RESUMO
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.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Rim/metabolismo , Tri-Iodotironina/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Animais , Transporte Biológico/efeitos dos fármacos , Western Blotting/métodos , Linhagem Celular , Membrana Celular/metabolismo , Cães , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Radioisótopos do Iodo , Nitrendipino/farmacologia , Piperidinas/farmacologia , Piridinas/farmacologia , Transfecção , Verapamil/farmacologiaRESUMO
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.
Assuntos
Coriocarcinoma/metabolismo , Iodetos/metabolismo , Placenta/metabolismo , Neoplasias Uterinas/metabolismo , Sequência de Bases , Transporte Biológico Ativo , Linhagem Celular Tumoral , DNA de Neoplasias/genética , Feminino , Humanos , Radioisótopos do Iodo , Microscopia Confocal , Modelos Biológicos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Simportadores/genética , Simportadores/metabolismoRESUMO
Integrated blood plasma levels of LH and FSH and their response to the iv administration of 100 mug synthetic LRF were studied in 29 normal subjects, 12 women with Stein-Leventhal syndrome, 8 subjects with primary gonadal failure, 7 women with Sheehan's syndrome, 20 subjects with pituitary tumors, 10 subjects with idiopathic gonadotropin deficiency and 5 subjects with hypothalamic tumors. Within each group there was considerable variation in the response of LH and FSH levels to LRF. In each group there was a statistically significant positive correlation between basal integrated gonadotropin levels and the response of the levels to LRF. Both within groups and between groups, the best indicator of the response to LRF was the basal levels of FSH and LH. In subjects with hypogonadotropic hypogonadism there was no significant difference in mean basal LH levels and mean response to LRF between patients with primarily pituitary disease (pituitary tumors or Sheehan's syndrome) and conditions which might represent hypothalamic disease (hypothalamic tumors or idiopathic gonadotropin deficiency). The response to an acute, single, injection of LRF appears to more directly reflect basal gonadotropin levels rather than disease category.
PIP: The diagnostic value of LRF was assessed by measuring the response of plasma levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH) to a single iv injection of synthetic LRF in 29 normal subjects, 12 women with Stein-Leventhal syndrome, 8 patients with primary gonadal failure, 7 women with Sheehan's syndrome, 20 patients with pituitary tumors, 10 subjects with idiopathic gonadotropin deficiency, and 5 patients with hypothalamic tumors. The response of plasma LH and FSH levels to LRF varied considerably within each group. Each group showed a positive correlation between basal LH and FSH levels and the response to LRF. There was no marked difference in basal LH values and the mean response to LRF between patients with pituitary tumors of Sheehan's syndrome and patients with hypothalamic tumors or idopathic gonadotropin deficiency. It is concluded that the response to a single, acute iv injection of LRF provides little information of disease states beyond that provided by basal gonadotropin levels.
Assuntos
Hormônio Foliculoestimulante/sangue , Hormônio Liberador de Gonadotropina/farmacologia , Hormônio Luteinizante/sangue , Adolescente , Adulto , Idoso , Criança , Feminino , Gonadotropinas/deficiência , Humanos , Hipogonadismo/etiologia , Hipogonadismo/fisiopatologia , Hipopituitarismo/fisiopatologia , Hipotálamo , Masculino , Menstruação , Pessoa de Meia-Idade , Neoplasias/fisiopatologia , Doenças da Hipófise/fisiopatologia , Neoplasias Hipofisárias/fisiopatologia , Síndrome do Ovário Policístico/fisiopatologiaRESUMO
Placental deiodination of T4 to rT3 has been proposed as the factor controlling materno-fetal transmission of T4. We investigated T4 transfer in the isolated perfused human placental lobule with and without addition of the deiodinase inhibitor, iopanoic acid. T4 (150 nmol/L) in protein-free medium was added to the maternal circuit. Without iopanoic acid, the appearance of T4 in the fetal circuit was very low, with fetal T4 levels reaching only 4.1 +/- 0.84 pmol/L at 6 h. Levels of rT3 rose progressively in both circuits, reaching 28.8 +/- 5.5 nmol/L in the maternal and 12.4 +/- 3.2 nmol/L in the fetal circuit by 6 h. No T3 could be measured in either circuit. Addition of 0.5 nmol/L iopanoic acid to maternal perfusate, however, resulted in significant reduction in the appearance of rT3 [maternal levels, 0.58 +/- 0.06 nmol/L (2% of control values); fetal levels, 0.33 +/- 0.03 nmol/L (2.7% of control values)] and a major (approximately 2700-fold) increase in T4 appearance in the fetal circuit, with fetal T4 levels reaching 10.1 +/- 3.4 nmol/L at 6 h. These results support the hypothesis that placental inner ring (type III) deiodination is a major factor controlling placental transmission of maternal T4.
