TRIIODOTHYRONINE AND THYROXINE NUCLEAR RECEPTORS IN LYMPHOCYTES FROM NORMAL, HYPER- AND HYPOTHYROID SUBJECTS

1977 ◽  
Vol 85 (1) ◽  
pp. 44-54 ◽  
Author(s):  
Th. Lemarchand-Béraud ◽  
A.-Ch. Holm ◽  
B. R. Scazziga
Keyword(s):  
Nuclear Receptors ◽  
Binding Sites ◽  
Intact Cell ◽  
Affinity Constant ◽  
Limited Capacity ◽  
Intact Cells ◽  
High Affinity ◽  
The Mean ◽  
Hypothyroid Patients ◽  
Hyperthyroid Patients

ABSTRACT In an investigation of thyroxine (T4) and triiodothyronine (T3) receptors in humans, the lymphocyte was chosen as the target cell. This study was performed to elucidate whether T3 and T4 bind to different receptors, if T4 is bound only after conversion into T3, and whether there is any modification of the receptors in hyper- and hypothyroidism. Lymphocytes were found to possess a high-affinity, limited-capacity binding sites for both T4 and T3. The mean equilibrium affinity constant (Ka) was 2.28 · 1010 ± 0.21 m−1 for T3, and 0.98 · 1010 ± 0.16 m−1 for T4. The mean number of saturable binding sites was 115 for T3, and 102 for T4. The binding capacities and affinities also determined in the lymphocyte nuclei isolated after incubation of the intact cell, were similar to those observed in the intact cells. In competition experiments, labelled T4 was as readily displaced by T3 as by T4 itself, whereas labelled T3 was displaced only by a 40 times higher concentration of T4 than T3. These observations suggest identical receptors for the two hormones and a binding of T4 as such, provided it is not in competition with T3. In lymphocytes from hyperthyroid patients, receptor affinities and numbers remained unchanged. In lymphocytes from hypothyroid patients, the affinity was normal, but the mean number of T3 binding sites was increased to 310 (P < 0.001), to return to normal after a few months of treatment.

HIGH-AFFINITY BINDING OF REVERSE T3 AND TETRAC IN NUCLEI OF HUMAN LYMPHOCYTES

1978 ◽  
Vol 87 (3) ◽  
pp. 516-524 ◽  
Author(s):  
T. Lemarchand-Béraud ◽  
A.-C. Holm ◽  
G. Bornand ◽  
A. Burger
Keyword(s):  
Human Lymphocyte ◽  
Binding Sites ◽  
Specific Binding ◽  
Human Lymphocytes ◽  
Affinity Constant ◽  
Reverse T3 ◽  
Intact Cells ◽  
High Affinity ◽  
Specific Binding Sites ◽  
Direct Measurements

ABSTRACT In a previous study, human lymphocyte nuclei were found to possess high affinity, low capacity binding sites for triiodothyronine (T3) and thyroxine (T4). The number of receptors per cell was similar for T3 and T4 (115±20), but the equilibrium affinity constant (Ka) for T3 (2.20±0.23 1010m−1) was twice that for T4 (1.05 ± 0.25 1010m−1). The present study shows that human lymphocyte nuclei also bind highly purified [125I]tetrac and [125I]rT3. The number of specific binding sites was 60 for tetrac and 40 for rT3. The Ka for tetrac (2.12 ± 0.29 1010m−1) was similar to that of T3, whereas that of rT3 (1.31 ± 0.2110m−1) was similar to that of T4. The Ka was the same when measured in intact cells and in nuclei isolated after incubation. Despite the similar Ka for tetrac, rT3 and T3, as obtained by direct measurements, tetrac had only 2 % and rT3 0.1 % of the T3 potency in T3 displacement studies. [125I] tetrac was displaced 50% by 20 fmol of T3 and [125I]rT3 by 8 fmol. These results show that tetrac and rT3 do bind as strongly to nuclear receptors as T3 and T4, but that when competing with T3 the apparent affinities decrease considerably for tetrac and rT3. Thus, the nuclear binding of these two analogues probably has no significance under physiological conditions, but may play some role under pathological conditions when the formation of T3 is decreased and that of rT3 and tetrac is increased. This may represent an adaptive mechanism in T4 inactivation.

