Binding of aldosterone to cytoplasmic and nuclear receptors of the urinary bladder epithelium of Bufo marinus

1978 ◽  
Vol 235 (3) ◽  
pp. C82-C89 ◽  
Author(s):  
M. Kusch ◽  
N. Farman ◽  
I. S. Edelman
Keyword(s):  
Urinary Bladder ◽  
Binding Capacity ◽  
Bufo Marinus ◽  
Scatchard Analysis ◽  
Type I ◽  
Bladder Epithelium ◽  
Nuclear Binding ◽  
Equilibrium Dissociation ◽  
Dissociation Constant Kd ◽  
Apparent Equilibrium

Binding of aldosterone to cytoplasmic and nuclear sites in urinary bladder epithelia of Bufo marinus (Dominican variant) is saturable and dependent upon steroid concentration. Scatchard analysis of specific cytoplasmic binding yielded a maximal binding capacity (N) of 14.5 X 10(-14) mol/mg protein and an apparent equilibrium dissociation constant (Kd) of 1.4 X 10(-8) M. Since Scatchard analysis of specific nuclear binding was complex, this binding was resolved by a computer-generated cirve-fitting technique which analyzed total aldosterone bound. Nuclear binding was resolved into three sites: a nonsaturable site that was linearly dependent upon aldosterone concentration, and two saturable sites (types I and II). Type I sites had relatively low capacity for aldosterone (N = 31 +/- 1 X 10(-14) mol/mg DNA) and high affinity (Kd = 2.5 +/- 0.5 X 10(-9 M); tffininty (Kd = 8.6 +/- 1.7 X 10(-7) M). Competition for [3H]aldosterone binding by dexamethasone, corticosterone, cortisol, progesterone, testosterone, and 17 beta-estradiol demonstrated that type I nuclear sites have higher affinity for aldosterone than for other steroids. The findings are consistent with the inference that the type I site is the mineralocorticoid receptor.

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 Validation of a High Throughput Scintillation Proximity Assay for 5-HydroxytryptaminelE Receptor Binding Activity

1997 ◽  
Vol 2 (1) ◽  
pp. 33-40 ◽  
Author(s):  
Steven D. Kahl ◽  
Frederick R. Hubbard ◽  
G. Sitta Sittampalam ◽  
Joseph M. Zock
Keyword(s):  
High Throughput ◽  
Binding Capacity ◽  
Binding Activity ◽  
Proximity Effects ◽  
Gene Encoding ◽  
Scintillation Proximity Assay ◽  
Receptor Binding Activity ◽  
The Stability ◽  
Equilibrium Dissociation ◽  
Dissociation Constant Kd

Membranes prepared from stable transfected cells expressing the human gene encoding a functional 5-hydroxytryptaminelE (5HT1E) receptor were used to investigate high-affinity [3H]serotonin ([3H]5-HT) binding with scintillation proximity assay (SPA) technology. In this nonseparation format, membranes are captured by WGA-coated SPA fluoromicrospheres for detection of receptor-bound radioligand. Total binding observed was approximately 2000 cpm compared to a nonspecific signal of 100 cpm determined in the presence of 10,uM unlabeled 5-HT. Non-proximity effects (NPE) for the radiolabel and SPA beads averaged less than 100 cpm. Saturation binding analysis yielded an equilibrium dissociation constant (Kd) of 5.38 ± 0.43 nM and a maximum binding capacity (Bmax) of 6.42 + 0.15 pmol/mg protein. Competition with unlabeled serotonergic compounds demonstrated a specificity of the assay with rank potencies (5-HT > a-Me-5-HT > 2-Me-5-HT > 5-CT) similar to those observed using traditional fitration techniques. The variability of the assay and the stability of all reagents were investigated to validate the assay for extended use throughout a typical high throughput screen operation lasting several months.

scholarly journals FVIIIa-mimetic bispecific antibody (Mim8) ameliorates bleeding upon severe vascular challenge in hemophilia A mice