Assuntos
Iodeto Peroxidase/metabolismo , Troca Materno-Fetal , Placenta/metabolismo , Tiroxina/metabolismo , Feminino , Meia-Vida , Humanos , Técnicas In Vitro , Iodeto Peroxidase/antagonistas & inibidores , Ácido Iopanoico/farmacologia , GravidezRESUMO
Propylthiouracil (PTU) is widely believed to cross the placenta less freely than methimazole (MMI) and is therefore regarded as the preferred drug for treatment of hyperthyroidism in pregnancy. Clinical studies comparing the two drugs show, however, no differences in maternal or fetal thyroid function. We investigated transfer from the maternal to the fetal circuit in the isolated perfused term human placental lobule of low and high doses of PTU (4 micrograms/mL and 40 micrograms/mL) and MMI (1.5 micrograms/mL and 15 micrograms/mL) in protein-free perfusate and low doses of both drugs with addition of 40 g/L of bovine albumin. Both drugs readily crossed the placenta, reaching equilibrium in all experiments in about 2 h. Drug concentrations in the two circuits fitted a two compartmental model. Transfer kinetics for the two drugs were similar, nonsaturable, and unaffected by addition of albumin. Clearances (mL.min-1.g-1, means +/- SD) of PTU from maternal to fetal circuits were: 0.229 +/- 0.110, 0.216 +/- 0.065, and 0.170 +/- 0.032; and for transfer of MMI: 0.165 +/- 0.025, 0.232 +/- 0.153, and 0.174 +/- 0.009 (for low doses without, low doses with, and high doses without albumin, respectively). Clearances of PTU from fetal to maternal circuits were: 0.147 +/- 0.072, 0.109 +/- 0.014, and 0.116 +/- 0.028; and for transfer of MMI: 0.095 +/- 0.029, 0.122 +/- 0.088, and 0.12 +/- 0.005 (in the same experiments). There was no significant difference between drugs or drug doses and no effect of addition of albumin. We conclude that PTU and MMI have similar placental transfer kinetics.
Assuntos
Antitireóideos/farmacocinética , Parto Obstétrico , Metimazol/farmacocinética , Placenta/metabolismo , Propiltiouracila/farmacocinética , Animais , Feminino , Humanos , Técnicas In Vitro , Troca Materno-Fetal , Modelos Biológicos , Perfusão , Gravidez , Albumina Sérica/metabolismo , Soroalbumina Bovina/metabolismoRESUMO
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.
Assuntos
Coriocarcinoma/metabolismo , Tiroxina/farmacocinética , Tri-Iodotironina/farmacocinética , Neoplasias Uterinas/metabolismo , Análise de Variância , Transporte Biológico/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Diazepam/farmacologia , Feminino , Moduladores GABAérgicos/farmacologia , Humanos , Radioisótopos do Iodo , Leucina/farmacologia , Ácido Meclofenâmico/farmacologia , Ácido Mefenâmico/farmacologia , Nifedipino/farmacologia , Nitrendipino/farmacologia , Fenilalanina/farmacologia , Fenitoína/farmacologia , Triptofano/farmacologia , Células Tumorais Cultivadas , Verapamil/farmacologiaRESUMO
We investigated the uptake of L-tri-iodothyronine (T3) by cultured human trophoblast cells. Uptake was time-dependent, initially linear and approaching equilibrium after 60 min with an approximate half-time of 13 +/- 4.5 min (mean +/- S.E.M., n = 4). It had a non-saturable component accounting for about 50% of total uptake. We demonstrated a single saturable T3 uptake mechanism with a calculated Michaelis constant (Km) of 755 +/- 145 nmol/l (n = 11-13) and a corresponding maximum velocity of 28.8 +/- 5.3 pmol/min per mg protein (n = 11-13). The Km value was similar to those reported in other tissues.