HUMAN LYMPHOCYTE BINDING AND DEIODINATION OF THYROID HORMONES IN RELATION TO THYROID FUNCTION

1975 ◽  
Vol 80 (4) ◽  
pp. 642-656 ◽  
Author(s):  
Ann-Charlotte Holm ◽  
Thérèse Lemarchand-Béraud ◽  
Bianca-Rosa Scazziga ◽  
Serge Cuttelod
Keyword(s):  
Thyroid Hormones ◽  
Thyroid Function ◽  
Human Lymphocyte ◽  
High Serum ◽  
Affinity Constant ◽  
Control Group ◽  
Binding Characteristics ◽  
The Mean ◽  
Hypothyroid Patients ◽  
Hyperthyroid Patients

ABSTRACT The human lymphocyte has been investigated regarding its function as a thyroid hormone target cell. Binding and deiodination of the thyroid hormones were determined after simultaneous incubation of 131I-labelled L-thyroxine (131I-T4) and 125I-labelled L-triiodothyronine (125I-T3) with lymphocytes from healthy subjects, from hyperthyroid and primary hypothyroid patients before and after treatment. The mean percentages of binding, 8.0 ± 0.5 (mean ± sem) for 131I-T4, and 9.7 ± 0.4 for 125I-T3 in the control group, were increased in the hyperthyroids to 10.1 ± 0.4 and 12.7 ± 0.6 respectively, and in the hypothyroids to 10.9 ± 0.7 and 12.8 ± 0.6. All elevated values returned to normal with successful treatment. The mean percentage of deiodination, 12.0 ± 1.7 for 131I-T4, and 6.5 ± 0.9 for 125I-T3 in the control group, showed a threefold increase in the hyperthyroid patients, to 35.9 ± 3.2 and 20.2 ± 1.9 respectively and remained unaltered in the hypothyroid patients. The values of successfully treated hyperthyroid patients were normal and those of the treated hypothyroid patients below normal. Human TSH added to the incubation medium stimulated the binding of both hormones, without influencing deiodination. Thus TSH might be active at the peripheral cellular level. This could contribute to the explanation of the increased binding by lymphocytes from primary hypothyroid patients with high serum concentrations of TSH. A preliminary analysis of the binding characteristics revealed an equilibrium affinity constant of 1.03·1010 m−1 for T3, and of 3.97· 109 m−1 for T4, with corresponding total numbers of binding sites of 1500 and 2000 per cell. It is concluded that, since lymphocyte activities closely reflect the thyroid function, these cells are well suited for studies on the peripheral fate of thyroid hormones and their cellular receptors.

Interaction of epidermal growth factor with receptors on human and porcine thyroid membranes

1984 ◽  
Vol 102 (1) ◽  
pp. 57-61 ◽  
Author(s):  
H. Humphries ◽  
S. MacNeil ◽  
D. S. Munro ◽  
S. Tomlinson
Keyword(s):  
Enzyme Activity ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Binding Sites ◽  
Affinity Constant ◽  
Maximal Inhibition ◽  
High Affinity ◽  
Epidermal Growth ◽  
And Function ◽  
Bovine Tsh

ABSTRACT Recent evidence suggests that epidermal growth factor (EGF) may play an important role in the regulation of thyroid growth and function. We have examined the interaction of murine EGF (mEGF) with human and porcine thyroid membranes and compared this with the binding of bovine TSH (bTSH) using 125I-labelled hormones as tracers. The characteristics of the binding of mEGF were found to be similar for human and porcine thyroid membranes. Epidermal growth factor bound with high affinity (affinity constant = 1·4 × 109 l/mol); the density of binding sites was low compared with the TSH receptor. At 37 °C, the binding of 125I-labelled EGF was maximal at 1 h and was saturable in the presence of unlabelled EGF; half-maximal inhibition was at 1 ng EGF/tube (0·5 nmol/l) using 0·5 mg membrane protein/tube. Unlabelled bTSH had no effect on the binding of labelled EGF. Similarly, unlabelled EGF did not affect the binding of labelled TSH; hence it was concluded that mEGF and bTSH bound to independent sites. Epidermal growth factor had no effect on adenylate cyclase activity in membranes prepared from human non-toxic goitre; increasing concentrations of EGF did not affect basal, TSH-stimulated or fluoride-stimulated enzyme activity. J. Endocr. (1984) 102, 57–61

scholarly journals Assay of Procarboxypeptidase U, a Novel Determinant of the Fibrinolytic Cascade, in Human Plasma

