Kinetics of T4 and T3 binding to plasma sites in salmonid teleost fish

The relative affinities and maximum capacities of the classes of L-thyroxine (T4)- and 3,5,3'-triiodo-L-thyronine (T3)-binding sites in plasma of three species of salmonid teleost fish were determined by saturation analysis on miniature Sephadex G-25 columns at 20-21 degrees and kinetic data were analyzed by the multiligand, multisite LIGAND computer program. In plasma of rainbow trout (Salmo gairdneri) homologous ligand displacement indicated that both thyroid hormones (TH) bound to a minimum of two classes of saturable sites and at least one nonsaturable site. For T4 (n = 13) the relative site affinities were 0.61 +/- 0.08 (SEM) X 10(7) M-1 and 0.86 +/- 0.11 X 10(5) M-1 and the site capacities were 8.3 +/- 1.16 X 10(-7) M and 5.15 +/- 1.34 X 10(-5) M, respectively; for T3 (n = 14) the relative site affinities were 1.8 +/- 0.16 X 10(7) M-1 and 1.7 +/- 0.17 X 10(5) M-1 and the site capacities were 7.8 +/- 1.3 X 10(-7) M, respectively. The greater affinities of T3 than T4 for plasma binding sites would explain the lower proportion of free T3 than free T4 in trout plasma. Two-site models with comparable values for TH-binding parameters were determined for brook trout (Salvelinus fontinalis) and arctic charr (Salvelinus alpinus). The TH-binding parameters were uninfluenced by severe hemoglobin contamination of plasma, bleeding of fish 24 hr previously, or 2 weeks starvation. Heterologous ligand displacement (T4 displaced by T3 or T3 displaced by T4) on rainbow trout plasma suggested two low-affinity, high-capacity sites, one binding predominantly T4 and one binding predominantly T3: a high-affinity, low-capacity site binding T3 exclusively: and a high-affinity, low-capacity site binding T4 predominantly but also binding T3 with low affinity.     

The contribution of local tissue thyroxine monodeiodination to the nuclear 3,5,3'-triiodothyronine in pituitary, liver, and kidney of euthyroid rats.

The contributions of local T4 monodeiodination and plasma T3 to the nuclear T3 of anterior pituitary, liver, and kidney were measured in euthyroid rats. After injection of [125I]T4, there was a gradual increase in the quantity of plasma [125I]T3 in excess of injected contaminant, which peaked at approximately 12 h after injection and remained a constant fraction of plasma [125I]T4 (2.8 X 10(-3) after that time. In the nuclei of anterior pituitary tissue, there was also a slow increase in locally produced [125I]T3 (in excess of that which could be accounted for by plasma [125I]T3) which appeared to peak at about 16 h after [125I]T4 administration. The ratio of nuclear [125I]T3 generated intracellularly to plasma [125I]T4 was constant at 18 and 24 h after T4 injection and was 13 +/- 2 X 10(-3) in nuclei of pituitary, 2.0 +/- 0.4 x 10(-3) in liver, and 0.47 +/- 0.1 x 10(-3) in kidney (all values are mean +/- SD). This locally generated T3 resulted in a markedly higher nuclear to plasma (N:P) ratio for [125I]T3 than for injected [131I]T3 in the same animals. The N:P ratio for [125I]T3 at equilibrium after injected T4 was 2.4 +/- 0.6, 0.47 +/- 0.09, and 0.10 +/- 0.03 (nanograms of T3 (mg DNA)-1/ng T3 ml-1) in pituitary, liver, and kidney. Comparable values for [131I]T3 N:P ratios were 0.47 +/- 0.14 (pituitary), 0.18 +/- 0.01 (liver), and 0.036 +/- 0.008 (kidney). Using RIA values for plasma T4 and T3 concentrations in these rats and maximal nuclear T3-binding capacities estimated in parallel experiments, the gravimetric quantities of nuclear T3 derived from plasma T3 and from local T4 to T3 monodeiodination were estimated and expressed as the percentage of saturation of T3 receptors. Seventy-eight percent of nuclear T3 receptor sites in anterior pituitary were occupied with one-half of the nuclear T3 derived directly from plasma T3 and the other half from intrapituitary T4 monodeiodination. Local T4 monodeiodination provided only 28% and 14%, respectively, of the nuclear T3 in liver and kidney, and the nuclear receptors of these tissues were about 50% saturated. Since our previous studies have shown that the occupancy of the pituitary nuclear T3 receptors may regulate TSH release, these data provide a mechanism by which TSH secretion could be altered by changes in either plasma T3 or T4, whereas nuclear T3 in liver and kidney is predominantly a function of the plasma T3 concentration.     