Assuntos
Tri-Iodotironina/farmacocinética , Trofoblastos/metabolismo , Células Cultivadas , Humanos , Radioisótopos do Iodo , Fatores de TempoRESUMO
We have studied the uptake of 125I-thyroxine (125I-T4) in the human choriocarcinoma cell line JAR. Uptake of 125I-T4 was time-dependent, stereospecific and reversible, with a saturable component of 33% after 120 min of incubation. Kinetic analysis of the initial specific uptake rates indicated the presence of a single uptake process with a Michaelis constant of 59.4 +/- 13.9 nM (n = 12) and maximum velocity of 0.29 +/- 0.06 pmol/min per mg protein. Uptake was dependent on intracellular energy as, in the presence of 2 nM potassium cyanide, saturable uptake was reduced to 60.6 +/- 8.5% (n = 4) of control uptake. Uptake was also temperature-dependent. Saturable 125I-T4 uptake after 60 min of incubation was 26.1 +/- 3.0% at 25 degrees C (n = 6) and 27.3 +/- 5.7% at 4 degrees C of control uptake at 37 degrees C. Ouabain did not inhibit 125I-T4 uptake indicating that the uptake was independent of the Na+ gradient across the cell membrane. Although T4 uptake was stereospecific, as D-T4 failed to inhibit 125I-L-T4 uptake, it was not specific for T4, as tri-iodothyronine (T3) and reverse T3 also inhibited 125I-T4 uptake. We conclude that JAR cells have a saturable, stereospecific and reversible membrane transport mechanism for T4 which is dependent on intracellular energy, but independent of the Na+ gradient across the cell membrane.
Assuntos
Coriocarcinoma/metabolismo , Tiroxina/metabolismo , Neoplasias Uterinas/metabolismo , Feminino , Humanos , Cinética , Ouabaína/farmacologia , Temperatura , Tiroxina/farmacologia , Fatores de Tempo , Tri-Iodotironina/farmacologia , Tri-Iodotironina Reversa/farmacologia , Células Tumorais CultivadasRESUMO
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.
Assuntos
Coriocarcinoma/metabolismo , Hormônios Tireóideos/metabolismo , Neoplasias Uterinas/metabolismo , Análise de Variância , Transporte Biológico , Transporte Biológico Ativo , Feminino , Humanos , Radioisótopos do Iodo , Tiroxina/metabolismo , Tri-Iodotironina/metabolismo , Tri-Iodotironina Reversa/metabolismo , Células Tumorais CultivadasRESUMO
The effects of cell swelling induced by hyposmotic shock on efflux of hybrid hormones and selected amino acids from human placental tissue were examined. Decreasing the osmolarity of external medium from 290 to 140 mOsm/kg stimulated release of taurine, tryptophan and glutamine from placental tissue fragments. The efflux rate constant for taurine increased from 0.0069 +/- 0.0012/min to 0.0646 +/- 0.0217/min (n = 6) (P < 0.001), for tryptophan from 0.016 +/- 0.0010/min to 0.0295 +/- 0.0016/min (n = 6) (P < 0.001), and for glutamine from 0.0267 +/- 0.0027/min to 0.0659 +/- 0.0043/min (n = 4) (P < 0.001). In contrast, hyposmotic challenge did not affect release of triiodothyronine, thyroxine and leucine. These results indicate that transport processes involved in the regulation of cellular volume are unlikely to facilitate efflux of thyroid hormones from placental tissue, and therefore are unlikely to mediate transfer of thyroid hormones across the placenta. In addition, it is unlikely that the transport system facilitating the release of amino acids from placental tissue during regulatory volume decrease is one of the known amino acid carriers.
Assuntos
Placenta/citologia , Placenta/metabolismo , Hormônios Tireóideos/metabolismo , Tamanho Celular , Feminino , Glutamina/metabolismo , Humanos , Soluções Hipotônicas , Leucina/metabolismo , Concentração Osmolar , Gravidez , Taurina/metabolismo , Tiroxina/metabolismo , Tri-Iodotironina/metabolismo , Triptofano/metabolismoRESUMO
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.
