Calcitriol receptors in rat thyroid follicular cells (FRTL-5)

1991 ◽  
Vol 125 (5) ◽  
pp. 574-580 ◽  
Author(s):  
Jens P. Berg ◽  
Peter A. Torjesen ◽  
Egil Haug
Keyword(s):  
Specific Binding ◽  
Sedimentation Coefficient ◽  
Scatchard Analysis ◽  
Rat Tissues ◽  
Camp Production ◽  
Follicular Cells ◽  
Single Class ◽  
Binding Data ◽  
Thyroid Follicular Cells ◽  
Rat Thyroid

Abstract. The FRTL-5 cell line is widely used as a model for normal thyroid follicular cells. These cells have retained their ability to alter cAMP production, cell proliferation, iodine uptake, and thyroglobulin synthesis in response to thyrotropin. We have previously shown that calcitriol attenuated both basal and TSH stimulated cAMP production dose-dependently in FRTL-5 cells. Cytosol fractions (105000 g, 60 min, 4°C) prepared from FRTL-5 cell homogenates possessed calcitriol-binding components with a sedimentation coefficient of approximately 3.7 S in high salt (0.3 mol/l KCl) sucrose gradients (5–20%). At 4°C, specific binding increased rapidly during the first 4 h and reached a plateau after 8 h. The specific binding (18 h, 4°C) was maximal at a [3H]calcitriol concentration of approximately 0.5 nmol/l. Scatchard analysis of the binding data indicated one single class of high affinity binding sites with Kd = 105±2 pmol/l and Bmax=38.5±4.7 pmol/g cytosol protein (mean ± sd, N=6). In conclusion, our results suggest that the FRTL-5 cells possess functional receptors for calcitriol with the same physicochemical properties as the receptors found in normal rat tissues.

Interaction of endothelin-1 with porcine thyroid cells in culture: a possible autocrine factor regulating iodine metabolism

1994 ◽  
Vol 142 (3) ◽  
pp. 463-470 ◽  
Author(s):  
T Tsushima ◽  
M Arai ◽  
O Isozaki ◽  
Y Nozoe ◽  
K Shizume ◽  
...  
Keyword(s):  
Binding Capacity ◽  
Specific Binding ◽  
Thyroid Cell ◽  
Scatchard Analysis ◽  
Camp Production ◽  
Iodide Uptake ◽  
Single Class ◽  
Thyroid Cells ◽  
Cells In Culture ◽  
Iodine Metabolism

Abstract Although endothelins were originally discovered as peptides with vasoconstrictor activity, recent studies have indicated a number of endothelin (ET)-induced hormonal functions in various tissues. We have studied the interaction of endothelins with porcine thyroid cells in culture. Specific binding of 125I-labelled ET-1 was demonstrated in porcine thyroid cells. The binding was displaced equally by unlabelled ET-1 and ET-2, but receptor affinity for ET-3 was lower than that for ET-1 and -2. Scatchard analysis of the data revealed a single class of high-affinity ET-1 receptors with a Kd of 0·45 nmol/l and a binding capacity of 2100 sites/cell. SDS-PAGE and autoradiography of 125I-labelled ET-1 cross-linked with thyroid cell membranes demonstrated ET-1 binding sites with an apparent molecular weight of 50 kDa. These results indicated that ET-1 receptors in thyroid cells are type A ET receptors. In association with the presence of ET-1 receptors, porcine thyroid cells responded to ET-1 and ET-2 with an increase in c-fos mRNA expression. Although ET-1 did not affect DNA synthesis stimulated by either EGF or IGF-I, it dose-dependently inhibited TSH-induced iodide uptake and also inhibited iodide uptake stimulated by forskolin and 8-bromo-cAMP. ET-1 had no effect on TSH-stimulated cAMP production. Thus, ET-1 inhibited TSH-induced iodine metabolism by acting at the steps distal to cAMP production. In agreement with a recent report, immunoreactive ET-1 was detected in medium conditioned by porcine thyroid cells. Antibody to ET-1 was found to increase TSH-induced iodide uptake. These results are compatible with the notion that ET-1 negatively regulates TSH-induced iodide uptake in an autocrine manner. Journal of Endocrinology (1994) 142, 463–470

scholarly journals Characterization of the high-affinity receptors on Swiss 3T3 cells which mediate the binding, internalization and degradation of the mitogenic peptide bombesin