1999 ◽  
Vol 45 (6) ◽  
pp. 807-813 ◽  
Author(s):  
Katinka A Schatteman ◽  
Filip J Goossens ◽  
Simon S Scharpé ◽  
Hugo M Neels ◽  
Dirk F Hendriks
Keyword(s):  
Human Plasma ◽  
Binding Sites ◽  
Hippuric Acid ◽  
Active Form ◽  
Reference Interval ◽  
Healthy Individuals ◽  
High Affinity ◽  
Lysine Residues ◽  
The Mean

Abstract Background: Procarboxypeptidase U (proCPU) is a novel proenzyme found in human plasma. The active form, carboxypeptidase U (CPU; EC 3.4.17.20), retards the rate of fibrinolysis through its ability to cleave C-terminal lysine residues on fibrin partially degraded by plasmin. This reduces the number of high-affinity plasminogen-binding sites on fibrin. Methods: We developed an assay to determine the proCPU concentration in human plasma. The assay involved quantitative conversion of proCPU to active CPU by thrombin-thrombomodulin, a very efficient activator of proCPU, followed by determination of the enzymatic activity of CPU with the substrate hippuryl-l-arginine, using an HPLC-assisted determination of the released hippuric acid. Using this method, we established a reference interval based on 490 healthy individuals. Results: The mean proCPU concentration, determined after activation of the zymogen in diluted plasma and expressed as CPU activity, was 964 U/L, with a SD of 155 U/L. The population showed a gaussian distribution. However, we noticed important differences related to age and the use of hormone preparations. Conclusions: The sensitivity and precision of the method make it suitable for routine clinical determinations and as a reference procedure.

Thyroxine in unextracted urine

1987 ◽  
Vol 114 (4) ◽  
pp. 503-508 ◽  
Author(s):  
I. Orden ◽  
J. Pie ◽  
M. G. Juste ◽  
J. A. Marsella ◽  
C. Blasco
Keyword(s):  
Urinary Excretion ◽  
Renal Clearance ◽  
Filtration Rate ◽  
Experimental Studies ◽  
Tubular Reabsorption ◽  
Enzyme Hydrolysis ◽  
Renal Handling ◽  
The Mean ◽  
Hypothyroid Patients ◽  
Hyperthyroid Patients

Abstract. The aim of this work was to estimate the daily urinary excretion of free and conjugated thyroxine using a direct radioimmunoassay and enzyme hydrolysis. The renal clearance of free T4 was also determined. The mean urinary values of free and total T4 (mean ± 1 sd) in 112 euthyroid controls were 1353 ± 496 and 1855 ± 651 pmol/24 h, respectively. Urinary excretion of free hormone in 13 hyperthyroid patients was 5552 ± 4320 pmol/24 h and total T4 was 8122 ± 7219 pmol/24 h. Urinary free T4 excretion was 223 ± 223 pmol/24 h in hypothyroid patients and total T4 was 542 ± 490 pmol/24 h. These results indicate that daily urinary T4 excretion is a good indicator of thyroid function. The mean renal clearance of free T4 was 52 ± 19 ml/min (mean ± 1 sd) in euthyroid patients, 53.7 ± 12.3 ml/min in hyperthyroid patients, and 67.6 ± 13.1 ml/min in hypothyroid patients. We estimated the endogenous creatinine renal clearance as a control of the renal filtration rate. The data suggest that there is T4 filtration of unbound T4 and partial tubular reabsorption. Further experimental studies will be necessary to clarify the renal handling of thyroxine as well as the fate of reabsorbed T4.