Counterregulation of nuclear 3,5,3'-triiodo-L-thyronine (T3) binding by oxidized and reduced-nicotinamide adenine dinucleotide phosphates in the presence of cytosolic T3-binding protein in vitro

The role of cytosolic T3-binding protein (CTBP) in the regulation of nuclear T3 binding was studied in vitro. Nuclear [125I]T3 binding was observed in the presence of 1.0 mM dithiothreitol (DTT). When the nuclei prepared from rat kidney were incubated with inactive form of CTBP which was also prepared from rat kidney, [125I]T3 binding to nuclei was not affected. When the nuclei were incubated with inactive form of CTBP in the presence of NADP, [125I]T3 binding to nuclei was increased, whereas binding was diminished when nuclei were incubated with CTBP in the presence of NADPH. The inactive form of CTBP was activated by NADPH. NADP also activated CTBP in the presence of DTT. Both active forms of CTBP were again inactivated by extraction with charcoal, and these inactive forms were reactivated by NADPH or by NADP and DTT, but not by NADP alone. Although the nuclei treated with 0.3 M NaCl lost the binding activity for [125I]T3 in the absence of NADP, the nuclei retained the binding activity for [125I]T3 in the presence of NADP and the inactive form of CTBP. Treatment of the nuclei with 0.5 M NaCl lost the binding activity for [125I]T3 not only in the absence but also in the presence of NADP and CTBP. These results suggested that NADP and NADPH play roles as counterregulatory factors for nuclear T3 binding in the presence of CTBP. Further, it was speculated that binding sites for the T3-CTBP complex, which is generated in the presence of NADP and DTT, are present in nuclei, and that binding sites for the complex are different from nuclear T3 receptors.     

Immersion of rainbow trout in 3,5,3'-triiodo-L-thyronine (T3): effects on plasma T3 levels and hepatic nuclear T3 binding

An immersion protocol was established for chronically administering thyroid hormone to fish. Rainbow trout were immersed in solutions of 3,5,3'-triiodo-L-thyronine (T3) at 11 degrees C for various periods. Most plasma T3 adjustment occurred by 24 hr and within 5 days consistent plasma T3 levels were observed. Availability of unoccupied hepatic nuclear T3-binding sites was assessed in two experiments by uptake of nuclear radioactivity in T3-immersed trout injected 12 hr previously with [125I]T3. At low ambient T3 levels over 92% of nuclear radioactivity was identified chromatographically and immunologically as T3, but at high ambient levels (25 micrograms/100 ml) biliary [125I]T3 metabolites may contaminate the nuclear fraction. Displacement of [125I]T3 from nuclei was first detected at 0.25 micrograms T3/100 ml water, corresponding to a plasma T3 level of 0.55 ng/ml; maximum displacement, depending on experiment, occurred at 1.25 and 3.8 micrograms T3/100 ml, corresponding to plasma T3 levels of 14 and 9 ng/ml. In conclusion, chronic physiologic T3 treatment of these trout can be achieved by immersion in T3 solutions of 0.1-5 micrograms T3/100 ml water.     

