scholarly journals Characterization of specific V1a vasopressin-binding sites on a rat mammary-tumour-cell line

1986 ◽  
Vol 240 (1) ◽  
pp. 189-196 ◽  
Author(s):  
G Guillon ◽  
C J Kirk ◽  
M N Balestre
Keyword(s):  
Cell Line ◽  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Mammary Tumour ◽  
Tumour Cell Line ◽  
Vasopressin Receptor ◽  
Single Class ◽  
Ligand Selectivity ◽  
Plot Analysis

WRK 1, a cloned cell line derived from a rat mammary tumour, carries specific vasopressin-binding sites. Specific binding of 2-tyrosine-3H-labelled [8-lysine]vasopressin ([3H]vasopressin) was time-dependent, saturable and reversible. Scatchard-plot analysis of hormone binding indicated the presence of a single class of receptors with an equilibrium dissociation constant of 12.7 +/- 0.2 nM. The maximal binding capacity was 75 +/- 6 fmol/10(6) cells, which corresponds to approx. 45,000 sites per cell. Oxytocin and a highly potent oxytocin analogue were able to inhibit completely [3H]vasopressin binding, but, in this respect, they were far less potent than vasopressin. This clearly demonstrates the vasopressinergic nature of this receptor. Pharmacological studies using a series of 14 vasopressin or oxytocin analogues indicated that the ligand selectivity of the vasopressin receptor found on WRK 1 cells resembles that of the rat hepatocyte. This signifies that this vasopressin receptor is of the V1a subtype. This conclusion was confirmed by the observation that vasopressin did not influence the production of intracellular cyclic AMP in WRK 1 cells.

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

Oxytocin receptors in rat kidney during development

1990 ◽  
Vol 259 (6) ◽  
pp. F872-F881 ◽  
Author(s):  
A. Schmidt ◽  
S. Jard ◽  
J. J. Dreifuss ◽  
E. Tribollet
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Rat Kidney ◽  
Distal Tubule ◽  
Juxtaglomerular Apparatus ◽  
Binding Studies ◽  
Single Class ◽  
Ligand Selectivity ◽  
Oxytocin Receptors ◽  
Two Stages

The development and characteristics of oxytocin (OT) receptors in the rat kidney were studied by light-microscopic autoradiography and on membrane preparations using the iodinated OT antagonist 125I-d(CH2)5[Tyr(Me)2,Thr4,Orn8,Tyr(NH2)9]OT. Specific binding was first detected by autoradiography at embryonic day 17 (E17) in both the cortex and the medulla. Cortical labeling was found thereafter at all ages examined including in the adult (postnatal day 90, PN90). It was localized on the distal tubule at the level of the juxtaglomerular apparatus. Medullary binding was detected only transiently during two stages of development, first, before PN6, and second, at the approximate time of weaning (PN20-PN30). Binding studies on crude membranes prepared from whole kidneys of animals aged between PN1 and PN15 showed a single class of high-affinity binding sites, with a dissociation constant of 0.13 +/- 0.08 nM. Thus transient OT binding sites expressed in the medulla do not differ from cortical OT binding sites; moreover, the ligand selectivity of kidney OT receptors regardless of location and age appears similar to that of previously characterized OT receptors. Our results suggest that OT may play a role in both renal development and renal function.

scholarly journals Substance P receptors in rat spleen: characterization and autoradiographic distribution

Blood ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1398-1401 ◽  
Author(s):  
CJ Wiedermann ◽  
K Sertl ◽  
CB Pert
Keyword(s):  
Substance P ◽  
Binding Sites ◽  
Marginal Zone ◽  
Specific Binding ◽  
Single Class ◽  
Ligand Selectivity ◽  
Tissue Sections ◽  
The Brain ◽  
Substance P Receptors ◽  
Red Pulp

Abstract The interaction of substance P with intact lymphatic tissue was quantified and autoradiographically visualized, using slide-mounted tissue sections of rat spleen. Radiolabeled substance P binds rapidly to an apparently single class of noninteracting high affinity sites (Kd = 2.4 nmol/L; Bmax = 9.4 fmol/mg protein). The ligand selectivity pattern suggests that substance P binding sites are similar to substance P receptors found in other tissues, including the brain, T lymphocytes, and macrophages. Substance P receptors are highly concentrated in the antigen-trapping spleen marginal zone, with low densities being found in the red pulp. No specific binding of radiolabel to T cell-dependent immunologic domains of the spleen is seen. The distribution of substance P receptors suggests that substance P is probably involved in the control of sensory functions of the immune system.