Blood ◽  
2021 ◽  
Author(s):  
Henrik Østergaard ◽  
Jacob Lund ◽  
Per Jr Greisen ◽  
Stine Kjellev ◽  
Anette Henriksen ◽  
...  
Keyword(s):  
Bispecific Antibody ◽  
Hemophilia A ◽  
Factor Ix ◽  
Factor X ◽  
Equilibrium Dissociation Constant ◽  
Biophysical Properties ◽  
Kd Values ◽  
Equilibrium Dissociation ◽  
Dissociation Constant Kd ◽  
Apparent Equilibrium

Hemophilia A (HA) is a bleeding disorder resulting from deficient Factor VIII (FVIII), which normally functions as a cofactor to activated Factor IX (FIXa) that facilitates activation of Factor X (FX). To mimic this property in a bispecific antibody (biAb) format, a screening was conducted to identify functional pairs of anti-FIXa and anti-FX antibodies, followed by optimization of functional and biophysical properties. The resulting biAb (Mim8) assembled efficiently with FIXa and FX on membranes, and supported activation with an apparent equilibrium dissociation constant (KD) of 16 nM. Binding affinity with FIXa and FX in solution was much lower, with KD-values for FIXa and FX of 2.3 and 1.5 µM, respectively. In addition, the activity of Mim8 was dependent on stimulatory activity contributed by the anti-FIXa arm, which enhanced the proteolytic activity of FIXa by four orders of magnitude. In hemophilia A plasma and whole blood, Mim8 normalized thrombin generation and clot formation with potencies 13 and 18 times higher than a sequence-identical analog of emicizumab, respectively. A similar potency difference was observed in a tail-vein transection model in hemophilia A mice, while reduction of bleeding in a severe tail-clip model was observed only for Mim8. Furthermore, the pharmacokinetics of Mim8 were investigated and a half-life of 14 days demonstrated in cynomolgus monkey. In conclusion, Mim8 is a FVIIIa-mimetic with a potent and efficacious hemostatic effect based on preclinical data.

Platelet 3H Ketanserin Binding in Migraine

Cephalalgia ◽  
2001 ◽  
Vol 21 (5) ◽  
pp. 567-572 ◽  
Author(s):  
R Shukla ◽  
VK Khanna ◽  
S Pradeep ◽  
M Husain ◽  
R Tandon ◽  
...  
Keyword(s):  
Dissociation Constant ◽  
Clinical Features ◽  
Binding Sites ◽  
Scatchard Analysis ◽  
Healthy Controls ◽  
Equilibrium Dissociation Constant ◽  
Binding Characteristics ◽  
Number Of Binding Sites ◽  
Equilibrium Dissociation ◽  
Dissociation Constant Kd

Platelet 3H ketanserin binding was studied in 33 patients of migraine and 30 healthy controls. The binding characteristics: equilibrium dissociation constant (Kd) and maximal number of binding sites (Bmax) determined by Scatchard analysis revealed a significant decrease in Kd and no change in Bmax in migraine cases. No correlation was observed between the Kd and Bmax with the clinical features of migraine. The findings of the present study show that there is a decreased affinity of platelet 5-HT2 receptors in migraine.

Effects of type I interferons on IGF-mediated autocrine/paracrine growth of human neuroendocrine tumor cells

2009 ◽  
Vol 296 (3) ◽  
pp. E559-E566 ◽  
Author(s):  
Giovanni Vitale ◽  
Peter M. van Koetsveld ◽  
Wouter W. de Herder ◽  
Katy van der Wansem ◽  
Joop A. M. J. L. Janssen ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Neuroendocrine Tumor ◽  
Binding Capacity ◽  
Type I Interferons ◽  
Scatchard Analysis ◽  
Type I ◽  
Receptor Mrna ◽  
Igf System ◽  
Igf I ◽  
Bon Cells