Binding of thyroid hormones in vivo by hepatic nuclei of Rana catesbeiana tadpoles

Thyroid hormone is essential for amphibian metamorphosis, and tadpoles develop responsiveness to exogenous T4 and T3 during the premetamorphic stage of development. The present studies were performed to investigate the receptors concerned with the initiation of this response. Premetamorphic tadpoles (stages VII-XV of Taylor and Kollros) were injected ip with [125I]T3 or [125I]T4 (0.001-10 nmol/tadpole). Twenty-four hours later, liver and serum were obtained, and organic 125I in liver nuclei and serum (shown to be unchanged hormone) was measured. Saturable binding sites for both T3 and T4 were present in the liver nuclei. Analysis of binding data indicated for T3 a mean value for Kd of 1.6 x 10(-12) M (moles of free T3 per liter plasma) and a mean value for maximum binding capacity of 0.1 ng/mg DNA. For T4, the mean Kd was 3.9 x 10(-15 M, and the mean maximum binding capacity was 0.5 ng/mg DNA. It was estimated that a significant fraction of these sites was not normally occupied by endogenous hormone. Properties of the T3-binding sites were similar in tadpoles at stages X and XV. Stable T4 and the acetic and propionic acid analogs of T3 competed with [125I]T3 for the sites almost as readily as did stable T3. The acetic acid analog of T4, D-T4, 3,5-diiodothyronine, and rT3, less active analogs, were relatively poor competitors. Binding of T3 to saturable but not to non-saturable nuclear binding sites was reduced in tadpoles kept at 4 C. On the basis of these findings, it is suggested that these nuclear binding sites are thyroid hormone receptors.     

Characterization of the 3,3',5-triiodo-L-thyronine-binding site on plasma membranes from human placenta

The binding of [125I]T3 to sites on human placenta plasma membranes was characterized, and the binding site was solubilized after affinity labeling with N-bromoacetyl-[125I]T3 (BrAc[125I]T3). Two classes of T3-binding sites were detected. One class has a high affinity (Kd = 2.0nM) and a low capacity (approximately 320 fmol/mg protein); the other has a low affinity (Kd = 18.5 microM) and a high capacity (approximately 2.2 pmol/mg protein). The binding sites were found to be specific for T3 in that other thyroid hormone analogs (D-T3, rT3, D-T4, and L-T4) were less effective or ineffective in displacing the bound [125I]T3. The affinity labeling ligand BrAc[125I]T3 was found to specifically label a protein with an apparent mol wt of 65,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The BrAc[125I]T3-labeled protein was solubilized with 2 mM 3-[( 3-cholamidopropyl)dimethylammonio]1-propane sulfonate. The apparent mol wt of the labeled protein was between 140,000 and 150,000 by Sephadex-G-200 gel filtration. These data demonstrate that a high affinity binding site specific for T3 is present on plasma membranes from human placenta and that the binding site is a protein, most likely a dimer, with a native mol wt between 140,000 and 150,000.     

High-affinity, limited-capacity triiodothyronine-binding sites in nuclei from various tissues of the rainbow trout (Salmo gairdneri)

High-affinity, limited-capacity 3,5,3'-triiodo-L-thyronine (T3)-binding sites were detected by in vitro saturation analysis in cell nuclei from liver, gill, kidney, brain, and erythrocytes (RBC), but not spleen. The sites were extracted from the purified nuclei using 0.4 M NaCl and incubated with [125]T3 in the presence of 0.2 M NaCl. In all tissues T3 binding approached equilibrium after 18 to 48 hr of incubation at 4 degrees and was reversible upon addition of excess unlabeled T3. The T3 association and dissociation rate constants (k+ and k-) were measured from the initial (4 hr) [125I]T3 association and dissociation rates for liver (k+ = 8.9 x 10(9) liters.mol-1.hr-1; k- = 0.067 hr-1) and for RBC (k+ = 1.9 x 10(8) liters.mol-1.hr-1; k- = 0.11 hr-1). The association constants (Ka) determined by saturation analysis were similar in all tissues investigated (average Ka = 2.8 x 10(9) liters.mol-1), except in RBC (Ka = 1.2 x 10(10) liters.mol-1). The Ka values calculated from the k+/k- ratio (1.4 x 10(11) liters.mol-1 and 1.8 x 10(9) liters.mol-1 for liver and RBC, respectively) differed substantially from those determined by saturation analysis. This discrepancy is likely due to nonsaturable T3 binding by coextracted nuclear proteins in the assay medium, altering the estimated k+. The maximal binding capacity of the nuclear sites varied widely between tissues (liver, 250; gill, 130; kidney, 63; brain, 30; and RBC, 10 fmol.(mg DNA)-1; spleen, below detection).(ABSTRACT TRUNCATED AT 250 WORDS)     

HPLC analysis of in vitro hepatic deiodination products of thyroid hormones in the rainbow trout, Oncorhynchus mykiss.