TRIIODOTHYRONINE BINDING TO LYMPHOCYTES FROM EUTHYROID SUBJECTS AND A PATIENT WITH PERIPHERAL RESISTANCE TO THYROID HORMONE

1976 ◽  
Vol 83 (1) ◽  
pp. 64-70 ◽  
Author(s):  
Kristian Liewendahl
Keyword(s):  
Thyroid Hormone ◽  
Binding Sites ◽  
Binding Capacity ◽  
Human Lymphocytes ◽  
Peripheral Resistance ◽  
Subsequent Treatment ◽  
Affinity Constant ◽  
Prolonged Incubation ◽  
Resistance To Thyroid Hormone ◽  
The Mean

ABSTRACT Triiodothyronine (T3) binding to Ficoll-Isopaque purified human lymphocytes was studied. During incubation of lymphocytes with [125I]T3 in a calcium-free medium at 37°C, maximal uptake of T3 in nuclei occurred after 2 h and declined after prolonged incubation. Incubation of lymphocytes with T3 concentrations ranging from 1 × 10−11 to 1 × 10−9 mol/l and subsequent treatment with Triton X-100 to strip off [125I]T3 bound with low affinity was used for the estimation of affinity and capacity of nuclear T3 binding sites. The mean equilibrium affinity constant (Ka) estimated with the Scatchard method in 11 euthyroid healthy subjects was 4.5 × 109 1/mol, and the mean maximal binding capacity 25 × 10−15 mol/100 μg DNA. In a female patient with peripheral resistance to thyroid hormone action, the estimated Ka was 3.5 × 109 1/mol and the number of T3 binding sites 37 × 10−15 mol/100 μg DNA. Although not statistically different from the mean value in euthyroid subjects, this Ka value was outside the range of control values observed and was considered presumptive evidence that the nuclear T3 receptors in this patient have abnormally low affinity for its ligand. The nuclear T3 binding capacity in this patient was significantly increased.

THE "EFFECTIVE THYROXINE RATIO" – NEW IN VITRO TEST OF THYROID FUNCTION AND ITS CORRELATION WITH FREE THYROXINE

1974 ◽  
Vol 76 (1) ◽  
pp. 83-88 ◽  
Author(s):  
Janusz Nauman ◽  
Alicja Nauman
Keyword(s):  
Pregnant Women ◽  
Normal Subjects ◽  
Free Thyroxine ◽  
In Vitro Test ◽  
High Reproducibility ◽  
Vitro Test ◽  
The Mean ◽  
Hypothyroid Patients ◽  
Hyperthyroid Patients

ABSTRACT The effective thyroxine ratio (ETR) and absolute concentration of free thyroxine (AFT4) were estimated in the sera of 31 normal subjects, 27 hyperthyroid patients, 12 hypothyroid patients and 21 euthyroid pregnant women. The mean ETR value in the controls was 1.0 ± 0.18, in the hyperthyroid patients 1.31 ± 0.25, in the hypothyroid patients 0.71 ± 0.21 and in normal pregnant women 0.99 ± 0.24. The mean AFT4 in the normal subjects was 3.0 ± 0.53 ng/100 ml, in the hyperthyroid patients 9.49 ± 2.44 ng/ 100 ml, in the hypothyroid patients 0.58 ± 0.15 ng/100 ml and in the pregnant women 2.84 ± 0.63 ng/100 ml, respectively. High reproducibility of ETR and a significant positive correlation between ETR and AFT4 with r = 0.96 suggest that ETR might be a suitable in vitro test for routine clinical evaluation of the thyrometabolic state.

Binding of phytohemagglutinin to porcine lymphocyte receptors. Time-course studies and comparative binding isotherms using whole cells or cellular receptors incorporated into phospholipid vesicles

1985 ◽  
Vol 63 (9) ◽  
pp. 1033-1043 ◽  
Author(s):  
Gilles Dupuis ◽  
Bânû Bastin
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Lymphocyte Activation ◽  
Mass Action ◽  
Affinity Constant ◽  
Computer Assisted ◽  
Intact Cells ◽  
Binding Process ◽  
High Affinity ◽  
Receptor Sites

