Thyroid oxidase (THOX2) gene expression in the rat thyroid cell line FRTL-5.

A cDNA encoding an NADPH oxidase flavoprotein was isolated from the rat thyroid gland. The predicted 1517-residue polypeptide was 82.5% identical to the human THOX2/DUOX2 and 74% similar to THOX1/DUOX1. Rat THOX2 lacks a stretch of 30 residues, corresponding to one exon in the human gene sequence. THOX2 mRNA was found to be expressed in cultured FRTL-5 cells. The level of THOX2 mRNA was increased by cAMP in these cells and it was decreased in the thyroids of rats treated with the antithyroid drug methimazole, unlike the TPO and NIS mRNAs. Since it was found in the intestine, duodenum, and colon, in addition to thyroid, we suggest that it be called LNOX, the new family of long homologs of NOX flavoproteins rather than THOX and/or DUOX.

Purification, molecular cloning, and functional expression of the human nicodinamide-adenine dinucleotide phosphate-regulated thyroid hormone-binding protein.

The kidney and several other thyroid hormone-responsive tissues contain a NADP-regulated thyroid hormone (TH)-binding protein (THBP), with an apparent molecular mass of 36 kDa on SDS-PAGE, responsible for most of the intracellular high-affinity T3 and T4 binding. THBP was purified to homogeneity from human kidney cytosol and used to generate proteolytic peptides. Microsequencing of four peptides revealed identity to amino acid sequences deduced from a human cDNA homolog to a cDNA encoding kangaroo mu-crystallin. This protein is a major structural kangaroo lens protein with no known function in other species. A full-sized cDNA (TH5.9) was isolated by 5'- and 3'-rapid amplification of cDNA ends using a human brain cDNA library and gene-specific PCR primers, confirming identity to the previously cloned human cDNA. The TH5.9 cDNA encodes a 314-residue protein (theoretical mol wt = 33,775) with significant homologies (40 to 60%) with two bacterial enzymes: lysine cyclodeaminase and ornithine cyclodeaminase. The TH5.9 cDNA was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Purified GST fusion protein, but not GST, bound T3 specifically with high affinity [dissociation constant (Kd) = 0.5 nM] in the presence of NADPH, and was labeled by UV-driven cross-linking of underivatized [(125)I]T3. T3 binding and photoaffinity labeling of GST fusion protein were activated by NADPH [activation constant (K[act]) = 10(-8) M], but not by NADH. The expressed protein displays the appropriate binding properties, indicating that TH5.9 cDNA encodes the NADP-regulated THBP characterized in human tissues.

Cytotoxicity of bilirubin for human fibroblasts and rat astrocytes in culture. Effect of the ratio of bilirubin to serum albumin.

The sensitivity of rat brain astrocytes and human fibroblasts in culture to unconjugated bilirubin was investigated. Medium containing 6 mumol/1 bilirubin and increasing concentrations of human serum albumin giving ratios of 0.5-1.5 that resulted in an increase of the free bilirubin concentrations. The LDH activity in the culture medium was an index of cytolysis and the MTT assay was used as an index of mitochondrial impairment. The ratios producing half-maximum cell lysis after 24, 48, and 72 h, were 1.1, 0.9 and 0.85, for astrocytes, and 1.2, 0.75 and 0.75, for fibroblasts. Mitochondrial activity decreased after 24 h for ratio = 0.7 and partly recovered at 48 h. Mitochondrial activity was more impaired in fibroblasts than in astrocytes above ratio = 0.7. The cytotoxic effects were linked to the free bilirubin concentration. We conclude that astrocytes are less sensitive to bilirubin cytotoxic effects than are fibroblasts.

High affinity thyroid hormone-binding protein in human kidney: kinetic characterization and identification by photoaffinity labeling.