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

scholarly journals Substance P receptors in rat spleen: characterization and autoradiographic distribution

Blood ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1398-1401
Author(s):  
CJ Wiedermann ◽  
K Sertl ◽  
CB Pert
Keyword(s):  
Substance P ◽  
Binding Sites ◽  
Marginal Zone ◽  
Specific Binding ◽  
Single Class ◽  
Ligand Selectivity ◽  
Tissue Sections ◽  
The Brain ◽  
Substance P Receptors ◽  
Red Pulp

The interaction of substance P with intact lymphatic tissue was quantified and autoradiographically visualized, using slide-mounted tissue sections of rat spleen. Radiolabeled substance P binds rapidly to an apparently single class of noninteracting high affinity sites (Kd = 2.4 nmol/L; Bmax = 9.4 fmol/mg protein). The ligand selectivity pattern suggests that substance P binding sites are similar to substance P receptors found in other tissues, including the brain, T lymphocytes, and macrophages. Substance P receptors are highly concentrated in the antigen-trapping spleen marginal zone, with low densities being found in the red pulp. No specific binding of radiolabel to T cell-dependent immunologic domains of the spleen is seen. The distribution of substance P receptors suggests that substance P is probably involved in the control of sensory functions of the immune system.

Specific binding sites for ovine prolactin in three amphibian cell lines

1985 ◽  
Vol 248 (1) ◽  
pp. C80-C87 ◽  
Author(s):  
M. Dunand ◽  
M. L. Aubert ◽  
J. P. Kraehenbuhl ◽  
B. C. Rossier
Keyword(s):  
Growth Hormone ◽  
Cell Lines ◽  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Human Growth ◽  
Functional Differentiation ◽  
Water Metabolism ◽  
Ovine Prolactin ◽  
Follicle Stimulating Hormones

Established cell lines (TB-6c and TB-M) obtained by continuous culture of epithelial cells from toad Bufo marinus urinary bladder, which, in culture, maintained a high degree of functional differentiation, exhibited a significant number of high-affinity (KA = 1-2 X 10(10) M-1) binding sites detected both with radioiodinated (125I) ovine prolactin (oPRL) and human growth hormone (hGH). Binding capacity was higher in the case of TB-6c cells (7,573 +/- 581 sites/cell) than with the TB-M cells (1,160 +/- 87). Similarly, binding sites for oPRL were characterized on Xenopus laevis kidney-derived cell line A6. With oPRL used both as tracer and standard, significant cross-reaction was observed with hGH, less with human or rat prolactin (PRL), and none with human chorionic somatomammotropin, bovine growth hormone, and rat luteinizing hormone or follicle-stimulating hormones. B. marinus pituitary extracts completely displaced the binding of 125I-oPRL to toad bladder binding sites. This finding of specific sites for PRL on amphibian bladder and kidney cells confirms that PRL exerts specific biological actions for the control of electrolyte and water metabolism in the amphibians.

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.

scholarly journals Identification and analysis of human erythropoietin receptors on a factor-dependent cell line, TF-1

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 375-380 ◽  
Author(s):  
T Kitamura ◽  
A Tojo ◽  
T Kuwaki ◽  
S Chiba ◽  
K Miyazono ◽  
...  
Keyword(s):  
Cell Line ◽  
Binding Sites ◽  
Bone Marrow Cells ◽  
Scatchard Analysis ◽  
Hematopoietic Growth Factors ◽  
Single Class ◽  
Gm Csf ◽  
Molecular Weights ◽  
Human Erythropoietin ◽  
Macrophage Colony

Abstract We have recently established a novel cell line, TF-1, from bone marrow cells of a patient with erythroleukemia, that showed an absolute growth dependency on each of three hematopoietic growth factors: erythropoietin (EPO) granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin 3 (IL-3). EPO stimulated the proliferation of TF-1 cells even at the physiologic concentration (0.03 U/mL). We performed binding experiments on TF-1 cells using radioiodinated EPO. The binding of radioiodinated EPO to TF-1 was specific, time- and temperature-dependent, and saturable. Scatchard analysis of the saturation binding data suggested the existence of a single class of binding sites (kd = 0.40 nmol/L; number of binding sites = 1,630 per cell). TF-1 cells were usually maintained in RPMI 1640 containing 10% fetal bovine serum and 5 ng/mL GM-CSF. The kd and the number of the EPO receptors were not changed by incubating the cells with IL-3, although culturing the cells in the presence of EPO resulted in down-modulation of EPO receptors. The chemical cross-linking study demonstrated that two molecules with apparent molecular weights of 105 kilodalton (Kd) and 90 Kd were the binding components of EPO. Present data suggest that human EPO receptors are very similar to the previously reported murine EPO receptors.