We have studied the binding of 125I-labeled phytohemagglutinin (PHA) to porcine splenic lymphocytes (107 cells∙mL−1) at 37 °C. Our data show that PHA binding is dependent on the incubation period and that a maximum quantity of 4.53 ± 0.13 μg is bound when 200 μg of PHA is added. The binding process is rapid and saturation can readily be achieved in less than 2 min. These observations suggest, in accordance with the mass-action principle, that the binding equilibrium can be rapidly displaced towards PHA–receptor complex formation when sufficient amounts of PHA are added. Our data further suggest that receptor sites are exposed at the cell surface and that putative cryptic receptor sites are unlikely to play a major role in the initial part of the binding process. We have studied this aspect by comparing Scatchard plots for PHA binding to receptors in intact lymphocytes and to lymphocyte-derived receptors incorporated into liposomes. In one case, partially purified plasma membranes were solubilized and incorporated into phosphatidylcholine–phosphatidylserine (PC–PS) vesicles prepared by detergent dialysis. In another case, PHA-receptor glycoproteins were purified by affinity chromatography and reassembled into PC–PS vesicles, using the same technique. Scatchard plot analyses showed nonlinear profiles with a concavity turned upward for PHA binding to receptors of intact cells or of PC–PS vesicles with plasma membranes, and a concavity turned downward for vesicles with purified glycoproteins. These observations suggest that PHA-receptor environment plays a determining role in the binding process of the lectin. Numerical data from binding experiments were obtained by computer-assisted nonlinear regression analysis, using a one ligand–two sites model. The (apparent) high-affinity constant (K1) for intact lymphocytes or partially purified plasma membrane components reassembled into liposomes was 4.6 × 107 M−1 and the (apparent) low-affinity constants (K2) were 4.4 × 106 ± 1.5 × 106 M−1 (intact lymphocytes) and 1.7 × 106 ± 0.6 × 106 M−1 (plasma membranes constituents reassembled into liposomes). The value obtained for reconstituted PHA-glycoprotein receptors (K) was 0.70 × 107 ± 0.10 × 107 M−1. The apparent number of sites was n1 = 0.19 × 106 ± 0.05 × 106 (high affinity) and n2 = 2.50 × 106 ± 0.50 × 106 (low affinity) sites per intact lymphocyte cell. In the case of the reconstituted systems, the number of sites per microgram protein of the PC–PS vesicles were n1 = 0.79 × 1010 ± 0.18 × 1010 and n2 = 31.8 × 1010 ± 8.5 × 1010 for the partially purified plasma membrane and n = 26.6 × 1010 ± 2.9 × 1010 for the purified PHA receptor glycoproteins. Data presented here are discussed in terms of the lymphocyte activation model of Prujansky and colleagues, which suggests that positively cooperative lectin binding is a sine qua non condition for lymphocyte activation.

Verlust von hochaffinen Prostacyclin-Bindungsstellen bei Patienten mit Morbus Basedow

1989 ◽  
Vol 28 (01) ◽  
pp. 17-20
Author(s):  
K. Weiss ◽  
M. Hermann ◽  
H. Sinzinger ◽  
R. Höfer ◽  
Irene Virgolini
Keyword(s):  
Binding Sites ◽  
Cell Function ◽  
Thyroid Tissue ◽  
Morbus Basedow ◽  
Thyroid Cell ◽  
Affinity Binding ◽  
Dependent Manner ◽  
Camp Production ◽  
High Affinity ◽  
Hyperthyroid Patients

Prostacyclin (PGI2) mediates like TSH its cellular effects through the interaction with specific binding sites associated with the adenylate cyclase-cAMP-system. Binding of PGI2 and the generation of cAMP induced by PGI2 was evaluated in thyroid tissue obtained intraoperatively from euthyroid and hyperthyroid patients with diffuse normofollicular colloid struma. Transformation of the binding data according to Scatchard revealed heterogeneity of the PGI2 binding sites in the tissue of euthyroid patients: the high-affinity binding sites were calculated to be 0.68 ± 0.18 pmol/mg protein (Ka = 16.2 ± 9.1 nM) and the low-affinity binding sites to be 5.4 ± 1.6 pmol/mg protein (Ka = 151 ± nM). In contrast, in the hyperthyroid patients the low-affinity binding sites were not demonstrable and the high-affinity sites were significantly (p <0.001) reduced (0.17 ± 0.05 pmol/mg protein, Ka = 83.5 ± 19.6 nM). The competition of the agonist for the PGI2 sites in hyperthyroid patients was significantly (p <0.005) diminished (IC-50-values: 0.98 ± 3.1 vs 46.9 ± 12.1 μM). PGI2 stimulated cAMP-production in a dose-dependent manner. However, the basal value was significantly lower also in the hyperthyroid patients (p <0.001). The evidence of reduced PGI2 sites as well as reduced PGI2-induced cAMP production in the thyroid gland of patients with Graves’ disease may indicate an important role for PGI2 to play in the modulation of thyroid cell function.

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