The binding of thyroid hormones and its regulation of NADPH and NADP+ were studied in human kidney cytosol, and a 38-kDa polypeptide (p38) was identified by photoaffinity labeling of cytosol with underivatized [125I]T3, SDS-PAGE, and autoradiography. The cytosolic thyroid hormone binding and p38 photolabeling were strongly activated by NADPH (maximum at 10(-7) M), whereas other nucleotides were less effective or ineffective. NADP+ did not activate T3 binding and p38 photolabeling, provided it was protected from conversion to NADPH by the addition of an exogenous oxidizing enzymatic system (oxidized glutathione plus glutathione reductase). Furthermore, NADP+ inhibited NADPH activation (half-maximum inhibitory effect at approximately 2 x 10(-5)M), and oxidation of NADPH to NADP+ induced dissociation of bound T3. The equilibrium dissociation constant (Kd) of the NADPH-activated cytosolic T3-binding sites was 0.3 nM, similar to the Kd of the nuclear T3 receptors. The kidney contained 200 times more cytosolic NADPH-activated thyroid hormone-binding sites than nuclear T3 receptors. Nonradioactive iodothyronines competed with [125I]T3 for both NADPH-activated binding and p38 photolabeling, with the following order of decreasing affinity: D-isomer of T3 > T3 > T4 > triiodothyroacetic acid > 3'-isopropyl-3,5-diiodothyronine > rT3. NADPH-activated T3 binding and photolabeled p38 were also detected in human heart and liver cytosols, but not in pancreas, cultured fibroblast and erythrocyte cytosols, or plasma. Rat kidney cytosol contained a 35-kDa photolabeled polypeptide homolog to human p38. The native molecular mass of the human photolabeled protein was 50 kDa, whereas that of the rat protein was 60 kDa, as determined by nondenaturing polyacrylamide gel electrophoresis. Two-dimensional PAGE of photolabeled p38 indicated an isoelectric point of 5.3. These findings describe the molecular properties of a NADPH/NADP+-regulated thyroid hormone-binding protein not previously identified in human and rat kidney cytosol.

Characterization of the thyroid hormone transport system of cerebrocortical rat neurons in primary culture.

The uptake of 3',3,5-triiodo-L-thyronine (T3) and L-thyroxine (T4) by primary cultures derived from rat brain hemispheres was studied under initial velocity conditions, at 25 degrees C. Uptake of both hormones was carrier mediated and obeyed simple Michaelis-Menten kinetics. The Km of T3 uptake was very similar to that of T4, and did not vary significantly from day 1 to 4 in culture (310-400 nM). The maximal velocity (Vmax) of T3 uptake nearly doubled between day 1 and 4 of culture (41 +/- 3 vs. 70 +/- 5 pmol/min/mg of DNA, respectively). The Vmax of T4 uptake did not change (28 +/- 8 and 31 +/- 4 pmol/min/mg of DNA on days 1 and 4, respectively). The rank order of unlabeled thyroid hormone analogues to compete with labeled T3 or T4 uptakes were the same (T3 > T4 > 3',5',3-triodo-L-thyronine > 3',3,5-triiodo-D-thyronine > triiodothyroacetic acid), indicating that the transport system is stereospecific. Unlabeled T4 was a stronger competitor of labeled T4 uptake than of labeled T3 uptake, whereas unlabeled T3 had the same potency for both processes. These results suggest that T3 and T4 are transported either by two distinct carriers or by the same carrier bearing separate binding sites for each hormone. They also indicate that the efficiency of T3 uptake increases during neuronal maturation.

Relationship between the thyroid hormone transport system and the Na(+)-H+ exchanger in cultured rat brain astrocytes.