Reverse-phase HPLC employing five different solvent systems was used to determine the 125I-labeled products formed in rainbow trout by in vitro incubation at 12 degrees of the hepatic microsome fraction with L-thyroxine (T4) labeled with 125I in the outer phenyl ring. Trout were starved for 2 weeks or fed a 2% ration. The only labeled products identified during incubation for 7.5-70 min over a T4 substrate range of 0.03-0.5 nM were 125I- and 3,5,[125I]3'-triiodo-L-thyronine (T3). These products in combination with the parent [125I]T4 accounted for over 96% of the total chromatographic radioactivity. Neither 3,[125I]3'-diiodo-L-thyronine nor 3,[125I]3',5'-T3 (reverse T3) was detected, suggesting negligible inner-ring deiodination of T4 or T3. Essentially equal production of 125I- and [125I]T3 validated the use of 125I- production as a measure of [125I]T3 generation in assays for hepatic 5'-monodeiodinase activity. However, in some experiments the 125I- level slightly exceeded the [125I]T3 level, indicating that outer-ring deiodination of T3 may occur to a limited degree. In conclusion, the present data for liver support earlier observations from in vivo studies in showing that for trout at 12 degrees deiodination pathways are geared primarily toward T4 outer-ring monodeiodination to produce T3 with undetectable inner-ring deiodination of T3 or T4 and limited outer-ring deiodination of T3.     

Differences in nuclear thyroid hormone receptors among species

Hepatic nuclear thyroid hormone receptors from rat, dog, chicken, and rainbow trout were compared. Receptor affinities for 3,5,3'-triiodo-L-thyronine (T3) were similar in preparations from rat, dog, and chicken, using isolated nuclei and nuclear extracts. Rainbow trout nuclear receptor showed a lower affinity for T3. Almost half of the receptors were released into the medium with rat and chicken nuclei, and 79.7 +/- 1.1% of the receptors were released with rainbow trout nuclei, when isolated nuclei were incubated with T3 at 22 degrees for 2 hr. The affinity constant of rat liver receptor for calf thymus DNA-cellulose at 0.17 M KCl, pH 7.4, was 3.98 +/- 1.47 x 10(5) M-1, when determined using DNA-cellulose columns. The number of salt bridges involved in DNA binding of the rat receptor was 5.73 +/- 0.38. When receptor-DNA interactions were compared among species, significant differences were found, but the receptors from dog and rainbow trout liver were similar. Sephacryl S-200 column chromatography showed that chicken receptor had a Stokes radius significantly smaller than that of rat receptor. Partial proteolysis of T3-receptor complex using trypsin alpha-chymotrypsin, elastase, and papain produced distinct T3-binding fragments in different species. Our data provide evidence that nuclear thyroid hormone receptors from different species have significant structural dissimilarities.     

Cytosolic 3,5,3'-triiodo-L-thyronine (T3)-binding protein (CTBP) regulation of nuclear T3 binding: evidence for the presence of T3-CTBP complex-binding sites in nuclei