Assuntos
Coriocarcinoma/metabolismo , Tiroxina/análogos & derivados , Neoplasias Uterinas/metabolismo , Transporte Biológico Ativo , Membrana Celular/metabolismo , Feminino , Humanos , Cinética , Modelos Biológicos , Placenta/metabolismo , Gravidez , Tiroxina/metabolismo , Células Tumorais CultivadasRESUMO
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.
Assuntos
Coriocarcinoma/metabolismo , Tri-Iodotironina/análogos & derivados , Neoplasias Uterinas/metabolismo , Transporte Biológico , Feminino , Humanos , Radioisótopos do Iodo , Cinética , Placenta/metabolismo , Gravidez , Tri-Iodotironina/metabolismo , Tri-Iodotironina/farmacologia , Células Tumorais CultivadasRESUMO
In the placenta the trophoblast cell layer separates maternal and fetal circulations and is involved in the active transport of selected substances across this barrier. We have used the JAR choriocarcinoma cell line to study aspects of trophoblast membrane transport. To determine whether JAR cells could be used in studies of vectorial transepithelial transport it was necessary to determine whether these cells were polarized and assembled tight junctions. In the present study we investigated JAR cells using a range of markers for specific cell surface domains combined with confocal laser scanning microscopy. Freshly isolated cells initially formed a confluent epithelial monolayer with recruitment of a tight junction-associated protein, ZO-1, and a cell adhesion molecule, E-cadherin, to the surface at sites of cell-cell contact. They did not, however, display cell surface polarization, as NaK-ATPase was not segregated in the basolateral domain, and a differentiated apical cell surface was not assembled. The monolayer stage was also unstable, as continued proliferation resulted in the formation of multilayered aggregates where ZO-1 and E-cadherin were lost from the cell surface. These results suggest that the JAR cell line is unlikely to be a suitable model for studies of transepithelial transport in the placenta.
Assuntos
Polaridade Celular , Junções Intercelulares , Trofoblastos/metabolismo , Transporte Biológico , Caderinas/análise , Divisão Celular , Coriocarcinoma , Epitélio/metabolismo , Epitélio/ultraestrutura , Fluoresceína-5-Isotiocianato/análogos & derivados , Humanos , Lasers , Proteínas de Membrana/análise , Microscopia Confocal , Fosfoproteínas/análise , ATPase Trocadora de Sódio-Potássio/análise , Trofoblastos/ultraestrutura , Células Tumorais Cultivadas , Aglutininas do Germe de Trigo , Proteína da Zônula de Oclusão-1RESUMO
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.
Assuntos
Coriocarcinoma/metabolismo , Tri-Iodotironina Reversa/metabolismo , Neoplasias Uterinas/metabolismo , Feminino , Humanos , Radioisótopos do Iodo , Cinética , Gravidez , Tiroxina/farmacologia , Tri-Iodotironina/farmacologia , Células Tumorais CultivadasRESUMO
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.
Assuntos
Proteínas de Transporte/genética , Expressão Gênica , Proteínas de Membrana/genética , Placenta/química , Simportadores , Anticorpos Monoclonais , Proteínas de Transporte/análise , Coriocarcinoma/química , Feminino , Doença de Graves/metabolismo , Humanos , Proteínas de Membrana/análise , Gravidez , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Glândula Tireoide/química , Distribuição Tecidual , Trofoblastos/química , Células Tumorais Cultivadas , Neoplasias Uterinas/químicaRESUMO
We studied uptake of L-triiodothyronine (T3) by the human choriocarcinoma cell line, JAR. Uptake was time dependent with a half-time of 56.2 +/- 7.2 min (mean +/- SEM, n = 4). A non-saturable component accounted for about 24% of total uptake. We found a single saturable uptake mechanism with a calculated Michaelis constant (Km) of 586 +/- 206 nM (n = 9) and a corresponding maximum velocity of 17.0 +/- 5.7 pmol/min per mg protein (n = 9), values similar to those we have described recently in cultured normal human trophoblast cells. Uptake was dependent on temperature and intracellular energy, being reduced at lower temperatures and in the presence of potassium cyanide. It was independent of the Na+ gradient across the cell membrane and the presence of Na+ in the external medium, but was affected by the cell membrane potential.