The entry of T3 and T4 into rat cultured astrocytes is mediated by a sterospecific saturable transport system. This study examines the effect of inhibiting the Na(+)-H+ exchanger and intracellular acidification on the initial velocity of [125I]T3 and [125I]T4 uptake. The resting intracellular pH (pHi) was approximately 7.15 in astrocytes exposed to CO2/HCO3(-)-free medium buffered with HEPES at pH 7.40 at 22 C. Isoosmotic replacement of extracellular sodium by mannitol or choline decreased the pHi by 0.15 pH unit and reduced uptake by about 20%. Replacing sodium with lithium had no effect on uptake. Amiloride, a specific blocker of the Na(+)-H+ exchanger, reduced pHi, as described above, and inhibited T3 and T4 uptake by about 35%. Acid loading the cells with a NH4+ pulse decreased the pHi by up to 1.2 pH units and the uptake of T3 and T4 by up to 50%. The maximum velocity of uptake was decreased, whereas the Km was unchanged. An isoosmotic increase in the extracellular K+ concentration to 50 mM had no effect on T3 uptake. The initial velocity of T3 uptake by acid-loaded cells was gradually restored by increasing the extracellular Na+ concentration. These results indicate that thyroid hormone transport into rat cultured astrocytes involves a mechanism linked to the activity of the Na(+)-H+ exchanger and the H+ concentration inside the cells.

Identification by photoaffinity labelling of a pyridine nucleotide-dependent tri-iodothyronine-binding protein in the cytosol of cultured astroglial cells.

High-affinity 3,3',5-tri-iodo-L-thyronine (T3) binding (Kd approximately 0.3 nM) to the cytosol of cultured rat astroglial cells was strongly activated in the presence of pyridine nucleotides. A 35 kDa pyridine nucleotide-dependent T3-binding polypeptide (35K-TBP) was photoaffinity labelled using underivatized [125I]T3 in the presence of pyridine nucleotides and the free-radical scavenger dithiothreitol. Maximum activations of T3 binding and 35K-TBP photolabelling were obtained at approx. 1 x 10(-7) M NADP+ or NADPH, or 1 x 10(-4) M NADH. NAD+ and other nucleotides were without effect. NADPH is the form which activates T3 binding and 35K-TBP photolabelling, since cytosol contains NADP(+)-reducing activity, and the activation of both processes in the presence of NADPH and NADP+ was prevented by an exogenous NADPH oxidation system. NADPH behaved as an allosteric activator of T3 binding. The NADPH oxidation system promoted the release of bound T3 in the absence of any change in the total concentration of the hormone. The 35K-TBP photolabelling and [125I]T3 binding were similarly inhibited by non-radioactive T3 (half-maximum effect at 0.5-1.0 nM T3). The concentrations of iodothyronine analogues that inhibited both processes were correlated (3,3',5-tri-iodo-D-thyronine > or = T3 > L-thyroxine > tri-iodothyroacetic acid > 3,3'5'-tri-iodo-L-thyronine). Molecular sieving and density-gradient centrifugation of cytosol identified a 65 kDa T3-binding entity, which included the 35K-TBP. These results indicate that 35K-TBP is the cytosolic entity involved in the pyridine nucleotide-dependent T3 binding, and suggest that the sequestration and release of intracellular thyroid hormones are regulated by the redox state of astroglial cell compartment(s).

Competitive inhibition of thyroid hormone uptake into cultured rat brain astrocytes by bilirubin and bilirubin conjugates.

Thyroid hormone (TH) metabolism is altered in cases of unconjugated hyperbilirubinemia. These effects might involve inhibition of TH uptake by their target cells. Astrocytes, which are in close contact with the membranes of brain capillaries, might be the first brain cells to come into contact with bilirubin. Cultured rat brain astrocytes were used as a model to study the effects of bilirubin and bilirubin analogues on TH uptake. The initial uptake of [125I]T3 and [125I]T4 was inhibited by unconjugated bilirubin, biliverdin, ditaurobilirubin and bilirubin glucuronides. The inhibition of T3 uptake by the bilirubin analogues was competitive. The Ki values were: unconjugated bilirubin (31 microM), biliverdin (48 microM), ditaurobilirubin (2.5 microM) and bilirubin glucuronides (1.2 microM). This last value is similar to the Km of T3 transport (0.4 microM), indicating that bilirubin glucuronides have a high affinity for the TH transport system. By contrast, the uptakes of [3H]tryptophan and ]3H]glutamine were not inhibited. These results suggest that the astrocyte plasma membrane bears specific bilirubin-interaction sites that are closely related to the TH transport system. However, uptake of [14C]bilirubin by cultured astrocytes was a non-saturable process. Binding of bilirubin to the astrocyte plasma membrane may inhibit the TH uptake and impair their metabolism and their action on the intracellular targets.