Effect of cytosolic 3,5,3'-triiodo-L-thyronine (T3)-binding protein (CTBP) on [125I]T3 binding to nuclei was investigated in vitro. CTBP and nuclei were prepared from rat kidney. CTBP was inactivated by incubating with charcoal and activated by incubating with NADPH or with NADP and dithiothreitol. Two complexes of CTBP and T3 [CTBP-T3(NADPH) and CTBP-T3(NADP)] were separately prepared, and the functions of these complexes were estimated. [125I]T3 binding to nuclei was not influenced by the inactive form of CTBP. NADP or NADPH alone did not modify [125I]T3 binding to the nuclei. However, the binding was markedly inhibited by NADPH in the presence of the inactive form of CTBP, but it was not inhibited by NADP in the presence of the inactive form of CTBP. The ability of nuclei to bind [125I]T3 was markedly diminished by pretreatment of the nuclei with 10(-8) M unlabeled T3. The diminished activity was not modified by adding NADPH or NADP. However, [125I]T3 bound to these nuclei in the presence of NADP and the inactive form of CTBP. Binding of [125I]T3 to these nuclei was not observed in the presence of NADPH and the inactive form of CTBP. When the nuclei that had previously been saturated with 10(-6) M unlabeled T3 were incubated with [125I]T3-CTBP(NADP) complex, radioactivity bound to the nuclei. The binding of radioactivity, however, was not observed when these nuclei were incubated with [125I]T3. The [125I]T3-CTBP(NADPH) complex did not bind to these nuclei. When the nuclei that had previously been treated with T3-CTBP(NADP) complex were incubated with [125I]T3, radioactivity bound to the nuclei. The binding of radioactivity, however, was not observed when these nuclei were incubated with [125I] T3-CTBP(NADP) complex. The [125I]T3-CTBP(NADPH) complex did not bind to these nuclei. These results suggested that 1) CTBP activated by NADP plays a role as a carrier protein for T3 from cytoplasm to nucleus; 2) there are binding sites for T3-CTBP(NADP) complex in rat kidney nuclei; and 3) these binding sites are different from nuclear T3 receptors.     

Thyroxine and 3,5,3'-triiodothyronine bind to the same putative receptor in hepatic nuclei of Rana catesbeiana tadpoles

Previous studies have provided conflicting evidence as to whether tadpole liver nuclei contain the same number of high affinity binding sites for T4 as they do for T3. Inability to prepare stable tadpole liver nuclei may have contributed to these inconsistencies. Thus, a study was carried out in which the in vivo binding of T4 and T3 by tadpole liver nuclei was reexamined with a greatly improved method for isolating liver nuclei. It was found that the maximum binding capacities of the nuclei for T3 and T4 were not significantly different [0.161 +/- 0.015 (+/- SEM) vs. 0.185 +/- 0.046 pmol/mg DNA]. However, the affinity of the binding sites for T3 was 2-3 times their affinity for T4 (Kd, 1.65 +/- 0.31 vs. 4.32 +/- 0.92 X 10(-12) M). Furthermore, the nuclear binding of [125I]T4 or [125I]T3 was decreased to comparable levels by administration of saturating amounts of T3 and T4 given either before or with the 125I-labeled hormone, indicating that both hormones were competing for the same set of sites. It is suggested that at least some of the previous conflicting findings related to the number of putative thyroid hormone receptors in tadpole liver nuclei are attributable to inadequate methodology which resulted in an overestimation of the maximum binding capacity for T4.     

Effects of catecholamines on plasma thyroid hormone levels in arctic charr, Salvelinus alpinus

Plasma levels of L-thyroxine (T4) and 3,5,3'-triiodo-L-thyronine (T3) were measured in arctic charr at 2, 6, or 24 hr after single intraperitoneal injection of epinephrine (E) or norepinephrine (NE). At a dose of approximately 1 microgram/g body wt (sufficient to cause a submaximal dermal melanophore pallor response) plasma T4 was usually elevated at 2 hr, consistently depressed at 6 hr, and unaffected at 24 hr. There was no effect of E on plasma [125I]T4 kinetics or [125I]T4 5'-monodeiodination to [125I]T3. Plasma T3 showed no consistent response to E or NE at any sampling time. At an E dose of 4 ng/g body wt (probably sufficient to cause a physiological elevation in plasma E level), neither plasma T4 nor T3 levels were altered at 6 hr. Acute depression in plasma T4 by the high doses of E and NE may reflect a local neurotransmitter role of catecholamines in inhibiting thyroidal T4 release through action at thyroidal, hypophysial, or hypothalamic levels.     