Assuntos
Coriocarcinoma/metabolismo , Tri-Iodotironina/metabolismo , Neoplasias Uterinas/metabolismo , Transporte Biológico/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Feminino , Humanos , Cinética , Troca Materno-Fetal , Potenciais da Membrana , Cianeto de Potássio/farmacologia , Gravidez , Sódio/farmacologia , Temperatura , Células Tumorais CultivadasRESUMO
We studied the effect of a number of amino acids on uptake of L-triiodothyronine (T3) in the human choriocarcinoma cell line, JAR. Tryptophan inhibited saturable T3 uptake by about 57% without any significant effect on the non-saturable uptake. Michaelis constant (Km) for T3 uptake was 1.06 +/- 0.15 microM (n = 15) with the corresponding maximum velocity (Vmax) of 24.2 +/- 3.1 pmol/min/mg cellular protein. For tryptophan uptake the Km was 1.31 +/- 0.26 microM (n = 7) and Vmax was 166.4 +/- 35.7 pmol/min/mg protein. The kinetic parameters for both uptake processes were similar to those reported in normal placenta. Uptake of T3 was inhibited by tryptophan but not phenylalanine, but tryptophan uptake was inhibited both by T3 and phenylalanine. Inhibition of T3 uptake by tryptophan was dose dependent, with an inhibition constant (Ki) of 2.9 +/- 0.5 mM. Similarly, tryptophan uptake was inhibited by T3 and phenylalanine in a dose dependent way with Ki values of 4.9 +/- 0.5 microM and 15.6 +/- 4.8 microM respectively. Km for T3 uptake was significantly increased to 1.86 +/- 0.42 microM (n = 4) in the presence of 3 mM unlabelled tryptophan and, similarly, Km for tryptophan uptake was significantly increased to 9.91 +/- 2.57 microM (n = 3) in the presence of 5 microM unlabelled T3. Efflux of T3 was progressively inhibited by increasing concentrations of both ligands, i.e. was saturable. We conclude that there is mutual competitive inhibition between uptake systems for T3 and tryptophan in JAR cells, but the kinetic parameters of cross-inhibition of uptake by the substrates suggest that the carriers are distinct. T3 may be transported in JAR cells by at least two transport systems with differing substrate specificities. We also demonstrated the presence of a saturable membrane carrier mediating the efflux of T3 from the cells which was subject to trans-inhibition by T3 and tryptophan.
Assuntos
Coriocarcinoma/metabolismo , Tri-Iodotironina/metabolismo , Triptofano/metabolismo , Neoplasias Uterinas/metabolismo , Transporte Biológico , Feminino , Humanos , Gravidez , Células Tumorais CultivadasRESUMO
Previous studies of the metabolism of 11 beta-hydroxy corticosteroids by placental tissue have indicated that the only product is the C11-oxidized metabolite. In the present study we have re-examined the metabolism of prednisolone in the isolated, perfused, dual recirculating human placental lobule, using a perfusate based on tissue culture medium 199. Four metabolites were identified in both the maternal and fetal compartments in 6 h perfusions by comparison of relative retention times measured by HPLC and capillary gas chromatography (GC) and of mass spectra recorded by capillary gas chromatography-mass spectrometry (GC-MS) with those of authentic reference standards. The steroids were derivatized as the MO-TMS ethers for mass spectral measurements. Analysis of samples from five perfusion experiments resulted in the following percentage conversions after 6 h perfusion (mean +/- SD, maternal and fetal perfusate, respectively): prednisone (49.1 +/- 7.8, 49.1 +/- 6.6), 20 alpha-dihydroprednisone (0.84 +/- 0.29, 0.81 +/- 0.35), 20 beta-dihydroprednisone (39.1 +/- 6.7, 39.2 +/- 5.9), 20 beta-dihydroprednisolone (6.8 +/- 2.7, 6.3 +/- 1.6) and unmetabolized prednisolone (4.1 +/- 1.8, 4.6 +/- 2.1). No evidence was found for metabolites formed by 6 beta-hydroxylation or cleavage of the C17-C20 bond.