1988 ◽  
Vol 252 (1) ◽  
pp. 227-235 ◽  
Author(s):  
K D Brown ◽  
M S Laurie ◽  
C J Littlewood ◽  
D M Blakeley ◽  
A N Corps
Keyword(s):  
Enzyme Inhibitors ◽  
Competitive Inhibition ◽  
Specific Binding ◽  
Scatchard Analysis ◽  
3T3 Cells ◽  
Single Class ◽  
High Affinity ◽  
Binding Data ◽  
Molecular Masses ◽  
Swiss 3T3

Bombesin and bombesin-related peptides such as gastrin-releasing peptide (GRP) stimulate DNA synthesis and proliferation of Swiss 3T3 cells in culture. We have used 125I-labelled [Tyr4]bombesin and 125I-labelled GRP to characterize and identify the receptors for these peptides on Swiss 3T3 cells. The binding of 125I-[Tyr4]bombesin, which retained full biological activity, was maximal between 20 and 30 min incubation at 37 degrees C, after which continued incubation led to a decline in cell-associated radioactivity. This decline was markedly slowed by the presence of lysosomal enzyme inhibitors. Specificity of the binding site was indicated by the competitive inhibition of binding by bombesin-related peptides, but not by unrelated peptides and growth factors. Scatchard analysis of binding data indicated a single class of high-affinity receptors. The calculated value for the dissociation constant (Kd) was 2.1 nM and each cell possesses approx. 240,000 receptors. Because [Tyr4]bombesin has no free amino group, 125I-GRP was used in chemical cross-linking studies. When disuccinimidyl suberate was used to covalently couple 125I-GRP to the cells, two major radiolabelled complexes were detected with molecular masses of approx. 80,000-85,000 and 140,000. The binding of 125I-[Tyr4]bombesin to the cells was pH-dependent with maximal binding at pH 6.5-7.5 and effectively no specific binding at pH values below 4.5. At 37 degrees C, cell-associated 125I-[Tyr4]bombesin quickly became resistant to removal by acidic buffers, suggesting its rapid transfer to an intracellular compartment. However, pre-incubation with unlabelled [Tyr4]bombesin did not induce down-regulation of bombesin receptors as measured by the subsequent binding of 125I-[Tyr4]bombesin. In contrast with the Swiss 3T3 cells, specific binding of 125I-[Tyr4]bombesin was not detectable in two cell lines which are biologically unresponsive to bombesin-related peptides.

Calcium and the Fc Receptor on Human Platelets

1987 ◽  
Vol 57 (03) ◽  
pp. 298-301
Author(s):  
William F Clark ◽  
Gerald J M Tevaarwerk ◽  
Bruce D Reid ◽  
Suzanne Hall ◽  
Anita Caveney ◽  
...  
Keyword(s):  
Specific Binding ◽  
Normal Subjects ◽  
Human Platelets ◽  
Fc Receptor ◽  
Normal Male ◽  
Scatchard Analysis ◽  
Normal Female ◽  
Apparent Difference ◽  
Binding Data ◽  
Direct Binding

SummaryWe have described the calcium dependence of the IgG Fc receptor (Fc-R) on human platelets by analyzing the direct binding of radiolabelled Fc fragments, monomers and dimers of IgG. Specific binding to platelets was undetectable at 37° C in a calcium-free preparation but readily detected when calcium was restored. Scatchard analysis of the binding data for the calcium-restored platelets permitted calculation of the available Fc-R and the Ka of binding for the different IgG ligands. The mean Ka of binding for 12 normal subjects varied from 107 to 108 L/M, with an equal receptor number measured by Fc fragments and dimers of IgG, but a lesser amount for monomeric IgG. There was no apparent difference in Fc-R number for platelets from 6 normal male versus 6 normal female subjects.At 4° C binding was detectable for dimers and polymers of IgG in a calcium-free preparation and this was markedly increased with recalcification. Thus, our data are consistent with an Fc receptor population on human platelets whose avidity for binding is significantly enhanced in a calcium-restored medium.

scholarly journals Hepoxilin A3-specific binding in human neutrophils

1996 ◽  
Vol 313 (2) ◽  
pp. 537-541 ◽  
Author(s):  
Denis REYNAUD ◽  
Peter DEMIN ◽  
Cecil R. PACE-ASCIAK
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Leukotriene B4 ◽  
Proteinase K ◽  
Human Neutrophils ◽  
Scatchard Analysis ◽  
Intact Cells ◽  
Specific Binding Sites ◽  
Binding Data ◽  
Hepoxilin A3