We recently demonstrated that interferon (IFN)-β has a more potent antitumor activity than IFN-α in BON cells, a neuroendocrine tumor (NET) cell line. The present study showed the role of type I IFNs in the modulation of the insulin-like growth factor (IGF) system in NETs. BON cells expressed IGF-I, IGF-II, IGF-I receptor, and insulin receptor mRNA. In addition, IGF-I and IGF-II stimulated the proliferation of BON cells and induced an inhibition of DNA fragmentation (apoptosis). As evaluated by quantitative RT-PCR, treatment with IFN-α (100 IU/ml) or IFN-β (100 IU/ml) inhibited the expression of IGF-II mRNA (−42% and −65%, respectively, both P < 0.001), whereas IGF-I receptor mRNA was significantly upregulated by IFN-α (+28%, P < 0.001) and downregulated by IFN-β (−47%, P < 0.001). Immunoreactive IGF-II concentration decreased in the conditioned medium during IFN-α (−16%, P < 0.05) and IFN-β (−69%, P < 0.001) treatment. Additionally, IGF-I receptor bioactivity was reduced (−54%) after IFN-β treatment. Scatchard analysis of 125I-labeled IGF-I binding to cell membrane of BON cells revealed a dramatic suppression of maximum binding capacity only in the presence of IFN-β. Finally, the proapoptotic activity of IFN-β was partially counteracted by the coadministration of IGF-I and IGF-II (both at 50 nM). In conclusion, these data demonstrate that the IGF system has an important role in autocrine/paracrine growth of BON cells. The more potent antitumor activity of IFN-β compared with IFN-α could be explained by several effects on this system: 1) both IFNs inhibit the transcription of IGF-II, but the suppression is significantly higher after IFN-β than IFN-α and 2) only IFN-β inhibits the expression of IGF-I receptor.

Somatostatin: occurrence in urinary bladder epithelium and renal tubules of the toad, Bufo marinus

Science ◽  
1980 ◽  
Vol 210 (4470) ◽  
pp. 644-646 ◽  
Author(s):  
J. Bolaffi ◽  
S Reichlin ◽  
D. Goodman ◽  
J. Forrest
Keyword(s):  
Urinary Bladder ◽  
Bufo Marinus ◽  
Renal Tubules ◽  
Bladder Epithelium ◽  
Toad Bufo

Neurotensin and neuromedin N stimulate mucin output from human goblet cells (Cl.16E) via neurotensin receptors

1992 ◽  
Vol 262 (3) ◽  
pp. G470-G476 ◽  
Author(s):  
C. Augeron ◽  
T. Voisin ◽  
J. J. Maoret ◽  
B. Berthon ◽  
M. Laburthe ◽  
...  
Keyword(s):  
Cell Membranes ◽  
Binding Capacity ◽  
Regulatory Peptides ◽  
Scatchard Analysis ◽  
Terminal Part ◽  
Neurotensin Receptors ◽  
Binding Data ◽  
Mucin Release ◽  
Neuromedin N ◽  
Dissociation Constant Kd

The stably differentiated human intestinal goblet cell line Cl.16E was used to study the effects of two structurally related regulatory peptides, neurotensin (NT) and neuromedin N (NN), on mucus secretion. NT and NN stimulated rapid release of mucins from filter-grown Cl.16E cells, this effect being dose related with a mean effective dose of 36 nM for NT and 422 nM for NN. The order of potency of NT, three NT fragments corresponding to the NH2-terminal part [NT-(1-11)] or to the COOH-terminal part [NT-(8-13) and NT-(9-13)], and NN in promoting mucin release and in inhibiting 125I-labeled NT binding to Cl.16E cell membranes was identical with NT greater than or equal to NT-(8-13) greater than NN greater than NT-(9-13) much greater than NT-(1-11) supporting the hypothesis that NT and NN stimulate mucin output through interaction with a common NT-preferring receptor. Scatchard analysis of equilibrium binding data showed one population of NT binding sites in Cl.16E cell membranes with the following characteristics: binding capacity (Bmax) was 141 fmol/mg of protein and dissociation constant (Kd) was 1.00 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