Assuntos
Perfusão , Placenta/metabolismo , Prednisolona/metabolismo , Cromatografia Gasosa , Cromatografia Líquida de Alta Pressão , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Técnicas In Vitro , Troca Materno-Fetal , GravidezRESUMO
Prednisolone is metabolized in the perfused human placental lobule to prednisone, 20 alpha-dihydroprednisone, 20 beta-dihydroprednisone and 20 beta-dihydroprednisolone. The pathway of metabolite formation was defined in perfusions of placental lobules using prednisone and 20 beta-dihydroprednisone separately as substrates and with prednisolone co-perfused with glycyrrhetinic acid, a potent inhibitor of the 11-oxidase component of the 11 beta-hydroxysteroid dehydrogenase enzyme system. The pattern of metabolites identified from 6 h samples indicated a reversible formation of prednisone from prednisolone, the production of the 20 alpha- and 20 beta-dihydro metabolites of prednisone from prednisone, the formation of 20 beta-dihydroprednisolone from 20 beta-dihydroprednisone only and no direct formation of 20 beta-dihydroprednisolone from prednisolone. Kinetic analysis at two substrate concentrations confirmed that the formation of three of the four steroid metabolites followed first order kinetics. In perfusions with an initial prednisolone concentration of 1 microgram/ml (n = 4) or 100 ng/ml (n = 3), the rate constants obtained were (mean +/- SD, maternal compartment, h-1): prednisone, 1.97 +/- 0.49 and 2.25 +/- 0.15, P > 0.1; 20 alpha-dihydroprednisone, 0.0006 +/- 0.0004 and 0.0017 +/- 0.0006, P < 0.1; 20 beta-dihydroprednisone, 0.15 +/- 0.022 and 0.15 +/- 0.0077, P > 0.1. In contrast, the rate constant for formation of 20 beta-dihydroprednisolone at an initial prednisolone concentration of 100 ng/ml (0.083 +/- 0.0095 h-1) was significantly (P < 0.01) greater than the corresponding rate constant at the higher initial prednisolone concentration (0.039 +/- 0.015 h-1). A significant increase (P < 0.05) was observed for the formation of 20 beta-dihydroprednisolone at the end of 6 h perfusions at the lower initial substrate concentration (11.2 +/- 1.9%) compared with the 1 microgram/ml concentration (6.0 +/- 2.5%).
Assuntos
Placenta/metabolismo , Prednisolona/metabolismo , 11-beta-Hidroxiesteroide Desidrogenases , Cromatografia Líquida de Alta Pressão , Feminino , Ácido Glicirretínico/farmacologia , Humanos , Hidroxiesteroide Desidrogenases/antagonistas & inibidores , Cinética , Perfusão , Prednisona/análogos & derivados , Prednisona/metabolismo , GravidezRESUMO
We have previously reported the placental metabolism of prednisolone to prednisone, 20alpha- and beta-dihydroprednisone and 20beta-dihydroprednisolone. In this study, the disposition of cortisol was investigated in vitro in the dual perfused, isolated human placental lobule after the addition of cortisol (1.2 micromol, n = 3 and 12 micromol, n = 4) to the maternal compartment. Analysis of 5 h maternal and fetal perfusate samples by high performance liquid chromatography-electrospray-tandem mass spectrometry (HPLC-ESI-MS/MS) revealed that cortisol was mainly metabolized to cortisone, but a significant production of 20alpha-dihydrocortisone, 20beta-dihydrocortisone, 20alpha-dihydrocortisol and 20beta-dihydrocortisol was also detected. Saturability of metabolism but not transfer was demonstrated. Metabolism was eliminated by co-perfusion with the potent 11beta-hydroxysteroid dehydrogenase (11beta-HSD) enzyme inhibitor 18beta-glycyrrhetinic acid (GA). The disposition of GA was analysed using HPLC-atmospheric pressure chemical ionisation-MS/MS (HPLC-APCI-MS/MS). GA was found to transfer from the maternal to the fetal circulations without detectable metabolism during 6 h of perfusion.