Triiodothyronine is a high-affinity inhibitor of amino acid transport system L1 in cultured astrocytes.

The relationship between the transport of thyroid hormones and that of amino acids was examined by measuring the uptake of amino acids that are characteristic substrates of systems L, A, and N, and the effect of 3,3',5-triiodo-L-thyronine (T3) on this uptake, in cultured astrocytes. Tryptophan and leucine uptakes were rapid, Na(+)-independent, and efficiently inhibited by T3 (half-inhibition at approximately 2 microM). Two Na(+)-independent L-like systems (L1 and L2), common to leucine and aromatic amino acids, were characterized kinetically. System L2 had a low affinity for leucine and tryptophan (Km = 0.3-0.9 mM). The high-affinity system L1 (Km approximately 10 microM for both amino acids) was competitively inhibited by T3 with a Ki of 2-3 microM (close to the T3 transport Km). Several T3 analogues inhibited system L1 and the T3 transport system similarly. Glutamine uptake and alpha-(methylamino)isobutyric acid uptake were, respectively, two and 200 times lower than tryptophan and leucine uptakes. T3 had little effect on the uptakes of glutamine and alpha -(methylamino)isobutyric acid. The results indicate that the T3 transport system and system L1 are related.

Triiodothyronine binding sites in the rat erythrocyte membrane: involvement in triiodothyronine transport and relation to the tryptophan transport System T.

The binding of L-triiodothyronine (T3) to rat erythrocyte membranes (ghosts and peripheral protein-depleted vesicles) was studied under equilibrium conditions. Ghosts contained high-affinity T3 binding sites whose dissociation constant (21 nM) was similar to the equilibrium-exchange Michaelis constant of T3 transport measured in ghosts. Each ghost contained about 8.10(3) high-affinity binding sites. The high-affinity T3 binding was stereospecific and was inhibited by L-tryptophan (Trp) but not by L-leucine. The iodothyronine and amino acid specificity of binding is therefore similar to that of System T, the erythrocyte T3/Trp transporter. These Trp-inhibitable high-affinity T3-binding sites were also present in peripheral protein-depleted membrane vesicles, indicating that they are integral part of the membrane. Ghosts prepared from human erythrocytes, which have very low System T transport activities, contained no detectable Trp-inhibitable high-affinity T3-binding sites. In rat erythrocyte ghosts, N-ethylmaleimide inactivated both the binding and the transport of T3. This inactivation was blocked by T3 and Trp with similar efficiencies. Phenylglyoxal, an arginine residue modifier, also inhibited both high-affinity T3 binding and System T transport activity. It is concluded that the Trp-inhibitable high-affinity T3-binding sites in the rat erythrocyte membrane are likely to be associated with System T.

Thyroid hormone concentrative uptake in rat erythrocytes. Involvement of the tryptophan transport system T in countertransport of tri-iodothyronine and aromatic amino acids.

The kinetic properties of transport system T, which is specific for uptake of aromatic amino acids, were studied in rat erythrocytes in the presence of leucine in order to block the neutral amino acid transport system L. Since the triiodothyronine (T3) transport system and system T are closely related, the trans effect of T3 and tryptophan on [3H]tryptophan transport and the trans effects of aromatic amino acids on [125I]T3 transport were studied. Equilibrium-exchange, zero-trans and infinite-trans studies of [3H]tryptophan transport indicated that system T in rat erythrocytes is a simple carrier with exchanging properties resulting in trans-acceleration of influx and trans-inhibition of efflux when tryptophan was present at the trans side of the membrane. In erythrocytes preloaded with unlabelled tryptophan, countertransport resulted in a 7-fold accumulation of labelled substrate inside the cells. T3 on the trans side of the membrane inhibited both influx and efflux of tryptophan, with Ki values similar to the Km values of the T3 transport system. Extracellular tryptophan trans-inhibited [125I]T3 efflux in a manner similar to [3H]tryptophan efflux. Preloading erythrocytes with tryptophan resulted in trans-acceleration of T3 uptake and a transient 5-fold accumulation of free T3 into erythrocytes. Phenylalanine and tyrosine (but not the D-isomer of tryptophan or non-aromatic amino acids) also produced trans-acceleration for T3 uptake and T3 countertransport. These results are compatible with a kinetic model assuming a common simple carrier of T3 and tryptophan transport and point to a countertransport pathway driving the uphill uptake of T3 by hetero-exchange with intracellular aromatic amino acids.