Dissociation of contraction and muscarinic receptor binding to isolated smooth muscle cells

1986 ◽  
Vol 251 (4) ◽  
pp. G546-G552 ◽  
Author(s):  
S. M. Collins ◽  
D. J. Crankshaw
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Binding Sites ◽  
Specific Binding ◽  
Muscle Cells ◽  
Muscarinic Agonists ◽  
Isolated Cells ◽  
Single Class ◽  
Antagonist Binding ◽  
Isolated Smooth Muscle Cells

We examined changes in [3H]QNB binding and cell length induced by muscarinic ligands in a suspension of single smooth muscle cells isolated from the canine stomach. Cells contracted following a brief (30 s) exposure to picomolar concentrations of muscarinic agonists and yielded ED50 values of 1.0 +/- 0.7 pM for oxotremorine, 12.5 +/- 1.8 pM for carbachol, and 16.0 +/- 2.9 pM for metacholine. Contraction was inhibited by atropine with a pA2 value of 10.2 +/- 1.1. The binding of [3H]QNB was rapid and reversible and was stereospecific and pharmacologically appropriate. Specific binding of [3H]QNB was saturable and bound with high affinity (KD 1.04 +/- 0.23 nM) to a single class of sites, of which there were approximately 200,000/cell. In competition experiments antagonist binding was generally homogeneous, whereas that of agonists was heterogeneous and subpopulations of binding sites with different affinities for agonists were identified. The Ki value of 8.1 +/- 1.1 nM for inhibition of QNB binding by atropine was greater than the pA2 of 10.2 +/- 1.1 derived from contraction studies. Furthermore, whereas picomolar concentrations of agonists induced cell contraction, substantially higher concentrations (10 nM to 10 mM) were required to inhibit [3H]QNB binding to the isolated cells.

Mineralcorticoid receptors along the nephron: [3H]aldosterone binding in rabbit tubules

1981 ◽  
Vol 241 (6) ◽  
pp. F605-F611 ◽  
Author(s):  
A. Doucet ◽  
A. I. Katz
Keyword(s):  
Binding Sites ◽  
Binding Capacity ◽  
Specific Binding ◽  
Rabbit Kidney ◽  
Scatchard Analysis ◽  
Thick Ascending Limb ◽  
Collecting Tubule ◽  
The Difference ◽  
Collecting Tubules ◽  
Cortical Collecting Tubule

To identify the site of mineralocorticoid action along the nephron, we measured the specific binding of [3H]aldosterone to nephron segments microdissected from aldosterone-deficient rabbits. Specific binding was defined as the difference between binding measured in the absence or in the presence of 2,000-fold excess of unlabeled hormone (in 10(-18) mol X cm tubule length-1 +/- SE). High specific binding capacity was found in the branched collecting tubule (108 +/- 4), the cortical collecting tubule (119 +/- 9), and the outer medullary collecting tubule (115 +/- 16), whereas specific binding was negligible in the proximal convoluted tubule (8 +/- 9), pars recta (2 +/- 6), medullary thick ascending limb (4 +/- 6), cortical thick ascending limb (6 +/- 2), and distal convoluted tubule (6 +/- 6). In cortical collecting tubules, Scatchard analysis of the specific [3H]aldosterone binding indicated a dissociation constant (KD) of 2.2 X 10(-9) M and a maximum number of binding sites of 157 X 10(-18) mol X cm tubule length-1. The steroid specificity was assessed from the competition of various steroids for [3H]aldosterone binding sites. Receptors from the cortical collecting tubule revealed the following sequence of affinities: aldosterone greater than DOCA greater than spironolactone greater than dexamethasone greater than 5 alpha-dihydrotestosterone = progesterone = 17 beta-estradiol, indicating that the binding sites in the collecting tubule are mineralocorticoid receptors. These results demonstrate significant [3H]aldosterone binding to receptors of high affinity and mineralocorticoid specificity only in the collecting tubule and suggest that this nephron segment is the target site of mineralocorticoid action in the rabbit kidney.

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