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

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.

Receptors for angiotensin I1 on platelets from man

1984 ◽  
Vol 66 (6) ◽  
pp. 725-731 ◽  
Author(s):  
Yuan Ding ◽  
Christopher J. Kenyon ◽  
Peter F. Semple
Keyword(s):  
Angiotensin Ii ◽  
Binding Capacity ◽  
Specific Binding ◽  
Venous Blood ◽  
Kinetic Studies ◽  
Free Fluid ◽  
Scatchard Analysis ◽  
Ang Ii ◽  
Temperature Dependent ◽  
Single Class

1. Platelets were prepared from peripheral venous blood on iso-osmotic density gradients of Percoll, resulting in a good recovery of cells (50–80%) which were relatively free of contaminating blood cells (erythrocyte <0.1%, leucocyte <0.1%). 2. At 22°C, specific binding of 125labelled angiotensin II (300 pmol/l) was time and temperature dependent, saturable, reversible and linear with cell concentration. 3. Scatchard analysis of saturation curves revealed a single class of binding sites with Kd 1.5 ± 0.4 × 10−10 mol/l and total binding capacity 6.3 ± 1.2 receptorslplatelet. Similar values (Kd 2.4 ± 0.7 × 10−10 mol/l and binding capacity 6.5 ± 1.0 receptors/cell) were obtained by displacement analysis. From kinetic studies the forward and reverse rate constants were 3.1 × 108 mol min−1 1−1 and 3.6 × 10−2/min giving a Kd of 1.2 × 10−10mol/l. 4. The relative binding potencies for angiotensin I1 and analogues were: [Sar1, Thr8]ANC II > ANG II > ANG III > [Sar1, Ala8]ANG II > ANG I. 5. Incubation with an extracellular marker (51Cr-labelled EDTA) demonstrated that binding of angiotensin II to platelets was not due to free fluid endocytosis.

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

1988 ◽  
Vol 117 (1) ◽  
pp. 116-124 ◽  
Author(s):  
Hirotoshi Nakamura ◽  
Hiroo Imura
Keyword(s):  
Rat Liver ◽  
Association Constant ◽  
Binding Capacity ◽  
Size Exclusion ◽  
Nuclear Proteins ◽  
Thyroid Cell ◽  
Scatchard Analysis ◽  
Thyroid Cells ◽  
Maximal Binding ◽  
Rat Thyroid

Abstract. 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 × 1010 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°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 × 1010 1/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.

Direct effect of endothelin-1 on the granulosa cells of the porcine ovary

1992 ◽  
Vol 134 (1) ◽  
pp. 59-66 ◽  
Author(s):  
S. Kamada ◽  
T. Kubota ◽  
Y. Hirata ◽  
M. Taguchi ◽  
S. Eguchi ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Binding Sites ◽  
Inositol Phosphate ◽  
Binding Capacity ◽  
Specific Binding ◽  
Scatchard Analysis ◽  
Single Class ◽  
Apparent Dissociation Constant ◽  
Endothelin 1 ◽  
Porcine Granulosa Cells

ABSTRACT Specific binding sites for endothelin-1 (ET-1), a novel potent vasoconstrictor peptide, as well as the effects of ET-1 on cytosolic free Ca2+ concentration ([Ca2+]i), intracellular total inositol phosphate (IP) generation and steroidogenesis were studied in cultured porcine granulosa cells. Scatchard analysis of a binding study using 125I-labelled ET-1 indicated the presence of a single class of high-affinity binding sites with almost equal affinity for ET-1 and ET-3: the apparent dissociation constant was 0·59 nmol/l and the maximal binding capacity was 1·84 pmol/mg protein. Affinitylabelling of 125I-labelled ET-1 to the membranes using disuccinimidyl tartarate as a cross-linker revealed one major and one minor band with the apparent molecular weights of 32 kDa and 49 kDa respectively. ET-1 dose-dependently (1−100 nmol/l) induced rapid and transient increases in [Ca2+]i in fura-2-labelled cells. ET-1 also dose-dependently stimulated total IPs in cells prelabelled with myo-[3H]inositol. ET-1 had a slight stimulatory effect on the secretion of progesterone but not of oestradiol from porcine granulosa cells. The present data clearly demonstrate the presence of a non-selective ET receptor (ETB) in porcine granulosa cells coupled with phosphoinositide hydrolysis and [Ca2+]i mobilization, and suggest that ET-1 may play some role in the production of progesterone by porcine granulosa cells. Journal of Endocrinology (1992) 134, 59–66

Effects of transforming growth factor α (TGF-α) on DNA synthesis and thyrotropin-induced iodine metabolism in cultured porcine thyroid cells