Hepoxilins have been shown to release calcium from intracellular stores in human neutrophils [Dho, Grinstein, Corey, Su and Pace-Asciak (1990) Biochem. J. 266, 63-68; Laneuville, Reynaud, Grinstein, Nigam and Pace-Asciak (1993) Biochem. J. 295, 393-397]. In this paper we report that tritium-labelled hepoxilin A3 (8S) binds to broken neutrophil membranes in a time-, substrate- and temperature-dependent fashion. Specific binding was displaced with unlabelled hepoxilin A3. Specific binding was greatest at 37 °C. Competitive binding was best observed with unlabelled hepoxilin A3 (8S); the glutathione conjugate, HxA3-C (8S or 8R), or 12(S)-hydroxyeicosatetraenoic acid was less active. Similarly inactive in displacing the bound radiolabelled hepoxilin A3 was leukotriene B4 as well as a variety of prostaglandins and thromboxane B2. Formylmethionyl-leucylphenylalanine was similarly inactive in competing for the hepoxilin binding sites. Specific binding was inhibited by pretreatment of the broken membranes during 30 min at 37 °C with proteinase K, while specific binding of the intact cells was unaffected. Scatchard analysis of binding data revealed a single population of binding sites with apparent KD and Bmax. of 79.3±9.1 nM and 8.86±1.4 pmol/ml per 2×106 cells (±S.E.M.) respectively reflecting approx. 2.67×106 sites/cell. These results demonstrate for the first time that neutrophils contain specific binding sites to hepoxilin A3.

Expression of insulin-like growth factor receptors in primary human thyroid neoplasms

1989 ◽  
Vol 121 (1) ◽  
pp. 112-120 ◽  
Author(s):  
Tohru Yashiro ◽  
Yoshito Ohba ◽  
Hitomi Murakami ◽  
Takao Obara ◽  
Toshio Tsushima ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Binding Capacity ◽  
Specific Binding ◽  
Human Thyroid ◽  
Scatchard Analysis ◽  
Type I ◽  
Single Class ◽  
Normal Tissues ◽  
Igf I ◽  
Cancer Tissues

Abstract. The presence of IGF-I receptors was demonstrated in normal and neoplastic tissues of human thyroid. Binding of [125I]IGF-I to thyroid membranes was dependent on time and temperature of incubation, and maximal binding was achieved at 4°C and 18 h of incubation. [125I] IGF-I binding was dose-dependently displaced by unlabelled IGF-I; half-maximal inhibition occurred at concentrations of 10–20 μg/l. IGF-II and insulin had relative potencies of 5 and 1% compared with IGF-I. Scatchard analysis of binding data revealed a single class of IGF-I receptors with high affinity (Ka: 1.2–8.6 × 109 1/mol) in normal thyroid tissues. Affinity cross-linking and autoradiography demonstrated the type I IGF receptors. Specific binding of [125I] IGF-I in thyroid cancer tissues (9.69 ± 2.07% per 200 μg protein; mean ± sem, N = 8) was significantly (p <0.05) higher than that in the surrounding normal tissues (3.03 ± 0.35%, N = 8). In contrast, there was no difference in the binding between adenoma tissues (4.19 ± 0.53%, N = 5) and the adjacent normal tissues (2.94 ± 0.24%, N = 5). The higher IGF-I binding in cancer tissues was due to an increase in the binding capacity without any change in the affinity. The presence of IGF-I receptors suggests a possible role of IGF-I and its receptors in the growth of thyroid cancer cells.

Stimulation of Na(+)-K(+)-ATPase by thyrotropin in cultured thyroid follicular cells

1995 ◽  
Vol 268 (5) ◽  
pp. C1252-C1258 ◽  
Author(s):  
T. A. Pressley ◽  
S. C. Higham ◽  
L. A. Joson ◽  
D. W. Mercer
Keyword(s):  
Thyroid Stimulating Hormone ◽  
Follicular Cells ◽  
Hormone Synthesis ◽  
Metabolic Turnover ◽  
Striking Increase ◽  
Thyroid Follicular Cells ◽  
A Cell ◽  
Messenger System ◽  
Rat Thyroid ◽  
Stimulation Of