Transport of thyroid hormones by human erythrocytes: kinetic characterization in adults and newborns.

The uptake of [125I]T3 and [125I]T4 by human erythrocytes was studied. The erythrocytes were obtained from adult subjects (28-41 yr old) and suspended in a protein-free medium. The half-times of equilibration for both T3 and T4 were 6 min. At equilibrium, T3 was concentrated 55-fold inside the cells, while T4 was concentrated 40 times, but these accumulations were not dependent on either cellular ATP or the transmembrane Na+ gradient. The amounts of cell-associated thyroid hormones were 20 times (T3) and 17 times (T4) higher than the amounts of free extracellular hormones at 5 X 10(9) erythrocytes/mL (the blood concentration). Oligomycin and phloretin inhibited T3-saturable transport (but not T4 transport) independently of cellular energy. We suggest that thyroid hormones are concentrated by intracellular trapping. The rates of T3 and T4 efflux from preloaded erythrocytes were similar to the influx rates. The initial velocities of T3 (but not T4) uptake and efflux were 70% saturable. The uptake was specific because the unlabeled analogs T4, triiodothyroacetic acid, rT3, D-T3, and D,L-thyronine inhibited [125I]T3 uptake 60, 125, 160, 190, and 1600 times less, respectively, than did unlabeled T3. The kinetic parameters of T3-saturable uptake, Km, and maximum velocity were determined for three groups of subjects: newborns, 28 to 41-yr-old adults, and 76 to 90-yr-old adults. The Km (67 nmol/L in 28 to 41-yr-old adults) was not age dependent, BUT the maximum velocity was significantly higher in newborns than in adults. We conclude that T3 transport across the human erythrocyte membrane is mediated mainly by facilitated diffusion, whereas T4 transport results from free diffusion. Human erythrocytes might act as a circulating pool of thyroid hormones, especially T3 in newborns.

Evidence for a close link between the thyroid hormone transport system and the aromatic amino acid transport system T in erythrocytes.

The transport of [125I]triiodothyronine ([125I]T3) and [3H]tryptophan ([3H]Trp) by washed rat erythrocytes was studied at 25 degrees C in the presence of leucine in order to block the neutral amino acid transport system L. Eadie-Hofstee plots of initial velocity data gave the following values of Km (micromolar) and Vmax (nanomole/min/10(8) cells): 0.122 +/- 0.007 and 0.140 +/- 0.021 for T3, and 558 +/- 28 and 17.4 +/- 2.3 for Trp (n = 5). The Trp transport system in rat erythrocytes is similar to the human erythrocyte aromatic amino acid-specific system T described by Rosenberg et al. (Rosenberg, R., Young, J. D., and Ellory, J. C. (1980) Biochim. Biophys. Acta 598, 375-384). Unlabeled aromatic amino acids (e.g. Trp, phenylalanine, tyrosine) competitively inhibited [125I]T3 uptake and unlabeled iodothyronine analogues (e.g. T3, D-T3, thyroxine, thyronine) competitively inhibited [3H]Trp uptake. The inhibition constants of these competitors measured with each labeled substrate were highly correlated. N-Ethylmaleimide irreversibly inhibited T3 and Trp transport and each substrate protected the transport system of the other from inactivation by N-ethylmaleimide. The Vmax of T3 and Trp transport by human erythrocytes were 500 and 120 times lower, respectively, than those of rat erythrocytes (0.30 and 126 pmol/min/10(8) cells, respectively). The T3 and Trp transport activities of sheep erythrocytes were undetectable. These results indicate that T3 and Trp either share a common multi-specific transport system or are transported by closely linked systems which interact in the erythrocyte membrane.