Transthyretin receptors on human astrocytoma cells

Transthyretin (TTR), a transport protein for T4 and retinol-binding protein, is the principal T4-binding protein of cerebrospinal fluid. Its function in regard to the delivery of its ligands and in other respects is unclear. The binding of [125I] TTR to cultured human astrocytoma cells was studied to determine whether these cells carry receptors for TTR. Scatchard analysis was consistent with a single class of binding sites with a Kd of 3 nM. No significant cross-reactivity with transferrin or serum albumin was observed. Internalization of TTR was temperature dependent and proportional to receptor occupancy. Dilutions of cerebrospinal fluid displaced [125I]TTR in proportion to their content of radioimmunoassayable TTR and in parallel with purified TTR. The uptake and internalization of TTR increased in the presence of high T4 or T3 concentrations. These results demonstrate that TTR binds to specific high affinity receptors on human astrocytoma cells. Receptor binding of TTR provides a potential mechanism for the delivery of its ligands within the central nervous system.     

Reverse T3 in rainbow trout, Salmo gairdneri

Plasma levels, peripheral metabolism and extrathyroidal in vivo production of reverse T3 (rT3 = 3,3',5'-triiodo-L-thyronine) were studied in rainbow trout at 12 degrees C. Plasma rT3 levels (less than 40 pg/ml) corresponded to the detection limit of the radioimmunoassay. By in vitro analysis, a high proportion (0.95%) of rT3 added to plasma existed in the free (dialyzable) form. Injected [125I]rT3 was cleared more rapidly from plasma (minimum MCR = 7.7 ml/hr/100 g) than T3. No phenolic (outer ring) rT3 deiodination was observed. rT3 rapidly entered the liver and up to 70% of the injected [125I]rT3 was lost via the biliary route, mainly as unidentified derivatives; about 10% of the biliary-excreted 125I label was identified as rT3 or its glucuronide conjugate. Using Sephadex column chromatography combined with specific antibody separations, it was not possible to demonstrate in vivo [125I]rT3 production from [125I]T4. It is concluded that in laboratory trout, in contrast to the situation in mammals, the T4 to rT3 pathway is not prominent and that iodothyronine deiodination is restricted to T3 formation. These findings may relate to differences in extrathyroidal iodine metabolism between trout and mammals.     

Existence of nuclear thyroid hormone receptors in the porcine thyroid gland and the rat thyroid cell line FRTL-5

We have investigated whether nuclear T3 receptors exist in the thyroid cell. Nuclear proteins extracted from porcine thyroid nuclei with 0.4 mol/l KCl were incubated with [125I]T3. The mixture was then analysed by sucrose density gradient ultracentrifugation which revealed that the T3-binding proteins migrated at the same position of 3.6 S as rat liver nuclear T3 receptors. Fractionation by high performance liquid chromatography using a size exclusion column and an ion exchanger column also demonstrated elution patterns of T3-binding similar to those of the rat liver receptor. Scatchard plots of crude nuclear extracts from porcine thyroid represented a curvilinear pattern. However, when the nuclear proteins partially purified by a DEAE column chromatography were analysed, a single binding component was found; the association constant was 4.1 x 10(10) l/mol and the maximal binding capacity was 602 fmolT3/mg protein. Displacement study with several T3 analogues showed a highly selective affinity for L-T3. Cultured rat thyroid cells of the FRTL-5 line also contained a single class of saturable, high affinity T3-binding site. Subconfluent cells in 100-mm dishes were incubated with increasing amounts of [125I]T3 at 37 degrees C for 3 h and radioactive T3 in isolated nuclei was counted. Scatchard analysis of data showed that the association constant and the maximal binding capacity were 3.44 +/- 0.63 x 10(10) l/mol and 63.7 +/- 17.8 fmolT3/mg protein, respectively. These results strongly suggest that there are nuclear T3 receptors, indistinguishable from the hepatic T3 receptors, in the porcine thyroid and rat FRTL-5 cells.     