1995 ◽  
Vol 132 (2) ◽  
pp. 242-248 ◽  
Author(s):  
Mariko Arai ◽  
Toshio Tsushima ◽  
Osamu Isozaki ◽  
Hiroshi Demura ◽  
Kazuo Shizume ◽  
...  
Keyword(s):  
Growth Factor ◽  
Dna Synthesis ◽  
Transforming Growth Factor ◽  
Dependent Manner ◽  
Camp Production ◽  
Iodide Uptake ◽  
Thyroid Cells ◽  
Transforming Growth Factor Α ◽  
Iodine Metabolism ◽  
Factor Α

Arai M, Tsushima T, Isozaki 0, Demura H, Shizume K, Emoto N, Miyakawa M, Nozoe Y, Murakami H, Ohmura E. Effects of transforming growth factor α (TGF-α) on DNA synthesis and thyrotropin-induced iodine metabolism in cultured porcine thyroid cells. Eur J Endocrinol 1995;132:242–8. ISSN 0804–4643 Transforming growth factor α (TGF-α) is a potent mitogen that is similar structurally to epidermal growth factor (EGF). As EGF is a potent growth stimulator and an inhibitor of iodine metabolism in cultured thyroid cells of several species, we studied whether TGF-α has similar effects using porcine thyroid cells in culture. Recombinant human TGF-α dose-dependently stimulated DNA synthesis of thyroid cells, with maximal stimulation (eight- to ninefold above basal) occurring at 2 nmol/l. The potency was approximately 50% that of mouse EGF and correlated with the ability to compete with EGF for receptor binding, suggesting that the action of TGF-α is mediated by interaction with EGF receptors. When thyroid cells were cultured for 3 days with thyrotropin (TSH) in the presence of TGF-α, TSH-induced iodide uptake was inhibited in a dose-dependent manner. The potency of TGF-α again was approximately 50% that of EGF. Transforming growth factor α did not inhibit TSH-stimulated cAMP production. Moreover, iodide uptake stimulated by either forskolin or 8-bromo-cAMP also was inhibited by TGF-α. Thus, we conclude that TGF-α inhibits TSH-induced iodine metabolism largely by acting at the steps distal to cAMP production. Northern blot analysis revealed expression of TGF-α mRNA in porcine thyroid cells. These observations suggest that TGF-α acts as an autocrine modulator of growth and differentiated functions in porcine thyroid cells. T Tsushima, Department of Medicine 2, Tokyo Women's Medical College, Kawadacho 8–1, Shinjukuku, Tokyo 162, Japan

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.

BINDING OF HUMAN CHORIONIC GONADOTROPHIN BY RAT TESTIS: EFFECT OF SEXUAL MATURATION, CRYPTORCHIDISM AND HYPOPHYSECTOMY

1975 ◽  
Vol 64 (1) ◽  
pp. 59-66 ◽  
Author(s):  
JOACHIM FROWEIN ◽  
WOLFGANG ENGEL
Keyword(s):  
Dissociation Constant ◽  
Sexual Maturation ◽  
Leydig Cells ◽  
Binding Capacity ◽  
Specific Binding ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin ◽  
Scatchard Analysis ◽  
Rat Testis ◽  
Relative Binding

SUMMARY The specific binding of 125I-labelled human chorionic gonadotrophin (HCG) by rat testicular homogenate as compared with isolated Leydig cells differs with respect to total binding capacity but not to the dissociation constant (KD) as revealed by Scatchard analysis. The maximal binding capacity for [125I]HCG of crude testicular homogenate was 95 ng/g rat testis. Hypophysectomy causes a decline in binding capacity within the first three days but on the 20th and 30th day after hypophysectomy the relative binding capacity no longer differs from that of controls. Binding capacity is enhanced in cryptorchid testes relative to normal, and increases during sexual maturation to a peak shortly before puberty.

Characterization of [3H]nifedipine binding to intact vascular smooth muscle cells

1988 ◽  
Vol 254 (1) ◽  
pp. C45-C52 ◽  
Author(s):  
K. Sumimoto ◽  
M. Hirata ◽  
H. Kuriyama
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Binding Capacity ◽  
Specific Binding ◽  
Smooth Muscles ◽  
Muscle Cells ◽  
Scatchard Analysis ◽  
Intact Cells ◽  
Porcine Coronary Artery ◽  
Control Value

