. Uptake of 125I-T3, 125I-T4 and 125I-rT3 was saturable and stereospecific, but not specific for T3, T4 and rT3, as unlabelled L-stereoisomers of the thyroid hormones inhibited uptake of each of the radiolabelled hormones. Efflux of 125I-T3 was also saturable and stereospecific and was inhibited by T4 and rT3. Efflux of 125I-T4 or 125I-rT3 was, in contrast, not significantly inhibited by any of the unlabelled thyroid hormones tested. A range of compounds known to interfere with receptor-mediated thyroid hormone uptake in cells inhibited uptake of 125I-T3 and 125I-rT3, but not 125I-T4. We conclude that in JAR cells uptake and efflux of 125I-T3 are mediated by saturable and stereospecific membrane transport processes. In contrast, the uptake, but not the efflux, of 125I-T4 and 125I-rT3 is saturable and stereospecific, indicating that uptake and efflux of T4 and rT3 in JAR cells occur by different mechanisms. These results suggest that in JAR cells thyroid hormones may be transported by at least two types of transporters: a low affinity iodothyronine transporter (Michaelis constant, Km, around 1 microM) which interacts with T3, T4 and rT3, but not amino acids, and an amino acid transporter which takes up T3, but not T4 or rT3. Efflux of T4 and rT3 appears to occur by passive diffusion in these cells.

Uptake of L-triiodothyronine sulphate by human choriocarcinoma cell line, JAr.

This study investigated uptake of triiodothyronine sulphate (T3S) and interactions between uptake of T3S and triiodothyronine (T3) using the human choriocarcinoma cell line (JAr) as a model of placental transport. Cells were incubated at 37 degrees C with 30 pM 125I-T3 for 2 min with unlabelled T3 (0-30 microM) or T3S (0-1 mM). Addition of an excess unlabelled T3 (30 microM) or T3S (1 mM) reduced the initial rate of 125I-T3 uptake by 69.3+/-3.6 per cent (P<0.0001) and 52.9+/-7.8 per cent (P<0.0001), respectively. The calculated Michaelis constant (Km) for T3 uptake was 0.378+/-0.133 microM (n = 3) with a corresponding maximum velocity (Vmax) of 15.4+/-6.9 pmol/min/mg protein. Uptake of 125I-T3 was inhibited in a dose-dependent way by the addition of unlabelled T3S (0-1 mM). The calculated inhibition constant (Ki) for the inhibition of 125I-T3 uptake by T3S was 121.8+/-35.2 microM (n = 6). Saturable uptake of 125I-T3S by JAr cells was negligible. The T3S preparation incubated with the cells contained about 0.1 per cent T3, sufficient to explain the apparent inhibition of 125I-T3 uptake by unlabelled T3S. These results suggest that, in contrast to T3 uptake in these cells, JAr cells do not have a saturable uptake mechanism for T3S, and that T3S does not interact with the T3 transporter in these cells.

Uptake of reverse T3 in the human choriocarcinoma cell line, JAr.

The uptake and efflux of reverse triiodothyronine (rT3) in JAr cells were investigated. Uptake of 125I-rT3 was time dependent and reversible with a saturable component of around 70 per cent of total uptake after 30 min of incubation. Efflux was not saturable. Kinetic analysis of the initial specific uptake rates revealed an uptake process with a Michaelis constant of 3.04+/-0.53 microM (mean+/-SEM, n=15) and a corresponding maximum velocity of 9.65+/-2.49 pmol/min/mg protein (n=15). Uptake of rT3 was stereospecific, but not specific for rT3, as unlabelled L stereoisomers of thyroid hormone analogues were more effective as inhibitors of 125I-rT3 uptake than rT3. Unlabelled T3 and thyroxine (T4) (10 microM) reduced cellular uptake of 125I-rT3 by around 82 and 74 per cent, respectively. The calculated inhibition constants Ki were 1.23+/-0.29 microM (n=4) and 0.66+/-0.19 microM (n=4) for T3 and T4, respectively. Similarly, rT3 reduced cellular uptake of 125I-T3 and 125I-T4 by 34 and 23 per cent, respectively. The calculated inhibition constants Ki were 1.75+/-0.55 microM (n=8) and 1.08+/-0.36 microM (n=8) for the inhibition of 125I-T3 and 125I-T4 uptake, respectively. Reverse T3 inhibited efflux of 125I-T3 from the cells by around 20 per cent, but did not inhibit efflux of 125I-T4. These results suggest that uptake of rT3 in JAr cells may occur via a single, saturable membrane carrier, which also interacts with T3 and T4, while efflux of rT3 may occur by passive diffusion.

Statistical moments for placental transfer of solutes in man.

The placental transfer of red blood cells and solutes in man has been investigated by statistical moment analysis, using the impulse-response technique. Model compounds of different lipophilicity (sucrose, water, antipyrine, propranolol and labetalol) were injected with a vascular reference (labelled red blood cells) as boluses into either the foetal or maternal circulation of a single-pass perfused placental lobule. Maternal and foetal venous outflow fractions were collected at intervals ranging from 1 to 600 s. Perfusion was conducted at maternal flow rates of 4 and 6 mL min(-1) and foetal flow rates of 2 and 3 mL min(-1), respectively, to yield a constant materno-foetal flow ratio of 2. The outflow concentration-time profile curves were analysed by statistical moment analysis. The sum of foetal and maternal recovery was close to 100% for red blood cells, sucrose, water and antipyrine, but lower for propranolol and labetalol. The mean transit time (MTT) values ranged from 20 to 500 s. The normalized variance (CV2) for red blood cells in the foetal and maternal circulation of the placenta were in the ranges 2.31 to 3.86 and 2.00 to 2.03, respectively. The shape of the outflow concentration-time profiles after bolus input is consistent with that of vascular residence time models such as the dispersion model. The heterogeneity in red blood cell transit times, as defined by CV2, is greater than in either the perfused leg or perfused liver.