Radioiodinated nondegradable gonadotropin-releasing hormone analogs: new probes for the investigation of pituitary gonadotropin-releasing hormone receptors.

Studies of pituitary plasma membrane gonadotropin-releasing hormone (GnRH) receptors using [125I]-iodo-GnRH suffer major disadvantages. Only a small (less than 25%) proportion of specific tracer binding is to high affinity sites, with more than 70% bound to low affinity sites (Ka = 1 x 10(6) M-1). [125I]Iodo-GnRH is also inactivated during incubation with pituitary plasma membrane preparations. Two superactive analongs of GnRH, substituted in positions 6 and 10, were used as the labeled ligand to overcome these problems. Both analogs bound to the same high affinity sites as GnRH on bovine pituitary plasma membranes, though the affinity of the analogs was higher than that of the natural decapeptide (Ka = 2.0 x 10(9), 6.0 x 10(9), and 3.0 x 10(8) M-1 for [D-Ser(TBu)6]des-Gly10-GnRH ethylamide, [D-Ala6]des-Gly10-GnRH ethylamide, and GnRH, respectively. The labeled analogs bound to a single class of high affinity sites with less than 15% of the specific binding being to low affinity sites (Ka approximately equal to 1 x 10(6) M-1). The labeled analogs were not inactivated during incubation with the pituitary membrane preparations. Using the analogs as tracer, a single class of high affinity sites (K1 = 4.0 x 10(9) M-1) was also demonstrated on crude 10,800 x g rat pituitary membrane preparations. Use of these analogs as both the labeled and unlabeled ligand offers substantial advantages over GnRH for investigation of GnRH receptors, allowing accurate determination of changes in their numbers and affinities under various physiological conditions.     

Purification and partial characterization of a novel thyroxine-binding protein (27K protein) from human plasma

T4-binding globulin (TBG) prepared from human plasma by the standard three-step procedure (T4-agarose affinity chromatography, anion exchange chromatography, and gel filtration) often shows in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in addition to the expected 54K band, another with a mol wt of 27,000 (27K protein). The two proteins can be separated after the three-step procedure by chromatofocusing (because of different isoelectric points, 4.2-4.8 for TBG and 5.0-5.2 for 27K protein) or by T4-aragose chromatography eluting with a linear gradient of T4 (TBG is eluted between 10(-10) and 10(-9) M T4, 27K protein between 10(-8) and 10(-7) M T4). The 27K protein does not appear to be a fragment of TBG since 1) it does not displace [125I]TBG bound to anti-TBG monoclonal antibodies; and 2) absorption of polyclonal antibody reacting with both TBG and 27K protein with sera from TBG-deficient patients completely prevents [125I]27K protein binding, while only slightly affecting [125I]TBG binding. On the other hand, 27K protein is not simply a contaminant devoid of biological activity, but is a T4-binding protein, as supported by the following findings: 1) it covalently binds [125I]T4 by photoaffinity labeling, and this binding can be almost completely prevented by excess T4; 2) equilibrium dialysis shows two equivalent T4-binding sites per 66K, with an association constant of 0.85 X 10(7) M-1, intermediate between albumin and prealbumin; and 3) tryptophanyl fluorescence analysis shows quenching of 37% of the fluorescence when the protein is titrated with T4. The 27K protein appears as a single 27K band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, pH 8.8, but under nondenaturing nonreducing conditions mostly remains at the origin of the gel; a fraction enters the gel and migrates slightly ahead of albumin. This electrophoretic pattern is distinct from those of albumin, prealbumin, and TBG. In immunoelectrophoresis in agar at pH 8.6, 27K protein moves slightly faster than TBG. The results of equilibrium sedimentation indicate a mol wt of 66,000, suggesting that the 27K protein might exist as a dimer. These data indicate that the 27K protein is a previously unrecognized T4-binding protein with a low affinity for the hormone. Further studies are required to clarify its physiological role in the transport of circulating thyroid hormones.     