Specific binding of the dihydropyridine Ca2+ antagonist [3H]nifedipine to dispersed smooth muscle cells of the porcine coronary artery was investigated and the findings were compared with the binding to microsomes of smooth muscles. Specific binding to intact cells was saturable and reversible. The dissociation constant was 1.93 +/- 0.42 nM and the maximal binding capacity was 59.6 +/- 12.4 fmol/10(6) cells, as assessed by Scatchard analysis of the equilibrium binding at 25 degrees C. The Kd value with intact cells was slightly higher than that observed with microsomes. Specific binding of [3H]nifedipine to intact cells was completely displaced by unlabeled dihydropyridine derivatives. Among other Ca2+ antagonists, verapamil and d-cis-diltiazem partially and flunarizine completely inhibited the binding. In the case of microsomes, d-cis-diltiazem stimulated the binding of [3H]nifedipine. These results suggest that there may be multiple binding sites for different subclasses of Ca2+ antagonists. Polyvalent cations had no effect on the binding to intact cells. In the case of ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-treated microsomes, the addition of CaCl2 and BaCl2 increased the Bmax, but the Kd value remained unchanged. MnCl2 and CdCl2 had stimulatory or inhibitory effects, depending on the concentrations, whereas LaCl3 had no effect. The effect of membrane depolarization on the binding was also examined. When the intact cells were incubated in high [K+]o solution for 60 min, the Kd was lowered to 1.4 nM from the control value of 2.0 nM, thereby indicating that [3H]nifedipine binds to Ca2+ channels, with a higher affinity, at depolarized states.

Prolactin Receptor in Human Mammary Carcinoma

1979 ◽  
Vol 65 (6) ◽  
pp. 695-702 ◽  
Author(s):  
Raffaele Di Carlo ◽  
Giampiero Muccioli
Keyword(s):  
Protein Concentration ◽  
Binding Capacity ◽  
Specific Binding ◽  
Prolactin Receptor ◽  
Scatchard Analysis ◽  
Breast Carcinomas ◽  
Human Breast ◽  
Human Mammary Carcinoma ◽  
Human Breast Carcinomas ◽  
Lactogenic Hormones

The specific binding of labelled human prolactin was determined in 83 human breast carcinomas. Twenty-seven tumors (32.5 %) contained specific binding for prolactin of at least 1 % and were considered prolactin receptor positive. The binding was found linearly related to membrane protein concentration and specific only for lactogenic hormones. By Scatchard analysis the dissociation constant appeared similar to that observed in other target tissues, with a low binding capacity.

Evidence for a direct action of GH on haemopoietic cells of a marine fish, the gilthead sea bream (Sparus aurata)

1995 ◽  
Vol 146 (3) ◽  
pp. 459-467 ◽  
Author(s):  
J A Calduch-Giner ◽  
A Sitjà-Bobadilla ◽  
P Álvarez-Pellitero ◽  
J Pérez-Sánchez
Keyword(s):  
Binding Sites ◽  
Sparus Aurata ◽  
Binding Capacity ◽  
Specific Binding ◽  
Head Kidney ◽  
Gilthead Sea Bream ◽  
Sea Bream ◽  
Single Class ◽  
Lower Vertebrate

Abstract Receptors for GH were characterized in the head kidney of gilthead sea bream (Sparus aurata), using radioiodinated and biotinylated ligands. The specific binding of radiolabelled recombinant gilthead sea bream GH (rsbGH) to head kidney membrane preparations was dependent on membrane concentration. Salmon prolactin, salmon gonadotrophin and carp gonadotrophin did not compete for 125I-labelled rsbGH-binding sites. Unlabelled rsbGH competitively displaced 125I-labelled rsbGH bound to head kidney membranes. Scatchard plots were always linear, denoting the presence of a single class of binding sites. The binding affinity (Ka=2·7 × 109 m−1) was equivalent to that found in liver membrane preparations, but the binding capacity (2·5 ±0·30 fmol/mg protein) was 50- to 75-fold lower. To identify the cells which express the GH receptor, head kidney smears were incubated with biotinylated rsbGH, followed by incubation with an avidin–biotin complex conjugated to alkaline phosphatase. The reaction with the new-fuchsin substrate gave a red precipitate, showing a specific and intense labelling in erythroblasts, polychromatophilic erythroblasts and myeloblasts. Noticeable binding was observed in myelocytes and immature granulocytes, tending to disappear at the latter stages of granulocyte maturation. Light but appreciable binding was also observed in monocytes, lymphocytes and acidophilic erythroblasts, whereas it was completely absent in proerythrocytes and erythrocytes. The proliferative action of rsbGH and recombinant human IGF-I on in vitro cultures of head kidney cells was demonstrated by a 5-bromo-2′-deoxy-uridine immunoassay. To our knowledge, this is the first report that provides suitable evidence for a role of GH as a haemopoietic growth and differentiation factor in lower vertebrate species. Journal of Endocrinology (1995) 146, 459–467

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