Erythrocyte-associated triiodothyronine in the rat: a source of hormone for target cells.

The accumulation of T3 by rat erythrocytes and its transfer to cultured rat hepatocytes were investigated. The amount of erythrocyte-associated T3 in whole rat blood was determined at 37 degrees C. The ratio of erythrocyte-associated T3 to plasma total T3 was 0.235 over a wide range of T3 concentrations, i.e. there is 25 times as much T3 in the erythrocyte compartment as free in the plasma. Influx and efflux of T3, which were shown previously to be carrier-mediated, proceeded rapidly (t 1/2 approximately 12-15 sec at 25 degrees C). These results suggest that erythrocytes are involved in the supply of T3 to target cells. This was checked by studying [125I]T3 uptake by cultured rat hepatocytes incubated either with erythrocyte suspensions pre-equilibrated with labelled T3 or with extracellular medium from the same erythrocyte suspensions. In protein-free medium, the initial velocity of T3 uptake was 1.5-fold faster in the presence of the erythrocyte suspension. Uptake was saturable, the apparent Km of T3 uptake (nmol/l) was 163 +/- 13 in the absence of erythrocytes and 102 +/- 6 in their presence. Vmax (fmol.min-1.well-1) was similar in both cases (477 +/- 26 and 511 +/- 20, respectively). In the presence of diluted plasma (1:16 dilution) and in the presence of the erythrocyte suspension, a 2-fold increase of initial velocity was obtained. Plasma by itself increased (4-5 times) the initial velocity of uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

The triiodothyronine carrier of rat erythrocytes: asymmetry and mechanisms of trans-inhibition.

The kinetic properties of the carrier-mediated transport of 3,5,3'-triiodo-L-thyronine (T3) in washed rat erythrocytes were investigated (1) by studying the effects of trans unlabelled T3 on influx and efflux of labelled substrate and (2) by testing some predictions of the theory of Lieb and Stein [1974) Biochim. Biophys. Acta 373, 165-177). The carrier was trans-inhibited by T3 on both sides of the membrane. Under zero-trans conditions, the carrier displayed asymmetrical properties, the Michaelis constant and the maximal velocity being more than 6-times higher for influx than for efflux. Under equilibrium-exchange conditions, the Michaelis constant was lower than the zero-trans values, as expected when trans-inhibition occurs. This kinetic behaviour is consistent with a carrier which is accessible to T3 simultaneously from both sides of the erythrocyte membrane.

Carrier-mediated transport of thyroid hormones into rat glial cells in primary culture.

The uptake of 3,3',5-[3'-125I]triiodo-L-thyronine ([125I]L-T3) and of L-[3',5'-125I]thyroxine ([125I]L-T4) by cultured rat glial cells was studied under initial velocity (Vi) conditions. Uptake of both hormones was carrier mediated and obeyed simple Michaelis-Menten kinetics. The following respective values of Km (microM) and Vmax (fmol/min/microgram of DNA) were obtained at 25 degrees C: 0.52 +/- 0.09 and 727 +/- 55 for L-T3 and 1.02 +/- 0.21 and 690 +/- 85 for L-T4. Ki values (microM) for the inhibition of [125I]L-T3 uptake by unlabeled analogues were as follows: L-T4, 0.88; 3,3',5'-triiodo-L-thyronine, 1.4; 3,3'-diiodo-L-thyronine, 2.9; 3,3',5-triiodo-D-thyronine, 4.8; and triiodothyroacetic acid, 5.3. These values indicate that the uptake system is stereospecific. Unlabeled L-T3 was a better competitor than unlabeled L-T4 for the uptake of [125I]L-T4, an observation suggesting that both hormones were taken up by a common carrier system. L-T3, and L-T4 uptake was pH dependent, a finding suggesting that the phenolic unionized form of the hormones was preferentially taken up. L-T3 uptake was studied in the presence of various inhibitors; the results suggest that uptake was independent of the transmembrane Na+ gradient and of the cellular energy. Compounds that inhibited cellular uptake but were without effect on L-T3 binding to isolated nuclei also inhibited L-T3 nuclear binding in intact cells, an observation suggesting that uptake could be rate limiting for the access of L-T3 to nuclear receptors when transport is severely inhibited.