Aldosterone receptor occupancy and sodium transport in the urinary bladder of Bufo marinus

1978 ◽  
Vol 235 (3) ◽  
pp. C90-C96 ◽  
Author(s):  
N. Farman ◽  
M. Kusch ◽  
I. S. Edelman
Keyword(s):  
Urinary Bladder ◽  
Short Circuit ◽  
High Capacity ◽  
Receptor Occupancy ◽  
Short Circuit Current ◽  
Bufo Marinus ◽  
Type I ◽  
Maximal Increase ◽  
Aldosterone Receptor ◽  
The Relationship

The concentration dependence of binding of [3H]aldosterone to cytoplasmic and nuclear receptors was evaluated in urinary bladder epithelial cells of Colombian toads. One class of specific sites (sensitive to displacement by excess aldosterone) was detected in the cytosol. However, two classes of specific nuclear [3H]aldosterone binding sites were evident. In the nucleus, high-affinity (Kd = 2.7 X 10(-9) M), low-capacity (N = 15 X 10(-14) mol/mg DNA) sites (type I) were completely saturated at approximately 4 X 10(-8) M aldosterone, a concentration which gave a maximal increase in short-circuit current (SCC). Occupancy of low-affinity (Kd = 4.6 X 10(-7) M), high-capacity sites (N = 150 X 10(-14) mol/mg DNA) (type II) occurred at higher steroid concentrations and did not correlate with further increase in SCC. Binding parameters of Colombian and Dominican variants of Bufo marinus were compared. In both variants, the SCC increase elicited by aldosterone correlated with accumulation of type I complexes in the nucleus, but the relationship was markedly nonlinear. Various alternatives were considered as the basis for a curvilinear dependence of the increment in Na+ transport on abundance of nuclear type I complexes.

Hypersensitivity to Thromboxane A2 in Cholesterol-Rich Human Platelets

1990 ◽  
Vol 64 (04) ◽  
pp. 594-599 ◽  
Author(s):  
Takuya Tomizuka ◽  
Kyohei Yamamoto ◽  
Aizan Hirai ◽  
Yasushi Tamura ◽  
Sho Yoshida
Keyword(s):  
Calcium Concentration ◽  
Binding Capacity ◽  
Thromboxane A2 ◽  
Cytosolic Calcium ◽  
Cholesterol Content ◽  
Human Platelets ◽  
Dissociation Rate ◽  
Platelet Membrane ◽  
Maximal Concentration ◽  
Equilibrium Dissociation

SummaryThe effect of changes in platelet membrane cholesterol content on thromboxane A2 (TXA2)-induced platelet activation was studied. Concentrations of 9,ll-epithio-ll,12-methano-TXA2 (STA2), a stable analogue of TXA2 which can cause half-maximal aggregation and release of [14C]serotonin in cholesterol-rich platelets were significantly lower than those in cholesterol-normal platelets. STA2-induced increase in cytosolic calcium concentration and [32P]phosphatidic acid formation in cholesterol-rich platelets were significantly greater than those in cholesterol-normal platelets. The maximal concentration of binding site (Bmax) for SQ29548 was significantly increased in cholesterol-rich platelets compared with cholesterol-normal platelets, while the equilibrium dissociation rate constant (Kd) for SQ29548 did not differ between cholesterol-rich and cholesterol-normal platelets. The present study suggested that sensitivity to TXA2 was increased by the incorporation of cholesterol into platelet membrane and that the cause of hypersensitivity to TXA2 in cholesterol-rich platelets may be partly explained by an increase in binding capacity for TXA2.

Initial effects of cyclophosphamide on urinary bladder epithelium in the rat

Pathology ◽  
1974 ◽  
Vol 6 (4) ◽  
pp. 343-350 ◽  
Author(s):  
Mary E. Schultz ◽  
Michael W. Weldon
Keyword(s):  
Urinary Bladder ◽  
Bladder Epithelium
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