The effects of sodium ipodate (ORAgrafin) on thyroid function in rainbow trout, Salmo gairdneri

Immature rainbow trout held at 12 +/- 1 degree were injected intraperitoneally with a fine saline suspension of sodium ipodate (5 mg/100 g body wt) every 3 days. Plasma 3,5,3'-triiodo-L-thyronine (T3) fell to 40% of control levels by Day 1 and remained at about this level for the duration of the study (22 days). Plasma L-thyroxine (T4) level was not altered on Day 1 but was lowered to 50% of control values by Days 7 and 22. Immersion of trout in T4 (2 micrograms/100 ml water) elevated plasma T4 but did not alter the ipodate suppression of plasma T3. Injection of control or ipodate-treated trout with [125I]T4, [125I]T3, or Na131I indicated that in addition to blocking T45'-monodeiodination to T3, ipodate also decreased plasma clearance of T4 and T3 and their removal by the bile. Ipodate did not alter the hepatosomatic index but did depress the hematocrit by 22 days, possibly due to the hypothyroid state. In conclusion, ipodate at a dose of 5 mg/100 g, approximately one-tenth of a lethal dose, is an effective acute and chronic hypothyroid agent to administer to trout.     

Receptors for corticotropin-releasing hormone in human pituitary: binding characteristics and autoradiographic localization to immunocytochemically defined proopiomelanocortin cells.

Using autoradiography combined with immunocytochemistry, we demonstrated that the target cells of CRH in the human pituitary were proopiomelanocortin cells. Scatchard analysis of [125I]Tyr0-oCRH saturation binding revealed the presence of one class of saturable, high affinity sites on pituitary tissue homogenate. The equilibrium dissociation constant (Kd) for [125I]Tyr0-oCRH ranged from 1.1-1.6 nM, and the receptor density was between 200-350 fmol/mg protein. Fixation of cryostat sections with 4% paraformaldehyde before tracer incubation improved both tissue preservation and localization of the CRH receptor at the cellular level. Additional postfixation with 1% glutaraldehyde inhibited tracer diffusion during subsequent immunocytochemistry and autoradiography. [125I]Tyr0-oCRH was found in cytoplasmic inclusions or at the cell periphery of ACTH/beta-endorphin cells in the anterior pituitary. Single cells of the posterior pituitary were also CRH receptor positive. Cells staining for PRL or GH were CRH receptor negative. We conclude that CRH binds only to high affinity receptors on ACTH/beta-endorphin cells in the human pituitary.     

Demonstration of putative thyroid hormone receptor in the brain nuclei of Singi fish, Heteropneustes fossilis (Bloch)

A single injection of [125I]triiodothyronine (T3) with or without stable T3 in Singi fish, Heteropneustes fossilis (Bloch), showed that the fish brain has saturable binding sites and that the specific uptake is 60-70% higher than the nonspecific uptake. The distribution kinetics of [125I]T3 in the serum, whole brain, and brain nuclei after a single injection of the labeled hormone showed that the removal of [125I]T3 from the serum was very rapid with the t1/2 of about 3.3 hr and the incorporation of the hormone into the brain and brain nuclei were very slow and achieve a maximal value after 4-6 hr of postinjection. The binding of [125I]T3 to the isolated brain nuclei of Singi fish was further studied in vitro. Binding was linearly increased with the increasing concentration of the DNA (nuclei). The binding achieved equilibrium between 15 and 20 min at 27 degrees and was stable at least for 1 hr. The binding was reversible in the presence of excess unlabeled T3. Scatchard analysis showed only a single class of binding sites. The mean dissociation constant (Kd) is 2.15 +/- 0.45 x 10(-10) M and maximum binding capacity (MBC) is 0.044 +/- 0.024 pmol/mg DNA. The relative binding affinities of thyroid hormone analogs for T3 sites were as follows: TRIAC greater than T3 greater than TETRAC greater than T4 greater than reverse T3 greater than T2. These findings were similar to those for other animals. Therefore, the nuclear binding sites in Singi fish brain, as demonstrated, may be regarded as thyroid hormone receptors.