Characterization of triiodothyronine transport and accumulation in rat erythrocytes.

The transport of L-T3 was studied in washed rat erythrocytes. L-T3 uptake was temperature sensitive: the initial velocity of uptake at low substrate concentration was 40 times higher at 37 C than at 0C whereas, at equilibrium, the ratio of cell-associated to extracellular L-T3 was about 7 times lower at 37 C than at 0 C. When [125I]L-T3-loaded erythrocytes were diluted into a serum albumin-containing medium, the efflux of L-T3 proceeded at a rate similar to that of influx. A large excess of unlabeled L-T3 in the medium blocked influx and efflux of labeled L-T3, indicating a saturable carrier-mediated transport process across the plasma membrane. the transport obeyed simple Michaelis-Menten kinetics with an apparent Km of 53 nM and a Vmax of 4.3 pmol/min.10(8) cells at 0 C. The Km increased only slightly with temperature whereas the Vmax was 100 times higher at 37 than at 0 C. The Arrhenius activation energy of uptake was 21 Cal/mol. The nonsaturable adsorption of L-T3 to the cells did not exceed 1% of the equilibrium levels at 0 C and 10% at 37 C. Uptake of L-T3 was very specific: unlabeled L-T4, D-T3, triiodothyroacetic acid, rT3, and DL-thyronine inhibited uptake with inhibition constant (Ki) values which were 35, 60, 65, 110, and 250 times, respectively, greater than the Km of L-T3. [125I]L-T4 uptake was negligible. L-T3 uptake and L-T4 inhibition of L-T3 uptake were pH dependent. It is suggested that only the unionized 4'-OH forms of the hormones were recognized by the transport system. At equilibrium, L-T3 was accumulated within the cell (apparent intracellular concentration approximately 50 times higher than that in the medium at 37 C). However, uptake was not dependent on the transmembrane Na+ gradient, suggesting facilitated rather than active transport. Analysis of L-T3 binding to erythrocyte cytosolic proteins suggested that they were implicated in the intracellular trapping of L-T3. At a concentration of 5 x 10(9) erythrocytes/ml (approximately the blood concentration), the amount of L-T3 accumulated in the cells was 13.5 times higher than the extracellular amount. We conclude that L-T3 is solely transported by a saturable, stereospecific, and Na+-independent carrier system. The intracellular accumulation and the rapid transmembrane movements of L-T3 suggest that erythrocytes might play a role in the interorgan transport of L-T3.

Induction of type II 5'-deiodinase activity by cyclic adenosine 3', 5'-monophosphate in cultured rat astroglial cells.

Cultured astroglial cells were found to contain a type II 5'-deiodinase (5'D) activity which was increased by 10(-3) M (Bu)2cAMP but not by 2 X 10(-3) M n-butyrate. 8-Bromo-cAMP (8-Br-cAMP) (10(-3) M) also increased this enzyme activity. Cycloheximide (2 micrograms/ml) inhibited the 8-Br-cAMP effect on 5'D activity. Forskolin (10(-5) M), cholera toxin (5 micrograms/ml), 10(-5) M isoproterenol, and 3 X 10(-6) M norephinephrine also increased the 5'D activity of astroglial cells. After a 4-h incubation these agents or cAMP analogs had maximal effect, and enzyme activities were 6- to 14-fold above control value. The stimulatory effects of isoproterenol and norepinephrine were almost completely reversed after 8 h incubation. The induction of 5'D activity by isoproterenol or norepinephrine was inhibited by the beta-adrenergic antagonist alprenolol (5 X 10(-6) M). The effect of norepinephrine was not significantly affected by the alpha 1-adrenergic antagonist, prazosin (10(-5) M). Thus, 5'D activity is controlled by agents increasing cAMP in astroglial cells, and in particular by the neurotransmitter, norephinephrine, via a beta-adrenergic mechanism.