Thyroid-stimulating hormone (TSH; thyrotropin) produces a pleiotropic response in the thyroid gland, accelerating nearly every aspect of metabolic turnover within the follicular epithelia. We examined the effects of TSH on expression of Na(+)-K(+)-ATPase in FRTL-5 cells, a cell line derived from rat thyroid. TSH (10 mU/ml) produced a nearly twofold increase in abundance of the mRNA encoding the catalytic alpha 1-subunit within 6 h of treatment. With the four mRNAs encoding the beta 1-subunit, TSH produced a striking increase in abundance, but this regulation was discoordinate, and some species increased more than others. Similar increases in mRNA abundance were elicited by activators of the adenosine 3',5'-cyclic monophosphate second messenger system. In contrast to the alpha 1- and beta 1-mRNAs, the abundance of the mRNA encoding the beta 2-subunit was unchanged with TSH after 6 h, indicating that the effects of thyrotropin were not universal or indiscriminate. Thyrotropin also caused a 76% increase in Na(+)-K(+)-ATPase activity and a 46% increase in pump-mediated transport after 48 h. These studies suggest that the changes in metabolic turnover initiated by TSH during hormone synthesis include upregulation of the N(+)-K+ pump.

The Binding Of 125I-Von Willebrand Factor To Human Platelets In The Presence Of Ristocetin

1981 ◽  
Author(s):  
P Silber ◽  
T H Finlay
Keyword(s):  
Von Willebrand Factor ◽  
Binding Sites ◽  
Binding Constant ◽  
Specific Binding ◽  
Human Platelets ◽  
Scatchard Analysis ◽  
Binding Data ◽  
Number Of Binding Sites ◽  
Von Willebrand ◽  
Willebrand Factor

The effect of ristocetin on the binding of 125I-porcine von Willebrand factor to human platelets was studied. Previously, we had shown that 125I-porcine von Willebrand factor binds to human platelets in the absence of ristocetin. The present work demonstrates that binding is stimulated by ristocetin and this stimulation is maximal at a ristocetin concentration of 2 mg/ml. At a ristocetin concentration of 0.5 mg/ml, Scatchard analysis indicates a binding constant of 5.18 × 10-9M and the presence of 105,000 binding sites. This compares with our previous finding, in the absence of ristocetin, of a binding constant of 2.92 × 10-7M and 4760 binding sites. These binding data assume the porcine von Willebrand factor to be a tetramer with a molecular weight of 9 × 105. This study indicates that ristocetin causes tighter binding and increases the number of binding sites on human platelets for porcine von Willebrand factor. Unlabelled porcine von Willebrand factor competitively inhibits the specific binding of the labelled protein and gives a binding constant of 0.17 × 10-9M. Similar results were obtained using human von Willebrand factor.

The rainbow trout skeletal muscle β-adrenergic system: characterization and signaling

2003 ◽  
Vol 284 (3) ◽  
pp. R689-R697 ◽  
Author(s):  
Michel B. Lortie ◽  
Thomas W. Moon
Keyword(s):  
Rainbow Trout ◽  
Binding Sites ◽  
Specific Binding ◽  
Adrenergic System ◽  
Camp Production ◽  
Binding Assays ◽  
Single Class ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Adrenergic Antagonist ◽  
Number Of Binding Sites

The presence and functionality of β-adrenoceptors (β-ARs) were examined in red (RM) and white muscle (WM) membranes isolated from the rainbow trout Oncorhynchus mykiss. Specific binding assays revealed the presence of a single class of binding sites with similar affinities in both muscle types ( K d in nM: 0.14 ± 0.03 and 0.18 ± 0.03 for RM and WM, respectively) but with a significantly higher number of binding sites in RM compared with WM (Bmax in fmol/mg protein: 3.22 ± 0.11 and 2.60 ± 0.13, respectively). Selective and nonselective β-adrenergic agonists (β-AAs) and antagonists indicated an atypical β-AR pharmacology. This result may represent a nonmammalian β-AR classification or, more likely, the presence of more than one β-AR subtype in trout muscles with similar affinities that could not be kinetically resolved. Adenylyl cyclase (ACase) assays showed a dose-dependent increase in cAMP production as concentrations of β2-AAs increased in both muscle membranes with significantly higher basal cAMP production in RM compared with WM (cAMP production in pmol cAMP · mg protein−1 · 10 min−1: 24.67 ± 3.06 and 9.64 ± 3.45, respectively). The agonist-induced increase in cAMP production was blocked by the β-adrenergic antagonist propranolol, while the ACase activator forskolin increased cAMP production by 7- to 14-fold above basal and ∼3-fold above all β-AAs tested. This study demonstrated the presence of atypical β2-ARs on RM and WM membranes of trout, suggesting that β2-AAs may be a tool to enhance protein accretion through this signaling pathway.

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