Thyroid hormone metabolism in neuron-enriched primary cultures of fetal rat brain cells.

The metabolism of thyroxine (T4) by cultures of embryonic-rat brain cells grown in a chemically defined medium was studied. Cells in these cultures were predominantly neurons, characterized by the developmental increase of the binding of [3H]flunitrazepam to the high-affinity (0.67 nM) benzodiazepine neuronal receptors. The cultures also contained astrocytes, characterized by immunological studies using an anti-glial fibrillary acidic protein (GFAp) and by the increase in glutamine synthetase (GS). Incubation of the cells, in situ, with 125I-labelled 3,5,3'-triiodothyronine (T3) showed the presence of a single class of high-affinity nuclear receptors for T3 with a maximal binding capacity of 270-470 fmol T3/mg DNA and a Kd of 63 +/- 13 pM. Cells incubated in situ with 50 pM [125I]T4 actively metabolized the hormone. The major metabolite, 3,3',5'-triiodothyronine (rT3) (159 +/- 43 fmol/4 h/mg DNA), was almost completely released into the medium. T3 was a minor metabolite (77 +/- 3 fmol/4 h/mg DNA), 75% of which accumulated in the cells. Of this T3, 35% was bound to the nuclear receptors after 4 h of incubation. In vitro assays showed that the 5'-deiodinase activity increased during culture and the 5-deiodinase decreased slightly. Cytosine-arabinoside (ARAc) treatment of the cultures reduced the DNA content per culture dish, corresponding to a fall in the number of GFAp-positive cells (astrocytes) and to a decrease in GS. A small increase in the number of benzodiazepine sites was observed. ARAc treatment markedly reduced the T3 production (14.5 +/- 0.7 fmol/4 h/mg DNA) and did not change the rT3 production. We suggest that T4 is metabolized to T3 in astrocytes, taken up by neurons and binds to their nuclear receptors.

Characterization of the thyroid hormone transport system of isolated hepatocytes.

Transport of 3,5-[3'-125I]triiodo-L-thyronine ([125I]T3) was studied in isolated rat liver hepatocytes. T3 transport was temperature-sensitive, the initial velocity of uptake, at low substrate concentration, was 60 times higher at 25 degrees C than at 0 degrees C. The activation energy of cellular uptake (26 kcal/mol) was different from that of binding to cytosolic proteins (6 kcal/mol), indicating that the latter was not the rate-limiting step. Uptake obeyed simple Michaelis-Menten kinetics, with an apparent Km of 0.34 microM and a Vmax of 0.15 fmol/min/cell at 25 degrees C. No simple diffusion occurred. Unlabeled T3, L-thyroxine (T4), 3,5,3'-triiodo-D-thyronine, and triiodothyroacetic acid inhibited T3 uptake with Kl values of 0.32, 1.4, 4.1, and 5.4 microM, respectively, indicating specificity of uptake which was different from specificity of intracellular binding sites. [125I]T4 was also taken up by a saturable process (Km = 0.65 microM and Vmax = 0.16 fmol/min/cell at 25 degrees C). T3 was a better competitor than T4 for the uptake of [125I]T4, indicating that both hormones were taken up by the same carrier system. Metabolic inhibitors had either no effect on T3 uptake or inhibitory effects unrelated to cellular ATP depletion. Uptake was not affected by modification of the membrane potential or the sodium ion gradient. T3 and T4 uptake was pH-dependent. It is suggested that the un-ionized 4'-OH form of the hormones was preferentially taken up. Inhibition of uptake by various compounds was compared to inhibition of thyroid hormone binding to transthyretin, nuclear receptor, and cytosolic-binding proteins. We conclude that, in freshly isolated hepatocytes, thyroid hormones are taken up by a saturable, stereospecific, Na+-independent carrier system.